Chapter One

1.1 Introduction

Serotonin (5-hydroxytryptamine, 5-HT), the ‘‘happy hormone,’’ has a

phylogenetically ancient role in neural transmission (Turlejski, 1996). Because the

serotonergic system has a widespread distribution in the CNS, it influences almost

every sphere of mammalian physiology, from cardiovascular regulation (Miyata et

al., 2000; Nebigil et al., 2000; Thorin et al., 1990), respiration, the gastrointestinal

system (Kato et al., 1999), pain sensitivity, and thermoregulation to more centrally

controlled functions. The latter include the maintenance of circadian rhythm,

appetite, aggression, sensorimotor activity, sexual behavior, mood, cognition,

learning, and memory. Hence drugs with serotonergic activity are used to treat the

a Vective disorders schizophrenia (AbiDargham et al., 1996; Breier, 1995; Kapur

and Remington, 1996; Meltzer, 2002; Ohuoha et al., 1993; Sodhi and Murray,

1997), anxiety (Gross et al., 2002), stress, eating disorders (Bray, 2000; GuyGrand,

1995; Halford, 2001; Heal et al., 1998; Heisler et al., 1998b; Hesselink and

Sambunaris, 1995; Jallon and Picard, 2001; Koponen et al., 2002; Luque and Rey,

1999; McNeely and Goa, 1998; Prasad, 1998; Weissman, 2001), and eliberate self-

harm (Holden, 1995). In addition, personality dysfunctions such as addictive

behaviors, aggression, psychopathic and sociopathic behavior, attention-deficit

hyperactivity, and autism are also associated with altered serotonergic

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transmission. Indeed, new serotonin receptor ligands are being explored as possible

treatments for Alzheimer’s disease, as they appear to improve memory (Sumiyoshi

et al., 2001), obesity (Bray, 2000; Rothman and Baumann, 2002; Stunkard and

Allison, 2003; Wechsler, 1998), and epilepsy (Chadwick et al., 1977; Chugani and

Chugani, 2003; Dailey et al., 1992; Deahl and Trimble, 1991; Fromm et al., 1977;

Heisler et al., 1998b; Lunardi et al., 1995; Monaco et al., 1995; Savic et al., 2001;

Statnick et al., 1996; Yan et al., 1994).

Although increasing knowledge of serotonergic function is propelling many

advances in the therapeutics of psychiatric and behavioral disorders, drugs in

clinical use often treat the disease symptoms instead of relieving or preventing the

causes. Moreover, treatment regimens are often lengthy or lifelong, sometimes

with severe side effects. As yet the causes of psychiatric disease are unknown,

therefore the role of serotonin in the etiology or progression of these disorders

requires exploration in order to facilitate improvements in medication and

prognoses. There is increasing support for the hypothesis that impaired

development and synaptic plasticity contribute to the etiologies of many central

nervous system (CNS) diseases.

1.2 The Discovery of Serotonin and Classification of Serotonin Receptors

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 The chemical 5-hydroxytryptamine (5-HT) was first isolated in serum and,

because of its powerful vasoconstrictive effects, was dubbed ‘‘serotonin’’ Rapport,

1948). Serotonin was later detected in the brain (Twarog and Page, 1953). In 1957,

Gaddum and Picarelli reported the existence of multiple serotonin receptor

subtypes, which they called 5-HT-M and 5-HT-D, after their antagonists, morphine

and dibenzyline, respectively. Peroutka and Snyder (1979) reclassified these

receptors based on radioligand-binding studies in brain homogenates. The 5-HT1

receptor was labeled by [3H]5-HT, whereas the 5-HT2 receptor (corresponding to

5-HT-D) was sensitive to the dopamine receptor ligand [3H] spiperone. By 1986,

the M receptors were renamed 5-HT3 receptors (Bradley, 1986), were found to be

the only ionotropic subtype of the 5-HTreceptor, and were detected to be at low

density in limbic and striatal areas (Abi-Dargham et al., 1993). [3H] Lysergic acid

diethylamide (LSD), a psychotomimetic compound with a structure similar to

serotonin, was found to have high affinity for serotonin receptors. Subsequently,

heterogeneity was revealed in the 5-HT1 receptor class; 5-HT1A receptors could

be distinguished from 5-HT1B receptors (equivalent to the human 5-HT1D

receptor) by the high affinity of the former for spiperone (Pedigo et al., 1981). The

use of receptor auto radiographic techniques demonstrated the existence of a third

5-HT1 receptor in the porcine choroid plexus, the 5-HT1C subtype (later renamed

5-HT2C), through its high affinity for [3H] mesulergine and [3H]5-HT (Pazos et

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al., 1984). The application of molecular cloning techniques in the late 1980s

revolutionized protein discovery and to date, 14 distinct subtypes of mammalian

serotonin receptors have been cloned. Both molecular structure and

pharmacological properties determine their classification, and under a revised

system, the serotonin receptors are now allocated to seven distinct families (Hoyer

et al., 1994).

1.3 Serotonergic Projections during Brain Development

The highest levels of serotonergic activity are detected early in development

(Lidov and Molliver, 1982). Serotonergic neurons form a superior and inferior

group of immature cells, which have distinct maturational and migration patterns

(Lidov and Molliver, 1982; Wallace and Lauder, 1983). In both groups,

serotonergic neurons form different subsets of cells, and the raphe nuclei from

which serotonergic fibers project in the brain all have different origins (Azmitia

and Gannon, 1986). In the human, serotonergic neurons can be detected when the

embryo is just 5 weeks old (Sundstrom et al., 1993), with rapid growth and

multiplication until the 10th week of gestation (Kontur et al., 1993; Levallois et al.,

1997; Shen et al., 1989). After 15 weeks, clustering of the serotonin cell bodies in

the raphe nuclei is observed. The synaptic density of biogenic amine systems in the

human cerebral cortex doubles from birth to 1-year-old, when it reaches a peak

value and decreases thereafter to adult levels by the age of 10 (Huttenlocher and
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Dabholkar, 1997). Similarly, levels of serotonin increase during the first 2 years

after birth and then decline to adult levels after the age of 5 (Hedner et al., 1986;

Toth and Fekete, 1986). An equivalent time course for the development of the

serotonergic system has been observed in the chicken (Kojima et al., 1988) and

rodents (Rubenstein, 1998) indicating that these changes are conserved in

evolution. The early arrival of the serotonergic system before other monoamines

indicates that it may be required to guide the development of other

neurotransmitter systems (Benes et al., 2000; Whitaker-Azmitia et al., 1996).

