Molecular Origins of Human Attention

The Dopamine-Folate Connection

by

Richard C. Deth Ph.D.

North Eastern University

Kluwer Academic Publishers

Boston/Dordrecht/London

1
2
3
To those paying for the gifts of others

4
FOREWORD

It has indeed been a strange trip. Back in 1994 I was a cardiovascular pharma-
cologist studying molecular mechanisms that cause blood vessels to contract. I had
reliable funding from the National Institutes of Health for this line of research. I lec-
tured pharmacy students on the treatment of hyper-tension, heart failure and renal
failure. My academic career path had been a logical, straight-line journey. I was
making progress in my chosen field, the field in which I was originally trained.
Where did I go wrong?
Something novel caught my attention. There was a structural pattern within
the family of neurotransmitter receptors we were studying, but there was this one
exception. Therein lies the story. The exception turned into a mystery about why
nature made this particular dopamine receptor, the D4 receptor subtype, different
from all the others. Pursuing the answer to that mystery has taken me far off the
straight-line route into dark and previously unexplored scientific realms, It may
sound a bit melodramatic, but take it from me, there were indeed a number of dark
moments. Literally, one thing led to another and soon my lab found that we were
working amidst tantalizing clues about mental illnesses such as schizophrenia and
autism. Some of the clues were dusty lines of biochemical investigation that seemed
clear and important but never quite connected to the core theories held by promi-
nent researchers in the field. As we investigated the relationship between this
unique dopamine receptor and the folic acid pathway we found the missing connec-
tions. Slowly we began to understand how problems in folate or methionine metab-
olism might impair the normal role of dopamine in attention and lead to psychiatric
problems including ADHD.
With our novel perspective we gradually explored mechanisms that control
folate-dependent methylation events in neural tissues. In doing so we uncovered
additional connections to psychiatric conditions. A case in point was the discovery
that insulin-like growth factor-1 (IGF-1) powerfully stimulated these reactions, pro-
viding a connection to developmental disorders and a new explanation for the age-
dependent onset of schizophrenia. The highly potent and specific inhibitory effects
of ethanol forged links to alcoholism. Discovery of copper’s critical role made a link
to Alzheimer’s disease. Perhaps most dramatic was the remarkably potent toxicity
of thimerosal, the ethyl-mercury releasing preservative found in vaccines, creating a
clear but sinister connection to autism.
Impaired attention plays a significant role in each of these psychiatric condi-
tions and it is the molecular basis of attention and attention-related learning that
binds them together. In writing this book I have attempted to synthesize and inte-
grate research from a number of areas in order to arrive at a reasonable and cohe-
sive description of the mechanism of attention. Of necessity it is a molecular

5
description, but our capacity for attention originate at the molecular level. Unique
molecular features of D4 dopamine receptors found only in humans and other pri-
mates provide a conceptual framework for attention-based learning. Using these
novel perspectives it is possible to appreciate how genetic and environmental fac-
tors cause disorders of attention. It is my hope that these molecular perspectives
might lead to more effective, mechanism-based treatment approaches for these dis-
orders.
I have tried to make these fascinating molecular events accessible to as many
readers as possible but, like every worthwhile mystery, it is a somewhat complex
story. In particular I hope that motivated parents who are seeking answers can
find some within these pages despite the somewhat technical vocabulary.
The process of writing this book has itself led to new insights and stimulated
new laboratory work. Specifically, the recognition that pieces of the autism and per-
vasive developmental disorders puzzle fit together within the unifying theme of
impaired DNA methylation was a direct outgrowth of writing Chapter 13. In subse-
quent collaborative studies we found that insulin-like growth factor-1, dopamine
and thimerosal all cause profound changes in DNA methylation.
I offer a disclaimer about my scientific limitations. My perspective is biased
by the particular research findings made in my lab and I view the molecular mecha-
nism of attention from this perspective. Opinions expressed herein are those of the
author. The molecular framework I describe is meant to be a provocative stimulus
for further investigation and confirmation. My personal expertise is based upon
knowledge of molecular function rather than clinical experience. This is actually a
good thing, since it is molecular-level information that holds the key to understand-
ing most mental illnesses. Fictional scenarios are used to illustrate several psychiat-
ric conditions. They are meant to emphasize that while the topic may be molecules,
the true arena is the human condition.
Finally, I urge you to consider wandering off the straight path every now
and then. Investigate something you find novel and interesting. Trust your
attention-based instincts and explore. You never know what you might find.
Richard.C.Deth
Professor.
North Eastern University,
Office: 312 Mugar Hall,
Voice: 617.373.4064,
Fax: 617.373.8886,
E-mail: r.deth@neu.edu.

6
ACKNOWLEGEMENTS

I am deeply grateful to the many graduate students and post-doctoral fellows

whose dedicated work in our laboratory not only generated data and got them their
degrees but also blazed a trail of discovery that I have been fortunate to share. Mar-
tin Kramer, Alok Sharma and Peter Wick were the first to embark down the murky
path leading to the secrets of the dopamine D4 receptor, starting in 1995. Marty
explored its unusual binding behavior and made initial connections with methio-
nine metabolism. Peter worked out a reasonably facile method for measuring the
movement of folate-derived methyl groups through the receptor. Alok used this
method to outline the basic features of the cycle of dopamine-stimulated phospho-
lipid methylation, including its pharmacological specificity and its regulation by
external factors. Alok provided a strong platform upon which others would build.
Mostafa Waly deserves special recognition. He first earned his M. S. degree
from Alexandria University in Egypt and quickly established himself as the resident
expert in nutrition and biochemistry within our pharmacology environment at
Northeastern University. Mostafa’s skill and energy in the laboratory were responsi-
ble for key findings about the potent effects of ethanol and the regulatory role of
insulin-like growth factor-1. He also demonstrated the toxic effects of heavy metals
and characterized the potent toxicity of thimerosal. Among many other students
making significant contributions to the D4 receptor story were Sung Shim, DaPei
Liu, Wenbo Tan, Ren Zhao, Suchitra Chan, Micki Nagata, Yuen Chen, Fran Power-
Charnitsky and Jorge Benzecry. Timur Turkdogman was brave enough to take on
the task of developing a computer simulation of dopamine stimulated phospholipid
methylation for his doctoral thesis in mathematics. He also provided wise advice
during the writing of this manuscript.
Collaborations were a vital in allowing the molecular events of attention
come into focus. Curtis DuRand, then at the VA Hospital in Bedford Massachusetts,
supervised clinical studies showing that defective phospholipid methylation was a
feature of schizophrenia. He also brought a great deal of methionine-related litera-
ture to my attention and fostered an optimistic outlook during early stages of the
work. Martha Teeter, his wife and x-ray crystallographer par excellence, mentored
me in the molecular modeling of dopamine receptors and gave me an appreciation
of how protein structure dictates function. During her several years in my lab
YaFang Liu made key observations about how the D4 receptor interacts with other
proteins via its primate-specific repeat segments. Ruma Banerjee, at the University
of Nebraska, generously agreed to measure methionine synthase activity, a non-
trivial task, and helped to show its remarkable dependence on P13-kinase signaling.
Sang Woon Choi and Joel Mason at the Tufts University/USDA Human Nutrition
Research Center used their newly developed mass spectrometry assay to demon-
strate changes in DNA methylation associated with P13-kinase interventions and

7
thimerosal. Barbara Malewicz and Malak Kotb carried out phospholipid NMR stud-
ies and methionine adenosyl-transferase assays respectively. Their generosity and
willingness to help is greatly appreciated.
I want to extend heartfelt thanks to Mimi Breed and to those at Kiuwer who
had enough interest (and bravery) to publish this book. Mimi prodded me along
through the too many months it took to finish writing and I needed every last e-
mail. The opportunity to assemble a scientific story in the scope allowed by a mono-
graph is a privilege that I don’t underestimate.
In my real life, outside of the office and the laboratory, my wife Maddy has
been the constant anchor, friend and realist that has nurtured me and kept me on
track. Had she not been there for me each of the past 34 years there is absolutely no
doubt that I would have wandered too far off the path in some hair-brained career
choice or enterprise. Together with our children Jessica and Josh we are a family
blessed with the wisdom and guidance of our parents, our grandparents and for that
we will remain eternally grateful.

8
1 THE GIFT OF ATTENTION

Attention. From the very first moments of our ex-utero existence it’s what we
want. Even more, it is what we absolutely need for survival. At the beginning we
have no attention to give and can only take from others. Early behavior appears to
be completely instinctual. However, in the ensuing weeks, subtle changes occur that
signal a transition from this primitive instinct-based existence to the signs of an
aware infant. Parents eagerly await this comforting evidence indicating that their
newborn can direct his/her attention to a particular voice or sound. It is an early
confirmation that those special human capabilities, which we take too much for
granted, are safe and sound within the next generation. At least for now the gift of
attention is alive and well. But, as we shall see, the journey is far from over.
What is really going on when a baby shifts its gaze to attend the source of
their mother’s voice? It clearly has more significance than a simple turn of the
head. Perhaps it’s more akin to tuning a radio to a certain frequency so that specific
information can now flow. More like a camera lens zooming in on a discrete por-
tion of the overall scene, elevating it to a higher priority than the unattended back-
ground information. At least for the moment, this is the information that really
counts; the information they are “paying attention” to. The word “paying” of course
implies the capability of giving out attention and the gift of a baby’s attention is one
of those transcendent joys which defines parenthood.
Learning is intimately associated with attention. Although we sense or take
in a vast amount of information, we essentially learn only that portion to which
we have paid attention. In this way attention serves as our discriminator between
passive and active experiences. It is a mechanism by which we can selectively priori-
tize certain sensory information above others. This ability to be selective no doubt
increases our learning efficiency.
Attention has a dynamic, time-dependent nature. When we first “shift
attention” to something fresh it marks the start of an ongoing process which either
wanes to an inattentive state or is terminated by a new episode of attention, There is
a natural tendency for the intensity of our attention to lessen within a second or
two, despite our best efforts to fully maintain it. One important determinant of
the duration and intensity is the familiarity of the object of our attention. Is it new,
interesting or even startling? Or, is it familiar, expected or even dull? Clearly it is
more difficult to sustain attention in the latter case. Novelty is a critical factor in
determining the quality and intensity of attention. Man’s interest in and pursuit
of novelty is one our most defining characteristics.
A particularly important aspect of attention is that we can, at least to some
extent, voluntarily control and direct it. That is to say, it is not a purely instinctual
activity. Of course under special circumstances we involuntarily attend to some-
thing (e.g. a loud noise or any other extraordinary sensory event) by instinct as a

9
part of a startle response. However, during most of our lives we actively choose the
objects of our attention, although our choices may be strongly driven by our per-
sonality, previous experience or other very individualized factors. We literally
choose to invest our attention much in the same manner as we invest other personal
resources. In doing so we shape the pathway of our mental development.
The nature of our immediate physical environment offers only a restricted
array of possible experiences for attention. Obviously we’re more likely to attend
to music in a home environment where music is played, more likely to learn farm-
ing on a farm etc. Advances in communications (especially television) have dramat-
ically increased the availability of virtual experiences, and contemporary life affords
more and more options that compete for our attention. Accordingly, the ability to
invest attention wisely and effectively has become a more important skill. Just think
of all those TV commercials, which are competing for your attention!
Not paying attention has its own usefulness. Imagine how difficult life
would be if you were compelled to notice everything in your environment with the
same high intensity normally reserved for attention. Being able to take things for
granted is extremely helpful for efficient functioning. It allows us to save our atten-
tion for those special things that provoke our interest. Actively choosing not to pay
attention (i.e. ignoring something) can also come in handy. Haven’t you walked
down a city street and averted your eyes and attention away from a needy street per-
son? Of course even as you walked on you really were paying attention, if only inter-
nally. Whether by obliviousness or active ignorance, we are also shaped by the act
of not paying attention.
Given that attention is so important and so uniquely human, you might think
that we should know a lot about it. While some pieces of the puzzle are known it is
surprising that at present there is no broadly held consensus on the critical sequence
of molecular events that leads to attention. Attention-Deficit-Hyper-Activity dis-
order (ADHD) has recently put attention on the public agenda. ADHD is the cen-
ter of a difficult, public debate on whether or not this condition is indeed a discrete
illness as opposed to a personal trait. Who should be treated? What are the long-
term consequences of treatment at a young age? This debate has added urgency to
our interest in understanding the molecular basis of attention, with the expectation
that once we understand the normal mechanism, we will be better able to address
the issue of ADHD as either a disease or a personality trait.
While a consensus molecular mechanism of attention may be lacking, there is
no shortage of relevant research observations, and several crucial elements in the
mechanism are known. Throughout these observations it is apparent that the neu-
rotransmitter dopamine plays a central role. For example, interventions, which
increase or decrease dopamine can increase or decrease attention respectively.
Methylphenidate (Ritalin®), the most common treatment for ADHD, blocks the
removal of dopamine at nerve endings, and the resultant increase in dopamine
levels is thought to be the basis for its clinical effectiveness. Amphetamines and

10
cocaine share this action and also act to increase attention. We drink coffee to
get the boost provided by caffeine’s increase of dopamine release. Dopamine
activity is obviously critical to one or more steps in the molecular mechanism of
attention.

Figure 1.1: The chemical structures of dopamine, norepinephrine and epinephrine.

A 3D Model of DOPAMINE

11
 Dopamine (C8H11NO2) is a very simple molecule, consisting of only
twenty atoms (Figure 1.1). Six carbon atoms form a phenyl ring to which two
hydroxyl (OH) groups are attached. This arrangement of dual hydroxyl groups next
to each other on a phenyl ring is termed a “catechol” group. A short two-carbon
chain is attached to the other end of the ring, and a positively-charged nitrogen at
the end of the chain forms an “amino” group (to qualify as an amine there must
be a connection to nitrogen). Together these features make dopamine a member
of the “catecholamine” family, which includes epinephrine (C9H13NO3 - also
known as adrenaline, the “fight or flight” stimulant) and norepinephrine
(C8H11NO3), the primary catecholamine neurotransmitter in the peripheral ner-
vous system (PNS as opposed to the central nervous system - the brain + spinal
cord). Indeed, in some nerves dopamine is a precursor for synthesis of norepineph-
rine, which in turn can be made into epinephrine (dopamine->norepinephrine-
>epinephrine) (Bob’s-Notes: Though food contains dopamine, it cannot cross the
blood-brain-barrier-BBB for use in the brain. So how does one “get” dopamine? It
is synthesized in the neurons in the brain and the adrenal glands atop the kidneys,
from L-Tyrosine and to a lesser extent from L-Phenylalanine and L-DOPA - all
three being amino-acids. L-Tyrosine is NOT an essential amino acid because it can
be made in the body from L-Phenylalanine-which is an essential amino acid; one
that must be eaten: found in many high-protein food products such as chicken, tur-
key, fish, peanuts, almonds, avocados, milk, cheese, yogurt, cottage cheese, lima
beans, pumpkin seeds, sesame seeds, bananas, and soy products. All nine essential
amino acids can only come from a food source. There you have it -give or take.)
During the past several years research from my laboratory has uncovered sev-
eral novel activities of dopamine that appear to be particularly important for its
role in the molecular mechanism of attention. These new insights reveal how
dopamine can modulate and amplify the synchronized electrical activity of brain
waves, leading to what we commonly refer to as attention. But this is just the first
step. Dopamine also triggers learning of attended information. It literally
shapes our brains, starting from the moment of birth, or maybe even
before. Our findings also have important implications for the origin of psychiatric
illnesses like autism and schizophrenia that are directly or indirectly related to the
mechanism of attention.
Attention doesn’t function exactly the same in everyone and DNA
sequences in the recently deciphered human genome hold many secrets about what
makes humans uniquely gifted among all creatures, current or past. From a knowl-
edge and understanding of the molecular events that allow for human attention we
can trace the factors that influence these dopamine stimulated events all the way
back to their genetic origins. In doing so we get a glimpse into how genes acquired
from our ancestors can shape our individual behavior, our personal identity and
our lives.

12
Chapter Summary:
• Attention allows the prioritization of selected information above a background of
awareness.
• The capacity for attention is a critical skill that is particularly well-developed in humans.
• Attention is closely linked to our ability to learn; we learn that to which we attend.
• How and when we use attention guides our personal development.
• The neurotransmitter dopamine plays a crucial role in the molecular mechanism of
attention.
• Person to person differences in the quality of attention are common and in some cases can
be traced to genetic origins.
• Disturbances in the molecular mechanism of attention can result in mental illness.

13
2 A MOLECULAR BASIS FOR ATTENTION

The molecular mechanisms that provide humans with the capacity for atten-
tion and attention-based learning are necessarily somewhat complex to understand.
If they were simple we would have figured them out long ago. That’s the bad news.
The good news is that the mechanisms aren’t so complex that we can’t learn to
understand them. Sure the brain is the most complexly organized structure in the
body and even the most sophisticated computers currently available don’t come
close to reproducing its abilities. Nonetheless we can harness our own powers of
attention to focus in on those particular aspects which specifically relate to
attention (Bob’s Note - introspection - and if and when we have sorted it all out
we can be considered to have reached moksha - It must be really powerful to con-
sistently know what you are thinking). There is a vast amount of neuro-scientific
knowledge providing clues for solving this puzzle and recent breakthroughs create a
new framework for understanding attention.
Since neuronal mechanisms are complex and the language of molecular sci-
ence is not shared by all, it is helpful to have the “Big Picture” in place right from the
beginning, providing a guide for the more detailed information that follows. With
that goal in mind, this chapter outlines the main elements of the molecular basis of
attention before we move on to explore the specific events in greater detail.
From Molecules to Behavior
First, let me assure you that there is indeed a molecular basis for attention,
as well as for every other ability we possess, for every thought we ponder and for
every action we take. On those rare occasions when we stop to think about it, most
of us understand and accept this concept, but we operate most efficiently when we
aren’t aware of what molecular events are occurring. The beauty of our molecular
system of attention and consciousness is that it is totally “transparent” to the
user. In this case ignorance is bliss and a lot less work. However, in those times
when these normally invisible systems go wrong and our autopilot fails, we are par-
ticularly drawn into the questions of why and how our brain operates the way it
does.
One of the primary functions of brain cells (i.e. nerves or neurons) is to
take in information via our sensory systems, convert it to an electrical form and
transmit this electrical activity to other brain cells, creating a useful internal rep-
resentation (mapping) of the world we experience. We use this representation to
guide both our thoughts and our actions. Under a microscope individual neurons
look generally the same, something like the example in Figure 2.1. They have
branches at one end for receiving information from other neurons (the dendrites).
They have a central cell body (the soma) that contains the nucleus with its DNA,
and typically they have a long extension coming out of the soma (the axon) which
travels for some distance before terminating in synaptic connections onto the next

14
target neuron(s). At a synapse the electrical activity (i.e. neuronal firing) triggers
release of neurotransmitter chemicals (e.g. dopamine or glutamate) that diffuse
across a small gap to the next neuron where they bind to specific receptor pro-
teins and trigger a response. The response may lead to electrical activation of den-
drites in the next nerve, sending the information forward another step.
Alternatively, some neurotransmitters act to modulate the flow of information
across the synapse by affecting release events at the presynaptic neuron or by alter-
ing the responsiveness of postsynaptic receptors..

Figure 2.1: A prototypical neuron. Information, in the form of electrical excitation,

flows from dendrites to the soma and then along the axon to the neurochemical
synapse with the next neuron (target cell).

15
16
 Of course all neurons are not the same at the molecular level. Nerve cells dif-
fer from one another because of the specific protein molecules they express both on
their surface and internally in their cytoplasm. Among the most important and
interesting proteins that distinguish particular neurons are the surface receptor
molecules for neurotransmitters such as dopamine. When a particular neuron has
receptors for dopamine on its surface it is ready and able to receive information in
the form of dopamine molecules released from other neurons. Not all neurons
express dopamine receptors and those that don’t are unable to “feel” dopamine and
are unaffected by its presence. Presynaptic neurons that express dopamine receptors
may release more or less neurotransmitter in response to the presence of dopamine
while postsynaptic neurons may be more or less responsive to their neurotransmit-
ters. Thus dopamine can modulate the flow of information at synapses whenever
neurons express dopamine receptors.
Nerves with the capacity to synthesize and release dopamine are called
dopaminergic neurons and they can converse with neurons containing dopa-
mine receptors. When dopaminergic neurons fire they release a small cloud of
dopamine molecules. As nearby neurons with dopamine receptors on their surface
bind the dopamine they in essence are receiving the information that the dopami-
nergic cell has just fired. So what? Who cares? What is the significance of this infor-
mation? Well, for some brain locations the release of dopamine is a chemical
indication that you are especially interested in the specific information passing
through this region and you would like to learn more about it. By causing the
release of dopamine in specific locations higher levels of the brain can amplify the
importance of particular neuronal activity to the detriment of other activity. The
reason driving the dopamine release could, for example, be the perceived novelty of
the information or perhaps dopamine release is the result of a voluntary effort to
learn more detail about a visualized object or a spoken word or phrase. In any case,
it is the presence of the neurotransmitter dopamine that initiates attention and facil-
itates attention-based learning during such an episode.
Attention is a behavior. It isn’t an obvious behavior like singing or walking,
partly because it commonly involves inaction rather than action. After all it is cer-
tainly more effective to attend to something when you’re not busy moving around
and having to organize and execute movements at the same time. However, you cer-
tainly are actively doing something when you are noticing the brilliant finger work
of the principal violinist during a Mozart concerto. You are attending. Attention is
the behavior you engage in when the molecule dopamine is released and active
someplace in your brain. The particular information being amplified by dopamine
has a more lasting impact and resonates in memory circuits to a greater extent than
passive, unattended information. Attended information may even reshape your
brain by initiating new synaptic connections, especially in the very same region
where the dopamine was released.

17
Images Arise From Synchronized Neuronal Firing
The firing activity of neurons is the mechanism by which they transmit infor-
mation and neurons exhibit differing firing rates. Some can fire as often as 200
times a second, but others may fire only once every several seconds. Firing of a neu-
ron sends information forward to all the target neurons it is in contact with (sort-of,
it is not in contact, physically speaking. If it were, then what is the need for dopa-
mine clouds, etc.). Because a neuron may synapse onto a thousand or more target
neurons, one single neuron can readily influence the activity of many other neu-
rons, It’s a wickedly complex network that could easily result in utter chaos if it
weren’t for the coordinated, synchronized firing of groups of neurons together.
Neuronal synchronization creates order much like an orchestra creates a sym-
phony from the coordinated activity of its individual members. When nerves fire
synchronously their separate information content merges together to form a higher
level of information that is more than the simple sum of its parts.
Something new emerges from this orchestrated activity of neurons and when
it does the individual bits of information blend seamlessly into a wholly conceived
image. When we see a book as a book, we combine all the individual sensory infor-
mation it provides (size, color etc.) together via neuronal synchronization to create
one single, unified object. It’s analogous to a video image emerging from the coordi-
nated presentation of individual pixels. This higher-order perception is certainly
convenient and helpful, since it facilitates our manipulation and utilization of the
whole larger object, rather than having to deal with the minutia from which it is
comprised. Moreover, we can also synchronize additional, non-visual information
such as the content and utility of a particular book. For instance a bible has certain
significance other than its physical appearance. Language, in the form of auditory
or written symbols is a means of conveying synchronized information. Words
bring meaning beyond their physical form. In fact once we have learned what a
word means it becomes quite difficult to regard that particular collection of letters
as mere black and white visual objects. Man and other primates have a highly
developed capacity for synchronizing and combining neuronal information and
this capacity may well be the single feature that most distinguishes us from our
more distant evolutionary cousins.
The process by which patterns of neuronal synchronization are created is
an example of associative learning. At some earlier point in our lives we learned
what a book is because when the book was moved (or our eyes moved) its visual
attributes remained together. We began to associate some information (e.g. color
and contrast) with other information (e.g. edge recognition, weight or texture).
Later we added an association with the presence of words within these objects, grad-
ually moving toward the mature concept of a book. Associations aren’t formed
instantaneously but require repetition, attention, and time before they are solid-
ified and carried forward (This is the root of chanting). At the neuronal level
associative learning takes the form of new synaptic connections between nerves car-

18
rying previously separate information. These synapses allow for synchronized neu-
ronal firing that was not possible before these connections were forged.
Interneuronal Networks Create Synchronization
Interneurons are a particular type of nerve cell whose role is to intercon-
nect other nerves, rather than to carry sensory or motor information themselves.
(Think One-To-Many Multiplexors - see: http://www.wisegeek.com/what-is-an-
interneuron.htm) Consistent with this role, interneurons frequently do not have
long axons, but they do make an extraordinary number of synaptic connections
(e.g. 10,000-15,000 per cell as opposed to 1000 or 2000 per cell). The growth and
development of interneurons is particularly important for associative learning
and for promoting synchronized activity. Indeed the timing and pattern of inter-
neuron development parallels our capacity for associative learning. Dopamine
receptors, particularly those designated as the D4 receptor type, are especially
dense on interneurons (1,2) and these receptors appear to play a critical role in
mediating the effects of dopamine during the molecular mechanism of attention
(3-8).
When they fire, interneurons typically inhibit the firing activity of their target
neurons. Neuronal circuits that contain inhibitory interneurons commonly exhibit
synchronized oscillations (9,10). The oscillations are a regular rise and fall of electri-
cal activity (firing rate) that can be measured in individual neurons as well as in the
synchronized firing activity of all the interconnected neurons within the circuit. The
frequency of the oscillatory activity is the time interval between each peak of firing
rate and different types of interneurons exhibit particular frequencies. For instance,
faster firing interneurons yield higher frequency oscillations (e.g. 40 Hz or 40 cycles
per second) while those with lower firing rates yield slower oscillations (e.g. 5 Hz);
Delta (0.1-4.0 Hz)... Theta (4.0-7.0 Hz)... Mu (8.0-13 Hz)... Beta (12-30 Hz).
During episodes of attention the prevalence of 40 Hz oscillations (so called
Gamma waves - 25 Hz to 100 Hz - but predominantly 40 Hz) increases (11) and it
has been suggested that synchronized oscillations at this frequency are particu-
larly important in the cognitive activity which occurs during attention (12).
Indeed 40 Hz activity may represent the electrical product of the associative
learning process. This concept is strengthened by the recent observation that the
ability of 8 month-old infants to discern visual objects as a single, whole image
was accompanied by the appearance of 40 Hz synchronized oscillations in partic-
ular brain regions (13). Dopamine D4 receptors are particularly enriched in those
interneurons giving rise to 40 Hz oscillations.
[Bob’s Notes: from: http://www.web-us.com/brainwavesfunction.htm
What is the function of the various brainwaves?
It is well known that the brain is an electrochemical organ; researchers
have speculated that a fully functioning brain can generate as much as 10 watts
of electrical power. Other more conservative investigators calculate that if all 10

19
billion interconnected nerve cells discharged at one time that a single electrode
placed on the human scalp would record something like five millionths to 50
millionths of a volt. If you had enough scalps hooked up you might be able to
light a flashlight bulb.
Even though this electrical power is very limited, it does occur in very
specific ways that are characteristic of the human brain. Electrical activity ema-
nating from the brain is displayed in the form of brainwaves. There are four
categories of these brainwaves, ranging from the most activity to the least
activity. When the brain is aroused and actively engaged in mental activities, it
generates beta waves. These beta waves are of relatively low amplitude, and are
the fastest of the four different brainwaves. The frequency of beta waves
ranges from 15 to 40 cycles a second. Beta waves are characteristics of a
strongly engaged mind. A person in active conversation would be in beta. A
debater would be in high beta. A person making a speech, or a teacher, or a
talk show host would all be in beta when they are engaged in their work.

The next brainwave category in order of frequency is alpha. Where beta

represented arousal, alpha represents non-arousal. Alpha brainwaves are
slower, and higher in amplitude. Their frequency ranges from 9 to 14 cycles
per second. A person who has completed a task and sits down to rest is often
in an alpha state. A person who takes time out to reflect or meditate is usually

20
in an alpha state. A person who takes a break from a conference and walks in
the garden is often in an alpha state.
The next state, theta brainwaves, are typically of even greater amplitude
and slower frequency. This frequency range is normally between 5 and 8 cycles
a second. A person who has taken time off from a task and begins to daydream
is often in a theta brainwave state. A person who is driving on a freeway, and
discovers that they can't recall the last five miles, is often in a theta state--
induced by the process of freeway driving. The repetitious nature of that form
of driving compared to a country road would differentiate a theta state and a
beta state in order to perform the driving task safely.
Individuals who do a lot of freeway driving often get good ideas during
those periods when they are in theta. Individuals who run outdoors often are in
the state of mental relaxation that is slower than alpha and when in theta, they
are prone to a flow of ideas. This can also occur in the shower or tub or even
while shaving or brushing your hair. It is a state where tasks become so auto-
matic that you can mentally disengage from them. The ideation that can take
place during the theta state is often free flow and occurs without censorship or
guilt. It is typically a very positive mental state.
The final brainwave state is delta. Here the brainwaves are of the great-
est amplitude and slowest frequency. They typically center around a range of
1.5 to 4 cycles per second. They never go down to zero because that would
mean that you were brain dead. But, deep dreamless sleep would take you
down to the lowest frequency. Typically, 2 to 3 cycles a second.
When we go to bed and read for a few minutes before attempting sleep,
we are likely to be in low beta. When we put the book down, turn off the lights
and close our eyes, our brainwaves will descend from beta, to alpha, to theta
and finally, when we fall asleep, to delta.
It is a well known fact that humans dream in 90 minute cycles. When
the delta brainwave frequencies increase into the frequency of theta brain-
waves, active dreaming takes place and often becomes more experiential to the
person. Typically, when this occurs there is rapid eye movement, which is char-
acteristic of active dreaming. This is called REM, and is a well known phenom-
enon.
When an individual awakes from a deep sleep in preparation for getting
up, their brainwave frequencies will increase through the different specific
stages of brainwave activity. That is, they will increase from delta to theta and
then to alpha and finally, when the alarm goes off, into beta. If that individual
hits the snooze alarm button they will drop in frequency to a non-aroused
state, or even into theta, or sometimes fall back to sleep in delta. During this
awakening cycle it is possible for individuals to stay in the theta state for an

21
extended period of say, five to 15 minutes--which would allow them to have a
free flow of ideas about yesterday's events or to contemplate the activities of
the forthcoming day. This time can be an extremely productive and can be a
period of very meaningful and creative mental activity.
In summary, there are four brainwave states that range from the high
amplitude, low frequency delta to the low amplitude, high frequency beta.
These brainwave states range from deep dreamless sleep to high arousal. The
same four brainwave states are common to the human species. Men, women
and children of all ages experience the same characteristic brainwaves. They
are consistent across cultures and country boundaries.
Research has shown that although one brainwave state may predominate
at any given time, depending on the activity level of the individual, the remain-
ing three brain states are present in the mix of brainwaves at all times. In other
words, while somebody is an aroused state and exhibiting a beta brainwave
pattern, there also exists in that person's brain a component of alpha, theta and
delta, even though these may be present only at the trace level.
It has been my personal experience that knowledge of brainwave states
enhances a person's ability to make use of the specialized characteristics of
those states: these include being mentally productive across a wide range of
activities, such as being intensely focused, relaxed, creative and in restful sleep.]
Dopamine, Attention and 40 Hz Oscillations
While the frequency of synchronized oscillations is determined by the type of
interneuron in a given circuit, the amplitude of the oscillation is a separate parame-
ter that can be modulated to increase or decrease, much like the volume control in a
radio increases the amplitude of a particular radio frequency. As amplitude
increases, the relative importance of information arising from the particular syn-
chronized circuits increases, as compared to information from other non-amplified
circuits. This increase in importance corresponds to attention, an enhancement of
particular selected information over and above other simultaneously occurring
unattended information. Attended information typically constitutes only a select
portion of the entire stream of current experience. Attention may be directed
toward only a fraction of a visual scene, perhaps just a portion of the information
previously conceived as a whole object, such as attending to only the frayed corner
of a book. Increasing the amplitude of selected information promotes the level of
attention and also promotes learning.
How is the amplitude of 40 Hz synchronized oscillations increased? Since
dopamine is centrally involved in attention, it is reasonable to propose that dopa-
mine amplifies 40 Hz oscillations by its action on inhibitory interneurons involving
one of the dopamine receptors. Among the five different types of dopamine recep-
tors, the D4 receptor has been linked to differences in attention-related behaviors
including the risk of ADHD (5,6) and the personality trait of novelty-seeking

22
(3,4). Since D4 receptors are enriched on the very interneurons that produce 40 Hz
oscillations (1), they may indeed be very important for producing attention.
In primates and man the dopamine D4 receptor protein contains special-
ized structural features that are highly variable from person to person and ape to
ape (14-17). These hyper-variable features are not found in lower animals suggest-
ing that they are important for the unique attention and cognitive capacities of pri-
mates and man. Indeed the D4 receptor gene is one of the most variable genes in
the entire human genome. Some of these genetic variants are distinctive for ethnic
groups from different regions of the world, indicating a pattern of evolution that
parallels the cultural evolution of man (16,17). These features make the dopamine
D4 receptor a worthy candidate for the maestro of attention and this book pro-
vides an in-depth exploration of this putative role.
Novel Signaling Activities of the Dopamine D4 Receptor
The D4 receptor has several unique functions that endow it with the capacity
to provide attention and to facilitate attention-related learning.
(1) Our laboratory first discovered one such unique activity about five
years ago as an outgrowth of our investigations into the signaling activity of a large
family of receptors called G protein-coupled receptors. These receptors normally
signal via their activation of GTP-binding proteins, hence the designation “G pro-
tein.” In a series of studies we found that D4 receptors can also signal via a previ-
ously unrecognized mechanism that only they can carry out (18-21). In this novel
mechanism the receptor transfers methyl groups (i.e. CH3 groups) from the folic
acid system to phospholipid molecules which make up the nerve cell membrane
around the receptor (Figure 2.2). Dopamine stimulates this process by causing a
change in shape of the D4 receptor. As a consequence of phospholipid methylation
(PLM), the membrane around the receptor becomes more fluid and less tightly
packed, which can modulate the activity of other receptors and membrane proteins
located nearby. We call this new signaling mechanism “solid-state signaling”
because it occurs entirely within the local cell membrane, as opposed to standard G
protein-coupled signaling, which involves the formation of second messenger mole-
cules that can diffuse throughout the cell. Because of its solid-state nature, dopa-
mine stimulated PLM is well suited for producing very rapid, localized changes in
neuronal activity.
(2) A second unique feature of the D4 receptor is its ability to specifically
locate itself at neuronal synapses among other membrane receptors and proteins
that become targets for solid-state signaling. When dopamine triggers activation
of the D4 receptor PLM-based solid-state signaling can modulate the efficiency of
synaptic transmission at sites on inhibitory interneurons where the receptor is pres-
ent in abundance. In this manner dopamine can amplify 40 Hz oscillations arising

23
from these interneuron circuits, causing selective attention to the information flow-
ing through those circuits.

Figure 2.2: The dopamine-folic acid connection. Methyl groups (CH3) bound to
folic acid are temporarily transferred to the dopamine D4 receptor, which then
further transfers them to phospholipid molecules of the membrane in a dopamine-
dependent manner. This overall process is called folate-dependent phospholipid
methylation (PLM) and the modulatory effect it produces on nerve function is
termed “solid-state signaling.”Bob’s note: folate and folic acid are the same thing.
Folate comes in food and folic acid is man-made.
(3) A third D4 receptor feature we found was that solid-state signaling
activity is highly dependent upon the metabolic status of nerve cells. This is
partly because the folate system that supplies methyl groups to the D4 receptor is
critically related to cellular metabolism and partly because signaling is sensitive to
the levels of ATP and adenosine. ATP is the primary source of metabolic energy
and adenosine is its energy-depleted product. As a consequence of these relation-
ships, the capacity for dopamine-stimulated, D4 receptor-mediated attention is
tightly linked to cellular energy status and to overall cellular metabolism. As we
shall see, changes in attention across the lifespan, which we commonly take for
granted, can now be understood in molecular terms based upon these relationships.
(4) Lastly, others and we have found that activation of D4 receptors also
provides a growth stimulus to neuronal cells, simultaneous with the activation of
solid-state signaling (22,23). There is evidence that this function is particularly

24
enhanced in man and primates and that it might contribute to our unique intellec-
tual gifts. When episodes of dopamine-mediated attention occur, the very same
interneurons involved in amplified 40 Hz oscillations receive a jolt of growth stimu-
lation as well. As they grow they form new synapses, thereby establishing connec-
tions that represent attention-related associative learning. Because attention and
associative learning are both driven via the local actions of dopamine, this mecha-
nism assures that we preferentially learn the information we pay attention to.
Psychiatric illnesses related to D4 receptor dysfunction
Our molecular mechanism of attention has evolved to a high level of effi-
ciency and reliability. Nonetheless, there is the inevitable occurrence of mutations
or environmental insults that can disrupt normal function. In fact the process of
evolution depends upon these occurrences, to some extent, as a mechanism to
investigate alternative genetic strategies that either fail or survive depending upon
their selective advantage. Using the new vantage point of D4 receptor-mediated
attention, it is possible to re-examine what is known about several different psychi-
atric illnesses to see whether these new findings might provide insights into their
causation or their treatment.
As noted above, certain structural and genetic variations in the dopamine D4
receptor and its gene have been associated with a higher prevalence of ADHD.
These variations, along with other identified risk factors can affect the receptor’s
signaling activity, leading to individual differences in the intensity of attention and
differences in attention-related learning. Do these differences constitute a disease or
a psychiatric illness or a disability? Alternatively, do they represent positions on a
continuous spectrum of individual variation in attention and learning styles? Are
these differences the hallmark of being human or some newly discovered mental ill-
ness? In our well-intended effort to optimize the success of our children within a
somewhat rigid framework of training and expectation, we run the risk of assigning
diagnostic labels to behavior that is noticeably out of the norm. As we learn more
about our genetic make-up and understand how it shapes our individual lives we
will have a more informed framework for addressing these difficult societal issues.

Chapter Summary:
• Attention is provided by molecular events that control neuronal firing.
• Neuronal firing represents a flow of information.
• Neurotransmitters such as dopamine modulate information flow at neuronal synapses.
• Neuronal firing is coordinated by synchronized oscillations.
• Synchronized firing provides a mechanism for unifying separate information into a more
complex whole.
• Inhibitory interneurons are important for creating synchronized oscillations of nerve
firing, which can exhibit specific frequencies.

25
• D4-type dopamine receptors (D4Rs) are located on interneurons where they can modulate
synchronized oscillations.
• Attention is associated with an increase in the amplitude of 40 Hz synchronized
oscillations.
• Dopamine stimulation of D4Rs causes phospholipid methylation (PLM), which can serve
as a modulatory signal at dopaminergic synapses.
• D4Rs can activate additional signaling pathways leading to new synapse formation and
associative learning.

26
3 HOW DOPAMINE RECEPTORS WORK

Neurotransmitter chemicals like dopamine allow the transfer of informa-

tion from one nerve cell to the next. These small signaling molecules are synthe-
sized and stored in nerve endings awaiting release. When the nerve is activated,
packets containing the neurotransmitter burst out of the nerve allowing the chemi-
cal to diffuse into the synaptic space, creating a temporary rise of about 100-fold in
its local concentration (Bob’s note: This is the so called dopamine cloud). A por-
tion of the released transmitter molecules collides with receptor protein molecules
on the surface of the nerve across the synapse and a portion of these collisions result
in specific binding to the receptors. Much of the released neurotransmitter diffuses
away or is metabolized (Bob’s note: meaning gone), while some is transported back
into the nerve ending (Bob’s note: This is called re-uptake) and is recycled for fur-
ther use.
Using a “lock and key” fitting mechanism, dopamine is specifically bound
and recognized by dopamine receptors. Recognition is accomplished by chemical
groups located within the mouth of the receptor that form precise bonds with dopa-
mine’s groups, including its positively charged amino group, its two hydroxyl
groups and its phenyl ring. Once bound, dopamine triggers a change in the shape of
the receptor protein (Bob’s note: the cell quivers lending credence to the phrase
“quivering jelly” that is used colloquially to describe brain matter - talk about “from
the mouths of babes), which serves as a signal that the receptor has successfully been
occupied by a dopamine molecule (Figure 3.1). While it might seem trivial, this
change in receptor shape (known as a change in receptor conformation) is the key
event in transferring the signal represented by the extracellular dopamine concen-
tration from the outside of the nerve cell to the intracellular compartment.
There are five different receptors that specifically bind and respond to dopa-
mine and these have been designated D1, D2, D3, D4 and D5 dopamine receptors,
in the order they were first discovered. Like the receptors for many neurotrans-
mitters and hormones, all five dopamine receptor proteins are constructed from
a long, single chain made up of 450 to 500 amino acids (24). This chain crosses
the (cell) membrane seven times and each time it takes on a helical shape as it
spans the cell membrane (Figure 3.2). These seven transmembrane helices are con-
nected to each other by loops, either outside the cell (extracellular) or inside the cell
(intracellular). The third intracellular loop is longer than the rest and it is the
most important region for intracellular signaling activity. The seven helices are
arranged in a more or less circular manner (like the staves in a barrel) forming a
cavity in the middle. It is within the mouth of this cavity that dopamine binds
(Bob’s note: imagine a key fitting into a lock) and is recognized by the receptor.
Individual amino acids from the receptor create binding opportunities

27
 Figure 3.1: The neurotransmitter dopamine (DA) binds to its receptor on the
outside of the cell resulting in a change of the receptor’s shape on the inside that
allows a G protein (G) to bind to the receptor.
that perfectly match the three-dimensional features of dopamine within the
binding cavity.

Figure 3.2: Dopamine receptors are formed from a single chain of amino acids that
creates seven transmembrane helices.
The molecular model in Figure 3.3 (below) illustrates how dopamine binds to
the D4 receptor. Transmembrane helices #3, #5 and #6 are particularly important in
binding and recognizing dopamine. A negative charge on helix #3 binds to its pos-
itively charged amino group while two hydroxyl groups on helix #5 of the recep-
tor form hydrogen bonds with dopamine’s two hydroxyl groups. These three

28
interactions serve to position dopamine’s phenyl ring squarely in the middle of the
binding cavity.

Figure 3.3: A molecular graphic model of dopamine binding to the dopamine D4

receptor. Dopamine is shown as the upper solid molecule. Methionine 313 on
transmembrane helix #6 is shown as the lower solid model.
Once dopamine is fixed in place, helix #6 rotates to form a bond with the
immobilized phenyl group of dopamine, constituting a dopamine dependent con-
formational change. While this rotational movement has not been directly con-
firmed for dopamine receptors, evidence from other similar receptors indicates that

29
rotation of helix #6 is a major part of the shape change that accompanies dopamine
occupation. Elegant studies carried out on the retinal receptor rhodopsin, by Dr.
Wayne Hubbell and colleagues at UCLA, provided direct evidence that helix #6
rotates about 23° upon activation by light (25). Studies with other catecholamine
receptors also suggest that helix #6 undergoes a rotation, as illustrated in Figure 3.4
(26,27). Since transmembrane helices are relatively rigid structural elements, the
rotation caused by dopamine binding to the outside portion of the receptor affects
the entire helix, including the portion that projects into the intracellular compart-
ment. This is the basic mechanism of operation for these types of receptors. Neu-
rotransmitter binding on the outer part of the receptor triggers rotation of one or
more helices, changing the exposure of the inside portion of the receptor and allow-
ing the signal to be transmitted to the cell interior. It is a simple but elegant way to
get the message from the outside across the cell membrane to the inside.

Figure 3.4: Molecular models of the R (left) and R* (right) conformational states of
the dopamine D4 receptor illustrating the rotated position of methionine #313 on
helix #6.
The standard intracellular message for dopamine receptors, as well as for
other seven helix receptors, involves their activation of GTP-binding G proteins.
Dopamine-occupied receptors activate G proteins by helping them to lose a previ-
ously bound molecule of GDP (a GTP that has lost one phosphate) allowing its
replacement by GTP. Once GTP has replaced GDP, the now activated G protein
separates into two parts, each of which can modulate enzymes or ion channel pro-
teins. This G protein-based signaling process serves to alter cellular activity for a
short period of time (e.g. a few seconds), until the GTP is hydrolyzed back to GDP,
setting the stage for another round of receptor activation. Cells continually update
their activity in response to the level of dopamine and other extracellular hormones
and neurotransmitters.
While the binding of dopamine promotes the critical receptor shape
change, there is a certain probability that the receptor could change shape all on

30
its own, resulting in the spontaneous activation of G proteins and further down-
stream signaling. This spontaneous activity alerts us to the fact that the binding of
dopamine or other neurotransmitters merely acts to increase the inherent probabil-
ity of the receptor being active. That is to say, receptors already have an intrinsic
capacity for being active, and neurotransmitters merely modulate the chance of this
happening in a positive manner.
The conventional designation for the activated shape of a receptor is R*, as
opposed to the inactive R shape or conformation. The preceding discussion can be
summarized schematically as follows:

Sometimes “hot spot” mutations occur naturally, resulting in abnormal

receptor signaling and causing diseases like hyper-thyroid-ism or premature
puberty. It was our interest in this particular “hot spot” location in the D4 recep-
tor that opened the door to discovery of a novel dopamine signaling mechanism
that is at the heart of the molecular mechanism of attention.
The first thing we noticed about the “hot spot” location on helix #6 was that it
has a unique spatial relationship with the amino acids further up the where dopa-
mine or other neurotransmitters bind. For example, the phenylalanine residue on
helix #6 that dopamine binds to is designated #331 in the D4 receptor while the
“hot spot” residue is eighteen places earlier in the sequence, at #313 (i.e. 331 - 313).
As it turns out, each amino acid in a helix is positioned 100o further around the
helix than the previous one. Thus eighteen amino acids add up to a total of 1800o

31
or 360o times 5. In other words the “hot spot” position is located exactly five full
turns directly below the place that dopamine binds on helix #6. Since we know that
dopamine binds in the middle of the receptor, the “hot spot” residue (and particu-
larly its side chain extending out from the helix) will be exposed toward the open
space in the middle of the receptor whenever dopamine binds to helix #6. In con-
junction with the information suggesting that helix #6 moves during receptor acti-
vation, this implies that exposure of the “hot spot” side chain toward the middle of
the receptor will be increased when the receptor is occupied by dopamine as com-
pared to the unoccupied state, as illustrated in Figure 3.4.
A comparison the amino acid sequence around the “hot spot” location in the
large family of seven helix receptors showed a pattern. Many of these receptors have
a type of amino acid at the “hot spot” that is capable of being modified by phosphor-
ylation (the addition of a phosphate group). Most commonly this amino acid was
threonine (T), but sometimes it was a serine residue (?? What is a serine residue),
both of which can receive phosphate groups.
Table 3.1 shows a comparison of the “hot spot” region in a number of these
receptors. Notice that, with the exception of the dopamine D4 receptor, each of
the receptors shown in bold print has a threonine (abbreviated as “T”) at the “hot
spot,” with two basic amino acid residues (either R or K) at two positions just pre-
ceding the threonine. The basic residues frequently serve to direct phosphorylation
to the following threonine. Observing this pattern led us to a realization: If nature
did use the “hot spot” as a site of phosphorylation, it would be just the same as if
this site had been mutated. It would be a naturally occurring temporary mutation
and would cause spontaneous receptor activity lasting until the phosphate was
removed. In fact this type of reversible phosphorylation is commonly employed by
nature to regulate other biochemical processes. The enzymes that add phosphate
groups are called protein kinases while the enzymes that catalyze their removal are
called protein phosphatases. The balance of kinase and phosphatase activities
determines the net level of phosphorylation.
Receptors that contain potential sites of phosphorylation at their “hot spot”
all signal via Gi/o-type G proteins and show an unusually high level of spontaneous
activity, as demonstrated for the alpha-2 adrenergic receptor (29). Phosphorylation
of the “hot spot” threonine by a protein kinase might therefore contribute to their
higher spontaneous activity. Indeed inhibition of protein kinase C (PKC), com-
pletely eliminates spontaneous activity of the alpha 2A adrenergic receptor (30).
This critical observation indicated that nature utilizes “hot spot” phosphoryla-
tion as a mechanism to increase spontaneous receptor activity. Moreover, activa-
tion of receptors by agonists such as dopamine [agents that stimulate receptors are
collectively known as agonists] could rotate helix #6 and increase the opportunity
for phosphorylation by exposing the “hot spot” threonine residue. Consequently,
the extent of spontaneous activity exhibited by a population of receptors would
be higher after a preceding period of agonist exposure, creating a sort of molecu-

32
lar memory of the earlier presence of agonist. This agonist dependent mechanism
for modulating the spontaneous activity of receptors may prove to be important in
understanding the regulation of the many receptors that can be phosphorylated at
their “hot spot” locus, although further research will be necessary to explore its
significance. Potentially interesting examples are opiate receptors that are activated
by morphine and other drugs and possess a “hot spot” threonine residue. Pretreat-
ment with a high concentration of morphine or other opiate agonists causes opiate
receptors to become spontaneously active, consistent with the effect of phosphory-
lation (31). Furthermore, inhibitors of protein kinase C (PKC) eliminate the signs of
morphine withdrawal (32), suggesting that phosphorylation induced spontaneous
activity might play a role in the mechanism of addiction and withdrawal for opiates.

Table 3.1: A comparison of amino acid sequences around the “hot spot” in several G protein-coupled
receptor families. Note the presence of a potential site for phosphorylation (the threonine residue (T)
preceded by two basic residues (R or K) at the -3 and -2 positions) in each of the bold-type receptors.
The single exception is the dopamine D4 receptor, which has a methionine residue at this “hot spot”
locus.

How does this discussion about “hot spot” phosphorylation relate to

attention and the dopamine D4 receptor? Well, sometimes the exception proves
to be more interesting than the rule. As shown in Table 3.1, the D4 receptor doesn’t
have a threonine at its “hot spot” location, although it is closely related to all the

33
other receptors in bold print. Instead it is only one of two receptors in the entire
family of over 2,000 members to have a methionine (M) residue at this position.
Intrigued by this anomaly we examined the D4 receptor and found that it does
indeed exhibit a very high level of spontaneous activity. Furthermore, we found that
we could eliminate spontaneous activity of the D4 receptor by inhibiting methio-
nine adenosyltransferase, an enzyme that normally acts on methionine. Through a
successive series of investigations our laboratory learned that nature makes special
use of the smethyl group from this uniquely positioned methionine in the dopamine
D4 receptor to trigger the molecular mechanism of attention.

Chapter Summary:
• Dopamine exerts its effects by binding to specific receptor proteins and causing them to
change shape.
• Dopamine receptors have seven transmembrane helices and rotation helix #6 is important
in their activation by dopamine.
• Mutation of a particular amino acid on helix #6, called a “hot spot”, can cause spontaneous
receptor activity.
• In many receptors the “hot spot” can be naturally mutated by addition of a phosphate
group.
• In the dopamine D4 receptor the “hot spot” amino acid is a methionine residue which can
donate its methyl group.
• Dopamine-stimulated, D4 receptor-dependent methyl donation is a critical event in
attention.

34
4 THE DOPAMINE D4 RECEPTOR

When it was initially discovered in 1991 by a team of researchers led by Dr.

Hubert Van Toi (33), the dopamine D4 receptor was assigned an important place
in the fabric of man’s molecular design. “Science” magazine printed a two-page
commentary speculating that the newly identified member of the dopamine recep-
tor family might “hold the key to schizophrenia”. This was a particular compliment
since the original D4 receptor paper had been published in Nature, their arch rival
in the competition for high-profile science stories. This intense early interest was
triggered by the fact that the newly discovered D4 receptor had a high binding affin-
ity for the uniquely effective antipsychotic drug clozapine used in the treatment of
schizophrenia. Since it is generally assumed that high affinity marks the molecular
site at which a drug works, this suggested that the D4 receptor might be responsible
for clozapine’s benefit in schizophrenia. By extension it also followed that the D4
receptor might play a special role in normal human cognition, which is impaired in
schizophrenia.
Initial interest in the D4 receptor was further fuelled by the subsequent find-
ing that a comparison of its DNA genetic sequence among different humans
revealed a striking number of variant forms (14,15). These genetic differences
implied that the D4 receptor protein itself was not always the same from person to
person. Indeed the D4 receptor is one of the most variable proteins (and genes) in
man. This is especially intriguing since these variant structure features were only
found in the D4 receptor of man and other primates, not in lower species such as
the rat or mouse. Could it be that these person to person D4 receptor differences
were associated with differences in man’s cognitive function?
This intense early interest in the D4 receptor triggered a frantic race among
drug companies to develop new drugs targeted to selectively block the receptor with
the expectation that they would improve treatment of schizophrenia. Clozapine,
while a remarkably effective drug, has serious, life threatening side effects that fur-
ther fuelled the search for a safer, perhaps even more effective replacement. In a rel-
atively short period of time Merck identified a highly D4 receptor-selective blocking
drug (L-745870) and, after pre-clinical testing, they initiated a clinical trial in per-
sons with schizophrenia. Much to the surprise of all parties, especially Merck, the
drug was a total failure. Psychotic symptoms were not improved at all by the drug
and in fact were slightly worse than with placebo treatment (34). This stunning
result cast a deep pall over research into the D4 receptor and essentially stopped fur-
ther interest in D4-related drug development, which largely continues to this day.
Nonetheless, the receptor’s unique primate-specific features remained to be under-
stood and its functional role unraveled. The D4 receptor became a shadowy bio-
medical mystery, a puzzle and a pariah, burdened by the very costly and
embarrassing clinical failure of a prototypical drug candidate.

35
Meet the dopamine D4 receptor
The general structure of the D4 receptor is shown in Figure 4.1. The first
thing to notice is the position of methionine #313 at the intracellular end of helix #6
in the hot spot locus. In this location it is adjacent to the inner surface of the cell
membrane, which is formed by an array of phospholipid molecules stacked side to
side against each other. Phospholipids are composed of two fatty acid tails that are
directed toward the center of the membrane and a headgroup that forms the
exposed surface of the cell membrane. Methionine #313 is positioned right next to
the headgroup of the phospholipids surrounding the D4 receptor. As we shall see,
this location allows the methyl group from methionine #313 to be transferred to
the phospholipid headgroup in a process known as phospholipid methylation
(PLM), a critical event in attention.

Figure 4.1: Structural features of the human dopamine D4 receptor. MET#313 is located on helix
#6, close to the inner membrane surface. Proline-rich regions allow for SH3 domain interactions with
other proteins. Humans and other primates have anywhere from two to eleven hyper-variable
repeated proline-rich regions in their D4 receptors.

The next thing to notice in the D4 receptor are several regions that are rich in
the amino acid proline located in the third intracellular loop. These proline-rich
regions provide a docking site for other proteins to attach to the D4 receptor if they
possess a structural domain capable of recognizing such proline-rich regions. This
structural domain is known as a SH3 (src homology 3) domain, named after the src
protein in which it was first discovered. When one protein is bound to another via

36
SH3 domain-mediated interactions, it increases their ability to work together, creat-
ing a team rather than operating randomly alone. In non-primates the D4 receptor
has only two proline-rich segments, located at either end of the third intracellular
loop, while primates (including humans) have anywhere from two to eleven addi-
tional segments, most commonly four (15,17,35). Thus the human D4 receptor
has a higher number of these protein-protein coupling sites. As we’ll see, this
ability to form multiple protein complexes adds a new level of receptor function
including combinatorial possibilities as segments are mixed and matched together.
Phospholipid Methylation(PLM)
Cell membranes are composed of a variety of phospholipid molecules, closely
packed one against the other with proteins either embedded in the membrane (like
the D4 receptor) or attached to it on either side (Figure 4.2).
[Omega3 in nature is available in two forms: phospholipid form (pro-
nounce: fos-fo-lipids) and the triglyceride form. Omega3 is by definition
“essential” because the human body cannot create it. It needs to be acquired
through diet]
[From: http://www.superbakrill.com/section.cfm?path=56&id=39
Omega-3 - essential fatty acids in diet: The long-chain omega-3 polyun-
saturated fatty acids DHA and EPA are popularly called omega-3. Supplemen-
tary intake of omega-3 is recommended in the western world, due to generally
low dietary intake and omega-3’s health-promoting benefits. Benefits attributed
to omega-3 include reduced risk and improved treatment outcomes regarding
cardiovascular disease and inflammatory joint diseases. Better brain and central
nervous system development, improved cognitive functioning, and improved
skin health are additional benefits. Research indicates that even more omega-3
benefits for individuals will be identified and that greater intake can lead to
better general health in western, industrialized cultures.
Antarctic krill – an important source of Omega-3: Aker BioMarine’s krill
oil — Superba™ — is extracted from the Antarctic krill species Euphausia
superba, which is rich in omega-3. Moreover, the omega-3 in krill oil is mainly
in the omega-3 phospholipid form, which research suggests is a preferred
dietary supplement when compared to omega-3 in triglyceride form. Marine
omega-3 in dietary supplements is mostly derived from fish, such as fish body
oil and cod liver oil, which provide omega-3 in triglyceride form. The omega-3
obtained from eating fatty fish such as salmon also provide some omega-3 in
the phospholipid form.
Phospholipids — key structural and functional roles: Phospholipid fatty
acids are key structural components of human cells and cell organelles and play
a vital role in membrane functioning. Systemic transport pathways and, espe-
cially, the transport of molecules across cell membranes and sub-cellular mem-

37
branes involve phospholipids. Thus, the functioning of the body’s cells, tissues,
and organs is affected by the bio-availability of various phospholipid fatty acids.

Changes in the Western diet - reduced intake of phospholipids: Food

sources and their nutrient values have changed tremendously over the past
century in western cultures. In fact, while the human genome most likely has
changed insignificantly over the past 10,000 years, the foods eaten in industrial-
ized countries are a far cry from the staples consumed when mankind evolved.
Reasonable assumptions are that “primitive” diets provided phospholipids in
greater abundance than the current Western diet and that humans evolved
such that phospholipids are the preferred source of omega-3. The current
Western diet provides only small amounts of phospholipids; dietary phospho-
lipids represent only 5 percent of total lipid intake, of which very little is in the
form of omega-3 phospholipids.
Krill oil - a rich source of omega-3 phospholipids: Omega-3 phospholip-
ids constitute a substantial proportion of the omega-3 in krill oil, of which
most is the phospholipid phosphatidylcholine. Research indicates that phospho-
lipid omega-3 fatty acids have greater bioavailability for cell growth and func-
tioning, compared with omega-3 triglycerides. Also, omega-3 utilization in

38
some tissues increases when delivered in the form of omega-3 phospholipids.
Such observations suggest that krill oil is a more bioefficient carrier of omega-
3 in humans.
Digestion and absorption of omega-3 phospholipids: Triglycerides and
phospholipids are digested and absorbed in different ways in the small intes-
tine. Omega-3 phospholipids follow simpler digestion and distribution routes
than omega-3 triglycerides in the human body. Greater bioavailability and
bioefficiency of omega-3 phospholipids relative to omega-3 triglycerides influ-
ence cellular absorption, functioning and distribution of omega-3.

Triglycerides are insoluble in water; their digestion by enzymes and sub-

sequent absorption in the small intestine requires emulsification by bile salts
via the formation of micelles.
Phospholipids are not dependent on bile for digestion; they can sponta-
neously form micelles and be conveyed in an aqueous environment. Phospho-
lipids can be absorbed without digestion in their intact form, or as
lysophosphatidylcholine after digestion by enzymes in the small intestine. Their
simpler digestion process in the small intestine before absorption is one of the
factors indicating that omega-3 phospholipids offer greater bioavailability in

39
the human body than omega-3 triglycerides. Another suggested factor is that
phospholipids are directly absorbed by intestinal epithelia for distribution via
blood plasma; considerable triglyceride transport involves facilitation by the
lymphatic system.

After absorption in the small intestine, lysophosphatidylcholine is reas-

sembled (via the addition of a fatty acid) to phosphatidylcholine. Phosphatidyl-
choline is an important structural component of cellular membranes and
participates in fatty acid transport in blood and across membranes. Because
fatty acids are water insoluble, they can not be transported in their free form
in blood; instead, lipoprotein assemblages act as a vehicle for fatty acid trans-
port in blood. Phospholipids located along the surface of lipoproteins play
important roles in fatty acid transport in blood. Human lipoproteins include
chylomicrons, HDL, LDL, and VLDL.

40
 The size of a lipoprotein assemblage varies according to the amounts and
types of fatty acids transported in it. Larger assemblages deliver fatty acids to
various tissues throughout the human body, while small lipoprotein assem-
blages serve as “housekeepers,” bringing excess fatty acids to the liver for deg-
radation. Omega-3 phospholipids in the lipoproteins can influence the
distribution of lipoproteins in the body and, hence, the availability of fatty
acids. Increased transport to and utilization of omega-3 by various tissues has
been demonstrated, when delivered as omega-3 phospholipids. For example,
elevated concentrations of omega-3 in target organs, such as the brain and
liver, are observed when delivered as omega-3 phospholipids. Thus, dietary
intake of omega-3 phospholipids appears to play a beneficial role in the distri-
bution of fatty acids to various body tissues.

41
 Omega-3 phospholipids – a more bioefficient source of Omega-3:
Increased intake of omega-3 is recommended due to its beneficial overall health
effects and its capacity to prevent and ameliorate disease. Krill oil delivers
omega-3 in the phospholipid form. Omega-3 phospholipids are more efficiently
absorbed via the small intestine and distributed to tissues, compared with
omega-3 triglycerides. Omega-3 phospholipids are suggested as preferred
sources of omega-3 for human cell structures and cell functions, as a result of
greater bioavailability and bioefficiency, compared with omega-3 triglycerides.
Thus, omega-3 phospholipids from krill are a more bioefficient source of
omega-3 than the omega-3 triglycerides derived from fish oil.
References:
Amate L, Gil A, Ramírez M. Feeding infant piglets formula with long-
chain polyunsaturated fatty acids as triacylglycerols or phospholipids influences
the distribution of these fatty acids in plasma lipoprotein fractions. J Nutr.
2001: 131 (4); 1250 – 5.
Lagarde M, Bernoud N, Brossard N|, et al. Lysophosphatidylcholine as a
preferred carrier form of docosahexaenoic acid to the brain. J Mol Neurosci.
2001; 16: 201-4.
Lemaitre – Delaunay D, Pachiaudi C, Laville M, et al. Blood compartmen-
tal metabolism of docosahexaenoic acid (DHA) in humans after ingestion of a
single dose of [(13)C]DHA in phosphatidylcholine. J Lipid Res. 1999; 40(10):
1867-74.
Ramirez M, Amate L, Gil A. Absorption and distribution of dietary fatty
acids from different sources. Early Hum Dev. 2001; 65 Suppl: S95-S101.
Tou JC, Jaczynski J, Chen Y-C. Krill for human consumption: nutritional
value and potential health benefits. Nutrition Nutr Rev. 2007; 65(2): 63-77.

Phospholipids for brain health: Like omega-3 fatty acids, phospholipids

are important for optimal brain health. Phospholipids are found in high con-
centrations in the lining of practically every cell of the body, including brain
cells. They help brain cells communicate and influence how well receptors
function. Although present in many foods, phospholipids are found in higher
concentrations in soy, eggs and the brain tissue (mostly DHA) of animals.
There may actually be a biochemical rational for the folk wisdom that says eat-
ing brain makes one smarter.
From: http://omega3phospholipids.krill-oil-reviews.com/
Omega-3 Phospholipids: Omega-3 phospholipids (pronounce: fos-fo-lip-
ids) are the key building blocks of cellular life. Without the unique properties
of phospholipids no life would have been possible. Currently, the only econom-

42
ical source for omega-3 phospholipids is krill oil. The best food source for
omega-3 phospholipids is raw or barely cooked fish (sashimi).
In the old days people would consume large quantities of omega-3 phos-
pholipids through the consumption of brain, liver, and other organ meats.
Mammalian brains are mainly fat, and the most common brain fat is DHA
(docosahexaenoic acid). DHA is with EPA (eicosapentaenoic acid) an essential
fatty acid which can not be made by your body in sufficient quantities. Essen-
tial means that the majority of these two fats must be consumed through diet.
Omega-3 deficiency on cellular level has a tremendous health impact. If
you wonder why krill oil and fish oil can be good for so many afflictions, then
it is only because omega-3 phospholipids are so essential to almost everything
in your body. Any omega-3 deficiency will influence a vast number of health
issues.
Why are phospholipids better? Phospholipids stick together in a unique
pattern, and form natural structures that allow oxygen and nutrients to enter
our cells, and which keep (most) toxins, viruses and bacteria out. Our cell
membranes are made from phospholipids, and the quality of the cell mem-
branes is determined by how much omega-3 is part of the phospholipid mole-
cule.
Fish oils don’t contain phospholipids, but do contain omega-3 triglycer-
ides. These fish oil molecules play no role in our cell membranes, and for any
omega-3 from fish oils to be used in cells, it must be converted in our liver to
a phospholipid. This conversion process is very inefficient and not every fish oil
omega-3 is guaranteed to play a role in our cellular health.
The formation of phospholipids in our liver is more a statistical process,
than a selection process. In other words, the consumption of large quantities of
bad fats will decrease the probability that good fish oil omega-3 will end up
playing a role in one of your cell membranes. (Fish Oil) Triglycerides are store
and transport molecules, whereas omega-3 phospholipids are the bricks in our
cellular walls.
Your body is very selective in which phospholipids it uses. For example,
our brain mainly uses DHA-phospholipids, because DHA is a high charged and
brain activity needs these highly charged molecules. Eye-retinas need even
more charged molecules to convert light rays to brain signals, so eye phospho-
lipids contain whenever possible two DHA lipid molecules. No wonder that
omega-3 plays an important role in brain health like ADHD, dementia, and
Alzheimer’s. Muscles are less picky, and will settle for lower quality fats if that’s
all that is available.
Omega-3 plays another important cellular role. DHA is a very long chain
fatty acid, and thus can have very specific forms and shapes. These shapes

43
seem to greatly impact the number and kind of receptors in the cell mem-
brane. For example, serotonin receptors, which influence mood and depression,
correlate to omega-3 levels in the phospholipid membrane.
Inflammation. Omega-3 (DHA and EPA) and omega-6 (AA) greatly
impact inflammation; omega 3 reduces inflammation, and in large quantities
omega-6 will increase inflammation. The ratio between omega 3 and 6 is there-
fore a good indication for the risk of many inflammatory diseases. If cells get
damaged, for example through oxidative stress, the fats that are attached to the
phospholipid are released (cleaved in jargon) and go down an inflammation or
anti-inflammation path. If the quantity of omega-6 and omega-3 is out of bal-
ance on a cellular level, the inflammatory processes can get out of control too.
Examples of inflammatory processes are cardiovascular disease, and arthritis.

Omega-3 fats are high unstable. For example, you would not consider
cooking French fries in fish oil. Consequently, industrial growers of fats (corn
oil, canola oil etc.) have an interest in breeding the omega-3 fats out of their
plants, and increasing the level of omega-6 fats. Their oils become more stable
as a result. Consumers are unaware that their nutrition is changing dramati-
cally without changing their diet. Because of these nutritional changes, and
because omega-6 fats are so much cheaper the historical balance of 1 to 1
between omega-6 and omega-3 has shifted in the US to 15 to 1 and for certain
poor populations which consume large quantities of cheap processed foods, the
ratio has shifted to 30:1 or worse.
Omega-3 phospholipids from krill: Krill oil contains 40% phospholipids
with bonded omega-3. The quantity of omega-3 (EPA and DHA) is far less than
in fish oils, but the phospholipid form is radically different.

44
 Krill Oil vs Fish Oil: What about other Krill oils besides NKO? Well, NKO
has the patent for gentle extraction. So if anyone claims to make it as gently as
NKO, they are breaking the law or doing something funny. Either way, nobody
knows because no one else has published their results.
The reason why this gentle process is so important is that the phospho-
lipid bond that makes Krill Oil superior is easily damaged by heat.
Once the phospholipid bond is damaged, then Krill oil is no different
than fish oil.
Heat damaged Krill oil is just really expensive, red-colored oil.
]
Individual phospholipids contain a charged polar headgroup that are ori-
ented toward the outer or inner membrane surface and two fatty acids that form the
non-polar core of the membrane.

Figure 4.2: The general organization of biological membranes.

It is well known that proteins embedded in the membrane can be profoundly
affected by any change in their surrounding membrane environment such as differ-
ences in the type of phospholipid or in the density of their packing. Many effects of
alcohol (ethanol), for example, are caused by its disruption of the normal mem-
brane environment around proteins. Phospholipid methylation, the addition of a
methyl group to certain phospholipids, reduces the packing density of membrane
phospholipids and can modulate the activity of embedded (integral) membrane
proteins.
Phospholipid methylation was initially described in the mid-1960’s (36),
although the work of Julius Axelrod and Fusao Hirata a decade later brought it to

45
prominence when they suggested that it was involved in receptor signaling pro-
cesses (37,38). While Axelrod did receive a Nobel Prize, it was not for his phospho-
lipid methylation studies but for his earlier pioneering work on the metabolism of
catecholamines. He and Hirata characterized enzymes that could methylate a spe-
cific phospholipid, phosphatidylethanolamine (PE), to form the fully methylated
phospholipid phosphatidylcholine (PC), as illustrated in Figure 4.3. The methyl
donor for this reaction is S-adenosylmethionine (also known as SAM or SAMe), an
activated form of the amino acid methionine. SAM is now sold as a non prescrip-
tion drug for the treatment of mild depression and its therapeutic action is probably
due to its ability to increase phospholipid methylation.

Figure 4.3: Methylation of phosphatidylethanolamine (PE). PE is successively

methylated to N-monomethylPE, N,N-dimethylPE and phosphatidylcholine by the
action of phospholipid methyltransferase (PLMT), using S-adenosylmethionine as
the methyl donor.
The main effect of adding one or more methyl groups to PE is that the phos-
pholipid molecules cannot pack as close to one another as before, resulting in a
loosening of the membrane structure or an increased membrane fluidity (Figure
4.4). Under this condition the fatty acids that make up the membrane core have
more space available to them so they can flex more easily instead of being tightly
constrained. The activity of receptors and other membrane proteins can be affected
by increased membrane fluidity, especially if they exhibit large conformational
movements that impinge on the protein/phospholipid interface. An increase in
phospholipid methylation therefore causes a change in the packing density of

46
the cell membrane that can affect the activity of membrane proteins. These mod-
ulatory effects of phospholipid methylation are terminated when phospholipase
enzymes subsequently remove the headgroup from methylated phospholipids.

Figure 4.4: Methylation of membrane phospholipids causes a decrease in packing

density and increased membrane fluidity.
Membrane proteins with phospholipids tightly held against their surface
respond to increased membrane fluidity with either an increase in their activity (e.g.
increased opening of receptor channels for chloride ions) or in other cases a
decrease in their activity (e.g. decreased opening of receptor channels for sodium
ions). The underlying cause for this effect of fluidity is that the two conformational
states of membrane proteins (e.g. active or inactive) have a differential affinity for
binding the surrounding phospholipids. As a result, changes in phospholipid pack-
ing density shift the energy balance between active and inactive states, thereby mod-
ulating membrane protein activity.
Phospholipid methylation based modulation is closely related to the mecha-
nism by which ethanol causes many of its effects. Ethanol easily dissolves in the
cell membrane and inserts itself at the interface between membrane proteins and
their surrounding phospholipids. In this location ethanol exerts its own modula-
tory effect on membrane protein activity. By increasing the activity of chloride
channel receptors (i.e. GABA or glycine receptors) (39) and decreasing the activity
of sodium (or calcium) channel receptors (such as nicotine or NMDA receptors)
(40, 41) the net effect of ethanol is to inhibit the firing of nerves, resulting in cen-

47
tral nervous system depression. These effects are readily observed at blood ethanol
levels of 0.08% to 0.10%, the legally defined concentration for being drunk. In other
words, the effects of changes in membrane fluidity are very much a part of our
everyday world, especially when we feel the effects of a drink or two (or more).
If changes in the membrane environment are such important modulators of
neuronal function and behavior why hasn’t nature, through so many years of evolu-
tion, incorporated this into its repertoire of control mechanisms? The answer is, it
has! The discovery of dopamine D4 receptor mediated phospholipid methylation
shows how localized control over membrane fluidity, linked to the concentration of
dopamine, can provide an elegant means of modulating neuronal activity, ulti-
mately providing us with the power of attention. We call this novel D4 receptor-
dependent mechanism of modulation “solid-state signaling” because it acts via
direct molecule to molecule contact within the plane of the membrane, as
opposed to diffusible intracellular second messenger signaling, the more common
mechanism for G protein-coupled receptors.
D4 Receptor-mediated Phospholipid Methylation
In work first published in May of 1999 we described the ability of dopamine
to stimulate phospholipid methylation (PLM) via its activation of D4 receptors
(D4Rs). The underlying molecular basis for D4R-mediated PLM involves the ability
of methionine #313 (MET313) at the hot spot location to be rotated into an accessi-
ble position during activation of the receptor by dopamine (i.e. in its R* state). This
increased exposure allows adenosylation of MET313, catalyzed by the enzyme
methionine adenosyltransferase (MAT). In the inactive R state MET313 is inacces-
sible to the enzyme, but in the active R* state MAT can transfer the adenosyl por-
tion of ATP to the sulfur atom of MET313. This reaction is the critical first step in
a four-step cycle of phospholipid methylation activity outlined in Figure 4.5.
Adenosylation of MET3 13 serves to weaken the bond holding the methyl
group (-CH3) to the sulfur atom, facilitating its transfer to the neighboring phos-
pholipid PE, creating N-methyl PE and increasing membrane fluidity. We provided
evidence for these reactions by directly showing that the D4 receptor was adenosyl-
ated and that adenosylation could be blocked by inhibiting MAT activity (18).
Inhibiting MAT also blocked the ability of dopamine to stimulate PLM. Further-
more, adenosylation of the receptor and dopamine-stimulated PLM were both
blocked by the D4 receptor antagonist clozapine and by other more selective block-
ers. Since antagonists like clozapine hold the receptor in its R state, this result indi-
cated that the R* state was required for adenosylation.

