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What neural mechanisms are responsible for long-term explicit memory mediated by the hippocampus and its associated structures in the medial temporal lobe of the mammalian brain? Unlike working memory, long-term storage of information by the hippocampus is not thought to depend on persistent neural firing but rather to involve long-lasting changes in the strength of synaptic connections.
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The hippocampus receives multimodal sensory and spatial information from the nearby entorhinal cortex. The major output of the hippocampus is through the pyramidal neurons in the CA1 region, which project back to the entorhinal cortex and to the subiculum, another medial temporal lobe structure. The critical importance of CA1 neurons in learning and memory is seen in the profound memory loss exhibited by patients with lesions in this region, which has been complemented by numerous studies in animal models. Information from the entorhinal cortex reaches CA1 neurons along two excitatory pathways, one direct pathway and one indirect. Together these inputs are termed the perforant pathways.
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The direct pathway has its origins in neurons of layer III of the entorhinal cortex. The axons of these neurons form synapses on the very distal apical dendrites of CA1 neurons (such perforant projections are also called the temporoammonic pathway). In the indirect pathway information from neurons of layer II of the entorhinal cortex reaches CA1 neurons through the trisynaptic pathway. In the initial leg of this pathway the axons of layer II neurons project through the perforant pathway to the granule cells of the dentate gyrus (an area considered part of the hippocampus). The granule cell axons project in the mossy fiber pathway to excite the pyramidal cells in the CA3 region of the hippocampus. Finally, the CA3 axons project through the Schaffer collateral pathway to make excitatory synapses on more proximal regions of CA1 pyramidal cell dendrites (Figure 67–2).
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The fact that CA1 pyramidal neurons receive cortical information through two pathways has led to the view that CA1 neurons compare information in the indirect circuit with sensory input from the direct pathway. Lesion studies indicate that both direct and indirect inputs to CA1 may be necessary for normal learning and memory. Lesions of the indirect Schaffer collateral pathway limit the ability of mice to perform a complex spatial learning and memory task, although some form of spatial learning remains intact. Lesions of the direct pathway to CA1 do not appear to alter initial formation of memory, but inhibit the ability of an animal to store those initial memories as long-term memory, a process termed consolidation. Genetic inactivation of the direct path also interferes with episodic memory, in which an animal must learn about the temporal relation between two or more events.
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In 1973 Timothy Bliss and Terje Lom⊘ discovered that the initial stage of the trisynaptic pathway—the perforant pathway from layer II of the entorhinal cortex to the dentate granule neurons—is remarkably sensitive to previous activity. A brief high-frequency train of stimuli (a tetanus) gives rise to long-term potentiation (LTP), a long-lasting increase in the amplitude of the excitatory postsynaptic potentials (EPSPs) in the dentate granule neurons. (In Chapter 66 we saw how a similar form of synaptic potentiation at synapses in the amygdala contributes to fear conditioning.) Subsequent studies showed that brief high-frequency trains of stimulation can induce forms of LTP at all three synapses of the trisynaptic pathway as well as at the direct perforant path synapses with CA1 neurons (Figure 67–3). Long-term potentiation can last for days or even weeks when induced in the intact animal using implanted electrodes. LTP can also be examined in slices of hippocampus and in cell culture, where it can last several hours.
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Studies in these different pathways have shown that LTP is not a single form of synaptic plasticity. Rather it comprises a family of processes that strengthen synaptic transmission at different hippocampal synapses through distinct cellular and molecular mechanisms. Indeed, even at a single synapse different forms of LTP can be induced by different patterns of synaptic activity. However, these distinct processes also share many important similarities.
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All forms of LTP are induced by synaptic activity in the pathway that is being potentiated—that is, LTP is homosynaptic. However, the various forms of LTP differ in the relative importance of different receptors and ion channels. In addition, different forms of LTP may recruit different second-messenger signaling pathways either in the presynaptic cell, altering transmitter release, or in the postsynaptic cell, altering its sensitivity to the neurotransmitter glutamate.
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The similarities and differences in the mechanisms of LTP at the Schaffer collateral, mossy fiber, and entorhinal inputs to CA1 can be seen by examining the role of the postsynaptic NMDA type of glutamate receptor in the induction of LTP in the three pathways. In all three pathways synaptic transmission is persistently enhanced in response to a brief tetanic stimulation. However, the contribution of the NMDA receptor to the induction of LTP differs in the three pathways.
