Interaction with the NMDA receptor locks CaMKII in an active conformation.

Calcium- and calmodulin-dependent protein kinase II (CaMKII) and glutamate receptors are integrally involved in forms of synaptic plasticity that may underlie learning and memory. In the simplest model for long-term potentiation, CaMKII is activated by Ca2+ influx through NMDA (N-methyl-D-aspartate) receptors and then potentiates synaptic efficacy by inducing synaptic insertion and increased single-channel conductance of AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid) receptors. Here we show that regulated CaMKII interaction with two sites on the NMDA receptor subunit NR2B provides a mechanism for the glutamate-induced translocation of the kinase to the synapse in hippocampal neurons. This interaction can lead to additional forms of potentiation by: facilitated CaMKII response to synaptic Ca2+; suppression of inhibitory autophosphorylation of CaMKII; and, most notably, direct generation of sustained Ca2+/calmodulin (CaM)-independent (autonomous) kinase activity by a mechanism that is independent of the phosphorylation state. Furthermore, the interaction leads to trapping of CaM that may reduce down-regulation of NMDA receptor activity. CaMKII-NR2B interaction may be prototypical for direct activation of a kinase by its targeting protein.     

An essential role for postsynaptic calmodulin and protein kinase activity in long-term potentiation

The phenomenon of long-term potentiation (LTP), a long lasting increase in the strength of synaptic transmission which is due to brief, repetitive activation of excitatory afferent fibres, is one of the most striking examples of synaptic plasticity in the mammalian brain. In the CA1 region of the hippocampus, the induction of LTP requires activation of NMDA (N-methyl-D-aspartate) receptors by synaptically released glutamate with concomitant postsynaptic membrane depolarization. This relieves the voltage-dependent magnesium block of the NMDA-receptor ion channel, allowing calcium to flow into the dendritic spine. Although calcium has been shown to be a necessary trigger for LTP (refs 11, 12), little is known about the immediate biochemical processes that are activated by calcium and are responsible for LTP. The most attractive candidates have been calcium/calmodulin-dependent protein kinase II (CaM-KII) (refs 13-16), protein kinase C (refs 17-19), and the calcium-dependent protease, calpain. Extracellular application of protein kinase inhibitors to the hippocampal slice preparation blocks the induction of LTP (refs 21-23) but it is unclear whether this is due to a pre- and/or postsynaptic action. We have found that intracellular injection into CA1 pyramidal cells of the protein kinase inhibitor H-7, or of the calmodulin antagonist calmidazolium, blocks LTP. Furthermore, LTP is blocked by the injection of synthetic peptides that are potent calmodulin antagonists and inhibit CaM-KII auto- and substrate phosphorylation. These findings demonstrate that in the postsynaptic cell both activation of calmodulin and kinase activity are required for the generation of LTP, and focus further attention on the potential role of CaM-KII in LTP.     

Regulation of distinct AMPA receptor phosphorylation sites during bidirectional synaptic plasticity

Bidirectional changes in the efficacy of neuronal synaptic transmission, such as hippocampal long-term potentiation (LTP) and long-term depression (LTD), are thought to be mechanisms for information storage in the brain. LTP and LTD may be mediated by the modulation of AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazloe proprionic acid) receptor phosphorylation. Here we show that LTP and LTD reversibly modify the phosphorylation of the AMPA receptor GluR1 subunit. However, contrary to the hypothesis that LTP and LTD are the functional inverse of each other, we find that they are associated with phosphorylation and dephosphorylation, respectively, of distinct GluR1 phosphorylation sites. Moreover, the site modulated depends on the stimulation history of the synapse. LTD induction in naive synapses dephosphorylates the major cyclic-AMP-dependent protein kinase (PKA) site, whereas in potentiated synapses the major calcium/calmodulin-dependent protein kinase II (CaMKII) site is dephosphorylated. Conversely, LTP induction in naive synapses and depressed synapses increases phosphorylation of the CaMKII site and the PKA site, respectively. LTP is differentially sensitive to CaMKII and PKA inhibitors depending on the history of the synapse. These results indicate that AMPA receptor phosphorylation is critical for synaptic plasticity, and that identical stimulation conditions recruit different signal-transduction pathways depending on synaptic history.     