48
 Figure 4.5: The four-step cycle of D4 receptor-mediated PLM.
In Step 1 MET313 is adenosylated in an ATP-dependent reaction
carried out by methionine adenosyltransferase (MAT).
In Step 2 the methyl group from MET313 is transferred to the
phospholipid PE creating increased membrane fluidity.
In Step 3 the adenosyl group is released to form adenosine in a
reversible reaction catalyzed by S-adenosylhomocysteine
hydrolase (SAHase).
In Step 4 MET313 is re-formed by the action of methionine
synthase (MET SYN) using 5-methyltetrahydrolfolate (5-
methylTHF) as methyl group donor.
We showed that inhibition of MAT activity with the drug cycloleucine
blocked spontaneous G protein activation by the D4 receptor. Since MET313 is in
the “hot spot” location, any naturally occurring mutation should produce increased
spontaneous activity and blocking the enzyme responsible for the mutation should
eliminate this activity. In this regard adenosylation of MET313 is analogous to
phosphorylation of the “hot spot” threonine residue in other spontaneously active
receptors. In fact each reaction uses a different piece of ATP to cause a temporary

49
mutation of this privileged “hot spot” location. In the case of threonine it is the
phosphate portion of ATP that is transferred while in the case of MET313 it is the
adenosyl portion with MAT serving a role analogous to a protein kinase.
In their earlier studies Axelrod and Hirata found that transfer of a methyl
group from S-adenosylmethionine (SAM) to PE required a phospholipid methyl-
transferase (PLMT) enzyme whose function is to simultaneously bind PE and SAMe
thereby facilitating methyl transfer (37). It does not appear that Step 2 of D4R-
mediated PLM requires such a PLMT enzyme. In the case of the D4 receptor, ade-
nosylated methionine (SAM313) is covalently attached to the receptor and the PE
headgroup is positioned immediately adjacent to the SAM residue. Thus there is no
need to bring the reactants together. Furthermore, amino acid sidechains in the D4
receptor near MET313 could facilitate transfer of the methyl group to PE, raising
the possibility that the receptor might catalyze this reaction. It is important to note
that the sidechain SAM313 must be pointing outward toward the surrounding
phospholipids for the methyl group to be transferred to PE rather than facing
inward as is required for adenosylation in the preceding step. Thus alternative con-
formations of the receptor may be required for different steps in the cycle of D4
receptor-mediated PLM.
After the receptor donates its methyl group to PE the next step (Step 3)
involves removal of the adenosyl group that is attached to the sulfur atom of S-ade-
nosylhomocysteine (SAH) 313. The enzyme S-adenosylhomocysteine hydrolase
(SAH hydrolase) reversibly catalyzes this step. Removal of the adenosyl moiety leads
to formation of homocysteine at position 313 in the D4R while the released adeno-
syl group becomes free adenosine. Evidence for SAH hydrolase involvement derives
from the ability of several different specific inhibitors of this enzyme to block dopa-
mine-stimulated PLM (18).
There are several particularly interesting aspects about SAH hydrolase. First
of all, it is a reversible enzyme and in fact it normally favors the synthesis of SAH
from adenosine and homocysteine (42). Only the rapid further metabolism of ade-
nosine and homocysteine keeps the reaction going in the direction of SAH hydroly-
sis. If the levels of adenosine or homocysteine increase, SAH is re-formed rather
than being broken down. This reversible feature causes the cycle of D4R-mediated
PLM to be inhibited at high levels of adenosine, due to difficulty in removing the
adenosyl group from its SAH state. Indeed we found that dopamine-stimulated
PLM is reduced by increasing adenosine levels either by adding extra adenosine or
by inhibiting the enzymes that metabolize it. There is important physiologic rele-
vance for this effect since it is well known that adenosine levels vary in a cyclic man-
ner (a circadian rhythm), and levels are highest at the onset of sleep. Furthermore,
adenosine formation is greatly increased in several psychiatric illnesses, including
some types of autism (43). Later we’ll discuss these implications in more detail.
Another interesting feature of SAH hydrolase is that it tightly binds a mole-
cule of copper (Cu2+) to each of its four subunits, and copper levels in turn regulate

50
the amount of SAH hydrolase enzyme that is produced (44,45). This has particular
implications for diseases of copper metabolism and possibly for Alzheimer’s dis-
ease, for which altered copper metabolism (46), elevated homocysteine concentra-
tions (47,48) and lower levels of SAM (49) have been described.
The fourth and final step in D4R-mediated PLM is the conversion of homo-
cysteine back to methionine so the cycle can start over again. This requires a new
methyl group to replace the one previously donated to PE, and the usual source of
this methyl group is the folic acid system in the form of 5-methyltetrahydrofolate
(5-methylTHF). The enzyme that transfers the methyl group from folate to homo-
cysteine is methionine synthase, famous for its vitamin B12 requirement and for
being a target for “laughing gas” or nitrous oxide.
The key to detecting D4 receptor-mediated PLM was the introduction of a
radiolabel into the methyl group of 5 -methylTHF. Based upon a suggestion by Dr.
Conrad Wagner (Vanderbilt University) we used [14C]-formic acid for this pur-
pose, since the carbon from formate becomes the methyl group of 5-methylTHF. As
illustrated in Fig. 4.6, human neuroblastoma cells gradually incorporate folate-
derived radiolabel into phospholipids starting after a 15 min delay. However, when
dopamine is present there is an extra increase due to D4 receptor activation. This
increase reflects methionine synthase activity directed toward the D4 receptor.

Figure 4.6: Dopamine stimulates the incorporation of radiolabel from [14C]-

formate into membrane phospholipids in human neuroblastoma cells.

51
 As elegantly described by Dr. Rowena Matthews and her colleagues at the
University of Michigan (50), methionine synthase has the ability to utilize either 5-
methylTHF or SAM as a source of methyl groups for conversion of homocysteine to
methionine. Under laboratory conditions, when an unlimited supply of 5-methyl-
THF and SAM are provided, 99 times out of 100 the enzyme uses 5-methylTHF. In
our studies of DA-stimulated PLM in nerve-like human neuroblastoma cells, we
only observed 5-methylTHF utilization. However, occasionally the cobalt ion in the
vitamin B12 becomes oxidized from its normal 1+ state to 2+ and the enzyme is
unable to continue using 5-methylTHF. This sets the stage for SAM to enter and
donate its methyl group to the enzyme while at the same time returning the cobalt
back to its 1+ state, allowing 5-methylTHF to again be used. In a sense SAM is a
backup, used only when needed to restart folate-dependent methionine synthase
activity. There is also an auxiliary enzyme, methionine synthase reductase that pro-
vides the reducing equivalents and assists in the SAM-dependent methylation of
cobalt (51).
The above performance features raise questions about how often SAM is
required under normal cellular conditions in the body and whether the availability
of 5-methylTHF ever becomes a limiting factor. Indeed, studies by Dr. Matthews
indicate that the oxidation of cobalt serves as a sort of timing device to trigger the
utilization of SAM under conditions when 5-methylTHF levels are low (50). Here’s
how it works: The rate of cobalt oxidation has a certain probability of occurring
per unit of time. If there is a high amount of 5-methylTHF present, it quickly
binds to the enzyme and transfers its methyl group to the cobalt before oxida-
tion occurs. However, if the concentration of 5-methylTHF is lower than nor-
mal, binding is slower and there is a greater chance that the cobalt will oxidize,
leading to the required utilization of SAM as methyl donor for the next cycle. If
the levels of 5-methylTHF are particularly low, SAM can serve as the source of
methyl groups for a number of cycles. Using SAM as the methyl donor to make
methionine is largely a waste for the methionine cycle, since it simply uses SAM
to remake itself, consuming an ATP in the process. However, with regard to
D4R-mediated PLM, SAM serves as back-up source of methyl groups for the
receptor cycle whenever 5-methylTHF levels are low. It appears that neuronal
cells are prepared to sacrifice their supply of SAM in order to meet the needs of
the D4R-mediated PLM cycle whenever the folate supply is inadequate. This can
prove to be important, since deficits in the formation of 5-methylTHF have been
linked to the occurrence of schizophrenia (52,53).
As mentioned earlier, SAM is marketed as a non-prescription nutritional
supplement for the treatment of mild depression and for symptoms of rheumatoid
arthritis. Unlike some claims for supplements, there is strong clinical trial-based
evidence for the benefits of SAM in depression (54-56). Although a direct link has
not been established, it is likely that SAM may work by augmenting the supply of
methyl groups to the methionine synthase reaction in Step 4, especially when the

52
supply of 5-methylTHF is low. As discussed more completely in later chapters,
depression can be viewed, at least in part, as suppressed attention to external
events. From this perspective the therapeutic benefits of SAM may at least partially
result from its effect on the molecular events that support attention.
A significant aspect of Step 4 is that it links dopamine-stimulated PLM to the
single-carbon folate pathway, which in turn serves as a dynamic source of single-
carbon groups to a number of fundamental metabolic processes, including the syn-
thesis of purines and thymidine, which are required for RNA and DNA. As a conse-
quence of this linkage the efficiency of D4R-mediated PLM can be affected by
changes in core metabolism, insofar as such changes might increase or decrease
availability of 5-methylTHF (20). Dopamine is certainly a key player in cognitive
function and its D4 receptor-mediated actions can now be recognized as being
closely linked to folate metabolism.
The ability of the D4 receptor to receive methyl groups from 5-methylTHF
was illustrated by a simple autoradiography experiment. By incubating human neu-
roblastoma cells in radiolabeled formic acid we introduced a label into the methyl
group of 5-methylTHF. After 30 minutes we used autoradiography to determine
which proteins were labeled as a result of receiving folate-derived methyl groups. As
shown in Fig. 4.7, only a single protein, the D4 receptor, was labeled and it only
became labeled when dopamine was added. Furthermore, the D4 receptor antago-
nist clozapine blocked labeling just like it blocked PLM, as did cycloleucine the
MAT inhibitor. Clearly the D4 receptor has a unique relationship with the folate
pathway.

53
 Figure 4.7: D4 receptors are labeled by radioactive 5-methylTHF.
SH-SY5Y human neuroblastoma cells were incubated in [14C]-
formic acid for 30 min. An autoradiograph of SDS-PAGE-
separated proteins shows selective labeling of the D4 receptor in
the presence of dopamine that is blocked by clozapine and
cycloleucine.
What happens to the methylated phospholipids after they are formed? How
long does the increased membrane fluidity they create last? These are issues that we
don’t know enough about at present. Successive methylations of PE ultimately form
phosphatidylcholine (PC), which is found primarily in the outer leaflet of the
plasma membrane bilayer (Fig. 4.8). Some of the N-methylPE and N,N-dimethylPE
generated by the D4 receptor may flip from the inner side of the membrane to the
outer side and be converted to PC. Another important fate of methylated phospho-
lipids may involve their hydrolysis by phospholipase enzymes acting on the inner
side of the membrane. Two of these phospholipases, phospholipase C(PLC) and
phospholipase D(PLD), remove the headgroup from phospholipids by hydrolyzing
the bond on either side of the phosphate group (Fig. 4.8). The freed headgroups are

54
released into the cytoplasm, while the fat-soluble products diacylglycerol and phos-
phatidic acid remain in the membrane.

Figure 4.8: Termination of phospholipid methylation.

Methylated phospholipids (e.g. PC) can flip to the outer leaflet of
the membrane or serve as substrates for phospholipases C and D,
which remove the methylated headgroup.
Through one or both of these pathways the membrane fluidizing effect of
DA-stimulated PLM can be terminated after a limited period of time. Since activa-
tion of G protein-coupled receptors like the D4R can lead to stimulated PLC and
PLD activity (57,5 8), dopamine may stimulate both the initiation and termina-
tion of solid-state signaling. However, solid-state signaling is very rapid in onset,
more rapid than G protein coupling-based signaling. Thus solid-state signaling
would occur before the slower onset PLC/PLD activity is fully activated while the
latter would limit the duration of PLM-associated attention.
Solid-state signaling, in the form of dopamine-stimulated PLM, is designed
to provide a localized influence on membrane structure that is capable of propagat-
ing the influence of dopamine to other membrane proteins that share the same
microenvironment. Its fast kinetics are well suited for rapid modulation of neuronal
activity at sites of synaptic transmission, as is required for a molecular mechanism
of attention.

55
 D4R-initiated solid-state signaling can exert important effects on the func-
tion of membrane proteins by influencing their ability to undergo conformational
changes. Examples of such proteins are ion channels and ion channel receptors as
well as transport proteins such as the calcium ATPase or calcium pump. All of these
membrane proteins contain transmembrane helices as an integral part of their
structure. During their on-off or open-closed activity cycles, one or more of their
helices can undergo a rotational movement in the membrane. These movements
constitute conformational changes in the membrane proteins, analogous to the
inactive R and active R* states of G protein-coupled receptors. For example, the
movement of transmembrane helices in ion channel receptors allows them to
change from their closed, inactive state to their open, active state. Surrounding
membrane phospholipids bind to the sides of these proteins, including the moving
helices. When the helices rotate, it changes their outer surface, the same surface to
which the phospholipids bind. If phospholipids bind more tightly to one state ver-
sus the other, their binding will tend to promote the lifetime of that state. This is the
same mechanism used by neurotransmitters to increase receptor activity, binding
with higher affinity to the active state. Phospholipids can have a similar influence on
activity, except that they bind on the side of the membrane protein whereas neu-
rotransmitters typically bind to the outside face.
In short, dopamine-stimulated PLM is capable of modulating a wide range of
membrane proteins by virtue of local changes in membrane packing density and its
consequent allosteric influence on protein conformation. This very rapid mode of
solid-state signaling appears to be a critical mechanism by which dopamine pro-
motes attention.

Chapter Summary:
• Dopamine D4 receptors have a methionine residue at the “hot spot” locus.
• D4 receptors in all species have proline-rich segments that allow it to bind other proteins.
• In primates and man the D4 receptor has an additional 2 to 11 praline-rich repeats that
provide for more complex simultaneous interactions with multiple proteins.
• Methylation of membrane phospholipid headgroups causes an increase of membrane
fluidity.
• Dopamine D4 receptors can be stimulated to carry out phospholipid methylation (PLM)
in response to dopamine.
• The effect of dopamine-stimulated PLM on other membrane proteins is called “solid-state
signaling.”
• Dopamine-stimulated PLM requires a four-step cycle in which a new methyl group is
provided from the folate pathway.
• S-adenosylmethionine (SAM) can serve as a reserve source of methyl groups when 5-
methylTHF levels are low.
• Adenosine levels are an important regulator of the PLM cycle.

56
5 D4 RECEPTOR-MEDIATED SOLID-STATE SIGNALING

In this chapter we begin to explore the function of dopamine stimulated

PLM, especially as it relates to neuronal mechanisms that are central to attention.
Under normal conditions D4R-mediated PLM activity is limited by the available
concentration of dopamine, which is required for changing the receptor to its R*
shape and initiating PLM. Nature designed the PLM system in this manner to be
responsive to changes in the level of dopamine. Dopamine-releasing nerves can
therefore bring about a localized change in membrane fluidity within D4R-contain-
ing target nerves across the synapse by causing the dopamine concentration to build
up in the synaptic cleft. In other words the design features of this system allow the
biophysical character of the post-synaptic membrane to be modulated whenever
dopamine is released from the presynaptic nerve. Effects of dopamine-induced
PLM on the post-synaptic nerve reflect D4R-mediated solid-state signaling.
The neuronal role of dopamine and other catecholamines is not to transmit
basic sensory information but rather to modulate the efficiency of information flow
through synapses. Thus most dopaminergic synapses in the brain can be considered
as three-way synapses, involving three neurons, as shown in Figure 5.1. In a three-
way synapse the primary information flow is carried by a neurotransmitter whose
post-synaptic receptor action directly controls neuronal firing via an electrical
mechanism. Typically these are fast- acting ion channel-based receptors that either
stimulate or inhibit firing of the post-synaptic nerve depending on which ion passes
through their channel. Stimulatory receptors increase firing rate, while inhibitory
receptors decrease firing rate. Stimulatory receptors include three different receptor
types that use glutamic acid (glutamate) as the neurotransmitter (NMDA, AMPA
and kainate receptors) as well as nicotinic cholinergic receptors that respond to the
neurotransmitter acetylcholine. Inhibitory receptors utilize GABA (gamma amino
butyric acid) or glycine as the neurotransmitter.
The presence of a third nerve in three-way synapses provides an opportunity
to modulate primary information flow. Dopamine-releasing nerves exert this type
of modulatory influence and the same applies to nerves releasing norepinephrine or
serotonin or other neurotransmitters that act via G protein-coupled receptors. In
this arrangement, the dopamine (or norepinephrine or serotonin etc.) does not
carry the primary information, but rather it affects the efficiency of basic informa-
tion flow. In a sense dopamine can add another level of secondary information in an
entirely different modality to the primary information. As illustrated in Figure 5.1
this effect can be exerted at the presynaptic neuron by influencing neurotransmitter
release or at the postsynaptic neuron by modulating receptor responsiveness or
even at both sites. The precise nature of the dopamine effect depends upon whether
dopamine receptors are present presynaptically or postsynaptically and also on
what type of dopamine receptors are present in each location.

57
 Secondary information can have either a positive or negative effect on the
trans-synaptic flow of primary information. For example, dopamine stimulation of
presynaptic D1-type receptors augments neurotransmitter release, while stimula-
tion of presynaptic D2-type dopamine receptors decreases neurotransmitter release.
Postsynaptic D4 receptors can exert an inhibitory influence on the response to
excitatory neurotransmitters such as glutamate, but may also exert presynaptic
effects as well.

Figure 5.1: A three-way neuronal synapse.

Dopamine (DA) can modulate the transfer of information across a
synapse by pre- and post- synaptic actions. Activation of
presynaptic D1 receptors can increase neurotransmitter (NT)
release while D2 receptor activation can inhibit release. D4R
stimulation can modulate postsynaptic or presynaptic response.
Secondary information affects the qualities of primary information. For
example, if the primary information is sensory in nature (e.g. a visual image of a
person), dopamine-mediated secondary information may confer a higher signifi-
cance to the particular sensory information as compared to information flowing
through other pathways unaffected by dopamine. Such an enhancing effect of dopa-
mine would comprise a neuronal mechanism for directing attention to a subset of
currently perceived sensory information. In other words, dopamine can cause
attention via its modulation of synaptic transmission. Conversely, anything that
interferes with these actions of dopamine can reduce our normal capacity for atten-

58
tion. Higher than normal levels of dopamine can promote attention and lower than
normal levels can reduce attention. A higher density of dopamine receptors can
increase dopamine effectiveness thereby augmenting dopamine dependent atten-
tion and vice versa. The same is true for any necessary component of the dopamine
signaling process. They all affect the quality and efficiency of attention.
Dopamine-stimulated, D4R-mediated PLM is a novel molecular mechanism
for bringing secondary information to bear upon a particular synapse or group of
synapses. Because D4 receptors appear to be particularly associated with attention,
discovery of D4R-mediated PLM opens the door to new thinking and new lines of
inquiry into how dopamine modulation of neurotransmission might cause atten-
tion.
D4 Receptor Repeats, Novelty-seeking Behavior and ADHD
In order to further probe the functional role of D4R-mediated PLM let us
turn our own attention to the proline-rich regions of D4 receptors. Recall that D4
receptors in lower species, with the exception of dogs (59), contain only two pro-
line-rich segments in the third intracellular loop as shown in Figure 4.1. Since pro-
line-rich regions allow formation of SH3 domain dependent complexes with other
proteins, this implies that the D4 receptor of lower species can only bind two pro-
teins at a time. However, in humans and other primates SH3 domain docking sites
have evolved to a much higher level of complexity. To be a member of the “primate
club” your D4 receptor must contain at least two additional proline-rich repeat seg-
ments. Receptors with two repeats are designated as D4.2 receptors, those with four
are D4.4 receptors etc. The most common form of the D4 receptor in humans is the
four-segment D4.4 receptor, although versions with up to eleven repeat segments
have been identified (15,35).
To fully appreciate the situation we have to remember that the structure of
the D4 receptor is specified at the level of its gene sequence, located near the end
of the short arm of chromosome 11. This DNA sequence gives rise to the protein
sequence through the intermediate role of messenger RNA (mRNA). Sequential
groups of three DNA bases [taken 3 at a time from the set of 4 (A (adenine), G (gua-
nine), C (cytidine) or T (thymidine))] specify a single amino acid in the D4R’s pro-
tein sequence; shown in Figure 5.2. For example, a DNA sequence of CCC would
specify a proline residue in the final protein product. Together the three DNA
nucleotides are called a codon because they code for a particular amino acid. The
first two nucleotides in a codon are the most important information and some-
times the third nucleotide can be different but still give the same amino acid (e.g.
CCA, CCG and CCT all specify proline). It is clear that the recent completion of the
human genome project will bring genetic information more directly into our lives,
hopefully for the better. A basic understanding of the DNA/mRNA/protein rela-
tionship will therefore become more and more important in the future.

59
 DNA exists as a paired helical chain in each chromosome and we have dupli-
cate copies of each of our 23 chromosomes. Therefore we get roughly half of our D4
receptor proteins from each of the chromosome 11 pair and each gene is called an
allele. Since the D4 receptor gene is not necessarily the same on each of the pair, it is
common that humans express a mixture of two different D4 receptor proteins, dif-
fering in the number of proline-rich repeats (e.g. both D4.4 and D4.7 receptors).
Thus it is more common to be heterozygous (i.e. have two different alleles) than
homozygous (have two of the very same allele). One chromosome is inherited from
your mother and the other from your father in a classical Mendelian manner.
Gregor Mendel was, of course, the famous pea-growing monk who first predicted
the function of a gene-like material.

Figure 5.2: An example of the 48 base repeat in the D4 receptor gene and the
resultant protein sequence the D4 receptor protein. Notice the presence of an
overlap at the ends since the repeat is out of frame with the codon sequence.
Each proline-rich D4 receptor repeat, also known as a variable number tan-
dem repeat or VNTR, consists of 16 amino acids, reflecting a 48-base repeat (16 X 3
bases/codon) at the level of DNA (Figure 5.2) (14). The first of the 48 bases is the
end of a codon and the last two bases are the start of a codon. While the proline-
rich segments are similar enough to each other to be considered as repeats, varia-
tions occur within the 48 bases giving rise to another whole layer of complexity.
This means that individual humans differ from each other not only in the number
of repeats in their two D4 receptors but also in the specific sequence within the indi-
vidual repeat segments. This is why the primate D4 receptor gene is considered to
be not just variable, but hyper-variable with important functional and evolutionary
implications.
A team of researchers led by Dr. Kenneth Livak at DuPont Merck identified
many of the D4R repeat sequences and initially assigned each of them a Greek letter
symbol (15). Later, as more variations were found, they ran out of letters and a

60
numbering system was applied (35). At present 35 variant sequences have been
identified, although others may yet be found. In D4.2 receptors the repeats are
almost always the same two, with the exception being Surui natives of South Amer-
ica. These two variants are retained as the first and last repeats in longer forms of
the receptor, with only limited exceptions. Worldwide, the D4.2 receptor accounts
for approximately 9% of the alleles in the human population (35). In a D4.3 receptor
the middle repeat could theoretically be any one of the other variants but in fact
only four variants are found in this position. In the D4.4 receptor, which is the most
common form (65% of the world total), the middle two repeats can be any of ten
possibilities, but two particular variants account for 95% of the total D4.4 receptors.
The second most common allele, the D4.7 receptor, accounts for about 19% of the
worldwide total and also exhibits one highly preferred set of variants, accounting
for almost 90% of its total.
Interestingly, different racial/ethnic patterns have been described for the
number of repeats. As described by studies from the lab of Dr. Kenneth Kidd at Yale
(17), the 7-repeat allele accounts for only 1% of the total among the Japanese,
while among the Surui and Ticuna people of South America it accounts for 70%.
Clearly, nature has done a great deal of tinkering with proline-rich segments of the
D4 receptor in humans and primates. As noted above, the end result (at least at this
moment in evolution) is that the D4 receptor is one of the most variable genes and
proteins in man, implying that its functional capacity may also be quite variable
from person to person.
Non-human primates (i.e. monkeys, chimpanzees, and gorillas) also have
48-base pair repeats in their D4 receptor genes that code for proline-rich segments
in the receptor protein (60). This immediately alerts us to the fact that some of the
performance features of D4Rs in man and apes are likely to be generally similar to
each other but quite distinct from non-primate species. In this regard, man and apes
are clearly part of the same D4R family, no matter what the laws of the State of Kan-
sas have to say about it.
In 1996 two papers in the journal Nature Genetics, by researchers from Israel
(4) and National Institute of Mental Health (3), reported that the presence of the 7-
repeat D4R allele was correlated with higher “novelty seeking” scores on several
personality inventory tests. This remarkable finding was touted in newspaper
reports as the first instance where a personality trait could be linked to a specific
genetic sequence. What prompted these researchers to consider the possibility of
such a link? In 1987, Cloninger proposed that major groups of personality traits
(e.g. temperament and character) were a reflection of the activity of specific neu-
rotransmitter systems (61). He suggested that variations in the activity of dopami-
nergic neurotransmission were responsible for the fact that some persons tended to
take more risks (i.e. were novelty seekers), as compared to others who placed a
higher value on harm avoidance. Within the context of contemporary science it
seems perfectly reasonable that aspects of our behavior are determined by the rela-

61
tive activity of particular neurotransmitter systems. At some level we know that
molecular events within the brain are responsible for what we feel and what we
do. Nonetheless it may be a bit unsettling to realize that a significant portion of our
personality is predetermined by genetic composition. Of course a dispassionate
comparison between our parents and ourselves often bears out the truth of this like-
lihood, implying a limit on self-determination and dominion over our own lives
and identity. Score one for mother nature in the ongoing controversy over nature
vs. nurture. However, the importance of a higher number of D4 receptor repeats in
causing higher novelty seeking scores in these two studies was limited to only about
10% of the total determinants, providing a comeback for nurture and leaving the
door open for other genetic determinants.
A number of follow-up studies attempted to replicate the novelty seeking/D4
receptor repeat relationship. Some of them did (62,62), but some of them didn’t
(64,65) leading to skepticism about the strength or validity of this relationship. It is
not clear what might account for conflicting results, but some possibilities include:

1. Difference in the expression of novelty seeking in different cultures.

2. Different definitions of novelty seeking in personality tests.
3.Confounding effects of drug use (e.g. alcohol and nicotine).

Overall, we can say that the linkage of a higher number of repeats with nov-
elty seeking is a provocative, but still controversial part of the D4 receptor story.
Recent results from Arinami and colleagues in Japan added a new and
intriguing twist to the D4 receptor/novelty-seeking issue (66). They found a strong
correlation between novelty seeking and a genetic polymorphism (variation)
located within the portion of the D4 receptor gene that controls how much of the
receptor protein is produced (i.e. the 5’ upstream promoter region of the gene). Spe-
cifically, higher novelty seeking scores were observed for persons homozygous for
the polymorphism that increased D4 receptor expression level. In contrast, lower
scores were found for persons who were homozygous for the polymorphism caus-
ing lower D4 receptor expression. In other words their work suggested that the
probability of the novelty seeking trait is also be influenced by how many D4 recep-
tors someone has available, in addition to differences in the type of D4 receptor they
produce. Clearly these two factors could work in concert to determine overall prev-
alence of this trait.
The influence of genetic predispositions can diminish with age, as the influ-
ence of experience and environmental factors becomes stronger. For this reason the
possible association of D4 receptor repeat length with temperament was examined
in two week-old neonates and one month-old infants using endpoints such as “neg-
ative emotionality”, “distress to limitations” and “adaptability”. Infants with the
longer 7-repeat allele had significantly lower scores for negative emotionality and

62
distress to limitations (67) and higher adaptability scores (68). When adaptability
was reassessed at 5 months of age, a significant relationship was not observed, sug-
gesting that experience and environmental factors had supervened genetic predis-
position at this time. At one year of age infants with the 7-repeat allele showed less
sustained attention in an information-processing task, a potential harbinger of
ADHD risk in later years (69).
One way to evaluate the physiological role of a particular protein is to remove
or inactivate its gene in mice and follow differences in their development or behav-
ior when that protein is “knocked out”. Knockout of the D4 receptor did not pro-
duce drastic alterations in the physical development of mice, but differences in their
behavior (70) and in their response to certain drugs (7) were observed. Interest-
ingly, D4R knockout mice exhibit a decrease in behaviors related to novelty seeking.
For example, when placed in a novel environment, these mice tended to show less
exploratory behavior. They preferred to avoid potential harm more than normal
mice did. In other tests, however, their overall motor activity was unimpaired. In
fact in a test of agility (keeping balance on a rotating rod) the knockout mice were
clearly superior to normal controls, perhaps because they were less easily distracted
(7). While it is perilous to extrapolate from mice to man, the knockout mice seem to
confirm a role for D4 receptors in how animals deal with novelty in their environ-
ment. It would appear that the presence of D4 receptors allows or encourages mice
to interact with a source of novelty. Remember that the D4 receptor in mice does
not have any repeats, but does have two proline-rich domains. If knocking out the
D4 receptor reduces novelty-seeking behavior, increased novelty seeking associated
with a high number of repeats in humans might represent the opposite condition.
D4 receptor knockout mice also showed exaggerated responses to the behav-
ioral effects of amphetamine and cocaine, both of which produce their effects by
increasing the levels of dopamine (70). They also were hyper-responsive to alcohol
(ethanol), which acts, at least in part, by producing changes in membrane fluidity.
This suggests that person to person differences in the activity of D4 receptors might
be a factor in determining our response to these commonly abused drugs, all of
which affect attention.
Another intriguing link between behavioral traits and the D4 receptor was
found in a recent comparison among breeds of dogs. Researchers in Japan com-
pared the D4 gene in two dog breeds: the docile and easily trained golden retriever
vs. the more aggressive and defensive shiba, a Japanese breed recognized for its use-
fulness as a watchdog (59). They found that the dog D4 receptor also contains
repeated proline-rich segments, although they are quite different from those found
in primates. Four different alleles (A-D) were identified, with A being the shortest
and D being the longest. All the golden retrievers had only A and/or B alleles, while
all the shibas had at least one C or D, but no A alleles. Thus a quieter and more read-
ily trainable demeanor was associated with the shorter repeat length. It is tempting,
but premature to extrapolate these findings to differences in human D4 receptors.

63
 About one year after the first papers on novelty seeking were published,
LaHoste and co-workers reported an association or the D4.7 receptor with ADHD
(5). They found that the percentage of children with at least one 7-repeat receptor
was more than twice as high in those diagnosed with ADHD as compared to non-
ADHD children. Put in other terms, the presence of the D4.7 receptor appeared to
be a risk factor for ADHD. A subsequent study suggested that homozygosity for the
seven repeat allele (i.e. presence on both chromosomes) greatly increased the likeli-
hood of ADHD (71). Follow-up studies have generally, but not unanimously, con-
firmed a link between a higher number of repeats and ADHD (6, 72-76). Logically,
ADHD studies have been carried out with a younger population, as compared to
novelty seeking studies. However, since both behaviors were associated with the
same D4.7 receptor allele, these results raise an intriguing question about how
ADHD and novelty seeking might relate to each other. This and other ADHD-
related aspects are discussed further in a later chapter.
The Functional Role of D4 Receptor Repeats
What is the functional significance of the D4 receptor’s proline-rich repeats
and how might this role fit in with the unique capacity of the D4R to carry out
PLM? Recognizing that the proline-rich nature of the repeats might lead to com-
plexes with other proteins, Dr. Hubert Van Tol used an artificial D4R expression
system to show that the human D4 receptor associates with a number of other pro-
teins, each of which possesses an SH3 domain (22). An SH3 domain is a specialized
region found in some proteins whose shape and amino acid composition allows it to
recognize and bind to proline-rich sequences in other proteins. Among the SH3
domain-containing proteins binding to the D4R were several so-called linker pro-
teins (e.g. Grb2, Nck and Crk) whose only role is to link one protein to another.
They also found the D4R to be associated with Ras GAP, a critical regulator of the
low molecular weight GTP-binding protein Ras, famous for its role in activating cell
growth and development. In neurons Ras controls the mitogen-activated protein
kinase (MAP kinase) signaling pathway which is important in learning. Dr. Ya Fang
Liu, working in our lab at Northeastern University, showed a similar pattern of D4R
interaction with SH3 domain-containing proteins, using human neuroblastoma
cells that express a native population of D4 receptors (23).
The preceding findings raised the exciting possibility that the human D4R
might be involved in activation of the MAP kinase pathway via its proline-rich
domains. In most cells, MAP kinase activation increases the expression of genes that
lead to cell division or mitogenesis. Since mature nerve cells don’t commonly divide
(a point of current controversy), MAP kinase activation plays a different role in
neurons. It still causes gene expression but in nerves its activation is associated with
the sprouting of new nerve endings called neurites (or neurite extensions) (77). The
tips of these neurites are capable of forming new synapses when they contact adja-
cent nerves. MAP kinase activation is also involved in the capacity for learning,
especially for creating spatial memory in novel environments (78). This learning

64
may be linked to forming new synapses and/or strengthening existing synapses.
Indeed, dopamine stimulation of D4 receptors does lead to MAP kinase activation,
as demonstrated by Van Toi and colleagues (22,79). They further showed that MAP
kinase activation did not occur after removal of the proline-rich segments. It is not
yet clear how dopamine occupation of the D4R specifically triggers activation, but
the ability of the receptor to bind Grb2 or RasGAP (or both) seems to be essential.
Taken together, these findings also suggest that dopamine stimulation of D4 recep-
tors may lead to increased neurite formation via MAP kinase activation.
D4 Receptors Bind to Synapse-associated Proteins
At excitatory synapses there is a dense collection of proteins just inside the
post-synaptic neuronal membrane so thick that it can be seen with a microscope.
This clump of proteins is called the post-synaptic density or PSD for short. It is a
collection of receptors, ion channels and protein kinases all held together by syn-
apse-associated-proteins or SAPs. SAPs are scaffolding proteins that serve as an
anchoring site for other proteins. A single SAP protein is able to directly co-localize
up to six different proteins in a cluster. The clustered proteins typically work
together in some manner, and by keeping them close together SAPs increase their
efficiency. This makes particular sense at a postsynaptic neurotransmission site
where all the necessary elements can be spatially organized for efficient synaptic sig-
naling.
There are four members of the SAP family: SAP9O, SAP97, SAP1O2 and
Rapsyn. As illustrated in Figure 5.3 they all share general structure features and all
have an SH3 domain, however, they can differ in the specific proteins they bind. All
SAPs contain three PDZ domains that recognize and bind the tail region of specific
proteins (e.g. NMDA-, AMPA- or kainate-type glutamate receptors, voltage-depen-
dent K+ channels, Ca2+ ATPase). They also contain a GK (guanylate kinase-like)
domain that binds GMP but apparently does not have catalytic activity. We investi-
gated whether SAP proteins might be able to bind D4 receptors.
Dr. John Marshall at Brown University first reported that one of the excit-
atory glutamate receptors, the kainate receptor, binds to SH3 domains of SAP9O
and SAP 102 but not SAP97 in studies conducted with rat brain. When I told John
about our D4 receptor findings and suggested a possible SAP/D4 receptor interac-
tion he agreed to take a look. The most common method for determining whether
two proteins bind to each other is a co immunoprecipitation or “co-IP” experiment.
These studies use an antibody that specifically binds to the protein to capture and
precipitate it, along with whatever else is attached to it. Proteins in the antibody pre-
cipitate are then separated from each other using gel electrophoresis and antibodies
specific for the attached proteins can be used to probe the gel. If the precipitate con-
tains detectable amounts of the other proteins it strongly suggests that the two are
complexed with each other. The co-IP results showed that SAP97 was associated
with the D4 receptor, while SAP9O and SAP 102 were not (80). This pattern was
just opposite of the results with kainate receptors, suggesting some sort of reciprocal

65
specificity. Another study, using immunofluorescence to assess co-localization, also
indicated that D4Rs and SAP97 were associated with each other. While only prelim-
inary, these findings were exciting because they placed the D4R, along with its
unique PLM activity, at a strategic position in the middle of the post-synaptic den-
sity complex among an array of other signaling proteins.

Figure 5.3: Synapse-Associated-Proteins (SAPs) provide for clustering of membrane

proteins via PDZ domain and SH3 domain interactions. They also possess a
guanylate kinase (GK) domain that binds but does not phosphorylate GMP.
It is important to recognize that these studies were carried out using material
from rat brain, which means that the D4R being studied was the non-primate recep-
tor containing only two proline-rich segments. Binding of the D4R to SAP97 can
therefore be attributed to one of these two more primitive segments. The other seg-
ment is presumably engaged with signaling protein such as those in the MAP kinase
pathway. In this configuration dopamine can simultaneously modulate postsynap-
tic responsiveness via PLM and initiate synapto-genesis via MAP kinase. Both of the
two non-primate proline-rich segments have a potential site of phosphorylation (i.e.
a serine (S) or threonine (T) residue) within them (e.g. PSGP and PQTP) (33) and
phosphorylation would likely disrupt SH3 domain-dependent complex formation.
This suggests that the ability of the D4R to bind other proteins could be regulated by
kinase-dependent phosphorylation. Coincidentally, MAP kinase specifically phos-
phorylates serine and threonine residues located near prolines. The presence of
D4Rs in the postsynaptic density might therefore depend upon the level of MAP
kinase activity.

66
 With its additional proline-rich repeats the primate D4R can simultaneously
engage additional signaling proteins as illustrated in Figure 5.4, perhaps including
SAPs other than SAP97. Determining the stoichiometry (i.e. the ratio) and specific-
ity of D4R/SAP interaction is an important but difficult task for the future. None-
theless, we can safely conclude that additional proline-rich segments in the primate
D4R allow interaction with a greater number of proteins.

Figure 5.4: Comparison of SAP binding by non-primate and primate forms of the
D4R. The non-primate D4R can bind only two proteins via proline-rich segments.
Repeat segments in primate receptors allow additional interactions with SAPs or
other proteins.

The Significance of D4R Repeat Number

Having learned more about the role of D4R repeats we can now re-examine
the enigmatic pattern of repeat distribution among the human and primate popula-
tion. Remember that two is the minimum number of repeats, but you can have up
to eleven. Four is by far the most common followed by seven and then two, a clearly
a discontinuous distribution. So why is it uncommon to have three, five or six
repeats? Why is four favored? How does D4R-mediated PLM relate to repeat num-
ber? We can now apply some analysis to these questions.

67
 We must recognize that when proteins bind to D4R repeats they take up
space and crowding within the membrane may become an issue. For example, the
size of each SAP is about twice the size of the D4R and each of the MAP kinase-
related proteins that bind to the D4R can be from half to twice as big as the D4R.
While each repeat adds a 16 amino acid chain to the D4R, there might not be
enough space for each additional repeat to bind an additional protein. With this
thought in mind we can suggest that the presence of two repeats may allow the
human D4R to bind one more additional protein than its non-primate counter-
parts. Since the first and last repeats are nearly always the same, this additional pro-
tein should the same for all persons with the D4.2 receptor. Expansion to three
repeats might or might not afford sufficient additional space for the binding of
another protein. In fact I would argue that the rarity of three repeats (less than 2%
worldwide) suggests that it does not in fact allow sufficient space and therefore was
not favored during evolution. Since the four repeat allele is most common, it pre-
sumably reflects some evolutionary advantage for survival, suggesting that an addi-
tional protein binding opportunity brings about some sort of improved
performance feature of the D4.4 receptor. in contrast, five and six repeats are rela-
tively rare (again less than 2%), suggesting they offer no particular advantage over
four or seven. The relatively high frequency of seven repeats (19% worldwide) is
consistent with an additional binding opportunity. More than seven repeats is a rare
occurrence, which might again reflect a lack of survival benefit.
This analysis is an obvious oversimplification of the complexity of evolution
but nonetheless it does follow directly from the protein binding capacity of proline-
rich repeat segments. From the perspective of evolutionary selection, differences in
the distribution of repeats among ethnic/racial groups can be expected to reflect dif-
ferences in the selective advantage conferred by repeat number within the context of
their historical development. Given the substantial evidence linking D4R repeat
number to personality traits such as attention or novelty seeking, it is likely that dif-
ferences in these behaviors may have led to the currently observed repeat distribu-
tion patterns. We will explore this concept further in a later chapter.
Finally, how does the number of repeats intersect with D4R-mediated PLM?
To approach this question it is helpful to visualize the microenvironment around
D4Rs, such as might occur within the postsynaptic density membrane (PSD).
Despite the fact that the PSD is crowded with proteins there must still be an intact
phospholipid membrane, a fact that is often overlooked when focusing on the activ-
ity of signaling proteins. As illustrated in Figure 5.5, the membrane microenviron-
ment around a D4R consists of a limited number of phospholipid molecules and a
limited number of proteins. Current knowledge allows us to estimate the space that
these different molecules occupy. For instance, phospholipids are about 70 Å2 in
cross-sectional area (an angstrom (Å) being 10-10 meters) and the D4R is about 700
Å2. Ion channel proteins are somewhat larger, occupying 1,000 to 1,500 Å2. As the
D4R binds more and more proteins, its microenvironment will accordingly contain

68
fewer and fewer phospholipids, with significant consequences for the efficiency of
D4R-mediated PLM. For example, with fewer phospholipids around, the D4R
might quickly run out of phospholipids to methylate and complete methylation
might be achieved sooner than would be the case if there were more phospholipids
and fewer proteins in the region. When a higher number of D4R repeats brings
more proteins into the microenvironment it squeezes out phospholipids and may
affect the dynamics of dopamine-stimulated solid-state signaling.

Figure 5.5: Comparison of the D4 receptor microenvironment, including

phospholipids and SAP-associated proteins, in D4.2 and D4.7 receptors.
By counting the number of receptors per cell and determining the number of
dopamine-stimulated phospholipid methylations per cell, we have estimated the
rate of D4R-mediated PLM. For the human neuroblastoma cells we normally use,
the rate is at least 20 methylations per second for each receptor or one methylation
every 50 milliseconds. Pretty fast, and the actual value could be even faster! Since it
takes no more than 15-20 phospholipids to encircle a D4R this implies that each
receptor could completely methylate its immediate environment within a second or
less. If other membrane proteins share that environment dopamine-stimulated

69
PLM could quickly change their environment as well. This is basis of solid-state sig-
naling.
Overall, variations in D4R function associated with different numbers repeats
could arise either from the activities of different proteins bound to the receptor or
from alterations in the protein/phospholipid ratio within the receptor’s microenvi-
ronment. Indeed both of these factors may be significant. Accordingly, an evolu-
tionary disadvantage to having a very high number of repeats becomes evident:
While the coupling of additional proteins to the D4R may allow more robust signal-
ing opportunities, it also brings a limitation in the efficiency of PLM-mediated
solid-state signaling. The trade-off between these consequences could be the evolu-
tionary driving force behind the predominance of the 4-repeat allele.
Dopamine Modulates NMDA Receptor Function
What specific membrane proteins might be targets for D4R-mediated solid-
state signaling? The obvious candidates would be proteins that are physically associ-
ated with the receptor either directly via SH3 domain interactions or indirectly via
mutual SAP binding. Candidate proteins may have been previously identified as
being regulated by dopamine in some manner. If their activity is indeed modulated
by PLM, candidate proteins might be recognized as being particularly sensitive to
changes in their membrane environment.
NMDA receptors are one of three ion channel-type receptors for glutamate,
the other two being AMPA and kainate receptors. Glutamate binding increases
opening of the NMDA receptor’s ion channel allowing calcium (Ca2+) and sodium
(Na+) ions to flow into nerve cells. This ion movement can trigger firing of the
nerve (providing excitation) and the rise in intracellular Ca2+ levels can also stimu-
late signaling pathways such as activation of calcium/calmodulin-dependent pro-
tein kinase II (CaM kinase II). The latter pathway is thought to be an important
mechanism for long-term memory induction (81). Indeed NMDA receptors are
widely distributed in the brain and are involved in memory and cognition as well as
in pathways that control neuronal survival (82).
A large number of studies have reported the ability of either dopamine or
dopamine receptor blocking drugs, such as neuroleptic drugs, to modulate NMDA
receptor activity. For example, Otmakhova and Lisman showed that dopamine
causes a strong reduction in NMDA receptor-mediated ion movements in the rat
hippocampus (83). Furthermore, this inhibitory action of dopamine was selectively
and completely blocked by the D4R-selective neuroleptic drug clozapine, suggesting
that the inhibitory effect of dopamine was mediated by D4Rs. Consistent with this
conclusion others have found that clozapine administration increases NMDA
receptor response (84). Clozapine was more potent in this regard than blockers of
other dopamine receptor subtypes. Thus D4R stimulation reduces NMDA response
while D4R inhibition increases it.

70
 Finally, NMDA receptors are well known for their high sensitivity to
increased membrane fluidity, particularly to the effects of ethanol and general anes-
thetics. Ethanol significantly inhibits NMDA receptor activation at concentrations
as low as 0.02%, five-fold below the legal definition of being drunk. Similar inhibi-
tory effects are observed with a number of general anesthetics, all of which increase
membrane fluidity. Taken together, these observations suggest that negative modu-
lation of NMDA receptors by dopamine may be caused by a stimulation of D4R-
mediated PLM and increased membrane fluidity in the microenvironment shared
by D4Rs and NMDA receptors (Figure 5.6).

Figure 5.6: D4R-mediated modulation of NMDA receptors. D4Rs and NMDA

receptors both bind to SAP proteins. Dopamine activation of PLM may inhibit
NMDA receptor response via changes in membrane fluidity.
While NMDA receptors are the most obvious target for solid-state signaling,
other membrane proteins that are bound to SAP proteins could also be affected.
Ca2+ ATPase, also known as the “calcium pump” transports calcium ions from the
inside of the cell to the outside, using ATP as a source of energy. This process
requires that Ca2+ ATPase change shape within the membrane during each trans-
port cycle, assuring that the surrounding phospholipids will be an important factor
in determining the efficiency of calcium transport. The calcium pump is highly sen-
sitive to ethanol, with low concentrations causing an increase in the rate of calcium
transport. Voltage dependent K+ channels also undergo a conformational change
during their activation cycle, and changes in phospholipid packing density could
also affect their activity. Dopamine-stimulated PLM can modulate the ensemble

71
activity of NMDA receptors, Ca2+ ATPase and K+ channels within excitatory post-
synaptic densities. This action can promote synchronized neuronal activity and
provide attention.

Chapter Summary:
• Dopamine modulates neurotransmission via the activation of dopaminergic receptors
located on both pre- and postsynaptic neurons.
• Dopaminergic modulation can promote attention to selected information.
• Proline-rich repeats in the D4R allow it to form complexes with SH3 domain containing
proteins, including synapse-associated proteins (SAPs).
• SAP binding positions the D4R in postsynaptic densities where its PLM activity can
modulate NMDA receptors.
• Additional proline-rich domains in primate D4Rs allow formation of multiple protein
complexes, including the binding of MAP kinase- regulating proteins.
• Activation of MAP kinase by dopamine may promote new synapse formation and
learning.
• A higher number of D4R repeats increases the potential for protein-based signaling, but
may limit the capacity for PLM based signaling.

72
6 INTERNEURONS, D4 RECEPTORS AND ATTENTION

The brain is a very complex and very busy place. To narrow our inquiry
about the connection between dopamine and attention it would be helpful to know
just where D4Rs are located and how their location relates to attention. D4Rs are
broadly distributed throughout the brain, rather than being highly localized in only
one or two brain regions. Studies from the lab of Dr. Patricia Goldman-Rakic at
Yale University showed that in the primate cortex D4Rs are particularly enriched in
certain specific types of inhibitory interneurons (1), providing an important clue
about where to look for the molecular events of human attention.
Cortical Interneurons
The capacity for attention appears to be a functional attribute of the cerebral
cortex, which is the largest portion of the human brain. The cerebral cortex is larger
in man than in lower primate species and is intimately involved in receiving and
processing sensory information. It integrates different elements and associates the
individual parts to help create a unified image or percept.
The cortex is divided into six layers, numbered from the outer surface (layer
I) to its innermost layer (layer VI) and, at a microscopic level, it can be seen to con-
tain a number of different types of nerve cells. The most common and most impor-
tant cell type for primary information flow is the pyramidal cell, named after the
pyramid-like shape of its cell body. Pyramidal cells traverse across cortical layers,
outside to in, but a single cell does not extend across all seven layers. Rather, one
pyramidal cell synapses onto others, forming an interlinked chain across the cortex,
gathering and exchanging information as the basic message moves through the lay-
ers. Pyramidal cells can also project laterally, conveying information in a side to side
manner. Like the prototypical neurons they are, pyramidal cells are composed of
highly divided dendritic extensions which receive input, a cell body or soma which
contains the nucleus and other centralized organelles and an extended axon which
carries the cell firing (action potential) forward to stimulate other neurons. Pyrami-
dal cells synthesize and release the neurotransmitter glutamate, which is how they
transfer excitation to the next cell. Bundled groupings of pyramidal cells carry simi-
lar or closely related information, forming columns of data flow across cortical lay-
ers. Commonly, incoming information first arrives in layer IV. Cells in the deeper,
more internal layers of the cortex (e.g. layers V and VI) project the information
downward toward the midbrain (e.g. thalamus), while more superficial neurons
(Layers I and II) project laterally across the cortex, spreading information to adja-
cent cortical columns.
As basic information flows through networked columns of pyramidal cells it
is subject to modulation by input from a variety of non-pyramidal cells. Most com-
mon among these are interneurons that interconnect pyramidal cells, either later-
ally within a cortical layer or between layers. Most interneurons are inhibitory,

73
meaning that when they fire their effect is to reduce the probability that their target
nerve will fire. They release the neurotransmitter GABA, whose binding to an ion
channel receptor allows negatively charged chloride ions (Cl-) to flow into the cell,
causing a decrease in its tendency to fire. Firing of inhibitory interneurons normally
provides a constant suppression of pyramidal cells, whose activity would otherwise
escalate into uncontrolled seizure activity. In other words pyramidal cells have an
intrinsic tendency to fire that is restrained by the activity of inhibitory interneurons.
Any reduction in that inhibitory tone (disinhibition) will promote increased pyra-
midal cell firing.
There are a wide variety of interneurons, that can be differentiated by their
anatomical features, by differences in their intrinsic firing rates and by specific cal-
cium-binding proteins they express (85). Presence or absence of these calcium-
binding proteins affects the firing properties of interneurons. The three major cal-
cium-binding proteins are parvalbumin, calbindin and calretinin, whose presence
can be detected by specific antibodies. Parvalbumin-positive GABAergic interneu-
rons (ppG interneurons) are also sometimes called basket or chandelier cells
because of the quasi-symmetrical distribution of extensions around their central cell
body, resembling a chandelier. These neurons display a high degree of post-natal
development starting in the deeper layers (V and VI) but gradually appearing in
more superficial layers (II-IV) (86). The extent of ppG interneuron development
appears to depend upon how much they are used, since the level of parvalbumin
expression rises in the visual cortex with the onset of visual function, while lower
than normal levels are observed when an eye is kept covered (87). It is particularly
intriguing that dopamine has recently been shown to stimulate the dendritic devel-
opment of ppG interneurons, which might be a significant aspect of its involvement
in attention-based learning.
ppG Interneurons are particularly rich in D4Rs (1), although other interneu-
ron types have also been reported to express D4Rs (2). This suggests that dopamine
activation of D4Rs might modulate the ongoing activity of ppG interneurons and/
or control their post-natal development. Indeed, in the first description of D4R
localization in ppG interneurons, Dr. Goldman-Rakic and colleagues suggested that
neuroleptic drugs like clozapine might bring about disinhibition of pyramidal cell
firing by reducing the firing rate of ppG interneurons (1). Presence of D4Rs is con-
sistent with an earlier study showing that dopaminergic nerves make synapses onto
ppG interneurons (88), providing a source of D4R activation. These dopamine
releasing nerves originate from the ventral tegmental area (VTA), a part of the brain
involved in attention and reward mechanisms. They provide the major source of
dopamine for stimulating D4Rs in ppG interneurons as well as for stimulating
dopamine receptors elsewhere in the cortex (e.g. D1 receptors located on pyramidal
cells) (89). When activated they provide simultaneous stimulation of all these dopa-
mine response systems.

74
 As we consider the source of stimulation for D4Rs we must appreciate
another of this receptor’s unique features. The D4R is an unusually promiscuous
receptor. In addition to responding to dopamine, it is also potently activated by nor-
epinephrine and epinephrine, whereas other dopamine receptors more strictly
require dopamine itself (18,90,91). D4R-mediated PLM can even be stimulated by
serotonin, which is not a catecholamine, albeit with low potency (18). This means
that D4R-mediated PLM is a somewhat promiscuous response, capable of being
activated by more than one neurotransmitter. As a consequence, D4R-mediated
PLM in ppG interneurons could also be stimulated by noradrenergic neurons
(releasing norepinephrine), which are actually more numerous in the cortex than
dopaminergic neurons. Epinephrine-releasing nerves are limited to one region in
the hypothalamus, and are unlikely to contribute to D4R activation elsewhere in the
brain.
Control of Interneuron Firing Rates
ppG Interneurons are notable for their very fast intrinsic rate of firing and are
sometimes referred to as “chattering” cells because of their extremely rapid spike
frequency. Different interneurons can be either fast firing, like ppG cells or slower
firing types. The underlying origin of intrinsic firing rate is the rhythmic interplay
between molecular systems (i.e. ion channels and ion pumps) which either promote
firing or inhibit firing, based upon their effects on the membrane potential (electri-
cal charge) across the nerve membrane. At rest the membrane potential is usually
negative, around -90 mV for example, but if it shifts to more positive (less negative)
levels (e.g. to -70 mV) by a process known as depolarization, the nerve’s electrical
properties become unstable and it responds by firing an action potential. An action
potential consists of brief (e.g. several millisecond) channel openings and ionic
fluxes which self-transmit throughout the nerve, starting from the point of original
depolarization (Figure 6.1). After an action potential occurs the neuron is repolar-
ized back to its resting level of -90 mV, but cannot immediately refire, due to a con-
straint on the ion channel proteins called “refractoriness”. Neurons have intrinsic
firing rates, which reflect the frequency with which they spontaneously depolarize
and recover from refractoriness, However, when they are in synaptic contact with
other neurons, their actual firing rate is subject to modulation, in either a positive or

75
negative direction, by the action of the neurotransmitters released by their synaptic
partners.

Figure 6.1: A neuronal action potential and its underlying ion channel components. A rapidly
repolarizing action potential allows rapid firing (A) while a slowly repolarizing action potential
reduces the firing rate (B). (C) Direction of sodium (Na+) and potassium (K+) gradients and ion
movements. Binding of calcium can delay the closing of some potassium channels and the level of
Ca2+ ATPase activity affects the duration of calcium binding.

Intrinsic firing rates of neurons are determined by their “pacemaker” activity,

resulting from the cyclic opening and closing of different ion channels with oppos-
ing effects on membrane potential. The outward movement of potassium (K)
through K channels normally serves to inhibit firing by maintaining a negative
membrane potential and any decrease in K channel opening has the ability to initi-
ate an action potential. Certain K channels are held open upon binding calcium ions
(Ca2+), resulting in a prolongation of the period before the nerve can re-fire (Figure
6.1). This mode of control serves to make the level of intracellular Ca2+ an impor-
tant regulator of intrinsic neuronal firing rates. When intracellular Ca2+ levels are
lowered, Ca2+ dissociates from its binding site on these K-channels and they close,
leading to depolarization and nerve firing. During each subsequent action potential
increases in Ca2+ levels again saturate the K+ channel binding site, contributing to
prolonged repolarization and inhibition of firing until the Ca2+ is once again
removed. Given this cyclic scenario, it is clear that the faster Ca2+ is removed from
neurons, the shorter will be the time-delay before next firing. In this manner, the

76
dynamics of intracellular Ca2+ levels are used to control the automaticity of neuro-
nal firing. An analogous but slower mechanism controls automaticity in cardiac
pacemaker cells.
In fast-firing interneurons such as the ppG-type, Ca2+ dynamics are poised
to provide a high intrinsic firing rate. That is to say, the rate of Ca2+ removal is so
fast that the next spontaneous action potential can occur quickly after the preceding
one. Indeed, differences in intrinsic firing rates, that are a hallmark of GABAergic
interneurons, reflect differences in their ability to control the intracellular Ca2+
level (92). Parvalbumin, found in fast-spiking ppG interneurons, binds Ca2+ with
very high affinity and helps to keep intracellular Ca2+ levels low. Calretinin and cal-
bindin are calcium-binding proteins expressed by other types of interneurons (85).
While calcium-binding proteins serve as static calcium buffers, the most
important dynamic mechanism for lowering intracellular Ca2 is activity of the Ca2+
pump (Ca2+ ATPase). As depicted in Figure 6.1, the Ca2+ pump binds Ca2+ ions at
the inner side of the cell membrane and transports them across the membrane to
the outside, where they are released. [Two protons (H+) move in the opposite direc-
tion to maintain electro-neutrality.] Because Ca2+ levels are much higher outside
the cell than inside, energy in the form of ATP is needed to drive the outward move-
ment of Ca2+, hence the Ca2+ pump is a Ca2+ -activated ATPase. Additional Ca2+
pumps, located within the neuron also lower the intracellular concentration by
transporting it into membrane-bound storage sites, the sarcoplasmic reticulum.
Activity of the cell surface Ca2+ pump is highly sensitive to its membrane
environment. This is no surprise, since it is largely composed of transmembrane
helices and movement of these helices within the membrane is a critical event dur-
ing a cycle of Ca2+ transport. The composition and packing density of membrane
phospholipids around the Ca2+ pump can be an important modulator of the rate at
which it is able to lower intracellular Ca2+. A number of studies have shown that
stimulation of PLM causes an increase in Ca2+ ATPase activity, associated with
faster rates of transport and a more rapid reduction of Ca2+ levels (93,94). Ethanol,
which increases membrane fluidity, also causes an increase in Ca2+ pump activity
(95,96). In GABAergic interneurons, stimulated PLM can increase the firing rate by
virtue of a more rapid removal of Ca2+. In other words, activation of PLM can
increase the probability that a particular interneuron fires at a faster rate. If we con-
sider firing rate to be the frequency of nerve activity, dopamine-stimulated solid-
state signaling can temporarily “tune” the frequency to a higher wavelength. By act-
ing at multiple sites, dopamine can simultaneously bring multiple loci to a similar
frequency of firing, resulting in a coordination of their information content.
Dopamine can most efficiently modulate Ca2+ pumping activity when the
Ca2+ ATPase protein is located very close to the D4R, close enough to be rapidly
influenced by dopamine-induced changes in phospholipid packing density. The cell
membrane Ca2+ pump protein contains certain amino acids in its carboxy-terminal
tail that cause it to be anchored via PDZ domain interactions to the same SAP pro-

77
teins that bind NMDA receptors and D4Rs (Fig. 5.3). However, co-localization of
the D4R and Ca ATPase has not as yet been demonstrated. Such a close physical
location is ideal for D4R-mediated PLM to regulate Ca2+ pump activity as well as
NMDA receptors and is poised for modulation of ppG interneuron firing.
Voltage-sensitive K+ channels are also bound to SAP proteins via one of the
three PDZ positions. There are many different types of K+ channels and the one
identified as binding to SAP proteins is designated as the “shaker” I type (97)
because defects in that channel cause shaking behavior in fruitflies, reflecting a loss
of its usual suppression of nerve firing. Shaker K channels are not regulated by Ca2+
levels. They are composed of transmembrane helices that rotate about 20o when the
channel opens. This raises the possibility that PLM-induced changes in membrane
packing density might also affect voltage-dependent K+ channel function. Indeed
ethanol exerts potent effects on different voltage-dependent K channels (98,99).
The firing rate of ppG interneurons in the cortex is importantly regulated by
excitatory stimulation provided by NMDA-type glutamate receptors. When stimu-
lated by glutamate these receptors allow calcium and sodium ions to enter, leading
to a small depolarization known as an excitatory post synaptic potential or EPSP.
Individual neurons receive inputs from many other neurons (up to 10,000/cell) and
if a sufficiently large number of EPSPs occur in an overlapping period of time they
summate to reach the threshold and initiate firing of the neuron. The firing rate
thereafter is a manifestation of the intrinsic tendency of that particular neuron to be
the fast-spiking or slower-spiking type. Interneurons also receive inhibitory inputs,
such as that provided by other GABAergic interneurons, that produce hyper-polar-
izations (IPSPs) and diminish the ability of EPSPs to reach threshold. The ability of
individual interneurons to receive and integrate a large number of EPSPs and IPSPs
is a key functional attribute.
Cortical Interneuron Circuits
As illustrated in the diagram in Figure 6.3, information-carrying pyramidal
cells activate inhibitory GABAergic interneurons which feedback onto the very
same pyramidal cells as well as onto adjacent pyramidal cells. In such a local loop
circuit the onset of sensory experience causes an initial increase of pyramidal cell
firing (Panel A). Pyramidal cell firing causes excitation of the interneurons, increas-
ing their inhibitory activity onto the very same pyramidal cells (Panel B), thereby
lowering pyramidal cell firing rate. This dynamic relationship is a form of negative-
feedback inhibition. But wait! That’s not the end of things. Reduced firing of the
pyramidal cell means less excitation of the interneuron, leading to reduced negative
feedback and a rise in pyramidal cell firing rate (Panel C). It becomes obvious that
such local negative-feedback circuits give rise to a rhythmic cycle of mutual excita-
tion (by pyramidal cells) and inhibition (by ppG interneurons). Indeed, experi-
ments have shown that when this sequence of changes repeatedly propagates
through a circuit, it gives rise to oscillatory fluctuations in firing rates (100),

78
 The temporal dynamics of closed-loop negative feedback circuits allow them
to display behavior elicited by a sudden increase in pyramidal cell firing. The pro-
pensity for oscillatory behavior is an intrinsic property of the circuit itself. It is an
“emergent” property, arising out of the circuit, rather than from the activity of a sin-
gle neuron. Since a single interneuron is in synaptic contact with many pyramidal
cells, its firing rate tends to synchronize oscillatory firing of all of its associated
pyramidal cells. In the example of Fig. 6.2 this means that the neurons carrying
color black information will fire in synchrony with neurons conveying long shape
information. Their simultaneous perception sets the stage for recognizing the black
pen on my desk. During attention dopamine can modulate the emergent property
of neuronal synchronization via its actions on post-synaptic density complexes.
Acting like the volume dial on a radio, it can increase the amplitude of synchronized
activity at a particular frequency (Fig. 6.3, Panel D). As described below, dopamine
apparently does this by recruiting more neurons to fire at a particular frequency
(e.g. 40 Hz). The more neurons firing in synchrony, the higher the amplitude at that
frequency.

79
 Figure 6.2: Synchronized oscillations in firing rate arise from cortical circuits
containing inhibitory interneurons. Arriving sensory information increases the
firing of pyramidal cells (Panel A). Subsequent activation of interneurons leads to
inhibition of pyramidal cell firing (Panel B). Lower pyramidal cell firing reduces
interneuron activation and increases pyramidal cell firing (Panel C). Dopamine,
acting at the pyramidal cell/interneuron synapse, can increase the amplitude of 40
Hz oscillations (Panel D).
The picture of a dual negative feedback circuit in Figure 6.3 is obviously woe-
fully oversimplified! To be more realistic imagine a multiplexed interneuron with
thousands of pyramidal cell connections, each representing a different feedback cir-
cuit with the potential for oscillatory behavior. Because they link so many neurons
together, fluctuations in interneuron firing can exert a powerful organizing effect
on the information flowing through its associated network. A sudden change in
activity of a single ppG interneuron can modulate the oscillatory activity in all of
these inter-linked circuits in a coordinated manner. The net result is a robust syn-

80
chronization of oscillations arising from neurons carrying many kinds of informa-
tion, with the interneuron being responsible for orchestrating the synchronization.
Synchronized Oscillations of Neuronal Firing
A number of recent studies have suggested that synchronized firing of nerves
is a key aspect of human attention and cognitive abilities (101-104). Synchroniza-
tion of firing rates for a large number of nerves may account for the “binding” of
their individual information content into a single coherent entity called a percept.
When a group of nerves fire in synchrony their informational content is unified. For
example, when arranged in parallel (e.g. a bundle of pyramidal cells) synchronized
firing can bring together or “bind” different types of information (e.g. edge, contrast
and depth) which together form a whole, more complex image. Clearly this is a very
powerful feature of interacting neuronal circuits. Synchronized oscillations in neu-
ronal activity also provide a vehicle for the spread of higher-order information
across the brain. While an individual bit of information is transmitted across a sin-
gle synapse, an oscillatory wave of synchronized activity can also move spatially as a
time-dependent variation in the probability of firing. In a linear arrangement of
nerve groups (e.g. A—>B-÷C—.D) synchronized oscillations can promote the
transfer of ensemble information from one brain region to another. Indeed differ-
ent regions can exhibit different frequencies or amplitudes in their oscillatory activ-
ity, and their information content can interact via the intersection of synchronized
oscillations. Waves of synchronized neuronal activity therefore represent a higher-
order level of organized information, with properties very different from the firing
activity of the individual nerves of which they are composed. Synchronization of
neuronal activity and its interplay with memory circuits appears to be very closely
related to consciousness (103, 105).
The existence of rhythmic neuronal firing patterns has long been recognized,
but technical advancements in the ability to measure very high frequency firing
rates with increased spatial discrimination have kindled new excitement about the
significance of neuronal synchronization. Recording the electrical activity of the
brain externally, through scalp-attached electrodes, yields electroencephalograms
(EEGs) whose patterns are used to identify seizure activity or other abnormalities
involving large numbers of neurons. Regular waves corresponding to alpha (slower)
or beta (faster) frequency rhythms are readily detected in EEGs.
In 1981, using a high level of EEG amplification and filtering, it was possible
to first identifS’ theta/gamma frequency synchronized brain activity in response to
an auditory stimulus (106). This relatively high frequency rhythmic brain activity
was called an event-related potential or ERP because its onset was temporally
related to the stimulus event. Subsequently, with technological improvements in
spatial resolution, magnetoencephalography (MEG) was developed to record the 3-
dimensional pattern of high frequency theta (5-10 Hz) and gamma (30-60 Hz)
range oscillations in response to auditory stimuli (107). An example of a theta/
gamma wave complex is illustrated in Figure 6.3. Theta refers to the frequency of

81
the slowly rising amplitude of the oscillation, while gamma refers to the higher fre-
quency component (e.g. 40 Hz) superimposed on the theta wave. Notice that differ-
ent frequency oscillations (theta and gamma) can occur simultaneously, combining
information from different circuits with each other. These complexes become
prominent in a time-locked manner in association with a subject’s attention to the
sensory stimulus, although they also occur spontaneously at lower amplitude.

Figure 6.3: Examples of Theta, Gamma and Theta/Gamma oscillations.

It is of particular interest that the amplitude of theta/gamma complexes is
high during both conscious attentive activity and during REM (rapid eye move-
ment) sleep but not during non-REM sleep (108). Since the REM portion of sleep is
the period when dreams commonly occur, these Theta Frequency (5-10 Hz) Theta/
Gamma Frequency Gamma Frequency (30-60 Hz) 100 milliseconds theta/gamma
complexes may be the cause of false sensory perceptions we associate with dream-
ing. During true sensory episodes, 40 Hz oscillations are found to occur in the spe-
cific brain areas that are appropriate for the particular type of stimulus being
attended to (e.g. auditory cortex for sound or visual cortex for light). As such these
synchronized waves are intimately associated with sensory experiences and our
attention to them. Indeed the events underlying amplification of synchronized
theta/gamma frequency activity in response to an attended sensory experience may
be the electrical analog of attention.

82
 Dopamine-induced Attention and Synchronized Oscillations D4Rs are stra-
tegically positioned for controlling the firing rate of ppG interneurons and modu-
lating synchronized oscillations in the firing of associated pyramidal neurons.
Evidence reviewed above indicates that D4Rs are enriched in ppG interneurons and
are associated with SAP complexes where they can modulate excitatory input. By
such a mechanism dopamine can selectively increase the synchronized activity of
certain tracts of pyramidal cells and enhance the information that they carry.
Indeed it has recently been reported that specific stimulation of D4 receptors modu-
lates the firing of pyramidal cells via involvement of GABAergic interneurons (109).
This increase in synchronized activity could form the mechanistic basis of attention,
which is the central hypothesis I wish to put forth:
“Attention is initiated by the ability of dopamine, acting via D4R- mediated
PLM within interneurons, to enhance the amplitude of synchronized pyramidal cell
firing. The onset of this effect of dopamine is temporally coupled to the episode of
attention and occurs in different brain regions specific to the modality of attended
information”
A selective increase in the amplitude of gamma frequency 40 Hz oscillatory
activity occurs during episodes of attention such as the presentation of a novel audi-
tory stimulus (11,12,104). Several studies have provided evidence that dopamine
increases the amplitude of 40 Hz synchronized oscillation during episodes of atten-
tion and that blockade of D4R-like receptors reduces the amplitude of 40 Hz oscilla-
tions. For example, the cocaine-induced release of dopamine increases
synchronized neuronal firing (110) while haloperidol, which blocks D4-like recep-
tors, causes a decrease in synchronized firing at 40 Hz (11). However, a specific role
for D4R-mediated PLM in promoting synchronization has yet to be directly dem-
onstrated.
Increased amplitude of 40 Hz synchronized oscillations in a given brain
region results from an increased number of neurons participating in synchronized
firing at this particular frequency. During attention dopamine could recruit partici-
pation of additional neurons in synchronized activity by virtue of the effect of D4R-
mediated PLM on the ensemble of SAP-associated ion channel and ion transport
proteins located at glutamatergic synapses on ppG interneurons. In essence, it can
be proposed that a consequence of dopamine-stimulated solid-state signaling is an
increased propensity for ppG interneuronlpyramidal cell circuits to exhibit 40 Hz
fluctuations in their firing rate. The particular frequency of 40 Hz represents the
resonant frequency of the ppG interneuron circuits under the condition of dopa-
mine exposure.
Temporal coordination of dopamine-induced effects on synchronized oscil-
lations with attention implies that dopamine release must be closely linked in time
to the original sensory perception that is the object of attention, Synchronization
must occur in the appropriate cortical region whose pyramidal cells carry the
attended information. For visual information involving, for example, attention to

83
the edge of an object, then edge-discriminating cells in the visual cortex should be
the sites of dopamine induced synchronization. Attention to low-pitched sounds
would require dopamine release in appropriate regions of the auditory cortex and
so forth.
The requirement for temporal coordination serves to emphasize that atten-
tion is a process that is initiated after the primary sensory event, not before or not
even at the exact same time. Thus the act of attention should not be confused with
the cognitive anticipation of an event, which is something quite different from the
attention episode itself. Anticipation of attention involves the activation of memory
processes that will be matched against actual experience when the episode of atten-
tion begins. We cannot direct our attention to something that we have not yet expe-
rienced, but we can use our memory to create a virtual analog of a previous
experience for comparison to current experience. In a complex visual scene we can
use our high-resolution foveal vision to direct attention toward a subset of the scene
(perhaps a word on the very page you are looking at), so that we can refine our per-
ception, interrogate the subset and gain further information. Even in the presence
of a preceding cognitive plan utilizing memory systems, this episode of visual atten-
tion requires initial “eye contact” (i.e. processing reflected photons of light from a
printed word) before active attention can be initiated. Similarly, when we first hear
someone’s voice, receipt of the initial sound precedes our re-directed attention.
However, once our attention is directed toward a particular sensory event, further
sensory input in that same modality (i.e. continued speech) receives continuous
attention. The molecular events of attention must therefore also be closely linked to
the processing of an initial sensory event and can be sustained after their initiation.
Indeed the molecular mechanism of attention must be very closely related to the
molecular mechanism for perception. It is not likely to be an entirely separate pro-
cess, but is more likely to be an amplifying modulation of the basic mechanism of
perception. In the putative D4R-mediated mechanism we are exploring, this implies
that dopamine release from VTA region-derived neurons begins some time after
the initial activation of a particular pyramidal cell pathway and remains continuous
for the duration of sustained attention.
If D4R-mediated PLM is indeed important for attention, the ability to sustain
attention may depend upon the ability to sustain D4R-mediated PLM throughout
the attention event. However, the quality of attention is normally not constant dur-
ing an episode of attention. We experience a degree of fade in the intensity of atten-
tion over time that might reflect a time-dependent decrease in the robustness of
DA-stimulated PLM or in its effectiveness. In lay terms the length of time before the
onset of this decrease defines our “attention span”. A gradual decrease in attention
to a particular set of information can be helpful in facilitating the smooth transition
of attention from one target to the next, although problems can arise. If the fade is
too rapid we may lose some of the benefits of attention such as attention-based

84
learning. On the other hand, if there were no fading we could exhibit perseverative
or compulsive attentive behaviors that might impair learning in a different manner.
Speaking of spans, we also generally recognize that the quality and duration
of our attention abilities change over the lifespan. In broad terms, children “notice”
things more readily but seem to have shorter attention spans than adults. For most
persons the capacity for attention seems to diminish as we join the “over-the-hill”
gang. Moreover, individual differences in the capacity for attention and in attention
span are more the rule than the exception. Of course attention is impaired in
ADHD, but it is also seriously impaired in a number of psychiatric illnesses includ-
ing autism and schizophrenia. The well-documented association of ADI{D with a
higher number of D4R repeats could therefore reflect a limitation in the ability to
initiate or sustain dopamine-stimulated PLM. Armed with a specific hypothesis
about its mechanism, we can now begin to explore the molecular basis for both nor-
mal and abnormal differences in attention capabilities.