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At the Schaffer collateral synapses with CA1 pyramidal neurons, the induction of LTP in response to a brief 100 Hz stimulation is completely blocked when the tetanus is applied in the presence of the NMDA receptor antagonist 2-amino-5-phosphonovaleric acid, (AP5 or APV). However, APV only partially inhibits the induction of LTP at the direct entorhinal synapses with CA1 neurons and has no effect on LTP at the mossy fiber synapses with CA3 pyramidal neurons (Figure 67–3). In the next two sections we consider the mechanisms of LTP in more detail, first in the mossy fiber pathway and then in the Schaffer collateral pathway.
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Long-Term Potentiation in the Mossy Fiber Pathway Is Nonassociative
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Glutamate released at the mossy fiber synapses binds to both the NMDA and AMPA type of glutamate receptors in the postsynaptic membrane of the CA3 neurons. However, under most conditions the NMDA receptors have only a minor role in synaptic transmission in this pathway. Moreover, as noted above, blocking these receptors has no effect on LTP (Figure 67–3C). Rather, LTP in the mossy fiber pathway is triggered by the large Ca2+ influx into the presynaptic terminals during a tetanus. In the presynaptic cell the Ca2+ influx activates a calcium/calmodulin–dependent adenylyl cyclase complex, thereby increasing the production of cAMP and activating protein kinase A. This leads to an increase in the release of glutamate from the mossy fiber terminals, resulting in LTP. Activity in the postsynaptic cell is not required for this form of LTP. Thus, mossy fiber LTP is nonassociative.
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The increase in transmitter release is thought to depend on the ability of protein kinase A to phosphorylate RIM1α, a synaptic vesicle protein that interacts with several other presynaptic proteins important for exocytosis (see Chapter 12). Thus mossy fiber LTP is abolished in mice in which the gene for RIM1α has been deleted through genetic engineering. The importance of presynaptic protein kinase A in mossy fiber LTP resembles aspects of the synaptic changes responsible for associative learning in the gill-withdrawal reflex of Aplysia and amygdala-based learned fear in rodents (see Chapter 66). Another similarity with the synaptic changes in Aplysia is that induction of mossy fiber LTP is under the control of a system of modulatory inputs. Just as the activation of adenylyl cyclase by serotonin is important for long-term facilitation in Aplysia, mossy fiber LTP is facilitated by the binding of norepinephrine to β-adrenergic receptors, enhancing the activation of adenylyl cyclase.
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Long-Term Potentiation in the Schaffer Collateral Pathway Is Associative
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Like the mossy fiber terminals in the CA3 region, glutamate released from the Schaffer collateral terminals activates both AMPA and NMDA receptors in the postsynaptic membrane of CA1 pyramidal neurons. However, unlike the mossy fiber system, LTP in the Schaffer collateral pathway requires activation of the NMDA receptors in the postsynaptic cell, which triggers a complex postsynaptic signaling cascade.
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The opening of the NMDA receptors, unlike the AMPA receptors, requires that two events occur simultaneously. First, like any ionotropic receptor, glutamate must bind to the NMDA receptor to open the channel. However, when the membrane is at the resting potential or only modestly depolarized by a weak synaptic input, glutamate binding by itself is not sufficient for the NMDA receptors to conduct ions because the pore of the receptor-channel is blocked by extracellular Mg2+ (Figure 67–4A; see Chapter 10). For the receptor to function efficiently, the postsynaptic membrane must undergo a significant depolarization to expel the bound Mg2+ by electrostatic repulsion. In this manner the receptor acts as a coincidence detector: It is functional only when action potentials in the presynaptic neuron release glutamate that binds to the receptor and the membrane potential of the postsynaptic cell is sufficiently depolarized.
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Because of the Mg2+ blockade of the NMDA receptors, at negative voltages near the resting potential EPSPs are largely generated by the opening of AMPA receptors. The burst of strong synaptic activity during induction of LTP opens a large number of AMPA receptors, generating an EPSP that is sufficient to trigger a postsynaptic action potential. The action potential generates a large depolarization that is able to expel Mg2+ from the pore of the NMDA receptor, permitting the receptor to conduct cations and contribute to the postsynaptic depolarization.
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Why are the NMDA receptors required to induce LTP if the AMPA receptors are sufficient to produce a large postsynaptic depolarization? The answer lies in the fact that in addition to conducting monovalent Na+ and K+ ions, similar to the conductance properties of the AMPA receptors, the NMDA receptors also have a high permeability to Ca2+. Thus activation of these receptors leads to a significant increase in the intracellular Ca2+ concentration in the postsynaptic cell. The Ca2+ elevation is vital to the induction of LTP; injection of a chemical chelator of Ca2+ into the postsynaptic CA1 cell blocks the induction of LTP. The increase in Ca2+ activates several downstream signaling pathways, including calcium/calmodulin–dependent protein kinase II (CaMKII), protein kinase C (PKC), and tyrosine kinases. These signaling pathways lead to changes that both enhance the response of the postsynaptic cell to glutamate and increase the amount of glutamate released from the presynaptic Schaffer collateral terminals (Figure 67–4B).