In the rat, axonal projections from the rostral raphe nuclei ascend to the

midbrain and forebrain, whereas those of the caudal nuclei descend to the spinal

cord

(Wallace and Lauder, 1983). The descending fibers enter the spinal cord by

embryonic day 14 (E14) and innervate the preganglionic sympathetic neurons and

somatic motor neurons. Here they start to form synapses at E17, with innervation

of dorsal horn neurons occurring later. The rostral projections are visible soon after

serotonin can be detected in the brain stem. The unbranched fibers grow in the

marginal zone as a fascicle in the median forebrain bundle, and by E15 they reach

the diencephalon, where they branch out. By E17, medial fibers from the medial

forebrain bundle project to the frontal pole of the telencephalon, while lateral fibers

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project to the hypothalamus and arrive at the rostral end of the brain, some crossing

the midline in the supraoptic commissure. There is a simultaneous entry by

serotonin fibers into the telencephalon, the majority passing through the diagonal

bands of Broca, the septal areas, and then projecting into the cerebral cortex. A

small number of serotonin fibers enter through the ganglionic eminences

(Rubenstein, 1998). Interestingly, after birth there are nonraphethalamocortical

fibers containing serotonin in the sensory neocortex. Because there is no serotonin

synthesis within these neurons, serotonin must be transported to neighboring

neurons from adjacent synapses (Lebrand et al., 1996), suggesting that all cells

found to contain serotonin may not necessarily manufacture it. This may indicate a

mechanism by which serotonin can act as a developmental signal in

nonserotonergic cells (discussed in Section III, C).

1.4 Growth Factors Influencing the Development of Serotonergic Neurons

Several growth factors that influence serotonergic development are also

important in synaptic plasticity events. These include the astroglial growth factor,

S100, levels of which are increased by serotonin, indicating that serotonergic

nerves can stimulate their own growth. Inhibition occurs via serotonergic receptors

at the nerve terminals. Serotonin not only regulates the growth and development of

its targets, but it is also autoregulatory (Whitaker-Azmitia, 2001). S100 is

described in greater detail in the review by Rothermundt, Ponath, and Arolt.

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 The serotonin system regulates its own different by sequential activation of

the 5-HT1A receptor, brain-derived neurotrophic factor and its receptor trkB (see

the review by Guillin and colleagues), CREB, CREM, and ATF-1, among many of

the signaling molecules under serotonergic influence (Herdegen and Leah, 1998).

In addition, Petl, an ETS domain transcription factor, is closely associated with

developing raphe serotonergic neurons. Other neurotrophins and growth factors

control the different of serotonergic neurons, including bone morphogenetic

protein, and ciliary neurotrophic factor. Furthermore, consensus binding motifs for

Petl have been detected in the 50-untranslated regions of the human and mouse

genes for the 5-HT1A receptor, serotonin transporter, aromatic l-amino acid

decarboxylase (dopadecarboxylase), and tryptophan hydroxylase. It follows that

Petl may also be critical for the regulation of serotonin levels (Hendricks et al.,

1999), which have profound effects on brain development.

The Role of Serotonin as a Growth Factor

The dietary precursor for serotonin, tryptophan, is found across the phylogenetic

spectrum from lower plants to higher mammals. It is therefore an evolutionarily

ancient chemical. Because tryptophan possesses an indole ring, it is capable of

absorbing light and is vitally important for energy production during plant

photosynthesis. Tryptophan can also be converted to auxin, which is a plant tropic

factor, guiding plant growth and cell different (reviewed by Azmitia, 2001). In
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animals, tryptophan is also the precursor for melatonin, a light-sensitive amine

vital for the control of circadian rhythm. It follows that if auxin is a plant growth

factor, then derivatives of tryptophan in animals could have similar functions.

Serotonin alters the morphology of many mammalian cell types, including skeletal

muscle (O’Steen et al., 1967), platelets (Leven et al., 1983), and fibroblasts

(Boswell et al., 1992). Once serotonergic terminals have developed in a target

region, serotonin release may influence neurogenesis (Lauder and Krebs, 1976,

1978), neuronal removal through apoptotic mechanisms, dendritic refinement, cell

migration, and synaptic plasticity (Chubakov et al., 1986, 1993; Lauder, 1990;

Lauder et al., 1981). When these events are combined, they could produce the

highly sophisticated organization of the hippocampus or the somatosensory maps

called ‘‘barrel fields’’ (discussed in Section III, E,2). At a later developmental

stage, serotonin directs dendritic growth. Serotonin activity alters the overall length

of dendrites, the formation of dendritic spines, and branches into the hippocampus

and cortex (Faber and Haring, 1999; Mazer et al., 1997; Okado et al., 1993; Wilson

et al., 1998; Yan et al., 1997a). As the animal matures, increased serotonin levels

inhibit normal spine formation, perhaps by a 5-HT1A receptor-induced mechanism

indicated in Fig. 3 (discussed in Section III, D,1). Depletion of serotonin levels

during the developmental years may cause a loss of synapses, which can be

corrected by adulthood. However, in the aged animal this may lead to an increase

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in the number of synapses, a ‘‘reactive synaptogenesis,’’ or perhaps a retention of

synapses normally lost (Whitaker-Azmitia, 2001). Therefore, early developmental

disturbance has the potential to alter brain structure and repair much later in life.

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L-tryptophan is a dietary precursor of brain serotonin. Serotonin synthesis occurs

as summarized above in the postganglionic serotonergic neurons.

Studies of neurotoxin-induced serotonergic lesions of dorsal and medial raphe

nuclei have detected changes in cell growth in basal ganglia regions and

hypothalamic nuclei of adult rats. Newly generated granule cells can be identified

by immunostaining for bromo-2 0-deoxyuridine (BrdU) and the polysialylated

form of neural cell adhesion molecule (PSA-NCAM). Decreases in PSA-NCAM

staining have been observed after the inhibition of serotonin synthesis induced by

parachlorophenylalanine (PCPA) administration, suggesting that serotonin may

reduce adhesion by acting on PSA-NCAM expression in its environment and thus

facilitate plasticity in adult brain. A normalization of PSA-NCAM staining two

months after neurotoxin lesions has been associated with a partial restoration in

serotonin fiber density in the nucleus accumbens and the supraoptic nucleus,

indicating that PSA-NCAM may facilitate the sprouting of serotonin fibers. As no

changes in striatal PSA-NCAM staining have been observed after selective lesions

of the dopaminergic pathway, serotonin appears to have a selective and critical role

in adult brain plasticity (Brezun and Daszuta, 1999).