Chapter Summary:
• Two primary types of neurons in the cortex are pyramidal cells and interneurons.
• D4Rs are particularly enriched on fast-spiking inhibitory interneurons.
• Neuronal circuits containing parvalbumin-positive GABAergic (ppG) interneurons give
rise to 40 Hz synchronized oscillations in the firing of pyramidal cells.
• Synchronized firing of neurons unifies their informational content and facilitates the
spatial flow of information.
• DA stimulation of D4R-mediated PLM on ppG interneurons may result in the modulation
of NMDA receptors, Ca2 ATPase activity and W channels, resulting in enhanced
probability of 40 Hz synchronized oscillations.
• Attention is a result of a DA-induced increase in 40 Hz synchronized oscillations.
• Attention occurs after the Initial processing cf sensory Information.
• Differences In the quality of attention across the lifespan and in various psychiatric
conditions can be caused by differences In DA-stimulated PLM.

85
7 ATTENTION-BASED LEARNING

There is more to attention than merely turning up the volume knob for par-
ticular sensory experiences. Attention is really a time-structured episode that
includes both the initial prioritization of specific information and the subsequent
learning process that accompanies this prioritization. In fact the relationship
between attention and learning is so intimate that in general we only learn from the
particular information which we attend to. Non-attended information is experi-
enced, but not necessarily learned, and the more frequently we attend to something
the more likely we are to learn it. Conversely, it is clear that deficits in attention can
lead to deficits in learning. We can therefore expect that the molecular mechanisms
facilitating the initiation of attention might be closely related to mechanisms that
support attention-based learning.
MAP Kinase and Learning
How might attention-based learning relate to the dopamine/D4 receptor
events just described? If you paid attention, you should recall that the MAP kinase
pathway is activated upon dopamine stimulation of D4Rs (22,23). Activation of the
MAP kinase pathway does not occur rapidly enough to mediate the onset of atten-
tion, as compared to DA-stimulated solid-state signaling. Rather, stimulation of the
MAP kinase pathway is important for learning. D4R-mediated stimulation of MAP
kinase may thus participate in the longer-term learning-related events that occur
after initiation of an episode of attention.
Like other protein kinases, MAP (Mitogen-Activated Protein) kinase takes a
phosphate group from ATP and attaches it to particular target proteins. This phos-
phorylation modulates activity of the target proteins, increasing or decreasing their
participation in a signaling pathway. Mitogens are substances that stimulate cells to
grow and divide, so MAP kinase is a particular protein kinase (actually a small fam-
ily of kinases) that is activated by growth-promoting mitogens mediating some of
their effects on neurons. Platelet-derived growth factor, insulin-like growth factor
and nerve-derived growth factor are examples of mitogenic hormones that can acti-
vate MAP kinase. When they bind to their receptors on the cell membrane, these
factors stimulate a sequential cascade of multiple steps, ultimately leading to
increased MAP kinase activity (Figure 7.1). Interestingly, the receptors that initially
bind these growth factors are also protein kinases and growth factor binding
increases their ability to phosphorylate each other as well as target proteins. Once
they are phosphorylated, these receptors serve as docking sites for proteins that
bind to them via their phosphorylation sites. Other proteins successively attach to
this growing signaling complex, ultimately leading to phosphorylation and activa-

86
tion of MAP kinase. Figure 7.1 illustrates an example of the most well characterized
MAP kinase signaling cascade.

Figure 7.1: The pathway leading from neuronal growth factors to MAP kinase activation. Binding to
the growth factor receptor causes its auto-phosphorylation and allows Grb2 binding. Other signaling
proteins successively associate, ultimately leading to the phosphorylation and activation of MAP
kinase.

D4Rs function as a docking site for components of the MAP kinase pathway,
similar to a growth factor receptor, using its proline-rich segments to bind SH3
domains of Grb2 and Ras GAP. Since exposure to dopamine stimulates MAP kinase
(22,23), some event triggered by dopamine must initiate activation of the pathway.
Dopamine stimulation of G proteins causes the release of their beta/gamma sub-
units that can activate MAP kinase. Indeed dopamine-induced activation of MAP
kinase is blocked by pertussis toxin, which interferes with G protein activation (22).
Active MAP kinase phosphorylates and activates target proteins that act as
DNA transcription factors, increasing DNA transcription (i.e. the synthesis of mes-
senger RNA from particular genes) leading to increased levels of selected proteins
whose actions lead to stimulated cell growth and/or cell division. In the case of fully
differentiated nerve cells, however, the potential for cell division is quite limited, so
the role of MAP kinase is restricted to increasing the growth and development of
mature neurons, rather than causing them to divide. However, during embryogene-
sis and early post-development, when neurons are capable of dividing, MAP kinase
may n important regulator of cell division. When an attended stimulus sufficiently
increases MAP kinase activity it increases neuronal development the specific region
that is involved in the episode of attention, increasing robustness of response during
future episodes.

87
 In differentiated neurons the activation of MAP kinase leads to an wase in the
number, length and complexity of dendritic extensions (77,111,112). Dendrites are
the multiple finger-like projections emanating ì the cell body which act as receivers
of input information, while the axon nsion carries output information away from
the cell body to target cells.based density of the dendritic arbor therefore allows a
neuron to receive a and broader range of input information as additional synapses
form new neurons. The process of forming new dendritic extensions is called togen-
esis and the activation of MAP kinase causes increased neuritogenesis.
Growth and development of nerve cells has a functional significance beyond
that of cell growth in other organs. Take the liver, for example. An increase in the
number of liver cells will result in a larger organ size and a proportionate increase in
its total metabolic capacity. The brain, however, is all about connections (i.e. syn-
apses) and relationships. When a neuron grows oes so by the formation of new syn-
apses. Each new synaptic connection creates new functionality and new
information within the brain, not merely a quantitative increase in what it was capa-
ble of doing before. A new synapse creates a “hard-wired” relationship that previ-
ously was not a feature of the brains capability. Stimulation of interneuron growth
and development by MAP kinase therefore controls the hard wiring of the brain.
Since synaptic connections represent relationships, activation of MAP kinase
is a prototypical example of how biochemical mechanisms are able to enode the
relationships we experience into our brain’s wiring diagram. For example, repeated
exposure to our “home” environment causes the repeated association of visualized
objects (e.g. chairs, tables and walls) in discrete spatial positions within that envi-
ronment. Learning these spatial relationships can take the form of new synapses,
created by neuronal growth under the influence of repeated activation of the MAP
kinase pathway. Once learned, these hard-wired relationships can be recalled more
or less at will with amazing accuracy and clarity. Repetition is very important. A
quick walk through someone else’s home for the first time certainly doesn’t yield the
same detailed recall of spatial relationships, but repeated exposure during an
extended stay for weeks or months gradually produces an internal map rivaling that
of our own home. Much of learning is based upon associative relationships between
information and synapse formation is the major mechanism for encoding these
relationships.
MAP Kinase Activation in ppG Interneurons
Fos (c-fos) is a transcription factor under the control of the MAP kinase path-
way and its levels increase soon after the activation of mitogenic receptors.
Increased Fos levels have been observed in association with the onset of learning in
several different experimental paradigms. One particularly interesting study was
carried out with young chicks (i.e. poultry) that can be trained to visually imprint
objects presented during a 1 hr training session, an attention-driven learning para-
digm (113). Imprinting resulted in measurably elevated Fos levels in a particular
region of the forebrain that was the source of the visual recognition memory, as

88
measured 1 hr after the imprinting session. In other words this example of atten-
tion-based learning was directly associated with increased Fos levels. A more
detailed examination of which nerve types showed the Fos response revealed that
95% of the Fos-positive neurons were interneurons containing parvalbumin and
GABA (i.e. ppG interneurons), whereas other types of interneurons did not show
increased Fos levels. Thus, at least in this paradigm, Fos activation within ppG
interneurons is a critical component of attention-based learning.
PpG interneurons display a remarkable pattern of postnatal development.
For example, in the visual cortex of monkeys the appearance of parvalbumin first
begins in late fetal life, but increases dramatically shortly after birth, reaching a peak
between one and two years of age (86). Levels then fall off, reaching a lower level at
about 4.5 years that is sustained throughout adulthood. In humans there is also evi-
dence of a postnatal rise in dopamine receptor-containing neurons and subsequent
fall to adult levels, with a peak occurring at about 8-9 yr. of age (114). As noted ear-
lier, the increase in parvalbumin expression is apparently linked to functional utili-
zation of the particular pathway, since deprivation of vision in animals (e.g. not
allowing one eye to open) causes a marked decrease in the development of ppG
interneurons (87). This observation implies use-dependent activation of an initially
undifferentiated, primitive interneuron that causes it to develop into a parvalbu-
min-expressing interneuron. It is therefore particularly noteworthy that recent
studies in slices of neonatal rat cortex found that exposure to dopamine causes an
increase in parvalbumin expression mediated by a D4-like receptor (115). Thus
dopamine released during episodes of attention may be a stimulus for the develop-
ment of ppG interneurons, especially during the first few years of life.
Not only does dopamine increase the number of parvalbumin expressing
interneurons, it also increases the density of their dendritic projections that reach
out and touch other nerves (115). While MAP kinase activation leads to the first
small protrusions (neurite extensions) that can eventually develop into dendrites,
other neurotrophic factors (nerve growth factor, brain-derived neurotrophic factor)
also play an important role. Formation of additional dendrites eventually leads to
the formation of additional synapses. The more synapses a single neuronal cell
makes onto other neurons, the more robust its spatial influence. [Note: It has been
estimated that a single ppG interneuron receives about 10,000 input synapses and
can make 1,000 output synapses onto other cells. (116)] In this manner activation of
MAP kinase during repetitive or prolonged attention can cause selected ppG inter-
neurons to increase their capability of producing synchronized activity during
future episodes of attention.
The ability of dopamine to both initiate attention via its promotion of 40 Hz
oscillations and stimulate synapse formation via MAP kinase is a remarkably ele-
gant mechanism for coordinating attention and attention-based learning. Taking an
even broader perspective, this coordination allows self-programming of the brain’s
wiring pattern into a gradually more complex network of connections, driven by the

89
extent of utilization in attention. Over time our personalized pattern of connectivity
reflects the summation of our previous attention-based experience.
It appears that during our early years there can be an increase in the absolute
number of ppG interneuron cells, whereas after a certain age the effects of MAP
kinase are restricted to the formation of new synapses for existing cells. Adverse
events that interfere with normal early-age development of ppG interneurons might
therefore have life-long consequences. An example of this is the effect of prenatal
cocaine exposure. Studies in rabbits showed that administration of cocaine to preg-
nant rabbits causes premature stimulation of ppG interneuron growth in the off-
spring (117), presumably caused by increased levels of dopamine induced by
cocaine. Since this artificially induced growth did not occur in conjunction with
episodes of sensory experience or attention or attention-based learning, its value is
dubious at best. More importantly, this premature development interferes with the
opportunity for normal attention-based synapse formation, providing an obvious
explanation for learning deficits associated with prenatal cocaine exposure. In a
more generalized form, we can expect that any drug or condition that increases
dopamine levels or otherwise prematurely activates the MAP kinase pathway in
utero may be likely to result in learning deficits.
There is evidence that anatomic changes in ppG interneurons are also tightly
linked to changes in the pyramidal cells they innervate. The time-course for
increased and decreased parvalbumin expression is closely paralleled by an increase
and decrease in the density of small spiny projections on the dendrites of adjacent
pyramidal cells (90). This relationship suggests that attention-initiated effects on
ppG interneurons may be propagated to other components of the oscillatory circuit.
Alternatively, D4 receptors or other receptor subtypes located on pyramidal cells
may directly initiate trophic responses (89,118,119).
An increase in synaptic complexity, stimulated by dopamine and MAP kinase
activation during an episode of attention is in fact a form of learning, in this case
attention-associated learning. Because the formation of a new neurite or new syn-
apse is not instantaneous, the probability of this type of learning will be increased by
prolonged or repeated activation of the same ppG interneuron. Higher, more
intense levels of dopamine activation will also increase it. Conversely, learning is
less likely for isolated episodes of attention, especially if they are of the low intensity
variety. These statements are so self-evident from our common experience that they
are trivial. However, armed with new information and a new molecular perspective
we are now in a position to visualize the precise events that account for these fea-
tures of attention.
We continuously make and break synapses in a dynamic ongoing manner. At
present more is known about the events which lead to synapse formation than
about the factors which trigger their disappearance. Nonetheless we can reasonably
assume that there is a balance of forces which promote formation of synapses vs.
forces which promote their loss, with attention being a process which favors syn-

90
apse formation. Continued involvement of an associated group of neurons in epi-
sodes of attention would favor retention of their synaptic connections, as compared
to synapses between under utilized neurons. The adage “Use it or lose it!” seems like
an apt description of this relationship, although here we are specifying that atten-
tion is the key element.
While formation of new synapses is a clear example of attention- related
learning, learning can take many other forms. Some may involve the MAP kinase
pathway while others involve alternative signaling mechanisms. Any change in the
functional properties of a neuronal pathway that occurs as a result of the passage of
information through that pathway can be considered to be a form of learning. This
notion appears to conflict with the common meaning of “learning”, which implies a
long-term mechanism for recognizing relationships, such as long-term memory.
But in fact learning occurs on all time scales, from milliseconds to years, with differ-
ent molecular mechanisms exhibiting characteristically shorter or longer time
frames. Some information (e.g. in a visual scene) is learned for only a second or two
and then it is gone. On the other hand, information that is fully attended to may last
a lifetime.
We have identified two different D4R-mediated signaling pathways, PLM
and MAP kinase activation, that are very different in the time-scale of their effects.
PLM provides a second to second modulation of prioritized information, while the
consequences of MAP kinase activation could, at least theoretically, last for years
and years. We must also not forget that dopamine stimulation of D4 receptors also
initiates signals via the activation of the traditional GTP-binding G protein path-
way. This path leads to increases or decreases in the concentration of several intra-
cellular second messenger molecules as well as the direct modulation of ion
channels by subunits of the G protein. The duration for these G protein-mediated
signaling events is typically in the seconds or minutes time frame. Thus during epi-
sodes of dopamine-stimulated attention, multiple signals are generated that can
alter the system for a wide range of time intervals. This has the net result of carrying
attention forward across the time domain and repetitive attentive episodes occur-
ring over the time domain favor longer lasting forms of learning. Indirect effects of
D4R stimulation on other signaling pathways also have the potential for contribut-
ing to attention-based learning. For example, activation of NMDA receptors leads
to long-term memory formation, and any influence of dopamine-stimulation PLM
on NMDA receptors could influence that process.
Putting It All Together
Just as the human brain uses synchronization to assemble and unify individ-
ual components to create a whole image, we now need to bind together the various
elements of the complex D4 receptor story in order to gain an overall view of its
unique role in attention.

91
 Attention involves prioritization of a selected subset of processed sensory
information and synchronized oscillations in the firing pattern of cortical neurons
are an important mechanism for binding their information together. The amplitude
of 40 Hz synchronized oscillations increases with the onset of attention. Attention
to specific information can be initiated by the release of dopamine from VTA-
derived neurons in the cortex that can stimulate dopamine D4 receptors located on
ppG interneurons, the pacemakers for 40 Hz oscillations. D4 receptors located
within post-synaptic complexes along with NMDA receptors, ion channels and
other proteins determine the amplitude of 40 Hz oscillations. By changing the D4
receptor conformation, dopamine initiates methylation of membrane phospholip-
ids in the receptor’s microenvironment, and thereby modulates the activity of
neighboring proteins. The net effects of D4 receptor stimulation are to amplify 40
Hz oscillations and enhance synchronization within the dopamine targeted circuits.
The particular information whose synchronization is enhanced by dopamine has a
higher probability of being transferred to working memory and short-term memory
circuits, yielding attention to this specific information. In a coordinated manner,
dopamine also activates the MAP kinase pathway in ppG interneurons that can ini-
tiate neuronal growth in early years, and increase synapse formation in mature
interneurons. The latter events determine which pathways will be available for syn-
chronized activity in the future, creating an attention-driven pattern of association
networks. These two D4 receptor-mediated events, PLM and MAP kinase activa-
tion, along with other dopamine-stimulated signaling pathways, provide for the
short-term initiation of episodes of attention and for attention-associated learning.
Increases in the number of D4 receptor-associated proteins, attached via pro-
line-rich repeat segments found only in primates, can affect the intensity and dura-
tion of dopamine-stimulated attention as the availability of phospholipids become
more scarce. Conversely, the presence of additional SH3 domain binding sites can
bring additional proteins involved in MAP kinase signaling or provide an opportu-
nity for multiple SAP interactions. These highly individualized D4 receptor features
can alter the nature and intensity of dopamine-elicited signals arising during atten-
tion. D4 receptor activation appears to play a unique and critical role in the well-
developed attention capabilities of humans and other primates. Heterogeneity in
the structure of D4 receptors among individual humans and between ethnic groups
may contribute to the variety in behavioral traits and attention qualities that are an
important characteristic of man.

92
Chapter Summary:
• Attention-related learning is mediated, at least in part, by the activation of MAP kinase.
• A number of growth factors stimulate neuronal growth and new synapse formation via
MAP kinase activation.
• New synapses create hard-wired associations between information.
• Dopamine stimulation of D4Rs leads to MAP kinase activation.
• Proline-rich repeats in the primate D4R allow docking of proteins ¡n the MAP kinase
pathway.
• Dopamine stimulates the development of ppG interneurons via a D4R-like receptor.
• PpG interneurons show a dramatic postnatal pattern of development that depends upon
their activation.
• Dopamine stimulation of D4Rs may lead to both the onset of attention via PLM-mediated
effects and the initiation of MAP kinase-mediated synapse formation.

93
8 AWARENESS, ATTENTION AND THE DETECTION OF
NOVELTY

We have finished describing a series of dopamine-triggered events that

together constitute a hypothesis on the molecular mechanism of attention and
attention-based learning. However, attention is not an all or none process. We con-
stantly utilize varying degrees of attention intensity to monitor and scan our exter-
nal physical environment and sometimes our internal environment as well. In the
case of vision, we may move our eyes in a visual saccade (a tracking motion) and
perhaps even make a facilitating turn of the head to redirect our best vision toward
specific locations we wish to attend to (foveal scanning). This is certainly high prior-
ity attention of the dopaminergic type we have been examining. But we also main-
tain a lower level of awareness for other elements in the visual scene that are not
focal points of high attention. This awareness has alternatively been termed “vigi-
lance” or “awareness” as well as other awareness-like descriptors. How might the
state of awareness relate to the synchronization-based attention theory just
advanced?
Let us again pose the theoretical question: Is it likely that there are totally sep-
arate systems or molecular mechanisms for states of awareness vs. states of attention
or might they represent variations on a single theme or even differences of intensity
for a single process? We don’t know the answer to this question, but I will argue that
the molecular mechanisms of attention and awareness are closely related. Even
within the D4R-based mechanism there is room for differences in the intensity of
attention. The most obvious example would be differences in the amount of dopa-
mine released. A higher level of dopamine release should produce a higher amount
of PLM, a more probable inhibition of NDMA receptors, perhaps involving more
synapses and/or more ppG interneurons. Differences in the intensity and/or dura-
tion of dopamine release could certainly provide graded differences in the quality of
attention. At the level of neuronal synchronization, this could be manifested as
higher amplitude 40 Hz oscillations, reflecting an increased number of neurons par-
ticipating in synchronous activity. Such a difference might be comparable to
increasing the strength of an incoming radio station signal from 10 to 10,000 watts,
but still maintaining the same frequency.
Our postulate of a central role for dopamine in the molecular mechanism of
attention was based upon the extensive literature implicating its involvement. How-
ever, alertness and vigilance have been more closely associated with adrenergic
mechanisms, involving release of the neurotransmitter norepinephrine (120). A
classic example is the heightened awareness associated with activation of the sympa-
thetic nervous system during moments of acute stress, better known as the “fight or
flight” response. At this juncture we can consider another feature of the D4R: its

94
unusual ability to potently bind and respond to norepinephrine and epinephrine, as
well as to dopamine. What is the functional importance of this promiscuity?
Dopamine vs. Norepinephrine
In our PLM studies we found that epinephrine was essentially equal in
potency to dopamine, and norepinephrine was only 5-fold less potent (18). This
relationship closely corresponds to earlier comparisons of their D4R-mediated G
protein responses that also found epinephrine and norepinephrine to be only
slightly less potent than dopamine (90,91). Clearly nature has designed the D4R to
be responsive to all three of these catecholamines. Norepinephrine-releasing nerves
are very common in the cerebral cortex, significantly more so than dopamine-
releasing nerves. Most of them originate from the reticular activating system, whose
activity is closely associated with our state of wakefulness and awareness. Thus nor-
epinephrine is poised to stimulate D4Rs in the cortex, although at equal concentra-
tions the effect of dopamine would produce a significantly stronger effect. In other
words norepinephrine would tend to produce a weaker stimulation of PLM, result-
ing in a more modest but still significant degree of 40 Hz synchronization. Such a
moderate degree of synchronization might correspond to a level of awareness while
being less intense than a dopamine-activated episode of attention. The unusual pro-
miscuity of D4Rs might be just right for producing multiple levels of PLM in
response to different neurotransmitters.
If we step back a bit and examine the overall D4R cycle of PLM, as outlined in
Figure 4.5, we can appreciate that the most critical design feature of the system is
that the rate of PLM is normally limited by the concentration of dopamine. When
its level is zero MET3 13 in the D4R remains hidden and nothing happens. But,
when dopamine levels are high, MET3 13 is exposed and becomes adenosylated, ini-
tiating a cycle of PLM. Thus availability of the “active” receptor shape is the rate-
limiting factor for D4R-mediated PLM. If receptor shape is so important for Step 1,
couldn’t it also be a factor in controlling the speed of succeeding steps in the PLM
cycle?
The second step in D4R-mediated PLM requires transfer of the methyl group
from the tip of MET3 13 to the nitrogen in the headgroup of the phospholipid PE.
Note in Figure 3.4 that when dopamine brings MET313 to the center of the receptor
for adenosylation the methyl group is moved farther away from the phospholipids
that surround the receptor on its outer surface. In this R’ position transfer of the
methyl group to PE would be very unlikely, as compared to the R conformation
where the methyl group is positioned close to the outer receptor surface. This situa-
tion suggests that the rate of Step 2 would be faster for the R state of the receptor,
just opposite to Step 1. In other words, while an agonist such as dopamine is essen-
tial for getting the cycle started (Step 1), its effect on receptor conformation may
prove to be detrimental for Step 2. For as long as dopamine stays on the receptor
and maintains the R* shape it will effectively prohibit Step 2. For PLM to proceed,
dopamine must come off, and the quicker the better!

95
 The speed of dopamine dissociation depends upon how tightly it is bound to
the D4R and this can be estimated from the concentration of dopamine required for
stimulating PLM to 50% of its full maximum (i.e. the EC50 for dopamine). For
many reactions this is also the concentration at which the receptor is 50% occupied,
known as the dissociation constant or KD. For a given binding reaction the KD is
determined by the ratio of the rate of dissociation (k2) to the rate of association (k1)
or k21k1. Since the rate of association is similar for a group of structurally similar
drugs binding to the same receptor, the ki value for dopamine and norepinephrine
is likely to be quite similar. The association rate for an ideal binding reaction, pre-
dicted by diffusion-limited collisions, would be about io M’sec’, while the actual
value for dopamine binding to the D4R would likely be at least five times slower
or2x 107M1 sec4.
The major determinant of a drug’s potency is its tendency to stay on the
receptor once it is initially bound (i.e. its dissociation rate or k2 value). We found an
overall EC50 value of 2 x 1 06 M for dopamine stimulation of PLM (18), which
would predict a k2 rate of 40 per sec if its k1 was 2 x iO. In other words once dopa-
mine occupies the D4R, on average it comes off in about 1140th of a second or 25
milliseconds (msec). Although our current methods only yield a rough estimate, we
calculated that the rate of dopamine stimulated PLM for a single receptor would,
when maximally stimulated by dopamine, cause methylation of at least 50 phospho-
lipids per second, or about one every 20 msec. The actual rate could be significantly
faster. Taken together these two calculations suggest that dopamine-stimulated
PLM can be very fast when dopamine concentrations are high and the rate at which
dopamine comes off the receptor could become a limiting factor under these condi-
tions.
From what we know about the approximate size of the D4R and phospholip-
ids, the receptor may be encircled by no more than 20 phospholipids. At a rate of
one methylation per 25 msec this means that about 500 msec would be required to
methylate the surrounding layer of phospholipids. Although these values are only
approximations they do provide a rough estimate of the potential time frame for the
molecular events involved in attention. Studies from the lab of Dr. Michael Posner
have estimated the time required for the initial processing of visual information fol-
lowed by an attention-requiring task (121). They found that 100-200 msec are
required for the primary processing step while the secondary semantic processing
began after 200 msec. The estimated rate of dopamine-stimulated PLM therefore
seems to be potentially fast enough to provide for a mechanism of attention that
occurs over a period of 100-300 msec.
Since dopamine has an EC50 for PLM that is 5-fold lower than that for nor-
epinephrine, the preceding relationships indicate that norepinephrine comes off the
D4R five times faster than dopamine. This increased rate of dissociation suggests
that step 2 of the PLM response to norepinephrine would occur five times faster for
norepinephrine than for dopamine. Thus when compared at the same concentra-

96
tion (e.g. 2 M) dopamine will produce a higher number of overall methylations per
sec, but after each of the adenosylation steps has occurred, the time required for
transfer of the methyl group to adjacent phospholipids (i.e. Step 2) would be faster
for norepinephrine. Taken together, this implies that norepinephrine could pro-
duce a weaker but more rapid PLM response and a correspondingly weaker but
more rapid level of neuronal synchronization. This kinetic profile makes norepi-
nephrine a good candidate for producing the rapid sensory scanning we associate
with awareness, as distinct from full attention.
It is mechanistically appealing to consider that different levels of 40 Hz
gamma frequency synchronization, produced by two different neurotransmitters,
might provide for graded levels of attention, occurring with different speeds. This
duality allows earlier synchronized information to serve as a prologue for the later
information. Indeed, doesn’t an episode of attention require a preceding awareness
of the sensory event?
From these relationships we can suggest that initial norepinephrine based
awareness information may guide subsequent development of dopamine-based
attention, as illustrated in Figure 8.1. According to this proposed circuit, the activity
of norepinephrine releasing nerves would promote synchronization of incoming
information, leading to a low-level, rough-draft type of awareness. This synchroni-
zation would be continuous and seamless, rather than episodic and focused like
attention. Its fleeting, evanescent nature is the beauty of such a system. It facilitates
scanning of the environment, unburdened by attachment to anything. Nonetheless,
a state of awareness means exactly that. We are aware of this fleeting information
and are constantly making comparisons with prior experience-based expectations.
If this vigilance reveals a lack of harmony or is incongruent with memory-based
expectations, the presence of novelty is recognized that can be the basis for activa-
tion of dopamine-dependent attention. This hypothetical sequence of events
implies that neurons involved in novelty detection are somehow able to cause dopa-
mine release, presumably by stimulating neurons arising from the VTA (ventral
tegmental area), the primary source of dopaminergic neurons in the cortex. The
proposed relationship also implies that dopamine-mediated attention involves a
second, more intense synchronization of at least a subset of the very same neuron
ensembles that norepinephrine originally acted on, Thus dopamine “re-enters” the
identical neuronal field, assuring a seamless transition from unattended awareness
to episodes of focused attention.

97
 Figure 8.1: Norepinephrine and dopamine-based synchronization could function sequentially to
provide awareness and attention. Novelty detection can result from of comparison of attended
information with memory-based information.

Novelty-stimulated Attention
An episode of productive attention in response to novelty can lead to learning
about the novel event, as depicted in Fig. 8.2. For example, if attention is directed
toward a novel visual object it leads us through a rapid series of comparisons with
physically similar objects we have earlier encountered, seeking to associate some
shared significance with the novel one. For example, perhaps you encounter a novel,
unfamiliar object which has a longish rod shape, similar in some respects to a pen-
cil, but lacking the typical yellow-orange surface color, and it has no black lead or
pink eraser. Visual system neurons that detect edge, depth and other shared attri-
butes of the overall shape would participate in the synchronized ensemble for both
the novel unknown object and for a pencil. However, neurons that normally detect

98
yellow or black or pink in the expected spatial positions of a pencil would not be
activated by the novel object and therefore would not be a part of the synchronized
ensemble it produces.
A comparison of memory-based synchronization patterns vs. currently active
synchronization patterns could therefore form the basis for novelty detection,
although at present there is no description of how recalled memory might allow
such a comparison. In our example of a pencil-like object, a comparison of the syn-
chronized firing patterns could reveal the absence of lead and eraser-related neuro-
nal elements, thereby triggering a declaration of novelty and initiating an episode of
attention. If attention leads to subsequent inquiry (e.g. asking the waiter in this vir-
tual Chinese restaurant what the novel object is used for), it might lead to learning
in the form of a satisfactory association of the novel object with now updated expec-
tations. Based upon its location and its utility you will have learned what a chopstick
is at this first encounter. Learning may take the form of associating the synchro-
nized visual ensemble created by a chopstick with the act of eating, mastered at
some earlier time. At your next encounter, your recall of the memory trace for a
chopstick will remove the “novel” label and you are now prepared to cognitively uti-
lize the object as a tool for eating pork-fried rice, assuming you can learn to effec-
tively manipulate them.
The Mechanism of Novelty Detection
It is easy to invoke a comparison of current and memory-based synchronized
neuronal ensembles as the basis for identification of novelty but it is not quite so
easy to specify the precise mechanism by which this comparison could take place.
To approach this issue we have to go back to basics and trace the pathway of sensory
information as it is successively processed through different areas of the brain.
While visual information serves as a useful example, other sensory modalities are
likely to utilize analogous mechanisms.
At the outset we should recognize that novel encounters are commonly
multi-modal. The combination of senses involved in evaluating a new food item
illustrates the potential complexity of starting information. Imagine, for example,
that you are invited to dine at a restaurant famous for its creative cuisine, and your
host has pre-selected your main course. The platter arrives, covered with a fancy sil-
ver top, and with great flair the waiter removes the top from the first dish, present-
ing it for your inspection and appreciation. After all the priming and anticipation
you have only two or three seconds to cognitively attend to the contents of the plat-
ter and formulate your reaction. Your sensory systems quickly respond to the pre-
sentation (visual), aroma (olfactory) and perhaps the sizzling sound (auditory) of
the dish. Based upon this initial information you formulate an immediate but tenta-
tive response, while being careful not to offend your host! Final judgment awaits
your evaluation of the taste (gustatory) and texture (touch) as you eat a sample of
this novel dish. Despite the simultaneous parallel processing of these independent
modalities, they are integrated into a unified experience. Did you like it or not?

99
Notice that despite the multiple components, we typically consider the whole as a
singular, unified gustatory experience. All the different sensory elements have com-
bined to a form single entity. Your overall evaluation may well have included a
quick memory-based comparison with previous dishes that looked somewhat like
the current one. It may also have included subjective information (e.g. “I usually
like Chinese food.”) or your appetite for novelty (e.g. “I’m not feeling very adven-
turous today.”). All of this diverse information must somehow interact to formulate
your final, lasting image of this novel experience.
Assembling small pieces of information into a more useful whole is one of
the most remarkable and important functions of the brain. Single nerves in our
individual sensory systems parse the event into separate, elemental components
(similar to pixels on a TV screen), and the brain then reassembles them by the coor-
dinated firing of an ensemble of neurons. Individual elements remain temporally
linked to each other through multiple levels of neuronal processing. The brain grad-
ually reassembles the components through a graded series of associations, using
synaptic connections laid down during previous experience. [Unlike computers
with pre-loaded operating systems, humans don’t come pre-programmed.]
Although the initial assemblage of information may yield only a primitive virtual
image, successive associations gradually refine and supplement the informational
content. What may have started out as a patch of orange becomes associated with
the edge detection of a circular shape and an association with food to gradually yield
our recognition of a slice of carrot (Figure 8.2). Even if our retina doesn’t take in
every reflected photon, prior experience provides the ability to fill in the entire con-
tinuous surface and perhaps even add the anticipation of depth although only the
top surface is seen. Silently we also associate the language symbol for this object (i.e.
carrot in English) just in case we need to communicate our perceptions to someone
else. As a result of these associations we can treat the carrot slice as a single, unified
object with all its attached cognitive attributes.
Somewhere in this hierarchical process of association and reassembly there
must exist a mechanism for comparing new, incoming information with pre-estab-
lished expectations, corresponding to the novelty detection process outlined in Fig.
8.2. Although the location and mechanism for novelty detection have not as yet
been elucidated, we can still consider its performance features and gain clues about
how it might function.
Comparison implies the simultaneous co-existence of two sets of
information: The observed and the expected. Congruence between these two
data sets affirms expectations and the currently observed information is treated as
non-novel. If we have previously learned enough about our immediate physical
environment we can operate in a purely cognitive mode, utilizing that congruence
to avoid having to continually interpret and learn about ongoing sensory experi-
ences, Incongruence, however, leads to a declaration of novelty and initiates a pro-
cess of attention, further investigation and learning. Attention to novelty serves to

100
amplify the detail and intensity of information available for investigation. For
example, we might increase our attention to information about the shape or color of
an unknown object. Note that such amplification is accompanied by a concomitant
loss of awareness intensity for unattended information, forming the basis for many
a magic trick and for a pickpocket’s livelihood. Attention increases the comparative
value of selected information while simultaneously decreasing the value of un-
selected information.

Figure 8.2: Creating a percept. Sensory objects are initially parsed into elemental
sensory information that is incrementally re-associated and supplemented with
non-sensory associations.
Once underway, the investigative process typically involves a series of mem-
ory-based comparisons in an attempt to match current novel experience with
expectations, which are now acknowledged to be incongruent, We want to clarify
specifics about the basis for this sense of novelty. During this process it can be useful
to partially deconstruct the experience in order to identify the novel components.
For example, perhaps our carrot slice looks the part visually, but the taste we experi-

101
ence is not what we expected. The investigational phase could then be directed
toward taste as the particular incongruent element, using a memory comparison to
tentatively inquire whether this was a taste associated with being rotten or, failing
that, whether this difference in taste was an indication of some exotic and unfamil-
iar vegetable. In a similar manner, if we see a car we don’t recognize, we immedi-
ately start to break down the whole into its parts (fenders, insignias etc.) in an
attempt to resolve the dilemma. Starting from this more primitive level, investiga-
tion can lead toward a new set of potential associations that are congruent with the
current observations. We develop tentative hypotheses to resolve this novelty-base
conflict (e.g. Perhaps this the new model year of a Toyota Celica?). If and when we
are satisfied with the newly developed rationalization we can move on. Learning has
occurred! We have successfully linked this unexpected information into a new uni-
fied concept that is no longer incongruent with experience, even though it may still
take the form of a temporary hypothesis. The next time that same car drives by its
properties will be entirely congruent with the memory pattern of this new concept
and it will no longer be novel.
The mechanism for novelty detection involves a comparison of current vs.
previous patterns of synchronized neuronal firing at different levels in the associa-
tive hierarchy. The detection of novelty at any level can initiate the formation of
new associations using the more fundamental information from a previous level to
create the new association. Thus it is necessary to regress to a more primitive level
in order to move forward again. Newborns represent the ultimate example of nov-
elty-driven detection and learning. To them everything is novel! Their visual associ-
ations have to start from the very basic. Repeated experience gradually leads to the
ability to define the edges of objects and to associate those edges with texture and
color in two-dimensional and then three-dimensional representations. They are
building their associative hierarchy from the bottom up.
Hierarchical integration of information, from simple to more complex per-
cepts, is an important factor in novelty detection. Thus a chair attached to the ceil-
ing or the occurrence of a refrigerator in a bathroom would be novel, but it is only
their context, not the objects themselves which are novel. Context is an example of
higher-order information. If we were able to somehow intercept these visual images
along their hierarchical pathway, novelty would be absent until we reached the
higher order expectation of spatial context. In this case the relationship between
already unified objects is the basis for a declaration of novelty. On the other hand, a
purple metal box resembling a refrigerator in the living room, could fail the congru-
ency test and be declared novel at a more primitive level. This suggests that a mech-
anism for novelty detection must be available at each successive level of association
as a unified percept is being formed, rather than being a separate process applied
only to the fully formed percept.
Memory sets our expectations for non-novel, already learned experiences. If
the coffee cup on my desk were to be observed in a location distinct from my most

102
recent expectations, this would be unexpected and novel and would reflect involve-
ment of short-term memory. Reading something that conflicted with what I learned
in the 4th grade would involve long-term memory. Because the biochemical mecha-
nisms for short-term and long-term memory are thought to be different, the system
that provides novelty detection cannot be linked to one particular form of memory
or one particular biochemical mechanism.
What form does memory take that allows it to compare current experience
with very recently processed information? In a process of hierarchical associations,
memory can take the form of a pattern of neurons participating in a percept. If the
current experience is identical to a previous experience, this pattern will be identi-
cal. If it is non-identical, different neurons will be active, creating a different pat-
tern. The question then becomes: How can memory create a pattern of active nerves
that allows comparison with the current pattern? This is a difficult question. One
general scheme could involve a prediction or anticipation of the pattern expected at
the next level of associative hierarchy. For example, if the simultaneous experience
of A and B always led to C in previous experience and C always led to D, E and so
forth, then a failure of E to occur after A, B and C would indicate the intervention of
novelty.

103
 Figure 8.3: The detection of novelty. Unexpected experiences lead to novelty
detection, attention and attention-based learning.
Figure 8.3 outlines a hypothetical example illustrating novelty detection,
attention and learning. As elemental information is assembled into percepts in a
hierarchical manner, the occurrence of unexpected information (represented by
dashed lines) leads to new intermediate percepts (P, Q and R) not previously associ-
ated with percept G. Either the absence of expected components (e.g. D, E and F) or
the presence of unexpected components (P,Q and R) results in an ensemble of neu-
ronal activity (i.e. a percept) that is distinct from predictions (i.e. percept S vs. the
expected G). This conflict evokes attention to the particular elements found to be
novel (P, Q and R), a process that involves disassembly of the higher order percept

104
into its components. Once the novel information is confirmed it is encoded and
associated with shared elements from the initially anticipated percept (i.e. P, Q and
R now become associated with A, B and C). Together they form a newly learned
percept S. In other words we use attention to convert novel experience into learned
associations.
The critical question still remains: What type of mechanism could allow
detection of unexpected information or the absence of expected information in a
percept? In the example shown in Fig, 8.4, memory-based expectations for neuronal
firing during the higher order percept G are partially derived from lower order per-
cepts A and B. When A and B project memory- based expectation forward to G, the
coincident arrival of C- and D-derived information via F confirms expectations and
indicates the absence of novelty. However, in the presence of unexpected sensory
information this confirmation does not occur. Neurons represented by G are there-
fore in a position to detect the occurrence of novelty. In other words, by splitting the
flow of information into parts and using a memory-based pathway to supply one
component further down the main pathway, bypassing the next step, it is possible to
construct a system with the ability to compare current experience with prior expec-
tations.

Figure 8.4: Memory-based expectations and actual experience allow the detection
of novelty. If primary percepts A/B elicit memory-based expectations at the level of
the higher order percept G the prediction can be compared with current experience
to yield a mechanism for novelty detection.
There are some neural circuits in the brain that are appropriately configured
to detect novelty. In the hippocampus, CAl cells appear to detect novelty by a mech-
anism similar to the model outlined above. As shown in Fig. 8.5, CAl cells receive
two main sources of input. One comes directly from superficial layers of the nearby
entorhinal cortex (the perforant pathway) and another input (the Schaeffer collat-

105
eral pathway) originates from the entorhinal cortex, but arrives only after being
processed by an intervening network of cells elsewhere in the hippocampus (dentate
gyms and CA3 cells). Lisman and Otmakhova proposed that CAl cells compare the
input information from these two pathways, thereby detecting the presence of nov-
elty (122). They further proposed that dopamine’s inhibition of the perforant path-
way input was an important aspect of novelty detection. One interesting experiment
would be to compare the oscillatory firing pattern of the two inputs, If they both
exhibit the same pattern of frequency components, there is the opportunity for syn-
ergy and entrainment of CAl cell firing. However, if this is not the case, the two
inputs will tend to interfere with each other and diminish their combined ability to
produce synchronized CAl cell firing. Output activity from CAl cells returns infor-
mation back again to the entorhinal cortex, but to deeper layers rather than the
superficial layers that first projected the information to the hippocampus. A further
comparison between new, incoming information and recent experience can there-
fore occur in the entorhinal cortex. Figure 8.5. Information processing by the hip-
pocampus. CAl cells receive dual inputs, one directly from the superficial entorhinal
cortex (perforant pathway) and one indirectly after processing by dentate gyms and
CA3 cells (Schaeffer collaterals). CAl cell output is directed back to deep layer neu-
rons of the entorhinal cortex.

Figure 8.5:
Recent studies from the laboratory of Dr. David Amaral show that D4Rs are
quite dense on neurons in the primate hippocampus, including both r CAl and CA3
cells (123). In this region, however, D4Rs are located primarily on pyramidal cells
rather than on GABAergic interneurons. This suggests a different role for these
D4Rs when they are activated by either dopamine or norepinephrine. It is interest-

106
ing that the structural organization of the hippocampus is more typical of the cortex
in more primitive species (e.g. reptiles and birds) in that it has only two layers vs.
the six layers found in the cortex of mammals. Entorhinal cortex, which connects
the sensory cortex with the hippocampus, exhibits an intermediate pattern in which
the six cortical layers merge into two primary layers (superficial and deep) as noted
above. These anatomic features suggest that the hippocampal formation is a more
primitive brain structure and that the pyramidal cell location of D4Rs in this region
may also reflect a more primitive role. The entorhinal cortex serves as a transitional
entry pathway as well as an exit path.
No matter the precise mechanism, our operating hypothesis requires that
novelty detection must eventually be reported to ventral tegmental area neurons so
that they are stimulated to increase their release of dopamine and cause attention.
Anatomic studies by Dr. Susan Sesack (University of Pittsburgh) have shown that
VTA neurons receive excitatory input from the frontal cortex (124), raising the pos-
sibility that awareness information may be sent directly from the prefrontal cortex
to the VTA where it could elicit dopamine-mediated attention. Moreover, the
release of dopamine must be spatially specific to the cortical region that is currently
processing the information targeted for attention. This implies that specific fibers
emanating from the VTA are activated by the novelty detection mechanism in a
spatially appropriate manner. For example, dopamine should be released only in the
auditory cortex if we are attending to a novel auditory experience.
From my personal experience, it is difficult to simultaneously pay focused
attention in a multi-modal manner. Rather it seems that there is a necessary reduc-
tion in attention to one sensory modality when another is being prioritized, in a sort
of competitive manner. It generally recognized that attention includes both an
amplification of target information and a suppression of the awareness of non-tar-
get information. In terms of synchronized oscillations, the increased amplitude of
40 Hz oscillations for attended information may be accompanied by a decrease in
the 40 Hz power of unattended information.
Once we have used attention to learn something novel, we generally don’t
want to waste time or energy re-learning the same information again, although we
certainly don’t want to totally ignore the information. We remain “aware” of
learned information on an ongoing basis in order to utilize it in a facile and trans-
parent manner. In the context of our PLM and MAP kinase based model this
implies that we remain able to generate PLM-induced awareness, while suppressing
MAP kinase-mediated learning. Since these are separate functions of the D4R, there
may well be a mechanism (e.g. an inhibitory phosphorylation that is triggered by
MAP kinase activation) that allows for selective regulation of signaling activity.
Such a mechanism would turn off learning, but permit attention, especially volun-
tary attention.

107
Variations in Attention to Novelty
The occurrence of novelty-stimulated attention is obviously related to age, to
environment and other factors. Newborns have the capacity to detect individual ele-
ments of objects in their world, but initially these features are not combined
together via synchronization, to form the whole, gestalt object. Bit by bit the repeti-
tive association of individual elements with each other allows the reconstruction of
the whole object as an ensemble of synchronized information. As individuals we
each must construct our own library of synapse-based associations from scratch.
Because the objects in our shared world possess intrinsic characteristics (size, shape,
color etc.) and we generally share the same detection systems, we typically end up
with the same association pattern for the physical appearance of objects in our
world. In other words objects reflecting light of a particular wavelength will result in
the association of a similar color (i.e. red in the English language) with ripe toma-
toes for all who view them. In contrast to this person to person consistency for
physical attributes, other associations may vary greatly from person to person. An
inviting, freshly picked tomato to one person may be a distasteful vegetable to
someone else. Subjective attributes can be associated with objects via the same syn-
chronization mechanism as objective attributes, but are less reliably shared among
different individuals.
The probability that one will experience novelty lessens with age. After all,
everything is novel to a newborn, in utero experience aside. As we get older novelty
is harder to come by and we may think that we’ve “seen it all”. However, the human
drive to create and explore novelty is a life-long trait although the value we individ-
ually place on novelty is subjective and commonly evolves with age. We assign an
especially high value to novelty when we’re young because it is our gateway to
understanding the world. For the elderly, novelty can be a nuisance, requiring pre-
cious energy for learning and adapting to change.
At any age there can be considerable variability in the value that individuals
attach to novelty, which can be expressed as differences in “novelty seeking” behav-
ior. Recall that persons with the seven repeat allele form of the D4R have been
reported to express a higher level of “novelty seeking” behavior (3,4) and a similar
increase was associated with a reduced density of D4Rs (66). Our analysis of the
influence of D4R repeats on PLM mediated attention suggests that a higher number
of repeats would increase the ratio of phospholipid to protein in the D4Rs microen-
vironment, placing a limit on the efficiency of PLM. Fewer D4Rs would also limit
PLM, leading to the question: How might a decreased capacity for dopamine-stimu-
lated PLM lead to increased “novelty seeking”, if indeed there is a connection at all?
First let’s attempt to further clarify the meaning of “novelty seeking”. The
Tridimensional Personality Questionnaire (TPQ) evaluates personality traits or
temperament in four areas: Novelty Seeking, Harm Avoidance, Reward Depen-
dence and Persistence based upon responses to a series of questions developed by
Cloninger and colleagues (61). They suggested that the presence of these traits was

108
related to the activity of specific neurotransmitter systems and their genetic deter-
minants. In the case of novelty seeking it was postulated that differences in dopa-
mine-mediated neurotransmission determined the extent to which these behaviors
were expressed. Individuals with higher scores in novelty seeking tended to be
impulsive, excitable, quick-tempered, extravagant, fickle and exploratory, while
those with lower scores were more reflective, stoic, slow-tempered, frugal, loyal and
rigid. Each individual trait is distinctive, but they all include a recurring element of
novelty and risk that is a favored behavior at one end of the spectrum (i.e. in “nov-
elty seekers) but disfavored at the other (i.e. in those we might call “contempla-
tors”). We can now recognize that the tag line “novel seeking” does not imply that
these are people who spend large amounts of their time actively trying to find new
experiences. Rather, it implies that the actions taken by these people in their ongo-
ing lives are less the result of a deliberate process of reasoned evaluation and more
the result of non-calculated instinctive behavior. The actions of novelty seekers are
more likely to reflect their initial reaction to a set of circumstances, while contem-
plators are more likely to respond in a manner that reflects more extensive process-
ing of the variables and possibilities. While novelty seekers approach the world with
a “Why not?” attitude, contemplators ask “Why?”. There is a sense that the influ-
ence of prior learned experience is more limited among extreme novelty seekers,
while it dominates over spontaneity in extreme contemplators.
In every continuous spectrum there is only one element that truly lies at each
extreme. Every other element is somewhere in between and so it is with the spec-
trum of novelty seekers vs. contemplators. Most likely we are somewhere in the
middle. What’s so interesting in this case is that it is the nature of our personal type
of DNA, provided by mom and dad and shaped by previous generations, that
apparently has a significant influence on where we lie on that spectrum.
Is it “good” to be at one end or the other of this spectrum? Is it “bad”? Of
course not! There is nothing immoral or illegal associated with variations in the six
personality traits that describe these behavioral differences. Sometimes different
just means different and this is a perfect example of the many variations that make
humans different one from the other. Would the world be a better place if we were
all narrowly distributed within a very limited set of personality options? More to the
point of genetic factors: Would the survival of man as a species be improved? [Such
issues are taken up in Chapter 14 when we consider the evolution of D4Rs.] These
are differences to celebrate and appreciate rather than differences to suppress. No
doubt there are specific circumstances in which advantages and disadvantages
accrue to novelty seekers vs. contemplators. Moreover, when asked to make a value
judgement, novelty-seekers will most likely attach positive value to their own
behavioral tendencies and contemplators will do the same. Perhaps this is another
situation where the “golden mean” applies. It may be advantageous to avoid the
extremes and be able to benefit, at least partially, from each behavioral option. It is

109
tempting to invoke the worldwide predominance of four repeats in the D4R (vs. two
or seven) as a manifestation of the wisdom of nature.
If novelty seeking is indeed a reflection of the limited influence of prior expe-
rience, how might this relate to a genetically determined limitation in D4R-medi-
ated PLM, caused either by increased repeat number or lower D4R expression level?
There are several possibilities to consider. In the case of higher repeat number, an
increased number of proteins attached to the receptor might leave fewer layers of
phospholipids separating the D4R from neighboring protein targets for its PLM
activity. Consequently, a smaller methylation signal could more readily influence its
targets. Following this line of thought, equivalent concentrations of norepinephrine
could produce a higher intensity, more dopamine-like synchronization at D4.7Rs
than at D4.2Rs, not because the D4.7R produces more methylations/sec, but
because responsiveness of the microenvironment is enhanced. Such a condition
could produced increased awareness or hyper-vigilance. However, another poten-
tial consequence of the decreased phospholipid to protein ratio would be a rapid
depletion of the supply of phospholipids for methylation. Thus the duration of an
episode of norepinephrine- or dopamine-stimulated synchronization might be
shortened. For example, if norepinephrine stimulation exhausted the available sup-
ply of PE, how much further effect could subsequently be produced by dopamine?
Probably not much! While only hypothetical, a limitation in the duration of D4R-
mediated PLM could lead to a shift in the balance between norepinephrine-based
vs. dopamine-based synchronization in favor of the former, resulting in impaired
attention and attention-related learning. In short it could lead to reduced “attention
span”.
While phospholipid methylation-based signaling may be limited for the
seven-repeat receptor this would not be true of other signaling activities. In fact the
presence of additional proline-rich SH3 binding domains brings an increased
potential for complexation with accessory proteins, facilitating the generation of
MAP kinase-based responses and other intracellular signals by the D4R. Thus while
neuronal synchronization by the seven-repeat receptor may be limited, it may
enhance learning and the creation of synapse-based associations for future use.
The presence of a greater or lesser number of D4Rs within the postsynaptic
density would also lead directly to differences in the ability of norepinephrine and
dopamine to initiate neuronal synchronization. A higher receptor density per
square micron of membrane surface would increase the probability that NMDA
receptors and other fluidity-sensitive membrane protein would be modulated by a
given concentration of either agonist and this should heighten both awareness and
attention responses. This effect would be independent of repeat-associated varia-
tions in efficiency, but the two genetic factors could summate in terms of their over-
all effect. In other words the presence of both an elevated level of D4R expression
and seven repeats might each enhance norepinephrine-induced awareness, result-
ing in an additive net effect. If we add genetic variations in the synthesis and release

110
of dopamine or differences in its rate of removal, we begin to get a more complete
view of the multi-factorial basis of the molecular events that provide for human
attention. It is no wonder that no two people are alike when it comes to how we
think.
Implicit in the proposed circuit of awareness and attention in Fig. 8.2 is an
“attention-less” existence in which sensory events arc perceived and internally rep-
resented via synchronized firing of neuronal groups in the absence of dopamine-
mediated attention. In this attention-less state all elements of an experienced scene,
including different modalities such as visual and auditory elements, have essentially
equal value and interest. Everything is detected, but nothing is special. The primary
apparati for receiving and processing the sensory information (e.g. the retina, the
optic nerve, and the primary visual cortex) are operating at a standard level of activ-
ity, but there is no particular interest or significance attached to the experience. This
state sounds akin to that of a zombie in a B-grade horror film; an automatic trance-
like existence unembellished by anything interesting. It certainly does not sound
appealing, but at the same time sounds frighteningly similar to many moments in
our lives: Times when we are doing things on “automatic-pilot” or times when we
are daydreaming and not paying attention to much of anything. Certainly we do not
need to be attentive every second or every minute of our lives. Such an existence, the
very opposite of being a zombie, could be aptly labeled mania. Together the
extremes of these states describe lives of depression and mania, and the regular
alternation between them fits the description of bipolar, manic-depressive disorder.
We will examine these clinical conditions in later chapters.

Chapter Summary:
• Awareness and attention may represent two intensity levels of neuronal synchronization
provided by norepinephrine and dopamine respectively.
• The lower affinity of norepinephrine for D4Rs may facilitate the speed of its PLM
response.
• Awareness precedes attention and allows the identification of novel experience.
• Novelty generally elicits attention and initiates attention-based learning.
• Comparisons between current experience and memory-based expectations provide
identification of novelty.
• Associative learning converts novelty into knowledge and novel experiences decrease with
age.
• Individual differences in D4 receptor density and repeat number, as well as other related
factors, cause person to person variation in the quality of attention.

111
9 ATTENTION AND METABOLISM

Most neurotransmitter receptors are relatively simple on/off switch mecha-

nisms, turned on by the binding of their neurotransmitter hormone and turned off
in its absence. Receptor channels, for example, are open when turned on and closed
when turned off, a straightforward and tidy arrangement. The cyclic process of D4
receptor-mediated PLM is, however, definitely different, with features designed to
make it responsive to and contingent upon prevailing metabolic conditions. For as
much as D4R-mediated PLM is involved in attention and attention-related learn-
ing, this implies that these mental capacities will be affected by changes in our
metabolism, an example of the “Mind/Body Connection” in action. In this Chapter
we’ll examine the molecular basis for this connection, with special attention to
changes that occur over the human lifespan.
The Folate Connection
The most obvious link between D4R-mediated PLM and metabolism is its
dependence upon the single-carbon folate pathway as a source of methyl groups
during ongoing PLM activity. The folate (folic acid) system is a core metabolic path-
way, providing single-carbon groups to several biochemical pathways involved in
cell maintenance and growth. Man cannot synthesize folate and its nutritional
requirement has been recognized for many years. Recently, dietary folate require-
ments have received greater attention after it was established that low folate during
pregnancy can lead to spina bifida (125), a failure of the spinal column to fully close.
Subsequently an elevated level of homocysteine, which can be caused by folate-defi-
ciency, was shown to be associated with increased cardiovascular mortality (e.g.
heart attacks) and the blockage of arteries. For these reasons, the U.S. government
mandated the addition of folic acid to flour and cereal grain products in order to
increase our routine daily intake. This resulted in a dramatic 10-fold decrease in the
prevalence of low folate levels and a significant reduction 0f 7% in homocysteine
levels (126).
The metabolic role of folic acid is to carry single carbon groups in different
oxidation states and supply them to biochemical reactions. It carries a carbon group
at either the 5-position or the 10-position or bridged between the two nitrogen
atoms at the 5 and 10 positions (Figure 9.1). The active form of folic acid is its tetra-
hydro-folate or THF form. In different oxidation states variable numbers of hydro-
gen or oxygen atoms are bonded to the carbon atom, making it suitable for different
reactions. For example, the most highly oxidized state is the formyl group in which
a double-bonded oxygen and one hydrogen are bonded to the carbon. The formyl
group can be attached to either the N-5 or N-10 nitrogen. The methenyl carbon has
a single hydrogen and forms a bridge by being single-bonded to N-5 and double-
bonded to N-10. In 5,10-methyleneTHF the bridge carbon is further reduced and
has two hydrogens. Further reduction adds two more hydrogens and breaks the

112
bridge, creating 5 -methylTHF, which is the single-carbon folate species required
for methylation of homocysteine and by the D4 receptor for PLM.

Figure 9.1: The structure of tetrahydrofolic acid and its single-carbon derivatives.

Activity of the D4 receptor-mediated PLM cycle can be affected by 5-methyl-

THF availability (20). As illustrated in Figure 9,2, nature designed the cycle to be
limited by the concentration of dopamine under normal conditions, but when the
level of 5-methylTHF is decreased it can become a limiting factor. The population
of D4 receptors normally exists mainly in the methionine state (D4 T) waiting for
dopamine to produce a conformational change in the receptor. This is the most effi-
cient situation for dopamine stimulated PLM. However, when the supply of 5-
methylTHF becomes limited, a large portion of the receptor population is waiting
for a new methyl group, stuck in its homocysteine (D4 state. In this condition the
effect of dopamine can be greatly reduced (20). Even a very high concentration of
dopamine will be unable to accelerate PLM since the shape of the D4 receptor is no
longer the rate-limiting event. Certainly the role of D4 receptor- mediated PLM in
attention and cognition would be impaired under this condition.
What then determines 5-methylTHF availability? Activity of 5,10-methy-
leneTHF reductase (MTHFR) is obviously critical for an adequate supply of 5-
methylTHF. While other single-carbon folates can be readily interconverted, for-
mation of 5-methylTHF by MTHFR is irreversible. In man there is a relatively com-
mon polymorphism in the MTHFR gene that causes the enzyme to exhibit lower
activity and be more sensitive to heat inactivation (i.e. be thermolabile) (127,128).
Persons homozygous for thermolabile MTHFR typically have elevated homocyste-
ine levels and increased cardiovascular risk, especially if they are low in folate,
Though it hasn’t been measured yet, we could expect that the D4 receptor would
also be found more in its homocysteine state in persons that are homozygous for
thermolabile MTHFR. Interestingly, several reports have found a correlation

113
between the low-activity thermolabile form of MTHFR and schizophrenia
(129,130). MTHFR activity depends upon the level of the enzyme, the concentration
of its substrate, 5,1 0-methyleneTFiF, adequate levels of the required co-factor
NAD(P)H as well as the levels of S-adenosylmethionine (SAM) which inhibits
enzyme activity. In this manner SAM provides negative feedback control.

Figure 9.2: The single-carbon folate pathway and its relationship to D4 receptor-
mediated PLM. Formic acid (formate) produced b’ mitochondria is the primary
source of carbon groups for the folate pathway. Radiolabeled [ 4C]fonnate allows
measurement of folate-dependent carbon transfers such as homocysteme
methylation. Folate-dependent PLM includes both non-receptor (right) and D4R-
dependent (left) cycles.
Another important determinant of 5-methylTHF levels is the rate at which
single-carbon folates are used by other metabolic processes. As shown in Figure 9.2,
there are four other pathways that compete for available single-carbon groups. In
the de novo purine synthesis pathway purines are synthesized piece by piece using a

114
series of small fragments to make the whole. In two different steps of the de novo
purine synthesis pathway 10-formylTHF donates its single-carbon group to the
growing purine structure. The purines adenine and guanine are perhaps best known
as two of the four building blocks of DNA and RNA and their sugar/phosphate
nucleotide forms (adenosine triphosphate (ATP) and guanosine triphosphate
(GTP)) are at the heart of cellular metabolism. A third purine, inosine, is not nor-
mally incorporated into RNA or DNA.
The demand for de novo purine synthesis depends upon the metabolic activ-
ity of cells. When metabolic activity is high, more single-carbon groups are diverted
for the all-important job of making purines. As the demand for purines lessens, the
carbon group of 10-formylTHF becomes available for other pathways. Control is
provided by the levels of adenosine, guanosine and inosine. When their levels are
high their monophosphate forms (AMP, OMP and IMP) bind to an enzyme in the
beginning of the pathway, causing inhibition of de novo purine synthesis, a classic
example of end-product negative feedback. We will revisit de novo purine synthesis
again in Chapter 13 since an inborn error in the pathway causes autism.
The second folate-requiring pathway involves the methylation of uracil in its
deoxyuridylate (dUMP) form to make deoxythymidine (dTMP), a reaction carried
out by the enzyme thymidylate synthase using 5,10-methyleneTliF (Figure 9.2).
Uracil is a pyrimidine component of RNA while thymidine is the equivalent pyrim-
idine component in DNA, During cell division, when new DNA strands are being
synthesized, single-carbon folates are diverted toward thymidine synthesis.
The third folate-dependent pathway involves conversion of the amino acid
glycine to serine by the multi-functional enzyme serine hydroxymethyltransferase
(SHMT). As shown in Figure 9.2 this cytoplasmic reaction also utilizes 5,10-methy-
leneTHF and requires vitamin B6 (pyridoxine) as a co-factor (131). SHMT also
exists within mitochondria but curiously that enzyme favors the reverse reaction,
converting serine into glycine with the formation of 5,1 0-methyl eneTHF (132).
Ultimately mitochondria release the single carbon from serine as formate which is
used to create 1O-formylTHF back in the cytoplasm of the cell.
The fourth pathway competing for single-carbon folates is a somewhat enig-
matic futile cycle. In addition to using 5, 10-methylene THF to make serine from
glycine, SHMT can also convert 5,l0-methyleneTHF to 5-formylTHF. 5-For-
mylTHF does not donate its single-carbon group to any reaction but instead regu-
lates folate dynamics by inhibiting de novo purine synthesis and also by inhibiting
SHMT. It causes this inhibition by competing with 10-formylTHF (133) and by
binding tightly to SHMT, blocking the access of 5,10-methyleneTHF (134). Via this
inhibition, 5-formylTHF regulates single-carbon utilization by three different path-
ways, including its own synthesis.
Across the lifespan, demand for de novo purine synthesis and thymdine syn-
thesis is high during infancy and throughout teenage growth years but abruptly
slows during post-pubertal years to the lower level J sustained through adulthood.

115
From adulthood through old age we experience a further, gradual metabolic slow-
ing. By our own experience and by observing the development of others we can
appreciate that both the quality of attention and the capacity for cognition are dra-
matically different across the lifespan. Although we may take these for granted as
normal features of human nature, there can be no doubt they reflect molecular
events occurring within the brain that are somehow linked to developmental
changes in metabolism. Recognition of the link between single-carbon folate metab-
olism and dopamine-stimulated PLM provides an opportunity to view these
changes in a more informed light. It can lead to novel hypotheses about the under-
lying causes of mental illnesses and new ways to treat them.
Single-carbon Metabolism in the Brain
There are tissue-specific differences in single-carbon metabolism that make
the brain more dependent upon the folate system than other organs in the body. In
some other cell types there are two different reactions that can carry out methyla-
tion of homocysteine, however, in brain tissue the 5-methylTHF-dependent methi-
onine synthase enzyme is the only option (135). Liver and kidney cells express a
second enzyme (betaine-homocysteine methyltransferase or BHMT) that utilizes
betaine as the source of the methyl group (Fig. 9.3). Therefore brain function may
be more sensitive to changes in folate metabolism than these other tissues. More-
over, since the brain is more dependent on methionine synthase, deficits of 5-
methylTHF will exert a more profound effect than in the body as a whole. Impor-
tantly, measurements of plasma homocysteine levels may be a poor indicator of
homocysteine status in brain tissue, since they are strongly influenced by liver func-
tion, Persons with normal plasma homocysteine levels could have a masked
decrease in neuronal tissues and those with an elevated plasma homocysteine could
be experiencing an even higher level within brain tissue. As we shall see, the lack of

116
BHMT has particular significance for the higher sensitivity of brain metabolism to
alcohol.

Figure 9.3: Dual pathways for methylation of homocysteine. Liver and kidney
express BHMT, which utilizes betame as methyl donor, as well as methionine
synthase (Met Syn). Neuronal cells rely solely upon methionine synthase activity.
The limited capacity of the brain to re-synthesize methionine from homocys-
teine does not create a shortage of methiomne as long as there is an abundance of
methionine available from the diet. Dietary methionine provides a ready source of
transferable methyl groups upon its adenosylation to form SAM. Since dietary
methionine reduces the need for homocysteine to be reconverted back to methio-
nine, it decreases the demand for folate-derived methyl groups (i.e. 5-methylTHF).
A steep age-dependent decline in the uptake of methionine by the brain has been
observed, using [‘1C]-labeled methionine in brain scan studies (136). This remark-
able finding was made serendipitously when [1 ‘C]-methionine was being used to
image brain tumors that take up methionine very rapidly since it is essential for
their aggressive growth. Investigators found that methionine uptake in the prefron-
tal cortex decreased by about 6-fold from 3 yrs. of age through about 20 yrs. after
which a steady level was maintained throughout adulthood. The decrease may
reflect a development-related reduction in the metabolic demand for methionine.
As brain metabolism makes a transition to adulthood methionine uptake is appar-
ently reduced, putting a greater demand on the folate system to provide for homo-
cysteine re-methylation. This maturation-dependent shift in brain metabolism
could be a critical factor in triggering the onset of schizophrenia that typically
occurs between 18-25 years of age in men and slightly later in women.
The D4 receptor and homocysteine itself compete for methionine synthase
and for available 5-methylTHF in neuronal cells. Availability of the BHMT system
in non-neuronal cells serves to buffer any tendency for homocysteine to accumulate
while in the brain D4 receptor function will be directly affected by such an increase.

117
In brain therefore the absence of BHMT assures an important role for homocyste-
ine as a regulator of D4 receptor-mediated PLM. Consequently, neuronal D4 recep-
tor-mediated PLM will be more sensitive to changes in folate metabolism. It is hard
to escape the conclusion that the lack of BHMT expression in the brain may have
evolutionary benefit because it introduces, or at least amplifies, metabolism-and
age-dependent changes in attention that are a hallmark of human development.
Vitamins and Co-factors
While we recognize that vitamins are essential for normal, healthy body func-
tion we rarely appreciate the specific role that vitamin molecules play when we
reach for that “One-A-Day®” or perhaps “Centrum Silver®”. In the case of D4R-
mediated PLM we have an opportunity to do just that, since several vitamins and
co-factors are required for this attention-related biochemical pathway. Vitamins are
small molecules that we do not have the capacity to synthesize ourselves but are
required for normal activity of enzymes and other functional proteins. Typically a
vitamin molecule is bound within the active site of an enzyme where it directly par-
ticipates in the reaction being carried out. However, during one cycle of enzymatic
activity the vitamin molecule is not used up. While the starting materials (sub-
strates) and products come and go, the vitamin molecule participates in many cycles
of activity, until it eventually dissociates and becomes a target for degradation or
elimination. Thus our diet must allow for adequate replenishment of vitamins, but
the amount required is small, compared to nutrients that are consumed during nor-
mal metabolism. The unique chemical features of vitamins account for their critical
role in a particular enzymatic reaction. For example, during a reaction cycle the
vitamin co-factor may be able to temporarily receive electrons or even atoms from
the substrate, causing it to be structurally modified. However, as an integral part of
the reaction the vitamin returns to its starting structure just as if nothing had hap-
pened. In some cases the vitamin co-factor may donate a proton or electron to a
reaction, requiring that it dissociate in order to replenish its supply.
All living organisms have a requirement for complementary molecules that
they themselves cannot produce. This can reach ridiculous extremes, as in the case
of viruses. Viruses have only a core of RNA or DNA surrounded by a protein coat.
For their life cycle, the host cell is a megastore of molecules that the virus can utilize
for a successful lifespan and for replication. Without a host cell, the virus cannot
replicate. Bacteria require vitamins and other co-factors from the surrounding envi-
ronment that determines their capacity to survive and divide. Evolution has left
man with certain molecular needs that we satisfy by our diet, taking in those plants
and animals that either produce or contain the co-factors we need.
Man cannot produce folie acid and we must therefore eat sufficient green
leafy vegetables, beans etc. to keep our folate levels in the range necessary for nor-
mal function. Folie acid has never been given a vitamin designation because it is
structurally altered during each reaction cycle and therefore is strictly speaking a co-
factor. For example, 5-methylTHF enters the methionine synthase reaction, but

118
111F comes out and is subsequently reconverted to 5-methylTHF by other reac-
tions. Thus the folate backbone merely serves as a carrier for single-carbon groups,
in a vitamin-like manner. The nutritional requirement for folie acid was first recog-
nized in relation to the occurrence of anemia when its levels were too low (137).
Subsequently, birth-defect spina bifida was linked to folate deficiency during preg-
nancy and more specifically to reduced levels of 5-methylTHF (125).
Low folate deficiency can lead to psychiatric disturbances including depres-
sion and schizophrenia (138,139). While folate deficiency can cause problems, hav-
ing a normal serum folate level does not guarantee that your single carbon folate
system is providing the needed methyl groups to support D4R-mediated PLM.
Remember that folie acid is only the carrier. What your metabolism does with folate
is just as important as having enough to begin with. Remember that total intracellu-
lar folate consists of the five major single carbon states of tetrahydrofolate (5-
methyl-, 5,1 0-methylene-, 5,1 0-methenyl-, s5-formyl- and 10-formylTHF) as well
as dihydrofolate. The relative amount of each type is determined by the activity of
enzymes involved in their formation and their utilization, and by the mitochondrial
synthesis of formic acid used to create 1O-formylTHF. In addition, intracellular
folates have multiple glutamic acid residues added to one end that increase their cel-
lular retention and their binding affinity for enzymes. Thus having enough folate
available is just the beginning. It’s what you do with it that really counts and several
vitamins are essential for normal single-carbon function.
Vitamin B6 (pyridoxine) plays an important role at two points in the folate/
single-carbon pathway. In its phosphorylated form (pyridoxal phosphate) it is a
required co-factor for both serine hydroxymethyltransferase (SHMT) and the
homocysteine-metabolizing enzyme cystathionine 3-synthase (CBS). As mentioned
above, SHMT interconverts the amino acids serine and glycine in concert with the
formation (when serine is converted to glycine) or utilization (when glycine is con-
verted to serine) of 5,10-methyleneTHF. 5, 10-methyleneTHF is created from THF
in mitochondria, while 5, 10-methyleneTHF serves as the single-carbon donor for
serine synthesis in the cytoplasm. CBS combines homocysteine and serine to form
cystathionine, a precursor of cysteine and glutathione. Consequently, vitamin B6
deficiency causes an accumulation of homocysteine that is associated with neuro-
psychiatrie and neurological symptoms including depression and seizures that
respond to vitamin B6 treatment (140,141). Isoniazid, an anti- tubercular drug,
blocks phosphorylation of vitamin B6 and can cause psychosis, mania and obses-
sive-compulsive disorder (142).
Nicotinic acid (niacin) in the form of nicotinamide adenine dinucleotide
phosphate (NADP), is a required co-factor for the enzyme that forms 5-methylTHF
(5, 10-methyleneTHF reductase or MTHFR). A deficiency of nicotinic acid there-
fore reduces the available level of 5-methylTHF. Persons who are homozygous for
the weak, thermolabile form of MTHFR will be extra sensitive to a Vitamin Bi defi-

119
cit. During the 1 960s and 1 970s nicotinic acid therapy was widely advocated for
schizophrenia but subsequent studies failed to substantiate its benefit (143)
Vitamin B2 (riboflavin), in the form of flavin adenine dinucleotide (FAD) is
an essential component of a reducing system which maintains activity of methio-
nine synthase. Vitamin B2 deficiency is associated with psychiatric symptoms
including depression.
Vitamin B 12 (cyanocobalamin) was first recognized as the factor that pre-
vents pernicious anemia. Vitamin B12 has a complex structure in which the most
important feature is a cobalt-containing corrin ring system. In the methionine syn-
thase reaction vitamin B12 is a central player in facilitating methyl transfer from 5-
methylTHF to homocysteine (50).
Methionine synthase has three separate structural domains that bind 5-
methylTHF, homocysteine and SAM respectively, as well as a central active site con-
taining vitamin B 12. During a primary reaction cycle the 5-methylTHF-containing
domain moves close enough to the active site to allow transfer of the methyl group
to the cobalt within vitamin B 12, transiently creating methylcobalamin (50). The 5-
methylTHF domain then moves away, allowing the homocysteine-containing
domain to enter the active site, facilitating transfer of the methyl group to the sulfur
atom of homocysteine. Vitamin B 12 therefore serves as a go-between for passing
the methyl group from 5-methylTHF to homocysteine. A direct transfer is energeti-
cally unfavorable. Clearly a vitamin B12 deficiency will directly impair the re-syn-
thesis of methionine although dietary methionine can mitigate the problem to some
extent. In contrast, the D4R PLM cycle in the brain is totally dependent upon
methionine synthase activity and is therefore more vulnerable to vitamin B 12 defi-
ciency. Lower vitamin B 12 levels are associated with psychiatric disturbances,
impaired cognition and de-myelination-associated neuropathies, especially in the
elderly, who often show a decrease in vitamin B 12 absorption (144). A combined
deficiency of vitamin B 12 and folate has been linked to the risk of Alzheimer’s dis-
ease (145).
The general anesthetic nitrous oxide (N2O or laughing gas) is an inhibitor of
methionine synthase by virtue of its ability to bind to the cobalt in vitamin B12, but
is this action does not appear to be the cause of anesthesia. Inhibition is slow in
onset and quite long-lasting, producing a cumulative effect during multiple expo-
sures that can result in a degenerative neuropathic syndrome similar to that seen in
vitamin B12 deficiency.
Sleep and Sleep Cycles
During a 24 hour day there are times when our capacity for attention is at its
best and times when it’s at its worst. For most individuals these times correspond to
early morning hours shortly after arising and late-night hours just before sleep.
When we feel tired there is a sense of energy depletion, accompanied by a loss of
mental acuity. When we’re ready for sleep our attention level is typically at its lowest

120
level of the day. Reciprocally, when we first wake up after a restful night’s sleep, we
may feel especially able to concentrate and get things done, even before that morn-
ing cup of coffee. While individual patterns and preferences may vary rest and/or
sleep improves attention.
Reduced activity during rest and sleep allows cellular metabolism to replenish
molecular energy sources such as the level of ATP. This is true of the brain as much
as any organ, although the brain remains quite active while we’re asleep. The pat-
tern of EEG recordings changes during sleep and overall activity is reduced but the
brain is certainly not inactive during sleep. REM (rapid eye movement) sleep is the
clearest example. Occurring more toward the later part of the sleep period, REM
sleep is characterized by a high level of gamma-frequency oscillations in the cortex
that are essentially identical to the complexes occurring during wakeful cognition
activity as discussed earlier (108). During REM sleep, however, these complexes do
not occur in coordination with external sensory information. Gamina frequency
complexes do not occur during non-REM sleep. Since REM sleep is the time when
dreaming is most common, it has been suggested that the totally internal thoughts
and experiences of a dream are a result of these complexes (108, 146). It’s no won-
der that dreams do such a good job of masquerading as reality, since they are caused
by the same neuronal events as actual experience, but are freed from the constraints
of the real world. The meaning of dreams is a matter for Freudians to debate, but
their non-random content suggests that the discharge of memory circuits that were
active during proceeding periods of wakefulness may trigger oscillations during
REM sleep.
What molecular events trigger sleep? Since we just finished outlining a novel
description of the events underlying attention, there is an opportunity to identify
novel links to current theories.
First of all, let’s be clear that unconsciousness is obviously different from
sleep. You only have to clap your hands once or twice next to someone who is asleep
and they will awake. This tells us that their capacity for attention has been greatly
attenuated during sleep, but it is not totally turned off How else did they hear your
clap? Clap your hands next to someone under full general anesthesia and nothing
will happen, due to an effective inhibition of the primary sensory experience, as well
as a loss of the capacity for attending to it. The effects of general anesthetics are
exerted at the interface between membrane proteins and surrounding phospholip-
ids, an action that is not unrelated to the mechanism of D4 receptor-mediated PLM.
In 1846 William Morton brought ether anesthesia to prominence with his
successful demonstration of its utility in surgery at the “Ether Dome” of Massachu-
setts General Hospital, an event commemorated by a statue located in a corner of
the Boston Public Garden. The molecular mechanism of general anesthesia is still
debated but most agree that it results from the anesthetic’s ability to dissolve in the
membrane and thereby affect the function of membrane proteins, GABA receptor
and NMDA receptor responses are : among the most sensitive to general anesthetics

121
(147,148) Anesthetics increase the inhibitory action of GABA receptors, causing a
decrease in nerve firing, mcluding cortical pyramidal cells Within our model of
attention, this action would oppose dopamine-induced disinhibition and thereby
interrupt active attention General anesthetics also inhibit NMDA receptor function,
similar to the effect of PLM. However, unlike the localized D4 receptor-initiated
response on ppG interneurons this action would also directly inhibit pyramidal cell
firing.
It is worthwhile to recognize that general anesthesia is not an “all or none”
phenomenon. For classical anesthetics such as halothane there are four stages of
anesthesia that reflect differences in sensitivity of various neuronal systems. These
include: Analgesia or loss of pain (Stage I); hyper-excitability and delirium (Stage
II); Surgical Anesthesia (Stage III) and Respiratory Arrest (Stage IV). As the level of
anesthetic builds up in the brain, patients move progressively from Stage I through
Stage Ill, and then reverse this order during re-emergence. With development of
newer anesthetic agents with differing mechanisms, these Stages don’t always apply.
What is the basis of natural sleep? Humans, like most animals, have an inter-
nal clock mechanism or circadian rhythm that controls our wakefulness with an
approximately 24-hr. cycle. This is evident as cyclic changes in the concentration of
a number of hormones and substances including melatonin, adenosine and cortisol.
A rise in melatonin synthesis occurs with the onset of darkness and contributes to
the induction of sleep as well as to the continued maintenance of the circadian cycle.
This leads to the utility of melatonin in re-setting the sleep cycle after changing
global time zones (i.e. treating jet lag). Interestingly, retinal synthesis of melatonin
in response to darkness is regulated by dopamine D4 receptors. Stimulation of the
D4 receptor is required for activation of melatonin synthesis and blocking the D4
receptor interrupts the darkness-induced increase of melatonin production (149).
The precise mechanism for this control has as yet not been elucidated.
Another circadian rhythm implicated in sleep induction involves the nucleo-
side adenosine. Serum and intracellular levels of adenosine rise in association with
the onset of sleepiness and sleep deprivation causes even greater increases in ade-
nosine that are reversed when sleep occurs (150, 151). Adenosine accumulates
under conditions of energy depletion and as ATP is re-synthesized its levels
decrease. Adenosine therefore appears to serve as an indicator of energy status, pro-
moting sleep when ATP levels are low and disfavoring sleep when ATP levels are
restored. These fluctuations occur initially within cells, where ATP is utilized and
re-synthesized, although adenosine can exit the cell, allowing it to act on adenosine
receptors located on the cell surface. This receptor mechanism has been proposed to
mediate the soporific (sleep-inducing) action of adenosine (152). However, discov-
ery of D4 receptor-mediated PLM and its reliance on SAH hydrolase affords a new
perspective on the role of adenosine.
An overview of the PLM cycle (Figure 9.2) shows that it is designed to be sen-
sitive to cellular energy status. Thus ATP is necessary for the MAT-catalyzed adeno-

122
sylation of methionine 313 and any significant decrease in ATP could hinder the
rate of PLM and limit the effectiveness of dopamine in stimulating the cycle. Fur-
thermore, Step 3 in the cycle, catalyzed by SAH hydrolase, is reversible with the net
direction being dictated by the prevailing concentrations of adenosine, homocyste-
ine and SAil. When adenosine levels rise, such as occurs at the onset of sleep, this
reaction favors SAH synthesis, effectively stopping the PLM cycle. The level of ade-
nosine is therefore poised to modulate the D4 receptor cycle. At higher levels of
adenosine the D4 receptor would accumulate in its D4 form, reducing the number
of receptors actively participating in the cycle. The ability of dopamine to augment
PLM would be progressively reduced with a concomitant decrease in the capacity
for attention.
Under normal conditions the concentration of adenosine is kept low by the
combined actions of two enzymes, adenosine kinase and adenosine deaminase (Fig.
9.4), thereby promoting D4 receptor cycle efficiency. In neurons adenosine kinase is
the more important determinant of adenosine levels (153), although the reverse is
true for other cell types. It uses ATP to convert adenosine to AMP, such that a
decrease in ATP will directly lead to an increase in adenosine. Conversely, the resto-
ration of ATP levels after a period of sleep or rest will lower adenosine levels,
increasing the effectiveness of dopamine in providing PLM-based attention.