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Neuroscientists often find it useful to distinguish between the mechanisms underlying the induction of LTP (the biochemical reactions activated by the tetanic stimulation) and those responsible for the expression of LTP (the long-term changes that take place at the synapse responsible for enhanced synaptic transmission). The mechanisms for the induction of LTP at the CA3-CA1 synapse are postsynaptic. What are the mechanisms involved in the expression of LTP at this synapse? Is the enhancement caused by an increase in transmitter release, an increased postsynaptic response to a fixed amount of transmitter, or some combination of the two?
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Recent studies suggest that the cellular mechanisms underlying the expression of LTP vary depending on the precise pattern of activity that induces LTP. In many cases LTP that is induced solely by Ca2+ influx through NMDA receptors appears to be largely caused by an increase in the response of the postsynaptic membrane of the CA1 neuron to glutamate. But other patterns of stimulation elicit other forms of LTP at the same synapse and these also have presynaptic effects that enhance transmitter release.
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One of the key pieces of evidence for a postsynaptic contribution to the expression of LTP at Schaffer collateral synapses comes from an examination of so-called "silent synapses" (Figure 67–5). In some recordings from pairs of hippocampal pyramidal neurons, stimulation of an action potential in one neuron fails to elicit a synaptic response in a second (postsynaptic) neuron when that neuron is at its resting potential (approximately –70 mV).
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This result is not surprising as any given hippo-campal presynaptic neuron is connected to only a small fraction of other neurons. However, what is surprising is that, in some neuronal pairs, when the second neuron is depolarized under voltage clamp to +30 mV, which removes the Mg2+ block from the NMDA receptors, stimulation of the presynaptic neuron elicits a large excitatory postsynaptic current (EPSC) in the postsynaptic neuron, mediated by the NMDA receptors. This result indicates that the two neurons were synaptically connected all along but the postsynaptic neuron contained only NMDA receptors at its synaptic contact with the presynaptic neuron. These connections are called silent synapses because they do not generate an EPSP at the normal resting potential of the cell as a result of the Mg2+ block of the NMDA receptors. Synapses from other presynaptic neurons on the same postsynaptic cell may have AMPA receptors in addition to NMDA receptors (nonsilent synapses).
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The key finding from these experiments is seen following the induction of LTP. Pairs of neurons initially connected solely by silent synapses now often exhibit large EPSPs at the resting potential mediated by AMPA receptors. These results indicate that LTP must involve an increase in the response of AMPA receptors to glutamate at the previously silent synapses, a process Roberto Malinow refers to as "AMPAfication."
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How does the induction of LTP increase the response of AMPA receptors at previously silent synapses? The strong synaptic stimulation used to induce LTP will trigger glutamate release at both silent and nonsilent synapses on the same postsynaptic neuron. This leads to the opening of a large number of AMPA receptors at the nonsilent synapses, which in turn produces a large postsynaptic depolarization. The depolarization will propagate throughout the neuron to relieve Mg2+ block of the NMDA receptors at both the nonsilent and silent synapses. At the silent synapses the Ca2+ influx through the NMDA receptors activates a biochemical cascade that ultimately leads to the insertion of clusters of AMPA receptors in the postsynaptic membrane from a pool of intracellular receptors stored in recycling endosomal vesicles. The fusion of these vesicles with the plasma membrane is triggered by the phosphorylation by protein kinase C of the cytoplasmic tail of the endosomal AMPA receptors (Figure 67–4B,C).
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As discussed earlier, LTP is not a unitary process even at a single synapse. At Schaffer collateral synapses LTP generated by a brief 100 Hz tetanus depends solely on Ca2+ influx through NMDA receptors, whereas LTP induced by a 200 Hz tetanus depends on Ca2+ influx through both NMDA receptors and L-type voltage-gated Ca2+ channels. (A similar mechanism contributes to LTP in the direct entorhinal pathway to CA1 neurons.) This high-frequency form of LTP is expressed both through presynaptic mechanisms that enhance glutamate release and through postsynaptic mechanisms that increase the membrane response to glutamate. Thus both the induction and expression of LTP depend on a family of presynaptic and postsynaptic processes.