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 Chapter Two

2.1 Serotonin

Serotonin (5-hydroxytryptamine, 5-HT) was discovered 60 years ago in

blood, peripheral tissues and central nervous system (1). It was first identified as a

vasoconstrictor substance that is released from platelets during the coagulation of

blood, and later as a monoamine neurotransmitter in the brain. It has been

established that gastrointestinal tract, blood platelets and brain were the main

locations of serotonin in the mammal’s body. In addition, serotonin could be also

found in plants (bananas, walnuts, tomatoes, hickories, pineapples), mushrooms,

octopuses, and in poison of insects (spiders, scorpions, wasps).

Serotonin is a tryptamine that consists of an indole ring with a hydroxide

group on the fifth C atom, and a carboxyl-amide side chain.

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 The chemical structure of serotonin or 3-(2-aminoethyl)-5-hydroxyindole.

The main precursor of serotonin is the essential amino acid L-tryptophan

that must be provided by food. L-tryptophan and the other precursor, 5-

hydroxytryptophan, are transported from blood to brain by the active carrier

system located in the blood-brain barrier (BBB). It is believed that serotonin, due

to its chemical properties, does not cross BBB, but new evidence (2) suggests that

serotonin might cross endothelial cells of the BBB using serotonin transporter. The

serotonin synthesis occurs in a two-step enzymatic procedure.

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 Synthesis and degradation of serotonin.

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 The first and rate-limited step is hydroxylation of the tryptophan to 5-

hydroxytryptophan. This reaction is catalyzed by tryptophan hydroxylase, a

specific enzyme located only in the serotonergic neurons. The 5-

hydroxytryptophan is decarboxylated by a nonspecific enzyme, aromatic amino

acid decarboxylase, into serotonin. Serotonin levels could be determined in the

brain and various body fluids including serum/plasma, platelets, cerebrospinal fluid

(CSF) and urine. The flavine-containing mitochondrial enzyme monoamine

oxidase (MAO) is the most important enzyme for degradation of serotonin. This

process has two steps: first step is a degradation of serotonin into 5-hydroxyindole

acetaldehyde, and the second one is degradation through aldehyde dehydrogenase

regulated conversion into 5-hydroxyinoleacetic acid (5-HIAA) as the main

metabolite of serotonin.

In the human body, serotonin is synthesized and located in two

compartments that are separated by BBB. The first compartment, called

»peripheral« compartment,

contains the majority (about 95%) of serotonin in the body. Peripheral serotonin is

synthesized in enterochromaffin cells of the gastrointestinal tract. The second, i.e.

»central« compartment of serotonin is the central nervous system that synthesizes

serotonin within serotonergic neurons. Recent studies (3, 4) revealed that

tryptophan
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hydroxylase exists in two isoforms with different location within the body of

mammals. Tryptophan hydroxylase type 1 (TPH1) is responsible for the synthesis

of the peripheral serotonin, while tryptophan hydroxylase type 2 (TPH2) is the

predominant isoform in the brain.

2.2 Serotonergic receptors

The complex functions of serotonergic system would be impossible without

a large number of serotonergic receptors 5). Serotonergic receptors are classified in

seven different groups or »families« called 5-HT1, 5-HT2, 5-HT3, 5-HT4, 5-HT5,

5-HT6, 5-HT7, and several subtypes that differ in terms of structure, action, and

location. Serotonergic receptors are distributed on the presynaptic and postsynaptic

neurons in the central nervous system and on the different peripheral cells and

organs. The majority of serotonin receptors are G protein-coupled receptors. The

exception is 5-HT3 receptor that belongs to the ligand-gated ion channel receptors.

Serotonergic receptors activate an intracellular second messenger (cAMP, IP3,

DAG) cascade and produce an excitatory or inhibitory response. Serotonin

receptors

are very important sites of action for different classes of psychotropic drugs, like

antidepressant drugs, atypical antipsychotic drugs (olanzapine, risperidone) and

psychoactive compounds (LSD, DMT).

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The serotonergic receptors.

2.3 Serotonin in the central nervous system

Serotonergic neurons are widely distributed throughout the mammalian brain and

serotonergic system is the largest single system in the brain. The nine groups of

serotonergic cell bodies are located mainly in the area of brain stem raphe nuclei.

Serotonergic nerve terminals could be found in nearly all other regions of the

central nervous system. The widespread distribution of the raphe projections

suggests a highly collateralized axon system. The communication of serotonergic

system with other important neurotransmitter systems like catecholaminergic

system is well established, although the mechanisms of interaction are not yet

completely understood. Significant amounts of data have demonstrated that these

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interactions are very important in the mechanisms of action of antidepressant and

anxiolytic drugs. Serotonin is implicated in many physiological (body temperature,

sleep, vomiting, sexuality, appetite), behavioral (aggression, mood) and cognitive

(learning, memory) functions. In addition, serotonin has an important role in the

growth of the central nervous system during development. It plays a critical role as

a growth factor in the immature brain, directing both proliferation and maturation.

This is supported by the higher serotonin turnover rate in the immature mammalian

brain than at any other time in life. Recent data suggested that an overload of

serotonin during cortical development could induce abnormal distribution and

incorrect positioning of cortical interneurons. There are several methods for the

determination of serotonin synthesis rate in vivo: a) pharmacological manipulation,

i.e. after the administration of compound that inhibits enzyme aromatic acid

decarboxylase like NSD-1015, b) the use of radiolabel tryptophan, c) the use of

radiolabel alpha-methyl tryptophan (alpha-MTrp) as an analogue of tryptophan.