Figure 9.4: The ratio of ATP to adenosine can regulate D4 receptor-mediated PLM.
ATP promotes Step 1 while high levels of adenosine inhibit Step 3. Adenosine levels
are determined by enzymes that contribute to its formation and to its removal.
Nothing is more central to cellular metabolism than ATP. Nature has
designed D4 receptor-mediated PLM to be sensitive to ATP status in a simple but
elegant manner. By using the ATP/adenosine ratio to control sleep/wake cycles this

123
mechanism assures restoration and renewal of energy-dependent functions, includ-
ing our capacity for attention.
There are a number of other circadian rhythm mechanisms. At least eight dif-
ferent genes have been identified that affect the internal clock mechanism (e.g.
Clock, Fer, Tim and Cry). These factors cooperate to cause rhythmic fluctuations in
their own synthesis in a light-dependent manner. Interestingly, cryptochrome pro-
teins (the products of Cry genes) use methenyltetrahydrofolate as an essential cofac-
tor. The supra-chiasmatic nucleus (SCN) region is thought to be the master
circadian location, setting the cycle for other parts of the brain and as a result con-
trolling the circadian rhythm of the whole organism. While non-SCN mechanisms
and SCN-based mechanisms can be separated from each other, their mutual depen-
dence on light/dark cycles normally keeps them functioning in a coordinated man-
ner.
Elevated adenosine and SAH levels can also exert an influence on gene tran-
scription via their effect on DNA methylation. Certain DNA bases (i.e. cytidine
located before guanine) are frequently methylated by DNA methyltransferase
enzymes that utilize SAM as the methyl donor and methylation generally turns off
transcription of the methylated gene. Sail interferes with DNA methylation by com-
peting with SAM, thereby regulating transcription. An increase of adenosine can
therefore affect transcription via the intermediate role of SAH. An adenosine-based
circadian rhythm might therefore involve SAH-mediated activation of one or more
genes (by reducing their methylation) whose expression leads to the lowering of
adenosine levels. Adenosine kinase and adenosine deaminase are candidates for the
latter activity and studies in rat brain found that both enzymes (as well as SAH
hydrolase) show higher activity during dark periods than during the light in these
nocturnal animals (154). Together these observations provide strong evidence for
an adenosine-based circadian rhythm involving SAH-modulated gene transcrip-
tion.
Meditation
Meditation is a general term applied to periods of wakeful rest for both the
body and the brain. We recognize the many forms of meditation, ranging from the
religious mediations of Buddhist monks, through yogi-led transcendental medita-
tion to the “Relaxation Response” promoted by Dr. Herbert Benson (155). While
they are all undertaken with the goal of self-betterment in mind the specific goal
may be very different for each form of meditation, perhaps spiritual fulfillment,
peaceful inner awareness or lowering of stress and blood pressure. Rather than
strictly representing a rest (i.e. the absence of activity) meditation is meant to
achieve an “altered consciousness” that is key to reaching the desired benefits. Some
might argue that these altered mental states can also be reached during strenuous
physical activity (e.g. the Shaker religion or the “runner’s high”). The state of hyp-
nosis is not very far from a meditative state.

124
 The common element of meditation is a wakeful separation from external
sensory stimuli, allowing internally derived representations to become dominant.
Clearly this state is distinct from sleep and, when effectively achieved, it is clearly
different from the eyes wide-open state of normal wakefulness. Repetitive activity of
some sort (e.g. chanting aloud, silently repeating a mantra, running or watching a
swinging pocket watch) is a useful vehicle for accessing the meditative state. The
question is: What happens next? From a meditator’s perspective the answer might
be: “Who cares, as long as I get there.” This quote can be attributed to my good
friend Dr. Jeremy Fields, a practitioner of transcendental meditation or TM. Okay
Jeremy, but since we are attempting to analyze attention and cognition-associated
molecular events, it might be worthwhile to apply our concepts to the altered con-
sciousness of meditation.
During our earlier examination of the role of D4Rs I invoked the concept that
attention is associated with increased amplitude synchronized gamma frequency
firing of cortical neurons and that dopamine increases synchrony by modulating an
excitatory synapse in a loop circuit. In this model, attention is a prioritization
applied to an ongoing sensory experience, such as viewing a specific object within a
scene. What happens when we take away this external sensory experience by closing
our eyes? There is a residual visual image for about a second or two before blackness
sets in. But almost immediately the importance of auditory information is enhanced
as attention shifts to the next available sensory modality. Where does attention shift
when it is perfectly quiet and your eyes are closed and you are not moving? While I
obviously can’t speak for everyone, for me internally derived “thoughts” start to
take over the center stage of attention. I become more aware and conscious of my
immediate existence. My thoughts turn to: Where am I located? What am I doing?
What am I feeling? I am now more attentive to self. This is, of course, no surprise.
We are more self-attentive when we are not busy attending to external sensory
experience. Meditation allows us to access self by reducing our prioritization of the
external world. To explore our molecular interest, however, we have to ask: Is the
attention to self that is experienced during meditation achieved via a similar mecha-
nism as attention to externally visualized objects? If not, end of story. However, if it
is, this leads to a further series of questions: Does it involve synchronized gamma
frequency oscillations? If they are not triggered by primary sensory experience,
what are they triggered by? If gamma frequency oscillations are not involved does it
reflect synchronization at another resonance frequency (e.g. delta, beta or alpha)? Is
dopamine involved? Are ppG interneurons involved?
An early French study reported that meditation during yoga was associated
with an initial increase of alpha frequency brain waves followed by faster beta and
gamma oscillations during deeper meditation (156). Subsequent work by R. Keith
Wallace and others confirmed the increase in alpha and theta waves during medita-
tion and suggested that EEG coherence (i.e. synchronized neuronal firing) was
related to consciousness (157,158). Another study found increased activation of

125
specific cortical regions during meditation, particularly in areas that participate in
imagery tasks (posterior sensory and associative cortices) (159). Notably, the VI
region that is normally involved in visual image formation was not activated.
Activity in a number of other cortical regions was reduced, particularly those
involved in sensory attention (e.g. frontal cortex and thalamus), The latter areas are
those that receive dopaminergic innervation from the VTA (Ventral Tegmental
Area), suggesting that meditation is associated with a decrease in dopamine-depen-
dent attention activity.
The intrinsic frequency of synchronized oscillations in a particular neuronal
loop circuit is determined by the firing properties of the pacemaker cells for that cir-
cuit. Thus ppG interneurons have been implicated in triggering gamma oscillations
(3 0-60 Hz) because of their high intrinsic firing rate, which can support the com-
paratively high frequency of gamma oscillations. In an analogous manner, alpha
frequency oscillations may therefore involve a different type of interneuron that is
characterized by a slower intrinsic firing rate (Figure 9.5). Within the same brain
region different interneurons can overlap in their patterns of synaptic connection,
allowing pyramidal neurons to participate in gamma and/or theta frequency oscilla-
tion. During attention gamma oscillations in a particular region could predominate
due to dopamine-induced recruitment. Elimination of gamma oscillations during
meditation would then allow slower frequencies to become more prominent.
We might then predict that the most important aspect of meditation is the
absence of dopamine-stimulated gamma oscillations, allowing the emergence of
alternative, lower frequency patterns of synchronized neuronal firing. Altered states
of consciousness experienced during meditation may therefore be akin to the inner
layers of an onion. These other states are always there but are only perceived after
the more dominant outermost layer is removed. In the case of brain wave synchro-
nization the potential for alpha frequency activity is always present, even during
active attentiveness. Accordingly, our normal conscious experience is a dynamic
mixture of synchronized information, reflecting the sum of a multitude of circuits,
firing at different resonance frequencies, competing with each other for short-term
dominance. Active dopamine-dependent attention allows the 40 Hz gamma fre-
quency information from ppG interneurons to predominate.

126
Figure 9.5: Different interneurons and neurotransmitters can yield different rates of
synchronized oscillations.
If dopaminergic systems are suppressed during meditation what is the mech-
anism that provides for synchronization at non-gamma frequencies? Any neu-
rotransmitter that modulates the firing rate of interneurons in recurrent local
circuits could serve the same role as dopamine does for ppG interneurons. Its mech-
anism of action need not (and probably is not) be PLM. For example, instead of
inhibiting NMDA receptor responsiveness a neurotransmitter might reduce the
release of glutamate from pyramidal cells or perhaps reduce the release of GABA
from interneurons. Either of these actions would cause disinhibition of pyramidal
cell firing and be capable of promoting synchronized activity. Because they express
a high density of D4Rs ppG interneurons are especially adapted to respond to dopa-
mine and to utilize PLM as a signaling mechanism. Other types of GABAergic inter-
neurons may have the specialized capacity to respond to serotonin, acetylcholine
etc. Indeed acetylcholine is well recognized for its ability to induce synchronized
neuronal firing in the hippocampus (160). While dopamine-releasing nerves from
the VTA subserve the process of sensory attention, nerves releasing these other
putative neurotransmitters may arise from brain regions associated with long-term
memory, emotional status, internal well-being, etc. Attending to something per-
ceived as dangerous can therefore result in a combination of converging informa-
tion that includes both sensory and subjective components.
A repetitive experience (e.g. a repeated sound or an internally repeated man-
tra) can assist in achieving different meditative states. This effect may result from
gradual desensitization of attention to a particular modality. The intensity of sen-
sory attention normally diminishes quite rapidly. For example, if we attempt to
stare continuously at an object the initial level of attention is difficult to sustain.

127
This feature is helpful for freeing our attention to move onto something else. Inter-
nal repetition of a mantra in a serene environment that is already low in external
stimuli facilitates the emergence of synchronized information from circuits that are
normally less represented. The act of breathing is a potential external distraction but
meditative methods that utilize a focus on the breath capitalize on its rhythmic
quality while diminishing its sensory importance.
Neuronal synchrony has only recently been appreciated as a potentially
important aspect of brain function. Advancing technology will allow exploration
of whether information flows from region to region in the form of electrical fields
rather than only in the form of synaptic transfer. Application of oscillatory fields to
the skull surface have recently been reported to improve reaction times and the
speed for certain calculations (161), giving new meaning to the phrase “put on your
thinking cap”. While this neuronal network phenomenon has at least some ear-
marks of consciousness we’ll have to wait for crucial experiments that can test such
a remarkable possibility.
Chapter Summary:
• The capacity for attention is linked to metabolism.
• Single-carbon folates support several core metabolic
• pathways including de novo purine synthesis, thymidine synthesis and methyl transfer
reactions such as PLM.
• Changes in growth status and development can affect the availability of methyl groups for
PLM.
• The brain is more dependent on the folate pathway for methyl groups than other tissues
such as the liver.
• A number of vitamins are critical for dopamine-stimulated PLM and vitamin deficiencies
result in neuropsychiatric symptoms.
• Circadian fluctuations in adenosine levels are associated with an altered capacity for
attention.
• Meditative techniques facilitate access to different states of consciousness in part by
eliminating sensory-based attention.
• States of consciousness may be associated with different patterns of synchronized
neuronal oscillations.

128
10 THE EFFECT OF DRUGS ON ATTENTION

It can be reasonably argued that one of the very first uses of drugs by man was
for the purpose of modulating attention. The intoxicating properties of alcohol
from fermented fruit were not lost on our primitive fore bearers and the systematic
production of beer is clearly described in writings from the tombs of ancient Egypt.
Not far behind are nicotine and caffeine, which, together with alcohol, form the
cornerstone of legitimate commerce in the modulation of attention that is no less
evident in our daily lives today than it was centuries ago. If we include cocaine, the
derivatives of opium (heroin, morphine etc.), marijuana and other illicit drugs we
begin to grasp the enormity of the connection between human social development
and the use or abuse of attention-altering drugs. In this chapter we will examine
how these agents impact the molecular mechanism of attention.
Given the central role of dopamine in providing for attention, it is no surprise
that any drug that increases or decreases its concentration will affect attention.
Indeed the primary strategy for treating a deficit in attention (i.e. ADHD) is to
increase dopamine levels with amphetamine-like drugs. Increasing the level of
dopamine can lead to elevated mood, increased awareness and heightened motor
activity. When produced at a moderate level these effects are generally desirable but
in excess these same effects turn nasty and become mania, anxiety and hyper-excit-
ability. Thus it is important to recognize that differences in the intrinsic potency
and time course of drug action make a world of difference in the type of effects they
produce and consequently in their social acceptance.
In many cases the mode of administration is a key factor in determining the
time course and intensity of drug action. For example, in its pure chemical form
nicotine makes an effective rat poison. However, when diluted in the dosage form
we know as cigarettes the amount of drug and the rate of administration are limited
and as a result the effect is greatly moderated. After all, how many cigarettes can you
smoke per minute? How many cups of coffee can you drink? Contrast these socially
acceptable dosage forms with the intravenous injection of the same drugs and both
their pharmacological and social profiles are vastly changed.
Another aspect to recognize when comparing drug effects is the potential for
multiple actions. Even though a number of drugs share the ability to increase dopa-
mine levels, each of them also has the potential for additional effects on other neu-
ronal systems that contribute to their overall effect. For example, the increase in
heart rate caused by caffeine and nicotine is not directly related to their ability to
increase dopamine, but is nonetheless part of their total effect. Such additional
actions may or may not be desirable. Because of dopamine’s involvement in reward
behaviors and addiction as well as attention, it is often difficult to distinguish which
factors motivate a drug’s continued use. Is it the desire to replicate the psychic

129
effects (positive reinforcement) or the desire to avoid an unpleasant abstinence syn-
drome (negative reinforcement)?
When dopamine-elevating drugs are administered systemically they non-
specifically increase its levels everywhere. Contrast this broad action with the mech-
anism of attention in which dopamine is selectively released in different regions of
the cortex. A generalized increase serves to create a state of heightened awareness,
shifting the starting point for dopamine-induced attention to a new, higher position
in its dose-response relationship. Such a generalized increase in the utilization of
methyl groups and ATP is needed to support the mechanism of PLM as well as
other dopamine receptor-mediated responses. When the drug effect wears off it can
leave behind a period of metabolic deficit during which the responsiveness of the
attention mechanism falls below the normal level as the system works to restore its
capabilities. High levels of dopamine also activate negative feedback pathways that
can remain active during the period of drug withdrawal. Together these events con-
tribute to the syndromes that accompany the acute and chronic use of attention-
altering drugs.
Drugs that artificially increase dopamine levels also stimulate learning, just
like dopamine does during attention-related learning. Frequently what gets
“learned” is the actual ritual or procedure of drug use or drug abuse. Such learning
is intimately related to the dopamine-mediated reward mechanism. For example,
learning to smoke a cigarette is facilitated because of the release of dopamine that
occurs during that process. Creation of new synapses or strengthening of existing
synapses in response to dopamine can lead to hard-wiring of the behavior, particu-
larly with frequent repetition. In this instance reinforcement of the drug-taking
behavior per se is not the intended goal, but once dopamine increases in association
with such an experience the molecular mechanisms of attention-related learning
record the episode as faithfully as a videocam recorder. It is subsequently available
for memory-based “replay” with the attendant potential for stimulating repeated
drug and abuse.
Caffeine
Whether in the dosage form of a cup of coffee or a canned cola drink, caffeine
is the all-purpose pick-me-up. We may enjoy the aroma of a freshly-brewed cafe
latte or a cup of joe with or without sugar or cream, but underneath it all the value
of a cup of coffee lies in its ability to recharge your psychic batteries and brace you
for the day (or night) ahead. Flavor and brand-name appeal are merely layered
upon the basic pharmacological reality that we (at least many of us) enjoy the stim-
ulant effects derived from the approximately 65 to 175 mg of caffeine we get from
each serving of coffee. Cola drinks provide 40-50 mg per 12 oz can. We use it to
reduce drowsiness and fatigue and to improve the speed and clarity of thought. The
average per capita intake of caffeine is about 200 mg/day in the U.S.

130
 Caffeine has several different actions. Due to its structural similarity to ade-
nosine it can block adenosine receptors and the resultant decrease in the effective-
ness of adenosine is responsible for most of its effects. For example, stimulation of
Al -type adenosine receptors on nerve endings normally causes a decrease in the
release of neurotransmitters including dopamine (162) and norepinephrine (163).
When caffeine occupies the receptor instead, this inhibitory action is blunted, lead-
ing to their increased release. It is not surprising, therefore, that a number of the
behavioral responses to caffeine can be antagonized by dopamine receptor blocking
drugs (164). In addition to blocking adenosine receptors caffeine also inhibits the
enzyme phosphodiesterase (PDE) that breaks down the intracellular second mes-
senger cyclic AMP (cyclic adenosine monophosphate). PDE inhibition raises cAMP
levels in nerve endings, which also contributes to the increased release of dopamine.
The effects of caffeine on the cardiovascular system (increased heart rate and blood
pressure and diuresis) are due to these same mechanisms.
Caffeine has a plasma half-life of 3-7 hrs so the effects of several cups of coffee
or cans of soda can easily last for 6-10 hrs. Interestingly, its duration can be consid-
erably increased during pregnancy due to slowed metabolism. Because it can easily
cross cell membranes and the “blood-brain barrier”, caffeine is rapidly absorbed
and the CNS stimulatory effects of caffeine are prompt, being detectable within 5-10
minutes. There is clear evidence for the development of both tolerance (reduced
effectiveness with repeated use) and dependence (the occurrence of adverse symp-
toms upon discontinuation) when caffeine is used regularly for prolonged periods
of time. While caffeine withdrawal symptoms (e.g. somnolence, irritability, and
headache) are milder than those for cocaine, they both reflect the consequence of
receiving a subnormal level of dopamine stimulation.
Cocaine
Natives of the Andean region of South America recognized the ability of
chewed leaves from the coca shrub to provide psychic stimulation and euphoria
many centuries ago. It remains an integral element of their culture where it is used
to increase the stamina and productivity of field workers. The active ingredient,
cocaine, has now become an unwelcome but integral element of the U.S. culture. Its
history of use and abuse intersects with cultural icons such as Sigmund Freud, who
used and studied its actions, and Coca-cola®, which included coca extract in its orig-
inal formulations.
Cocaine is a powerful agent for increasing the concentration of dopamine in
the brain. It has a very rapid effect, particularly when the free base (crack) form is
smoked, due to its rapid transfer across the lungs into the blood and equally rapid
movement from the blood to the brain. Its primary action is to bind to and block the
dopamine transport (DAT) system that returns dopamine to nerve endings after its
release (165). Inhibition of DAT activity causes an accumulation of dopamine
across the whole brain wherever it is released. Cocaine can also inhibit re-uptake of
norepinephrine and serotonin by their selective transporters but to a lesser extent

131
than dopamine. The increased levels of dopamine are associated with arousal,
increased awareness and vigilance and an increased sense of self-confidence and
well being, that can reach a state of euphoria. Improvements in attention last longer
than the feelings of euphoria.
The cocaine “high” is brief, giving way to a desire for more drug only 10-30
minutes after the previous use (166). Addiction to cocaine use is, of course, all too
common. Because it blocks the re-uptake of dopamine, frequent repeated use of
cocaine depletes nerve endings of their usual supply and the amount of dopamine
available for release drops below the normal level. In this case the desire for re-
administration is driven not only by the interest in experiencing another “high” but
also by an interest in avoiding the negative feelings that are inescapably associated
with depleted dopamine stores. Withdrawal symptoms include dysphoria, depres-
sion, sleepiness and fatigue, just the opposite of the effects experienced when the
drug is first administered. However, when cocaine is administered at longer inter-
vals (e.g. once a day) the response to each dose becomes measurably greater reflect-
ing “sensitization” (167).
Because they bind so tightly and specifically to the dopamine transporter,
analogues of cocaine are used in PET (positron-emission tomography) studies to
quantitate the density of DATs. In Parkinson’s disease, for example, their number is
greatly decreased due to the death of dopamine-releasing neurons (168). It is of spe-
cial interest that the density of DATs is reportedly increased in persons with ADHD
(169). A higher DAT density would result in the more rapid uptake of dopamine,
weakening dopamine signal intensity.
Amphetamines
Drugs known collectively as amphetamines are structural analogues of dopa-
mine lacking the ability to activate receptors. Similar to cocaine they act on nerve
endings to increase dopaminergic stimulation but their mechanism is, however,
somewhat different. Instead of blocking DAT activity amphetamines inhibit the
storage of dopamine within the nerve terminal where it is normally kept within
small membrane sacs called vesicles. After amphetamines release stored dopamine
from these vesicles the dopamine is transported out of the nerve by reversed activity
of the DAT system (170). The net effect is an increased level of dopamine in the syn-
apse and, depending upon the dose and dose interval, a depletion of stored dopa-
mine in the nerve ending.
The D-isomer of amphetamine (dextroamphetamine) causes effects similar
to cocaine (and caffeine) including increased awareness, decreased fatigue, eleva-
tion of mood and increased self-confidence. “Speed” or methamphetamine is aptly
named for the increased level of activity (speech and motor) and the apparently
increased rate of mental processing it produces, but the latter is often accompanied
by an increased error rate. Repeated use of amphetamines leads to a gradual
decrease in its effectiveness (called tachyphylaxis) as the supply of dopamine avail-

132
able for release falls. This occurs because the rate of synthesis cannot keep pace with
the rate at which dopamine is being lost from the nerve terminal. Drug abusers typ-
ically escalate the dose in an attempt to overcome tachyphylaxis. Stopping after the
use of higher doses is inevitably associated with depression and fatigue (i.e. crash-
ing), as the level of dopamine release falls below the normal level, due to its deple-
tion. When used to postpone sleep, amphetamine stimulation eventually gives way
to a prolonged period of sleep to make up for the deficit.
Amphetamines are used clinically for the treatment of ADHD and narco-
lepsy, as well as for appetite control. In addition to d-amphetamine (Dexidrine®) the
amphetamine family also includes methamphetamine (Desoxyn®), methylphenidate
(Ritalin®) and pemoline (Cylert®). Their role in treating ADFID is further discussed
in Chapter 11.
Nicotine
Smoking a cigarette delivers both nicotine and other pharmacologically
active materials that act together to cause its stimulatory effects in the cortex and
elsewhere in the brain. In addition nicotine has a number of effects outside the cen-
tral nervous system, particularly on the cardiovascular system. Suspended in the
vapor phase of smoke, nicotine’s transfer from the lungs to the brain is so rapid that
detectable increases in its brain level are produced between each successive puff (i.e.
within 10 sec). Smoking produces an elevation of mood, an increased level of atten-
tion, and an increased rate of cognitive processing. It increases both the rate and
force of heart contractions as well as increasing vascular constriction, leading to
increased blood pressure.
Nicotine itself acts by potently stimulating ion channel-type receptors bear-
ing its name (nicotinic-type cholinergic receptors) and acetylcholine is the neu-
rotransmitter for these receptors. When activated, they allow sodium and/or
calcium ions to enter the neuron, leading to either an increase in firing rate or, if
they are located on nerve terminals, an increase in the release of neurotransmitter.
In the brain, a prominent action of nicotine is to stimulate the release of dopamine
by virtue of stimulating nicotinic receptors on dopaminergic nerve endings (171).
This occurs not only in attention areas such as the cortex, but also in reward centers
(nucleus accumbens) and in movement centers (corpus striatum). There appears to
be a particular association of nicotinic receptors with dopaminergic neurons.
Administration of nicotine has been demonstrated to increase dopamine levels in
the frontal cortex reflecting activation of the same VTA-derived dopaminergic neu-
rons that trigger attention (172). It is no wonder, therefore, that nicotine augments
attention. There is evidence that a particular type of nicotinic receptor, the alpha-7
subtype, is involved in activation of VTA neurons via a nicotine-triggered increase
of glutamate release and stimulation of NMDA receptors (173). The alpha-7 nico-
tinic receptor has been linked to schizophrenia (174) and the incidence of smoking
is very high among people with schizophrenia.

133
 Cigarette smoke contains a complex mixture of pharmacologically active sub-
stances, of which nicotine is only one. Pertinent to the mechanism of attention is
the presence of substances that reduce the rate of dopamine metabolism by inhibit-
ing the enzyme monoamine oxidase (MAO). There are two major forms of this
enzyme, MAO A and MAO B, and cigarette smoking has been shown to produce
significant inhibition of both forms, including measurements in the brains of smok-
ers vs. non-smokers (175,176). Together with the ability of nicotine to stimulate the
release of dopamine it appears that cigarette smoking produces a sort of “double
dopamine whammy”, not only releasing dopamine but also extending its duration
of action. Inhibition of MAO can also increase the levels of other biogenic amine
neurotransmitters including norepinephrine and serotonin.
Opiates
Morphine, heroin and other opium poppy-derived opiates cause a dose-
dependent dulling of attention, mental clouding and drowsiness, along with a
depressant action on many other functions including respiratory, cardiovascular
and endocrine systems. Their therapeutically important ability to relieve pain is
commonly, but not always, associated with a feeling of euphoria. Interactions
between opiates and dopamine neurotransmission are thought to mediate addictive
behavior associated with their prolonged use.
Opiates act by stimulating specific G protein-coupled receptors (mu, delta
and kappa types) that are normally targets for endogenous opiod peptides such as
endorphins, enkephalins and dynorphins. Release of endogenous opiods is linked to
stressful physiological conditions and appears to be part of an adaptive response
mechanism that includes an increased threshold for pain. Mu and delta receptors
are responsible for the analgesic actions of morphine. When stimulated, opiate
receptors act to decrease neurotransmitter release, accounting for the inhibitory
nature of opiate effects. Analgesia results from inhibition of neurotransmitter
release at spinal neurons that carry pain information toward the brain and also at
neurons arising from the midbrain that set peripheral pain thresholds. At higher
doses than needed for analgesia, morphine and other opiates interfere with atten-
tion and cognition, by affecting the release of dopamine and/or GABA within inter-
neuronal circuits that subserve attention (177,178).
Addiction to opiates, as well as to other drugs, has been linked to their effects
on the nucleus accumbens, part of the reward circuitry in the brain. The nucleus
accumbens receives extensive dopaminergic input from the VTA and has a high
density of dopamine receptors as well as mu and delta receptors. Activation of these
opiate receptors increases the release of dopamine (179) and is thought to be critical
for the development of the motivational effects of opiate use. Attention networks,
including the VTA and frontal cortex neurons, play an important role in the devel-
opment of addiction behaviors. The nucleus accumbens receives input from the
frontal cortex, which is activated during attention (180). During the development of
addiction, repetitive attention to drug effects leads to strong associations and

134
“learning” of abuse-related behaviors. Agents like opiate that increase dopamine
levels in the nucleus accumbens carry a high risk for developing addiction.

Nucleus Accumbens visible in RED

Medial surface, person facing to the left.

Nucleus accumbens is very roughly in the area labeled 34.

135
Marijuana
Smoking marijuana (cannabis) distorts attention and alters awareness. It
modulates sensory experience and can foster a feeling of self-enlightenment. These
actions are mediated, at least in part, by the active ingredient A9-tetrahydrocannab-
inol or THC. A cannabinoid-binding receptor protein that selectively recognizes
THC has been identified (181) followed by the discovery of naturally occurring can-
nabinoid substances that can activate the receptor (182). These endogenous canna-
binoids, such as anandamide, are synthesized from phosphatidylethanolamine (PE),
the same phospholipid methylated by dopamine D4 receptors. Furthermore, dopa-
mine causes a large increase in the release of anandamide (183) and blockade of
anandamide uptake inhibits dopamine responses (184). These factors suggest that
endogenous cannabinoids may be an integral component of the dopamine based
mechanism of attention.
The structure of anandamide and its simple two-step biosynthetic pathway
are illustrated in Fig. 10.1. The first reaction involves attachment of arachidonic
acid (a 20-carbon fatty acid with four double bonds) to the amino group of PE,
meaning that D4R-mediated PLM and anandamide synthesis reactions compete for
PE as a substrate. In the second step N-arachidonoylPE is cleaved by phospholipase
D to yield N-arachidonylethanolamide (anandamide). There are other naturally
occurring cannabinoid substances including, for example, 2-arachidonylglycerol. In
addition, the omega-3 fatty acid DHA can substitute for arachidonic acid to form an
anandamide-like compound containing six additional carbons (185).
Unlike standard neurotransmitters, anandamide is not fully pre- synthesized
and stored for future release. Rather, its precursor N arachidonylPE is present in the
cell membrane and anandamide formation is triggered by the activation of phos-
pholipase D. In freely moving rats, stimulation of dopamine receptors with the ago-
nist quinpirole increased anandamide release by eight-fold, reflecting the ability of
D4-like receptors to activate phospholipase D (186). Under normal conditions,
therefore, effects of anandamide occur after the initial actions of dopamine.
Once they are formed by phospholipase D activity inside the cell, endogenous
cannabinoids move freely across the membrane and act on G protein-coupled
receptors outside the cell or diffuse away to act on nearby cells. Activation of canna-
binoid receptors leads to reduced neurotransmitter release (e.g. glutamate or dopa-
mine) (187,188). A specific uptake system transports cannabinoids back into nerve
terminals, in a manner analogous to the dopamine transporter, and agents that

136
block this transport produce effects similar to anandamide. Inside the cell anan-
damide is hydrolyzed to inactive products.

Figure 10.1: Biosynthesis of the endogenous cannabinoid Anandamide from arachidonic acid and PE.
Activity of phospholipase D is rate limiting and can be increased by activation of D4-like dopamine
receptors. Note that phospholipid methylation (PLM) and Anandamide synthesis pathways compete
for PE.

Anandamide causes catalepsy, a response commonly associated with inhibi-

tion of D2-like receptors (189) and also interferes with motor responses to the D4/
D2 receptor agonist quinpirole (190). These actions contribute to the sense of iner-
tia commonly experienced when smoking marijuana, and serve to illustrate func-
tional antagonism of dopamine responses by endogenous cannabinoids. During
episodes of dopamine-induced attention, the local production of anandamide can
lead to inhibition of D2 and/or D4R-mediated responses. Exogenous cannabinoids
(e.g. marijuana) exaggerate this normal inhibitory component, producing a general-
ized dampening of attention and awareness. By interfering with dopaminergic
mechanisms, marijuana allows other neurotransmitter systems to become more
important, which can enhance the subjective quality of an experience such as plea-
surable tastes or smells. These effects are not hallucinations since they are merely
alterations of the normal sensory experience. However, when dopaminergic systems
are more extensively suppressed the dominant basis of conscious thought can move
from external sensory input to internal memory-based input, leading to hallucina-
tion-like experiences.

137
Alcohol
Alcohol (i.e. ethanol), produced by the fermentation of carbohydrate-rich
foods, creates profound and complex effects on mood and attention. Its use for this
purpose is an ancient and time-honored tradition. Whether in the form of a glass of
cask-mellowed burgundy or a pint of well-crafted lager beer, the consumption of
alcohol has a respected place in our world. In the form of alcoholism, however, its
use is a scourge of vast proportions, affecting 5-10% of American men and 3-5% of
American women. Alcohol is addicting and causes important damage to the liver,
heart, brain and other organs. The difference between these two perspectives on
alcohol use is intertwined with the frequency of drinking episodes as well as the
context of its use. Alcohol produces profound changes in our capacity for attention.
While normally considered a depressant drug (causing impaired attention
and slower reflexes), lower concentrations of ethanol can cause signs of CNS excita-
tion (elevated mood, increased talkativeness). These socially desirable effects are
typically part of the initial response to drinking, as the alcohol concentration just
starts to rise and are generally attributed to a depressant effect on inhibitory mecha-
nisms that leads to excitation. Persons who experience this mild euphoria in a pro-
nounced manner are more likely to develop alcohol addiction while those who
respond only with a depressive reaction are not. With increasing time and increas-
ing dosage, the effects of ethanol become uniformly depressant in nature. Attention
is first impaired and then lost completely as stupor and delirium take hold, eventu-
ally giving way to loss of consciousness.
The duration and intensity of ethanol’s effects are greatly influenced by its
rate of metabolism. Alcohol is first converted to acetaldehyde and then to acetic acid
by the sequential actions of alcohol dehydrogenase and aldehyde dehydrogenase
enzymes. Metabolism starts in the stomach, but the liver is the major site of ethanol
metabolism. Humans express a number of alcohol dehydrogenases that differ in
their specificity for ethanol and their efficiency in converting it to acetaldehyde.
Genetic variants in alcohol dehydrogenase and aldehyde dehydrogenase are respon-
sible for individually different rates of alcohol metabolism (191). Most notably,
about 50% of Asians have a less active form of acetaldehyde dehydrogenase that
causes them to accumulate high levels of acetaldehyde, resulting in uncomfortable
effects such as flushing, headache and hypotension. Presence of this genetic factor
tends to reduce the incidence of chronic alcoholism (192).
Ethanol is soluble in both aqueous and lipid environments. When dissolved
in cell membranes it increases membrane fluidity, contributing to many of its
actions. The most prominent and potent effects of alcohol are those produced on
membrane proteins such as ion channel receptors. Different lines of research have
suggested that the small ethanol molecule could either fit into a small cavity in the
receptor protein itself or could affect the conformational changes membrane pro-
teins undergo as they change between active and inactive states (193,194). The latter
effect may occur at the interface where membrane proteins make contact with sur-

138
rounding phospholipids. Ethanol inhibits excitatory NMDA and nicotinic receptors
(40,41) while activating inhibitory GABA and glycine receptors (39) and activating
certain potassium channels (98,99). Together these effects account for its general-
ized depressant activity.
Since D4R-mediated solid-state signaling utilizes membrane fluidity as a
means of producing attention we undertook studies to investigate the effect of etha-
nol on D4R-mediated PLM using [14C]formate to label the single-carbon folate
pool. We found that 0.1% ethanol rapidly inhibited basal PLM to less than half of its
normal level. The concentration producing half-maximal inhibition was approxi-
mately 0.025 % (Fig. 10.2), making folate dependent PLM among the most ethanol-
sensitive processes. [A blood alcohol level of 0.08 to 0.1% is legally drunk.] For
someone weighing 180 lbs., it takes only 1-2 drinks to achieve a blood alcohol level
of 0.025%. Remarkably, the effect of ethanol was completely specific for folate
dependent PLM and no significant effect was observed when PLM was measured by
the more classical method of labeling the methyl group of methionine, even at 0.5%
ethanol. This suggested that ethanol specifically affects the folate-dependent, methi-
onine synthase-mediated re-methylation of homocysteine..

Figure 10.2: Effect of Ethanol on folate-dependent PLM in cultured human

neuroblastoma cells. The concentration of ethanol required for half-maximal
inhibition was approximately 0.025%
Earlier studies found that ethanol reduced methionine synthase activity in
both rats (195,196) and pigs (197). Investigators at the University of Nebraska
found that ethanol administration to rats caused a 50% decrease in hepatic methio-

139
nine synthase activity (195). This decrease was accompanied by a reciprocal
increase in the activity of betaine-homocysteine methyltransferase (BHMT), the
alternative system for re-methylating homocysteine that does not rely on the folate
pathway for methyl groups. Increased BHMT activity was therefore able to com-
pensate for an ethanol-induced reduction in folate-dependent methylation, at least
in the liver. However, since the brain does not express BHMT it cannot compensate
by increasing its activity. As a consequence, brain function will be more vulnerable
to the inhibitory effect of ethanol on methionine synthase than liver. Inhibition of
folate-dependent methylation by ethanol leads to the accumulation of homocyste-
ine (198,199). One study from the Tufts University/U.S.D.A. Nutrition Research
Center in Boston found that prolonged administration of ethanol caused a signifi-
cant increase of over 40% in the serum level of homocysteine (198). Another study
reported a similar increase in the level of 5-methylTHF (200). All these effects are
indicative of impaired methionine synthase activity.
Further investigation of the mechanism underlying ethanol’s inhibition of
folate-dependent PLM led to our discovery that its action results from interference
with a signaling pathway stimulated by the insulin-like growth factor-1 (IGF-1)
receptor (202). As its name implies, IGF-1 (earlier known as somatomedin-C) is a
powerful regulator of cellular growth and survival. The liver is the main source of
IGF-1 in the bloodstream and growth hormone (somatotropin, secreted by the pitu-
itary gland) is responsible for stimulating its synthesis and release. However, IGF-1
is also locally produced in the brain where it contributes to the growth, survival and
function of neurons (203). As illustrated in Figure 10.3, IGF-1 binding causes auto-
phosphorylation of the IGF-1 receptor allowing the binding of insulin response
substrate-i (IRS-i) or IRS-2, which in turn binds phosphatidylinositol 3-kinase
(P13-kinase), an enzyme that phosphorylates phosphatidylinositol (PI) phospholip-
ids at their 3-position (204). This phosphorylation creates an anchoring site for pro-
tein kinases such as Akt/PKB and phosphoinositide-dependent protein kinase-1
(PDK1) to dock at the inner side of the membrane where they can be more active.
Akt/PKB is activated upon phosphorylation by PDK1, initiating several signaling
pathways, including one that leads to increased methionine synthase activity. The
latter increase is evident as an IGF-i-induced increase of folate dependent PLM.
Ethanol interferes with the ability of IGF-1 to activate P13-kinase (205,206).
As a consequence it interferes with P13-kinase-dependent activity of methionine
synthase. In collaborative studies, Dr. Ruma Banerjee at the University of Nebraska
directly measured the enzyme activity of methionine synthase and found that a 60
min incubation of human neuronal cells with 0.1% ethanol reduced activity to an
unmeasurably low level (202). In these same studies IGF-1 increased methionine
synthase activity about three-fold and ethanol pretreatment completely blocked the
increase. Dopamine can also stimulate methionine synthase activity via the P13-
kinase pathway, as a consequence of G protein activation. Ethanol blocked the effect
of dopamine as well. Although ethanol has many effects on many different systems,

140
it seems likely that its potent inhibition of methionine synthase makes a significant
contribution to the loss of the capacity for attention during alcohol ingestion.

Figure 10.3: IGF-1 signaling and its relationship to folate-dependent homocysteine

re-methylation. Binding of IGF-1 initiates autophosphorylation of the IGF-1
receptor, allowing docking of IRS-1. IRS-1 binds P13-kinase and facilitates its
phosphorylation of inositol containing phospholipids, creating a membrane binding
site for Akt/PKB and PDK1. Phosphorylation of Akt/PKB mediates the IGF-1-
induced increase in homocysteine methylation, leading to decreased SAH and
increased DNA methylation. Ethanol interferes with the P13-kinase pathway and as
a result inhibits methionine synthase activity.
Is methionine synthase activation important for the growth-regulating role of
IGF-1? The answer seems to be yes and the ability to affect DNA methylation seems
to be the mechanism. S-adenosylhomocysteine (SAH) is an inhibitor of DNA meth-
ylation and increased methylation of homocysteine by methionine synthase causes a
decrease in SAH levels, as illustrated in Figure 10.3. Thus IGF-1 (or dopamine) can
increase DNA methylation by stimulating methionine synthase activity. By inhibit-
ing P13-kinase signaling and methionine synthase, ethanol will decrease DNA
methylation. In collaboration with Dr. Joel Mason at the Tufts University/USDA
Center for Human Nutrition and Aging we evaluated the effect of ethanol on DNA
methylation. A four-day exposure to 0.1% ethanol caused a 50% reduction in the
extent of global DNA methylation (202). Gene expression is sensitive to DNA meth-

141
ylation. In general DNA winds more tightly around histone proteins when it is
methylated, decreasing gene expression. Both IGF-1 and ethanol can therefore reg-
ulate gene expression through their effects on methionine synthase. The importance
of DNA methylation in developmental disorders is discussed more fully in Chapter
13.
Although ethanol is used and abused for its effects on attention its concomi-
tant inhibition of IGF-1 receptor signaling and methionine synthase may be an
important source of toxicity, particularly during chronic use or when used during
pregnancy. IGF-1 is critical for normal fetal development of organs, as evidenced by
the 74% reduction in total body weight and 38% reduction in brain weight when the
gene for IGF-1 is knocked-out in mice (206). Since ethanol potently blocks IGF-1
response, fetal alcohol syndrome may reflect, at least in part, the consequences of
ethanol’s inhibition of methionine synthase activity and resultant impairment of
DNA methylation. As illustrated in Fig. 10.3, ethanol’s interference with IGF-1 sig-
naling can account for observed increases in homocysteine and SAH (198,199) as
well as decreased DNA methylation (207). Moreover, IGF-1 also promotes neuronal
survival by blocking programmed cell death (apoptosis) while ethanol interferes
with this protective action (208). Clearly the short-term attention-modulating
effects of ethanol can develop into genotoxic and neurotoxic consequences with
longer-term use and abuse.
Chapter Summary:
• Most commonly abused drugs directly or indirectly exert effects on attention.
• Using different molecular mechanisms, nicotine, caffeine, cocaine and amphetamines
increase the synaptic concentration of dopamine, leading to augmented attention.
• By inhibiting the enzyme phosphodiesterase and raising neuronal levels of cAMP, caffeine
increases the release rate of dopamine and other neurotransmitters.
• Cocaine inhibits the transporter protein that is responsible for lowering synaptic
dopamine levels, increasing the intensity and duration of receptor stimulation.
• Nicotine depolarizes dopaminergic nerve endings, increasing the release of dopamine,
while other materials in cigarette smoke inhibit dopamine metabolism.
• Morphine and other opiates inhibit the release of dopamine and other neurotransmitters,
thereby reducing attention.
• Cannabinoids selectively interfere with dopaminergic receptor responses and can distort
attention experiences. Ethanol enhances the activity of inhibitory GABAergic ion channel
receptors, and inhibits excitatory ion channel receptors such as NMDA receptors. In
addition ethanol potently and selectively inhibits folate-dependent homocysteine
methylation, but augments D4R-mediated PLM, which may contribute to its affects on
attention.
• Insulin-like growth factor-1 stimulates folate-dependent homocysteine methylation.
Ethanol’s interference with this action can result in impaired DNA methylation.

142
11 ATTENTION-DEFICIT HYPERACTIVITY DISORDER

As they sat listening to Mrs. Salzman, their son Jason’s third grade teacher,
Marcie and Kevin Bagnold were not caught completely off-guard. After all who
knew their son better than they did? Jason had been a handful from the time he first
began to crawl. He was almost constantly in motion, eagerly exploring the room for
all the adventurous possibilities it held. He was into everything. He would move
chairs to be able to climb on the kitchen table and then would scramble from the
table onto the countertop to get into the flour container. There would be a big wide
smile on his face when mom or dad finally discovered what he was up to. Child
prooflng their house had been a contest of wits between Jason and his parents. Of
course along with this mischievous behavior came a bright, happy, lovable toddler
who learned to talk at a very early age. Later he learned to count almost on his own
and was reading well before kindergarten. Sure there were issues about his behavior.
In first and second grade he had more than a few confrontations with his class-
mates, but Marcie and Kevin chalked this up to his being “all boy”. There could be
no doubt about that.
But this teacher’s conference was taking on a different tone than they had
anticipated. Mrs. Salzman’s comments about Jason’s reading, writing and math
skills centered around the same theme: “While he’s doing alright, he ‘could do so
much better if he would just stay on task. It seems as if he gets tired of the lesson
very quickly and then starts to get antsy. While the other kids are carefully working
through an exercise, Jason has already moved onto something else. Looking back at
his work, it is sloppy and there are many careless mistakes that are just a result of
having zipped through things too quickly. When Jason finishes early and starts to
fidget, he tends to bother his neighbors, trying to get them to join in. These are not
just isolated incidents. It has been an every day routine for the past several months.”
Mrs. Salzman then shifted position and her voice modulated toward a notice-
ably more serious tone. “This behavior is not just a problem for Jason, but for the
class as a whole. I have to spend far too much time managing just one student and
the others suffer as a result. In the eight years I’ve been teaching I’ve had a number
of children with these same issues. To me it’s a classic example of ADHD, attention-
deficit hyper-activity disorder. I wanted to let you know that I’m recommending
Jason be evaluated by the school psychologist as soon as possible. If he agrees with
me, and I’m relatively sure that he will, we would like you to have Jason seen by
your pediatrician or a behavioral specialist who can help him. In my experience kids
who are recognized early and started on drugs like Ritalin® do extremely well. Its
almost a miracle to see how quickly they change and how their schoolwork really
improves. While it’s a difficult choice I’m sure you’ll make the decision that is best
for Jason.”

143
 The message was starting to sink in. Marcie and Kevin were being asked to
medicate their son, not only to help him in his schoolwork, but also to help create a
more efficient learning environment for his whole third grade class. They had seen
all the TV stories about the rising incidence of ADHD and how Ritalin® was being
used at younger and younger ages than ever before. They had even talked to each
other about whether Jason might have ADHD, but while the hyper-activity label
might fit it seemed to them that he was a lively, normal young boy. Now things were
different. In the intimate surroundings of Room 28 in Ridgefield Elementary
School, right next door to the art room, they had just been told that their son, their
pride and joy, has a psychiatric illness.
Several facets of this too common scenario are hard to understand. Why is a
third grade teacher the primary diagnostician for this illness? Is it a disease of the
classroom? Shouldn’t the doctor’s opinion come first rather than last? Why didn’t
Marcie and Kevin think that their son had a disorder? Were they just fooling them-
selves? Under what circumstances should a growing eight year-old boy be given
drug treatments that will likely alter his personality? Will it change the type of per-
son he becomes? If so, will it necessarily be for the better? How will Jason react to
the label of having ADHD or to the need to take medicine in order to do better in
school? Is he taking the medicine for himself or for the rest of the class or for the
teacher?
All these questions and more surround the 5-15% of children in the U.S. who
are identified as having ADHD (209,210), with the application of more rigorous cri-
teria yielding the lower prevalence estimates (209). Since the incidence of ADHD is
three- to four-fold higher in boys, 8 to 24% of our male population may be affected
with this attention disorder. The ADHD incidence in the U.S. is at least 3-fold
higher than the rest of the world taken as a whole. Clearly it is critically important
for our society to understand the process of attention in sufficient detail to inform
the public debate over ADHD. Like all serious debates there are several sides to con-
sider, each with its own merits and its own zealous proponents. Knowledge about
the molecular and genetic events that influence the quality of attention and atten-
tion-based learning can inform all sides of the debate. Such knowledge could allow
us to view this debate with a more detached perspective rather than taking sides in a
hot button controversy that is closely linked to our contemporary lifestyle.
While important in its own right, the controversy over ADHD is even bigger
when considered in terms of our society’s willingness to address social problems
with technology in the form of drug therapy. In light of ADHD’s current prevalence
are we using neurochemical therapy to optimize the intellectual capabilities of a sig-
nificant portion of our population? Are we using the same therapy to improve social
order within our chosen system of education? What other disorders will be next?
What other behaviors will have “fixed” with their own treatments? If our road to the
future travels down a slippery slope greased by the quick fix of drugs, where are we
headed? Initial concerns over the use of Prozac® largely faded as our society adapted

144
and accepted the now widespread use of SSRIs (selective serotonin reuptake inhibi-
tors) for treatment of depression. Extensive use of Ritalin® is just another junction
on the very same road.
Defining ADHD
The most authoritative source for defining psychiatric illnesses id the Diag-
nostic and Statistical Manual of Mental Disorders (DSM) published by the Ameri-
can Psychiatric Association (211). The fourth edition of this manual (DSM-IV) lists
five broad criteria for ADHD. The most important criterion lists nine specific
potential behaviors of inattention and nine potential behaviors of hyper-activity-
impulsivity (Table 11.1). A diagnosis of ADHD is considered if at least six of the
nine behaviors in either group are present. If six or more behaviors from both
groups are present the correct classification is ADHD, Combined Type. If only six
from the inattentive list are present the classification is ADHD,
Predominately Inattentive Type. Six behaviors from the hyper-activity-
impulsivity list gives rise to the Predominately hyper-active Impulsive Type. In
addition to meeting this major criterion, the other four criteria which must be satis-
fied include: onset prior to age 7, occurrence both at home and at school, presence
of a significant impairment in function and absence of other confounding disor-
ders.
Other groups have weighed in on the diagnosis of ADHD, including the
American Academy of Pediatrics, which recently published guidelines for practitio-
ners (212). Six specific recommendations were made that are essentially identical to
the DSM-IV guidelines, making it clear that the Academy of Pediatrics is on the
same wavelength as the American Psychiatric Association, but there is the potential
for turf issues. Since these two groups, pediatricians and psychiatrists, are the most
likely physicians to be involved in the medical diagnosis of ADHD, there is the
potential for professional rivalry over who is more appropriate and qualified. In the
end there is also the important question of which group will financially benefit from
the ADHD business. If 5-15% of school-age children have ADHD we are talking
about millions of initial consultations, millions of follow-up visits and millions of
prescriptions. Professional organizations can assist their membership by promot-
ing “ownership” of diseases and ADHD is no exception. Although this is certainly
not the primary reason for promulgating diagnostic criteria it is a very practical out-
come nonetheless.
To carry this financial perspective a bit further we should recognize that the
initial designation of the behaviors in Table 11.1 as a psychiatric disorder has
important financial ramifications. For example, the American Psychiatric Associa-
tion’s decision that Fidgets with hands or feet or squirms in seat” or “Has difficulty
waiting turn” may be grounds for declaring the presence of mental illness opens the
door to a vast financial boon for its membership. In essence these diagnostic criteria
set the standard for the boundaries of normal and abnormal behavior and people

145
with abnormal behavior are candidates for medical visits and medical treatment.
The more such abnormal young people, the better the business for psychiatrists and
pediatricians. It’s an overly crass view of ADHD, but true nonetheless. In this seg-

146
ment of our economy the vendors have the ability to determine the size of their
market.

Table 11.1: Diagnostic criteria for Attention-Deficit hyper-activity Disorder from DSM-IV (211).
Criteria A-D must all be satisfied.

147
 The recent alarming increase in the incidence of ADHD is not driven solely
by definitions in DSM-IV or by the availability of willing health professionals. There
are important societal and parental factors as well. The behaviors in Table 11.1,
especially those listed under hyper-activity impulsivity, are a synopsis of “bad”
behaviors that have been typical of boys, and to a lesser extent girls, forever. These
bad behaviors have been dealt with in different ways by generations of parents,
according to the prevailing societal norms. At home parents set the standards for
their children’s behavior with combinations of discipline and positive reward. At
school group-based rules and regimentation are coupled with the awarding of
grades in order to shape behavior in parallel to curriculum-based learning. Over the
past three to four decades our American society has greatly restricted the use of cor-
poral punishment as a method for dealing with bad behavior. These days striking a
child is a good way for a parent to end up in court or for a teacher to get fired. Gone
are the days when a rap on the knuckles with a ruler or the threat of “I’m going to
get my belt” was used to assure “good” behavior and attention. While we don’t
regret the societal decision to eliminate violence against kids, what behavioral tools
do we have to fill this void?
Enter the pharmaceutical age. Much of behavior is a manifestation of neuro-
chemical actions in the brain and we utilize neurochemical drugs to control abnor-
mal behavior or to improve dysfunctional behavior. The original purpose of
neurochemical drugs was to treat obvious and serious mental illnesses such as
schizophrenia, epilepsy, bipolar disorder and depression. ADHD is none of these.
However, if “Fidgets with hands or feet or squirms in seat” or “Has difficulty await-
ing turn” are declared a mental illness it opens the door for using neuro-chemicals
to control “bad” behavior. With the rising concern over poor discipline in the class-
room and prohibitions against the use of punishment or intimidation, the path for
expansion of ADHD to a disorder of epidemic proportions was smooth and invit-
ing. Our society has now taken that path.
Another important factor promoting the incidence of ADHD is the natural
desire of parents and teachers to want to optimize the behavior and learning of chil-
dren. This means sitting in your seat, paying attention to the lessons of the day and
getting better grades on your report card. In the bigger picture it may also mean not
getting into trouble, going to college (or going to a better college) and living a more
“normal” life. Medical treatment of ADHD can and does lead to these very desirable
outcomes in many cases. In a matter of days the effects of drugs like Ritalin® can
produce remarkable improvements in behavior and school performance for some
kids. Treatment really does work! If the only barrier to better performance is a doc-
tor’s visit and a prescription, who wouldn’t be interested in the possibility? When
considering the child, the parent, the teacher, the doctors, the pharmacists and the
pharmaceutical industry it’s a win-win-win-win-win-win situation. Indeed, having
10% of our school-age population take drugs to improve their learning and behav-

148
ior may be a good thing to do. It is in fact the approach our current society feels is
the best way to deal with these behaviors.
It has also been proposed that the recent rise in ADHD may be linked to the
administration of vaccines. This intriguing possibility is discussed in the context of
autism and other developmental disorders in Chapter 13.
Do children grow out of ADHD when they reach their late teens? Some stud-
ies have suggested exactly that while others found that the same traits and behaviors
continue to be a part of the personality, albeit in adult-appropriate form (213-5).
After all, how many adults have difficulty with squirming in their seats? This ques-
tion again alerts us to changes in attention behavior that are integral parts of the
maturation process and the lifespan in general. Dopamine and norepinephrine-
based attention systems, which operate in a highly dynamic manner during youth,
exhibit a gradual restraint during late puberty leading to the quasi-steady level of
adulthood. It is therefore no surprise that symptoms of ADHD might ameliorate
during this same time frame. In adulthood, the level of attention-related activity
may be limited by a different set of biochemical factors than during juvenile years.
For example the availability of methyl groups from the folate pathway may become
more important during adulthood whereas dopamine concentrations may be more
important during youth. In many cases ADHD is only problematic for a limited
number of years, raising further questions about the necessity for its treatment and
its validity as a bona fide psychiatric diagnosis.
Individual Variations In Attention
Identical twins share exactly the same DNA sequences, although separate
experience may allow “epigenetic” differences such as patterns of DNA methylation
to occur. A cloned animal is genetically identical to other clones and we are on the
threshold of human cloning experiments. Person to person variation in genetic
make-up creates human diversity, not only in skin pigment and eye color, but in
behavioral traits as well. In the first portion of this book I offered a detailed molecu-
lar description of attention and attention-based learning in which dopamine D4
receptors play a central role. Genes encoding for each of the proteins in this process
are not constant among humans. Although some are more crucial than others are,
one can identify at least twenty different proteins that can exert a significant influ-
ence over the capacity for attention. Mutations and polymorphisms in each of these
can influence the abundance or scarcity of their protein products or affect the com-
petency of the protein to carry out its normal function. Recently it has been esti-
mated that there are about 12 single-nucleotide polymorphisms(SNPs) per gene, a
much higher level of individual variability than previously expected (216). It is no
wonder that there are so many variations in attention. They are a natural expression
of variation in the genes that dictate the efficiency and character of molecular events
during attention.

149
 Let’s assume that there is a naturally occurring range of attention abilities
around some average value. In other words some persons exhibit “better” skills
while others exhibit “poorer” skills. Perhaps “better” and “poorer” reflect differ-
ences in the duration of attention to a reading task among seven year-old children.
The so-called “normal” distribution of observed values around a mean would be a
typical finding for such a condition. Values lying more than two standard devia-
tions from the mean values (i.e. the 5-6% at each extreme) can be arbitrarily defined
as statistically high or low, but they are contiguous and continuous extensions of the
normal distribution. The 5-15% incidence of ADHD suggests that we may be using
a surprisingly rigorous standard to define abnormal attention. This is especially the
case if one considers only male children. On the other hand, attention skills may not
be distributed normally. There may be a second peak or group that is not a continu-
ous or contiguous extension of the normal distribution. Such a finding would
clearly identify a distinctive population and would support the presence of a psychi-
atric disorder. To my knowledge there is noevidence for such a bimodal distribution
of attention skills that would lead to the definition of an ADHD population. More-
over, at this time the diagnosis of ADHD is generally not based upon quantitative
measures of any sort. Given these circumstances it seems that our society has under-
taken a massive drug treatment program to adjust the activity level and learning
skills of a significant fraction of individuals within the normal range of behavior.
At this very point in the history of mankind we have gained the ability to
assess individual differences in genetic sequences that can explain normal and path-
ological variations in biochemical and behavioral abilities. In the case of ADHD the
first genetic link was to the dopamine D4 receptor. Initial studies by LaHoste and
colleagues at the University of California at Irvine in 1996 reported that the fre-
quency of having a D4 receptor gene with 7 or 8 proline-rich repeat segments was
29% in an ADHD population vs. 12 % in a control group (5). Increased frequency of
the 7-repeat form (allele) was associated with a decreased frequency of the 4- or 5-
repeat forms, from 76% in the control group to 53% in the ADHD group. This ini-
tial finding has since been confirmed in a number of independent studies (6,62-
3,217,218), although several other studies failed to find an association of the seven-
repeat form with ADHD (64,65). At this time there is no adequate explanation for
this discrepancy although the nature of the criteria for defining ADHD leave room
for significant differences in the severity and type of ADHD exhibited by subjects.
In addition, the contribution of the seven-repeat form of the D4 receptor to overall
risk of ADHD appears to be relatively small and therefore not easily detected. One
study found a positive association of the 7-repeat allele in an adult ADHD popula-
tion (21% frequency vs. 10% in the control group), along with a trend toward
increased transmission of the 7-repeat allele from heterozygous parents to ADHD-
affected offspring (218). In other words there was evidence that the occurrence of
ADHD was associated with a child receiving the 7-repeat gene from parents who
had both 7-repeat and non-7-repeat genes.

150
 While these associations link ADHD and the D4R gene, the number of
repeats is not the dominant cause of ADHD symptoms. However, if the D4R is
somehow involved in attention, variations in other parts of the gene might also con-
tribute to differences in attention and/or risk of ADHD. Indeed the D4R gene is one
of the most polymorphic in the human genome. Besides variation in the number
and type of repeats there is a polymorphic locus within the upstream region, a -521
C/T single nucleotide polymorphism, and more D4R protein is produced when the
C variant is present (66). Two studies reported an association between novelty-seek-
ing behavior and the C variant(66,219) while another study linked it to parental
detachment anxiety in infants (220). A 12 base-pair repeat affecting the first trans-
membrane helix of the D4 receptor is associated with a delusional disorder when
absent (221). Variations are also found in intron I, a region of the gene that does not
contribute to the final structure of the receptor but affects the structure of its mes-
senger RNA (222). A study from the lab of Dr. James Kennedy at University of
Toronto found that particular combinations of these variant sites appeared to con-
fer either risk or protection from ADHD (223).
If variations in the structure or density of dopamine D4 receptors can lead to
a risk of ADHD it seems likely that variations in the level of dopamine could also be
a factor. Consistent with this expectation, both positron-emission tomography
(PET) and genetic analysis studies have implicated the dopamine transporter pro-
tein as another risk factor for ADHD. The dopamine transporter (DAT) protein is
responsible for the re-uptake of dopamine from the neuronal synapse after it is
released (Fig, 11.1). As such it is a primary mechanism for lowering the synaptic
level of dopamine and terminating its stimulation of postsynaptic receptors. This
transmembrane protein binds dopamine on the outer surface of the nerve mem-
brane then undergoes a conformational change that allows the dopamine molecule
to be released on the inner side of the membrane. As noted earlier, the DAT protein
is the target of cocaine, amphetamine and Ritalin-like drugs used in the treatment of
ADHD, all of which cause an increase in dopamine levels by blocking its re-uptake.
A colleague at Northeastern University, Dr. Robert Hanson, synthesized a
labeled analogue of cocaine that could be visualized in PET studies (224). This agent
was then used to estimate the density of DAT proteins in the brains of six adults
with ADHD and found that each patient exhibited a higher level of the transport
protein as compared to their age-matched control group (225). It was also interest-
ing that the density of transporters showed a downward trend with age, being about
50% lower in the 50-60 year-old group, compared to the 20-30 year-old group.
When averaged together the DAT increase in ADHD subjects amounted to about
70%. In other words persons with ADHD had a significantly higher density of the
cellular machinery for lowering the level of synaptic dopamine. This potentially sig-
nificant finding reinforces the idea that a lower level of dopamine contributes to the
symptoms of ADHD. It also suggests that PET imaging could provide a potentially
useful diagnostic test to confirm the diagnosis of ADHD in a quantitative manner.

151
Furthermore, this result makes a strong case that beneficial effects observed during
Ritalin® treatment reflect its inhibition of a process that is overactive in ADHD. On
the downside, this initial study had only a few subjects and needs to be replicated in
a larger number of individuals, including younger persons, in order to establish the
prevalence of increased DAT levels in ADHD.

Figure 11.1: Mechanisms for terminating the synaptic action of dopamine. Dopamine (DA) is
synthesized in the presynaptic nerve terminal from the amino acid tyrosine (Tyr) via the
intermediate dihydroxyphenylalanine (DOPA) and stored in vesicles for release. Synaptic dopamine
can diffuse away, be transported back into nerve terminals by the dopamine transporter (DAT)
protein, or be metabolized by the enzymes monoamine oxidase (MAO) and catechol-O-
methyltransferase (COMT).

An ADHD-associated increase in the density of DAT proteins suggests an

increased level of messenger RNA for DAT and possibly an increase in the rate at
which the mRNA is transcribed from the DAT gene. In humans, the DAT gene con-
tains a variable number tandem repeat (VNTR) in the 3-un-translated region
(UTR) of the gene (226), the region immediately following the DNA sequence that
codes for the protein. Each repeat is 40 bases long and anywhere from 3 to 11
repeats occur that can affect gene transcription and DAT density. Interestingly, sev-

152
eral studies have found an association between occurrence of the 10-repeat DAT
gene and ADHD (227-8) and the 10-repeat allele is associated with higher DAT
expression levels (229). This suggests that the higher number of repeats in the 3’-
UTR of the DAT gene may increase the efficiency of DAT mRNA leading to higher
DAT density. Of further interest is the fact that non-primates do not have a similar
VNTR repeat in their DAT gene (230) such that the presence of repeats is a pri-
mate-specific feature, similar to the D4 receptor VNTR.
Obviously there is a striking parallel between genetic variations in the DAT
gene and variations in the D4R gene, although only in the D4R do these variations
affect the structure of the final protein product (i.e. as the presence of seven
repeats). With the DAT polymorphism affecting the strength and duration of dopa-
mine stimulation and D4R polymorphisms affecting the receptor response the two
could provide additive risk of ADHD. Given thehigher incidence of ADHD in
males, the presence of a Y-chromosome (or thepresence of only one X-chromo-
some) might be considered as a third genetic risk.
D4R Function In ADHD
We can now conceptualize how the molecular events of attention and atten-
tion-based learning might operate differently in ADHD. If attention is due to a
dopamine-induced increase in 40 Hz synchronized oscillations brought about by
D4R-mediated PLM, an increase in DAT density will decrease the duration of PLM
by lowering the dopamine concentration more rapidly. Thus the duration and pos-
sibly the amplitude of the 40 Hz event will be reduced and the episode of attention
will be more brief and perhaps less intense. If the seven-repeat form of the D4R is
present, the number of phospholipids available for methylation might be limiting,
placing a further constraint on the duration and intensity of the PLM signal. When
both DAT and D4R polymorphisms combine, a more drastic reduction in the qual-
ity of dopamine-initiated attention could occur.
Similar restrictions would also apply to vigilance and awareness, which I ear-
lier proposed to be mediated by a weaker nor adrenergic stimulation of D4Rs. How-
ever, limitations in the local density of phospholipids might not be as quantitatively
important for a weaker stimulus. Furthermore, if norepinephrine not dopamine is
responsible for awareness, increases in DAT levels might not affect the duration or
intensity of noradrenergic stimulation. For example, the ability to initially notice
something would be normal but the subsequently triggered episode of dopamine-
dependent attention would be truncated. Moreover, an abbreviated period of
focused attention provides the opportunity to return more quickly to the scanning
mode of awareness. In essence there would be a shift in the balance between aware-
ness and attention in favor of the former. This scenario is consistent with the symp-
toms and diagnostic criteria of ADHD, especially insofar as impulsive behavior
might be linked to decreased time spent in evaluating alternatives before acting.
Variations in the balance between awareness and attention are probably a very nor-

153
mal feature of humans, helping to define our personalities and releasing us from the
monotony of clonal behavior.
How would these same factors play out during attention-based learning, for
instance during the construction of attention-directed interneuronal networks? For
as much as sustained dopamine stimulation of MAP kinase is needed for neuronal
arborization and new synapse formation, increased DAT protein density could
blunt the development of such networks by limiting synaptic dopamine levels. The
rate at which new associations are made would be reduced and the rate of attention-
based learning could be impaired. While this has an ominous sound, akin to mental
retardation, we are not talking about that level of severity. Rather I am suggesting
that a reduced intensity of attention may result in a more gradual development
toward the same level of knowledge and understanding. In ADHD it may be more
common that multiple exposures to the same information are required but learning
does occur. This style of delayed learning may be reflected as “immaturity” when
compared to peers who are better at paying attention. Although no one wants to be
immature, there is a flip side to this coin. A delay in creating our hardwired view of
the world allows more time for further adjustments and flexibility with added expe-
riences. In the extreme case it may extend the “wonder years” of youth when our
ability to acquire new information is at its peak.
Let’s digress for a moment to consider what learning is about. Of course there
are different types of learning. When we are exposed to an experience or a concept
for the very first time it can constitute a revelation and we have the opportunity for
learning something entirely new. In a more mundane form learning commonly
consists of recognizing associations or connections between information that we
have already experienced. When you crack open five eggs in a row and each of them
yield a yellow yolk, we learn to expect a yolk out of the sixth one. Over time this
association becomes so strong that we create a mental image of the yolk inside the
egg as we view it from the outside. This learning is certainly handy when it comes to
making omelets. However, what has simultaneously occurred is that we have
restricted the range of possibilities of things that might be held within an egg. What
if the sixth egg yielded a pearl instead of a yolk? In other words associative learning
is as much a process of eliminating possibilities as it is gaining new information.
The quicker we learn, the quicker we eliminate other possibilities. Stronger associa-
tions provide a greater exclusion of other possible associations. In the latter case
there is a chance for the world to be viewed in terms of black or white absolutes.
Alternatively, there is also room in the world for “fuzzy logic” where a modicum of
uncertainly is retained just in case revision is needed. Certainly a lifetime brings
many occasions when we need to revise our concepts and be open to new learning.
Under these circumstances weaker associations may be easier to revise. Perhaps
ADHD confers some beneficial learning traits along with some constraints on the
rate of classical associative learning.