Potentiation of synaptic transmission in the hippocampus by phorbol esters

Protein kinase C (PKC), a calcium-dependent phospholipid-sensitive kinase which is selectively activated by phorbol esters, is thought to play an important role in several cellular processes. In mammalian brain PKC is present in high concentrations and has been shown to phosphorylate several substrate phosphoproteins, one of which may be involved in the generation of long-term potentiation (LTP), a long-lasting increase in synaptic efficacy evoked by brief, high-frequency stimulation. Since the hippocampus contains one of the brain's highest levels of binding sites for phorbol esters and is the site where LTP has been most thoroughly characterized, we examined the effects of phorbol esters on hippocampal synaptic transmission and LTP. We found that phorbol esters profoundly potentiate excitatory synaptic transmission in the hippocampus in a manner that appears indistinguishable from LTP. Furthermore, after maximal synaptic enhancement by phorbol esters, LTP can no longer be elicited. Although the site of synaptic enhancement during LTP is not clearly established, phorbol esters appear to potentiate synaptic transmission by acting primarily at a presynaptic locus since changes in the postsynaptic responses to the putative transmitter, glutamate, cannot account for the increased synaptic responses induced by phorbol esters. These findings, in conjunction with previous biochemical studies, raise the possibility that, in mammalian brain, PKC plays a role in controlling the release of neurotransmitter and may be involved in the generation of LTP.     

Protein kinase C injection into hippocampal pyramidal cells elicits features of long term potentiation

Long-term potentiation (LTP) in the hippocampus is an interesting example of synaptic plasticity because of its induction by physiological discharge rates and its long duration. Of the possible biochemical mechanisms that regulate prolonged changes in cell function, protein phosphorylation is a particularly attractive candidate. We have therefore examined the effect of intracellular injection of calcium/diacylglycerol-dependent protein kinase (protein kinase C (PKC] in CA1 pyramidal neurones in hippocampal slices. Injection of the active enzyme elicited long-lasting enhancement of synaptic transmission, similar to LTP, whereas inactivated kinase failed to do so. The observed changes included an increased amplitude of the excitatory post-synaptic potential (e.p.s.p.) and an increased probability of firing and a reduced latency of the associated actin potential.     

Long-term potentiation in the hippocampus is blocked by tyrosine kinase inhibitors.

Long-term potentiation (LTP) in the hippocampus is thought to contribute to memory formation. In the Ca1 region, LTP requires the NMDA (N-methyl-D-aspartate) receptor-dependent influx of Ca2+ and activation of serine and threonine protein kinases. Because of the high amount of protein tyrosine kinases in hippocampus and cerebellum, two regions implicated in learning and memory, we examined the possible additional requirement of tyrosine kinase activity in LTP. We first examined the specificity in brain of five inhibitors of tyrosine kinase and found that two of them, lavendustin A and genistein, showed substantially greater specificity for tyrosine kinase from hippocampus than for three serine-threonine kinases: protein kinase A, protein kinase C, and Ca2+/calmodulin kinase II. Lavendustin A and genistein selectively blocked the induction of LTP when applied in the bath or injected into the postsynaptic cell. By contrast, the inhibitors had no effect on the established LTP, on normal synaptic transmission, or on the neurotransmitter actions attributable to the actions of protein kinase A or protein kinase C. These data suggest that tyrosine kinase activity could be required postsynaptically for long-term synaptic plasticity in the hippocampus. As Ca2+ calmodulin kinase II or protein kinase C seem also to be required, the tyrosine kinases could participate postsynaptically in a kinase network together with serine and threonine kinases.     

Presynaptic induction of heterosynaptic associative plasticity in the mammalian brain

The induction of associative synaptic plasticity in the mammalian central nervous system classically depends on coincident presynaptic and postsynaptic activity. According to this principle, associative homosynaptic long-term potentiation (LTP) of excitatory synaptic transmission can be induced only if synaptic release occurs during postsynaptic depolarization. In contrast, heterosynaptic plasticity in mammals is considered to rely on activity-independent, non-associative processes. Here we describe a novel mechanism underlying the induction of associative LTP in the lateral amygdala (LA). Simultaneous activation of converging cortical and thalamic afferents specifically induced associative, N-methyl-D-aspartate (NMDA)-receptor-dependent LTP at cortical, but not at thalamic, inputs. Surprisingly, the induction of associative LTP at cortical inputs was completely independent of postsynaptic activity, including depolarization, postsynaptic NMDA receptor activation or an increase in postsynaptic Ca2+ concentration, and did not require network activity. LTP expression was mediated by a persistent increase in the presynaptic probability of release at cortical afferents. Our study shows the presynaptic induction and expression of heterosynaptic and associative synaptic plasticity on simultaneous activity of converging afferents. Our data indicate that input specificity of associative LTP can be determined exclusively by presynaptic properties.     

RIM1alpha is required for presynaptic long-term potentiation.