The limitation of the pharmacological methods is that the effect of the tested

compound on the serotonin synthesis could be in part influenced by the pre-

pharmacological manipulation itself. Radiolabel tryptophan is an essential amino

acid incorporated in proteins, while radiolabel tryptophan metabolites, like

serotonin and 5-HIAA, are lost very rapidly from the brain. The use of alpha-MTrp

labelled with 3H or 14C and the determination of serotonin synthesis by an auto

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radiographic method (10) permits the measurement of serotonin synthesis in the rat

brain with high anatomical resolution and without any pharmacological pre-

treatment. The disadvantages of this method are the need for special equipment and

a long procedure. The alteration of the serotonergic system has been related with

the aetiology of different neurological (migraine, Alzheimer’s disease, epilepsy)

and psychiatric (depression, schizophrenia, mood disorders, alcoholism, ADHD,

PTSD) disorders.

2.4 Peripheral serotonin

Peripheral serotonin is synthesized in enterochromaffin cells of the

gastrointestinal tract. The synthesis is regulated by TPH1 (4). From gut serotonin is

released in the blood stream and then stored mostly in blood platelets. The other

peripheral cells that contain serotonin are macrophages and mast cells. Peripheral

serotonin is metabolized in the liver by a MAO-A to 5-HIAA. The 5-HIAA is

filtrated and excreted by the kidney. The vast increase in urine excretion of 5-

HIAA was found in carcinoid syndrome, due to the pronounced production of

serotonin by carcinoid cells. In humans a direct association between

neurotransmitters in the brain and those excreted in the urine is not yet defined.

New evidence suggests that neurotransmitters excretion in the urine might be used

as possible biomarkers of the central nervous system activity. The study in rats

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treated with 5-hydroxytryptophan has shown simultaneous changes in the activity

of brain serotonergic system and urinary serotonin levels.

Serotonin has an important role in the vascular biology. It is involved in the

control of vascular resistance, blood pressure, haemostasis and platelet function

(11). One of the most important functions of the peripheral serotonin is the

promotion of platelet aggregation and blood clotting. Activated 5-HT-2A receptors

on platelet membrane also stimulate platelet activation and aggregation.

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 Chapter Three

3.1 Function of Brain Serotonin

The function of brain serotonin is an enigma. There have been several

attempts to identify a unifying function of dopaminergic transmission in the brain

(Berridge and Robinson, 1998; Schultz, 2010; Schwartenbeck et al., 2014) and

similar attempts have been made for serotonin (Andrews et al., 2015; Azmitia,

2007; Branchi, 2011; Dayan and Huys, 2009; Deakin, 1998). Most researchers

acknowledge that the function of the 5-HT system remains ‘elusive’ (Dayan and

Huys, 2009) and ‘a puzzle’ (Cools et al., 2008; Dayan and Huys, 2015; Seymour et

al., 2012) and it is argued here that this may be due to the special diversity and

complexity of the serotonin system with its many receptor subtypes (Hoyer et al.,

1994), extensive innervation of the brain and paracrine style of transmission

(Hornung, 2003; Jennings, 2013). The notion that 5-HT is an enigma among

neuromodulators (said to be ‘involved in everything but responsible for nothing’

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(Muller and Homberg, 2015)) is relevant here, and it is argued that the riddle of 5-

HT can only be solved by focusing on its individual receptor subtypes.

Accordingly, given the inherent complexity of the serotonin system, one

strategy for understanding its functioning is to focus on a select number of receptor

subtypes that have been particularly well characterized. From this foundation, one

might then consider whether other serotonin receptor subtypes can be incorporated

into the associated model, or whether one or more additional models are required

to cover the full range of functions associated with brain serotonin transmission.

Following this approach, we have chosen to concentrate on the 5-HT1A and 5-

HT2A receptors. Our reasons for doing this are (at least) three-fold, and include:

(1) the prevalence of their expression in the human brain and specific localisation –

e.g. in stress circuitry (5-HT1AR) and high-level cortex (5-HT2AR) (e.g. Beliveau

et al., 2016); (2) compelling evidence for their involvement in the pharmacology of

different psychiatric disorders and medications (Celada et al., 2004); and (3) their

apparent functional pre-eminence and opposition – as has been noted by others

(Azmitia, 2001). Following on from this last point, the 5-HT1A and 5-HT2A

receptors show diametrically opposite responses to their endogenous ligand, with

5-HT1A receptor signalling being inhibitory and 5-HT2A receptor signaling being

excitatory (Araneda and Andrade, 1991; Azmitia, 2001; Charig et al., 1986;

Fletcher et al., 2007). This stark functional opposition is intriguing – and motivates

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us to ask why this should be the case, and what purpose it serves? We suggest that

inherent diversity within the serotonergic system relates to its capacity for flexibly

and adaptably responding to different degrees of adversity and challenge in the

organism’s environment, with distinct responses mediated by distinct serotonergic

pathways. As noted above, an obvious caveat here is that 5-HT receptors we do not

specifically focus on in the present review may complement one or the other of

these two pathways – and may also modulate unrelated physiological and

behavioural functions. For example, signalling at 5-HT receptors other than the 2A

receptor has been associated with neuroplasticity (Kraus et al., 2017) – and thus,

may also feed into pathway 2 (below). Similarly blockade of certain 5-HT

receptors (e.g. 5-HT2C, 5-HT7 and even 5-HT2A) may complement pathway 1

(below). However, a thorough coverage of this matter is beyond the scope of this

article. In what follows, focus is directed to 5-HT1A and 5-HT2A receptor

signalling and research pertaining to their associated functions. It is argued that

studying potent serotonergic compounds such as rapid-acting, highly effective 5-

HT releasers (such as 3,4-methylenedioxymethamphetanine, MDMA (Baumann et

al., 2008; Heifets and Malenka, 2016)) and direct 5-HT2AR agonist psychedelic

drugs such as psilocybin and lysergic acid diethylamide, LSD (Glennon et al.,

1984; Vollenweider et al., 1998), can be particularly informative about the function

of serotonergic transmission in the brain because their acute and longer-term

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effects are especially marked and novel (Griffiths et al., 2008; Mithoefer et al.,

2013), and there is a growing literature on human research with such drugs,

including an increasing number of neuroimaging studies (Carhart-Harris et al.,

2013b, 2015b; Muthukumaraswamy et al., 2013) and clinical trials (Bogenschutz et

al., 2015; Carhart-Harris et al., 2016a; Gasser et al., 2014; Griffiths et al., 2016;

Grob et al., 2011; Mithoefer et al., 2011; Ross et al., 2016; Sanches et al., 2016) –

see Carhart-Harris and Goodwin (2017) for a review.

