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Long term potentiation (LTP) is believed to be the molecular basis for learning in the brain.
Describe the molecular events leading to LTP and evidence that this may be occurring in
vivo.

The molecular basis for long term potentiation (LTP) in the brain has been investigated and
studied repeatedly as psychologists have tried to understand the basic biological processes
involved in learning. Activity-dependent synaptic plasticity is generally accepted as an
essential element of memory formation in the brain (Lynch, 2004), and as a result the
phenomenon of LTP has received substantial attention: however, there is still considerable
debate about the exact mechanisms involved in LTP as well as their relative importance to
the process. Fortunately a general consensus has been reached on certain areas and types
of LTP, so that it is still possible to use the current evidence for LTP to understand the
fundamental molecular events that cause LTP to occur. This analysis will firstly attempt to
accurately convey the molecular events that lead to LTP and will then move on to discuss
the evidence that LTP occurs in vivo and is not merely an artificial concept that has no
bearing on the study of learning in living creatures.

It should be noted that this review will focus primarily on LTP in hippocampal synapses
without discussing long term depression (LTD), LTP in other areas of the brain or associative
LTP. LTP processes in areas of the brain can vary according to the individual structures and
other processes that take place locally: hippocampal LTP has been the most investigated
and thus best-understood form of LTP (Lynch, 2004) due to the perceived role of the
hippocampus in memory formation, which means that accounts of molecular LTP processes
are most applicable to it compared to other, less-studied forms of LTP. The account of LTP
given here will be general and elements of it can be applied universally to LTP, but it is
intended solely to account for processes at hippocampal synapses. The discussion of
evidence for in vivo LTP will discuss evidence for LTP processes in general and should show
that studies have collected results that are consistent with LTP theory but that ultimately
the process of LTP has not been conclusively demonstrated in vivo.

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LTP begins shortly after the stimulation and associated activity of postsynaptic receptors by
glutamate: a-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors bind
glutamate and permit an influx of Na into the cell through ligand-gated ion channels, and
repeated stimulation causes depolarisation of the postsynaptic neuron (Abel and Lattal,
2001). Depolarisation, combined with the presence of glutamate (as observed by
coincidence detectors according to Abel and Lattal, 2001), causes the Mg block in Nmethyl-D-aspartate (NMDA) receptors to be released, permitting the influx of Ca through
ion channels into the intracellular environment of the postsynaptic neuron (Lynch, 2004).
The influx of Ca is a crucial underlying element of LTP and triggers a series of events that
lead to increases in synaptic efficiency (Lynch, Muller, Seubert and Larson, 1988). The more
immediate effects of the Ca influx will be discussed first, followed by the long-term
consequences on the pre- and postsynaptic cells.

The influx of Ca activates calmodulin-dependent kinase II (CaMKII) and protein kinase C

(PKC): both become phosphorylated and remain activated independently of Ca for a
period following phosphorylation (Schafe, Nader, Blair and LeDoux, 2001). CaMKII then
autophosphorylates a number of other targets, which can include adenylyl cyclase (through
G-protein-coupled receptors) and threonine: phosphorylated adenylyl cyclase raises levels
of cyclic adenosine monophosphate (cAMP) and thus activates protein kinase A (PKA) (Abel
and Lattal, 2001) which phosphorylates cAMP response element binding protein (CREB)
(Schafe et al, 2001). The phosphorylated CREB forms complexes with other gene
transcription factors, which bind with cAMP-dependent response element (CRE), which
mediates gene expression, to promote the creation of new transcriptionally-linked genes
and proteins (Abraham and Williams, 2003). The role of these new proteins will be discussed
further when looking at the long-term molecular events supporting LTP. Meanwhile, the
phosphorylated threonine causes AMPA receptor autophosphorylation, leading to an
increased excitatory current into the cell and therefore greater stimulation of NMDA
receptors (Schafe et al, 2001). New AMPA receptors also appear to be inserted into the
postsynaptic membrane, with the insertion associated with NMDA and CaMKII activation:
this increases the strength of the response of the postsynaptic cell to glutamate even
further (Lynch, 2004).
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Simultaneously, a retrograde messenger (possibly nitric oxide produced by binding activated

calmodulin to nitric acid synthase, or arachidonic acid) appears to provide feedback to the
presynaptic cell, stimulating guanlyl cyclase into producing cyclic guanosine monophosphate
(cGMP), which in turn increases the presynaptic output of glutamate (Abraham and
Williams, 2003). Other rapid changes brought about by Ca influx include the movement of
ribosomes into the dendritic spines in preparation for local protein synthesis, the breaking
down of adhesion molecules, and the production of synaptic tags at potentiated synapses to
allow them to capture newly-produced proteins in the dendritic spines (Abraham and
Williams, 2003).

Short-term changes in the presynaptic and postsynaptic neurons significantly increase the
efficiency of synaptic communication, making the connection between the two neurons
stronger. However, this short-term, early-phase LTP (E-LTP) cannot be sufficient for memory
formation, as memories often last for more than an hour or so (i.e. longer than the
immediate effects of the described events remain active) (Lynch, 2004). For the molecular
events forming the basis of long-phase LTP (L-LTP) the synthesis of new proteins and the
activity of the adhesion molecules must be examined.