Two main forms of long-term potentiation (LTP)-a prominent model for the cellular mechanism of learning and memory-have been distinguished in the mammalian brain. One requires activation of postsynaptic NMDA (N-methyl d-aspartate) receptors, whereas the other, called mossy fibre LTP, has a principal presynaptic component. Mossy fibre LTP is expressed in hippocampal mossy fibre synapses, cerebellar parallel fibre synapses and corticothalamic synapses, where it apparently operates by a mechanism that requires activation of protein kinase A. Thus, presynaptic substrates of protein kinase A are probably essential in mediating this form of long-term synaptic plasticity. Studies of knockout mice have shown that the synaptic vesicle protein Rab3A is required for mossy fibre LTP, but the protein kinase A substrates rabphilin, synapsin I and synapsin II are dispensable. Here we report that mossy fibre LTP in the hippocampus and the cerebellum is abolished in mice lacking RIM1alpha, an active zone protein that binds to Rab3A and that is also a protein kinase A substrate. Our results indicate that the long-term increase in neurotransmitter release during mossy fibre LTP may be mediated by a unitary mechanism that involves the GTP-dependent interaction of Rab3A with RIM1alpha at the interface of synaptic vesicles and the active zone.     

Presynaptic enhancement shown by whole-cell recordings of long-term potentiation in hippocampal slices

Long-term potentiation (LTP) of synaptic transmission in the hippocampus is a widely studied model system for understanding the cellular mechanisms of memory. In region CA1, LTP is triggered postsynaptically by Ca2(+)-dependent activation of protein kinases, but the locus of persistent modification remains controversial. Statistical analysis of synaptic variability has been proposed as a means of settling this debate, although a major obstacle has been the poor signal-to-noise ratio of conventional intracellular recordings. We have applied the whole-cell voltage clamp technique to study synaptic transmission in conventional hippocampal slices (compare refs 28-30). Here we report that robust LTP can be recorded with much improved signal resolution and biochemical access to the postsynaptic cell. Prolonged dialysis of the postsynaptic cell blocks the triggering of LTP, with no effect on expression of LTP. The improved signal resolution unmasks a large trial-to-trial variability, reflecting the probabilistic nature of transmitter release. Changes in the synaptic variability, and a decrease in the proportion of synaptic failures during LTP, suggest that transmitter release is significantly enhanced.     

Calcium stores regulate the polarity and input specificity of synaptic modification

Activity-induced synaptic modification is essential for the development and plasticity of the nervous system. Repetitive correlated activation of pre- and postsynaptic neurons can induce persistent enhancement or decrement of synaptic efficacy, commonly referred to as long-term potentiation or depression (LTP or LTD). An important unresolved issue is whether and to what extent LTP and LTD are restricted to the activated synapses. Here we show that, in the CA1 region of the hippocampus, reduction of postsynaptic calcium influx by partial blockade of NMDA (N-methyl-D-aspartate) receptors results in a conversion of LTP to LTD and a loss of input specificity normally associated with LTP, with LTD appearing at heterosynaptic inputs. The induction of LTD at homo- and heterosynaptic sites requires functional ryanodine receptors and inositol triphosphate (InsP3) receptors, respectively. Functional blockade or genetic deletion of type 1 InsP3 receptors led to a conversion of LTD to LTP and elimination of heterosynaptic LTD, whereas blocking ryanodine receptors eliminated only homosynaptic LTD. Thus, postsynaptic Ca2+, deriving from Ca2+ influx and differential release of Ca2+ from internal stores through ryanodine and InsP3 receptors, regulates both the polarity and input specificity of activity-induced synaptic modification.     

Postsynaptic NMDA receptor-mediated calcium accumulation in hippocampal CA1 pyramidal cell dendrites

In the CA1 hippocampal region, intracellular calcium is a putative second messenger for the induction of long-term potentiation (LTP), a persistent increase of synaptic transmission produced by high frequency afferent fibre stimulation. Because LTP in this region is blocked by the NMDA (N-methyl-D-aspartate) receptor antagonist AP5 (DL-2-amino-5-phosphonovaleric acid) and the calcium permeability of NMDA receptors is controlled by a voltage-dependent magnesium block, a model has emerged that suggests that the calcium permeability of NMDA receptor-coupled ion channels is the biophysical basis for LTP induction. We have performed microfluorometric measurements in individual CA1 pyramidal cells during stimulus trains that induce LTP. In addition to a widespread component of postsynaptic calcium accumulation previously described, we now report that brief high frequency stimulus trains produce a transient component spatially localized to dendritic areas near activated afferents. This localized component is blocked by the NMDA receptor antagonist AP5. The results directly confirm the calcium rise predicted by NMDA receptor models of LTP induction.     