We acknowledge that MDMA also releases dopamine (DA) and

noradrenaline (NA) (Baumann et al., 2008) but its 5-HT releasing properties are

many times greater than its catecholamine releasing properties, e.g. 5-HT release in

the frontal cortex is approximately 5 times that of DA release (Golembiowska et

al., 2016), preferential 5-HT versus DA and NA release is unusual for an

amphetamine, and MDMA’s subjective effects are also distinct from those of other

more conventional amphetamines (Bedi et al., 2014).

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Regional distribution of serotonin 1A (left) and 2A receptors (right) in healthy

volunteers as measured using PET imaging and radioligands selective for the 5-

HT A and 2A receptors. Pathway 1 refers to the ‘passive coping’ pathway

hypothesized to be mediated by 5-HT1AR signalling and concerned with passive

endurance, and ‘pathway 2’ refers to the ‘active coping’ pathway hypothesised to

be mediated by 5-HT2AR signalling and concerned with an active change in

outlook and/or behaviour. Images reproduced from (Beliveau et al., 2016) with

permission. Note: The dense expression of the 5-HT1AR in medial temporal lobe

regions and particularly the hippocampus is not clearly evident in the relevant

maps shown here (left) but can be seen in values presented in the paper itself, as

well as others (Pazos and Palacios, 1985; Pazos et al., 1987).

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3.2 Psychological functions associated with brain 5-HT

1. Impulsivity and aggression: One of the most reliable behavioural effects of

reducing 5-HT transmission in the brain is to increase impulsive and aggressive

behaviours (Audero et al., 2013; Brown et al., 1979; Duke et al., 2013; Mosienko

et al., 2015; Soubrie, 1986). Indeed, some of the earliest hypotheses on the

function of 5-HT in the brain proposed that it serves to suppress behavioural

response to pain (Harvey et al., 1975), anxiety (Wise et al., 1970) and aversive

stimuli more generally (Deakin and Graeff, 1991; Soubrie, 1986) and these ideas

continue to have traction (Deakin, 2013; Yanowitch and Coccaro, 2011). The anti-

aggression effects of 5-HT enhancing compounds led to them being called

‘serenics’ (Olivier and Moss, 1990), a fitting term in our view, and one that is also

apt in relation to the subjective effects of MDMA, a particularly potent 5-HT

releaser. Related to these hypotheses, is the notion that 5-HT transmission enables

a person to better tolerate delay (Soubrie, 1986), and the patience-promoting

properties of 5-HT have recently received significant experimental support

(Fonseca et al., 2015; McDannald, 2015; Miyazaki et al., 2012, 2014; Ranade et

al., 2014). Low concentrations of the serotonin metabolite (5-HIAA), implying low

central 5-HT function, have been associated with impulsivity (Fairbanks et al.,

2001), aggression (Brown and Linnoila, 1990) and suicidal behaviour (Asberg et

al., 1976), and tryptophan depletion (a diet-based approach that produces a

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transient depletion of central 5-HT) has also been found to enhance impulsivity

and aggression (Dougherty et al., 1999, 2010). In contrast, tryptophan

supplementation (Duke et al., 2013), acute MDMA administration (Ramaekers and

Kuypers, 2006; van Wel et al., 2012), acute fenfluramine (Cherek and Lane, 2001)

and chronic 5-HT reuptake inhibitor administration (Butler et al., 2010; Wolff and

Leander, 2002), all of which are known to increase central 5-HT function, have all

been found to reduce impulsivity and aggression. For a more in-depth discussion of

the complexities of the relationship between brain 5-HT and aggression, including

some contradictory findings to the rule that low synaptic 5-HT is associated with

increased aggression, see this review (Mitchell, 2005).

5-HT1AR signalling, impulsivity and aggression. There are solid grounds to

believe that the anti-aggression and impulsivity effects of 5-HT are mediated by

postsynaptic 5-HT1A receptor signalling (Sanchez and Hyttel, 1994; Schreiber and

De Vry 1993), with some contribution from postsynaptic 5-HT1B receptors

(Ramboz et al., 1996; Sijbesma et al., 1991). Assessing the functional effects of 5-

HT1A receptor manipulations is complicated, however, owing to the opposing

influences of pre- and postsynaptic 1A receptor activation. Prior to a time-

dependent 5-HT1A autoreceptor desensitisation by reuptake blockers (Le Poul et

al., 1995), stimulation of these presynaptic 5-HT1A receptors reduces serotonin

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efflux, whereas postsynaptic 5-HT1A receptor activation is an important (and often

clinically desirable) consequence of increased serotonin efflux (Artigas, 2013b).

Moreover, selective 5-HT1AR antagonists or full 5-HT1AR agonists are not

available for human use (beyond the very low doses used in PET imaging), and so

cannot be used to incisively inform on this matter. With these caveats, it can be

relatively safely inferred that (postsynaptic) 5-HT1AR agonism appears to reduce

aggressive and impulsive behaviours (de Boer and Koolhaas, 2005; Olivier et al.,

1989; Popova et al., 2007; Sanchez and Hyttel, 1994; White et al., 1991; Wolff and

Leander, 2002). Note, however, that many 5-HT1A receptor agonists are in fact,

only partial agonists; thus, their impact on net 5-HT1AR signalling is dependent on

basal 5-HT efflux and competition with the full agonist endogenous ligand, 5-HT

itself (Mitchell, 2005).

It has been claimed that the 5-HT1AR is the most prevalent and well-

distributed 5-HT receptor in the brain (Paterson et al., 2013; Varnas et al., 2004).