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Because induction of LTP requires Ca2+ influx into the postsynaptic cell, the increase in transmitter release during LTP implies that the presynaptic cell must receive information from the postsynaptic cell that LTP has been induced. There is now evidence that Ca2+ -activated second messengers in the postsynaptic cell, or perhaps Ca2+ itself, cause the postsynaptic cell to release one or more chemical messengers that diffuse to the presynaptic terminals to enhance release (see Figure 67–4B,C and Chapter 11). Importantly, these diffusible retrograde signals appear to affect only those presynaptic terminals that have been activated by the tetanic stimulation, thereby preserving synapse specificity.
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Long-Term Potentiation in the Schaffer Collateral Pathway Follows Hebbian Learning Rules
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The NMDA receptors endow LTP in the Schaffer collateral pathway with several interesting properties that have direct relevance to learning and memory. First, LTP in this pathway requires the near simultaneous activation of a large number of afferent axons, a feature called cooperativity (Figure 67–6). This requirement stems from the fact that relief of Mg2+ block of the NMDA receptor requires a large depolarization.
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The second important property of LTP in the Schaffer collateral pathway is that it is associative. A weak synaptic input normally does not produce enough postsynaptic depolarization to induce LTP. However, if that weak input is coactivated or paired with a strong synaptic input that does produce suprathreshold depolarization, then the large depolarization will be able to propagate to the synapse with weak input, leading to relief of the Mg2+ blockade of the NMDA receptors in the postsynaptic membrane at that site and the induction of LTP.
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The third key property of LTP is that it is synapse specific. If a particular synapse is not activated during a period of strong synaptic stimulation, the NMDA receptors at that site will not be able to bind glutamate and thus will not be activated despite the strong postsynaptic depolarization. As a result, that synapse will not undergo LTP.
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Each of these three properties of cooperativity, associativity, and synapse specificity underlies key components of memory storage. Cooperativity ensures that only events of a high degree of significance, those that activate sufficient inputs, will result in memory storage. Associativity, like associative Pavlovian conditioning, allows an event (or conditioned stimulus) that has little significance in and of itself to be endowed with a higher degree of meaning if that event occurs just before or simultaneously with another more significant event (an unconditioned stimulus). Finally, synapse specificity ensures that inputs that convey information not related to a particular event will not be strengthened to participate in a given memory.
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The finding that the induction of LTP in the Schaffer collateral pathway requires that presynaptic activity be strong enough to elicit firing in the postsynaptic neuron provides evidence for Hebb's rule, proposed in 1949 by the psychologist Donald Hebb as a theoretical mechanism for how neuronal circuits are modified by experience: "When an axon of cell A … excites cell B and repeatedly or persistently takes part in firing it, some growth process or metabolic change takes place in one or both cells so that A's efficiency as one of the cells firing B is increased." A similar principle is involved in fine-tuning synaptic connections during the late stages of development (see Chapter 56).
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The Hebbian nature of LTP is best illustrated by the phenomenon of spike timing-dependent plasticity. Under most circumstances hippocampal neurons do not produce the high-frequency trains of action potentials typically used to induce LTP. However, a form of LTP can be induced by pairing a single presynaptic stimulus with the firing of a single action potential in the postsynaptic cell. In fact, this coincidence detection is very strict. In agreement with Hebb's postulate, the pairing protocol produces LTP only if the postsynaptic cell fires a few milliseconds after the EPSP. That is, the presynaptic cell must fire before the postsynaptic cell. If the postsynaptic cell fires just before the EPSP, a long-lasting decrease in the size of the EPSP occurs (this long-term depression is described more fully below.) If the action potential occurs more than a hundred milliseconds before or after the EPSP, the synaptic strength will not change.
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The pairing rules of spike timing-dependent plasticity result in large part from the cooperative properties of the NMDA receptor. If the postsynaptic spike occurs during the EPSP, the spike is able to relieve the Mg2+ blockade of the receptor at a time when the NMDA receptor-channel has been activated by the binding of glutamate. This leads to a large influx of Ca2+ through the receptor and the induction of LTP. However, if the postsynaptic action potential occurs prior to glutamate release, any relief from the Mg2+ block will occur when the gate of the receptor-channel is closed, because of the absence of glutamate. As a result there will be little influx of Ca2+ through the receptor to induce LTP.
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These studies of the Schaffer collateral pathway indicate that two sequential associative mechanisms ensure that the induction of LTP is restricted to those synapses at which there is both presynaptic and postsynaptic activity, in accord with Hebb's learning rule. The first mechanism is the associative property of the NMDA glutamate receptor. The second is the selective action of retrograde messengers released from the postsynaptic cell at only those presynaptic sites that are active. As we saw in Chapter 66, these two associative mechanisms in series also contribute to associative classical conditioning in Aplysia and in the amygdala. Thus mechanisms of synaptic plasticity important for learning and memory have been conserved throughout evolution of the species at broad classes of synapses and for distinct forms of learning.