154
 Inquiry and the opportunity for discovery can be severely limited by the con-
straints of associative learning. In his classic analysis entitled “The Structure of Sci-
entific Revolutions” Thomas Kunn described how a number of important
discoveries emerged from conditions where the discoverer was not constrained by
the prevailing dogma that blinded others (231). He found a common thread in the
breakthroughs made by Copernicus, Darwin, Einstein and others. In each case a
truly open mind was brought to a problem, allowing a freedom and creativity of
thought that led to truly novel perspectives on thorny issues in their respective
fields. By entering an entirely new field of study or endeavor at an advanced age one
avoids the limiting influence inherent in the classical training paradigm for the field.
Surely there are losses from the absence of the traditional training route, but often
these are balanced by gains in terms of unrestrained possibilities.
Like other aspects of American society, our system of education is highly
structured and relatively homogenous across the entire country. We have embraced
a body of knowledge about human history, math, English and science and have set-
tled on a reasonably successful method of training successive generations to under-
stand this knowledge so they can participate in sustaining our culture. The
particular method of education we utilize is no doubt better suited to some styles of
learning than others. It is best suited to young persons who engage fully with their
teachers and their textbooks, but less well suited to kinetically active individuals
who only engage in a sporadic manner. What we are now recognizing is that genetic
variations among children can lead to differences in learning style and abilities.
Some are outright resistant to the standard learning method. In such cases it may be
useful to consider changing the learning system so that it adapts to the individual
rather than using drug therapy to adapt the individual to the system.
Why are a higher number of proline-rich D4R repeats associated with
ADHD? As described earlier, these repeats provide the opportunity for the receptor
to directly or indirectly link with other proteins. A higher number of repeats there-
fore implies a more protein-rich environment around the D4R, and a correspond-
ingly phospholipid-depleted environment (See Fig. 5.5). The good news about this
reciprocal relationship is that there are a greater number and a greater variety of
protein targets for modulation by PLM-mediated solid-state signaling. However,
there have been no comparative studies on the quantity or variety of proteins asso-
ciated with D4.2 vs. D4.4 or D4.7 receptors, so for the time being this remains only a
hypothesis. Nonetheless we can propose that additional signals may originate from
the seven-repeat receptor as compared to the two-repeat form. During episodes of
dopaminergic attention the function of interneurons expressing D4Rs can therefore
be modulated in a more complex manner. Some of these signaling cascades, like the
MAP kinase pathway, can produce long-lasting effects (e.g. by effects on DNA tran-
scription) laying the groundwork for associative memory. With adequate dopamine
stimulation a higher number of D4R repeats may therefore activate a more robust
array of longer-term learning avenues.

155
 The bad news about having a high number of repeats is that the number of
phospholipids available to the D4R for generating synchronized oscillations is
restricted. In a similar manner to excess DAT proteins this could restrict the robust-
ness of the dopamine-induced PLM response. This limitation particularly comes
into play when norepinephrine-stimulated PLM precedes dopamine-stimulated
PLM. For example, if there are only twenty molecules of phosphatidylethanolamine
available to a D4R and norepinephrine activation methylates ten of them, only ten
more will remain for the subsequent episode of attention. In this case attention will
not be able to rise much above the level of awareness. The dopamine-induced
increase in the ensemble of synchronized neurons (i.e. the amplitude of 40 Hz oscil-
lations) will be less significant. On the other hand, if there is a pool of thirty mole-
cules of phosphatidylethanolamine available to the D4R, twenty will still be
available for dopamine-induced attention, yielding a more extensive response.
Another predicted consequence of having fewer phospholipids and more proteins
around the D4R would be a decrease in the time required for signal transfer from
the D4R to the neighboring target proteins. In other words if there were only a sin-
gle layer of phospholipids separating the receptor from NMDA receptors or calcium
pumps, the latter proteins could be quickly affected by even the first few methyl-
ations. However, if there were several layers, the signal would have to work through
the first layer into the second layer in order to start modulating other proteins. This
theoretical difference could, for example, contribute to differences in reaction time.
Polymorphisms in DAT and D4R genes are only two examples of how
genetic variations can promote differences in how the dopamine dependent mecha-
nism of attention performs. We can anticipate that other polymorphisms in genes
regulating other components of the overall process will also be discovered. Variants
in the dopamine synthesis pathway (e.g.tyrosine hydroxylase or DOPA decarboxyl-
ase) or in the enzymes that metabolize dopamine (catechol-O-methyltransferase
(COMT) and monoamine oxidase (MAO)) are candidates. Indeed a polymorphism
in COMT has been linked to schizophrenia (232), but not ADHD (233). Polymor-
phisms in NMDA receptors, calcium pumps or other target proteins may affect
their efficiency or make them more or less sensitive to the effect of PLM. A poly-
morphism in the calcium pump protein has been linked to spontaneously hyper-
tensive rats (234) that are used as an animal model of ADHD (235). Commonly
occurring variations in the enzyme 5,10-methylenetetrahydrofolate reductase can
affect the ability of the folate pathway to maintain a supply of 5-methyltetrahydro-
folate for the PLM process (52-3,236). Variations that affect the level of adenosine
or ATP could also impact the efficiency of D4R-mediated PLM and its role in atten-
tion. With such an abundance of opportunities for diversity it is no wonder that sib-
lings frequently differ significantly in their attentive and learning styles. It is a
reflection of natural and normal variety in one of the most human of attributes.

156
Prescription Drug Treatment of ADHD
A number of prescription drugs are approved by the FDA for treatment of
ADHD. Others are in various stages of development and there are several non-pre-
scription drug treatment approaches. Methylphenidate(Ritalin®) is the most widely
recognized drug treatment and its use has been at the center of public debate about
treating ADHD. It is important, however, to recognize the existence and value of
non-drug treatments for ADHD. Specialized learning approaches and individual-
ized learning environments may be more effective than traditional classroom set-
tings. Behavioral therapy such as a token reward system can be employed in the
home and in the classroom. These strategies should be considered primary therapy
much like dietary management is a primary therapy for obesity and/or hyper-cho-
lesterolemia. Drug treatment should not be the knee-jerk response to a diagnosis of
ADHD, even recognizing that drug treatment provides a response rate which is
superior to behavioral approaches (237). Given the molecular focus of my own
expertise, I cannot comment on non-drug therapeutic approaches but I do hope
that psychiatrists, psychologists, schoolteachers and parents will appreciate the con-
tribution of biochemical and genetic factors to the condition of ADHD as they con-
sider different interventions to help affected children and adults. In many cases they
are dealing with a genetically-based personality trait rather than a mental illness.
While the trait may be have a significant negative impact on the individual’s social
and academic development let us reserve the label of mental illness for more drastic
disorders.
methylphenidate (Ritalin®) and amphetamine are stimulant drugs that are
structurally related to dopamine and to each other (Fig. 11.2). Amphetamine exists
as a pair of mirror-image “d” and “1” enantiomers that differ only in the three-
dimensional position of atoms around an asymmetric carbon atom (denoted by * in
Fig. 11.2). d-Amphetamine is 3-4 times more potent than 1-amphetamine. Methyl-
phenidate also has an asymmetric carbon, giving rise to d and 1 enantiomers that
are present as a 50:50 mixture in the commonly used formulation, with the d isomer
again being more potent. Amphetamine-like drugs have an effectiveness rate of
about 70% in both juvenile and adult forms of ADHD (237,238).
When administered at lower doses, d-amphetamine and methylphenidate
reduce locomotor activity and are useful for treatment of hyper-activity symptoms
associated with ADHD, although higher doses can actually increase locomotor
activity. Performance on complex visual-memory search tasks is also improved in a
dose-dependent manner. When the task is not too difficult methylphenidate

157
improves performance at low and high doses, but with higher demand tasks only
higher doses produce an improvement (239).

Figure 11.2: Chemical structures of dopamine, amphetamine and methylphenidate

(Ritalin). Asterisk (*) indicates the location of asymmetric carbon atoms.
At therapeutic doses methylphenidate and amphetamines enter dopaminer-
gic nerve terminals via the DAT protein and inside the terminal they can displace
dopamine from vesicular storage sites. Displaced dopamine can then exit the nerve
terminal via reversed activity of the transporter and cause a higher concentration of
dopamine in the synapse (240). This mechanism would provide a generalized
increase of dopamine levels. However, when they bind to the DAT protein methyl-
phenidate and amphetamines impair its ability to transport dopamine, similar to
the action of cocaine. Dr. Nora Volkow and colleagues at Brookhaven Laboratories
estimated that a 0.5 mg/kg oral dose of methylphenidate (e.g. a 20-mg tablet for an
88-lb. child) occupies and blocks about 60% of DAT proteins (241). This blockade
increases the synaptic lifetime of action potential-released dopamine during epi-
sodes of attention, leading to greater stimulation of dopamine receptors located
across the synapse. The dopamine increase can augment D4 receptor stimulation
and its modulation of glutamate receptors(e.g. NMDA and AMPA receptors) as
illustrated in Fig. 11.3. Interestingly, Dr. Volkow’s study found a clear decrease in
the size of methylphenidate response with age, being only one-quarter as effective in
40 yr-old subjects as in 20 yr.-olds (241).

158
 It has been estimated that therapeutic doses of amphetamine-like drugs cause
about a 6-fold increase in the resting level of dopamine (which is normally around 5
X lO-9) but at most a doubling of the typical 60-fold rise achieved during stimulated
dopamine release (242,243). Their main effect may therefore be to increase baseline
dopamine activity rather than to augment bursts of activity occurring during epi-
sodes of attention. These elevated levels of dopamine can lead to long-term changes
in the density of both dopamine receptors and DAT proteins that contribute to the
net drug response.

Figure 11.3: Methylphenidate enters the nerve terminal and displaces dopamine
(DA) and also blocks the re-uptake of dopamine by the dopamine transporter.
Together these actions increase synaptic dopamine and augment stimulation of
postsynaptic dopamine receptors such as the D4 receptor increasing its modulation
of glutamate receptors.
Augmenting D4 receptor activation may be an important action of methyl-
phenidate and amphetamines, contributing to both improved attention and
decreased hyper-activity. For ADHD individuals with an excess of DAT proteins,
increasing dopamine levels is a particularly appropriate mechanism of action, coun-
tering the underlying cause of the disorder. A specific role for D4 receptors in
ADHD is strongly supported by recent studies using an animal model of ADHD. In
this model a selective loss of dopaminergic neurons was produced in neonatal rats
and their activity level was monitored after they matured (e.g. at 10 weeks of age)
(244-6). When placed in a novel cage environment, these lesioned animals showed a

159
particularly high level of motor activity that did not rethrn to normal over time, as
was the case for untreated or sham-treated animals. This form of hyper-activity can
therefore be attributed to the loss of usual dopamine stimulation at some type of
receptor. Administration of specific D4R agonists was very effective at eliminating
this hyper-activity behavior, while agonists that stimulate other dopamine receptors
(e.g. D1 or D2 receptors) were ineffective (244-6). Thus, at least in this dopamine
deficiency model, D4 receptors are the most critical target for hyper-activity.
A number of amphetamine-like drugs compete for the lucrative ADHD mar-
ket. Some have been designed to improve on methylphenidate and d-amphetamine,
for example by optimizing the time-course of their effects. After oral dosage methyl-
phenidate levels peak in the brain at about two hours and decline to sub-therapeutic
levels within 4-6 hrs. In real life this means that kids can take one dose at home
before leaving for school but require a second dose during school hours. This also
means a disruptive trip to the school nurse’s office or to some other medication
supervisor, including the psychological impact of public treatment. In the case of d-
amphetamine, one approach has been the development of a dosage form containing
a mixture of various salts with differing absorption rates (e.g. Adderall®). This pro-
duces a broader, less pronounced peak in drug levels and extends the overall dura-
tion of action. Methylphenidate dosage forms with delayed activity have also been
developed (e.g. Metadate CD®, Concerta®) that allow children to make it through the
school day with a single morning dose, a school nurse-friendly idea. A long-acting
transdermal dosage form for methylphenidate (MethyPatch®) also has been clini-
cally tested and may be available soon. Insomnia due to prolonged stimulant effects
can be a common side-effect, especially when Ritalin® or other drugs are adminis-
tered later in the day. Methylphenidate has a half-life of 4.5 hrs and a 2 PM dose
should therefore wear off before bedtime. Longer lasting dosage forms bring greater
chance of an excessively long duration of action. Nervousness, headache, stomach-
ache and loss of appetite are also common side effects during methylphenidate ther-
apy (247).
Widespread use of amphetamine-like drugs for the treatment of ADHD has
increased the opportunity for their abuse. They are classified as Schedule II con-
trolled substances by the FDA. Methamphetamine (“speed”) and cocaine have a
similar mechanism of action to methylphenidate so it is no surprise that ADHD
drugs are abused. Their slower rate of transfer to the brain and lower potency limit
the extent of “high” they produce but their ready availability tends to offset these
factors, increasing their choice for abuse.
Anti-depressant drugs have also been tested in ADHD and some have been
found to be effective. Like amphetamines, these agents work by blocking the re-
uptake of neurotransmitters back into the nerve terminals from which they were
released. Different drugs may or may not be selective for inhibition of specific neu-
rotransinitter transporters. Of course the most well known examples are the selec-
tive serotonin re-uptake inhibitors (SSRIs) including Prozac®, that increase synaptic

160
levels of serotonin with little effect on synaptic levels of norepinephrine or dopa-
mine. SSRIs are effective anti-depressants but are not effective in ADHD, Bupro-
pion (Wellbutrin®) blocks the re-uptake of dopamine, norepinephrine and
serotonin and has shown effectiveness in treating ADHD, as has the norepineph-
rine-specific agent atomoxetine (248). The effectiveness of the latter agent under-
scores the involvement of norepinephrine in the mechanism of attention. Other
less-selective drugs that have been used in the treatment of depression (e.g. desipra-
mine or nortriptyline) are also effective in reducing ADHD symptoms, but their
safety is a major concern, based on four cases of unexplained sudden death in chil-
dren being treated with these agents (249).
A number of non-amphetamine-like drugs have also been used in ADHD but
none of them appears to have as high a level of effectiveness. One such drug is cloni-
dine, a drug used for the treatment of hyper-tension and for reducing the symptoms
of opiate withdrawal. By partially stimulating alpha 2-type adrenergic receptors on
nerve endings, clonidine causes a decrease in the release of neurotransmitters asso-
ciated with a generalized sedative effect. Although this is a negative side effect when
treating hyper-tension, it can be put to use in the treatment of heroin addiction or
ADHD. Clonidine and a similar agent, guanfacine, are effective at reducing hyper-
activity and impulsivity components of ADHD (250). A meta-analysis of 39 cloni-
dine studies concluded that it is less effective than stimulant drugs with a number of
side effects and should be considered as a second-tier treatment option (251).
New, non-amphetamine-like drugs are currently under Development for
treatment of ADHD. Abbott Laboratories is testing a nicotine-like agent (ABT-418)
which stimulates the opening of nicotinic acetylcholine channels and has particular
selectivity for a specific type of channel (alpha4beta2) while sparing the channels in
peripheral tissues (252). This distinction significantly reduces the occurrence of car-
diovascular effects as compared to nicotine. In animal models ABT-418 increased
cognitive performance, presumably because of its ability to stimulate the release of
dopamine (253), leading to a clinical trial in adults with ADHD. ABT-418 caused
improvement of symptoms in 40% of subjects vs. 13% for placebo, indicating some
effectiveness, but less than typically occurs for amphetamine-like agents (254).
Another new agent, Perceptin®, is a blocker of histamine H-3 receptors and has
recently been given FDA approval for Phase II clinical trials. Such preliminary trials
are meant to examine different dosage levels for possible effectiveness. At present it
is unclear whether Perceptin® will be effective in ADHD, or what role histamine H-
3 receptors might play in attention.
Non-prescription Treatments For ADHD
Prescription drugs remain the mainstays of ADHD therapy but some non-
prescription treatments have also been found to be effective. Several act by aug-
menting membrane fluidity, in keeping with the importance of this event in atten-
tion. For example, a small study published in 1990 reported that S-
adenosylmethionine (SAM), in doses of 1200 mg to 2400 mg/day caused “moderate

161
to marked improvement” in ADHD symptoms in an adult population (255). SAM,
of course, is a donor of methyl groups similar to the D4R and is widely used as a
non-prescription treatment for depression. By promoting phospholipid methyla-
tion, supplemental SAM increases membrane fluidity thereby setting a higher base-
line for dopamine-induced increases. In other words SAM produces additive effects
with D4R stimulation that may be particularly helpful when dopamine stimulation
is weaker than normal. This action may be the basis for its beneficial effect in
ADHD in adults. However, its relatively high cost and frequent dosage requirement
make SAM a less favorable treatment option.

Figure 11.4: Structure of the omega-3 unsaturated fatty acids DHA and linolenic
acid. DHA is a major component of the brain and is found in cold water fish oil.
Linolenic acid, which is more common in the diet, can be converted to DHA via a
multi-step elongation pathway.
Another non-prescription treatment approach is supplementation with
highly unsaturated “essential” fatty acids, also known as omega-3 fatty acids. Cold-
water fish (the smelly kind like mackerel, salmon or tuna) are rich in omega-3 fatty
acids, so fish oil is a common source for the commercial products. These fish utilize
omega-3 fatty acids as an adaptation to their cold environment. To function effec-
tively, they must maintain normal levels of membrane fluidity in their cells despite
the tendency of cold temperatures to cause solidification of fats and phospholipids.
To accomplish this they preferentially accumulate and utilize long-chain, highly
unsaturated fatty acids for their membrane phospholipids. As illustrated in Figure
11.4, the presence of extra double (unsaturated) bonds in these fatty acids causes
them to assume a more kinky shape. Consequently they do not pack as close to one
another as saturated or less unsaturated fatty acids, meaning fewer phospholipids
per unit of membrane area. Because of this, membranes constructed from phospho-

162
lipids containing omega-3 fatty acids are comparatively more fluid at a given tem-
perature, allowing cold-water fish to function normally at lower temperatures.
A remarkable study by Drs. Stevens and Burgess and colleagues at Purdue
University analyzed the levels of various fatty acids in the plasma and red blood cells
of 53 boys with ADHD compared to 43 age-matched controls (256). They found
significantly lower levels of several individual omega-3 fatty acids in both plasma
and cells including docosahexaenoic acid (DHA), which has 22 carbons and six
double bonds. Eicosapentaenoic acid (EPA), which has 20 carbons and 5 double
bonds, was also significantly lower in the plasma samples. Furthermore, there was a
correlation between the severity of ADHD, as rated by parents, and the degree of
essential fatty acid deficiency. Breast milk is relatively rich in essential fatty acids,
and this study also found that ADHD subjects were much less likely to have been
breast-fed. If they were breast-fed the duration was much shorter. Prenatal and
perinatal deficiency of omega-3 fatty acids causes life-long behavioral effects in lab-
oratory animals (257). Taken at face value, this potentially important study suggests
that lower levels of essential fatty acids may be a part of the risk of ADHD and that
reduced breast feeding may play a role in introducing this risk. Of course it is
entirely possible that ADHD children are more difficult to breast feed, so causation
is not established. These findings could be dismissed as some sort of esoteric meta-
bolic anomaly, but since membrane fluidity seems to be a critical element in the
molecular mechanism of attention, they might reflect an important and under-
appreciated illustration of how attention abilities are related to diet and metabolism.
If there were a relationship between low levels of omega-3 fatty acids and
ADHD, their administration might improve ADHD symptoms. Indeed there have
been recent reports describing beneficial effects of omega-3 fatty acid supplementa-
tion in ADHD, particularly when associated with dyslexia (258-260). While these
results are encouraging, earlier placebo-controlled, double blind studies of DHA
and gamma-linolenic acid supplementation in ADHD found no beneficial effect on
other ADHD symptoms (261,262). There is a need for further studies to assess the
importance of fatty acid deficiencies in ADHD and to evaluate the benefit of supple-
mentation as a treatment approach. Since they are dietary supplements, omega-3
fatty acid preparations are non-prescription and cannot be patented. As a conse-
quence there is little incentive for companies to spend the large sums of money
needed for such clinical trials.
It is important to recognize that when they are used, omega-3 fatty acids have
to be given in much larger amounts than traditional medicines, since they represent
an alternative food source rather than a drug. To be effective they have to replace a
significant proportion of the total fatty acid pool. When starting omega-3 fatty acid
supplementation the changeover is necessarily gradual, requiring several weeks at a
minimum. DHA and EPA are the most common omega-3 fatty acids, and a com-
bined daily dose of about 1 gm is typically recommended. Many fish oil prepara-
tions contain only about 30% DHA/EPA, so it may be necessary to take three large 1

163
gm capsules in order to obtain the needed amount. Fatty acids comprise 30% of the
brain’s weight and the brain has a higher proportion of omega-3 fatty acids than
other tissues. In neurons, DHA is particularly enriched in the so-called “growth
cones” which creep outward to contact other nerves and begin the process of syn-
apse formation (264). Lower levels of DHA may therefore impede this process.
The omega-3 story actually starts with blue-green algae. Like other animals,
man cannot create omega-3 fatty acids, which is why they are considered an essen-
tial part of the diet, or essential fatty acids. Cold-water fish don’t make them either.
They get them in their diet by eating blue-green algae, which have the ability to syn-
thesize them. Fish eat the algae and man eats the fish, in a classic food chain sce-
nario. The trouble is that humans, especially in the U.S. are not eating enough cold-
water fish or other omega-3sources (e.g. fiaxseed, hemp seed, black currant seed,
pumpkin seed and soy bean oils) to meet their needs, although increased use of
canola oil has made some improvement. It is estimated that currently in the U.S. the
omega-3 intake is still less than one-quarter of the recommended level (264). As
food production in our culture became more and more industrialized over the past
150 years, the intake of omega-3 fatty acids has drastically decreased, with increased
use of cereal grains to feed domestic livestock. The diet of prehistoric man is
thought to have included an abundance of omega-3 fatty acids. A comparison of
various cultures around the world shows an inverse correlation between the intake
of omega-3 fatty acids and the frequency of depression (265-267). ADHD may be
provoked in part by insufficient omega-3 fatty acids in our diet and the dramatic
rise in the incidence of ADHD during the past several decades might be a manifes-
tation of unhealthy dietary trends. Risks from genetic background and diet may
combine to place individuals at greatest risk of ADHD.
Long-term Effects of ADHD Treatments
One of the most concerning issues surrounding ADHD is whether drug treat-
ment causes long-term problems that are not as yet fully appreciated. Methylpheni-
date has been used in the treatment of ADHD for over 50 years, providing several
generations of real-life experience and there is no evidence of calamitous drug-
induced effects that express themselves only after a period of years, although weight
gain and growth rate may be decreased. Even though its use has greatly escalated in
the past decade, there is no reason to expect this general record of safety to change.
The focus of concern should then be on the significance of the therapeutic interven-
tion itself. Across the lifespan, does it matter whether we use drugs to raise the level
of dopamine six to eight hours a day for a period of perhaps 10 years? Sure it mat-
ters, both in good ways and bad ways.
For as much as a child learns better and is not subjected to negative sanctions
from bad grades or bad behavior, he or she may obtain benefits that are long-term
and life-long. Self-confidence and self-esteem may improve and can carry over into
all aspects of life. Within neuronal circuits of the brain the extra dopamine may pro-
mote formation of useful synapses, helping to codify associative relationships and

164
facilitating higher, more effective levels of attention (and synchronized gamma fre-
quency oscillations). Indeed, it may make a child or adult more efficient and a “bet-
ter” person in many ways. One follow-up study of boys with normal intelligence
who were earlier diagnosed with ADHD found that they completed significantly
fewer years of schooling (about 2 yrs. on average) and had lower ranking occupa-
tional positions than a control group of similar intelligence without ADHD (268).
Notably, these individuals did receive treatment, so negative outcomes were not
avoided by drug therapy. One could speculate that these differences might have
been even greater without treatment.
There are also downsides to the drug treatment of ADHD. If therapy is effec-
tive it creates a slightly different person. Children have been“optimized” to a greater
or lesser extent. With the help of a temporary neurochemical adjustment they may
be a better fit for their home and school environments. When asked to fill out med-
ical history forms some time in their future, they may (or may not) volunteer that
they suffered from a psychiatric illness when they were young. They might think of
themselves as having a disorder, which fortunately could be effectively treated, and
be comforted by the fact that so many others in their school suffered from the same
malady. When the time comes for them to face the same decision as their parents,
regarding whether or not to treat their own children for ADHD (after all the genetic
linkage makes this a likely scenario) will their own experience increase the chance
that they will opt for drug treatment? Is it possible that the recent upturn in ADHD
diagnosis and drug treatment might induce a second and third generation of accep-
tance among a genetically related group of affected individuals?
The diagnosis and treatment of ADHD brings a significant expense to our
nation’s annual medical bill. For ADHD diagnosis and treatment the additional
medical costs per year have been estimated to be $375 (269) while others have esti-
mated the cost for a 9-year interval of treatment for ADHD to be about $2,400
(270). If 5% of our children are treated for an average of 9 years this amounts to an
annual cost of $850 million. If the current incidence is closer to 10%, it rises to over
$1.5 billion annually. While “money is no object” when the health and well-being of
children are concerned, there is reason to be concerned that the overzealous diagno-
sis of ADHD places an important burden on an already strained national health
budget.
Reflecting on ADHD
Is there a “correct” view of ADHD when we consider its diagnosis, its origins
and some of the current treatment options? Probably not. There will continue to be
different opinions, depending upon whose perspective we adopt. The mother who is
tearing out her hair in desperation as she deals with a hyper-active child has a differ-
ent perspective from the prescription drug benefits manager at the HMO or from
the drug companies whose bottom line depends upon their ADHD business. My
own perspective is that ADHD is a trait shared by a significant proportion of our
society and probably by similar proportions of many societies around the world.

165
Expression of this trait is promoted by certain genetically based factors (including
sex), but it is not exclusively genetic in origin. Differences in pre- and perinatal
events such as maternal nutrition and breast-feeding may be important, as well as
differences in the home and school environments that guide behavior and nurture
personal development. Neurotoxic insults, including lead poisoning and mercury
exposure can also cause ADHD. Within the proportion of people who share this
trait there is substantial variation in its level of expression, which probably reflects
the variable presence of multiple risk factors, genetic and otherwise. Individuals
with ADHD symptoms experience difficulties meeting the behavioral expectations
of our society, both in and out of the home environment and face limitations in
their efficiency of learning within the structure of our educational system. As a con-
sequence, learning via non traditional modalities (e.g. experimentation, trial and
error) may be more effective and common in these individuals, With maturation,
the magnitude of the differences that constitute the ADHD trait typically lessen and
the importance of the limitations decrease, partly as a result of normal age-depen-
dent changes in the biochemical mechanisms that support attention. In addition,
other skills and factors become more significant determinants of success and failure
when we move outside of the highly structured educational environment of youth.
Nonetheless, there is a price to be paid for lowered academic achievement, both in
terms of a loss of lowered horizons for advancement as well as the limitations that
accrue when someone has not fully benefited from a fundamental education. Posi-
tive personality traits such as high energy and inquisitiveness may counterbalance
some or all of the downside traits that comprise ADHD diagnostic criteria, espe-
cially in adulthood.
When all is said and done much of the ADHD debate is centered upon indi-
vidual differences in one of the most highly developed human abilities, the capacity
to selectively attend to information and to learn from it. Diversity in the way we
execute this ability is the rule rather than the exception, evidenced by the naturally
occurring diversity in the genes and proteins that are critical for attention. Any
large-scale effort to “normalize” differences in attention via drug therapy would, of
course, be a misguided adventure.Nonetheless, well-intentioned participants in the
educational and medical sectors of contemporary American society could fuel such
an effort. In many cases drug therapy is an all too easy solution to behavioral prob-
lems and for parents trying to promote high academic achievement.
There is an obvious need for greater restraint and selectivity in deciding to
use drugs for the treatment of ADHD. A nature-based perspective tells us to respect
and understand differences in attention-related behavior. However, it is just as
obvious that there remains a place for the medical treatment of those who suffer
from extreme expression of the traits comprising current ADHD diagnostic criteria
and for those whose behavior may harm others. Drug treatment is, however, only
one avenue of approach, and the number of instances which justify drug treatment
should be greatly reduced from current levels. Developmental strategies, including

166
early parental involvement, appropriate discipline, alternative learning modalities
and possibly dietary supplementation are all recommendable options and may serve
as useful alternatives to drug therapy. We should question the limits of our “one size
fits all” educational system and instead recognize the value of natural diversity in
modes of attention. This recognition should motivate us to find new approaches for
reducing the conflict between our social and educational structure and the virtues of
individualism.
Chapter Summary:
• ADHD affects approximately 10% of children in the U.S., with a 3 to 4-fold higher
prevalence in boys than girls.
• There is high frequency of variation in attention-related genes that is especially evident in
humans, giving rise to individual differences in the capacity for attention and in the risk of
ADHD.
• The seven-repeat form of the D4R and an increased density of dopamine transporters are
both associated with increased risk of ADHD.
• ADHD is most commonly treated with stimulant drugs like methylphenidate and d-
amphetamine that release dopamine from neuronal storage sites and increase its synaptic
levels.
• Antidepressant drugs that block the uptake of dopamine, norepinephrine and serotonin
are also used to treat ADHD, as well as adrenergic agonists such as clonidine.
• Non-prescription drug treatment options include S-adenosylmethionine and fish oil-
derived omega-3 fatty acids.

167
12 SCHIZOPHRENIA

As he read the Boston Globe in the living room he shared with eight other
residents, Frank Toth didn’t know exactly what to make of the attention currently
being directed toward schizophrenics like him. It was the best of times and the
worst of times. The front page story about the Oscars said “A Beautiful Mind” was
racking up award after award including “Best Picture” and “Best Director” for its
portrayal of Dr. John Nash and his emergence from the depths of psychosis to claim
the 1994 Nobel Prize. Russell Crowe was acclaimed for his dynamic and realistic
performance. It seemed like schizophrenia was starting to be viewed with more sen-
sitivity and compassion. While he knew it was only the world of Hollywood enter-
tainment, Frank thought this was a good news story. However, on the front page of
the “Regional News” section there was a story about the upcoming trial of Michael
McDermott who shot seven people dead up in Wakefield. He was going to claim an
insanity defense and inevitably schizophrenia would again be linked to homicide,
fear and suspicion. This was the real world and clearly bad news.
Frank thought: “What does all this attention to schizophrenia mean?” He
concluded that it meant a tricky situation for him over the next couple of weeks.
Nobody at the nursing home where he worked in the kitchen knew that he was a
schizophrenic, not even the couple of people that he could call friends. After all, it
was none of their business. They just knew Frank as the cook’s helper and clean-up
guy who worked three afternoons and evenings a week for the last eighteen months.
Even when he had taken three weeks off for medical reasons Frank managed to get
by just saying that he had a “neurological problem”, consistent with the doctor’s
note to his supervisor. Since his antipsychotic meds caused his hands to tremble and
made him move kind of awkwardly, the neurological excuse seemed to fit and
wasn’t a problem like telling people he was a schizophrenic. Only Frank knew that
he had in fact suffered a psychotic break and was back on the ward for a week while
they readjusted his meds before allowing him back to the community house. It was a
rough time and he wasn’t at all sure that be was going to be okay again. Indeed his
whole life was unsure. He never felt far from the chaos and bewilderment that con-
sumed him for the first eight years of his life as a schizophrenic.
These thoughts made Frank nervous. Lighting up another cigarette he
remembered, as best he could, his own life story. It wasn’t a movie script and it
wasn’t on the front page of any paper. The only thing that was important about his
story was that it happened to him and therefore it was real in a way that made the
experiences of the rest of the world unimportant.
Actually his first twenty years seemed like somebody else’s life. He was a nor-
mal kid, even better than normal, or so he thought. Maybe he had been fooling him-
self all along. Maybe there were some signs of impending trouble that he and his
parents had missed. But he had been an honors student, particularly gifted in math

168
and science. He was a little socially uncomfortable but nothing serious. Looking
back it was clear that things had started to go downhill after he started at Boston
University. His freshman year was a difficult adjustment. Dorm living wasn’t all it
was cracked up to be. It was tough to fit in with all the strangers. Sure he did have
one friend, his roommate Ralph, but everybody else on his floor were jerks. He felt
increasingly isolated but attributed these feelings to being out on his own. His
grades weren’t good, probably because he cut lots of classes, but at least he survived
his first year. Understandably his parents weren’t happy, but they just didn’t know
how tough it was to get things done in college.
Frank was desperate to move out of the dorms and find a place where he
could be alone. A small studio apartment near Fenway Park was his refuge. He
didn’t really need to be around anyone, He liked having a smaller world, going out
to eat and to buy food but otherwise keeping to himself. Gradually, in a way that he
didn’t even notice, Frank’s world retreated to within his mind, but his life changed
forever in November of his sophomore year when his ex-roommate Ralph visited.
Frank’s worried parents asked Ralph to check on him after they had a ram-
bling, largely nonsensical phone conversation with him the night before. They
thought maybe he was on drugs. When Ralph first arrived he was shocked by
Frank’s appearance and by the filth in his apartment. It was hard to figure out which
was more responsible for the stench that assaulted his nostrils. Frank immediately
launched into a highly animated and confusing explanation of the amazing things
that were happening in his life. He told Ralph about being contacted by agents from
the Federal Immigration Bureau who wanted him to keep an eye on the family in
the house next door. They appeared to be ordinary people but were really a front for
the Iranian government and were plotting to assassinate the President. Ralph was
shocked and skeptical but Frank spent more than an hour trying to convince him
that he needed to join him in thwarting their evil plan. Ralph wanted to give him the
benefit of the doubt, but when Frank said he received instructions from the Feds
only in the bedroom closet via radio waves that were broadcast to the precise fre-
quency of his brain he knew something was seriously wrong. After Frank showed
him the pages and pages of wrinkled notes he had accumulated about the every
movement in and out of the suspect house for the past three weeks Ralph under-
stood why he hadn’t seen him in classes for a while. While Frank was in the other
room talking to himself Ralph called his parents and together they agreed that tak-
ing Frank to the student health office was a good idea.
Frank wasn’t at all interested in the suggestion that they go to the health cen-
ter and talk to someone. Ralph tried to coax and cajole him but this only infuriated
Frank. He broke into an angry rant about how he never really trusted Ralph. A sec-
ond call to the health office brought a visit by the campus police. Eventually an
ambulance was sent to his apartment and, over his strenuous objections, Frank was
on his way to Boston City Hospital. At the emergency room they drew blood and
gave him a shot to calm him down (his first drug treatment for psychosis he thought

169
in retrospect) and he was interviewed by several different nurses and doctors. Frank
did his best to put them at ease about the situation without revealing the secret
aspects of his critical assignment. After all, how could he expect them to under-
stand? He had to be careful whom to trust. It wasn’t easy to think clearly, especially
as the drug started to kick in. After about an hour his parents arrived, met with the
doctors and Frank was moved to the psych ward for the first of what would prove to
be many visits.
The intervening eight years had been a surreal journey through varying levels
of managed and un-managed madness, punctuated by psychotic episodes that
required hospitalization, readjustment of his neuroleptic drug dosage and in some
instances switching to a new drug altogether. He was constantly watched and moni-
tored by a confusing number of doctors, nurses and ward attendants, not to men-
tion the other patients and his parents. How ironic this was for someone who had
wanted so desperately to be alone. Gradually Frank came to understand that he was
experiencing multiple realities. At times his reality was shared with those around
him and that made his care givers happy. At other times his reality was not shared.
He had two-way conversations in the absence of people. The voices he heard were as
clear and normal as when people were present. To Frank they were real voices, but
this reality didn’t make anybody happy, including Frank. When the anti-psychotic
drugs were effective he was able to distinguish between the two realities and the hal-
lucinations became less prominent, but they were never far away. Again Frank felt
the unsettling vulnerability of a relapse that he knew would be with him for the rest
of his life.
As one of the lucky ones Frank had been able to move into a community-
based, supervised residential program after six years. His family had been very sup-
portive and was dedicated toward helping him achieve as much independence as
possible. He was making progress. Sadly, Frank recalled those that were not so
lucky. Several patients he had come to know committed suicide. Not really surpris-
ing, especially when the drugs don’t work and you don’t have a future. Frank was
doing pretty well, thanks to his Clozaril treatment that he was switched to after
being on other drugs that didn’t work nearly as well. He figured his life as a schizo-
phrenic lay somewhere between the Nobel Prize and mass murder.
What is schizophrenia?
Schizophrenia is the prototypical psychotic disorder. The term “psychotic”
can be loosely applied to the symptoms of a range of psychiatric illnesses (e.g.
Alzheimer’s disease, depression, mania), but when used in a specific manner it
implies the presence of hallucinations, delusions, disorganized speech or disorga-
nized or catatonic behavior. Of these, hallucinations are the hallmark symptom of
schizophrenia. It involves a diminution of normal cognitive abilities accompanied
by the substitution of alternative internally-derived experience.

170
 Cognition, the capacity to effectively experience and comprehend one’s envi-
ronment, is such a reliable and transparent ability that 99% of the world population
operates in relative ignorance of its true importance and its fragility. The 1% suffer-
ing from schizophrenia wage an ongoing internal battle to regain or sustain their
precarious hold on normal cognitive function. Is there anyone who would argue
against the notion that cognition is the most defining characteristic of man? It
therefore follows that the loss of cognitive abilities, especially for those who once
enjoyed its benefits, must be one of the most threatening and frightful experiences
that man can endure. Approximately 10% of persons with schizophrenia ultimately
commit suicide. In the final analysis we may conclude that the 1% who suffer from
schizophrenia are paying the price for the cognitive gifts enjoyed by the 99% of
unaffected individuals.
Although there is still much to be learned about schizophrenia, we can be
sure that it is caused by aberrant molecular processes and it is these molecular
events that require our attention. A significant portion of the risk for developing
schizophrenia arises from genetic causes (twin studies suggest about 50% of total
risk), while untoward events (e.g. fetal, perinatal or postnatal insults) may also con-
tribute to its occurrence. Schizophrenia clearly is a purely emotional response to an
overbearing mother or to other Freudian factors which are the grist of psychoana-
lytical mills. Although extreme stress can be associated with and even trigger acute
psychotic episodes, it is the molecular consequences of stress that are responsible
for this influence, In keeping with the theme of this volume, we will sift through the
available molecular evidence for clues to the origin(s) of schizophrenia, and by now
it should surprise no one that dopamine and the pathway of D4R-mediated PLM
will be found to be prime suspects.
DSM IV defines schizophrenia as a psychotic disturbance of at least six
months duration including one month with two active symptoms (defined as “delu-
sions, hallucinations, disorganized speech, grossly disorganized or catatonic behav-
ior, negative symptoms”) (211). Catatonic behavior means a lack of movement or
reactivity to the environment. Negative symptoms include a profound lack of inter-
est in events and surroundings (flattened affect), a poverty of speech (alogia) and a
failure to initiate or sustain activities (avolition). By contrast, hallucinations, delu-
sions and disorganized speech are examples of positive symptoms. If the distur-
bance lasts less than six months and causes less interference with normal function, it
is termed schizophreniform disorder. There are other closely related categories
including schizoaffective disorder, delusional disorder etc.
One of the most confounding features of schizophrenia is its typical age of
onset in the late teens through the late twenties in persons who otherwise were pre-
viously normal. Less commonly, schizophrenia can occur in childhood. While the
overall incidence is similar for males and females, males are more likely to have an
earlier onset, suggestive of a protective role for estrogens. The occurrence of peri-
menopausal and postpartem psychotic episodes is consistent with a loss of this pro-

171
tection (271,272). The disease is typically chronic with the potential for life-long
symptoms, although rare cases of apparent remission are reported. Initial stages
tend to be dominated by negative symptoms while hallucinations and other positive
symptoms become more prominent as the disease progresses. The severity of symp-
toms can be episodic, with periods of relatively few signs and normal function alter-
nating with weeks or even months of exacerbation. Response to treatment with
neuroleptic, antipsychotic drugs is quite variable, with a significant proportion of
patients showing little benefit. The latter pattern strongly suggests that there are a
number of specific causes of schizophrenia, of which only some respond to dopa-
mine receptor blocking drugs.
Theories on the Cause of Schizophrenia
Because it is the prototype psychotic illness, multitudes of theories have been
advanced to explain the origins of schizophrenia. By far the most widely accepted
theory revolves around dysfunction in dopaminergic neurotransmission, including
a critical role for D2-like (i.e. D4-like) dopamine receptors. This “Dopamine
Hypothesis” is, however, far from explicit in explaining schizophrenia and is still
evolving. Its origin primarily lies with the recognized effectiveness of D2-like recep-
tor blocking drugs in treating schizophrenia. As a class these agents are called neu-
roleptic drugs and they arc also used in the treatment of a number of other
psychiatric conditions, including Tourette’s syndrome, mania, autism and more. In
recognition of their effectiveness, the “Dopamine Hypothesis”, first put forth in
general terms by Dr. Steven Matthysse in 1973 (273) and later amplified by Dr.
Arvid Carlsson (274), proposed that schizophrenia was caused by a hyper-dopami-
nergic state. Hence the therapeutic utility of dopamine receptor blockers could be
rationalized. In addition it was recognized that high doses of amphetamines, which
increase synaptic dopamine levels, could elicit schizophrenia-like symptoms, sup-
porting the concept that there was an excess of dopamine or at least an excessive
response to dopamine. Despite the logic of this proposal there is little or no direct
evidence for increased levels of dopamine per se. Measurements of dopamine itself
or its metabolites in cerebrospinal fluid have failed to find a consistent increase
(275), leaving a cloud over the validity of the Dopamine Hypothesis, particularly
over its proposal that schizophrenia is caused by higher levels of dopamine stimula-
tion. Nonetheless, D2-like receptor blocking drugs do work in most cases and the
main paradigm within the pharmaceutical industry has been to develop incremental
improvements in neuroleptic drug properties.
Several recent findings point toward the need to revise the Dopamine
Hypothesis. Dr. Daniel Weinberger and colleagues examined the association
between polymorphic alleles of catechol-O-methyltransferase (COMT) and schizo-
phrenia. COMT inactivates dopamine and norepinephrine, by adding methyl
groups onto their catechol hydroxyl groups. [Note: SAM is the source of methyl
groups for this reaction.] Surprisingly they found a significant association between
the more active form of COMT and schizophrenia. In other words, faster metabo-

172
lism of dopamine was associated with a greater risk of schizophrenia. This is oppo-
site to what might have been expected if schizophrenia was caused by an elevated
level of dopamine. Indeed, one might expect that higher COMT activity would exert
a protective effect if higher dopamine levels were causing schizophrenia. Con-
versely, this important finding suggests that too low a level of dopamine is a risk for
schizophrenia and requires a significant shift in the Dopamine Hypothesis.
A related finding about neuroleptic drugs also calls for rethinking the Dopa-
mine Hypothesis. Dr. Phillip Seeman, who has made a number of important contri-
butions to understanding the role of dopamine receptors to schizophrenia, recently
analyzed the binding affinities of neuroleptic drugs in relation to their clinical effec-
tiveness (276). He pointed out that the so-called “atypical” neuroleptics, such as clo-
zapine, olanzapine, quetiapine and risperidone, are distinguished by their lower
affinity for D2-like receptors, including the D4R. Atypical neuroleptic drugs are
clinically superior to typical agents (such as haloperidol, thioridazine etc.) in two
respects: 1. They cause significantly less side-effects, particularly extrapyramidal
symptoms such as tremor and rigidity, that can be extremely debilitating conse-
quences of treatment with typical neuroleptics. 2. They can bring about greater
improvement in cognitive function. If indeed schizophrenia were simply due to
excessive dopaminergic stimulation of D2-like receptors, then higher affinity agents
should be clinically superior, not the other way around. As Seeman points out, the
low affinity of these agents allows dopamine to break through their receptor block-
ade and stimulate D2-like receptors. This feature appears to be desirable and impor-
tant, both for their efficacy in treating the cognitive symptoms of schizophrenia and
for reducing extremely nasty side effects. By strongly blocking D2-like receptors,
typical neuroleptics are effective in reducing the positive symptoms of schizophre-
nia, such as hallucinations, indicating that these cardinal symptoms are associated
with activity of these receptors. However, cognition (or attention) is certainly not
improved, but is in fact impaired by typical agents. Together these recent findings
require a major revision of the Dopamine Hypothesis to a form that is consistent
with the actions of typical and atypical neuroleptic drugs.
Among his many contributions toward understanding the role of dopamine
receptors, Dr. Seeman’s lab found differences in D4R binding in post-mortem brain
samples from persons with schizophrenia (277). They used a controversial method
for determining D4R binding, subtracting the D2 and D3 receptor binding from the
binding of a drug that binds to D2, D3 and D4 receptors. A six-fold higher density
of D4R sites was found in samples from persons with schizophrenia, as compared to
either normal controls or to persons with other neurological diseases (e.g. Alzheim-
ers or Parkinson’s Disease). Such a large increase suggested that the utility of neuro-
leptic drugs might result from blocking excess D4Rs. Other studies replicated this
observation using different techniques (278-280), although some did not (281,282).
The cause and significance of this apparent increase remains uncertain at present. A
confounding issue is the fact that different states of the D4R during the PLM cycle

173
(i.e.. D4ÆT, D4 SAM, D45 and D4 HCY) can exhibit different binding properties.
Schizophrenia-associated differences in D4R binding could therefore reflect a dif-
ferent status of the cycle.
The “Dopamine Hypothesis” of schizophrenia was preceded by a “Single-carbon
Hypothesis” put forth in 1966 by Dr. John Smythies (283). He proposed that “the
biochemical fault in one of the schizophrenias may lie in the biochemical mecha-
nism itself of transmethylation: the one carbon cycle, in which methionine, S-ade-
nosylmethionine (SAM) and folic acid are involved”. A year earlier a case report
from Johns Hopkins University was published entitled: “Schizophrenia in a patient
with a defect in methionine metabolism” in which the particular defect was an
increased level of homocysteine (284). In 1955 Dr. Seymour Kety showed that the
administration of methionine to persons with schizophrenia caused an acute exac-
erbation of their illness while having no effect in normal individuals (285). This
classic observation has since been replicated in at least ten subsequent studies (286).
Clearly there is some connection between methionine metabolism and schizophre-
nia. In 1975 Dr, S. Harvey Mudd and colleagues reported that a defect in 5, 10-
methylenetetrahydrofolate reductase (MTHFR), the enzyme that makes 5-methyl-
THF, can cause schizophrenia (287). More recently several studies found an associ-
ation between schizophrenia and a commonly occurring polymorphism in the
MTHFR gene (288,289) while another did not (290). Persons with the weaker, ther-
molabile form of the enzyme showed a higher incidence of schizophrenia, suggest-
ing that impaired formation of 5-methylTHF might be a risk factor for
schizophrenia. A much lower level of methionine metabolism in isolated lympho-
cyte (291) and whole-body studies (292), along with reports of reduced methionine
adenosyltransferase (MAT) activity in schizophrenia (293,294), lend support to the
“Single-carbon Hypothesis”. Unfortunately at the time these observations were
made there was no obvious connection between these metabolic observations and
dopaminergic neurotransmission. Thus the “Single-carbon Hypothesis” has lan-
guished in relative obscurity for a number of years. Recently, however, presence of
the thermolabile MTHFR polymorphism was found to be associated with a higher
rate of neuroleptic drug success (295), forging a link between dopaminergic recep-
tor blockade and a reduced capacity for 5-methylTHF production. Our discovery of
folate dependent, dopamine-stimulated PLM provides a new mechanistic frame-
work for understanding how dopamine and single-carbon theories might fit
together.
Recent appreciation of the importance of DNA methylation has revived
interest in a possible link between methylation and schizophrenia. For example,
transcription of the gene for reelin, an extracellular protein produced by GABAer-
gic interneurons, is controlled by its methylation status (296), and reel in expression
levels are reduced by up to 50% in schizophrenia (297). An underlying problem
with single-carbon metabolism may cause this decrease in reelin production. Reelin
is found associated with postsynaptic densities on pyramidal cells, particularly on
their dendritic spines, and plays a role in the formation of synapses with GABAergic

174
interneurons (298). Reduced reelin production could manifest itself as impaired
interneuronal function, including impaired 40 Hz oscillations. Another prominent
theory of schizophrenia involves NMDA receptors and the neurotransmitter gluta-
mate. It is well recognized that drugs such as ketamine or phencyclidine (PCP),
which block the NMDA receptor, can cause hallucinations (299) and that can be
reduced by neuroleptic drugs (300). An NMDA receptor link to schizophrenia was
thus suggested.
Another aspect of the “Glutamate Theory” is based upon the fact that exces-
sive stimulation of NMDA receptors by glutamate can lead to destruction of neu-
rons via a process termed excitotoxicity. At various stages of development NMDA
receptor-mediated excitotoxicity and associated neuronal death is entirely normal,
for example the “pruning” of unneeded neurons during adolescence (301). How-
ever, when it occurs at the wrong time, such as after a stroke, it can lead to a loss of
brain function.
Dr. Daniel Weinberger and colleagues showed that when neonatal rats were
subjected to excitotoxic damage they showed a hyper-activity that was only mani-
fested two months later, corresponding to years later in human development (302).
Furthermore, treatment with the neuroleptic agent haloperidol blocked hyper-
activity. It was therefore suggested that the development of schizophrenia might be
the delayed result of an earlier, perhaps neonatal, insult such as oxygen deprivation
during delivery. Another intriguing observation was that reducing the level of one
of the NMDA receptor subunits in mice produced a neurological syndrome that
could be reversed by the D4R-selective neuroleptic drug clozapine, even though
nothing had been done to dopaminergic systems (303). This finding emphasizes the
close functional relationship between NMDA receptors and D4-like dopamine
receptors.
Abnormalities involving the cell membrane have been documented in schizo-
phrenia, leading to a “Membrane Hypothesis” (304,305). Differences in the relative
concentrations of phospholipid species have been reported, using either freshly
obtained blood cells, cultured fibroblasts or post-mortem brain samples (305-310).
In all tissue samples the findings have been consistent. Persons suffering from
schizophrenia were found to have significantly reduced brain levels of phosphati-
dylethanolamine (PE), which is, of course, the required substrate for PLM. Levels of
phosphatidyicholine (PC), the product of PLM, were also reduced in most studies.
We compared PLM in white blood cells of persons with schizophrenia and found an
almost a four fold lower level compared to controls (Fig. 12.1). Thus a decrease in
the membrane level of PE is a broadly confirmed feature of schizophrenia and the
decrease appears to occur throughout the entire body.
Magnetic resonance spectroscopy brain imaging studies in subjects with
schizophrenia also reveal altered phospholipid dynamics indicative of a membrane
disorder that is present at the initial onset of the disease (311-313). Reduced levels of
membrane fluidity-promoting polyunsaturated fatty acids have also been reported

175
in schizophrenia (314,315). These differences suggest that the baseline level of
membrane fluidity may be low in the neurons of persons with schizophrenia. Lower
availability of PE would reduce the efficiency of dopamine-stimulated PLM. These
deficits could synergize with any limitation in the ability of the folate system to pro-
vide single-carbon groups for PLM, yielding a substantial impairment of the overall
pathway.

Figure 12.1: Comparison of phospholipid methylation in lymphocytes of schizophrenia patients with

levels in control subjects. PLM was almost four-fold lower in the patient group (18).

Anatomic differences have also been described in brains from persons with
schizophrenia (316-319). One of the most prominent differences is an increase in
the size of ventricles, the fluid-filled spaces separating various lobes from each
other. Larger ventricle size implies a smaller neuronal tissue mass in schizophrenia,
perhaps due to early neuronal damage or accelerated neurodegenerative loss
although some studies have found no such decrease (320). A number of studies
describe schizophrenia-associated differences in parvalbumin-positive GABAergic
intemeurons, including differences in density, dendritic arborization and in the
number of specialized nerve ending structures called cartridges (321-326). Recall
that these interneurons, which develop starting after 3-6 months (325), are critical
for generating 40 Hz synchronized oscillations. Any reduction in their number
would therefore affect the capacity for producing 40 Hz oscillations. Since the devel-
opment of parvalbumin-positive intemeurons is stimulated by dopamine, acting via
a D4-like receptor, these schizophrenia-associated anatomic differences might
reflect a result of abnormal dopaminergic signaling.

176
 The prevalence of cigarette smoking is very high (> 90%) in persons with
schizophrenia, unless they are in a setting where access is prohibited. Moreover,
persons with schizophrenia can experience a re-emergence or worsening of psychi-
atric symptoms when they attempt to stop smoking. These observations suggest that
cigarette smoking is a form of self-medication, implying that nicotine or other
ingredients of cigarette smoke might be producing beneficial effects. As noted ear-
lier, nicotine stimulates the release of dopamine and glutamate from nerve endings
(171-173). Increased dopamine release may be helpful in stimulating D4R-mediated
PLM and also ameliorating the side effects of neuroleptic drugs. In addition ciga-
rette smoke contains compounds that inhibit both A and B forms of the enzyme
monoamine oxidase (MAO) (176,176). This enzyme oxidizes dopamine and nor-
epinephrine, and MAO inhibitors were widely used for the treatment of depression
before the development of selective serotonin uptake blocking drugs. Inhibition of
MAO due to cigarette smoking would augment the effect of nicotine-induce dopa-
mine and might form the basis for self-medication in schizophrenia. Since expres-
sion of the alpha-7 subtype of nicotinic receptors is decreased in schizophrenia,
smoking may compensate for a deficit in cholinergic-induced dopamine release
(327).
There are a number of other theories about the cause of schizophrenia,
including possible involvement of an as yet unidentified virus (328), a relationship
to stress and even a link to the seasons (there is a small excess of Winter births
among schizophrenic individuals). Perhaps with time and more research we will
find confirm how each of these observations relates to the origin of this human
scourge. When challenged, even seasoned biomedical researchers often throw up
their hands, shrug their shoulders and mutter something about the unfathomable
complexity of the human brain. After reading the preceding chapters, you may
share my personal optimism that the long sought after solution to this riddle of
human suffering lies within the D4R/folate pathways.
A Unified Theory of Schizophrenia
At this point in time schizophrenia research does not need another brand-
new theory as much as it needs to understand the relationship among already exist-
ing findings and existing theories. What is needed is a unifying theory capable of
integrating the myriad of clues and results, including those theories briefly reviewed
above. Although there are obvious dangers, let’s give it our best shot.
Major theories on schizophrenia can be incorporated into a single “Unified
Theory” based upon the role of D4R-mediated PLM in attention and related effects
on DNA methylation. While each earlier schizophrenia theory addresses a part of
the overall puzzle, they are individually incapable of adequately addressing the dis-
order as a whole. In a sense these theories need to be synchronized so that their
individual information content can be combined to create a more complete view of
the underlying genetic and biochemical events in schizophrenia. None of them are
wrong and we shouldn’t view them as competing with each other for the honor of

177
being right. We need to see how they can be harmonized into a single, credible
description of this devastating medical enigma.Schizophrenia appears to be a psy-
chiatric disorder resulting from multiple, synergistic deficits in the biochemical
pathway supporting normal attention and cognition, including, but not limited to,
dysfunctional D4R-mediated PLM in GABAergic interneurons. At the genetic level
humans exhibit an extraordinary number of variants and polymorphisms in genes
that code for the principal proteins of this pathway. These include proteins involved
in the folate pathway, the methionine cycle, catecholamine synthesis, release, re-
uptake and metabolism, the D4R itself, and enzymes involved in phospholipid
metabolism. To this list we can add other components of the system that produces
40 Hz synchronized oscillations, such as elements that provide glutamatergic and
GABAergic neurotransmission, calcium pumps and potassium channels. At its core
schizophrenia appears to be a disorder of the capacity for producing synchronized
oscillations. Normal variations in the efficiency of individual components in this
system give rise to the rich diversity in how humans utilize the gift of attention.
Over millennia this diversity has allowed primates, including humans, to escape the
clonal existence which typifies most other species. Schizophrenia may be a manifes-
tation of the downside risk inherent in such diversity.
We all carry and harbor greater or lesser portions of the genetic risk of
schizophrenia, a risk that is estimated to contribute 50% of the ultimate determina-
tion of whether you will or will not develop the disease. If we compiled a list of the
key proteins required for 40 Hz oscillations, there would easily be 25 or more
entries, and each of their corresponding genes probably exhibits several polymor-
phisms, either in the coding region for the protein itself or in promoter regions that
regulate protein expression level. These polymorphisms introduce either more or
less activity for a given step, thereby modulating attention and introducing diver-
sity. The random distribution of positive and negative variants provides a broad
range of satisfactory function, but the system can only tolerate a reasonable number
of negative variants before function is compromised. Moreover, if several of these
multiple negative variants happen to impinge upon the same or successive steps,
their combined impact can be greatly magnified.
With this combinatorial risk perspective we can begin to ask how many
adverse variants in a pathway would be required to produce the 1% incidence of
schizophrenia? For example, if a single negative variant reduced the efficiency of
one step by 30%, leaving 70% of normal activity, a combination of two such steps in
a row would reduce overall function to 49% (70% times 70%), three would reduce it
to 34%, four to 24% etc. At what level might impaired function lead to clinical
symptoms? Of course we don’t really know, but we might expect that a system oper-
ating at one-third to one-quarter of normal efficiency should make itself evident. If
so, only three or four 30% negative variants would be necessary to yield symptoms.
If a homozygous polymorphism giving rise the negative variant was present in 20%
of the population, that particular step would be affected in only. 1 out of 5 individu-

178
als. The risk of two such steps being affected would be 4% and the risk of three steps
being affected would be about 1%. In the reality of cellular biochemistry, when defi-
ciencies occur they give rise to compensatory responses that dampen their actual
impact. We might then speculate that the unfortunate occurrence of four to five
negative genetic polymorphisms might be sufficient to give rise to schizophrenia.
Non-genomic insults (e.g. perinatal hypoxia, drug-induced neuronal damage, and
stress) are superimposed upon this background of genetically determined risk.
Folates and the Age-dependent Onset of Schizophrenia The hallmark of
schizophrenia is its age-dependent onset, earlier in males (18-25 yrs.) than in
females (24-30 yrs.). This pattern is consistent with a growth and development-
related triggering event, along with a partial protective effect of estrogens. Linear
growth normally ceases in the late teens with the calcification of bone growth plates,
defining the achievement of physical maturity. Prior to this point in time the cells in
most tissues and organs are rapidly dividing in association with body growth. To
support this growth the folate pathway has to be very active, in order to supply sin-
gle-
carbon groups for de novo purine synthesis (e.g. for DNA, RNA and ATP
synthesis) and thymidine synthesis (for DNA synthesis). However, when growth
ceases the demand for these activities is greatly curtailed since only lower levels of
purines and thymidine synthesis are required for the steady state demands of the
mature body. Thus the period of vulnerability for onset of schizophrenia corre-
sponds to a period of diminished demand for single carbon folates by these two
nucleotide synthesis pathways. As a result the potential number of single-carbon
groups available for 5-methylTHF synthesis is increased after maturation, with
potential consequences for D4R-mediated PLM and other methylation reactions.
Growth hormone produces many of its trophic actions via IGF-1 and
the signaling activity of IGF- 1 in the brain decreases upon maturation due to
a decrease in both IGF-1 and IGF-1 receptor levels (329). As described earlier, IGF-
1 normally acts to increase methionine synthase activity. A fall-off in IGF-1 signal-
ing activity will therefore introduce a restriction on the efficiency of folate-depen-
dent homocysteine methylation and the efficiency of D4R-mediated PLM. A
maturation-associated decrease in methionine synthase activity, mediated by
decreased IGF-1 signaling, would be an appropriate mechanism to offset potential
adverse effects of elevated 5-methylTHF. Of course it is apparent that the success of
such a transition would depend upon maintaining the right balance between the
levels of 5-methylTHF and residual IGF-1 activity. Too little 5-methylTHF synthe-
sis or too great a decrease in IGF- 1 could lead to impaired D4R-mediated PLM
function and to an increase in homocysteine levels.
As noted earlier, a remarkable age-dependent decrease in the uptake of
methionme by the frontal cortex has been documented starting from the earliest age
measured (2-4 yrs) until approximately 20 yrs (135). After age 20 methionine
uptake remained stable at this lower level throughout adulthood. As the uptake of

179
dietary methionine by the brain diminishes, the folate pathway becomes more sig-
nificant as a source of methyl groups. Moreover, the D4R PLM mechanism faces
stronger competition for the folate-derived methyl groups it needs. Other methyla-
tion reactions (DNA methylation, catecholamine methylation etc.), that had previ-
ously relied upon dietary methionine for the synthesis of S-adenosylmethionine,
will increasingly depend upon methionine formed by the folate-dependent re-
methylation of homocysteine. Under these circumstances any latent deficit in the
capacity to provide 5-methylTHF will make itself evident as IGF-1 signaling activity
decreases in association with cessation of growth.
In light of the evidence for a single-carbon folate deficit in schizophrenia and
the dependence of D4R-mediated PLM on folate-derived methyl groups, it seems
likely that in many cases schizophrenia develops due to the combined effects of an
inadequate supply of 5-methylTHF and a development-related decrease IGF-1 sig-
naling. Together these lead to impaired methionine synthase activity. Vulnerability
to an inadequate 5-methylTHF can be associated with the presence of the low-activ-
ity thermolabile form of MTHFR or to other factors that restrict its availability, such
as reduced levels of its precursor 5, 10-methyleneTHF. These risks remain dormant
until the developmental transition to maturity when the folate pathway is down-
regulated and methionine synthase activity decreases. Additional risks can arise
from anywhere in the 40 Hz pathway, but the age dependent onset of schizophrenia
strongly suggests that the most important triggering event is a growth-related
diminished capacity for methylation of homocysteine and the homocysteine state of
the D4 receptor. Deficiencies in required enzyme co-factors (e.g. vitamin B6 for
MTHFR, vitamin B 12 for methionine synthase, folie acid etc.) can further lower 5-
methylTHF levels and/or impair homocysteine methylation.
Inadequate methiomne synthase activity can be caused by either reduced cat-
alytic capacity of the enzyme (e.g. due to insufficient IGF- 1 activation) or to
impaired availability of the 5 -methylTHF co-factor or by some combination of
both. This leads to important consequences for the effectiveness of neuroleptic
drugs. For example, drugs that block the D4 receptor will reduce the demand for 5-
methylTHF and can be helpful in compensating for an inadequate supply of 5-
methylTHF. However, if the primary limitation is the intrinsic catalytic activity of
methionine synthase, there will be no great benefit of neuroleptic drugs, accounting
for “treatment resistant” forms of schizophrenia. Accordingly the effectiveness of
neuroleptic drug treatment is correlated with presence of the mutated thermolabile
form of MTHFR which is less efficient in producing 5-methylTHF (295).
A critical question is “What controls the dynamic activity of the single-car-
bon folate pathway?” Folate researchers have fingered one of the important players,
5-formylTHF. This folate species was an enigma for many years because, unlike all
the other single-carbon folates, it does not donate its carbon to any biological reac-
tion. Why then does nature bother to make it? Apparently it is made for the purpose
of controlling the folate pathway.

180
 Dr. Verne Schirch, at Virginia Commonwealth University, who has been an
immense figure in detailing folate biochemistry, made the crucial initial observa-
tions, along with Dr. Patrick Stover (Cornell University), who did his doctoral
research in Schirch’s lab. First they found that 5-formylTHF is synthesized by the
enzyme serine hydroxymethyltransferase (SHMT), using 5, 10-methenylTHF as a
substrate (330). Furthermore they found that after 5-formylTHF is synthesized it
turns around and strongly inhibits SHMT (331), the very enzyme that gave it life! In
doing so it tightly regulates SHMT activity and critically controls folate dynamics.
There are two closely related types of SHMT, one in the cellular cytoplasm and one
in mitochondria. Mitochondria are the cell’s energy source, based upon their ability
to produce ATP, and are also the source of single-carbon groups for the folate path-
way, based upon their production of formic acid (formate). In the cytoplasm SHMT
uses 5,10-methylene THF and glycine to make serine, effectively siphoning
off single-carbon groups from the folate pathway and decreasing the synthe-
sis of 5-methylTHF (Fig. 12.2). By inhibiting SHMT, 5-formylTHF keeps single-car-
bon groups in the central pathway, favoring their conversion to 5-methylTHF. 5-
FormylTHF also inhibits an enzyme within the de novo purine synthesis pathway
(332), further reducing the diversion of single-carbons out of the folate pathway.
Too little 5-formylTHF can lead to higher cytoplasmic SHMT activity along with
reduced levels of 5-methylTHF (333) and can contribute to the onset of schizophre-
nia.
Schirch made a further observation about 5-formylTHF and metabolic con-
trol. They followed the level of 5-formylTHF in the fungus Neurospora crassa,
which undergoes a dormant spore stage during its lifecycle (334), and found that 5-
formylTHF levels were very high during the spore stage, accounting for more than
85% of total single-carbon folates. However, as soon as the organism leaves its
spore stage and re-initiates growth, levels of 5-formylTHF fall to very low levels
(<10%). This dramatic change suggests that very high levels of 5-formylTHf may be
one way to induce or maintain metabolic dormancy, a perfectly reasonable thought,
since the folate pathway is essential for synthesis of purines and thymidine, support-
ing the synthesis of RNA and DNA. While human adulthood is not exactly a dor-
mant state, it seems possible that nature utilizes the level of 5-formylTHF to
modulate folate dynamics and restrain metabolism.
Genetic studies carried out in yeast by Dr. Ian Dawes have led to a similar
conclusion. He found that 5, 10-methyleneTHF binds to a transcriptional regula-
tory factor controlling the expression level of many enzymes in the folate and
methionine cycle pathways (335), High levels of 5,10-methyleneTHF suppress gene
expression while low levels turn it on. His work also showed that 5-formylTHF con-
trols 5, 10-methyleneTHF levels by virtue of its inhibitory effect on SHMT. When 5-
formylTHF levels are low, the higher SHMT activity lowers 5,i0-methyleneTHF and
this leads to increased gene expression. Under normal conditions, this relationship

181
would serve to stabilize activity of the folate and methionine cycles at a desirable
intermediate level.

Figure 12.2: Role of 5-formylTHF in regulating the single-carbon folate pathway. By

inhibiting both de novo purine synthesis and serine synthesis, 5-formylTHF keeps
single-carbon groups within the folate pathway and favors formation of 5-
methylTHF
There is another important player in the 5-formylTHF story, namely the
enzyme responsible for its breakdown, methenylTHF synthase or MTHFS for short.
MTHFS catalyzes the ATP-dependent conversion of 5-formylTHF back to methen-
ylTHF, from whence it originally came. Together, SHMT and MTHFS form an
apparently futile cycle in which 5-formylTHF is made and then unmade, so to
speak, as illustrated in Fig. 12.1. This cycle would not only be futile, but would also
be wasteful of ATP if 5-formylTHF were not such an important regulatory mole-
cule. Overexpression of MTHFS causes 5-formylTHF levels to plummet, as would
be expected, but, significantly, the concentration of 5-methylTHF also falls to very
low levels (333). This is because 5-formylTHF is no longer plugging up the exits
from the single-carbon folate pathway! Consequently we must conclude that if 5-
formylTHF is so important in controlling the level of 5-methylTHF, MTHFS is
equally important because it controls 5-formylTHF concentration.
Working even further backwards in an effort to understand control of the
folate pathway, we may ask how MTHFS activity is regulated. Little is known at
present, but we can anticipate a role for promoters and suppressors at the level of

182
DNA transcription. In one of those coincidental findings that are just too tantaliz-
ing to ignore, Dr. Stover’s laboratory recently reported that when they purified
MTHFS they found it contained a tightly bound small molecule. Upon further anal-
ysis the molecule turned out to be the oxidized quinone form of dopamine (336),
although the functional importance, if any, of this binding is not currently known.
Since MTHFS carries out a reduction as it converts 5-formylTHF to 5, 1O-methen-
ylTHF, it needs a source of reducing equivalents. Wouldn’t it be amazing if dopa-
mine served as a regulatory co-factor in this reaction? An increased intracellular
concentration of oxidized dopamine could accelerate the catabolism of 5-for-
mylTHF, lowering the levels of 5 -methylTHF and putting a brake on D4R-medi-
ated PLM.
Another tantalizing clue concerning folate regulation was the discovery that
MTHFS binds to a phosphorylated tyrosine residue on the IGF-1 receptor (337), the
very same receptor that controls methionine synthase activity. When this phos-
phorylated segment of the IGF-1 receptor was used as bait in a scientific fishing
experiment, only a single protein was found to take the bait (i.e. bind to the seg-
ment). Sequence analysis of the protein revealed it to be MTHFS. This is a particu-
larly surprising result since MTHFS does not contain known structural domains
with the ability to bind to phosphotyrosine residues. Nonetheless, this finding is
another link between IGF-1 signaling and the single-carbon folate pathway. For
example, when the IGF-1R is activated, phosphorylation of this tyrosine residue
could promote MTHFS binding to the receptor, sequestering it and reducing its
participation in the 5-formylTHF futile cycle. The consequential rise in 5-for-
mylTHF levels would augment 5-methylTHF levels and facilitate homocysteine
methylation and D4R-mediated PLM. If confirmed, this mode of control over
folate-dependent homocysteine re-methylation would complement IGF-1 stimu-
lated, P13-kinase-dependent stimulation of methioninc synthase.
Together these pieces of information support the hypothesis that the onset of
schizophrenia is commonly triggered by an age-dependent decrease in the signaling
activity of the 10F-1 receptor. While such a deficit in folate dependent methylation
events is the most likely triggering event for schizophrenia, it alone cannot fully
account for the disorder. When other significant risk factors are present, age-depen-
dent limitations in methionine synthase activity or in the availability of 5-methyl-
THF may be sufficient to initiate symptoms. Indeed, even a “normal” age-
dependent decrease in the supply of folate-derived methyl groups might be suffi-

183
cient to elicit symptoms if there is a strong enough “genetic loading” at other critical
points in the pathway.

Figure 12.3: Most prominent theories on the cause of schizophrenia can be related
to the mechanism of dopamine-dependent attention.
What are the other factors that make an age-dependent down-regulation of
folate-dependent methylation such a big risk for some individuals? We have already
met some of the likely suspects in the form of competing schizophrenia theories. As
summarized in Figure 12,3, they include impaired singe-carbon dynamics,
increased dopamine metabolism by catechol-O-methyltransferase, reduced levels of
phosphatidylethanolamine, reduced levels of NMDA receptors, reduced levels of
unsaturated fatty acids, a reduced density of GABAergic interneurons. In short, a
portion of the risk arises from each of the many elements that must work together
to provide dopamine-dependent 40 Hz synchronized oscillations. The dopamine
hypothesis, the single-carbon hypothesis, the membrane hypothesis, the NMDA
hypothesis are all correct, just as their underlying data suggests The critical aspect to
recognize is that no single hypothesis alone is adequate to account for schizophre-
nia. As mentioned earlier, the answer does not lie in a competition among hypothe-
ses but rather in understanding how a multitude of observations are inter-related.
No doubt there are many other additional risk factors remaining to be identified,
but if the “Unified Hypothesis” offered here is correct, they may also be accommo-
dated somewhere within the molecular mechanism that supports synchronized
oscillations.

184
 At the time of this writing a “DNA Methylation Theory of Schizophrenia” has
just being formulated (296) that appears to complement and extend the “Unified
Hypothesis”. Deficits in the molecular mechanism of attention and in DNA methyl-
ation can both arise from impaired single-carbon folate metabolism, triggered by
the same fall-off in IGF- 1 signaling activity. They are inter-related manifestations
of a single illness. Global hypo-methylation of DNA can result in a wide variety of
functional and morphological alterations and may account for many of the observa-
tions associated with schizophrenia. We can anticipate that research activity during
the next several years will greatly illuminate the role played by DNA methylation.
How do antipsychotic neuroleptic drugs work? If schizophrenia can be
caused by a deficiency in D4R-mediated PLM, it seems illogical and counter intui-
tive that drugs which block the D4R should be mainstays of treatment. Shouldn’t
they make things worse, not better? To make sense of this we’ll have to go back to
basic principles or at least back to the cycle of D4R-mediated PLM. The D4R PLM
cycle normally operates with the concentration of dopamine being rate-limiting.
When the level of dopamine rises sufficiently to occupy the D4R and promote a
change in receptor conformation, PLM is triggered. What would happen to the
cycle if levels of 5 -methylTHF in the methionine synthase step became rate limit-
ing? First of all, the population of receptors would be found primarily in the homo-
cysteine state awaiting a methyl group, rather than in the methionine state waiting
for dopamine to bind. This will introduce a delay and a decrease in dopamine
response. When the level of dopamine (or norepinephrine) rises during an episode
of attention, it will be less efficient in stimulating PLM and less efficient in promot-
ing 40 Hz synchronized oscillations, Moreover, dopamine stimulation will further
increase the demand for methyl groups, further aggravating the problem with their
scarcity. Secondly, as the homocysteine form of the receptor accumulates during a
5-methylTHF shortage, it may be re-converted backwards to its S-adenosylhomo-
cysteine form, because of the reversible nature of SAH hydrolase. This will further
reduces the amount of receptor in the methionine form that is needed for dopa-
mine-stimulated PLM.
What steps could be taken to remedy the above situation? Obviously one
approach would be to restore levels of 5-methylTHF to normal, but we’ll get to that
later. Another approach would be to reduce the demand for methyl groups so that
the limited supply will be sufficient to meet demand. How could a decrease in
demand be accomplished? Well, one way would be to employ D4 receptor blocking
drugs to reduce the level of dopamine stimulation. Of course this approach would
be potentially problematic insofar as it would interfere with already weakened D4
receptor-mediated PLM. By blocking D4 receptors, inhibitors would allow the
receptors to accumulate in the methionine state and would reduce the proportion of
receptors in both the homocysteine and S-adenosylhomocysteine states. However,
when it comes time for dopamine to stimulate PLM these receptor blockers would
be in the way. Ideal blockers would therefore be drugs that rapidly come off the

185
receptor so that a rise in dopamine levels can quickly occupy the receptor and trig-
ger increased PLM. As it turns out, this is exactly what we now know: The best neu-
roleptic drugs are those with the most rapid rate of receptor dissociation.
Drugs with a fast “off rate” are generally weak and less potent, precisely
because of their rapid dissociation from the receptor. Typically they have to be
administered at higher doses. Traditional drug discovery efforts have focused on
finding agents with the slowest rate of dissociation, finding agents with the highest
possible potency. The more potent a drug is, the lower the concentration required
to produce receptor blockade and the lower the required dose. The cost to drug
manufacturers is also reduced, since treatment requires lesser amount of drug.
However, in the case of D4R-mediated PLM, the goal of therapy is not to completely
shut down the cycle, but rather to partially reduce the demand for methyl groups
until it matches supply. Thus conventional wisdom about finding the highest
potency drug does not apply in the case of the D4 receptor cycle. In fact such agents
could actually make matters worse by strongly interfering with dopamine-stimu-
lated PLM. Indeed this was the case for the first D4R-selective antagonist to reach
clinical trials (338). The drug, named L-745870 from Merck, was the product of a
crash drug discovery program initiated after discovery of the D4R and recognition
of its higher affinity for clozapine. L-745870 has at least a 100-fold higher affinity for
the D4R than clozapine, because it stays on the receptor 100 times longer. Specifi-
cally, the dissociation constant (KD) of L-745870 is 2 X 10-10 M(the concentration
required for occupying 50% of the receptor pool) while that of clozapine is about 4
X 10-10 M. This means that when the concentration of dopamine goes up, it has to
wait almost 100 times as long for the L-745870 to come off the receptor than it does
for clozapinc. The molecular mechanism of attention simply cannot wait that long.
L-745870 will require more than 10 seconds to dissociate from 50% of the receptors,
while attention occurs within 100-500 msec. Clozapine, on the other hand, dissoci-
ates in about 50 msec, allowing quicker dopamine access to the receptor. When
tested in the clinic, L-7458780 unfortunately worsened patient’s symptoms rather
than improving them (338). In hindsight, one can sec that this drug does not have
the right profile to be an effective neuroleptic drug, but at the time it was developed
D4 receptor-mediated PLM was unknown. Even more importantly, the very public
clinical failure of L-745870 was interpreted by many in the drug industry to mean
that agents acting on D4 receptors could not be useful antipsychotic drugs, damp-
ening interest in D4-related drug development and research.
Within the past several years, Dr. Phillip Seeman and colleagues have published sev-
eral papers in which they point out that atypical neuroleptic drugs are distinguished
by their rapid dissociation from D2-like receptors (276, 339,340). ‘While the
emphasis of these studies is mainly on the lower occurrence of Parkinsonism-like
side-effects with atypical agents, a very important issue, the improved therapeutic
response to atypical agents, especially improved cognitive function, can likewise be
attributed to their lower receptor affinity. It is my hope that even more effective
neuroleptic drugs may be identified using D4R-mediated PLM as the test system.