Serotonin has a high affinity for this receptor subtype (Peroutka and Snyder, 1979),

serotonergic projections densely innervate 5-HT1AR-rich regions (Hornung, 2003)

and 5-HT concentrations may be higher in 5-HT1AR-rich subcortical/limbic

regions than in the 5-HT2AR-rich cortex during basal conditions (Bose et al.,

2011; Erritzoe et al., 2010; Kirby et al., 1995; Rueter and Jacobs, 1996), although

see Adell et al. (1991) and Hjorth and Sharp (1991). These factors imply that
27 | P a g e
manipulation of synaptic 5-HT concentrations will significantly impact on

postsynaptic 5-HT1AR signalling and limbic functioning. With this in mind, it is

telling that 5-HT lesions and depletion both tend to promote impulsivity and

aggression (Audero et al., 2013; Dougherty et al., 1999), whereas stimulating

serotonin function tends to reduce these behaviours (Miyazaki et al., 2014). It is

also relevant that the potent 5-HT releaser, MDMA, has marked pro-social, pro-

empathy, anti-aggressive effects during the acute phase (Bedi et al., 2010, 2014;

Frye et al., 2014; Hysek et al., 2012, 2014a; Kamboj et al., 2015; Kirilly et al.,

2006; Schmid et al., 2014; Stewart et al., 2014), perhaps via an inhibitory action on

activity in limbic regions (Carhart-Harris et al., 2015b), and some of these effects

in rodents’ can be attenuated by pre-treatment with a 5-HT1A receptor antagonist

(Hunt et al., 2011).

The table above summarizes findings that support various associations between

5-HT, signalling at its post-synaptic 5-HT1A and 5-HT2A receptors and relevant

28 | P a g e
psychological phenomena. A number of these associations require qualification,

e.g. 5-HT2AR agonism can have opposite acute and longer-term effects (Carhart-

Harris et al., 2016c). To account for this, we use the acronyms ‘ST’ and ‘LT’ for

acute (short-term) and long-term outcomes respectively, where we feel

disambiguation is required. Also, receptor signalling may increase plasticity in one

region but decrease it in the other (e.g. Vaidya et al., 1997). As this matter is most

relevant in relation to molecular markers of plasticity in the hippocampus and

cortex, we use the acronyms ‘hip’ and ‘cx’ to provide the necessary

disambiguation. Regarding plasticity, we use ‘general plasticity’ (gP) to refer

simply to an increased capability for change and ‘regional plasticity’ (rP) when we

are specifically referring regional changes in molecular markers of plasticity such

as trophic factors. It is important to stress that the effects of 5-HT2AR agonism are

highly context sensitive (see Figure 2), e.g. the effects of 5-HT2AR signalling on

mood and mental health are likely highly sensitive to the quality of the

environment in which a 5-HT2AR-mediated experience occurs (Johnson et al.,

2008), and this rule may also apply for treatment with an SSRI (Branchi, 2011)

perhaps due to increased 5-HT2A receptor signalling through increased synaptic 5-

HT.

2. Anxiety and stress

29 | P a g e
 5-HT1AR signalling, anxiety and stress. Related to the hypothesis that 5-HT

functions to moderate aversive mental states (Deakin and Graeff, 1991) and

promote patience (McDannald, 2015) is the notion that 5-HT plays an important

role in negatively modulating anxiety (Piszczek et al., 2015). Selective reductions

of 5-HT in the forebrain have been found to enhance anxiety-related behaviours

(Pum et al., 2009; Tu et al., 2014), whereas chronically administered SSRIs have

been found to reduce anxiety (Blanco et al., 2013). Like impulsivity and

aggression, anxiety appears to be negatively modulated by 5-HT1AR stimulation

(Heisler et al., 1998; Parks et al., 1998; Schreiber and De Vry, 1993; Toth, 2003),

and although there are some contradictory findings (File et al., 1996), this effect

appears to be mediated by postsynaptic 5-HT1AR signalling (Celada et al., 2013a;

Gross et al., 2002; Piszczek et al., 2015; Stefanski et al., 1993; Tauscher et al.,

2001; Tu et al., 2014; Zhou et al., 2008, 2014). Postsynaptic 5-HT1A receptors are

densely expressed in limbic regions and particularly the hippocampus (Pazos and

Palacios, 1985; Varnas et al., 2004), which is known to be involved in anxiety

(Gray, 1983; Tu et al., 2014). Serotonin 1A receptors are highly expressed on

excitatory neurons in the hippocampus (Pompeiano et al., 1992) and 5-HT1AR

stimulation has an inhibitory influence on pyramidal neuron activity (Andrade,

2011). Hippocampal hyperactivity is strongly associated with states of anxiety and

stress (Engel et al., 2009) and 5-HT appears to quell limbic hyperactivity via the

30 | P a g e
inhibitory action of postsynaptic 5-HT1ARs (Dong et al., 1998; Tada et al., 2004).

This mechanism could explain the reduced metabolism and blood flow observed in

limbic regions with acutely administered MDMA (Carhart-Harris et al., 2015b;

Gamma et al., 2000), buspirone (Friston et al., 1991), fenfluramine (thalamus and

temporal cortex (Meyer et al., 1996)) and chronically administered SSRIs

(Mayberg et al., 2000) – as well as reduced cortico-limbic reactivity to negative

stimuli with MDMA (Bedi et al., 2009; Carhart- Harris et al., 2014d) and SSRIs

(Arnone et al., 2012; Ma, 2015). The improved ability to tolerate negative stimuli

with both acute MDMA (Carhart-Harris et al., 2014d; Mithoefer et al., 2011, 2013)

and chronic SSRI treatment (Corchs et al., 2009; Mineur et al., 2015) may be due

to elevated levels of synaptic 5-HT activating inhibitory postsynaptic 1A receptors

in stress-sensitive limbic regions. It is also likely to explain the use of SSRIs and

direct 5-HT1AR agonists such as buspirone, as anxiolytic medications. There is

also compelling evidence through 5-HT1AR knock out studies that this receptor is

involved in the moderation of anxiety (Chattopadhyay, 2007).

3. Learning and cognition

5-HT1AR signalling learning and cognition. Postsynaptic 5-HT1AR

stimulation is generally considered to be a desirable property of anxiolytic and

antidepressant medications (Artigas, 2015), and the postsynaptic 5-HT1AR is

thought to be the principal (therapeutic) site of action of SSRIs (Artigas, 2013a,

31 | P a g e
2015; Samuels et al., 2015). Chronic treatment with SSRIs has been associated

with increased neurogenesis (Boldrini et al., 2009), particularly in the hippocampus

(Boldrini et al., 2009, 2012) and some improvements in learning and cognition

(Bui et al., 2013), albeit with some contradictory findings (Deakin et al., 2004).