In order for L-LTP to be reached, CREB must be phosphorylated indirectly by phosphorylated

adenylyl cyclase (Abel and Lattal, 2001). The formation of new proteins by the influence of
CREB and other basic leucine zipper (bZIP) transcription factors on the activity of CRE (and
on genes including brain-derived neurotrophic factor (BDNF) and early growth response 1)
(Schafe et al, 2001) provides proteins as either raw material or as direction for synaptic
augmentation and the creation of new dendritic spine synapses (once picked out by the
synaptic tag to ensure growth occurs only at potentiated synapses) (Abraham and Williams,
2003). These proteins provide the basis for widespread morphological changes in the
synapse to increase the contact area of the potentiated synapse, with new receptors and
enzymes arriving at the synapse following the installation of the structural proteins
(Abraham and Williams, 2003). Once the synapse has ceased to be altered proteases are
used to break down old adhesion molecules in the synapse so that newly synthesised ones
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can be inserted to replace those broken down and to fix the new morphological alterations
of the synaptic structure in place (Abraham and Williams, 2003). At this stage the
restructuring of the pre- and postsynaptic cells with the postsynaptic cell undergoing the
majority of alterations is preserved so that the strengthened synapse can continue to
function long after the initial induction of its potentiation, forming the basis for L-LTP.

The molecular events that form the basis for hippocampal LTP have been outlined and
discussed, but much of the evidence that supports the theory of LTP originates from in vitro
studies on hippocampal slices (Pike, Molden, Paulsen and Moser, 2001). In vivo evidence is
required in order to provide support for the theory of LTP and the argument that it is an
accurate model for the molecular activity that supports learning.

In vivo evidence for LTP is known to be difficult to acquire: it is almost impossible for
experimenters to track individual synapses and monitor them for potentiation when
observing the neural activity of a living animal (Pike et al, 2001). This has led research into
LTP to focus almost exclusively on synapses in layers removed from the hippocampus
(causing disruptive changes in the synapse) and artificial, electrically stimulated potentiation
provoked by high frequency stimuli (HFS): this focus has led to arguments that LTP is simply
an artificial way of persuading synapses to alter their strength rather than a natural process
(Jeffrey, 2001, p. 120). There have, however, been a number of studies that have managed
to provide results that do not necessarily prove the existence of LTP in vivo but are
consistent with the theory and support it to some extent. Charpier and Deniau (1997)
managed to demonstrate that with tetanic stimulation of the facial motor cortex of
anesthetised rats LTP could be induced in vivo, and despite their study was of striatal
synapses and the artificiality of the experiment the potentiation observed is significant in
that it demonstrates that HFS can generate LTP in vivo, even if HFS itself does not occur
naturally. The study also demonstrated that striatal LTP seems to be dependent on the
availability of calcium in the postsynaptic cell by showing that by exposing calcium to the
calcium chelator BAPTA LTP was prevented (Charpier and Deniau, 1997). Schulz, Siemer,
Krug and Hollt (1999) investigated the phosphorylation of CREB in vivo in hippocampal LTP
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and showed that CREB phosphorylation relied on NMDA receptor activation and that it only
occurred when the tetanic stimulation was sufficient to cause non-decremental LTP: their
results indicate the importance of CREB transcription factors in L-LTP and the link between
CREB phosphorylation and persistent potentiation of the hippocampal synapse.

Both studies provide important information about the importance of different elements of
LTP in in vivo brain activity, but they are both limited by their use of tetanic stimulation to
induce LTP: the criticism remains that HFS is not a realistic way of inducing LTP as it does not
appear to occur naturally. Similarly, although Messaoudi, Ying, Kanhema, Croll and
Bramham (2002) achieved results that indicate that BDNF can trigger transcriptiondependent L-LTP independently of NMDA activation in vivo the study still relied on tetanic
stimulation, undermining the usefulness of the results to in vivo LTP. A more natural
experimental method is required to provide strong evidence for LTP. Attempts have been
made to produce a natural method of stimulation for LTP experiments: Pike, Meredith,
Olding and Paulsen (1999) attempted to mimic naturally occurring theta bursts, which have
been linked to L-LTP (Lynch et al, 1988), by using lower-frequency stimuli at 5 Hz and
demonstrated that the theta bursts were able to yield synaptic potentiation when pre- and
postsynaptic activity coincided. The results of the study suggest that theta bursts may
provide a natural form of stimulation that induces LTP without the need for tetanic stimuli
and that LTP can occur naturally. However, as the experiment itself does not take place in
vivo it needs to be repeated with living subjects so that the real value of theta bursts in
providing evidence for LTP can be assessed. At present there is little evidence that LTP is
occurring in vivo, though there is evidence that it can occur in vivo if stimulated artificially
and that in this case at least some of the important molecular events of LTP appear to take
place as predicted.

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There are a number of complex molecular events that take place in order to produce LTP,
and researchers have made substantial progress in understanding these events. Elements of
LTP including NMDA activation, Ca influx and the phosphorylation of CREB have been
demonstrated by in vitro studies of LTP, primarily in hippocampal slices, and these studies
have improved the theoretical understanding of LTP as a process. Regrettably, in vivo
evidence for LTP remains scarce: there is some evidence of artificially stimulated LTP taking
place in living brains as well as hopes of finding a more natural form of experimental
stimulation in theta bursts, but actual in vivo evidence that LTP occurs naturally is rare and
appears almost impossible to obtain.

Word count: 1926.

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