186
 Neuroleptic drugs can also improve DNA methylation in cells expressing the
D4R. By reducing dopamine-stimulated PLM they decrease the ongoing demand
for 5-methylTHF by the D4R cycle. As a consequence the limited amount of 5-
methylTHF will be diverted more toward methylation of homocysteine, lowering
the concentration of homocysteine as well as the concentration of SAH. This will
have the net effect of augmenting DNA methylation in, for example, GABAergic
mterneurons expressing the D4R.
Another important feature of most atypical neuroleptic drugs is their ability
to inhibit serotonin receptors as well as D2 and D4 receptors. Again, clozapine is the
prototypical example. It is 5-fold more potent at inhibiting the 5-1-if lA serotomn
receptor than D4 receptors, such that clinically used doses will also produce a par-
tial blockade of these serotonin receptors. Olanzapine, risperidone, ziprasidone and
quetiapine also exhibit some degree of serotonergic blockade. This has led to the
suggestion that at least part of the improved therapeutic profile of atypical agents
might be due to serotonin blockade. Their lower affinity for D2/D4 receptors
increases the likelihood that atypical drugs will be relatively non-selective at clinical
doses.
Alternative Treatments for Schizophrenia
While neuroleptic drugs are the overwhelming first choice treatment for
schizophrenia, other treatments have been reported to be at least partially effective.
For example, administration of the amino acid glycine has been shown to augment
the therapeutic effects of typical neuroleptic drugs (341,342), particularly improving
negative symptoms, although it is not effective when administered with clozapine
(343). Glycine is an inhibitory neurotransmitter at glycine receptors, but its benefi-
cial effects in schizophrenia have been attributed to it role as a positive allosteric
regulator of NMDA receptors (341). Glutamate is relatively ineffective at activating
NMDA receptors if there is not sufficient glycine present. D-cycloserine, a drug
originally used to treat tuberculosis, is also active at the glycine regulatory site on
NMDA receptors and exerts beneficial effects when added to typical neuroleptic
drug therapy (344), but not when added to clozapine (345).
Glycine is also the main source of single-carbon groups for the folate path-
way. In mitochondria, the ATP-producing organdies within cells, glycine is broken
apart by the glycine cleavage system and converted into formic acid (formate) (333).
As described earlier, formate exits mitochondria into the cytoplasm where it is cou-
pled to an empty molecule of tetrahydrofolate (THF) to make 10-formylTHF. Suc-
cessive reductions of 10-formylTHF yield 5,10-methenylTHF, 5,10-mcthyleneTHF
and finally 5-methyITFiF such that the methyl group carbon in 5-methylTHF
comes from glycine. Glycine is therefore an important source of methyl groups for
PLM. Another role of glycine involves the enzyme serine hydroxymethyltransferase
(SHMT), which, in mitochondria, catalyzes the conversion of serine to glycine while
simultaneously converting THE to 5-methylTHF (333). The cytoplasmic form of
the enzyme catalyzes the reverse reaction, however, forming serine from glycine and

187
using up 5-methylTHF in the process. D-cycloserine is also an inhibitor of SHMT
and could work to increase cytoplasmic levels of 5-methylTHF. It therefore remains
to be seen whether these folate-related actions of glycine and D-cycloserine might
contribute to their therapeutic benefits in schizophrenia.
Not surprisingly folie acid supplementation (added to standard neuroleptic
drug therapy) has been shown to improve schizophrenia in some patients, particu-
larly those with a folate deficiency (< 200 jtg/ml). Since the diet of many schizo-
phrenic individuals is not the best, folate deficiency is not an uncommon finding.
Two different surveys found folate deficiency in 17 to 36% of schizophrenia patients
and 31-43% of patients with major depression (346,347). Treatment with 5-methyl-
THF was especially effective, reducing residual symptoms by almost 50% in neuro-
leptic-treated patients (348). Taking folie acid only assures a sufficient supply of the
carrier of single-carbons, but does not necessarily assure a sufficient supply of the
single-carbon groups (e.g. methyl groups) themselves. 5-MethylTHF provides at
least one load of methyl groups and is preferentially transported into the brain. 5-
MethyTHF is currently used as a nutriceutical (a drug-like nutritional supplement)
in Europe, and should soon be available in the U.S. Since it cannot be patented, it
will have to find its way through the mental health marketplace without the assis-
tance of traditional prescription drug merchandizing. It seems obvious that persons
with schizophrenia or major depression should be given 5-methylTHF as matter of
routine, just to ensure an adequate folate supply.
Several studies have reported beneficial effects of omega-3 fatty acid supple-
mentation in treating schizophrenia (349-352), lending therapeutic significance to
the finding of reduced levels of polyunsaturated fatty acids (PUFAs) in red blood
cells (353) and in post-mortem brain (354). In the initial study reporting their effec-
tiveness, the aim was actually to see whether PUFAs could reduce movement-asso-
ciated side-effects of neuroleptic drug therapy. However, researchers were surprised
to also find significant improvements in memory and other measures of cognitive
function (349). A second study likewise found improvements in schizophrenia
symptoms as well as lessened tardive dyskinesia (350).
As discussed earlier in relation to ADHD, PUFAs are incorporated into phos-
pholipids and form the hydrophobic core of the membrane bilayer. The extra dou-
ble bonds in PUFAs introduce extra kinks in the long chain of carbon atoms,
making it more difficult for the phospholipids to pack close to one another. As a
consequence there are fewer phospholipids per square area of membrane, creating a
more fluid membrane. This effect is similar to that produced by methylation of the
phospholipid headgroups during dopamine stimulated PLM and increased levels of
PUFAs can affect the conformation-dependent activity of membrane proteins like
NMDA receptors and Ca+2 ATPase. However, dopamine is only released under
specific circumstances (i.e. during attention) and its transient effects are informa-
tional, while PUFA based fluidity is the steady background upon which the effect of

188
dopamine is superimposed. The level of this background is extremely important, as
is evident by the exceptionally high level of PUFAs (i.e. DHA) in the brain.
The incidence of alcohol abuse is high among people with schizophrenia
(355). If schizophrenia is indeed a disorder whose origin lies within the faulty regu-
lation of membrane fluidity, this might reflect a form of self-medication. Ethanol
increases membrane fluidity and, as described earlier, it shifts the flow of folate-
derived methyl groups toward the D4 receptor cycle, away from the non-receptor
pathway. A generalized increase in membrane fluidity could bring dopamine-
induced PLM into a more effective range for solid-state signaling. Increasing the
flow of 5-methylTHF to the receptor pathway could also be beneficial, especially
when its levels are low to begin with. Persons with schizophrenia don’t necessarily
elect to drink more alcohol as a medical decision, but they may derive a stronger
positive reinforcement from its use than do unaffected individuals. In addition they
may experience stronger withdrawal symptoms as its fluidizing effects wear off and
their own ability to restore normal fluidity shows its limitations. In any case, it
seems important to recognize that alcoholism and alcohol abuse may be related to
the underlying metabolic pathology of schizophrenia rather than to poor moral
judgement.
Hallucinations
As the hallmark symptom of schizophrenia, we should pay special attention
to hallucinations. What are they? Where do they come from? Does a “Unified The-
ory” of schizophrenia shed new light on their origins? Hallucinations appear to be
false perceptions caused by abnormal neuronal activity occurring within the very
same brain locations that give rise to normal perceptions. For example, during audi-
tory hallucinations the brain regions that are active are the same ones that are active
during the processing of real auditory events (356). This is perfectly reasonable.
Sound wave information is converted to the activity of particular nerves in a multi-
step processing system that provides us with the ability to perceive sounds. If, for
some reason, these very same neurons were activated either spontaneously or by
some mechanism of internal origin, it would generate the perception of a sound,
even it there was none.
When we dream during REM sleep, the mental images we experience are not
triggered externally, but they can create a very good imitation of the real thing. We
all dream and think nothing of it. If those same dreams occurred while we were
awake, they would be a hallucination. In other words we are all capable of halluci-
nating, but there is an active mechanism which suppresses this ability. This suppres-
sive mechanism is commonly weakened or ineffective in schizophrenia, although
not everyone with schizophrenia experiences hallucinations.
One of the more effective ways of revealing our shared capacity for halluci-
nating is to interfere with or even eliminate our normal sensory experience. The
classical example is a sensory deprivation chamber where light, sound and other

189
sensory modalities are eliminated. As the inner mind is freed from contact with the
external world, it gradually begins to create its own perceptual experiences, a pro-
cess that can progress to overt hallucinations. Sleep deprivation can produce similar
effects. Persons who meditate for hours at a time also report experiencing hallucina-
tions. During religious meditation such hallucinations may take on a spiritual sig-
nificance.
The occurrence of hallucinations in otherwise normal individuals tells us that
ongoing sensory contact with our environment is part of the mechanism by which
hallucinations are suppressed. Our brains have the intrinsic capability to hallucinate
Specialists in the study of hallucination have proposed that one of the contributing
causes is a loss of the usual dominance of sensory experience (357). Even Emil
Kraeplin, the “Father of schizophrenia” who first gave the syndrome its name,
described its primary feature as a “loosening of associations” (358). Indeed, a loos-
ening of the mind’s association with the external world can set the stage for an inter-
nally generated alternative reality that is freed from the usual constraints of physical
world experience.
Synchronized oscillations are prominent during active attention and con-
scious experience and also during REM sleep. They may well be the electrical repre-
sentation of percepts arising from different circuits, joining together and flowing
from region to region in the brain. 40 Hz theta-gamma frequency complexes are
generated during attention to external stimuli and when they occur, even during
REM sleep, they are interpreted as reflecting sensory experience. Schizophrenic
individuals are not efficient in the production of 40 Hz synchronized oscillations in
response to, for example, an attended auditory tone (359). This deficit can be attrib-
uted to limitations in their capacity to initiate dopamine-stimulated PLM or a
reduction in its influence on the interneuronal systems that generate 40 Hz oscilla-
tions. No matter what the precise cause, this implies that the dominance of external
sensory information is compromised in schizophrenia and the capacity for atten-
tion is not what it should be. Another manifestation of this deficit is the well-docu-
mented reduction in the so-called P300 wave, which normally occurs in EEGs about
300-500 milliseconds after introduction of a novel sound or stimulus. It is signifi-
cantly reduced in schizophrenia (360) indicating a loosening of the usual associa-
tion of neuronal firing with external stimuli. It’s not that schizophrenics don’t
perceive the sound, it’s that they don’t have the ability to attend to it in the normal
manner.
Hallucinations may be associated with a loss of the capacity for attention to
external stimuli. False percepts that constitute a hallucination may arise from inter-
nally driven oscillatory circuits that dominate conscious thought because of the
weakened effectiveness of externally driven percepts. This implies a kind of compe-
tition or interplay between internally and externally derived information, bringing
us to one of those really big questions about our minds: Where does that internal
voice come from anyway?

190
 Truth is, of course, that we don’t know the origins of our inner voice. It is
clearly a “normal” feature, at least until it goes astray and becomes a feature of
insanity. Isn’t it interesting how rarely we acknowledge the presence of these voice-
like internal conversations? Often it is the voice of “self’, a manifestation of our very
core identity that we guard and cherish as “me”. Our internal voice gets turned off
during periods of active attention and the absence of this internal voice is a hall-
mark feature of high-quality attention to external events. When we are caught up in
a particularly absorbing activity, such as playing or listening to a piece of music, we
enjoy periods of mindless engagement that can refresh our thought process and ele-
vate our spirits. We can become similarly distracted and engaged by much more
mundane events like active involvement in daily tasks and jobs. Imagine being less
able to engage in such invigorating distraction and instead being constantly engaged
in internal discourse and struggling self-analysis. Perhaps that is a small part of
schizophrenia.
Neuroleptic drugs reduce hallucinations, suggesting that there is a role for
dopamine and D4-like receptors in causing internally generated percepts. Typical
neuroleptics, like haloperidol for example, also interfere with externally generated
percepts, so they are not selective for hallucination-associated events. In normal
persons these drugs suppress 40 Hz oscillations (361) and, at higher doses, cause
dissociation from the surrounding environment without loss of consciousness (i.e.
neuroleptic anesthesia). Clozapine, an atypical agent, also reduces hallucinations
but with less impairment or even an improvement of cognitive function, presum-
ably because of its rapid dissociation from the D4 receptor. In this sense, clozapine
is more selective for reducing hallucinations because it does not suppress attention
to external stimuli as much as typical neuroleptics. To my knowledge there is no
evidence that hallucinations are due to internally generated 40 Hz oscillations, but
no evidence that they are not either. Since REM sleep dreams are associated with 40
Hz activity (108), hallucinations may be as well.
All that is necessary to cause an inappropriate attentive episode is for clopa-
mine to be released in inappropriate areas of the brain. Dopamine is released from
fibers emanating from the VTA (ventral tegmental area), whose activity is in turn
governed by glutamate-releasing nerves originating from the hippocampus. False
attention could then be generated by misguided activation of VTA-derived neu-
rons, initiated by similarly misguided outputs from the hippocampus. The hippo-
campus is most renowned for involvement in memory, both short-term and long-
term, and the discharge of these memory circuits could drive internally generated
40 Hz activity during hallucinations, under conditions where external inputs to the
hippocampus (e.g. via the entorhinal cortex) are weakened.
Paying the Price for “The Gift of Attention”
Nature and evolution are clever craftsmen, but there is an element of imper-
fection inherent in all biological events, including the molecular mechanism of
attention. This built-in imperfection leads to DNA mutations and naturally occur-

191
ring genetic experimentation that continues to this very moment. Over millions of
years mutational events have developed and refined human capabilities and the
window for error-based experimentation remains open. It is this intrinsic error rate
that sets the stage for schizophrenia. It seems that the least we should do in return is
to acknowledge the value of this gift by comforting and, if possible, curing those
who pay the price for our most precious gifts.
Personally, I believe that there is great hope for finding a real “cure” for
schizophrenia or at least for finding medications that are vastly superior to current
neuroleptic drugs. My optimism is based upon the fact that many or most persons
who develop schizophrenia have previously demonstrated their ability to function
normally. Since schizophrenia occurs as a transition from normal to abnormal, we
should be able to understand the events that occurred during this transition and
reverse them, turning the developmental clock backwards, so to speak. What parent
of a 30 year-old with schizophrenia doesn’t wish to return to the years before the
disease took hold? From the unique perspective I have gained via my laboratory’s
research in the past several years, I believe that the key to turning back the clock lies
with understanding the age-dependent modulation of the IGF-1 signaling pathway
as we transition from juvenile status to adulthood. It is reasonable to expect that this
knowledge will lead to totally new treatment approaches that more directly address
the fundamental underlying cause of schizophrenia.

Chapter Summary:
• Schizophrenia is a heterogeneous psychotic disorder that occurs in about 1% of the
worldwide population, equally in men and women, with a typical onset in late teens and
the twenties.
• While the cause(s) of schizophrenia are not established there is an important genetic
component and multiple risk factors contribute to its overall incidence.
• Major theories on the cause of schizophrenia focus on abnormalities in dopamine
signaling, single-carbon folate metabolism, glutamate receptor function or membrane
structure.
• A “Unified Hypothesis” can be formulated that recognizes the contribution of individual
components to normal attention.
Neuroleptic drugs used to treat schizophrenia act by blocking dopamine receptors,
including the D4 receptor subtype, which can serve to reduce the demand for folate-
derived methyl groups.
• Paradoxically, neuroleptic agents with lower receptor affinity have a superior clinical
profile because they allow access of dopamine to its receptors.
• Some studies have found clinical benefit from omega-3 fatty acid or folate
supplementation as well as treatment with glycine.
• Improved understanding of the biochemical basis of schizophrenia should lead to
treatment approaches that are more effective and less debilitating than currently employed
neuroleptic drugs.

192
13 AUTISM

It was 10:30 PM. Margery collapsed into her La-Z-Boy chair and took in a
deep breath, a very deep breath, after another exhausting day. Thank God she made
it through. With a little luck Keshawn would sleep through the night. Of course
without a little luck he could be up for hours on end like last Thursday night when
the two of them kept company until almost 2 AM. That was a bad one. She had
heard four year-old Keshawn moving around in his room at midnight and found
him on the floor, repetitively hitting his right leg while clutching his favorite stuffed
animal with his left hand, rocking back and forth the whole time. This certainly
wasn’t unusual, and Margery could have just left him there, but she didn’t want to
face the next day if Keshawn didn’t get at least some sleep. Moreover, she knew that
this perseverative behavior could go on for hours unless she did something. She
tried reading him a story but stopped after a few minutes without having distracted
him a bit. It was like she wasn’t even there, another example of their one-way rela-
tionship. When she finally picked Keshawn up and put him back into bed he threw
a particularly nasty tantrum, kicking and screaming for almost a full hour. When he
finally tired himself out, Margery dragged herself back to bed knowing that she
would be worthless at work tomorrow.
Now, a few days later, Margery felt back to “normal”. Of course normal was
no picnic. Normal meant hoping that Keshawn didn’t wake up smelling from urine
and feces. Thankfully they had been making some progress with potty training,
using pieces of chocolate as a reward but even at four years it was still a lucky day
when he would go accident-free. Breakfast was sometimes a struggle and frequently
a mess. Luckily Keshawn’s appetite had picked up as he grew so that getting him to
eat was less of a problem. They were off to the Sunshine Center by 8 o’clock and
Margery typically made it to work by 9. Her mom usually picked Keshawn up at 3
o’clock, although her sister helped out once or twice a week. For now, at least, they
had a system worked out. Of course it would be a lot easier if her husband Donnell
was still around, but their relationship just couldn’t stand up to the extra pressure of
Keshawn’s autism. At least he gave her a break by taking care of Keshawn every
other Saturday.
While getting through each day was her main concern, Margery couldn’t help
worrying about what the future held for Keshawn. She loved him with an intensity
that secretly surprised her. Maybe it was because he was so much in need of mother-
ing. Maybe it happened because they needed each other so much. He only had a few
phrases that he could use to communicate and in this shared silence Margery had
learned to anticipate his thoughts and needs as best she could. She couldn’t imagine
her life without him, but sooner or later that was bound to happen. Would he ever
be able to function on his own? What happens after school years? Would he have to
be placed in an institution? Knowing what a struggle it had already been to get the

193
medical insurance company and the government to provide financial help, would
autism commit her to a life of poverty? They say that whatever doesn’t kill you
makes you stronger and Margery hoped and prayed that she would have the
strength and the energy to provide Keshawn the life he deserved.
Thank God for the chat groups that gave Margery so much support and
information about autism. Shortly after the doctor gave them the diagnosis for her
son’s developmental delay, Margery when online to Yahoo and found a group
focused on autism in children. It connected her with other mothers dealing with the
same issues and in many cases they had already “been there and done that”. She
learned about how to set up a regular routine that would work the best for both Kes-
hawn and her. She heard a sometimes bewildering array of treatment recommenda-
tions that might lead to at least a small gain in function (a gluten-free diet, B
vitamins, metal chelation therapy etc.). These seemed to be the only hope since the
doctor had said there wasn’t any real drug treatments for autism unless there was a
problem with seizures or something like that. Most importantly she gained personal
strength from the courage shown by others as they coped with autism and many
times she was just thankful that Keshawn wasn’t as bad as some of the other kids
with autism that she was hearing about.
From time to time Margery wondered about what caused Keshawn’s autism.
He had seemed perfectly normal at birth. It was only at his eighteen-month check-
up that the pediatrician expressed concern about whether he was meeting the
expected milestones for a toddler. He hadn’t started talking yet and his motor devel-
opment was also a bit delayed, but there was no real reason for concern, He was a
just a little late. Several of the autism websites that Margery used had a lot to say
about vaccinations being a cause of autism. She even saw an article in Newsweek
saying that parents were concerned about mercury poisoning and about the mer-
cury-containing preservative thimerosal that was in vaccines. Margery didn’t have
much of an opinion one way or the other about these speculations. As far as she was
concerned vaccinations were safe. Autism was probably caused by some random
accident, like a birth defect that just isn’t discovered until later on. Everybody gets
vaccinated. It’s the law. Everybody doesn’t get autism.
Autism is one of a group of “Pervasive Development Disorders” that includes
Rett Syndrome, Asperger’s Syndrome, Childhood Disintegrative Disorder and Per-
vasive Development Disorder Not Otherwise Specified, as classified in the DSM IV
(211). The movie “Rainman “, in which Dustin Hoffman played the role of an autis-
tic individual with a savant talent for memorizing trivia, provided the public a view
of this still mysterious disorder.
Likewise Doug Flutie, football quarterback extraordinaire and father of an
autistic child, has stimulated public awareness of autism and worked to raise for
parents and for research. Other parents and activists have worked hard to intensify
government and private sector support in an attempt to better and even cure

194
autism. As a disease with early childhood onset, it is not hard to share their fervor
and their frustration.
Impaired social interaction (joint attention) and communication are hall-
marks of autism (362). Affected individuals also tend to have a limited array of
activities and interests. In some cases they express extraordinary abilities in selected
skill areas, hence the identification as a savant. These individuals show incredible
gifts that seem to be the result of operating within a narrower behavioral bandwidth
than normal. Unfortunately these special gifts are overbalanced by gaps in other
abilities. Nonetheless they alert us to the potential for unique creativity and talent
that mysteriously shadows mental illness.
Autism most commonly shows itself during the second or third year of life.
Before that time development may appear to be normal or signs are too subtle to be
appreciated. Early signs can include a failure to respond to facial cues or to affec-
tionate attention, or even failure to respond to parental voices. These are the first
indications of isolation and internalized attention that become more evident as the
normal peer group gains their social skills. Many autistic children never develop
language. Commonly they don’t bond to parents or siblings and progressively
develop behavioral difficulties, which can include self-injury due to repetitive
actions, exaggerated responses to sensory stimuli, short attention span and hyper-
activity. Sleeping patterns are often disturbed. In general these symptoms indicate
that attention and attention-based learning problems are an important component
of the symptoms of autism.
Autism has a strong genetic component, evidenced by the very high concor-
dance among identical twins (90%) and six-fold higher incidence among siblings
(363). The incidence is about four-fold higher in boys than girls (parallel to
ADHD), raising a question about possible protective genes on the X-chromosome
or risk-conferring genes on the Y-chromosome. The actual incidence of autism has
recently become a point of controversy. Earlier estimates reported a prevalence of 5-
10 per 10,000 children or 0.05% (364). However, it has been suggested that this may
be a significant underestimate. For example, a prevalence of 38 per 10,000 or 0.3 8%
was found when a data set from 1970 was reviewed with contemporary criteria
(365). Although the diagnosis of autism is being made more frequently at present
largely because of revised criteria, evidence indicates that the incidence of autism
has been dramatically rising.
The unfortunate truth is that little is known about the cause of autism. Cur-
rent treatments are generally symptomatic and ineffective and there is no consensus
theory about underlying molecular mechanisms. Nonetheless, there are a number
of isolated observations that point the way toward where we should look for
answers.
One of the clearest points to build upon involves a rare, genetic cause of
autism that was first discovered in 1984 by Belgian scientists Jacken and Van den
Berghe (366). They analyzed the blood and urine from three autistic siblings and

195
found high levels of two unusual metabolic intermediates. The two metabolites were
succinyladenosine (SA) and succinylaminoimidazole carboxamide ribotide
(SAICAR), which would be expected to accumulate if the enzyme adenylosuccinase
lyase (ASL) was deficient (Fig. 13.1), but surprisingly, red blood cell (RBC) levels of
the enzyme were not reduced compared to control subjects. Significant levels of
both compounds were found in the patient’s plasma, but not in control plasma.
Moreover, concentrations of the two compounds in cerebrospinal fluid (CSF) were
20-fold higher than plasma levels, suggesting that the brain form of ASL was proba-
bly abnormal, even though the RBC form of the enzyme was normal. Subsequent
studies confirmed this pattern and identified several different mutations in ASL,
each of which is associated with autism (367-369). Although it is only a rare cause of
autism, the fact that ASE mutations cause autism is a significant clue.
ASL catalyzes two steps in the de novo purine synthesis pathway that synthe-
sizes adenosine monophosphate (AMP) and the precursor purine inosine (Fig.
13.1). A decrease in ASE activity not only leads to lower levels of AMP, but can also
reduce the levels of its higher energy diphosphate (ADP) and triphosphate (ATP)
forms (370). Activity of the de novo purine synthesis pathway is controlled by nega-
tive feedback of its products, (AMP, IMP and GMP) such that when ASL is mutated
and less active there is a reduced level of feedback inhibition, stimulating the rate of
steps leading up to the ASE block. This causes a massive build-up of SA and
SAICAR in the brain, the plasma and eventually in the urine, as discovered by
Jaeken and Van den Berghe (366).
Acceleration of de novo purine synthesis has important ramifications for
folate metabolism and for folate-dependent PEM. 10-FormylTHF is a cofactor for
de novo purine synthesis, contributing single-carbon groups to both the third and
eighth steps in the pathway. The build-up and urinary loss of SA and SAICAR
means that a large number of single-carbon groups are wasted because they never
made it down the pathway all the way to adenosine. Moreover, the flow of available
single-carbon groups is diverted toward de novo purine synthesis, decreasing their
availability for other folate dependent reactions such as dopamine-stimulated PEM.

196
Indeed our lab showed that D4R-mediated PLM is greatly reduced under condi-
tions where de novo purine synthesis is increased (20).

Figure 13.1: The role of adenylosuccinase lyase (ASL) in de novo purine synthesis. Mutations in ASL
can cause autism, associated with the urinary loss of high amounts of the precursor intermediates,
including a folate-derived formyl group.

Since CSF levels of SA and SAICAR are 20-fold higher than plasma levels, it
appears that most of the wasted single-carbons are coming from the brain and the
amount of loss is truly impressive. The concentration of each of the two wasted
metabolites in urine was found to be about 1.7 millimoles per liter (366). Since SA
contains one folate-derived carbon and SAICAR has two, together they represent
over 5 millimoles per liter of folate-derived carbons. Daily urinary output is
between one to two liters, so the loss could be as much as 10 millimoles per day.
Since a millimole is 6 x 1020 atoms, this equals 60 x 1020 lost single-carbons per
day, or 7 x 1016 per second. You get the point. By diverting carbon atoms to de
novo purine synthesis, ASL mutations can lead to a deficiency of 5-methylTHF and
to an impaired capacity for folate-dependent attention. Other folate-dependent pro-
cesses would also be adversely affected.
Tetrahydrobiopterin (BH4) is another important metabolic co-factor whose
level is reportedly reduced in autism (371). BH4 is essential for reactions in which a
hydroxyl group is added to a phenyl ring, and is required for the synthesis of dopa-
mine and norepinephrine as well as serotonin. Accordingly, BH4 is localized in
dopaminergic, adrenergic and serotonergic neurons, where it is synthesized from
GTP by the action of GTP cyclohydrolase and other enzymes. Low BH4 can reduce

197
the levels of all three of these biogenic amines, consistent with at least one study
which found lower dopamine levels in the prefrontal cortex in autism (372). Treat-
ment with BH4
produced some improvement in some autistic subjects, associated with a nor-
malization of D2-like receptor density (373). Any deficit in BH4 activity could easily
synergize with a folate deficit to produce more dramatic reductions in dopamine-
stimulated, folate-dependent PLM.
Dr. Ted Page and his colleagues at the University of California at San Diego
have made several significant biochemical findings that provide further clues about
the molecular basis of autism and pervasive developmental disorders. For example,
they measured the rate of de novo purine synthesis in fibroblast cells from seven
autistic patients using [14C]-labeled formic acid, the same approach we have used
for measuring folate-dependent PLM (374). They found a greater than 3-fold higher
rate of entry of the folate-derived single-carbon group into newly synthesized
purines in autistic patients as compared to normal controls. In other words a higher
than normal proportion of single-carbon groups was being used for purine synthe-
sis decreasing the amount available for D4R-mediated PLM. Together with mutant
ASL findings, these results provide very strong support for a link between autism
and the diversion of single-carbon folates away from the molecular mechanism of
attention.
Page and colleagues also studied a group of four patients with Pervasive
Development Disorder (PDD), a disease category which includes autism as well as
other related disorders. These individuals exhibited profound speech delay, extreme
hyper-activity, inability to focus and a “delirious” affect and abnormal social inter-
actions (43,375). Unlike the autistic patients described above, these individuals had
significantly lower rates of de novo purine synthesis than controls. Even more strik-
ing, however, was a six- to ten-fold increase in the activity of the enzyme 5’-nucleo-
tidase (5’-NTase). 5’-NTase catalyzes the formation of adenosine from AMP,
implying that these individuals produce adenosine at a much higher rate than nor-
mal, which would raise adenosine concentrations significantly, with significant con-
sequences for D4R-mediated PLM.
Remember that adenosine is a part of the cycle of dopamine stimulated,
folate-dependent methylation cycle. It is released from S- adenosylhomocysteine
(SAH) by SAH hydrolase in a reversible reaction (Fig. 13.2). Because this reaction is
reversible any increase in the products adenosine and homocysteine (or the homo-
cysteine form of the D4R) will cause the reaction to occur in the opposite direction.
This means that a build up of adenosine will inhibit D4R-mediated, folate-depen-
dent PLM and will increase the concentration of SAH. In subjects with excessive 5’-
Ntase activity, we would therefore expect to find impairment of dopamine stimu-

198
lated PLM with adverse consequences for attention and attention-related learning
and behaviors .

Figure 13.2: Activity of 5’-nucleotidase is increased in some individuals with autism,

while in others the activity of adenosine deaminase is decreased. Each of these
changes promotes a higher level of adenosine and impairs homocysteine
methylation as well as D4 receptor-mediated PLM.
Dr. Page and colleagues also found that administration of uridine caused a
significant clinical improvement in each of the four patients with elevated 5’-NT
activity (375). We conducted an analogous test on PLM using cultured human neu-
roblastoma cells, adding adenosine to mimic the effect of increased 5’-NTase, which
caused a modest decrease in dopamine-stimulated PLM. When uridine was added
under normal conditions it had no significant effect, but when added to the high
adenosine group, it restored normal PLM activity. Uridine therefore showed an in
vitro activity that paralleled its beneficial effect in autistic individuals with elevated
5 ‘-NTase activity.
Uridine’s ability to promote PLM depends upon the presence of a high level
of adenosine. This indicates that the mechanistic basis for uridine’s therapeutic
effect most likely results from the ability of UMP, uridine’s monophosphate deriva-
tive, to compete with AMP for hydrolysis by 5 ‘-NTase, thereby reducing adenosine
formation (Fig. 13.3). Under normal cell culture conditions the importance of 5’-
NTase for PLM is relatively minor, since the adenosine level is kept low by the
actions of adenosine kinase and adenosine deaminase (Fig. 13.2). However, when
adenosine levels are increased, UMP inhibition of AMP hydrolysis expresses itself
as an increase in the rate of folate-dependent PLM. This action of uridine points the

199
way toward novel opportunities to treat this type of pervasive development disor-
der, and may also be useful in other attention-related disorders.

Figure 13.3: Inhibition of adenosine formation by uridine. Since both AMP and
UMP compete for the enzyme 5’-NTase, administration of uridine can decrease
adenosine formation when it is converted into UMP.
Another link between adenosine metabolism and autism was provided by
studies of adenosine deaminase (ADA). ADA catalyzes removal of an amino group
from adenosine and its replacement by oxygen to yield inosine. Lower ADA activity
is associated with autism (376). The ADA gene exhibits a number of polymor-
phisms and mutations. Two alleles, designated ADA1 and ADA2, differ by only one
nucleotide, leading to the switch of an asparagine residue for an aspartic acid resi-
due in the enzyme. This change reduces ADA activity such that persons who are
heterozygous (ADA1/ADA2) have about a 20% lower enzyme activity, as measured
in blood cells (377). ADA1 accounts for 96% of total alleles and ADA2 for only 4%
in control populations. In the Italian-descent population studied, 7.9% of controls
were heterozygous and none (out of 152) was homozygous for ADA2 (378). In their
sample of autistic patients they found a significantly higher overall frequency of
ADA2 alleles (8.8%), a higher frequency of heterozygotes (17.6%) and one individ-
ual (out of 91) who was homozygous for ADA2. This association between the lower
activity allele of ADA and autism is consistent with a risk from an excess adenosine
similar to increased 5 ‘-NTase activity. As illustrated in Fig. 13.4 both of these meta-
bolic abnormalities, as well as ASL mutations, would have an adverse effect on the

200
efficiency of homocysteine re-methylation and folate-dependent PLM. If they
occurred together they could synergize to cause even more severe inhibition.

Figure 13.4: In autism impaired de novo purine synthesis or altered adenosine

metabolism could adversely affect dopamine-stimulated PLM, thereby producing
deficits in attention.
If the preceding observations of autism-associated metabolic defects are so
obvious, why haven’t they been followed-up? After all, its been eighteen years since
Jaeken and Van den Berghe first identified ASL as a likely cause of one form of
autism and the field of autism research has been desperate for solid clues. Well,
that’s a very big and a very important question that concerns the large number of
observations continually being made by scientists, which can lead to a large number
of highly divergent hypotheses. How do we know which new findings will turn out
to be most significant and most worthy of follow-up? In short, we don’t know. For
me, however, the key factor is how well such new findings integrate with other
already existing findings. Do they bridge a gap in knowledge or do they just stand
alone in isolation? For the most part, all well conducted scientific studies yield cor-
rect results, describing some particular aspect of a disease process. Given that confi-
dence, the trick is to understand the relationships that bring all these correct
observations into a singles. cohesive framework, a framework that, is relevant to the
disease in question. In the case of mutated ASL, observations of increased de novo
purine synthesis, increased 5’-NTase activity and decreased ADA activity, their rela-
tionship to impaired attention and attention-related learning, the hallmarks of

201
autism, was not obvious until the discovery of D4R-mediated, folate-dependent
PLM. Similar to schizophrenia, the critical breakthrough in thinking is to recognize
how these metabolic processes relate to neurotransmission and to dopamine-
dependent neurotransmission in particular.
There are a number of other significant observations that may bear upon the
cause(s) of autism, although a consensus hypothesis has not as yet emerged. These
have focused on increased brain size, altered serotonergic neurotransmission,
immune dysfunction, G! peptidase deficits, effects of the peptide secretin as well as
adverse effects of vaccines and/or their ethylmercury-containing preservative thi-
merosal. The Institute of Medicine, at the request of the Centers for Disease Control
and the National Institutes of Health, conducted a study of possible relationships
between thimerosal containing vaccines and neuro developmental disorders. Their
report, released to the public in October of 2001, found credible evidence of neuro-
logical damage due to vaccine-related ethylmercury exposure (379). Moreover, a
remarkable breakthrough article entitled: “Autism: A Novel Form of Mercury
Poisoning”, specifically proposed that the recent rise in autism was caused by
the mercury in vaccines, based upon striking similarity between autism symptoms
and those associated with mercury toxicity (380). Thimerosal is now removed from
most vaccines in the U.S. as a result of Food and Drug Administration actions in
1999.
During the writing of this book our lab was in the midst of investigating the
ability of IGF-1 and the P13-kinase system to regulate folate dependent methylation
of homocysteine, a project that remains ongoing. We were growing our human
neuroblastoma cells in two different incubators, one of which was lined with cop-
per. A graduate student, Jorge Benzecry complained that his cells gave different
responses when grown in the copper-clad incubator and the possibility became a
point of group discussion. It didn’t seem to make sense that the metal walls could
affect cells growing inside petri dishes, but we decided to investigate anyway.
Mostafa Waly, who was doing the IGF-1 work, added different amounts of copper
sulfate or copper chloride to cells and was surprised to find that it caused a modest
stimulation of folate dependent PLM. Further studies showed that removal of cop-
per inhibited the stimulation by IGF-1 and that other metal ions also potently inter-
fered with the IGF-1 response. As it turns out they also interfered with dopamine
stimulated PLM because they were reducing the activity of methionine synthase.
Among the metals we examined, mercury and lead were particularly potent,
producing a decrease of folate-dependent PLM at nanomolar (nM) concentrations,
far below the serum levels that are commonly used to define mercury or lead poi-
soning. Mercury levels above 1 nM produced a progressive inhibition of the IGF-1
response, Remarkably, the presence of copper (Cu2+) protected against the inhibi-
tory action of mercury, suggesting that the two metal ions competed for a particular
site. If mercury was this potent, how might thimerosal compare? As shown in Fig.
13.5, thimerosal was more than 100-fold more potent than mercury in blocking

202
basal and IGF-1-stimulated PLM. This result indicates that the ethylmercury
released from thimerosal is a much more potent inhibitor than mercury itself.
Although more work is needed to confirm the relationship of this striking observa-
tion to autism, the exquisite sensitivity of folate-dependent PLM to thimerosal
strongly supports the theory that mercury-containing vaccines might contribute to
autism.

Figure 13.5: Dose-dependent inhibition of basal and IGF-1 stimulated PLM by

thimerosal. The threshold for inhibition is 0.01 nM and half-maximal inhibition
occurs at about 3 nM. Vaccination produces blood levels between 10-8 and 10-7
(202).
By interfering with the activity of the P13-kinase signaling system thimerosal
and heavy metals lead to diminished methionine synthase activity and consequently
to the accumulation of homocysteine. We found that exposure of neuronal cells to
10 nM thimerosal for 60 min completely inhibited methionine synthase activity
(202). Lower methionine synthase activity will also impair D4R-mediated PLM and
the potential for a negative impact on the mechanism of attention is obvious. In
addition, accumulation of homocysteine and its interconversion product S-adeno-
sylhomocysteine can adversely affect other methylation reactions including DNA
methylation. Indeed in cultured cells thimerosal, at a concentration produced by
vaccination (i.e. 10 nM), reduced global DNA methylation by over 30% (202). Such
a drastic change is likely to affect gene expression and could therefore affect the
course of brain development. Indeed we found that as little as 0.1 nM thimerosal
caused dramatic changes in the development and morphology of SH-SY5Y cultured

203
human neuroblastoma cells. As shown in Fig. 13.6, thimerosal treatment caused
these normally spindle-shaped cells to condense and round up. After thimerosal
was washed out, cells resumed normal growth and appearance. These findings offer
a ready explanation for the loss of normal attention and the failure of neurological
development that characterizes autism.

Figure 13.6: Thimerosal exposure (0.1 nM for 4 days) induces changes in the
morphology of SH-SY5Y human neuroblastoma cells.
The chance of developing autism in response to mercury poisoning is partic-
ularly high in individuals who carry genetic risk factors adversely affecting path-
ways that are important for DNA methylation such as adenosine metabolism or de
novo purine synthesis. At this time it is unclear whether the toxic effects of thimero-
sal are limited to neuronal cell types and whether it only affects cells at certain stages
of development. More research is needed to answer these and many other ques-
tions. Nonetheless, these extremely potent effects of thimerosal indicate that folate
related methylation events and DNA methylation in particular are the likely arena
for the origin of some types of autism.
Could the rise in ADHD during the past several decades also have been
caused by vaccine-associated thimerosal exposure? Instead of producing the severe
symptoms of autism, ethylmercury might produce milder consequences in individ-
uals who are not genetically loaded for autism risk. Such milder symptoms would
still be expected to involve impaired attention and developmental delay. Individuals
with genetic risk factors in the attention pathway (e.g. the seven repeat form of the
D4 receptor) could be at greater risk from a mercury or ethylmercury insult.

204
 Since mercury and ethylmercury are slowly cleared from the body (379) we
might expect the toxic effects of neonatal exposure to be transient. As yet we have
not characterized the reversibility of their inhibitory influence on methionine syn-
thase, but these certainly would be critical studies. Nonetheless, repeated adminis-
tration of thimerosal-containing vaccines over an extended period of time could
produce alterations in the pattern of gene expression that could have very long con-
sequences. During early years there are windows of development that open and
then close. Growth and development of interneurons is one example. Abnormalities
in the molecular events of attention and attention-related learning during these
periods can be manifested as life-long deficits.
Drug Treatments for Autism
Neuroleptic drugs are a mainstay of current therapy for autism 381). They
lessen the severity of repetitive self-injurious ‘behaviors and decrease hyper-activity
and aggressive outbursts, but are not effective therapy for the major symptoms such
as impaired language development or attention deficits. Serotonin uptake inhibitors
(Prozac®-like drugs) are also used to combat obsessive behaviors, depression and
aggressiveness. Methylphenidate and other stimulants are used to improve atten-
tion and treat hyper-activity. Secretin administration, as part of a GI diagnostic test,
prompted a brief episode of improved attention and spontaneous speech in a very
limited number of cases (382), but most follow-up studies have unfortunately not
yielded any evidence of efficacy in autism (383-5). One recent study did report ben-
efit, particularly in patients who exhibited significant diarrhea, which is common in
autism (386). It of interest that an over-the-counter nutriceutical, dimethylglycine
(DMG), has been reported to be beneficial in autism by some parents and on line
sources, but controlled clinical trials failed to find any effect (387-8). Interest arises
from the fact that dimethylglycine serves as a source of formic acid when it is con-
verted to sarcosine, and formic acid is the source of single-carbon group for the
folate pathway. Thus DMG could theoretically be a way to fortify the supply of sin-
gle-carbon folates.
The previously described deficits in purine metabolism associated with
autism are no doubt present from the earliest stages of fetal development, since at
least some of them are the result of documented genetic mutations and polymor-
phisms. Consequently their detrimental effects may cause fundamental abnormali-
ties in pre-natal anatomic development as well as a failure to develop normal post-
natal skills and behaviors. Under these conditions, the earliest possible detection
and intervention is particularly important, since many developmental milestones
may be irreversibly passed. The cluster of purine- and folate-related metabolic
abnormalities provides an opportunity for pre-natal and early post-natal screening.
Since autism has such a clear pattern of genetic risk, such screening is doubly
important for pregnancies and births when there is a positive family history of
autism. The presence of one or more of these deficits could predict autism, setting
the stage for close observation and early therapeutic intervention.

205
 Novel treatment strategies should emerge from this new biochemical per-
spective on autism. Moreover, the varied nature of purine- and folate related abnor-
malities alerts us to the likelihood that there are many different individual causes of
pervasive developmental disorders, so that we should not expect any one treatment
to benefit everyone. This is of particular concern when testing the efficacy of treat-
ments, since interventions that are very useful and appropriate for some might not
help a sufficient number of others to create a significant outcome for the entire
autistic population. Accurate diagnostic testing and sub-grouping will then be the
key to customizing and optimizing drug therapy. In the case of isolated ASL muta-
tions, it would seem to make sense to supply an exogenous source of purines, which
should provide negative feedback to the de novo purine synthesis pathway and
reduce diversion of single-carbon folates. In the case of excess 5 ‘-NTase activity, it
is obvious that selective inhibitors of the cytoplasmic, neuronal form of this enzyme
should produce benefit, parallel to the action of uridine. The modest 20% reduction
of ADA activity in persons carrying the ADA2 allele may not be of sufficient magni-
tude to cause autism itself, but may synergize with elevated 5 ‘-NTase activity or
some other deficit (e.g. decreased adenosine kinase activity). In any case, inhibition
of 5 ‘-NTase activity would also be potentially helpful in such cases.
Clearly there is a need to further investigate the prevalence of purine and
folate metabolism-related deficits in autism, as well as deficits in other elements of
the folate-dependent methylation pathway. While schizophrenia appears to be the
manifestation of aberrant mid-life adjustments in folate metabolism, autism and
related developmental disorders may reflect the consequences of similar events in
early life stages. For example, early disruption of the dopamine-dependent mecha-
nism of attention will interfere with the coordination between learning and atten-
tion. In such a condition, the importance of external stimuli as the basis for growth
of GABAergic interneuron networks may be diminished, allowing a more dominant
role for internal, memory-based stimuli. Over time the consequence of this shift will
be a more involuted existence, perhaps accompanied by extraordinary capabilities
in selected domains because of developmental failure in other domains. The
remarkable talents of savant autistic individuals, expressed as a gift for learning and
creating music or as a gift for memorizing numbers or text, are attributable to this
unusual allocation of sensory and memory resources. Smaller differences in these
abilities, observed among “normal” persons, may reflect similar individual limita-
tions that reciprocally lead to individual strengths in other abilities.
A Further Clue from Rett Syndrome Rett syndrome is a relatively rare neuro-
logical disorder, exclusively affecting girls, that is typically first observed after six to
twelve months of seemingly normal development. There is a progressive slowing of
growth, deterioration of any communication skills and loss of purposeful hand use,
accompanied by characteristic repetitive hand movements. Because Rett syndrome
is restricted to females, its cause was anticipated to be related to the X chromosome.
In 1999 Dr. Hoda Zoghbi and colleagues identified a gene that accounts for over

206
80% of cases (389). The culprit turned out to be the gene coding for a DNA-binding
protein, MeCP2, that specifically attaches to methylated cytosine/guanine pairs in
regions of DNA that are modified by S-adenosylmethionine-dependent DNA meth-
yltransferases. Methylation of DNA, an example of epigenetic regulation, is a mech-
anism for suppressing for silencing the activity of particular genes. MeCP2 plays a
critical role by binding to the methylated CpG locations, allowing the binding of
other proteins that together provide stable repression of the gene. Genetic studies of
children with Rett syndrome revealed a large number of different mutations, all
clustered in the MECP2 gene on the X-chromosome (390). These mutations inter-
fere with the usual effects of DNA methylation, allowing normally silenced genes to
be continuously transcribed into RNA and protein products.
Association of Rett syndrome, a predominately CNS-based disorder, with
abnormal DNA methylation events leads to provocative questions about how
abnormal folate-dependent methylation might cause pathological events in other
conditions. For example, if Rett syndrome is caused by one type of DNA methyla-
tion abnormality, what other conditions would result if DNA methylation failed
due to other causes? Is the brain-specific nature of Rett syndrome an indication that
DNA methylation in the brain has a unique role to play in developmental disorders?
Metabolic abnormalities that adversely affect folate-dependent methylation
reactions can cause autism and other pervasive development disorders. While
decreased attention and attention-related learning are one consequence, purine-
and folate-related abnormalities can also lead to impaired DNA methylation and
altered gene expression. Recent studies have found associations between DNA
hypo-methylation and other mental illnesses, such as Down syndrome (391-2), and
defects in folate metabolism have long been recognized to contribute to develop-
mental and psychiatric problems (393). By recognizing the folate connection to de
novo purine synthesis, adenosine metabolism and IGF-1 signaling we can postulate
with some confidence that many of the symptoms of autism and other pervasive
developmental disorders may primarily result from defective DNA methylation. In
broader terms, normal development of all tissue types appears to depend upon a
progressive program of neuronal gene silencing and gene expression controlled by
DNA methylation. This program is initiated upon conception and affects the well-
being of the organism throughout its lifespan. Any insult to normal DNA methyla-
tion events, arising genetically or by environmental exposure, has the potential for
causing disease.
DNA methylation in nerve cells may be particularly vulnerable to folate dys-
function since the re-methylation of homocysteine is more highly dependent upon
folate-derived methyl groups, due to the absence of betaine homocysteine methyl-
transferase (BHMT), which is itself apparently silenced in a tissue-specific manner.
As a consequence of this absolute folate dependence, availability of methionine for
SAM synthesis may be limited if 5-methylTHF levels are low. Inborn errors of
metabolism involving the folate pathway are therefore particularly evident as neu-

207
rological disorders. Because
of the ability of homocysteine to recombine with adenosine, any failure of methio-
nine synthase to efficiently re-methylate homocysteine results in accumulation of S-
adenosylhomocysteine (SAH). SAH is a powerful inhibitor of DNA methylation
(Fig. 13.7) because it retains significant affinity for the DNA methyltransferase
enzyme after SAM-dependent methyl transfer. In fact the affinity of methyltransfer-
ase enzymes for SAH is frequently higher than their affinity for SAM (394).

Figure 13.7: S-adenosylmethionine (SAH) regulates DNA methylation and gene

expression. SAH levels are dependent upon the metabolism of homocysteine and
adenosine. Activation of P13-kinase by IGF-l or dopamine can influence DNA
methylation by augmenting methionine synthase activity while thimerosal and
mercury block this pathway. Availability of 5-meTHF is also critical and can be
affected by other pathways that utilize single-carbon folates.
The prevailing SAH concentration is determined by a number of factors
including the rate of its formation by methylation reactions, the activity of SAH
hydrolase, and by the concentrations of homocysteine and adenosine. Thus any
slowing of homocysteine re-methylation by methionine synthase, slowing of ade-
nosine removal by adenosine kinase or adenosine deaminase or increased adenos-
ine formation by 5’-NTase will cause SAH to increase and DNA methylation to
decrease. Faulty DNA methylation and failure of D4 receptor-based attention in
neuronal tissues may be the common thread that links Rett syndrome, autism and
other pervasive developmental disorders.

208
 In general, developmental disorders are neurological disorders. There is no
parallel problem with liver or cardiac development, indicating that brain function is
particularly vulnerable to disordered DNA methylation. Why would nature allow
such vulnerability to survive evolution? Perhaps because the brain is held in a devel-
opmentally immature state until birth in order to assure that its hard-wiring will be
responsive to postnatal sensory experience. If development were complete in utero
the ability to adapt and customize neuronal connections would be compromised. A
prime example is the postnatal development of interneurons. By holding off their
development until dopamine-stimulated attention can be used as a guide, nature
assures that brain connectivity is determined by actual experience. The unfortunate
downside of this delay is the greater vulnerability of brain development to postnatal
insults that affect DNA methylation. If attention is impaired then interneuron
development will not be driven by external input and associative learning will suf-
fer. Under these circumstances hard-wiring may be driven by internally-derived
stimuli to a greater extent. The fact that attention and neuronal development are
both controlled by the activity of methionine synthase (Fig. 13.8) assures their inte-
gration.

Figure 13.8: Dual consequences of impaired folate-dependent methylation.

Methionine synthase activity is critical for both re-methylation of the D4 receptor
and for conversion of homocysteine to methionine. Impairment of its activity
therefore adversely affects dopamine dependent attention and attention-related
learning as well as DNA methylation-dependent neuronal development.

Chapter Summary
• Metabolic abnormalities involving the single-carbon folate pathway and/or the
methionine cycle are a feature of autism and other pervasive developmental disorders.
• Deficits in attention and attention-based learning in autism may be due to elevated levels
of homocysteine or adenosine.
Increased levels of homocysteine and/or adenosine can lead
• to S-adenosylhomocysteine formation and inhibition of DNA methylation.
• Impaired DNA methylation may be a common feature of developmental disorders.

209
14 OTHER PSYCHIATRIC DISORDERS

Dysfunctional phospholipid methylation and folate metabolism are certainly

not the underlying cause of all mental illnesses. That having been said, these two
factors may make significant contributions to a number of such illnesses. In this
Chapter we will explore these possibilities, making connections wherever possible,
with the hope of provoking new thinking and stimulating exploration of new possi-
bilities.
There are a few unifying themes to consider. For example, different disorders
may result depending upon the stage of life during which dysfunctional folate-
dependent methylation becomes evident. If it were to start during fetal development
we might expect anatomic consequences to be evident at birth with serious lifelong
impairments that could include mental retardation. If the onset is post-natal the
consequences could be characterized as the developmental disorders we have just
discussed. Schizophrenia seems to reflect the risk of a metabolic transition from
juvenile to adulthood. In old age, we might expect consequences relating to the loss
of skills and abilities such as Alzheimer’s disease. In other words there are metabolic
transitions across the entire lifespan that can introduce age-dependent risks to our
molecular mechanism of attention. Another theme arises from the complex, multi-
step nature of folate-dependent biochemical pathways. There are many different
ways in which this system can go wrong and as a result many different syndromes
may occur, although they all share the common thread of impaired attention.
Mental Retardation
Mental retardation is defined as “significantly sub-average general intellec-
tual functioning with limitations in adaptive functioning” (211). Generally this cor-
relates with an ¡Q (intelligence quotient) of < 70. Further ; gradations are related to
IQ scores: mild (50 to 70), moderate (40 to 50), severe (20 to 40) or profound (< 20),
In several instances the underlying cause of retardation has been specifically identi-
fied and these examples are briefly described for comparative purposes.
Down syndrome is the most common genetic cause of mental retardation,
having an incidence of 1 in every 1,000 live births at a maternal age of 30 years but 9
out of 1,000 at a maternal age of 40 years. (395). It is caused by the presence of three
copies of chromosome 21 (trisomy), rather than the normal two and includes char-
acteristic anatomic abnormalities that can involve the heart and GI tract as well as
facial features. Trisomy occurs during meiosis (early egg or sperm formation) when
paired chromosomes that normally separate from each other fail to do so, creating
an egg or sperm with two copies of chromosome 21 instead of one. In 95% of the
cases, the extra chromosome is maternal in origin (396). Mental limitations and
anatomic features are all presumed to result from the increased representation of
the genes present on chromosome 21. Individual genes are likely to be more impor-
tant for some features of the syndrome than others, and those responsible for men-

210
tal retardation have not yet been specifically identified, although there is recent
evidence that abnormal folate metabolism may contribute to the occurrence of tri-
somy.
In 1999, Dr. Jill James and colleagues at the National Center for Toxicological
Research reported a significant over-representation of the 677C —> T MTHFR
polymorphism in mothers of Down syndrome children (44%) as compared to con-
trols (30%) (397). Synthesis of 5-methylTHF is reduced by 35% in persons who are
heterozygous for the 677T enzyme and reduced by 70% in persons who are homo-
zygous. Folate-deficiency can further reduce the availability of 5-methylTHF. As
would be expected, homocysteine levels were significantly increased in the mothers
that carried the mutant MTFIFR, since 5-methylTHF is necessary for its conversion
to methionine. However, elevated homocysteine was also found in Down syndrome
mothers who did not have the mutant MTHFR, indicating a role for other factors. A
follow-up study re-confirmed the MTHFR-related risk and found that a detrimental
polymorphism in methionine synthase reductase, the enzyme responsible for keep-
ing methionine synthase in its active state, also increased the risk of Down syn-
drome, even more than the mutant MTHFR (398). When present together, the two
polymorphisms increase the risk by more than 4-fold. Presence of the methionine
synthase reductase polymorphism also contributes to elevated homocysteine levels.
These findings strongly suggest that hypo-methylation of DNA sets the stage for tri-
somy, similar to the way low folate during pregnancy can lead to neural tube defects
such as spina bifida.
Dr, James and colleagues also examined folate and methionine cycle metabo-
lism in lymphocytes from children with Down syndrome. They found reduced lev-
els of all four elements of the methionine cycle, homocysteine, methionine, SAM
and SAH, and also found hyper-methylation of their DNA (399). This is in direct
contrast to the Down syndrome mothers, who had elevated homocysteine and
hypo-methylation of DNA. The culprit seems to be an extra copy of one of the genes
on chromosome 21, cystathionine beta synthase (CBS), which converts homocyste-
ine to cystathionine, taking it out of the methionine cycle. Supplementation of the
lymphocytes with methionine, folinic acid (5-formylTHF), vitamin B12, thymidine
or dimethylglycine (DMG) helped to normalize their metabolism, The fact that
DNA was hyper-methylated despite lower levels of SAM serves to emphasize the
importance of SAH as the key regulator.
Fragile X syndrome is the second most common genetically-based cause of
mental retardation, affecting 1 in 4,000 males and about half as many females (400).
Since its first description in 1969 (401), knowledge about the origin of fragile X has
gradually accrued until at present only a few remaining elements remain a mystery.
As its name implies, the critical problem is the presence of a particularly weak DNA
sequence on the long-arm of the X-chromosome that can break, although actual
breakage is not necessary for the syndrome to develop. In fact there are several frag-
ile sites located close to each other on the X chromosome (FRAXA, FRAXE and

211
FRAXF) and two other sites on chromosomes 11 (FRA 1 lB) and 16 (FRA 16A).
Each of these sites contains a repeated trinucleotide sequence (e.g. CCG) and it is
the presence of a high number of repeats which creates the fragility. For example, at
the FRAXA site less than 54 repeats is not a problem (normal 6 to 54), but more
than this can lead to instability of the region. The presence of more than 80 repeats
brings particular risk (called a pre-mutation) and causes the number of repeats to
greatly increase (e.g. to over 200) either during meiosis in females (i.e. egg forma-
tion) or during the early stages of cell division after fertilization. Both male sperm
and female eggs can carry pre-mutations, but for some reason they only expand to
full mutations in the female gametes (402). Expansion seems to occur because of the
redundant, repeated sequences themselves, causing slippage during the replication
process (403).
After a full mutation develops the region (rich in CpG dinucleotides)
becomes hyper-methylated. Methylation results in silencing of this gene which is
the proximal cause of the resultant mental retardation. In the case of FRAXA, the
adjacent gene is called fragile-site mental retardation-i (FMR1) and codes for a pro-
tein (FMRP) which appears to be involved in the formation and elimination of syn-
apses. FMRP has three RNA-binding domains, enabling it to crosslink different
RNAs to form active zones of protein synthesis in the small neuronal spines that
develop into synapses (404). In the absence of FMRP the number of mature spine
synapses was about half of normal. This anatomic finding may correlate with the
reduced capacity for learning.
Of further interest to our folate theme is the fact that actual breakage of the
fragile sites is promoted by low folie acid levels (405), probably because low folate
increases the need for DNA repair, which increases the chance of breakage. One
more good reason to make sure that folate intake is adequate.
Depression and Bipolar Disorder
Depression and bipolar disorder are interrelated mood disorders. Depression
is a unipolar condition of depressed mood while bipolar disorder is characterized by
the presence of manic episodes, mixed with depressive episodes. Taken together
they define a spectrum of dysfunctional mood with extremes of depression and
mania. Because almost everyone experiences at least some significant fluctuations of
mood, the term dysfunctional is vitally important in recognizing the presence of a
major impairment in abilities that distinguishes a psychiatric disorder. There are
elements from studies on the etiology of these disorders that suggest possible con-
nections to methylation events that are the focus of this section, notwithstanding
many other important factors that might also be involved.
Let me start by suggesting that the level of attention directed toward external
stimuli may be closely linked to mood. When we are actively engaged in attending
to something happening around us it adds to the quality of our existence. We are an
active part of the experiential world. Frequently this attention provides the motiva-

212
tion for what we decide to do or think next. It initiates action, physical or mental, as
a reaction to the attention experience. The experience helps to shape who we are, or
who we will be. However our motivation and our ability to be involved with the
external world is not constant. On a daily basis we experience a quieter mood as
evening turns to night and sleep approaches. We turn off the lights, turn off the tele-
vision and hope that the non-stimulating environment will bring rejuvenating
slumber. If sleep is deep and effective, we may wake and greet the new day with
energy and perhaps even with the highest level of optimism that we can muster. In
colder climates like Boston, an approaching Winter season may bring downward
shifts in mood which help us to be in harmony with the scaled down opportunities
for outdoor activity, perhaps a vestigial homage to hibernation. We take heart in the
confidence that sunny days of Spring will eventually lift our spirits and invite us out
into the active world. Of course some people aren’t able to sleep as well as others,
and some don’t greet the new day with optimism. Others may have particular diffi-
culty with seasonal adjustments. Some may not be drawn to the outside world, pre-
ferring to ponder their situation with a primarily internal dialog. The latter
descriptors may simply reflect the choice of a more introspective and self-contained
lifestyle, or, in the presence of sufficient dysfunction, they can be signs of mild
depression. Coupled with feelings of despair and hopelessness they can progress to
immobility, catatonia and signs of more severe depression with contemplation of
suicide and attempts at suicide and/or suicide as extreme outcomes.
At the other extreme of attention we find distraction progressing toward
mania. You move freely from one overly brief episode of attention to the next with a
briefer than normal interval for processing and respite. Your productivity is
increased and you feel you’re on a roll. Life is good! Maybe it’s that double-shot cafe
latte you had at lunch, but whatever the cause you’re full of energy. Confidence
abounds. Your thoughts are truly inspiring, at least to you. The ideas flow so quickly
that you can hardly keep up with them. You’re witty and entertaining. There’s cer-
tainly no trouble maintaining your end of the conversation, in fact you can easily
keep up both ends. Perhaps you’ll have a little trouble getting to sleep tonight. In the
back of your mind you know that you’ll crash sooner of later, but for now you can
just enjoy the ride. As described by Jameson (406) and others the state of hypoma-
nia apparently has its appeal. However, the extremes of full-blown maniac episodes,
lasting for weeks at a time, are not pleasurable. They are exhausting and are associ-
ated with increasing irritability, hostility and reckless behavior. Social and occupa-
tion dysfunction become obvious and hospitalization may be required. As a part of
bipolar disorder these episodes may be either followed by, or preceded by, periods
of deep depression and 10-15% of persons affected by bipolar disorder commit sui-
cide.
If depression is related to diminished capacity for attention and mania an
expression of excess attention, we might expect to find pharmacological connec-
tions to the mechanism of dopamine-stimulated PLM, and furthermore we might

213
further expect these connections to be opposite for each disorder. Certainly if you
wanted to produce flat affect (i.e. a depression-like state) you could give someone a
neuroleptic drug like haloperidol that tightly blocks D4-like receptors. It drastically
reduces interaction with the outside world, even to the point of producing catatonia
when higher doses are used. Conversely, if you want to elevate someone’s mood, or
cause mania, give them a dopamine releasing drug, such as caffeine, nicotine or
amphetamines. Indeed we abuse these drugs for the specific purpose of elevating
our mood. The link between dopamine and mood is obvious.
There is strong evidence for the importance of membrane fluidity in the etiol-
ogy of both depression and bipolar disorder. Not surprisingly, much of the evidence
derives from studies of omega-3 fatty acids. A decrease of omega-3 fatty acid levels
(measured either in red blood cells or in serum phospholipids) is associated with
depression (407). This difference persisted despite anti-depressant therapy, includ-
ing treatment with selective serotonin uptake inhibitors (SSRIs, or Prozac®-like
drugs), indicating that membrane abnormalities probably represent a separate and
distinct risk factor for depression. A number of studies have found a significant cor-
relation between higher consumption of fish and lower incidence of neuropsychiat-
ric illness when comparing diets from different countries around the world. For
example, a worldwide study of schizophrenia outcomes found that differences
could be attributed to the origin of fats in the diet (408). Fish and vegetable sources
were associated with better outcomes vs. land animal sources. A Finnish study
found that the incidence of depressive symptoms was significantly higher among
infrequent fish consumers than among frequent consumers (409), Despite these
observations, there have been no controlled studies showing a therapeutic benefit of
omega-3 fatty acid supplementation in depression.
Postpartum depression provides a special example of how a deficit of omega-
3 fatty acids can cause depressive symptoms. The growing fetus and the breast-feed-
ing newborn infant place a high demand on the mother for DHA, the most abun-
dant omega-3 fatty acid in the brain. It alone accounts for more than 20% of the
brain’s weight! The placenta has a special mechanism for transporting DHA from
the maternal circulation to the fetus to provide for the rapidly growing brain, result-
ing in a lowering of serum levels in the mother (410). This depletion continues dur-
ing breast-feeding, when maternal serum levels of DHA are reported to decrease by
more than 30% (411). The decrease was found to be even greater during subsequent
pregnancies, as compared to the first (412). The situation is further exacerbated by
the transfer of vitamins and folie acid from the mother to the fetus, as well as the
dramatic fall-off in estrogens and progesterone that accompany the end of preg-
nancy. The combination of these biochemical changes sets the stage for serious
postpartum depression if there has not been enough nutritional support during
pregnancy.
Omega-3 fatty acids have recently been shown to have remarkable therapeu-
tic effects in bipolar disorder. Dr. Andrew Stoll, from the McLean Hospital, Harvard

214
Medical School, conducted a clinical trial in which bipolar patients were given 9.2
gm/day (14 capsules) of a fish oil-derived mixture of EPA and DHA or an olive oil
placebo and were followed up for 120 days (413). Patients also received their usual
medication regimens. During this interval the rate of relapse (i.e. the frequency of
having a manic episode) was 60% in the group receiving olive oil, but only 12% in
the group receiving the omega-3 fatty acid-rich supplement. This is a truly remark-
able level of effectiveness for any drug therapy, let alone for a nutrient-based inter-
vention. Additional studies are underway to verify this finding and to explore the
mechanism for omega-3 fatty acid benefit, however, it seems that changes in nerve
cell membranes brought about by these fish-oil constituents have a powerful anti-
manic, mood-stabilizing effect.
Using human neuronal cells we found that a four-day supplementation of the
cell culture medium with DHA caused basal and dopamine-stimulated D4R-medi-
ated PLM to increase by over 100% (414). IGF-1-stimulated PLM was also increased
to a similar extent by DHA. Its longer chain length and extra double bonds may
make DHA-containing PE a more efficient substrate for methylation. High levels of
DHA in brain tissue therefore promote PLM and its depletion will have the opposite
effect. EPA and DHA also inhibit the activity of several protein kinases, including
protein kinase C (a phospholipid-dependent protein kinase), CAM kinase II
(important in memory formation), cAMP-dependent protein kinase A and MAP
kinase (415). These actions, along with their effects on PLM may contribute to their
therapeutic action in bipolar disorder.
S-adenosylmethionine (SAM) is widely used as an over-the-counter treat-
ment for depression. Its effectiveness has been documented in clinical trials, includ-
ing blinded, placebo-controlled studies (416-8). SAM’s level of effectiveness can be
quite substantial, rivaling and/or exceeding that of prescription antidepressants
(416). In most cases it is purchased and paid for by consumers themselves after
either self-diagnosis or professional diagnosis. Since SAM is rather cost expensive
(about $50 per week for 1,600 mg/day), this tends to limit its utilization. SAM is rel-
atively safe to use, although it can trigger episodes of mania, particularly in persons
with bipolar disorder.
The beneficial effect of SAM in depression is, no doubt, a result of its methyl-
ation of some target, either a protein, DNA or, most interesting to our discussion,
the membrane phospholipid PE. By promoting basal PLM and increasing mem-
brane fluidity, SAM could provide a generalized enhancement of synchronized neu-
ronal activity, increasing the baseline upon which episodes of dopamine-induced
(or norepinephrine-induced) activity are superimposed. By providing an alternative
source of methyl groups it could increase the effectiveness of folate-dependent,
D4R-mediated PLM, especially under conditions where the supply of 5-methylTHF
was not sufficient to meet demand. In this fashion exogenous SAM could give the
folate system a breather. The occurrence of manic episodes as a side effect of SAM

215
lends strong support to the concept that depression and mania are extremes in a
continuum of phospholipid methylation status.
Selective serotonin re-uptake inhibitors (SSRIs such as Prozac®, Zoloft® etc.)
are the most widely prescribed treatment for depression. By blocking the re-uptake
of serotonin back into nerve terminals, they increase stimulation of serotonin recep-
tors. Cortical neurons, including GABAergic interneurons, form synapses with
serotonin-releasing nerves, such as those arising from the dorsal raphe nucleus.
Serotonin may therefore exert a modulatory influence on interneuron firing rate
and synchronized oscillations. Serotonin can activate D4R-mediated PLM, albeit it
with a significantly weaker potency than either dopamine or norepinephrine (18).
Its lower potency means that, at best, serotonin would contribute a weak level of
PLM activation, not comparable to the level associated with norepinephrine medi-
ated awareness or dopamine-induced attention. However, a low background of
serotonin-based PLM could contribute to mood.
Lithium therapy is a mainstay of bipolar treatment options, because of its
ability to terminate episodes of mania. Although its mechanism of therapeutic
action is not fully established, lithium is thought to act by inhibiting the inositol
phosphate signaling pathway. Specifically, lithium inhibits inositol- 1-phosphate
phosphatase, the enzyme responsible for removing the phosphate from the sugar
inositol, making if available for recycling back into the phosphatidylinositol (PI)
cycle (419). By blocking this enzyme, lithium reduces the level of phosphatidylinosi-
tol-type phospholipids, which include PI and its phosphorylated derivatives PIP,
PIP2 and PIP 3. The PI family of phospholipids is important in two general ways: 1.
In response to receptor activation the phospholipids can be hydrolyzed to form
intracellular second messenger signaling molecules such as inositol triphosphate
(1P3) and diacylglycerol (DAG). 2. Polyphosphorylated inositol phospholipids (e.g.
PIP3) can serve as membrane anchoring sites for proteins containing pleckstrin
homology (PH) domains. P13-kinase, which was earlier discussed in relation to its
ability to increase methionine synthase, plays a key role in creating PIP3. Because it
reduces the availability of PI, lithium will reduce the effectiveness of P13-kinase,
acting similar to a P13-kinase inhibitor. PI derived second messengers promote
activation of protein kinase C (PKC), which is essential for dopamine-stimulated,
folate-dependent PLM (19), among many other actions. In addition to reducing Pl
synthesis, lithium also inhibits glycogen synthase kinase 3 (GSK3), an action that
mimics the effect of IGF-1.
Newer options for treating bipolar disorder include trans cranial magnetic
stimulation, atypical antipsychotic drugs such as olanzapine and anticonvulsant
drugs such as lamotrigine, gabapentin, valproic acid and carbamazepine. Lamotrig-
ine was originally developed as an anti-folate drug and is used to treat several types
of seizures, including those resistant to other anti epileptic drugs. Its therapeutic
action in bipolar disorder is not, however, thought to involve its anti-folate activity.

216
In general anti epileptic drugs increase inhibitory GABA receptor activity or reduce
excitatory responses, which can be useful in limiting episodes of mania.
Repetitive trans cranial magnetic stimulation (TMS) has shown success in
treating both depression and bipolar disorder. TMS produces oscillatory magnetic
fields, similar to those produced by synchronized neuronal firing, that are strong
enough to penetrate the skull. There appears to be an interaction between these
externally applied fields and internally generated fields, and cognitive improve-
ments can be measured during the application of oscillatory fields in the gamma
frequency range (161,420). Repeated TMS treatments using a similar frequency
range reportedly lessen the symptoms of depression (421), but TMS is still consid-
ered to be an experimental treatment option. How TMS works remains unclear, but
some have compared it to electro-convulsant shock therapy, which, while it helps, is
a horrendous mode of therapy to endure.
Alzheimer’s Disease
Alzheimer’s disease is a progressive neuro-degenerative disorder with a typi-
cal onset in late life (over age 65), characterized by a significant loss of brain cells,
particularly cholinergic interneurons, leading to impaired memory and progressive
mental incapacitation. Initially, short-term memory is most affected, but there is a
gradual loss of other cognitive skills including the ability to recognize objects and
persons, to carry out simple calculations or to perform basic tasks. With the recent
extension of expected lifespan in the developed world, the importance of
Alzheimer’s disease has dramatically increased. It is now recognized as a major bar-
rier to the goal of living a healthy and productive older life. Although the initiating
events of Alzheimer’s disease are still unknown, a number of significant observa-
tions have been made, many of which converge on an abnormally high formation of
amyloid deposits and neurofibrillary tangles, which may trigger neuronal death.
Amyloid deposits are formed from amyloid beta peptide (abbreviated Ap), a cleav-
age product of a intracellular transmembrane protein, amyloid precursor protein
(APP) (Fig. 14.1). Amyloid deposits particularly accumulate when the A peptide is
slightly longer than normal (42 amino acids rather than 40). A is formed by the
action of beta and gamma proteases on APP while alpha secretase activity elimi-
nates AB formation. Gamma secretase activity, located within the membrane inte-
rior, determines whether An-40 or An-42 will be produced.
Genetic mutations in the APP protein can yield higher levels of the
Alzheimer's disease-related An-42, as can mutations in the gamma secretase
enzyme (422). This makes sense since mutations in APP can shift its position in the
membrane, resulting in a different point of cutting by gamma secretase. Similarly,
mutations in the gamma secretase enzyme can affect either its position in the mem-
brane relative to APP or its mode of binding APP, leading to an increase of the An-
42 product. A higher risk of developing Alzheimer’s disease is also found in persons
carrying the apolipoprotein (Apo) E4 gene, and the accelerated rate of cognitive
decline is particularly evident in women (423). Apolipoproteins serve as organizing

217
centers for large complexes of lipids, including cholesterol and triglycerides, and
facilitate their transfer between cells and between membrane systems. A recent
study found increased deposition of beta-amyloid in mice expressing both Apo E4
(but not Apo E3) and a mutant form of APP (424), indicating that they are additive
risk factors. The importance of Apo E4 may lie in the structure of membranes that it
promotes, with certain membrane constituents favoring formation of An-42. Pres-
ence of the Apo E4 allele has also been associated with schizophrenia. It was found
that the age of schizophrenia onset was 4 years earlier in women carrying one or two
Apo E4 alleles and their risk of negative symptoms was 4-fold greater (425). Inter-
estingly, Apo E4 did not appear to be a significant risk factor for men.