There is evidence to suggest that increased neurogenesis (at least in the

hippocampus) is a 5-HT1AR-mediated effect (Gould, 1999; Huang and Herbert,

2005; Malberg et al., 2000; Santarelli et al., 2003); however, other 5-HT receptors

(e.g. the 5-HT4 and 5-HT2A) are also thought to contribute (Azmitia, 2001; Imoto

et al., 2015; Jha et al., 2008; Kraus et al., 2017). Despite this association between

5-HT1AR signalling and

neurogenesis, there is a body of evidence to suggest that postsynaptic 5-HT1AR

stimulation is impairing to learning and cognition (Ogren et al., 2008), so how can

we reconcile these things? One possibility is that the observed pro-cognitive effects

of SSRIs are actually mediated by other (non-1A) 5-HT receptors (Boulougouris et

al., 2008; Furr et al., 2012; Imoto et al., 2015), and another is that improvements in

cognition in patients treated with SSRIs is an epiphenomenon of improvements in

mood (Chepenik et al., 2007). It is also important to note that the evidence that

SSRIs

improve cognition is relatively weak (Beheydt et al., 2015; Knorr, 2012; Knorr et

al., 2011; Siepmann et al., 2003) and their modest ability to address cognitive
32 | P a g e
symptoms in depression is considered one of their limitations (Popovic et al.,

2015). 5-HT2AR signalling, learning and cognition. The relationship between the

5-HT2AR and cognition is somewhat different to that of the 5-HT1AR. As

discussed above, activation of postsynaptic 5-HT1ARs is associated with cognitive

and learning impairments (Ogren et al., 2008), whereas 5-HT2AR activation is

associated with improvements in certain aspects of cognition and learning (Gimpl

et al., 1979; Harvey, 1996, 2003; Harvey et al., 2004, 2012; King et al., 1974;

Romano et al., 2006, 2010; Welsh et al., 1998; Zhang and Stackman, 2015; Zhang

et al., 2016) as well as an unlearning or ‘extinction’ learning (Zhang et al., 2013).

Serotonin

2A receptor activation has also been associated with neurogenesis (Catlow et al.,

2013; Cavus and Duman, 2003; Frankel and Cunningham, 2002; Gewirtz et al.,

2002; Jones et al., 2009; Meller et al., 2002; Niitsu et al., 1995; Vaidya et al.,

1997), particularly in the cortex (Gewirtz et al., 2002; Jones et al., 2009; Vaidya et

al., 1997) (but not in the hippocampus (Vaidya et al., 1997)), which may explain

the type of cognitive and learning enhancements that are associated with its

functioning (e.g. associative learning). Specifically, a number of studies have

shown enhancements of associative learning with 5-HT2AR agonism and

impairments with its blockade (Barre et al., 2016; Harvey, 1996, 2003; Harvey et

al., 2004; Romano et al., 2000, 2006; Welsh et al., 1998).

33 | P a g e
 Cognitive flexibility in humans is thought to be positively modulated by 5-

HT2AR functioning (Boulougouris et al., 2008) and there is evidence to suggest

that 5-HT2AR agonists (such as LSD and psilocybin) enhance cognitive flexibility

and creative thinking (Frecska et al., 2012; Harman et al., 1966; Janiger and

Dobkin de Rios, 1989; King et al., 1974; MacLean et al., 2011; McGlothlin et al.,

1967; Sessa, 2008), potentially in an enduring way (MacLean et al., 2011).

Serotonin depletion and inactivation has been shown to impair cognitive flexibility

(Clarke et al., 2004, 2007; Matias et al., 2017) and there is evidence that this may

be due to decreased basal activation of 5-HT2ARs (Boulougouris et al., 2008; Furr

et al., 2012). Serotonin neurons have been found to activate when animals

experience a surprising violation of assumptions, independent of its reward-related

implications (Matias et al., 2017), supporting the association between 5-HT,

environmental sensitivity and adaptability (Branchi, 2011). Our argument here is

that 5-HT2AR signalling is the key mediator of this effect. Promotion of plasticity

via 5-HT2AR signalling is central to our thesis that, along with improving stress-

tolerance, a key function of brain serotonin transmission is to engage processes

necessary for change, when change is necessary. Note: although we acknowledge it

would be pertinent and potentially valuable, a more in-depth discussion of the 5-

HT2AR and animal and human behavioural measures of cognitive flexibility is

beyond the scope of this paper.

34 | P a g e
 4. Serotonin, depression and mood

Serotonin was first isolated and named in the late 1940s (Rapport et al.,

1948) and subsequently found in the brain in the early 1950s (Gaddum, 1953;

Twarog and Page, 1953). At the same time, scientists were beginning to identify

interactions between serotonin and the recently discovered lysergic acid

diethylamide (LSD) (Gaddum, 1953; Shaw and Woolley, 1956). Struck by LSD’s

remarkable potency (psychoactive in doses as low as 20 μg) and powerful

modulatory effects on mood and cognition (Busch and Johnson, 1950; Hofmann,

1980), it was speculated that abnormal serotoninergic functioning may underlie

certain mental disorders (Gaddum, 1957; Woolley and Shaw, 1954). Although the

‘psychotomimetic’ (mimicking psychosis) properties of LSD and related

psychedelics were recognized in the 1950s and 60s (Isbell et al., 1959), as they are

today (Carhart-Harris et al., 2013a, 2016c), these compounds were also used

extensively as psychotherapeutic aids for the treatment of a range of disorders,

including depression and anxiety (Grinspoon and Bakalar, 1979; Sandison, 1954;

Sandison and Hopkin, 1964).

The earliest and most direct evidence for the involvement of monoamines in

mood regulation however, came with the observation that reserpine, which

depletes 5-HT and noradrenaline in the brain (Pletscher et al., 1955), also induces

depressed mood in some individuals (Achor et al., 1955) – see also (Antkiewicz-
35 | P a g e
Michaluk et al., 2014). This observation was closely followed by the discovery of

the antidepressant properties of the monoamine oxidase inhibitors (MAOIs)

(Udenfriend et al., 1957) and subsequently the tricyclic antidepressants (TCAs)

(Axelrod and Inscoe, 1963; Kuhn, 1958) – both of which increase synaptic

monoamines (Gur et al., 1999; Matos et al., 1990). More specific evidence for the

involvement of 5-HT in depression came from studies showing a combined

antidepressant effect with an MAOI plus tryptophan, the biochemical precursor to

5-HT (Coppen et al., 1963; Hess and Doepfner, 1961; Pare, 1965).