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Long-Term Potentiation Has Early and Late Phases
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Long-term potentiation has two phases. One train of action potentials produces a phase of LTP lasting 1 to 3 hours called early LTP. This component, which is the phase we have been considering up to now, does not require new protein synthesis, cAMP, or PKA activation. However, four or more trains of synaptic stimulation induce a late LTP that lasts up to 24 hours; this late LTP does require cAMP and PKA, as well as changes in gene transcription and the synthesis of new proteins (Figure 67–7).
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Although the mechanisms for early LTP in the Schaffer collateral and mossy fiber pathways are quite different, the mechanisms for late LTP in the two pathways appear similar. In both pathways late LTP recruits the cAMP and PKA signaling pathway, which recruits the cAMP response element binding protein (CREB) transcription factor, leading to the synthesis of new mRNAs and proteins.
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How do the synaptic mechanisms for the expression of early and late LTP differ? Quantal analysis (see Chapter 12) was used to examine synaptic transmission between a single presynaptic CA3 neuron and a single postsynaptic CA1 cell (Figure 67–8).
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Prior to LTP a CA3 neuron typically forms only one functional synapse with a CA1 neuron. At this synapse a presynaptic action potential releases with low probability a single vesicle of transmitter. This weak connection between a single CA3 and single CA1 neuron means that a large number of CA3 neurons must be co-activated to trigger a spike in the postsynaptic CA1 cell. Following induction of early LTP, the probability that a presynaptic action potential will release a vesicle is increased (Figure 67–8C).
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Induction of the late phase of LTP by direct application of a chemical analog of cAMP dramatically changes the response to synaptic stimulation. Under these conditions a presynaptic action potential elicits a very large EPSP through the release of multiple quanta of transmitter (Figure 67–8D). Because each release site (active zone) in the presynaptic terminal is thought to release at most one vesicle in an all-or-none fashion, the increase in the number of quanta indicates that late LTP recruits new presynaptic release sites apposed to new clusters of AMPA receptors in the postsynaptic membrane. Moreover, the formation of new synapses requires new protein synthesis, consistent with the idea that late LTP involves a growth process. Light microscopic imaging studies of live neurons in hippocampal slices provide direct evidence that LTP induces the formation of new dendritic spines, the sites of new excitatory synaptic input.
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Like sensitization of the gill-withdrawal reflex in Aplysia, late LTP in the Schaffer collateral pathway is synapse specific. When two independent sets of synapses in the same postsynaptic CA1 neuron are stimulated using two electrodes spaced some distance apart, the application of four trains of tetanic stimulation to one set of synapses induces late LTP only at the activated synapses; synaptic transmission at the second set of nonstimulated synapses is not altered. However, Uwe Frey and Richard Morris found that if a single tetanus is applied to the second set of synapses soon after the four tetani are applied to the first set, the single train is able to induce late LTP at the synapses it activates. This phenomenon is similar to the synapse-specific capture of long-term facilitation at the sensory-motor neuron synapses in Aplysia (see Chapter 66). At the Schaffer collateral synapses the single tetanus somehow marks the activated synapses allowing them to respond to, or capture, the new proteins synthesized in response to signals from the synapses that received the four tetani.
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How can a few brief trains of synaptic stimulation produce such long-lasting increases in synaptic transmission? Studies from Todd Sacktor have shown that the maintenance of late LTP depends on a novel isoform of protein kinase C termed PKMζ (PKM zeta). Most isoforms of PKC contain both a regulatory domain and a catalytic domain (see Chapter 11). Binding of diacylglycerol, phospholipids, and Ca2+ to the regulatory domain of PKC relieves its inhibitory binding to the catalytic domain, which is then free to phosphorylate its protein substrates. In contrast, PKMζ lacks a regulatory domain and so is constitutively active.
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Levels of PKMζ in the hippocampus are normally low. Tetanic stimulation that induces LTP leads to an increase in synthesis of PKMζ through enhanced translation of its mRNA. This mRNA is present in the CA1 neuron dendrites, enabling its local translation to rapidly alter synaptic strength. Blockade of PKMζ with a specific inhibitor does not block early LTP but does block late LTP. Moreover, application of the PKMζ blocker several hours after the LTP induction protocol can reverse late LTP after it has been established. This result indicates that the maintenance of late LTP requires the persistent and ongoing activity of PKMζ, which leads to the persistent increase in insertion of AMPA receptors in the postsynaptic membrane (Figure 67–9).
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