Figure 14.1: Amyloid peptide (A3) is formed from amyloid precursor protein (APP) by the combined
actions of beta and gamma secretase.

Folate-related homocysteine metabolism is impaired in Alzheimer’s disease.

A number of studies have found significantly higher plasma homocysteine levels
and/or lower folate levels in Alzheimer’s disease patients (426-428). Indeed elevated
homocysteine appears to be an independent source of risk for developing
Alzheimer’s, according to recently published data from the Framingham Study
(429). Investigators followed changes in serum homocysteine levels in 1,100 persons
during an eight-year period and correlated them with the initial development of
dementia, which occurred in 111 persons, including 83 with Alzheimer’s. They
found that the risk of developing Alzheimer’s was related to the amount of increase
in plasma homocysteine during the eight-year period of observation. The average
baseline homocysteine level was 13.1 .tM at the start of the study and there was a
clear trend for higher levels in older individuals (e.g. 11.5 j.tM for the 65-69 yr.
group vs. 22.3 1.tM for the 90-94 yr. group). An increase of 5 1.M during the study

218
period was associated with a 40% increase in the risk of Alzheimer’s for both men
and women. Elevation of homocysteine can lead to an increase of S-adenosylhomo-
cysteine and inhibition of DNA methylation that might contribute to neuronal
degeneration in Alzheimer’s disease.
What might cause homocysteine levels to be higher in Alzheimer’s disease? A
likely culprit would be a folic acid deficiency or lower levels of 5-methylTHF. One
particularly interesting study examined the serum levels of a number of nutritional
factors in nuns, members of the School Sisters of Notre Dame religious congrega-
tion, who generously agreed to donate their brains for study after they died (430). In
1993, blood samples were drawn from 95 sisters at their convent in Minnesota and
basic mental function exams were administered. They all ate out of one kitchen so
they shared a basically similar diet, although some took folic acid-containing multi-
vitamins and some did not. By 1999, 30 sisters had died, and their brains were ana-
lyzed for signs of Alzheimer’s disease, including cortical atrophy, amyloid plaques
and neurofibrillary tangles, which were present in about 50% of the subjects. A very
strong correlation was found between the severity of cortical atrophy and previously
determined serum folate levels. No such correlation was found for 18 other nutri-
ents or nutritional markers. Atherosclerosis of brain blood vessels was also elevated
in association with low folate levels. Furthermore, among the 15 sisters with signs of
Alzheimer’s there was a significant correlation between cognitive function and
folate levels, as might be expected from cortical atrophy. This data supports the con-
cept that single-carbon donations from the folate pathway are important for main-
taining normal brain cell mass and for avoiding degenerative losses during
Alzheimer’s disease. Taking a folate supplement may be a big help, but the geneti-
cally determined ability to maintain a robust and dynamic supply of single-carbon
groups may be even more important.
Copper and zinc have also been proposed to play a role in Alzheimer’s dis-
ease. Serum concentrations of both metals are increased in persons carrying the
Apo E4 gene (431) and A binds copper and reduces it from its 2+ to its 1+ state
(432). Moreover, copper’s toxicity toward cultured neurons is greater when APP is
present than when it was absent (433). Zinc supplementation, along with a low cop-
per diet, causes a decrease in APP levels in women (434) and mice lacking the APP
protein have elevated levels of copper in their cerebral cortex and liver (435). Cop-
per and zinc both enhance the rate of A fibril formation and the presence of ApoE4
further enhances this process (436).
S-Adenosylhomocysteine (SAH) hydrolase binds copper with very high affin-
ity and copper levels regulate the level of enzyme activity. In 1993 Dr. Murray
Ettinger and colleagues at the State University of New York at Buffalo first purified
a copper-binding protein from mouse liver and proceeded to characterize its struc-
ture and properties (44). Cloning of its gene showed that the copper-binding pro-
tein was in fact SAH hydrolase. SAH hydrolase contains four identical subunits and
each binds copper with a KD of 10.16 to io’ M, indicative of extremely high affinity.

219
Decreases in copper levels caused a decrease in SAH hydrolase levels (45), but it
remains unclear whether increases of copper can cause elevated activity. Our dis-
covery of it’s essential role in the P13-kinase signaling pathway (202) means that
there are two points where copper exerts critical control over methylation-related
events. Furthermore, we found that monovalent Cu acted as an inhibitor of Cu2,
implying that Ar-induced reduction of Cu2 converts a positive effector into a nega-
tive one. An increase in the amount of A13 (i.e. an increase in amyloid deposits)
could therefore lead to impairment of the IGF-1/P13 -kinase signaling pathway.
Similar to some of the other disorders we have discussed, there appears to be
a deficit of omega-3 fatty acids in Alzheimer’s disease. For example, serum levels of
both EPA and DHA were found to be significantly lower, not only in Alzheimer’s
disease patients, but also in patients with other types of dementia or cognitive
impairment (437).
It is possible to construct an integrated hypothesis of pathological events dur-
ing Alzheimer’s disease that incorporates most of the major theories. As illustrated
in Fig. 14.2, altered membrane structure (e.g. associated with ApoE4 or lowered lev-
els of DHA) could increase the proportion of A13-42 that is formed, leading to
enhanced amyloid fibril formation. The copper binding and reducing activity of the
amyloid fibrils could then lead to a lower concentration of intracellular Cu2 and a
corresponding decrease in P13-kinase-dependent methionine synthase activity. The
resultant increase in homocysteine and S-adenosylhomocysteine could impair DNA
methylation leading to decreased neuronal survival. While clearly speculative, this
mechanism gains strength from its inclusiveness and can be directly tested. Because
of its multi-step nature, there are many components that could contribute to overall
risk, only some of which are likely to have been identified. For example, it is unclear
why cholinergic neurons would be particularly vulnerable to these events.

220
 Figure 14.2: An integrated hypothesis for the etiology of Alzheimer’s disease.
Formation of the 40 and 42 amino acid forms of beta amyloid (A13) from amyloid
precursor protein (APP) results in the accumulation of amyloid plaques. Mutations
in secretase enzymes or in APP can increase plaque formation. Altered membrane
structure can affect gamma secretase activity to increase formation of the
amyloidogenic AJ3-42. Once formed, amyloid plaques carry out the reduction of
Cu2’ to Cu leading to a loss of P13-kinase-dependent signaling and reduced
methionine synthase activity. The consequential rise in homocysteine and S-
adenosylhomocysteine (SAR) leads to impaired DNA methylation and neuro
degeneration. Reduced P13-kinase activity also activates apoptotic cell death
pathways.
At present the available drug treatments for Alzheimer’s are aimed at aug-
menting levels of the neurotransmitter acetylcholine, in an attempt to counteract
the prominent loss of cholinergic neurons. The four drugs currently approved by
the FDA (tacrine (Cognex®), donepezil (Aricept®), galantamine (Reminyl®) and riv-
astigmine (Excelon®) are all inhibitors of acetylcholinesterase, the enzyme that
breaks down acetyicholine. They increase the neurotransmitter’s effectiveness as the
number of cholinergic neurons dwindles. While some measurable improvement in
memory can be demonstrated for these agents, the effect is not large and is generally
transient as progression of the disease continues. Moreover, it is clear from their
mechanism that these agents do not address the underlying cause of Alzheimer’s.

221
Memantine is an experimental drug with a different mechanism of action that has
been reported to slow disease progression (43 8-9). It acts via inhibition of NMDA
receptors, suggesting that stimulation of these receptors might play a role in the
pathophysiology of Alzheimer’s disease. It is well recognized that over-stimulation
of NMDA receptors can cause neuronal death, so this may be a promising
approach.
Non-steroidal anti-inflammatory drugs (NSAIDs) such as ibuprofen report-
edly exert a protective effect against developing Alzheimer’s disease. A prospective
study of 7,000 individuals 55 years of age or older found that long-term NSAID use
was associated with a remarkable 80% lower frequency of developing Alzheimer’s
dementia (440). At the same time dementia due to vascular disease was not affected
by NSAID use. The large size of this effect suggests that persons at high risk for
Alzheimer’s (e.g. those with a strong family history or homozygosity for the Apo E4
allele) could derive benefit from long-term NSAID therapy. The mechanism under-
lying NSAID benefit is unclear, but may involve increased clearance of beta amyloid
deposits (441) or a decrease in their formation (442).
Currently there is a massive effort underway to identify new treatments for
Alzheimer’s disease focused on the central paradigm of reducing amyloid plaques
but the jury is still out on whether this overall strategy will be successful. If more
evidence accumulates for the importance of elevated homocysteine and reduced
folate levels as risk factors, new treatment strategies may emerge that will be based
upon their role in underlying pathologic mechanisms.

Chapter Summary
• DNA methylation events are important to the etiology of both Down syndrome and
Fragile X syndrome.
• Disorders of mood, such as depression and bipolar disorder, are associated with altered
attention.
• In some cases depression and bipolar disorder can be effectively treated with interventions
that alter membrane fluid properties, including omega-3 fatty acid-rich fish oils (both
depression and bipolar disorder) and SAM (depression only).
• Activity of the folate pathway and the methionine cycle are reduced in Alzheimer’s
disease.
• Amyloid-induced reduction of copper may impair P13-kinase signaling pathways and
affect DNA methylation.

222
15 AN EVOLUTIONARY PERSPECTIVE

The forces of evolution are constantly revising both the human genome and
the human organism it creates and sustains. This can be a difficult concept to grasp
since we are talking about changes across the entire human species, the population
of the world, integrated over time, through the passage of many millennia. From the
self-centered perspective of our individual lives, in the narrow slit of time within
which we exist, evolution can feel like something that “happened” (emphasis on the
past tense) rather than something that “is happening”. Those of us who accept Men-
delian views of inheritance and Darwinian views of evolution must pause to recog-
nize and respect the current and ongoing remodeling of the human genome and its
potential implications.
Among the myriad of genes and traits that could be subjected to analysis, the
human dopamine D4 receptor and the human capacity for attention to novelty are
outstanding examples, worthy of our own time and attention. After plowing
through the preceding chapters, I hope you will agree that it is this capacity for
attention and attention-related learning, invested by each of us under different cir-
cumstances and in different ways that is the underpinning of man’s individuality
and uniqueness. It distinguishes us one from another and distinguishes our species
from all others. It is our gift. Creationism aside, this cognitive gift has evolved dur-
ing millions of years in the time domain and its development has been closely linked
to the social evolution of man. While other more physical traits (e.g. blue eyes, red
hair, and height) are also subject to evolutionary revision, cognition-related traits
are more closely linked to man’s social evolution.
Let’s start with an illustration of evolution, focusing on the habitat and habits
of man as compared to, for example, ants. Imagine a prehistoric moment when a
hunter-gatherer Homo sapiens (or even Homo erectus) was foraging for food in the
African veldt when he happened to notice a colony of ants that had built a sizable
anthill, a mound of sandy soil penetrated by tunnels, filled with scurrying ants. He
paused to eat a few ants before returning to his own crudely fashioned shelter of
branches and leaves. Now fast-forward that image to 6,000 or so years ago when a
successor Homo sapiens, having migrated to a verdant middle-eastern valley,
notices a similar anthill as he starts to prepare a small plot of land for cultivation of
beans and wheat. Not at all tempted to eat the ants, he knocks down the annoying
anthill before returning to his house, built from stone and clay, complete with a
sealed wooden roof. Fast-forward again to the present, as a middle-aged suburban-
ite in Westwood, Massachusetts ventures out into his backyard and notices a similar
anthill, essentially identical to the prehistoric version, which is threatening to blem-
ish his otherwise immaculate lawn. Having paid plenty to his gardener to create a
perfect backyard scene, complementing his five-bedroom, six-bathroom Tudor-
style manse, he immediately calls the gardener on his cell phone. Next day the gar-

223
dener arrives, sprays the anthill with 2-(1 -methylethoxy) phenol methylcarbamate
and the ants are dead. Now that’s evolution!
While the ant’s habitat and habits have not evolved very much during this
time span, man’s habitat and habits have clearly been on the move. We create new
physical and social environments and raise new generations within these evolving
novel environments. Each generation accepts the existing environment as normal,
but at the same time each generation further modifies and advances the human
environment, creating the dynamic social evolution recounted in history books over
the past several millennia. What drives man to this dynamic existence? Why aren’t
we satisfied with the status quo? I would suggest that our intrinsic attention to nov-
elty is a key element of our unique make-up and that attention to novelty leads
directly to our enhanced capacity for learning. The evolutionary origin of this
capacity may lie within primate-specific genetic features of dopaminergic signaling,
including those evident within the dopamine D4 receptor.
Are you a man or a mouse?
Man is no better than other species at noticing novel things. In fact, we may
be worse than most species at being vigilant or aware of unusual events in our sen-
sory environment. We are no match for rats and mice when it comes to reaction
time in response to a stimulus. What does distinguish us humans is our ability to
readily learn and use novel attended information. Species differences in the D4
receptor reflect this distinguishing ability.
The D4 receptor in rats and mice contains the essential “hot spot” methio-
nine residue at the same location as in the human receptor and the ten amino acids
preceding it and the ten amino acids after it are identical in all three species (443-5).
Thus the D4 receptor is well conserved with regards to its capacity for carrying out
dopamine-stimulated PLM and initiating attention. Those portions of the D4 recep-
tor involved in specifically binding dopamine (i.e. the seven transmembrane heli-
ces) are also very well conserved across species, differing by only one or two amino
acids in each of the helices. The big difference is, of course, in the third intracellular
loop region, where the human D4 receptor has extra proline-rich amino acid
repeats that the rat and mouse do not.
Protein sequences containing the sequence PXXP, where P = proline and X =
any other amino acid, can be used as a binding site by other proteins containing
SH3 (rc-homology-3) domains (446). It allows a lock and key type of binding and
the residues between the two prolines, as well as those on either side, determine the
ability of specific SH3 domains to bind to a particular PXXP (447). The third cyto-
plasmic loop of the human D4 receptor is the supreme example of a multiple SH3
binding region as shown for the 7-repeat, D4.7 receptor in Fig. 15.1. Note that this
particular repeat sequence is very common, accounting for almost 90% of all 7-
repeat alleles. The first and last 20 amino acids of the loop (not shown) are involved
in G protein activation and do not contain any prolines. The middle segment con-

224
sists of 142 amino acids, 54 of which are prolines (38%). There are a total of 33 pos-
sible PXXP segments, 13 of which are unique in this example. Many of the PXXP
motifs overlap each other. For example, the sequence PGPPSPTPP has three PXXP
motifs. This implies that SH3 domain-containing proteins may compete for PXXP
binding sites on a first-come first-served basis. The size of SH3 domain-containing
proteins places an upper limit on how many can simultaneously bind to a single D4
receptor. It seems unlikely that more than five or six could bind at once.
The first five PXXP motifs in the D4.7 receptor do not involve repeats, but are
similar to proline-rich sequences in lower species such as rat and mouse (Fig. 15.1).
Indeed the first three PXXP sequences are identical in man, rat and mouse recep-
tors, suggesting that all three species bind similar SH3 domain-containing proteins
using this region. The rat receptor has a total of six PXXP motifs while the mouse
has nine. If we equate the number of potential binding sites with some form of
attention-related learning mechanism, perhaps mice have more such abilities than
rats.
Without repeats, man would have only one more PXXP site than the mouse,
however, with repeats we have anywhere from five (the D4.2 receptor) to forty more
(the D4. 11 receptor) sites. While quantity may count for something, the specific
binding preference of the additional SH3 domain proteins can make a critical differ-
ence, perhaps paving the way for a different type of neuronal response that is coor-
dinated with attention. A world-wide sample of D4 receptor genes indicates that
there are eleven unique PXXP motifs present within repeats that complement the
six PXXP combinations found before and after the repeats. Out of the total of seven-
teen, twelve of the PXXP combinations are found in man but not in rat or mouse.
Thus when it comes to docking with SH3 domain proteins, human D4 receptors
provide both an increased quantity and greater diversity of binding opportunities.
As primates, we humans share a considerable number of genetic and behav-
ioral features with gorillas, chimpanzees, orangutans, baboons and monkeys,
including our D4 receptors. As detailed in studies by Dr. Kenneth Livak and col-
leagues, all five of these species possess 48-base-pair repeats in their D4 receptor
genes that give rise to multiple PXXP motifs similar to or identical to human
sequences (60). For example, the first 48-base-pair repeat in the orangutan gene
codes for exactly the same amino acid sequence as the first repeat in humans. In
their very limited sample in non-human primates. Livak et al. (60) found four to
nine repeats with five repeats being the most common. The pattern of D4 receptor
genetic. diversity among primates indicates that man is no more closely related to
any one of the other species than they are to each other. This leads to the proposal

225
that variations in the number and nature of repeats evolved independently in each
lineage, starting some 6-9 million years ago (448).

Figure 15.1: Amino acid and corresponding DNA sequences for residues #234-376 in the third
cytoplasmic loop of the most common human D4.7 dopamine receptor allele (448). Arrowheads
indicate the beginning and end of each 48 base-pair repeat. Variations from repeat #1 are indicated
in bold print. Note the presence of multiple PXXP motifs in non-repeat sequences found in lower
species as well as in primate-specific repeat sequences.

How have humans and other primates developed different numbers of

repeats? Obviously more than individual mutations were involved. The DNA
sequence can change if a gene segment from one chromosome switches places with
its equivalent (homologous) partner on the other paired chromosome in a process
called homologous recombination or crossing over. After fertilization recombina-
tion allows the mixing of maternal and paternal genetic features, which otherwise
would just stay together on the single chromosome they came from. Swapping of
the same number of bases is most typical, yielding equal recombination, but some-
times more move in one direction than the other, yielding unequal recombination.
It has been proposed that unequal recombination is responsible for the variation in
number of repeats in primate D4Rs (448). For example, a pair of chromosomes each

226
carrying a four-repeat receptor could end up as six- repeat and two-repeat chromo-
somes. Since the first repeat is identical in 99.9% of humans and the last repeat is
identical in 94%, the swapping basically involves just the internal repeats, with the
first and last segments acting as stable anchors. This guarantees that at least two
repeats will remain after recombination, even if it is unequal. A chromosome with
four repeats therefore has two internal repeats available for recombination, while a
chromosome with two repeats has none available to give away and can only receive
repeat sequences from another chromosome.
As proposed by Ding et al. (448), the existence of repeat numbers 2, 3, 5 and 6
can readily be explained by postulating a progenitor allele containing four repeats
that unequally recombined in different ways to yield the other observed repeat
sequences. However, unequal recombination cannot account for all of the
sequences found in seven-repeat alleles, since certain of their internal repeat
sequences (e.g. the fourth repeat in the D4.7 receptor shown in Fig. 14.1) could not
be directly formed by recombination of the four-repeat allele. Moreover, these
sequences have not been found in any four-repeat alleles. Consequently, these
authors concluded that the seven-repeat allele arose in a distinctive manner, involv-
ing either multiple mutations or other gene transfer events. Further analysis of
nearby genetic features indicated that the seven-repeat allele showed signs of posi-
tive selection, meaning that its frequency is greater than would be predicted simply
by random occurrence. This rather striking and provocative conclusion suggests the
presence of an evolutionary force that is moving the human genetic pool toward
seven repeats. It is all the more provocative considering the link between seven
repeats and increased risk of ADHD.
Unequal homologous recombination may also have played a hand in the ini-
tial creation of progenitor D4 receptor repeats in primates and proline rich
sequences found in non-primate genes (e.g. mouse and rat) may have been the
starting material. As illustrated in Fig. 14.1, the primate-specific repeats are inserted
within the non-primate proline-rich segment. In the rat gene the proline-rich seg-
ment stretches across 198 base-pairs (#682 to 879) of which 91 are cytidine (C), 51
guanine (G), 30 thymidine and 26 adenine. The excess of C (46% vs. 25% if it was
random) is associated with the presence of 19 prolines out of the 66 amino acids
(198 ÷ 3) coded by this segment, since the codons CCC, CCG, CCT and CCA each
code for proline. Homologous recombination relies upon sequence similarity
(homology) between two gene sequences to align the two chromosomal strands and
facilitate exchange of segments. As a result of the skewed predominance of cytosine,
the success of recombinant events in this segment is increased, since the probability
of finding a homologous or identical match on the other strand is greater. In other
words, this highly cytosine-enriched region favors recombination. The length of the
cytosine-enriched segment in the non-primate receptor (198 bases) is sufficiently
long to allow unequal recombination of a middle section of either 48 or 96 bases
(the size of one or two primate repeats) while leaving both ends intact. Alterna-

227
tively, gene duplication could have given rise to an ancestral primate-like gene. The
D4 receptor in dogs exhibits a cytosine-enriched 39-bp unit that also can occurs as
separate 27- and 12-bp segments (449). A Korean wolf has a similar repeat struc-
ture. This suggests that unequal recombination is a general feature of D4 receptor
genes across different species. Indeed, the rat and mouse genes themselves may have
gained their multiple PXXP domains via such a mechanism.
There are two general driving forces for genetic evolution. The first is based
upon the chemistry of the genetic molecule itself. The second is based upon the
qualities or traits of the resultant organism, especially as they might favor or disfa-
vor reproduction thereby influencing extension and expansion of the germline.
Increased vulnerability of the D4 receptor to unequal recombination is clearly an
example of the former type of molecular evolution. Effects of D4 receptor repeats on
behavior, personality traits or cognitive function would fall in the latter category.
DNA methylation provides another example of how the chemical makeup of
DNA can be a driving force for evolution. Cytosine residues in DNA are commonly
methylated when they are next to a guanine (i.e. CpG). The genome contains far
fewer CpG pairs than would be expected from chance, indicating an active process
that suppresses their occurrence. Methylated cytosine residues have a higher proba-
bility of undergoing oxidative de-amination than does the unmethylated form.
Deaminated cytosine closely resembles uracil, which is not normally found in DNA
and is excised by nucleases, increasing the opportunity for mutations and inser-
tions. Thus CpG-rich regions can be “hotbeds” for evolution and the excess of cyto-
sine in the repeat region creates an increase in CpG sites in the D4 receptor (e.g. 45
are present in the most common seven-repeat allele). This may be an important fac-
tor in the evolution of the D4 receptor. To date there have been no studies on the
methylation status of the D4 receptor.
Because the D4 receptor is directly involved in folate-dependent methylation,
its activity can influence the level of DNA methylation. When the receptor is highly
active, it becomes the major consumer of folate derived methyl groups and the
methylation of homocysteine slows down. The resultant increase in homocysteine
concentration leads to a rise of S-adenosylhomocysteine and to inhibition of DNA
methylation. In this way dopamine can affect gene expression and the development
of cells that express the D4 receptor. Further studies are needed to determine the
functional significance of this intriguing relationship.
Diet can also be an evolutionary factor. The human diet is evolving much
faster than the human genome and the preceding chapters are testimony to the crit-
icality of nutrition for optimal mental performance. What are your daily sources of
folie acid, pyridoxine (Vitamin B6), nicotinic acid, methylcobalamin (Vitamin B
12), EPA and DITA? From our hunter-gatherer origins we evolved to individual
growers and cultivators. As systems of trade and commerce developed we soon
began to depend upon specialized farmers for our food and we tended to eat what-
ever they chose to grow and make available. Preservation of food by drying, salting

228
and canning brought further convenience and encouraged industrialization of the
food supply. Today industrialized farming is predominant and prepared foods have
further increased our reliance on other people’s standards for our personal nutri-
tion. Too often the goal for the food industry is lowered production costs rather
than nutritional adequacy. As dollar conscious and convenience-craving consum-
ers, nutrition is often our third or fourth priority when choosing our food. The
USDA helps by encouraging a healthy diet and a safe food supply. In particular their
recently enacted requirement for folie acid supplementation of flour and pasta
products should prove to be a real boon for mental health in the United States.
Genetic evolution typically occurs over hundreds and thousands of genera-
tions. Six thousand years of recorded human history represents only three hundred
generations of twenty years each. Within the last four to five generations our diets
and sources of food have undergone significant changes. Our biochemical pathways
are poorly equipped to handle such major shifts in the nutrients they receive and
this is especially the case for micronutrients like vitamins, and essential co-factors.
In the end there is really only one person (besides your wife or your mother) that
you can count on to have your best nutritional interests at heart, and that’s you.
The more repeats the merrier?
What is the significance of having a greater number and variety of PXXP
domains? We earlier discussed two specific examples of SH3 domain-containing
proteins, synapse-associated proteins (SAPs) and Grb2. SAPs serve to locate the
D4R within synaptic complexes to increase solid-state signaling effectiveness, while
Grb2 links it to MAP kinase pathways that stimulate neuronal growth and synapse
formation. D4R interaction with SAP97 was demonstrated in rat brain (80), and six
of the seven PXXP sequences in the rat D4 receptor are present in all human recep-
tors (33,443,444), so it is quite likely that SAP interaction is a feature of all human
D4 receptors as well. At present we don’t know which particular PXXP is responsi-
ble for SAP interaction. There is a family of SAP proteins and it is possible that
some of the human-specific PXXP sequences may enable the human receptor to
bind to other SAP proteins. Although the ability of rat D4 receptors to activate MAP
kinase has not been specifically demonstrated it is probably important for attention-
related learning. Two other Grb2-like linker proteins, Nck and Crk, also bind to the
human D4 receptor and may facilitate signals other than MAP kinase activation
(22).
Dopamine simultaneously initiates multiple intracellular signals via the D4
receptor including solid-state signaling, G protein-based signaling, MAP kinase
activation and more. One recent study showed that D4 stimulation also activates the
Akt/NFKB pathway (450). Akt is a protein kinase downstream of P13-kinase while
NFKB is a nuclear regulatory protein, implying that dopamine can also affect gene
transcription through this pathway. Another study found that stimulation of the D4
receptor activates the platelet-derived growth factor (PDGF) receptor, even in the
absence of PDGF (451). This list of signaling pathways is growing and the complex-

229
ity of D4 receptor PXXP motifs suggests that other signals still remain to be discov-
ered. Each different signal means that an episode of attention will provide a
different type of neuronal activation leading to neurite outgrowth (sprouting), new
synapse formation, strengthening of existing synapses, altered neurotransmitter
synthesis or release, altered expression of receptors, metabolic changes etc. Since
these changes are synchronized, via attention, with particular sensory experiences,
they provide different modalities of attention-related learning and different ways to
shape our brain. In simple terms, the greater the variety of signals that are produced
in coordination with attention, the broader the array of neuronal responses and the
greater the diversity of learning. This diversity is our repeat-related gift.
Although I am obviously prejudiced, I will assert that methylation dependent
solid-state signaling is the most fundamentally important D4 receptor-generated
signal. Solid-state signaling determines which particular information will be ampli-
fied via 40 Hz oscillations to dominate our thoughts, while the other pathways cre-
ate hard wired associations and additional stable manifestations of learning. Other
G protein-coupled receptors can generate most of the same signals as the D4 recep-
tor, but they lack the capacity for PLM. Nature has selected the D4 receptor for
some of its most elegant tinkering. The fact that D4 receptors in lower species share
the capacity for dopamine-stimulated PLM argues for the fundamental importance
of solid-state signaling, Because of its reliance on folate-derived single-carbon
methyl groups, solid-state signaling affects the levels of homocysteine and adenos-
ine, triggering effects on DNA methylation and circadian rhythms. But perhaps the
most compelling testimony for the importance of solid-state signaling comes from
those who suffer from schizophrenia and related mental illnesses when the methyla-
tion process fails.
Is it better to be homozygous for seven repeats rather than for two or four?
The increased risk of ADHD immediately answers: No. Let’s then rephrase the
question to: “Is it different to be homozygous for seven repeats?” The answer to this
query appears to yes, since differences in temperament can be detected from birth
through adulthood. But being different is just that. It does not necessarily imply bet-
ter, or worse. Nonetheless, in certain settings temperament can make a difference.
I have used the following scenario to frame the issue: Let’s say that you are far
out to sea on a ship that unfortunately sinks and you are cast into the ocean, in peril
of drowning. Then, in a moment of good luck, you spot a lifeboat nearby. You swim
over to the boat and, using your last bit of energy, your drag yourself aboard and are
surprised to find that there is someone else already inside. There is a good chance
that the temperament of this individual will be an important factor in your mutual
survival. How many D4 receptor repeats would you like him/her to have? Would
you be better off finding someone who was highly focused and could row for hours
on end without being distracted, or would your chances be better with someone
who notices everything out of the ordinary and might be imaginatively resourceful?
It’s a tough call, and the answer greatly depends upon what could happen in the

230
future, A greater demand for adaptability might favor the latter talents, but consid-
erations of endurance would favor the former. There is potential value for both tem-
peraments and you should probably just be thankful you aren’t alone. Perhaps you’ll
find yourselves compatible because you’re so similar, or maybe your differing tal-
ents will complement each other. Attempting to force your new found companion
into being a strong and steady rower, when in fact he/she is better suited to being a
lookout would be a recipe for disaster. When the future is unknown, which by
nature it is, adaptability is a tremendous asset for success and survival and the most
important place to look for adaptability is within yourself, not in others.
While molecular evolution might have had a heavy hand in molding the
human D4 receptor, the behavioral traits exhibited by people with different num-
bers of repeats could also be a factor. And what behaviors hath evolution wrought?
Again the answer seems to be DIVERSITY! Under the dictum “survival of the fit-
test” we are left to conclude that man’s survival has been well served by the remark-
able degree of variety in the D4 receptor gene and in the receptor protein it encodes.
Divergence, not convergence, has been the evolutionary trend. Diversity can be
manifested as differences in the intensity of attention (e.g. casual and brief vs. deep
and sustained) or as differences in the quality of attention-related learning (e.g. cre-
ating a greater variety of associations). At moments when our prehistoric ancestors
were confronted with novel challenges, diversity might have been particularly valu-
able in surviving or adapting, thereby increasing chances for procreation and for
passing their particular D4 receptor gene to the next generation.
Because we carry only two alleles of any gene, the opportunity to reap bene-
fits from genetic diversity is mainly manifested across populations. One person
alone cannot represent much diversity. If everyone in a tribe or extended family dis-
played only one or two haplotypes on both their D4 receptor alleles, we would
expect a common, clonal temperament across that culture, as compared to a more
genetically diverse population. In that narrower world everyone would tackle new
problems with more or less the same attitude and outlook, diminishing the chances
for creative, successful solutions. In the presence of effective communication, how-
ever, individual differences in perspective and outlook can be shared for the poten-
tial benefit of the whole social structure. Under these circumstances diversity then
becomes a powerful asset for survival and for evolutionary dominance. Throughout
the development and social evolution of our human species, inventions and helpful
adaptations arose from experimentation with different ways of doing things. In our
culture many humans strive to be the first, to be different and to try out something
new. Scientific research is an organized prima fade example of this behavior, while
technology and technological advances are the products. Moreover, we are driven
by an almost religious confidence that finding something new will be worth the
effort and worth the failures that inevitably accompany a quest for innovation.
However, the drive for novelty and adaptation is not equally shared by all
persons or by all cultures, Change per se is not intrinsically desirable and diversity is

231
not necessarily valuable, especially when the need for adaptation is not great. When
humans migrated across the globe, facing new challenges of weather or the need for
food and survival, adaptation was doubtless high on the list of helpful attributes. As
described by Jared Diamond in Guns, Steel and Germs (451), the ability to carry
seeds and grow crops in new locations was a critical adaptive skill for developing
and migrating man, and many founder crops (e.g. beans) were rich sources of folic
acid. The novel conditions and challenges they encountered may have favored those
individuals with diversity in temperament and cognitive function. Conversely, cul-
tures that remain in a single, supportive locale have less need for adaptation to nov-
elty, and diversity may be less rewarded.
Of course there is a practical limit to how different one’ s attentive and cogni-
tive abilities can be while still falling within the realm of diversity rather than the
realm of dysfunction. The inability of others to share and find value in an exces-
sively divergent perspective often leads to isolation and to sanctions. These practical
limits to diversity bring us intriguing close to a working definition of being mentally
ill. It is therefore not coincidental that disorders involving D4 receptor-related
molecular mechanisms manifest themselves as excessive mental diversity (i.e. men-
tal illness).
What may the future hold?
Our evolution is ongoing and it seems that we are entering a new realm of
both risk and opportunity thanks in large measure to decoding of the human
genome and to the availability of tools for manipulating genetic events. Our particu-
lar branch of the human race, meaning technology-based cultures present on all
continents, has demonstrated the ability to clone various animal species and it
appears inevitable that Homo sapiens will be cloned in the foreseeable future. This
is an awesome and scary prospect whose wisdom is unclear. Nonetheless human
cloning falls within the realm of things that will be done because they can be done.
When coupled with knowledge of the human genome there is a ghastly potential for
eugenics and Orwellian competition among genetically improved clonal groups of
humans. We could tinker with our genes in the same manner as hot rod enthusiasts
soup-up their Fords and Chevies. These horrible possibilities are balanced against
the potential benefits that could arise from gene therapy to treat diseases such as
schizophrenia, autism, Parkinson’s disease, diabetes and cancer. Having humans in
charge of their own evolution is a dicey situation at best.
The term “gene therapy” is laden with negative connotation, but there are
graded levels of gene therapy, including many clearly useful and safe opportunities
that exist below the threshold event of altering germline (i.e. egg and sperm) DNA
sequences. We are already conducting a modest form of gene therapy when we use
drugs that modulate the transcription of DNA into messenger RNA (mRNA), such
as the use of retinoic acid for the treatment of acne or estrogens for birth control.
The next step will be to use new drug-like molecules to seek out and bind to specific
DNA loci and modulate their role in transcription for the purpose of treating dis-

232
ease. Anti-sense RNA will be used to interfere with the translation of mRNA into
proteins in an analogous approach. The targeted delivery of stabilized mRNA offers
hope of increasing the expression of proteins that are either missing or inactive
because of genetic mutations. As it pertains to the molecular mechanism of atten-
tion and associated mental illnesses, we can hope that perinatal genetic testing will
allow sufficiently early intervention to thwart developmental disorders. Recently
developed proteomic techniques can be applied to blood cells like lymphocytes to
monitor levels of key enzymes, receptors and other factors, particularly in persons
who are identified as being at high risk. Genetic monitoring will become a routine
part of check-ups much like current blood tests, except that it will be highly individ-
ualized, based upon a genetic profile that can be obtained early in fetal develop-
ment. By anticipating and even warding off serious mental illnesses we can not only
improve the lives of individuals at risk and their families but also achieve major sav-
ings for the healthcare delivery system.
By defining mental illnesses in molecular tenus we will place them on a par
with other major illnesses (e.g. diabetes, heart failure, cancer) for whom this defini-
tion is already well accepted. For centuries the brain’s complexity has served as a
veil, obscuring our knowledge of its inner workings. This overwhelming complexity
served to reduce expectations for the biomedical and psychiatric communities to
effect cures. We are now poised to remove that veil and offer mechanism-based
drug therapies that will address many of the underlying causes of mental illness. The
lesson of schizophrenia should be a clear reminder that most psychiatric disorders
will ultimately be recognized as being caused by biochemical abnormalities rather
than by unfathomable frailties of the human condition. It was not long ago that
overbearing mothers were commonly thought to be the cause of schizophrenia and
cold mothers the cause of autism. Through the pioneering work of Seymour Kety
and others the concept of a molecular basis for schizophrenia gradually took hold
and is now the dominant thought. We can look forward to a time not far in the
future when detailed knowledge of these molecular events will translate into truly
effective treatments that are tailored to meet individual needs.

233
Chapter Summary:
• Evolution continues to shape the human genome, including the D4 receptor.
• Humans demonstrate an exceptional ability to adapt to novel circumstances that translates
into social evolution.
• Repeat sequences in the primate D4 receptor can facilitate attention-related learning and
evolution has created an exceptional level of variety ¡n repeat sequences.
• Unequal recombination starting from a founder sequence may have given rise to different
numbers of repeats and different repeat sequences.
• There is evidence suggesting increased predominance of the seven-repeat allele of D4
receptors.
• Methylation of the D4 receptor gene may increase mutations and promote recombination
events.
• Variety in repeat sequences leads to variety in dopamine initiated signaling responses and
ultimately to variety in attention-related learning and behavior.
• Evolution has promoted diversity in the D4 receptor gene and diversity in the behaviors
and abilities it sub-serves.

234
16 THE GIFT OF WISDOM

Thankfully, an overwhelming proportion of Homo sapiens enjoys the gifts of

attention, attention-related learning and normal cognitive function. These pro-
cesses function so transparently that we normally don’t need to recognize or fully
appreciate their molecular origins or their tenuous nature. But, as Joni Mitchell put
it: “You don’t know what you got ‘til its gone.” Our priceless gifts are inextricably
linked to the mentally ill who function beyond the limits of acceptable cognitive and
affective diversity. In a sense it is they who make our gift possible. They pay the
price.
In 1995 first I encountered a Chinese painting which shows a goddess
bestowing a pearl of wisdom upon the world, representing an allegory for the cause
of schizophrenia. The painting left me puzzled and confused. How could schizo-
phrenia possibly be considered a gift? Schizophrenia is a curse, not a gift. Gradually
I began to grasp the incredible clarity of this allegorical depiction. Of course schizo-
phrenia and wisdom are related. It is clear that the very same gift that bestows wis-
dom to most of us also brings a curse to some. The two are inseparable.
Nature evolved an elegant molecular mechanism, driven by the lowly folate-
derived methyl group, that enables all animals to amplify selected information in a
process that we call attention. During post-natal development dopamine, the neu-
rochemical trigger for attention, helps to create a hard-wired associative neuronal
network that learns from experience. Nature has taken care to preserve and even
nurture diversity within this mechanism of attention and attention-related learning,
particularly in man and in his closest relatives. Had our mechanism of attention
been designed with less diversity its reliability could no doubt be improved, but with
what loss? If there is a single lesson to be learned from the molecular mechanism of
attention it is the value and the risks of human genetic diversity. Let us have the wis-
dom to respectfully direct our gift of attention and our appreciation toward those
among us whose gifts are understandably less perfect than our own.

235
Reference List

1. Mrzljak L, Bergson C, Pappy M, Huff R, Levenson R, Goldman-Rakic PS. Localization of

dopamine D4 receptors in GABAergic neurons of the primate brain. Nature (1996)
381:245-8.
2. Wedzony K, Chocyk A, Mackowiak M, Fijal K, Czyrak A. Cortical localization of
dopamine D4 receptors in the rat brain-immunocytochemical study. J Physiol Pharmacol
(2000) 51:205-21.
3. Benjamin J, Li L, Patterson C, Greenberg BD, Murphy DL, Hamer DH. Population and
familial association between the D4 dopamine receptor gene and measures of Novelty
Seeking. Nat Genet (1996) 12:81-4.
4. Ebstein RP, Novick O, Umansky R, Priel B, Osher Y, Blame D, Bennett ER, Nemanov L,
Katz M, Belmaker RH. Dopamine D4 receptor (D4DR) exon III polymorphism associated
with the human personality trait of Novelty Seeking. Nat Genet (1996) 12:78-80.
5. LaHoste GJ, Swanson JM, Wigal SB, Glabe C, Wigal T, King N, Kennedy JL. Dopamine D4
receptor gene polymorphism is associated with attention deficit hyper-activity disorder.
Mol Psychiatry (1996) 1:121-4.
6. Swanson J, Oosterlaan J, Murias M, Schuck S, Flodman P, Spence MA, Wasdell M, Ding Y,
Chi HC, Smith M, Mann M, Canson C, Kennedy JL, Sergeant JA, Leung P, Zhang YP,
Sadeh A, Chen C, Whalen CK, Babb KA, Moyzis R, Posner MI. Attention deficit/hyper-
activity disorder children with a 7-repeat allele of the dopamine receptor D4 gene have
extreme behavior but normal performance on critical neuropsychological tests of
attention. Proc Natl Acad Sci U S A (2000) 97:4754-9.
7. Dulawa SC, Grandy DK, Low MJ, Paulus MP, Geyer MA. Dopamine D4 receptor-knock-
out mice exhibit reduced exploration of novel stimuli. J Neurosci (1999) 19:9550-6.
8. Zhang K, Tarazi FI, Baldessarini RJ. Role of dopamine D(4) receptors in motor hyper-
activity induced by neonatal 6- hydroxydopamine lesions in rats.
Neuropsychopharmacology (2001) 25:624-32.
9. Cobb SR, Buhl EH, Halasy K, Paulsen O, Somogyi P. Synchronization of neuronal activity
in hippocampus by individual GABAergic intemeurons. Nature (1995) 378:75-8.
10. Traub RD, Spruston N, Soltesz I, Konnerth A, Whittington MA, Jefferys GR. Gamma-
frequency oscillations: a neuronal population phenomenon, regulated by synaptic and
intrinsic cellular processes, and inducing synaptic plasticity. Prog Neurobiol (1998)
55:563-75.
11. Tiitinen H, Sinkkonen J, Reinikainen K, Aiho K, Lavikainen J, Naatanen R. Selective
attention enhances the auditory 40-Hz transient response in humans. Nature (1993)
364:59-60.
12. Fries P, Reynolds JH, Rorie AE, Desimone R. Modulation of oscillatory neuronal
synchronization by selective visual attention. Science (2001) 29 1: 1560-3.
13. Csibra G, Davis G, Spratling MW, Johnson MH. Gamma oscillations and object
processing in the infant brain. Science. (2000) 290:1582-5.
14. Van Tol RH, Wu CM, Guan HC, Ohara K, Bunzow JR, Civelli O, Kennedy J, Seeman P,
Niznik HB, Jovanovic V. Multiple dopamine D4 receptor variants in the human
population. Nature (1992) 358:149-52.
15. Lichter IB, Barr CL, Kennedy JL, Van Tol FIN, Kidd KK, Livak KJ. A hyper-variable
segment in the human dopamine receptor D4 (DRD4) gene. Hum Mol Genet (1993)
2:767-73.
16. Nakatome M, Honda K, Tun Z, Wakasugi C. Detection and analysis of the human D4
dopamine receptor gene (DRD4) sequence variant in the Japanese and Mongolian
population. Biochem Biophys Res Commun (1996) 220:773-6.

236
17. Chang FM, Kidd JR, Livak KJ, Pakstis AJ, Kidd KK. The world-wide distribution of allele
frequencies at the human dopamine D4 receptor locus. Hum Genet (1996) 98:91-101.
18. Sharma A, Kramer ML, Wick PF, Liu D, Chan S, Shim S, Tan W,
Ouellette D, Nagata M, DuRand CJ, Kotb M, Deth RC. D4 dopamine receptor-mediated
phospholipid methylation and its implications for mental illnesses such as schizophrenia.
Mol Psychiatry (1999) 4:235-46.
19. Sharma A, Waly M, Deth RC. Protein kinase C regulates dopamine D4 receptor-mediated
phospholipid methylation. Eur J Pharmacol (2001) 427:83-90.
20. Zhao R, Chen Y, Tan W, Waly M, Sharma A, Stover P, Rosowsky A, Malewicz B, Deth RC.
Relationship between dopamine-stimulated phospholipid methylation and the single-
carbon folate pathway. J Neurochem (2001) 78:788-96.
21. Deth RC, Sharma A, Waly M. “Dopamine-stimulated solid-state signaling: A novel role
for single-carbon folates in human attention.” In Proc. 12th Intl. Symp. Chem. Pteridines
and Folates. Kluwer Academic Publishers, 2002.
22. Sharma A, Liu Y-F, Deth RC. Effects of pertussis toxin B-oligomer on D4 dopamine
receptor-mediated MAP kinase activation. Soc Neurosci Abstr (2000) 26:1334.
23. Oldenhof J, Vickery R, Anafi M, Oak J, Ray A, Schoots O, Pawson T, von Zastrow M, Van
Tol RH. SH3 binding domains in the dopamine D4 receptor. Biochemistry (1998)
37:15726-36.
24. Hartman DS, Civelli O. Molecular attributes of dopamine receptors: new potential for
antipsychotic drug development. Ann Med (1996) 28:2 1 1-9.
25. Farrens DL, Altenbach C, Yang K, Hubbell WL, Khorana HG. Requirement of rigid-body
motion of transmembrane helices for light activation of rhodopsin. Science (1996)
274:768-70.
26. Ghanouni P, Steenhuis JJ, Farrens DL, Kobilka BK. Agonist-induced conformational
changes in the G-protein-coupling domain of the beta 2 adrenergic receptor. Proc Nati
Acad Sci U S A (2001) 98:5997-6002.
27. Ballesteros JA, Jensen AD, Liapakis G, Rasmussen SG, Shi L, Gether U, Javitch JA.
Activation of the beta 2-adrenergic receptor involves disruption of an ionic lock between
the cytoplasmic ends of transmembrane segments 3 and 6. J Biol Chem (2001) 276:29171-
7.
28. Kjelsberg MA, Cotecchia S, Ostrowski J, Caron MG, Lefkowitz U. Constitutive activation
of the alpha 1 B-adrenergic receptor by all amino acid substitutions at a single site.
Evidence for a region which constrains receptor activation. J Biol Chem (1992) 267:1430-
3.
29. Tian WN, Duzic E, Lanier SM, Deth RC. Determinants of alpha 2-adrenergic receptor
activation of G proteins: evidence for a precoupled receptor/G protein state. Mol
Pharmacol (1994) 45:524-31.
30. Zhu Q, Qi L-J., Abou-Samra A, Shi A and Deth RC. Protein kinase C- dependent
constitutive activity of Œ2AID-adrenergic receptors. Mol Pharmacol (Submitted).
31. Liu JG, Prather PL. Chronic exposure to mu-opioid agonists produces constitutive
activation of mu-opioid receptors in direct proportion to the efficacy of the agonist used
for pretreatment. Mol Pharmacol (2001) 60:53-62.
32. Wang Z, Bilsky EJ, Porreca F, Sadee W. Constitutive mu opioid receptor activation as a
regulatory mechanism underlying narcotic tolerance and dependence. Life Sci (1994)
54:PL339-50.
33. Van Tol FTH, Bunzow JR, Guan HC, Sunahara RK, Seeman P, Niznik HB, Civelli O.
Cloning of the gene for a human dopamine D4 receptor with high affinity for the
antipsychotic clozapine. Nature (1991) 350:610-4.
34. Kramer MS, Last B, Getson A, Reines SA. The effects of a selective D4 dopamine receptor
antagonist (L-745,870) in acutely psychotic inpatients with schizophrenia. D4 Dopamine
Antagonist Group. Arch Gen Psychiatry (1997) 54:567-72.
35. Ronai Z, Guffman A, Nemocla Z, Staub M, Kalasz H, Sasvari-Szekely M. Rapid and
sensitive genotyping of dopamine D4 receptor tandem repeats by automated ultrathin-
layer gel electrophoresis. Electrophoresis (2000) 21:2058-61.

237
36. Artom C. Methylation of phosphatidyl monomethylethanolanine in liver preparations.
Biochem Biophys Res Commun (1964) 15:201-6.
37. Hirata F, Tailman IF, Henneberry RC, Mallorga P, Strittmatter WJ, Axelrod J.
Phospholipid methylation: a possible mechanism of signal transduction across
biomembranes. Prog Clin Biol Res (1981) 63 :3 83-8.
38. Strittmatter WJ, Hirata F, Axe lrod J. Regulation of the beta-adrenergic receptor by
methylation of membrane phospholipids. Adv Cyclic Nucleotide Res (1981) 14:83-91.
39. Mihic SJ, Ye Q, Wick MJ, Koltchine VV, Krasowski MD, Finn SE, Mascia MP, Valenzuela
CF, Hanson KK, Greenblatt EP, Harris RA, Harrison NL. Sites of alcohol and volatile
anaesthetic action on GABA(A) and glycine receptors. Nature (1997) 389:385-9.
40. Narahashi T, Aistrup GL, Marszalec W, Nagata K. Neuronal nicotinic acetylcholine
receptors: a new target site of ethanol. Neurochem Tnt (1999) 35:131-41.
41. WeightFF, LovingerDM, White G. Alcohol inhibition of NMDA channel function.
Alcohol Alcohol Suppl (1991) 1:163-9.
42. Ueland PM. Pharmacological and biochemical aspects of S adenosyihomocysteine and S-
adenosylhomocysteine hydrolase. Pharmacol Rev (1982) 34:223-53.
43. Page T, Yu A, Fontanesi J, Nyhan WE. Developmental disorder associated with increased
cellular nucleotidase activity. Proc Natl Acad Sci U SA (1997) 94:11601-6.
44. Bethin KE, Petrovic N, Ettinger MJ. Identification of a major hepatic copper binding
protein as S-adenosylhomocysteine hydrolase. J Biol Chem (1995) 270:20698-702.
45. Bethin KE, Cimato TR, Ettinger MJ. Copper binding to mouse liver S
adenosylhomocysteine hydrolase and the effects of copper on its levels.J Biol Chem (1995)
270:20703-11.
46. Strausak D, Mercer IF, Dieter HI-I, Stremmel W, Multhaup G. Copper in disorders with
neurological symptoms: Alzheimer’s, Menkes, and Wilson diseases. Brain Res Bull (2001)
55:175-85.
47. Clarke R, Smith AD, Jobst KA, Refsum H, Sutton L, Ueland PM. Folate, vitamin B 12, and
serum total homocysteine levels in confirmed Alzheimer disease. Arch Neurol (1998)
55:1449-55.
48. McCaddon A, Davies G, Hudson P, Tandy S, Cattell H. Total serum homocysteine in
senile dementia of Alzheimer type. Tnt J Geriatr Psychiatry (1998) 13:235-9.
49. Morrison ED, Smith DD, Kish SJ. Brain S-adenosylmethionine levels are severely
decreased in Alzheimer’s disease. J Neurochem (1996) 67:1328-31.
50. Goulding CW, Postigo D, Matthews RG. Cobalamin-dependent methionine synthase is a
modular protein with distinct regions for binding homocysteine, methyltetrahydrofolate,
cobalamin, and adenosylmethionine. Biochemistry (1997) 36:8082-91.
51. Leclerc D, Wilson A, Dumas R, Gafuik C, Song D, Watkins D, Heng FIR, Rommens 1M,
Scherer SW, Rosenblatt DS, Gravel RA. Cloning and mapping of a eDNA for methionine
synthase reductase, a flavoprotein defective in patients with homocystinuria. Proc Nati
Acad Sci U S A (1998) 95 :3059-64.
52. Arinami T, Yamada N, Yamakawa-Kobayashi K, Hamaguchi H, Toru M.
Methylenetetrahydrofolate reductase variant and schizophrenia/depression. Am J Med
Genet (1997) 74:526-8. 225 53.
53. Regland B, Germgard T, Gottfries CG, Grenfeldt B, Koch-Schmidt AC. Homozygous
thermolabile methylenetetrahydrofolate reductase in schizophrenia-like psychosis. J
Neural Transm (1997) 104:931-41.
54. Bottiglieri T, Laundy M, Crellin R, Toone BK, Carney MW, Reynolds EH. Homocysteine,
folate, methylation, and monoamine metabolism in depression. J Neurol Neurosurg
Psychiatry (2000) 69:228-32.
55. Fetrow CW, Avila JR. Efficacy of the dietary supplement S-adenosyl-L methionine. Ann
Pharmacother (2001) 35:1414-25.56.
56. Fava M, Giannelli A, Rapisarda V, Patralia A, Guaraldi GP. Rapidity of onset of the
antidepressant effect of parenteral S-adenosyl-L-methionine.Psychiatry Res (1995)
56:295-7.

238
57. Felder CC, Jose PA, Axelrod J. The dopamine-1 agonist, SKF 82526, stimulates
phospholipase-C activity independent of adenylate cyclase. J Pharmacol Exp Ther (1989)
248:171-5.
58. Senogles SE. The D2s dopamine receptor stimulates phospholipase D activity: a novel
signaling pathway for dopamine. Mol Pharmacol (2000) 58:455-62.
59. Niimi Y, Inoue-Murayama M, Murayama Y, Ito S, Iwasaki T. Allelic variation of the D4
dopamine receptor polymorphic region in two dog breeds, Golden retriever and Shiba. J
Vet Med Sci (1999) 61:1281-6.
60. Livak KJ, Rogers J, Lichter JB. Variability of dopamine D4 receptor (DRD4) gene sequence
within and among nonhuman primate species. Proc Natl Acad Sci U S A (1995) 92(2):427-
31.
61. Cloninger CR, Svrakic DM, Przybeck TR. A psychobiological model of temperament and
character. Arch Gen Psychiatry (1993) 50:975-90.
62. Faraone SV, Doyle AE, Mick E, Biederman J. Meta-analysis of the association between the
7-repeat allele of the dopamine D(4) receptor gene and attention deficit hyper-activity
disorder. Am J Psychiatry (2001) 158: 1052-7.
63. Curran S, Mill J, Sham P, Rijsdijk F, Marusic K, Taylor E, Asherson P. QTL association
analysis of the DRD4 exon 3 VNTR polymorphism in a population sample of children
screened with a parent rating scale for ADUD symptoms. Am J Med Genet (2001)
105:387-93.
64. Eisenberg J, Zohar A, Mei-Tal G, Steinberg A, Tartakovsky E, Gritsenko I, Nemanov L,
Ebstein RP. A haplotype relative risk study of the dopamine D4 receptor (DRD4) exon III
repeat polymorphism and attention deficit hyper-activity disorder (ADHD). Am J Med
Genet (2000) 96:258-61.
65. Hawi Z, McCarron M, Kirley A, Daly G, Fitzgerald M, Gill M. No association of the
dopamine DRD4 receptor (DRD4) gene polymorphism with attention deficit hyper-
activity disorder (ADHD) in the Irish population. Am J Med Genet (2000) 96:268-72.
66. Okuyama Y, Ishiguro H, Nankai M, Shibuya H, Watanabe A, Arinami T. Identification of
a polymorphism in the promoter region of DRD4 associated with the human novelty
seeking personality trait. Mol Psychiatry (2000) 5:64-9.
67. Auerbach J, Gelier V, Lezer S, Shinwell E, Belmaker RH, Levine J, Ebstein R. Dopamine
D4 receptor (D4DR) and serotonin transporter promoter (5-HTTLPR) polymorphisms in
the determination of temperament in 2-month-old infants. Mol Psychiatry (1999) 4:369-
73.
68. De Luca A, Rizzardi M, Torrente I, Alessandroni R, Salvioli GP, Filograsso N, Dallapiccola
B, Novelli G. Dopamine D4 receptor (DRD4) polymorphism and adaptability trait during
infancy: a longitudinal study in 1- to 5-month-old neonates. Neurogenetics (2001) 3:79-
82.
69. Auerbach JG, Benjamin J, Faroy M, Geller V, Ebstein R. DRD4 related to infant attention
and information processing: a developmental link to ADHD? Psychiatr Genet (2001)
11:31-5.
70. Rubinstein M, Phillips TJ, Bunzow JR, Falzone TL, Dziewczapolski G, Zhang G, Fang Y,
Larson JL, McDougall JA, Chester JA, Saez C, Pugsley TA, Gershanik O, Low MJ, Grandy
DK. Mice lacking Dopamine D4 receptors are supersensitive to ethanol, cocaine, and
methamphetamine. Cell (1997) 90:991-1001.
71. Sunohara GA, Roberts W, Malone M, Schachar RJ, Tannock R, Basile VS, Wigal T, Wigal
SB, Schuck S, Moriarty J, Swanson TM, Kennedy IL, Barr CL. Linkage of the dopamine D4
receptor gene and attention deficit hyper-activity disorder. J Am Acad Child Adolesc
Psychiatry (2000) 39:1537-42.
72. Faraone SV, Biederman J, Weiffenbach B, Keith T, Chu MP, Weaver A, Spencer TJ,
Wilens TE, Frazier J, Cieves M, Sakai J. Dopamine D4 gene 7-repeat allele and attention
deficit hyper-activity disorder. Am J Psychiatry (1999) 156:768-70.
73. McCracken JT, Smalley SL, McGough JJ, Crawford L, Del’Homme M, Cantor RM, Liu A,
Nelson SF. Evidence for linkage of a tandem duplication polymorphism upstream of the
dopamine D4 receptor gene (DRD4) with attention deficit hyper-activity disorder
(ADHD). Mol Psychiatry (2000) 5:53 1-6.

239
74. Holmes J, Payton A, Barrett 1H, Hever T, Fitzpatrick H, Trumper AL, Harrington R,
McGuffin P, Owen M, Oilier W, Worthington J, Thapar A. A family-based and case-
control association study of the dopamine D4 receptor gene and dopamine transporter
gene in attention deficit hyper-activity disorder. Mol Psychiatry (2000) 5:523-30.
75. Roman T, Schmitz M, Poianczyk G, Eizirik M, Rohde LA, Hutz MH. Attention-deficit
hyper-activity disorder: a study of association with both the dopamine transporter gene
and the dopamine D4 receptor gene. Am J Med Genet (2001) 105:471-8.
76. Faraone SV, Doyle AE, Mick E, Biederman J. Meta-analysis of the association between the
7-repeat allele of the dopamine D(4) receptor gene and attention deficit hyper-activity
disorder. Am J Psychiatry (2001) 158:1052-7.
77. Sano M, Kitajima S. Activation of microtubule-associated protein kinase in PCi 2D cells in
response to both fibroblast growth factor and epidermal growth factor and concomitant
stimulation of the outgrowth of neurites. J Neurochem (1992) 58:837-44.
78. Selcher JC, Atkins CM, Trzaskos JM, Paylor R, Sweatt JD. A necessity for MAP kinase
activation in mammalian spatial learning. Learn Mem (1999) 6:478-90.
79. Oak TN, Lavine N, Van Toi fill. Dopamine D(4) and D(2L) Receptor Stimulation of the
Mitogen-Activated Protein Kinase Pathway Is Dependent on trans-Activation of the
Platelet-Derived Growth Factor Receptor. Mol Pharmacol (2001) 60(1):92-103.
80. Deth RC, Mehta S, Tan W, Liu YF, Marshall J. D4 Dopamine receptors co-localize with
the synaptic scaffolding protein SAP-97 and glutamate receptors in rat brain extracts. Soc
Neuroscience Abstracts (1999) 25 :22 14.
81. Lisman J, Schulman H, Cime H. The molecular basis of CaMKII function in synaptic and
behavioural memory. Nat Rev Neurosci (2002) 3:175-90.
82. Sattler R, Tymianski M. Molecular mechanisms of glutamate receptor-mediated
excitotoxic neuronal cell death. Mol Neurobiol (2001) 24:107-29.
83. Otmakhova NA, Lisman JE. Dopamine selectively inhibits the direct cortical pathway to
the CA 1 hippocampal region. J Neurosci (1999) 19: 1437-45.
84. Arvanov VL, Liang X, Schwartz J, Grossman S, Wang RY. Clozapine and haloperidol
modulate N-methyl-D-aspartate- and non-N-methyl-D aspartate receptor-mediated
neurotransmission in rat prefrontal cortical neurons in vitro. J Pharmacol Exp Ther
(1997) 283:226-34.
85. Whiting KP, Restau CJ, Brain PF. Steroid hormone-induced effects on membrane fluidity
and their potential roles in non-genomic mechanisms. Life Sci (2000) 67(7):743-57.
86. Anderson SA, Classey ID, Conde F, Lund JS, Lewis DA. Synchronous development of
pyramidal neuron dendritic spines and parvalbumin immunoreactive chandelier neuron
axon terminals in layer III of monkey prefrontal cortex. Neuroscience (1995) 67:7-22.
87. Cellerino A, Siciliano R, Domenici L, Maffei L. Parvalbumin immunoreactivity: a reliable
marker for the effects of monocular deprivation in the rat visual cortex. Neuroscience
(1992) 51 :749-53 88.
88. Sesack SR, Snyder CL, Lewis DA. Axon terminals immunolabeled for dopamine or
tyrosine hydroxylase synapse on GABA-immunoreactive dendrites in rat and monkey
cortex. J Comp Neurol (1995) 363:264-80.
89. Goldman-Rakic PS. The “psychic” neuron of the cerebral cortex. Ann NY Acad Sci (1999)
868:13-26.
90. Lanau F, Zenner MT, Civelli O, Hartman DS. Epinephrine and norepinephrine act as
potent agonists at the recombinant human dopamine D4 receptor. J Neurochem (1997)
68:804-12.
91. Newman-Tancredi A, Audinot-Bouchez V, Gobert A, Millan MJ. Noradrenaline and
adrenaline are high affinity agonists at dopamine 4 receptors. Eur J Pharmacol (1997)
319:379-83.
92. Chard PS, Bleakman D, Christakos S, Fuilmer CS, Miller RJ. Calcium buffering properties
of calbindin D28k and parvalbumin in rat sensory neurones. J Physiol (1993) 472:341-57.
93. Ganguly PK, Panagia V, Okumura K, Dhalla NS. Activation of Ca2+-stimulated ATPase
by phospholipid N-methylation in cardiac sarcoplasmic reticulum. Biochem Biophys Res
Commun (1985) 30:472-8.

240
94. Panagia V, Okumura K, Makino N, Dhalla NS. Stimulation of Ca2+ pump in rat heart
sarcolemma by phosphatidylethanolamine N methylation. Biochim Biophys Acta (1986)
856:383-7.
95. Cervino V, Benaim G, Carafoli E, Guerini D. The effect of ethanol on the plasma
membrane calcium pump is isoform-specific. J Biol Chem (1998) 273:29811-5.
96. Benaim G, Cervino V, Lopez-Estrano C, Weitzman C. Ethanol stimulates the plasma
membrane calcium pump from human erythrocytes. Biochim Biophys Acta(1994)
1195(1):141-8.
97. Kim E, Niethammer M, Rothschild A, Jan YN, Sheng M. Clustering of Shaker-type K+
channels by interaction with a family of membrane-associated guanylate kinases. Nature
(1995) 378:85-8.
98. Dopico AM, Widmer H, Wang G, Lemos JR, Treistman SN. Rat supraoptic magnocellular
neurones show distinct large conductance, Ca2+-activated K+ channel subtypes in cell
bodies versus nerve endings. J Physiol (1999) 519:101-14.
99. Nakamura K, Kouchi H, Ohe T, Namba M. Increase in beating rate of cultured chick
cardiac myocytes by ethanol and inhibition of the increase by antiarrhythmic drugs.
Alcohol Clin Exp Res (1999) 23:81S-84S.
100. Cobb SR, Buhi EH, Halasy K, Paulsen O, Somogyi P. Synchronization of neuronal activity
in hippocampus by individual GABAergic intemeurons. Nature (1995) 378:75-8.
101. Canu MH, Rougeul A. Nucleus reticularis thalami participates in sleep spindles, not in
beta rhythms concomitant with attention in cat. C R Acad Sci 111(1992) 3 15:513-20.
102. Murthy VN, Fetz EE. Coherent 25- to 35-Hz oscillations in the sensorimotor cortex of
awake behaving monkeys. Proc Natl Acad Sci USA (1992) 89:5670-4.
103. Fries P, Roelfsema PR, Engel AK, Konig P, Singer W. Synchronization of oscillatory
responses in visual cortex correlates with perception in interocular rivalry. Proc Natl Acad
Sci USA (1997) 94:12699-704.
104. Tiitinen H, May P, Naatanen R. The transient 40-Hz response, mismatch negativity, and
attentional processes in humans. Prog Neuropsycho pharmacol Bio! Psychiatry (1997)
21:751-71.
105. Singer W. Consciousness and the binding problem. Ann N Y Acad Sci (2001) 929:123-46.
106. Galambos R, Makeig S, Talmachoff PJ. A 40-Hz auditory potential recorded from the
human scalp. Proc Nati Acad Soi USA (1981) 78:2643-7.
107. Ribary U, Ioannides AA, Singh KD, Hasson R, Bolton IP, Lado F, Mogilner A, Limas R.
Magnetic field tomography of coherent thalamocortical 40-Hz oscillations in humans.
Proc Nati Acad Sci USA (1991) 88:11037-41.
108. Limas R, Ribary U. Coherent 40-Hz oscillation characterizes dream state in humans. Proc
Nati Acad Sci U S A (1993) 90:2078-8 1.
109. Onn SP, Grace AA. Dopamine D4 receptor-dependent regulation of PFC pyramidal
neuron activity is mediated via GABAergie interneurons. Soc. Neuroscience Abstracts
(2001) 27:2189.
110. Harris GC, Hausken ZE, Williams JT. Cocaine induced synchronous oscillations in central
noradrenergic neurons in vitro. Neuroscience (1992) 50:253-7.
111. Robb ins DJ, Cheng M, Zhen E, Vanderbilt CA, Feig LA, Cobb MH. Evidence for a Ras-
dependent extracellular signal-regulated protein kinase (ERK) cascade. Proc Natl Acad Sci
USA (1992) 89(15):6924-8.
112. Kimpinski K, Mearow K. Neurite growth promotion by nerve growth factor and insulin-
like growth factor-i in cultured adult sensory neurons: role of phosphoinositide 3-kinase
and mitogen activated protein kinase. JNeurosci Res (2001) 63:486-99.
113. Ambalavanar R, McCabe BJ, Potter KN, Horn G. Learning-related fos like
immunoreactivity in the chick brain: time-course and co-localization with GABA and
parvalbumin. Neuroscience (1999) 93:1515-24.
114. Seeman P, Bzowej NH, Guan HC, Bergeron C, Becker LE, Reynolds GP, Bird ED, Riederer
P, Jellinger K, Watanabe S, et al. Human brain dopamine receptors in children and aging
adults. Synapse (1987) 1:399-404.

241
115. Porter LL, Rizzo E, Hornung JP. Dopamine affects parvalbumin expression during cortical
development in vitro. J Neurosci (1999) 19:8990-9003.
116. Megias M, Emri Z, Freund TF, Gulyas AI. Total number and distribution of inhibitory and
excitatory synapses on hippocampal CAl pyramidal cells. Neuroscience (2001) 102:527-40.
117. Wang XH, Jenkins AO, Choi L, Murphy EH. Altered neuronal distribution of
parvalbumin in anterior cingulate cortex of rabbits exposed in utero to cocaine. Exp Brain
Res (1996) 112:359-71.
118. Wedzony K, Chocyk A, Mackowiak M, Fijal K, Czyrak A. Cortical localization of
dopamine D4 receptors in the rat brain-immunocytochemical study. J Physiol Pharmacol
(2000) 5 1:205-21.
119. Lezcano N, Mrzljak L, Eubanks S, Levenson R, Goidman-Rakic P, Bergson C. Dual
signaling regulated by calcyon, a Dl dopamine receptor interacting protein. Science (2000)
287:1660-4.
120. Aston-Jones G, Chiang C, Alexinsky T. Discharge of noradrenergic locus coeruleus
neurons in behaving rats and monkeys suggests a role in vigilance. Prog Brain Res (1991)
88:501-20.
121. Posner MI. Attention: the mechanisms of consciousness. Proc Natl Acad Sci USA (1994)
91(16):7398-403.
122. Lisman JE, Otmakhova NA. Storage, recall, and novelty detection of sequences by the
hippocampus: elaborating on the SOCRATIC model to account for normal and aberrant
effects of dopamine. Hippocampus (2001) 11551-68.
123. Loftus M, Amral DG. The dopamine system in the hippocampal formation of the Maca
fascicularis monkey. Soc Neuroscience Abstracts (2000) 26:1431.
124. Carr DB, Sesack SR. Projections from the rat prefrontal cortex to the ventral tegmental
area: target specificity in the synaptic associations with mesoaccumbens and mesocortical
neurons. J Neurosci (2000) 20:3 864-73.
125. van der Put NM, Thomas CM, Eskes TK, Trijbels FJ, Steegers Theunissen RP, Mariman
EC, De Graaf-Hess A, Smeitink JA, Blom HJ. Altered folate and vitamin B12 metabolism
in families with spina bifida offspring. QJM (1997) 90:505-10.
126. Selhub J, Jacques PF, Bostom AG, Wilson PW, Rosenberg 1H. Relationship between
plasma homocysteine and vitamin status in the Framingham study population. Impact of
folic acid fortification. Public Health Rev (2000) 28:117-45.
127. Kang SS, Wong PW, Zhou JM, Sora J, Lessick M, Ruggie N, Grcevich G. Thermolabile
methylenetetrahydrofolate reductase in patients with coronary artery disease. Metabolism
(1988) 37:611-3.
128. Frosst P, Blom HJ, Milos R, Goyette P, Sheppard CA, Matthews RG, Boers GJ, den
HeijerM, Kluijtmans LA, van den Heuvel LP, et al. A candidate genetic risk factor for
vascular disease: a common mutation in methylenetetrahydrofolate reductase. Nat Genet
(1995) 10:111-3.
129. Arinami T, Yamada N, Yamakawa-Kobayashi K, Hamaguchi H, Toru M.
Methylenetetrahydrofolate reductase variant and schizophrenia depression. Am J Med
Genet (1997) 74(5):526-8.
130. Regland B, Germgard T, Gottfries CG, Grenfeldt B, Koch-Schmidt AC. Homozygous
thermolabile methylenetetrahydrofolate reductase in schizophrenia-like psychosis. J
Neural Transm (1997) 104:931-41.
131. Stover P, Schirch V. Enzymatic mechanism for the hydrolysis of 5,10-
methenyltetrahydropteroylglutamate to 5-formyltetrahydro-231 pteroylglutamate by
serine hydroxymethyltransferase. Biochemistry (1992) 3 1:2155-64.
132. Stover PJ, Chen LH, Suh JR. Stover DM, Keyomarsi K, Shane B. Molecular cloning,
characterization, and regulation of the human mitochondrial serine
hydroxymethyltransferase gene. J Biol Chem (1997) 272:1842-8.
133. Bertrand R, Jolivet J. Methenyltetrahydrofolate synthetase prevents the inhibition of
phosphoribosyl 5-aminoimidazole 4-carboxamide ribonucleotide formyltransferase by 5-
formyltetrahydrofolate polyglutamates. J Biol Chem (1989) 264:8843-6.

242
134. Stover P, Schirch V. 5-Formyltetrahydrofolate polyglutamates are slow tight binding
inhibitors of serine hydroxymethyltransferase. J Biol Chem (1991) 266:1543-50.
135. Sunden SL, Rencluchintala MS, Park El, Miklasz SD, Garrow TA. Betaine-homocysteine
methyltransferase expression in porcine and human tissues and chromosomal localization
of the human gene. Arch Biochem Biophys (1997) 345:171-4.
136. O’Tuama LA, Phillips PC, Smith QR, Uno Y, Dannals RF, Wilson AA, Ravert HT, Loats S,
Loats l{A, Wagner HN Jr. L-methionine uptake by human cerebral cortex: maturation
from infancy to old age. J Nucl Med (1991) 32:16-22.
137. Wills L. Treatment of “pernicious anaemia of pregnancy” and “tropical anaemia” with
special reference to yeast extract as a curative agent. Br Med J(1931) 1:1059-64.
138. Bottiglieri T. Folate, vitamin B12, and neuropsychiatric disorders. Nutr Rev (1996) 54:382-
90.
139. Susser E, Brown AS, Klonowski E, Allen RH, Lindenbaum J. Schizophrenia and impaired
homocysteine metabolism: a possible association. Biol Psychiatry (1998) 44:141-3.
140. Wyatt KM, Dimmock PW, Jones PW, Shaughn O’Brien PM. Efficacy of vitamin B-6 in the
treatment of premenstrual syndrome: systematic review. BMJ (1999) 318: 1375-81.
141. McCarty MF. High-dose pyridoxine as an ‘anti-stress’ strategy. Med Hypotheses (2000)
54:803-7.
142. Alao AO, Yolles JC. Isoniazid-induced psychosis. Ann Pharmacother (1998) 32:889-91.
143. Lindenbaum J, Healton EB, Savage DG, Brust JC, Garrett TJ, Podell ER, Marcell PD,
Stabler SP, Allen RH. Neuropsychiatric disorders caused by cobalamin deficiency in the
absence of anemia or macrocytosis. N Engl J Med (1988) 318:1720-8.
144. Wang HX, Wahlin A, Basun H, Fastbom J, Winbiad B, Fratiglioni L. Vitamin B( 12) and
folate in relation to the development of Alzheimer’s disease. Neurology (2001) 56:1188-94.
145. Ban TA. Nicotinic acid in the treatment of schizophrenias. Practical and theoretical
considerations. Neuropsychobiology (1975) 1:133-45. 232
146. Perez-Garci E, del-Rio-Portilla Y, Guevara MA, Arce C, Corsi-Cabrera M. Paradoxical
sleep is characterized by uncoupled gamma activity between frontal and perceptual
cortical regions. Sleep (2001) 24(1): 118-26.
147. Belelli I, Pistis I, Peters JA, Lambert JJ. General anaesthetic action at transmitter-gated
inhibitory amino acid receptors. Trends Pharmacol Sci (1999) 20:496-502.
148. Weight FF, Lovinger DM, White G, Peoples RW. Alcohol and anesthetic actions on
excitatory amino acid-activated ion channels. Ann N Y Acad Sci (1991) 625:97-107.
149. Zawilska JB, Nowak JZ. Does D4 dopamine receptor mediate the inhibitory effect of light
on melatonin biosynthesis in chick retina?Neurosci Left (1994) 166:203-6.
150. Chagoya de Sanchez V, Hernandez-Munoz R, Diaz-Munoz M, Villalobos R, Glender W,
Vidrio S, Suarez J, Yanez L. Circadian variations of adenosine level in blood and liver and
its possible physiological significance. Life Sci (1983) 33:1057-64.
151. Basheer R, Porkka-Heiskanen T, Strecker RE, Thakkar MM, McCarley RW. Adenosine as
a biological signal mediating sleepiness following prolonged wakefulness. Biol Signals
Recept (2000) 9:319-27.
152. Elliott KJ, Todd Weber E, Rea MA. Adenosine Al receptors regulate the response of the
hamster circadian clock to light. Eur J Pharmacol (2001) 414:45-53.
153. Pak MA, Haas HL, Decking UK, Schrader J. Inhibition of adenosine kinase increases
endogenous adenosine and depresses neuronal activity in hippocampal slices.
Neuropharmacology (1994) 33:1049-53.
154. Chagoya de Sanchez V, Hernandez Munoz R, Suarez J, Vidrio S, Yanez L, Diaz Munoz M.
Day-night variations of adenosine and its metabolizing enzymes in the brain cortex of the
rat--possible physiological significance for the energetic homeostasis and the sleep-wake
cycle. Brain Res (1993) 612:115-21.
155. Benson H. The Relaxation Response. New York, New York: HarperCollins, 2000.