The idea that serotonergic mechanisms are involved in the pathogenesis and

treatment of depression was controversial in the 1960s (Coppen, 1969, 1967);

however, it gradually gained traction in the 1980s and into the 1990s with the

development and licensing of the SSRIs (Carlsson, 1981; Cowen and Browning,

2015) and particularly fluoxetine (Bremner, 1984). When chronically administered,

SSRIs increase concentrations of synaptic 5-HT (Smith et al., 2000) by blocking its

reuptake (Carlsson, 1981), show superior efficacy to placebo in depression (Horder

et al., 2011; Hieronymus et al., 2016; Barth et al., 2016) and are safer than MAOIs

and TCAs (Pletscher, 1991). Another important finding supporting the

involvement of serotonin in depression was the observation that acute tryptophan

depletion can induce a (transient) relapse in symptoms in formerly depressed

patients (Smith et al., 1997) and plasma tryptophan levels have been found to be

36 | P a g e
low in patients with severe depression (Anderson et al., 1990), potentially owing to

inflammation related

mechanisms (Wichers et al., 2005).

A two-part or ‘bipartite’ model of brain serotonin function. Model proposes that

brain serotonin mediates adaptive responses to adversity via two distinct

mechanisms: one mediated by postsynaptic 5-HT1AR signalling in aid of stress

moderation (pathway 1) and the other mediated by 5-HT2AR signalling is aid of

more substantial adaptive changes (pathway 2). SSRIs and other conventional

antidepressant medications work on and can enhance pathway 1, whereas pathway

2 can be enhanced by 5-HT2AR agonist psychedelic drugs such as psilocybin.

Note: it is hypothesised that active coping can be most effectively implemented if

37 | P a g e
the window of plasticity afforded by 5-HT2AR agonism is complemented by

supportive psychotherapy that promotes a willingness to confront and work

through sources of stress (Watts et al., 2017).

5. Plasticity and the entropic brain

The proposal that psychedelics induce a plastic state is consistent with the

‘entropic brain’ hypothesis, introduced by us in 2014 (Carhart-Harris et al., 2014b).

This idea emerged out of observations of consistencies between neuroimaging

findings on the action of psychedelics (Carhart-Harris et al., 2014b;

Muthukumaraswamy et al., 2013) and a sense that their physical (brain) effects

recapitulate their psychological effects – and vice versa. Inspired by Karl Friston’s

Free-Energy principle (Friston, 2010), the information theory-based measure of

entropy was applied to the psychedelic state in an effort to capture its essential

phenomenological and neurophysiological qualities. Entropy is formally both

uncertainty and unpredictability (Ben-Naim, 2007) – and not coincidentally, these

terms possess meaning in both a mechanistic and subjective sense. A growing

number of analyses are now endorsing the principle that the brain exhibits

increased entropy under psychedelics (Atasoy, 2017; Carhart-Harris et al., 2014b;

Lebedev et al., 2016; Schartner et al., 2017; Tagliazucchi E, 2014; Viol, 2016) (see

38 | P a g e
also Gallimore, 2015) and countless other human and animal studies by

independent teams, despite not formally measuring entropy, report findings that are

consistent with the entropic brain principle (Celada et al., 2013b;

Muthukumaraswamy et al., 2013; Riba et al., 2004, 2014; Wood et al., 2012).

Entropy exists most purely as an index of uncertainty (Ben- Naim, 2007) but

its origins lie in thermodynamics (Ben-Naim, 2007, 2008). Entropy is perhaps

most familiar to people in the context of thermodynamics and specifically how it

relates to the second law: that isolated systems tend towards disorder, or exhibit

increased entropy over time (i.e. decay). The relationship between information

theory-based entropy and thermodynamic entropy is a formal one, with the latter

being merely an applied and contextualized version of the former (Ben-Naim,

2007, 2008).

In the context of 5-HT2AR signalling and how this may inform on the

function of brain serotonin, one may think of enhancing 5-HT2AR signalling as

analogous to increasing the temperature or excitability) of the brain; indeed, the

excitatory effect of 5-HT2AR signalling has long been recognised (Aghajanian and

Marek, 1999; Celada et al., 2013b). Extending this analogy to the process of

annealing (i.e. whereby a metal is heated to make it more malleable) – one may

think of 5-HT2AR signalling as functioning to induce an entropic state

39 | P a g e
characterised by enhanced flexibility and malleability during which work can be

done that, upon

cooling, may leave a lasting change (Gopnik, 2010). Viewed through the lens of

the popular Bayesian brain model of brain function (Knill and Pouget, 2004), one

could see this 5-HT2ARmediated entropic state as working to ‘reset’ reinforced

prior’s in

depression – such as pessimistic beliefs and negative self-perceptions (Moutoussis

et al., 2014). See Carhart-Harris et al. (2017b) for recent neurobiological support

for this idea.

40 | P a g e
 Chapter Four

4.1 Conclusion

Since serotonin discovery in the gastrointestinal tract (1), the comprehensive

investigations enlarge its first role as a hormone to the neurotransmitter function in

the central nervous system that has a myriad of central and peripheral functions.

However, serotonin has an important role in the modulation the effects of other

neurotransmitters. In the words of Thomas Carew, a Yale researcher, Serotonin is

only one of the molecules in the orchestra. But rather than being the trumpet or the

cello player, it is the band leader who choreographs the output of the brain.

Research spanning more than five decades has shown that serotonergic projections

in the brain have a widespread distribution and that these projections interact with

other neurotransmitter systems, thereby influencing many, if not all, physiological

41 | P a g e
unctions. Evidence from biochemical, pharmacological, and clinical studies

demonstrates the huge importance of an intact serotonergic system to support brain

development and neurogenesis in the maintenance of normal CNS function.

Serotonin acts as a growth factor and influences other growth factors during

development. The high level of serotonin function during these crucial stages of

brain development and the pharmacological evidence for impaired serotonergic

function in several disparate brain disorders underpins the importance of

understanding this highly complex system. Increased insight may facilitate real

progress in drug development, with the ultimate goal of preventing and even curing

these debilitating illnesses.

42 | P a g e
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