243
156. Das NN, Gastaut H. Variation de l’activite electrique du cerveau, du coeur et des muscles
squelletiques au cours de la meditation et de l’extase yogique. Electrocenceph Clin
Neurophysiol (1957) Suppl 6:211-219.
157. Wallace RK. Physiological effects of transcendental meditation. Science (1970) 167:1751-
4.
158. Orme-Johnson DW, Haynes CT. EEG phase coherence, pure consciousness, creativity,
and TM--Sidhi experiences. mt j Neurosci (1981) 13:211-7.
159. Lou HC, Kjaer TW, Friberg L, Wlldschiodtz G, Hoim S, Nowak M. A 150-H20 PET study
of meditation and the resting state of normal consciousness. Hum Brain Mapp (1999)
7:98-105.
160. Fellous JM, Sejnowski TJ. Cholinergic induction of oscillations in the hippocampal slice in
the slow (0.5-2 Hz), theta (5-12 Hz), and gamma (35-70 Hz) bands. Hippocampus (2000)
10:187-97.
161. Boroojerdi B, Phipps M, Kopylev L, Wharton CM, Cohen LG, Grafman J. Enhancing
analogic reasoning with rTMS over the left prefrontal cortex. Neurology (2001) 56(4):526-
8.
162. Okada M, Kiryu K, Kawata Y, Mizuno K, Wada K, Tasaki H, Kaneko S. Determination of
the effects of caffeine and carbamazepine on striatal dopamine release by in vivo
microdialysis. Eur J Pharmacol (1997) 321: 181-8.
163. Jackisch R, Fehr R, Hertting G. Adenosine: an endogenous modulator of hippocampal
noradrenaline release. Neuropharmacology (1985) 24:499-507.
164. Garrett BE, Holtzman SG. Dl and D2 dopamine receptor antagonists block caffeine-
induced stimulation of locomotor activity in rats. Pharmacol Biochem Behav (1994)
47:89-94.
165. Ritz MC, Lamb RJ, Goldberg SR, Kuhar MJ. Cocaine receptors on dopamine transporters
are related to self-administration of cocaine. Science (1987) 237:1219-23.
166. Volkow ND, Wang GJ, Fowler JS, Logan J, Gatley SJ, Wong C, Hitzemann R, Pappas NR.
Reinforcing effects of psychostimulants in humans are associated with increases in brain
dopamine and occupancy of D(2) receptors. J Pharmacol Exp Ther (1999) 291:409-15.
167. Kalivas PW, Duffy P. Effect of acute and daily cocaine treatment on extracellular
dopamine in the nucleus accumbens. Synapse (1990) 5:48-58.
168. Allard P0, Rinne J, Marcusson JO. Dopamine uptake sites in Parkinson’s disease and in
dementia of the Alzheimer type. Brain Res (1994) 637:262-6.
169. Dougherty DD, Bonab AA, Spencer TJ, Rauch SL, Madras BK, Fischman AJ. Dopamine
transporter density in patients with attention deficit hyper-activity disorder. Lancet (1999)
354:2132-3.
170. Jones SR, Gainetdinov RR, Wightman RM, Caron MG. Mechanisms of amphetamine
action revealed in mice lacking the dopamine transporter. JNeurosci (1998) 18:1979-86.
171. Giorguieff MF, Le Floc’h ML, Glowinski J, Besson MJ. Involvement of cholinergic
presynaptic receptors of nicotinic and muscarinic types in the control of the spontaneous
release of dopamine from striatal dopaminergic terminals in the rat. J Pharmacol Exp Ther
(1977) 200:535-44.
172. Toth E, Sershen H, Hashim A, Vizi ES, Lajtha A. Effect of nicotine on extracellular levels
of neurotransmitters assessed by microdialysis in various brain regions: role of glutamic
acid. Neurochem Res (1992) 17:265-7 1.
173. Schilstrom B, Fagerquist MV, Zhang X, Hertel P, Panagis G, Nom ikos GG, Svensson TH.
Putative role of presynaptic alpha7* nicotinic receptors in nicotine stimulated increases of
extracellular levels of glutamate and aspartate in the ventral tegmental area. Synapse
(2000) 3 8:375-83 .
174. Leonard S, Adams C, Breese CR, Adler LE, Bickford P, Byerley W, Coon H, Griffith JM,
Miller C, Myles-Worsley M, Nagamoto HT, Rollins Y, Stevens KE, Waldo M, Freedman R.
Nicotinic receptor function in schizophrenia. Schizophr Bull (1996) 22:431-45.
175. Fowler JS, Volkow ND, Wang GJ, Pappas N, Logan J, MacGregor R, Alexoff D, Shea C,
Schlyer D, Wolf AP, Warner D, Zezulkova I, Cuento R. Inhibition of monoamine oxidase
B in the brains of smokers. Nature (1996) 379:733-6.

244
176. Fowler JS, Volkow ND, Wang GJ, Pappas N, Logan J, Shea C, Alexoff D, MacGregor RR,
Schlyer DJ, Zezulkova I, Wolf AP. Brain monoamine oxidase A inhibition in cigarette
smokers. Proc Nati Acad Sci U S A (1996) 93:14065-9.
177. Cohen GA, Doze VA, Madison DV. Opioid inhibition of GABA release from presynaptic
terminals of rat hippocampal interneurons. Neuron (1992) 9:325-35.
178. Johnson SW, North RA. Opioids excite dopamine neurons by hyper-polarization of local
interneurons. J Neurosci (1992) 12:483-8.
179. Gerrits MA, Petromilli P, Westenberg HG, Di Chiara G, van Ree JM. Decrease in basal
dopamine levels in the nucleus accumbens shell during daily drug-seeking behaviour in
rats. Brain Res (2002) 924:141 -50.
180. Tzschentke TM. Pharmacology and behavioral pharmacology of the mesocortical
dopamine system. Prog Neurobiol (2001) 63:241-320.
181. Devane WA, Dysarz FA 3rd, Johnson MR, Melvin LS, Howlett AC. Determination and
characterization of a cannabinoid receptor in rat brain. Mol Pharmacol (1988) 34:605-13.
182. Devane WA, Hanus L, Breuer A, Pertwee RG, Stevenson LA, Griffin G, Gibson D,
Mandelbaum A, Etinger A, Mechoulam R. Isolation and structure of a brain constituent
that binds to the cannabinoid receptor. Science (1992) 258:1946-9.
183. Giuffrida A, Parsons LH, Kerr TM, Rodriguez de Fonseca F, Navarro M, Piomelli D.
Dopamine activation of endogenous cannabinoid signaling in dorsal striatum. Nat
Neurosci (1999) 2:358-63.
184. Beltramo M, de Fonseca FR, Navarro M, Calignano A, Gorriti MA,Grammatikopoulos G,
Sadile AG, Giuffrida A, Piomelli D. Reversal of dopamine D(2) receptor responses by an
anandamide transport inhibitor. JNeurosci (2000) 20:3401-7.
185. Bisogno T, Delton-Vandenbroucke I, Milone A, Lagarde M, Di Marzo V. Biosynthesis and
inactivation of N-arachidonoylethanolamine (anandamide) and N-
docosahexaenoylethanolamine in bovine retina. Arch Biochem Biophys (1999)
370(2):300-7.
186. Giuffrida A, Parsons LH, Kerr TM, Rodriguez de Fonseca F, Navarro M, Piomelli D.
Dopamine activation of endogenous cannabinoid signaling in dorsal striatum. Nat
Neurosci (1999) 2:358-63.
187. Gerdeman G, Lovinger DM. CB 1 cannabinoid receptor inhibits synaptic release of
glutamate in rat dorsolateral striatum. J Neurophysiol (2001) 85 :468-7 1.
188. Kathmann M, Bauer U, Schlicker E, Gothert M. Cannabinoid CB 1 receptor-mediated
inhibition of NMDA- and kainate-stimulated noradrenaline and dopamine release in the
brain. Naunyn Schmiedebergs Arch Pharmacol (1999) 359:466-70.
189. Watanabe K, Matsunaga T, Nakamura S, Kimura T, Ho 1K, Yoshimura H, Yamamoto I.
Pharmacological effects in mice of anandamide and its related fatty acid ethanolamides,
and enhancement of cataleptogenic effect of anandamide by phenylmethylsulfonyl
fluoride. Biol Pharm Bull (1999) 22:366-70.
190. Beltramo M, de Fonseca FR, Navarro M, Calignano A, Gorriti MA, Grammatikopoulos G,
Sadile AG, Giuffrida A, Piomelli D. Reversal of dopamine D(2) receptor responses by an
anandamide transport inhibitor. JNeurosci (2000) 20:3401-7.
191. Agarwal DP. Molecular genetic aspects of alcohol metabolism and alcoholism.
Pharmacopsychiatry (1997) 30:79-84.
192. Shen YC, Fan IR, Edenberg HJ, Li TK, Cui YH, Wang YF, Tian CH, Zhou CF, Zhou RL,
Wang J, Zhao ZL, Xia GY. Polymorphism of ADH and ALDH genes among four ethnic
groups in China and effects upon the risk for alcoholism. Alcohol Clin Exp Res (1997)
21:1272-7.
193. Harris RA. Ethanol actions on multiple ion channels: which are important? Alcohol Clin
Exp Res (1999) 23:1563-70.
194. Covarrubias M, Vyas TB, Escobar L, Wei A. Alcohols inhibit a cloned potassium channel
at a discrete saturable site. Insights into the molecular basis of general anesthesia. J Biol
Chem (1995) 270:19408-16.
195. Barak AJ, Beckenhauer HC, Tuma DJ, Badakhsh S. Effects of prolonged ethanol feeding
on methionine metabolism in rat liver. Biochem Cell Biol (1987) 65:230-3.

245
196. Barak AJ, Beckenhauer HC, Kharbanda KK, Tuma DJ. Chronic ethanol consumption
increases homocysteine accumulation in hepatocytes. Alcohol (2001) 25:77-8 1
197. Halsted CH, Villanueva J, Chandler CJ, Stabler SP, Allen RH, Muskhelishvili L, James SJ,
Poirier L. Ethanol feeding of micropigs alters methionine metabolism and increases
hepatocellular apoptosis and proliferation. Hepatology (1996) 23:497-505.
198. Stickel F, Choi SW, Kim Yl, Bagley PJ, Seitz HIC, Russell Rivi, Seihub J, Mason JB. Effect
of chronic alcohol consumption on total plasma homocysteine level in rats. Alcohol Clin
Exp Res (2000) 24:259-64.
199. Barak AJ, Beckenhauer HC, Kharbanda KK, Tuma DJ. Chronic ethanol consumption
increases homocysteine accumulation in hepatocytes.Alcohol (2001) 25(2):77-81.
200. Hidiroglou N, Camilo ME, Beckenhauer HC, Tuma DJ, Barak AJ, Nixon PF, Seihub J.
Effect of chronic alcohol ingestion on hepatic folate distribution in the rat. Biochem
Pharmacol (1994) 47:1561-6.
201. 201. Kenyon SH, Nicolaou A, Gibbons WA. The effect of ethanol and its metabolites upon
methionine synthase activity in vitro. Alcohol (1998) 15:305-9.
202. Waly M, Olteanu H, Banerjee R, Choi SW, Mason J, Benzecry J, Power V, Deth RC. P13-
Kinase Regulates Methionine Synthase: Activation by IGF- 1 or Dopamine and Inhibition
by Heavy Metals and Thimerosal. Submitted.
203. Bondy CA, Lee WH. Patterns of insulin-like growth factor and IGF receptor gene
expression in the brain. Functional implications. Ann N Y Acad Sci (1993) 692:33-43.
204. Myers MG Jr, Grammer TC, Wang LM, Sun XJ, Pierce JH, Blenis J, White MF. Insulin
receptor substrate-i mediates phosphatidylinositol Y-kinase and p7OS6k signaling during
insulin, insulin-like growth factor-i, and interleukin-4 stimulation. J Biol Chem (1994)
269(46):28783-9.
205. Resnicoff M, Sell C, Ambrose D, Baserga R, Rubin R. Ethanol inhibits the
autophosphorylation of the insulin-like growth factor 1 (IGF- 1) receptor and IGF- 1-
mediated proliferation of 3T3 cells. J Biol Chem (1993) 268(29):21777-82.
206. Liu W, Baker J, Perkins AS, Robertson EJ, Efstratiadis A. Mice carrying null mutations of
the genes encoding insulin-like growth factor I (Igf- 1) and type 1 IGF receptor (Igflr). Cell
(1993) 75:59-72.
207. Choi SW, Stickel F, Baik 11W, Kim Yl, Seitz HK, Mason JB. Chronic alcohol consumption
induces genomic but not p53-specific DNA hypo-methylation in rat colon. J Nutr (1999)
129:1945-50.
208. Zhang FX, Rubin R, Rooney TA. Ethanol induces apoptosis in cerebellar granule neurons
by inhibiting insulin-like growth factor 1 signaling. J Neurochem (1998) 71:196-204.
209. Barbaresi WJ, Katusic SK, Colligan RC, Pankratz VS, Weaver AL, Weber KJ, Mrazek DA,
Jacobsen SJ. How common is attention-deficit/hyper-activity disorder? Incidence in a
population-based birth cohort in Rochester, Minn. Arch Pediatr Adolesc Med (2002)
156:217-24.
210. Rowland AS, Umbach DM, Stallone L, Naftel AJ, Bohlig EM, Sandier DP. Prevalence of
medication treatment for attention deficit/hyper-activity disorder among elementary
school children in Johnston County, North Carolina. Am J Public Health (2002) 92:231-4.
211. American Psychiatric Association: Diagnostic and Statistical Manual of Mental Disorders,
Fourth Edition. Washington D.C.: American Psychiatric Association, 1994.
212. American Academy of Pediatrics: Clinical Practice Guideline: Diagnosis and Evaluation of
the Child With Attention- Deficit/hyper-activity Disorder. Pediatrics (2000) 105:1158-
1170.
213. Mannuzza S, Klein RG, Addalli KA. Young adult mental status of hyper-active boys and
their brothers: a prospective follow-up study. J Am Acad Child Adolesc Psychiatry (1991)
30:743-5 1.
214. Hill JC, Schoener EP. Age-dependent decline of attention deficit hyper-activity disorder.
Am J Psychiatry (1996) 153:1143-6.
215. 215. Hofstra MB, van der Ende J, Verhulst FC. Child and adolescent problems predict
DSM-IV disorders in adulthood: a 14-year follow-up of a Dutch epidemiological sample. J
Am Acad Child Adolesc Psychiatry (2002) 41:182-9.

246
216. Stephens JC, Schneider JA, Tanguay DA, Choi J, Acharya T, Stanley SE, Jiang R, Messer
CJ, Chew A, Han 1H, Duan J, Carr JL, Lee MS, Koshy B, Kumar AM, Zhang G, Newell
WR, Windemuth A, Xu C, Kalbfleisch TS, Shaner SL, Arnold K, Schulz V, Drysdale CM,
Nandabalan K, Judson RS, Ruano G, Vovis GF. Haplotype variation and linkage
disequilibrium in 313 human genes. Science (2001) 293 :489-93.
217. Holmes J, Payton A, Barrett 1H, Hever T, Fitzpatrick H, Trumper AL, Harrington R,
McGuffin P, Owen M, Ollier W, Worthington J, Thapar A. A family-based and case-
control association study of the dopamine D4 receptor gene and dopamine transporter
gene in attention deficit hyper-activity disorder. Mol Psychiatry (2000) 5:523-3 0.
218. Muglia P, Jam U, Macc iardi F, Kennedy JL. Adult attention deficit hyper-activity disorder
and the dopamine D4 receptor gene. Am J Med Genet (2000) 96:273-7.
219. Ronai Z, Szekely A, Nemoda Z, Lakatos K, Gervai J, Staub M, Sasvari Szekely M.
Association between Novelty Seeking and the -521 C/T polymorphism in the promoter
region of the DRD4 gene. Mol Psychiatry (2001) 6:35-8.
220. Lakatos K, Nemoda Z, Toth I, Ronai Z, Ney K, Sasvari- Szekely M, Gervai J. Further
evidence for the role of the dopamine D4 receptor (DRD4) gene in attachment
disorganization: interaction of the exon III 48-bp repeat and the -521 C/T promoter
polymorphisms. Mol Psychiatry (2002) 7:27-31.
221. Catalano M, Nobile M, Novelli E, Nothen MM, Smeraldi E. Distribution of a novel
mutation in the first exon of the human dopamine D4 receptor gene in psychotic patients.
Biol Psychiatry (1993) 34:459-64.
222. Petronis A, O’Hara K, Barr CL, Kennedy IL, Van Tol HH. (G)n mononucleotide
polymorphism in the human D4 dopamine receptor (DRD4) gene. Hum Genet (1994)
93:719.
223. Barr CL, Wigg KG, Bloom S, Schachar R, Tannock R, Roberts W, Malone M, Kennedy IL.
Further evidence from haplotype analysis for linkage of the dopamine D4 receptor gene
and attention-deficit hyper-activity disorder. Am J Med Genet (2000) 96:262-7.
224. Elmaleh DR, Fischman AJ, Shoup TM, Byon C, Hanson RN, Liang AY, Meltzer PC,
Madras BK. Preparation and biological evaluation of iodine- 125-IACFT: a selective
SPECT agent for imaging dopamine transporter sites. JNucl Med (1996) 37:1197-202.
225. Dougherty DD, Bonab AA, Spencer TJ, Rauch SL, Madras BK, Fischman AJ. Dopamine
transporter density in patients with attention deficit hyper-activity disorder. Lancet (1999)
354:2132-3.
226. Vandenbergh DJ, Persico AM, Hawkins AL, Griffin CA, Li X, Jabs EW, Uhi GR. Human
dopamine transporter gene (DAT 1) maps to chromosome 5pl5.3 and displays a VNTR.
Genomics (1992) 14:1104-6.
227. Cook EH Jr, Stein MA, Krasowski MD, Cox NJ, Olkon DM, Kieffer JE, Leventhal BL.
Association of attention-deficit disorder and the dopamine transporter gene. Am J Hum
Genet (1995) 56:993-8.
228. Barr CL, Xu C, Kroft J, Feng Y, Wigg K, Zai G, Tannock R, Schachar R, Malone M,
Roberts W, Nothen MM, Grunhage F, Vandenbergh DJ, Uhl G, Sunohara G, King N,
Kennedy IL. Haplotype study of three polymorphisms at the dopamine transporter locus
confirm linkage to attention-deficit/hyper-activity disorder. Biol Psychiatry (2001)
49:333-9.
229. Fuke S, Suo S, Takahashi N, Koike H, Sasagawa N, Ishiura S. The VNTR polymorphism of
the human dopamine transporter (DAT 1) gene affects gene expression.
Pharmacogenomics J(2001) 1:152-6. 230. Miller GM, De La Garza RD 2nd, Novak MA,
Madras BK. Single nucleotide polymorphisms distinguish multiple dopamine transporter
alleles in primates: implications for association with attention deficit hyper-activity
disorder and other neuropsychiatric disorders. Mol Psychiatry (2001) 6:50-8.
230. Kuhn, TS. The Structure of Scientiftic Revolutions. Chicago, Illinois: The University of
Chicago Press, 1962.
231. Egan MF, Goldberg TE, Kolachana BS, Callicott TEl, Mazzanti CM, Straub RE, Goldman
D, Weinberger DR. Effect of COMT Va1108/158 Met genotype on frontal lobe function
and risk for schizophrenia. Proc Natl Acad Sci U S A (2001) 98(12):6917-22.

247
232. Barr CL, Wigg K, Malone M, Schachar R, Tannock R, Roberts W, Kennedy IL. Linkage
study of catechol-O-methyltransferase and attention-deficit hyper-activity disorder. Am J
Med Genet (1999) 88:710-3.
233. Ohno Y, Matsuo K, Suzuki H, Tanase H, Ikeshima H, Takano T, Saruta T. Genotypes of
sarco(endo)plasmic reticulum Ca(2+)-dependent ATPase II gene in substrains of
spontaneously hypertensive rats. J Hypertens (1996) 14:287-91.
234. Sagvolden T. Behavioral validation of the spontaneously hyper-tensive rat (SUR) as an
animal model of attention-deficit/hyper-activity disorder (AD/HD). Neurosci Biobehav
Rev (2000) 24:31-9.
235. Joober R, Benkelfat C, Lal S, Bloom D, Labelle A, Lalonde P, Turecki G, Rozen R, Rouleau
GA. Association between the methylenetetrahydrofolate reductase 677C-->T missense
mutation and schizophrenia. Mol Psychiatry (2000) 5(3):323-6.
236. A 14-month randomized clinical trial of treatment strategies for attention-deficit/hyper-
activity disorder. The MTA Cooperative Group. Multimodal Treatment Study of Children
with ADHD. Arch Gen Psychiatry (1999) 56:1073-86.
237. Spencer T, Biederman J, Wilens T, Faraone S, Prince J, Gerard K, Doyle R, Parekh A,
Kagan J, Bearman SK. Efficacy of a mixed amphetamine salts compound in adults with
attention-deficit/hyper-activity disorder. Arch Gen Psychiatry (2001) 58:775-82.
238. Berman T, Douglas VI, Barr RG. Effects of methylphenidate on complex cognitive
processing in attention-deficit hyper-activity disorder. J Abnorm Psychol (1999) 108:90-
105.
239. Jones SR, Gainetdinov RR, Wightman RM, Caron MG. Mechanisms of amphetamine
action revealed in mice lacking the dopamine transporter. JNeurosci (1998) 18:1979-86.
240. Volkow ND, Wang GJ, Fowler JS, Logan J, Franceschi D, Maynard L, Ding YS, Gatley SJ,
Gifford A, Zhu W, Swanson SM. Relationship
241. between blockade of dopamine transporters by oral methylphenidate and the increases in
extracellular dopamine: therapeutic implications. Synapse (2002) 43:181-7.
242. Kuczenski R, Segal D. Concomitant characterization of behavioral and striatal
neurotransmitter response to amphetamine using in vivo microdialysis. J Neurosci (1989)
9:2051-65.
243. Suaud-Chagny MF, Buda M, Gonon FG. Pharmacology of electrically evoked dopamine
release studied in the rat olfactory tubercle by in vivo electrochemistry. Eur J Pharmacol
(1989) 164:273-83.
244. Zhang K, Tarazi FI, Baldessarini RJ. Role of dopamine D(4) receptors in motor hyper-
activity induced by neonatal 6-hydroxydopamine lesions in rats.
Neuropsychopharmacology (2001) 25:624-32.
245. Zhang K, Tarazi FI, Davids E, Baldessarini RJ. Plasticity of dopamine d(4) receptors in rat
forebrain. Temporal association with motor hyper-activity following neonatal 6-
hydroxydopamine lesioning. Neuropsychopharmacology (2002) 26:625-33.
246. Zhang K, Davids E, Tarazi FI, Baldessarini RJ. Effects of dopamine D(4) receptor-selective
antagonists on motor hyper-activity in rats with neonatal 6-hydroxydopamine lesions.
Psychopharmacology (Berl) (2002) 161:100-106.
247. Goodman and Gilman ‘s The Pharmacological Basis of Therapeutics, Tenth Edition. New
York: McGraw-Hill, 2001.
248. Spencer T, Biederman J, Heiligenstein J, Wilens T, Fanes D, Prince J, Faraone SV, Rea J,
Witcher J, Zervas S. An open-label, dose-ranging study of atomoxetine in children with
attention deficit hyper-activity disorder. J Child Adolesc Psychopharmacol (2001) 11:251-
65.
249. Riddle MA, Geller B, Ryan N. Another sudden death in a child treated with desipramine. J
Am Acad Child Adolesc Psychiatry (1993) 32:792-7.
250. Scahill L, Barbon L, Farkas L. Apha-2 agonists in the treatment of attention deficit hyper-
activity disorder. J Child Adolesc Psychiatr Nurs (1999) 12:168-73.
251. Connor DF, Fletcher KE, Swanson JM. A meta-analysis of clonidine for symptoms of
attention-deficit hyper-activity disorder. J Am Acad Child Adolesc Psychiatry (1999)
38:1551-9.

248
252. Sullivan IP, Donnelly-Roberts D, Briggs CA, Anderson DJ, Gopalakrishnan M, Xue IC,
Piattoni-Kaplan M, Molinari E, Campbell JE, McKenna DG, Gunn DE, Lin NH, Ryther
KB, He Y, Holladay MW, Wonnacott S, Williams M, Arneric SP. ABT-089 [2-methyl-3-
(2-(S)-pyrrolidinylmethoxy)pyridine]: I. A potent and selective cholinergic channel
modulator with neuroprotective properties. J Pharmacol xp Ther (1997) 283:235-46.
253. McGaughy J, Decker MW, Sarter M. Enhancement of sustained attention performance by
the nicotinic acetylcho!ine receptor agonist ABT-4 18 in intact but not basal forebrain-
lesioned rats. Psychopharmacology (Berl) (1999) 144:175-82.
254. Wilens TE, Biederman J, Spencer TJ, Bostic J, Prince J, Monuteaux MC, Soriano J, Fine C,
Abrams A, Rater M, Polisner D. A pilot controlled clinical trial of ABT-4 18, a cholinergic
agonist, in the treatment of adults with attention deficit hyper-activity disorder. Am J
Psychiatry (1999) 156:1931-7.
255. Shekim WO, Antun F, Hanna GL, McCracken JT, Hess EB. S-adenosyl L-methionine
(SAM) in adults with ADHD, RS: preliminary results from an open trial.
Psychopharmacol Bull (1990) 26:249-53.
256. Stevens U, Zentall SS, Deck JL, Abate ML, Watkins BA, Lipp SR, Burgess JR. Essential fatty
acid metabolism in boys with attention- deficit hyper-activity disorder. Am J Clin Nutr
(1995) 62:76 1-8.
257. Strupp BJ, Levitsky DA. Enduring cognitive effects of early malnutrition: a theoretical
reappraisal. J Nutr (1995) 125:2221S-2232S.
258. Stordy BJ. Dark adaptation, motor skills, docosahexaenoic acid, and dyslexia. Am J Clin
Nutr (2000) 71:323S-6S.
259. 259. Arnold LE, Pinkham SM, Votolato N. Does zinc moderate essential fatty acid and
amphetamine treatment of attention-deficit/hyper-activity disorder? J Child Adolesc
Psychopharmacol (2000) 10:111-7.
260. Richardson AJ, Pun BK. A randomized double-blind, placebo.controlled study of the
effects of supplementation with highly unsaturated fatty acids on ADFID-related
symptoms in children with specific learning difficulties. Prog europsychopharmacol Biol
Psychiatry (2002) 26:233-9.
261. Aman MG, Mitchell EA, Turbott Sil. The effects of essential fatty acid supplementation by
Efamol in hyper-active children. J Abnorm Child Psychol (1987) 15:75-90.
262. Arnold LE, Kleykamp D, Votolato NA, Taylor WA, Kontras SB, Tobin K. Gamma-
linolenic acid for attention-deficit hyper-activity disorder: placebo-controlled comparison
to D-amphetamine. Biol Psychiatry (1989) 25:222-8.
263. Auestad N, Innis SM. Dietary n-3 fatty acid restriction during gestation in rats: neuronal
cell body and growth-cone fatty acids. Am J Clin Nutr (2000) 71:312S-4S.
264. Kris-Etherton PM, Taylor DS, Yu-Poth S, Huth P, Moriarty K, Fishell V, Hargrove RL,
Zhao G, Etherton TD. Polyunsaturated fatty acids in the food chain in the United States.
Am J Clin Nutr (2000) 7 1:179S-88S.
265. Hibbeln JR, Umhau JC, George DT, Salem N Jr. Do plasma polyunsaturates predict
hostility and depression? World Rev Nutr Diet (1997) 82:175-86.
266. Tanskanen A, Hibbeln IR, Tuomilehto J, Uutela A, Haukkala A, Viinamaki H, Lehtonen J,
Vartiainen E. Fish consumption and depressive symptoms in the general population in
Finland. Psychiatr Serv (2001) 52:529-3 1.
267. Locke CA, Stoll AL. Omega-3 fatty acids in major depression. World Rev Nutr Diet (2001)
89:173-85.
268. Mannuzza S, Klein RG, Bessler A, Malloy P, Hynes ME. Educational and occupational
outcome of hyper-active boys grown up. J Am Acad Child Adolesc Psychiatry (1997)
36:1222-7.
269. Guevara J, Lozano P, Wickizer T, Mell L, Gephart H. Utilization and cost of health care
services for children with attention-deficit/hyper-activity disorder. Pediatrics (2001) 108(1
):7 1-8.
270. Leibson CL, Katusic SK, Barbaresi WJ, Ransom J, OBrien PC. Use and costs of medical
care for children and adolescents with and without attention-deficit/hyper-activity
disorder. JAMA (2001) 285:60-6.

249
271. Herzog A, Detre T. Psychotic reactions associated with childbirth. Dis Nerv Syst (1976)
37:229-35.
272. Hafner H, Riecher-Rossler A, An Der Heiden W, Maurer K, Fatkenheuer B, Loffler W.
Generating and testing a causal explanation of the gender difference in age at first onset of
schizophrenia. Psychol Med (1993) 23:925-40.
273. Matthysse S. Antipsychotic drug actions: a clue to the neuropathology of schizophrenia?
Fed Proc (1973) 32:200-5.
274. Carlsson A. Does dopamine play a role in schizophrenia? Psychol Med (1977) 7:583-97.
275. Widerlov E. A critical appraisal of CSF monoamine metabolite studies in schizophrenia.
Ann N Y Acad Sci (1988) 537:309-23.
276. Kapur S, Seeman P. Does fast dissociation from the dopamine d(2) receptor explain the
action of atypical antipsychotics?: A new hypothesis. Am J Psychiatry (2001) 158:360-9.
277. Seeman P, Guan HC, Van Toi HH. Dopamine D4 receptors elevated in schizophrenia.
Nature (1993) 365:441-5.
278. Murray AM, Hyde TM, Knabie MB, Herman MM, Bigelow LB, Carter JM, Weinberger
DR, Kleinman JE. Distribution of putative D4 dopamine receptors in postmortem
striatum from patients with schizophrenia. J Neurosci (1995) 15:2186-91.
279. Seeman P, Guan HC, Van Toi HH. Schizophrenia: elevation of dopamine D4-like sites,
using [311]nemonapride and [1 251] epidepride. Eur J Pharmacol (1995) 286:R3-5.
280. Marzelia PL, Hill C, Keks N, Singh B, Copolov D. The binding of both [3H]nemonapride
and [3H]raclopride is increased in schizophrenia. Biol Psychiatry (1997) 42(8):648-54.
281. Reynolds GP, Mason SL. Absence of detectable striatal dopamine D4 receptors in drug-
treated schizophrenia. Eur J Pharmacol (1995) 281 :R5-6.
282. Tang SW, Helmeste DM, Fang H, Li M, Vu R, Bunney W Jr, Potkin S, Jones EG.
Differential labeling of dopamine and sigma sites by [3H]nemonapride and
[3H]raciopride in postmortem human brains. Brain Res (1997) 765(1):7-12.
283. Smythies JR. Recent advances in the biochemistry of psychosis. Guy’s Gazette (1966) May
14, 2-7.
284. Spiro HR. Schimke RN, Welch JP. Schizophrenia in a patient with a defect in methionine
metabolism. J Nerv Ment Dis (1965) 141:285-90.
285. Park LC, Baldessarini RJ, Kety SS. Methionine effects on chronic schizophrenics. Arch
Gen Psychiatry (1965) 12:346-35 1.
286. Cohen SM, Nichols A, Wyatt R, Pollin W. The administration of methionine to chronic
schizophrenic patients: a review of ten studies.Biol Psychiatry (1974) 8:209-25.
287. Freeman JM, Finkelstein JD, Mudd SH. Folate-responsive homocystinuria and
“schizophrenia”. A defect in methylation due to deficient 5,1 0-methylenetetrahydrofolate
reductase activity. N Engi J Med (1975) 292:491-6
288. Arinami T, Yamada N, Yamakawa-Kobayashi K, Hamaguchi H, Toru M.
Methylenetetrahydrofolate reductase variant and schizophrenialdepression. Am J Med
Genet (1997) 74:526-8.
289. Regland B, Germgard T, Gottfries CG, Grenfeidt B, Koch-Schmidt AC. Homozygous
thermolabile methylenetetrahydrofolate reductase in schizophrenia-like psychosis. J
Neural Transm (1997) 104:931-41.
290. Kunugi H, Fukuda R, Hattori M, Kato T, Tatsumi M, Sakai T, Hirose T, Nanko S. C677T
polymorphism in ethylenetetrahydrofolate reductase gene and psychoses. Mol Psychiatry
(1998) 3:435-7.
291. Ismail L, Sargent T 3rd, Dobson EL, Pollycove M. Altered metabolism of the methionine
methyl group in the leukocytes of patients with schizophrenia. Biol Psychiatry (1978) 1
3(6):649-60.
292. Sargent T 3rd, Kusubov N, Taylor SE, Budinger TF. Tracer kinetic evidence for abnormal
methyl metabolism in schizophrenia. Biol Psychiatry (1992) 32:1078-90.

250
293. Kelsoe JR Jr, Tolbert LC, Crews EL, Smythies JR. Kinetic evidence for decreased
methionine adenosyltransferase activity in erythrocytes from schizophrenics. J Neurosci
Res (1982) 8:99-103.
294. Smythies IR, Alarcon RD, Morere D, Monti JA, Steele M, Tolbert LC, Walter-Ryan WG.
Abnormalities of one-carbon metabolism in psychiatric disorders: study of methionine
adenosyltransferase kinetics and lipid composition of erythrocyte membranes. Biol
Psychiatry (1986) 21: 1391-8.
295. Joober R, Benkelfat C, Lal S, Bloom D, Labelle A, Lalonde P, Turecki G, Rozen R, Rouleau
GA. Association between the methylenetetrahydrofolate reductase 677C-->T missense
mutation and schizophrenia. Mol Psychiatry (2000) 5:323-6.
296. Costa, E, Chen Y, Davis J, Dong E, Noh JS, Tremolizzo L, Veldic M, Grayson DR, Guidotti
A. Reelin and schizophrenia: A disease at the interface of the genome and epigenome. Mol
Intervent (2002) 2:47-57.
297. Impagnatiello F, Guidotti AR, Pesold C, Dwivedi Y, Caruncho H, Pisu MG, Uzunov DP,
Smalheiser NR, Davis JM, Pandey GN, Pappas GD, Tueting P, Sharma RP, Costa E. A
decrease of reelin expression as a putative vulnerability factor in schizophrenia. Proc Nati
Acad Sci USA (1998) 95:15718-23.
298. Rodriguez MA, Pesold C, Liu WS, Kriho V, Guidotti A, Pappas GD, Costa E.
Colocalization of integrin receptors and reelin in dendritic spine postsynaptic densities of
adult nonhuman primate cortex. Proc Natl Acad Sci USA (2000) 97:3550-5.
299. Javitt DC, Zukin SR. Recent advances in the phencyclidine model of schizophrenia. Am J
Psychiatry (1991) 148:1301-8.
300. Castellani S, Giannini AJ, Adams PM. Physostigmine and haloperidol treatment of acute
phencyclidine intoxication. Am J Psychiatry (1982) 139:508-10.
301. Johnston MV. Neurotransmitters and vulnerability of the developing brain. Brain Dey
(1995) 17:30 1-6.
302. Lilirank SM, Lipska BK, Weinberger DR. Neurodevelopmental animal models of
schizophrenia. Clin Neurosci (1995) 3:98-104.
303. Mohn AR, Gainetdinov RR, Caron MG, Koller BH. Mice with reduced NMDA receptor
expression display behaviors related to schizophrenia. Cell (1999) 98:427-36.
304. Horrobin DF, Glen AI, Vaddadi K. The membrane hypothesis of schizophrenia.
Schizophr Res (1994) 13:195-207.
305. Rotrosen J, Wolkin A. Phospholipid and prostaglandin hypotheses of schizophrenia. In:
Meltzer H (ed). Psychopharmacology: The Third Generation of Progress. Raven Press:
New York, 1987, pp 759-764.
306. Smythies JR, Alarcon RD, Morere D, Monti JA, Steele M, Tolbert LC, Walter-Ryan WG.
Abnormalities of one-carbon metabolism in psychiatric disorders: study of methionine
adenosyltransferase kinetics and lipid composition of erythrocyte membranes. Biol
Psychiatry (1986) 21:1391-8.
307. Mahadik SP, Mukherjee S, Correnti EE, Kelkar ilS, Wakade CG, Costa RM, Scheffer R.
Plasma membrane phospholipid and cholesterol distribution of skin fibroblasts from
drug-naive patients at the onset of psychosis. Schizophr Res (1994) 13:239-47.
308. Fenton WS, Hibbeln J, Knable M. Essential fatty acids, lipid membrane abnormalities, and
the diagnosis and treatment of schizophrenia. Biol Psychiatry (2000) 47:8-21.
309. Yao 1K, Leonard S, Reddy RD. Membrane phospholipid abnormalities in postmortem
brains from schizophrenic patients. Schizophr Res (2000) 42:7-17.
310. Schmitt A, Maras A, Petroianu G, Braus DF, Scheuer L, Gattaz WF. Effects of
antipsychotic treatment on membrane phospholipid metabolism in schizophrenia. J
Neural Transm (2001) 108:1081-91.
311. O’Callaghan E, Redmond O, Ennis R, Stack J, Kinsella A, Ennis JT, Larkin C, Waddington
IL. Initial investigation of the left temporoparietal region in schizophrenia by 31 P
magnetic resonance spectroscopy. Biol Psychiatry (1991) 29:1149-52.
312. Pettegrew JW, Minshew NJ. Molecular insights into schizophrenia. J Neural Transm
Suppl (1992) 36:23-40.

251
313. Jensen JE, Al-Semaan YM, Williamson PC, Neufeld RW, Menon RS, Schaeffer B,
Densmore M, Drost DJ. Region-specific changes in phospholipid metabolism in chronic,
medicated schizophrenia: (31 )P MRS study at 4.0 Tesla. Br J Psychiatry (2002) 180:39-44.
314. Mahadik SP, Mukherjee S, Horrobin DF, Jenkins K, Correnti EE, Scheffer RE. Plasma
membrane phospholipid fatty acid composition of cultured skin fibroblasts from
schizophrenic patients: comparison with bipolar patients and normal subjects. Psychiatry
Res (1996) 63:133-42.
315. Peet M, Laugharne J, Rangarajan N, Horrobin D, Reynolds G. Depleted red cell membrane
essential fatty acids in drug-treated schizophrenic patients. J Psychiatr Res (1995) 29:227-
32.
316. Andreasen NC, Amdt S, Swayze V 2nd, Cizadlo T, Flaum M, O’Leary D, Ehrhardt JC, Yuh
WT. Thalamic abnormalities in schizophrenia visualized through magnetic resonance
image averaging. Science (1994) 266:294-8.
317. Nopoulos P, Torres I, Flaum M, Andreasen NC, Ehrhardt JC, Yuh WT. Brain morphology
in first-episode schizophrenia. Am J Psychiatry (1995) 152:1721-3.
318. Wright IC, Rabe-Hesketh S, Woodruff PW, David AS, Murray RM, Builmore ET. Meta-
analysis of regional brain volumes in schizophrenia. Am J Psychiatry (2000) 157:16-25.
319. Shenton ME, Dickey CC, Frumin M, McCarley RW. A review of MRI findings in
schizophrenia. Schizophr Res (2001) 49:1-52.
320. Thune JJ, Uylings HB, Pakkenberg B. No deficit in total number of neurons in the
prefrontal cortex in schizophrenics. J Psychiatr Res (2001) 35(l):15-21.
321. Kalus P, Senitz D, Beckmann H. Altered distribution of parvalbumin immunoreactive
local circuit neurons in the anterior cingulate cortex of schizophrenic patients. Psychiatry
Res (1997) 75 :49-59.
322. Beasley CL, Reynolds GP. Parvalbumin-immunoreactive neurons are reduced in the
prefrontal cortex of schizophrenics. Schizophrenia Res (1997) 24(3):349-55.
323. Danos P, Baumann B, Bernstein HG, Franz M, Stauch R, Northoff G, Krell D, Faikai P,
Bogerts B. Schizophrenia and anteroventral thalamic nucleus: selective decrease of
parvalbumin-immunoreactive thalamocortical projection neurons. Psychiatry Res (1998)
82:1-10.
324. Reynolds GP, Zhang ZJ, Beasley CL. Neurochemical correlates of cortical GABAergic
deficits in schizophrenia: selective losses of calcium binding protein immunoreactivity.
Brain Res Bull (2001) 55 :579-84.
325. Reynolds GP, Beasley CL. GABAergic neuronal subtypes in the human frontal cortex--
development and deficits in schizophrenia. J Chem Neuroanat (2001) 22:95-100.
326. Lewis DA, Cruz DA, Melchitzky DS, Pierri iN. Lamina-specific deficits in parvalbumin-
immunoreactive varicosities in the prefrontal cortex of subjects with schizophrenia:
evidence for fewer projections from the thalamus. Am J Psychiatry (2001) 158:1411-22.
327. Leonard S, Adler LE, Benhammou K, Berger R, Breese CR, Drebing C, Gault J, Lee MJ,
Logel J, Olincy A, Ross RG, Stevens K, Sullivan B, Vianzon R, Virnich DE, Waldo M,
Walton K, Freedman R. Smoking and mental illness. Pharmacol Biochem Behav (2001)
70:561-70.
328. Yolken RH, Karlsson H, Yee F, Johnston-Wilson NL, Torrey EF. Endogenous retroviruses
and schizophrenia. Brain Res Brain Res Rev (2000) 3 1:193-9.
329. LeRoith D, Werner H, Fana TN, Kato H, Adamo M, Roberts CT Jr. Insulin-like growth
factor receptors. Implications for nervous system function. Ann N Y Acad Sci (1993)
692:22-32.
330. Stover P, Schirch V. Serine hydroxymethyltransferase catalyzes the hydrolysis of 5,1 0-
methenyltetrahydrofolate to 5-formyltetrahydrofolate. J Biol Chem (1990) 265:14227-33.
331. Stover P, Schirch V. 5-Formyltetrahydrofolate polyglutamates are slow tight binding
inhibitors of serine hydroxymethyltransferase. J Biol Chem (1991) 266:1543-50.
332. Bertrand R, Jolivet J. Methenyltetrahydrofolate synthetase prevents the inhibition of
phosphoribosyl 5-aminoimidazole 4-carboxamide ribonucleotide formyltransferase by 5-
formyltetrahydrofolate polyglutamates. J Biol Chem (1989) 264:8843-6.

252
333. Girgis S, Suh IR, Jolivet J, Stover PJ. 5-Formyltetrahydrofolate regulates homocysteine re-
methylation in human neuroblastoma. J Biol Chem (1997) 272:4729-34.
334. Kruschwitz HL, McDonald D, Cossins EA, Schirch V. 5-
Formyltetrahydropteroylpolyglutamates are the major folate derivatives in Neurospora
crassa conidiospores. J Biol Chem (1994) 269:28757-63.
335. Piper MD, Hong SP, Ball GE, Dawes 1W. Regulation of the balance of one-carbon
metabolism in Saccharomyces cerevisiae. J Biol Chem (2000) 275:30987-95.
336. Stover PF. Personal communication.
337. Dumenil G, Rubini M, Dubois G, Baserga R, Fellous M, Pellegrini S. Identification of
signalling components in tyrosine kinase cascades using phosphopeptide affinity
chromatography. Biochem Biophys Res Commun (1997) 234:748-53.
338. Kramer MS, Last B, Getson A, Reines SA. The effects of a selective D4 dopamine receptor
antagonist (L-745,870) in acutely psychotic inpatients with schizophrenia. D4 Dopamine
Antagonist Group. Arch Gen Psychiatry (1997) 54:567-72.
339. Kapur S, Seeman P. Antipsychotic agents differ in how fast they come off the dopamine
D2 receptors. Implications for atypical antipsychotic action. J Psychiatry Neurosci (2000)
25:161-6.
340. Kapur S, Seeman P. Does fast dissociation from the dopamine d(2) receptor explain the
action of atypical antipsychotics?: A new hypothesis. Am J Psychiatry (2001) 158:360-9.
341. Javitt DC, Zylberman I, Zukin SR, Heresco-Levy U, Lindenmayer IP. Amelioration of
negative symptoms in schizophrenia by glycine. Am J Psychiatry (1994) 15 1:1234-6.
342. Heresco-Levy U, Javitt DC, Ermilov M, Mordel C, Silipo G, Lichtenstein M. Efficacy of
high-dose glycine in the treatment of enduring negative symptoms of schizophrenia. Arch
Gen Psychiatry (1999) 56:29-36.
343. Potkin SG, Jin Y, Bunney BG, Costa J, Gulasekaram B. Effect of clozapine and adjunctive
high-dose glycine in treatment-resistant schizophrenia. Am J Psychiatry (1999) 156:145-7.
344. Goff DC, Tsai G, Manoach DS, Coyle JT. Dose-finding trial of D cycloserine added to
neuroleptics for negative symptoms in schizophrenia. Am J Psychiatry (1995) 152:1213-5.
345. Goff DC, Henderson DC, Evins AE, Amico E. A placebo-controlled crossover trial of D-
cycloserine added to clozapine in patients with schizophrenia. Biol Psychiatry (1999)
45(4):512-4.
346. Godfrey PS, Toone BK, Carney MW, Flynn TG, Bottiglieri T, Laundy M, Chanarin I,
Reynolds EH. Enhancement of recovery from psychiatric illness by methylfolate. Lancet
(1990) 336:392-5.
347. Carney MW, Chary TK, Laundy M, Bottiglieri T, Chanarin I, Reynolds EH, Toone B. Red
cell folate concentrations in psychiatric patients. J Affect Disord (1990) 19:207-13.
348. Bottiglieri T, Hyland K, Laundy M, Godfrey P, Carney MW, Toone BK, Reynolds EH.
Enhancement of recovery from psychiatric illness by methylfolate. Lancet (1990)
336:1579-80.
349. Vaddadi KS, Gilleard CJ, Soosai E, Polonowita AK, Gibson RA, Burrows GD.
Schizophrenia, tardive dyskinesia and essential fatty acids. Schizophr Res (1996) 20:287-
94.
350. Peet M, Laughame ID, Mellor J, Ramchand CN. Essential fatty acid deficiency in
erythrocyte membranes from chronic schizophrenic patients, and the clinical effects of
dietary supplementation. Prostaglandins Leukot Essent Fatty Acids (1996) 55:71-5.
351. Pun BK, Richardson AJ, Horrobin DF, Easton T, Saeed N, Oatridge A, Hajnal JV, Bydder
GM. Eicosapentaenoic acid treatment in schizophrenia associated with symptom
remission, normalisation of blood fatty acids, reduced neuronal membrane phospholipid
turnover and structural brain changes. mt j Clin Pract (2000) 54:57-63.
352. Maidment ID. Are fish oils an effective therapy in mental illness--an analysis of the data.
Acta Psychiatr Scand (2000) 102:3-11.
353. Assies J, Lieverse R, Vreken P, Wanders Rl, Dingemans PM, Linszen DH. Significantly
reduced docosahexaenoic and docosapentaenoic acid concentrations in erythrocyte

253
 membranes from schizophrenic patients compared with a carefully matched control
group. Biol Psychiatry (2001) 49:510-22.
354. Yao 1K, Leonard S, Reddy RD. Membrane phospholipid abnormalities in postmortem
brains from schizophrenic patients. Schizophr Res (2000) 42:7-17.
355. Cantor-Graae E, Nordstrom LG, McNeil TF. Substance abuse in schizophrenia: a review
of the literature and a study of correlates in Sweden. Schizophr Res (2001) 48:69-82.
356. Silbersweig DA, Stern E, Frith C, Cahill C, Holmes A, Grootoonk S, Seaward J, McKenna
P, Chua SE, Schnorr L, et al. A functional neuroanatomy of hallucinations in
schizophrenia. Nature (1995) 378: 176-9.
357. Slade PD. An investigation of psychological factors involved in the predisposition to
auditory hallucinations. Psychol Med (1976) 6:123-32.
358. Kraeplin E. Zur diagnose und prognose der dementia praecox: Heidelberger verslamm
lung 26/27, November 1898. Zschr Neurol Psychiatr (1899) 56:254.
359. Kwon JS, O’Donnell BF, Wallenstein GV, Greene RW, Hirayasu Y, Nestor PG, Hasselmo
ME, Potts GF, Shenton ME, MeCarley RW. Gamma frequency-range abnormalities to
auditory stimulation in schizophrenia. Arch Gen Psychiatry (1999) 56:1001-5.
360. Faux SF, Torello MW, McCarley RW, Shenton ME, Duff’ FH. P300 in schizophrenia:
confirmation and statistical validation of temporal region deficit in P300 topography. Biol
Psychiatry (1988) 23:776-90.
361. Ahveninen J, Kahkonen S, Tiitinen H, Pekkonen E, Huttunen J, Kaakkola S, Ilmoniemi RJ,
Jaaskelainen IP. Suppression of transient 40-Hz auditory response by haloperidol suggests
modulation of human selective attention by dopamine D2 receptors. Neurosci Left (2000)
292:29-32.
362. Fein, D, Joy, S, Green LA, Waterhouse L. Autism and Pervasive Developmental Disorders.
In: Fogel BS, Schiffer RB, Rao SM (eds.) Neuropsychiatry. Willams and Wilkins,
Baltimore: 1996, pp 571-614.
363. Bailey A, Le Couteur A, Gottesman I, Bolton P, Simonoff E, Yuzda E, Rutter M. Autism as
a strongly genetic disorder: evidence from a British twin study. Psychol Med (1995) 25:63-
77.
364. Powell JE, Edwards A, Edwards M, Pandit BS, Sungum-Paliwal SR, Whitehouse W.
Changes in the incidence of childhood autism and other autistic spectrum disorders in
preschool cildren from two areas of the West Midlands, UK. Dey Med Child Neurol
(2000) 42:624-8.
365. Heussler H, Polnay L, Marder E, Standen P, Chin LU, Butler N. Prevalence of autism in
early 1 970s may have been underestimated. BMJ (2001) 323:633.
366. Jaeken J, Van den Berghe G. An infantile autistic syndrome characterised by the presence
of succinylpurines in body fluids. Lancet (1984) 2:1058-61.
367. Stone RL, Aimi J, Barshop BA, Jaeken J, Van den Berghe G, Zalkin H, Dixon JE. A
mutation in adenylosuccinate lyase associated with mental retardation and autistic
features. Nat Genet (1992) 1:59-63.
368. Salerno C, Siems WG, Crifo C. Succinylpurinemic autism: increased sensitivity of
defective adenylosuccinate lyase towards 4-hydroxy-2-nonenal. Biochim Biophys Acta
(2000) 1500:335-4 1.
369. Marie S, Race V, Nassogne MC, Vincent MF, Van den Berghe G. Mutation of a nuclear
respiratory factor 2 binding site in the 5’ untranslated region of the ADSL gene in three
patients with adenylosuccinate lyase deficiency. Am J Hum Genet (2002) 71:14-21.
370. Salerno C, lotti S, Lodi R, Crifo C, Barbiroli B. Muscle energy metabolism in human
adenylosuccinase deficiency. An in vivo 3 1P-NMR spectroscopy study. Adv Exp Med Biol
(1998) 43 1:275-8.
371. Tani Y, Fernell E, Watanabe Y, Kanai T, Langstrom B. Decrease in 6R-5,6,7,8-
tetrahydrobiopterin content in cerebrospinal fluid of autistic patients. Neurosci Left
(1994) 181:169-72.
372. Ernst M, Zametkin AJ, Matochik JA, Pascualvaca D, Cohen RM. Low medial prefrontal
dopaminergic activity in autistic children. Lancet (1997) 350:638.

254
373. Fernell E, Watanabe Y, Adolfsson I, Tani Y, Bergstrom M, Hartvig P, Liija A, von
Knorring AL, Gillberg C, Langstrom B. Possible effects of tetrahydrobiopterin treatment
in six children with autism--clinical and positron emission tomography data: a pilot study.
Dey Med Child Neurol (1997) 39:313-8.
374. Page T, Coleman M. Purine metabolism abnormalities in a hyper-uricosuric subclass of
autism. Biochim Biophys Acta (2000) 1500:291-6.
375. Page T, Yu A, Fontenessi J, Nyhan W. A syndrome of seizures and pervasive
developmental disorder associated with excessive cellular nucleotidase activity. Adv Exp
Med Biol (1998) 431:789-92.
376. Stubbs G, Lift M, Lis E, Jackson R, Voth W, Lindberg A, Lift R. Adenosine deaminase
activity decreased in autism. J Am Acad Child Psychiatry (1982) 21:71-4.
377. Bottini N, De Luca D, Saccucci P, Fiumara A, Elia M, Porfirio MC, Lucarelli P, Curatolo P.
Autism: evidence of association with adenosine deaminase genetic polymorphism.
Neurogenetics (2001) 3:111-3.
378. Persico AM, Militerni R, Bravaccio C, Schneider C, Melmed R, Trillo S, Montecchi F,
Palermo MT, Pascucci T, Puglisi-Allegra S, Reichelt KL, Conciatori M, Baldi A, Keller F.
Adenosine deaminase alleles and autistic disorder: case-control and family-based
association studies. Am J Med Genet (2000) 96:784-90.
379. Institute of Medicine: Immunization Safety Review: Thimerosal containing Vaccines and
Neurodevelopmental Disorders, eds. Stratton K, Gable A, McCormick MC, Washington,
D.C.: National Academy Press, 2001.
380. Bernard S, Enayati A, Redwood L, Roger H, Binstock T. Autism: a novel form of mercury
poisoning. Med Hypotheses. 2001, 56(4):462-71.
381. Aman MG, Langworthy KS. Pharmacotherapy for hyper-activity in children with autism
and other pervasive developmental disorders. J Autism Dcv Disord. (2000) 30(5):451-9.
382. Horvath K, Stefanatos G, Sokolski KN, Wachtel R, Nabors L, Tildon JT. Improved social
and language skills after secretin administration in patients with autistic spectrum
disorders. J Assoc Acad Minor Phys. (1998) 9(1):9-15.
383. Sandler AD, Sutton KA, DeWeese J, Girardi MA, Sheppard V, Bodfish JW. Lack of benefit
of a single dose of synthetic human secretin in the treatment of autism and pervasive
developmental disorder. N Engl J Med. (1999) 341(24):1801-6.
384. Owley T, McMahon W, Cook EH, Laulhere T, South M, Mays LZ, Shernoff ES, Lainhart J,
Modahi CB, Corsello C, Ozonoff S, Risi S, Lord C, Leventhal BE, Filipek PA. Multisite,
double-blind, placebo- controlled trial of porcine secretin in autism. J Am Acad Child
Adolesc Psychiatry. (2001) 40(11): 1293-9.
385. Coniglio SJ, Lewis ID, Làng C, Burns TG, Subhani-Siddique R, Weintraub A, Schub H,
Holden EW. A randomized, double-blind, placebo-controlled trial of single-dose
intravenous secretin as treatment for children with autism. J Pediatr. (2001) 138(5):649-
55.
386. Kern JK, Van Miller S, Evans PA, Trivedi MH. Efficacy of porcine secretin in children with
autism and pervasive developmental disorder. J Autism Dey Disord. (2002) 32(3): 153-60.
387. Bolman WM, Richmond JA. A double-blind, placebo-controlled, crossover pilot trial of
low dose dimethylglycine in patients with autistic disorder. J Autism Dey Disord. (1999)
29(3): 19 1-4.
388. Kern JK, Miller VS, Cauller PL, Kendall PR, Mehta PJ, Dodd M. Effectiveness of N,N-
dimethylglycine in autism and pervasive developmental disorder. J Child Neurol. (2001)
16(3): 169-73.
389. Amir RE, Van den Veyver IB, Wan M, Tran CQ, Francke U, Zoghbi HY. Rett syndrome is
caused by mutations in X-linked MECP2, encoding methyl-CpG-binding protein 2. Nat
Genet. (1999) 23(2): 185-8.
390. Van Den Veyver IB, Zoghbi HY. Genetic basis of rett syndrome. Ment Retard Dey Disabil
Res Rev. (2002) 8(2):82-6.
391. Pogribna M, Melnyk S, Pogribny I, Chango A, Yi P, James SJ. Homocysteine metabolism
in children with Down syndrome: in vitro modulation. Am J Hum Genet. (2001) 69(1):88-
95.

255
392. O’Leary VB, Pane-McDermott A, Molloy AM, Kirke PN, Johnson Z, Conley M, Scott JM,
Mills JL. MTRR and MTHFR polymorphism: link to Down syndrome? Am J Med Genet.
(2002) 107(2):151-5.
393. Greenblatt JM, Huffman LC, Reiss AL. Folie acid in neurodevelopment and child
psychiatry. Prog Neuropsychopharmacol Biol Psychiatry. (1994) 18(4):647-60.
394. Clarke S and Banfield K. “S-adenosylmethionine-dependent Methyltransferases: Potential
Targets in Homocysteine-linked Pathology.” In Homocysteine in Health and Disease, R
Carmel and D Jacobsen eds., Cambridge University Press, Cambridge, England, 2001.
395. Hook EB, Cross PK, Schreinemachers DM. Chromosomal abnormality rates at
amniocentesis and in live-born infants. JAMA (1983) 249:2034-8.
396. Antonarakis SE. Parental origin of the extra chromosome in trisomy 21 as indicated by
analysis of DNA polymorphisms. Down Syndrome Collaborative Group. N EngI J Med
(1991) 324:872-6.
397. James SJ, Pogribna M, Pogribny IP, Melnyk S, Hine RJ, Gibson JB, Yi P, Tafoya DL,
Swenson DH, Wilson VE, Gaylor DW. Abnormal folate metabolism and mutation in the
methylenetetrahydrofolate reductase gene may be maternal risk factors for Down
syndrome. Am J Clin Nutr (1999) 70:495-501.
398. O’Leary VB, Pane-McDermott A, Molloy AM, Kirke PN, Johnson Z, Conley M, Scott 1M,
Mills IL. MTRR and MTHFR polymorphism: link to Down syndrome? Am J Med Genet
(2002) 107:151-5.
399. Pogribna M, Melnyk S, Pogribny I, Chango A, Yi P, James SJ. Homocysteine metabolism
in children with Down syndrome: in vitro modulation. Am J Hum Genet. 2001
Jul;69(1):88-95.
400. Turner G, Webb T, Wake S, Robinson H. Prevalence of fragile X syndrome. Am J Med
Genet (1996) 64:196-7.
401. Lubs HA. A marker X chromosome. Am J Hum Genet (1969) 21:23 1-44.
402. Sherman SL, Jacobs PA, Morton NE, Froster-Iskenius U, Howard Peebles PN, Nielsen KB,
Partington MW, Sutherland GR, Turner G, Watson M. Further segregation analysis of the
fragile X syndrome with special reference to transmitting males. Hum Genet (1985)
69:289-99.
403. Richards RI, Sutherland GR. Dynamic mutation: possible mechanisms and significance in
human disease. Trends Biochem Sci (1997) 22:432-6.
404. Weiler IJ, Irwin SA, Klintsova AY, Spencer CM, Brazelton AD, Miyashiro K, Comery TA,
Patel B, Eberwine J, Greenough WT. Fragile X mental retardation protein is translated
near synapses in response to neurotransmitter activation. Proc Natl Acad Sci USA (1997)
94:5395-400.
405. Giraud F, Ayme S, Mattei iF, Mattei MG. Constitutional chromosomal breakage. Hum
Genet (1976) 34:125-36.
406. Jam ison KR. Touched With Fire: Manic Depressive Illness and the Artistic Temperament.
Free Press, New York, NY, 1993.
407. Maes M, Christophe A, Delanghe J, Altamura C, Neels H, Meltzer HY. Lowered omega3
polyunsaturated fatty acids in serum phospholipids and cholesteryl esters of depressed
patients. Psychiatry Res (1999) 85 :275-9 1.
408. Christensen O, Christensen E. Fat consumption and schizophrenia. Acta Psychiatr Scand
(1988) 78:587-91.
409. Tanskanen A, Hibbein IR, Tuomilehto J, Uutela A, Haukkala A, Viinamaki H, Lehtonen J,
Vartiainen E. Fish consumption and depressive symptoms in the general population in
Finland. Psychiatr Serv (2001) 52:529-3 1.
410. Haggarty P, Ashton J, Joynson M, Abramovich DR, Page K. Effect of maternal
polyunsaturated fatty acid concentration on transport by the human placenta. Biol
Neonate (1999) 75:350-9.
411. Jensen CL, Maude M, Anderson RE, Heird WC. Effect of docosahexaenoic acid
supplementation of lactating women on the fatty acid composition of breast milk lipids
and maternal and infant plasma phospholipids. Am J Clin Nutr (2000) 71(1 Suppl):292S-
9S.

256
412. Hornstra G. Essential fatty acids in mothers and their neonates. Am J Clin Nutr (2000)
71(5 Suppi): 1262S-9S.
413. Stoll AL, Severus WE, Freeman MP, Rueter S, Zboyan HA, Diamond E, Cress KK,
Marangell LB. Omega 3 fatty acids in bipolar disorder: a preliminary double-blind,
placebo-controlled trial. Arch Gen Psychiatry (1999) 56:407-12.
414. Waley M and Deth R. Unpublished observation.
415. Mirnikjoo B, Brown SE, Kim HF, Marangell LB, Sweatt JD, Weeber EJ. Protein kinase
inhibition by omega-3 fatty acids. J Biol Chem (2001) 276:10888-96.
416. Agnoli A, Andreoli V, Casacchia M, Cerbo R. Effect of s-adenosyl-l methionine (SAMe)
upon depressive symptoms. J Psychiatr Res (1976) 13:43-54.
417. Bell KM, Plon L, Bunney WE Jr, Potkin SG. 5-denosylmethionine treatment of depression:
a controlled clinical trial. Am J Psychiatry (1988) 145:1110-4.
418. Bottiglieri T, Hyland K, Reynolds EH. The clinical potential of ademetionine (S-
adenosylmethionine) in neurological disorders. Drugs (1994) 48:137-52.
419. Hallcher LM, Sherman WR. The effects of lithium ion and other agents on the activity of
myo-inositol- 1 -phosphatase from bovine brain. J Biol Chem (1980) 255:10896-901.
420. Little JT, Kimbrell TA, Wassermann EM, Grafman J, Figueras S, Dunn RT, Danielson A,
Repella J, Huggins T, George MS, Post RM. Cognitive effects of 1- and 20-hertz repetitive
transcranial magnetic stimulation in depression: preliminary report. Neuropsychiatry
Neuropsychol Behav Neurol (2000) 13:119-24.
421. Triggs WJ, McCoy KJ, Greer R, Rossi F, Bowers D, Kortenkamp S, Nadeau SE, Heilman
KM, Goodman WK. Effects of left frontal transcranial magnetic stimulation on depressed
mood, cognition, and corticomotor threshold. Biol Psychiatry (1999) 45:1440-6.
422. Gandy S, Petanceska S. Regulation of Alzheimer beta-amyloid precursor trafficking and
metabolism. Biochim Biophys Acta (2000) 1502:44-52.
423. Mortensen EL, Hogh P. A gender difference in the association between APOE genotype
and age-related cognitive decline. Neurology (2001) 57:89-95.
424. Carter DB, Dunn E, McKinley DD, Stratman NC, Boyle TP, Kuiper SL, Oostveen JA,
Weaver U, Boller JA, Gurney ME. Human apolipoprotein E4 accelerates beta-amyloid
deposition in APPsw transgenic mouse brain. Ann Neurol (2001) 50:468-75.
425. Martorell L, Virgos C, Valero J, Coil G, Figuera L, Joven J, Pocovi M, Labad A, Vilella E.
Schizophrenic women with the APOE epsilon 4 allele have a worse prognosis than those
without it. Mol Psychiatry (2001) 6:307-10.
426. Clarke R, Smith AD, Jobst KA, Refsum H, Sutton L, Ueland PM. Folate, vitamin B 12, and
serum total homocysteine levels in confirmed Alzheimer disease. Arch Neurol (1998)
55:1449-55.
427. Postiglione A, Milan G, Ruocco A, Gallotta G, Guiotto G, Di Minno G. Plasma folate,
vitamin B( 12), and total homocysteine and homozygosity for the C677T mutation of the
5,10-methylene tetrahydrofolate reductase gene in patients with Alzheime?s dementia. A
case-control study. Gerontology (2001) 47:324-9.
428. Wang HX, Wahlin A, Basun H, Fastbom J, Winblad B, Fratiglioni L. Vitamin B( 12) and
folate in relation to the development of Alzheimer’s disease. Neurology (2001) 56(9):
1188-94.
429. Seshadri S, Beiser A, Selhub J, Jacques PF, Rosenberg 1H, D’Agostino RB, Wilson PW,
Wolf PA. Plasma homocysteine as a risk factor for dementia and Alzheimer’s disease. N
Engl J Med. (2002) 346:476-83.
430. Snowdon DA, Tully CL, Smith CD, Riley KP, Markesbery WR. Serum fo late and the
severity of atrophy of the neocortex in Alzheimer disease: findings from the Nun study.
Am J Clin Nutr (2000) 71:993-8.
431. Gonzalez C, Martin T, Cacho J, Brenas MT, Arroyo T, Garcia-Berrocal B, Navajo JA,
Gonzalez-Buitrago JM. Serum zinc, copper, insulin and lipids in Alzheime?s disease
epsilon 4 apolipoprotein E allele carriers. Eur J Clin Invest (1999) 29:637-42.

257
432. Multhaup G, Schlicksupp A, Hesse L, Beher D, Ruppert T, Masters CL, Beyreuther K. The
amyloid precursor protein of Alzheimer’s disease in the reduction of copper(II) to
copper(I) Science (1996) 271:1406-9.
433. White AR, Multhaup G, Maher F, Bellingham S, Camakaris J, Zheng H, Bush AI,
Beyreuther K, Masters CL, Cappai R. The Alzheimer’s disease amyloid precursor protein
modulates copper-induced toxicity and oxidative stress in primary neuronal cultures. J
Neurosci (1999) 19:9170-9.
434. Davis CD, Milne DB, Nielsen FH. Changes in dietary zinc and copper affect zinc-status
indicators of postmenopausal women, notably, extracellular superoxide dismutase and
amyloid precursor proteins. Am J Clin Nutr (2000) 71:781-8.
435. White AR, Reyes R, Mercer IF, Camakaris J, Zheng H, Bush AI, Multhaup G, Beyreuther
K, Masters CL, Cappai R. Copper levels are increased in the cerebral cortex and liver of
APP and APLP2 knockout mice. Brain Res (1999) 842:439-44.
436. Moir RD, Atwood CS, Romano DM, Laurans MH, Huang X, Bush AI, Smith JD, Tanzi RE.
Differential effects of apolipoprotein E isoforms on metal-induced aggregation of A beta
using physiological concentrations. Biochemistry (1999) 38:4595-603.
437. Conquer JA, Tierney MC, Zecevic J, Bettger WJ, Fisher RH. Fatty acid analysis of blood
plasma of patients with Alzheimer’s disease, other types of dementia, and cognitive
impairment. Lipids (2000) 35:1305-12.
438. Fleischhacker WW, Buchgeher A, Schubert H. Memantine in the treatment of senile
dementia of the Aizheimer type. Prog Neuropsychopharmacol Biol Psychiatry (1986)
10:87-93.
439. Winbiad B, Poritis N. Memantine in severe dementia: results of the 9M-Best Study
(Benefit and efficacy in severely demented patients during treatment with memantine). mt
j Geriatr Psychiatry (1999) 14:135-46.
440. in ‘t Veld BA, Launer U, Hoes AW, Ott A, Hofman A, Breteler MM, Stricker BH. NSAIDs
and incident Alzheimer’s disease. The Rotterdam Study. Neurobiol Aging (1998) 19:607-
11.
441. Lim GP, Yang F, Chu T, Gahtan E, Ubeda O, Beech W, Overmier JB, Hsiao-Ashec K,
Frautschy SA, Cole GM. Ibuprofen effects on Alzheimer pathology and open field activity
in APPsw transgenic mice. Neurobiol Aging (2001) 22:983-91.
442. Blasko I, Apochal A, Boeck G, Hartmann T, Grubeck-Loebenstein B, Ransmayr G.
Ibuprofen decreases cytokine-induced amyloid beta production in neuronal cells.
Neurobiol Dis (2001) 8:1094-101.
443. O’Malley KL. Harmon S, Tang L, Todd RD. The rat dopamine D4 receptor: sequence,
gene structure, and demonstration of expression in the cardiovascular system. New Biol
(1992) 4:137-46.
444. Asghari V, Schoots O, van Kats S, Ohara K, Jovanovic V, Guan HC, Bunzow JR, Petronis
A, Van Toi HF!. Dopamine D4 receptor repeat: analysis of different native and mutant
forms of the human and rat genes. Mol Pharmacol (1994) 46:364-73.
445. Fishburn CS, Carmon S, Fuchs S. Molecular cloning and characterisation of the gene
encoding the murine D4 dopamine receptor. FEBS Lett (1995) 361:215-9.
446. Yu H, Rosen MK, Shin TB, Seidel-Dugan C, Brugge JS, Schreiber SL. Solution structure of
the SH3 domain of Src and identification of its ligand-binding site. Science (1992)
258:1665-8.
447. Rickles RJ, Botfield MC, Zhou XM, Henry PA, Brugge JS, Zoller MJ. Phage display
selection of ligand residues important for Src homology 3 domain binding specificity. Proc
Nati Acad Sci USA. (1995) 92:10909-13.
448. Ding YC, Chi HC, Grady DL, Morishima A, Kidd IR, Kidd KK, Flodman P, Spence MA,
Schuck S, Swanson JM, Zhang YP, Moyzis RK. Evidence of positive selection acting at the
human dopamine receptor D4 gene locus. Proc Natl Acad Sci USA (2002) 99:309-14.
449. Niimi Y, Inoue-Murayama M, Kato K, Matsuura N, Murayama Y, Ito S, Momoi Y, Konno
K, Iwasaki T. Breed differences in allele frequency of the dopamine receptor D4 gene in
dogs. J Hered (2001) 92:433-6.

258
450. Zhen X, Zhang J, Johnson GP, Friedman E. D(4) dopamine receptor differentially
regulates Akt/nuclear factor-kappa b and extracellular signal-regulated kinase pathways in
D(4)MN9D cells. Mol Phannacol (2001) 60:857-64.
451. Oak TN, Lavine N, Van Tol.H.H. Dopamine D(4) and D(2L) Receptor Stimulation of the
Mitogen-Activated Protein Kinase Pathway Is Dependent on trans-Activation of the
Platelet-Derived Growth Factor Receptor. Mol Pharmacol. 2001 Jul;60(1):92-103.
452. Diamond J. Guns, Germs, and Steel: The Fates of Human Societies. W.W. Norton &
Company, New York, NY 1999.

259
INDEX

http://www.raysahelian.com/phospholipids.html
http://www.lef.org/magazine/mag2006/jun2006_report_ashwa_01.htm
http://www.whale.to/a/deth.pdf
http://www.ssreg.com/images/classes/princeton/files/
Serotonin%20motor%20activity%20and%20depression%20related%20disorders.pd
f
http://ieet.org/index.php/IEET/more/pellissier20110719 - Ashkenazi
http://www.slate.com/articles/arts/everyday_economics/2003/06/
why_jews_dont_farm.html
http://nymag.com/news/features/ashkenazi-jews-2011-11/
http://www.nytimes.com/2006/01/14/science/14gene.html

260