LTP promotes formation of multiple spine synapses between a single axon terminal and a dendrite

Structural remodelling of synapses and formation of new synaptic contacts has been postulated as a possible mechanism underlying the late phase of long-term potentiation (LTP), a form of plasticity which is involved in learning and memory. Here we use electron microscopy to analyse the morphology of synapses activated by high-frequency stimulation and identified by accumulated calcium in dendritic spines. LTP induction resulted in a sequence of morphological changes consisting of a transient remodelling of the postsynaptic membrane followed by a marked increase in the proportion of axon terminals contacting two or more dendritic spines. Three-dimensional reconstruction revealed that these spines arose from the same dendrite. As pharmacological blockade of LTP prevented these morphological changes, we conclude that LTP is associated with the formation of new, mature and probably functional synapses contacting the same presynaptic terminal and thereby duplicating activated synapses.     

Temporally distinct pre- and post-synaptic mechanisms maintain long-term potentiation

Long-term potentiation (LTP) in the hippocampus is widely studied as the mechanisms involved in its induction and maintenance are believed to underlie fundamental properties of learning and memory in vertebrates. Most synapses that exhibit LTP use an excitatory amino-acid neurotransmitter that acts on two types of receptor, the N-methyl-D-aspartate (NMDA) and quisqualate receptors. The quisqualate receptor mediates the fast synaptic response evoked by low-frequency stimulation, whereas the NMDA receptor system is activated transiently by tetanic stimulation, leading to the induction of LTP. The events responsible for maintaining LTP once it is established are not known. We now demonstrate that the sensitivity of CA1 neurons in hippocampal slices to ionophoretically-applied quisqualate receptor ligands slowly increases following the induction of LTP. This provides direct evidence for a functional post-synaptic change and suggests that pre-synaptic mechanisms also contribute, but in a temporally distinct manner, to the maintenance of LTP.     

Local, persistent activation of Rho GTPases during plasticity of single dendritic spines

The Rho family of GTPases have important roles in the morphogenesis of the dendritic spines of neurons in the brain and synaptic plasticity by modulating the organization of the actin cytoskeleton. Here we used two-photon fluorescence lifetime imaging microscopy to monitor the activity of two Rho GTPases-RhoA and Cdc42-in single dendritic spines undergoing structural plasticity associated with long-term potentiation in CA1 pyramidal neurons in cultured slices of rat hippocampus. When long-term volume increase was induced in a single spine using two-photon glutamate uncaging, RhoA and Cdc42 were rapidly activated in the stimulated spine. These activities decayed over about five minutes, and were then followed by a phase of persistent activation lasting more than half an hour. Although active RhoA and Cdc42 were similarly mobile, their activity patterns were different. RhoA activation diffused out of the stimulated spine and spread over about 5 μm along the dendrite. In contrast, Cdc42 activation was restricted to the stimulated spine, and exhibited a steep gradient at the spine necks. Inhibition of the Rho-Rock pathway preferentially inhibited the initial spine growth, whereas the inhibition of the Cdc42-Pak pathway blocked the maintenance of sustained structural plasticity. RhoA and Cdc42 activation depended on Ca(2+)/calmodulin-dependent kinase (CaMKII). Thus, RhoA and Cdc42 relay transient CaMKII activation to synapse-specific, long-term signalling required for spine structural plasticity.     

Repeated cocaine exposure in vivo facilitates LTP induction in midbrain dopamine neurons

Drugs of abuse are known to cause persistent modification of neural circuits, leading to addictive behaviours. Changes in synaptic plasticity in dopamine neurons of the ventral tegmental area (VTA) may contribute to circuit modification induced by many drugs of abuse, including cocaine. Here we report that, following repeated exposure to cocaine in vivo, excitatory synapses to rat VTA dopamine neurons become highly susceptible to the induction of long-term potentiation (LTP) by correlated pre- and postsynaptic activity. This facilitated LTP induction is caused by cocaine-induced reduction of GABA(A) (gamma-aminobutyric acid) receptor-mediated inhibition of these dopamine neurons. In midbrain slices from rats treated with saline or a single dose of cocaine, LTP could not be induced in VTA dopamine neurons unless GABA-mediated inhibition was reduced by bicuculline or picrotoxin. However, LTP became readily inducible in slices from rats treated repeatedly with cocaine; this LTP induction was prevented by enhancing GABA-mediated inhibition using diazepam. Furthermore, repeated cocaine exposure reduced the amplitude of GABA-mediated synaptic currents and increased the probability of spike initiation in VTA dopamine neurons. This cocaine-induced enhancement of synaptic plasticity in the VTA may be important for the formation of drug-associated memory.     

Synaptic scaling mediated by glial TNF-alpha

Two general forms of synaptic plasticity that operate on different timescales are thought to contribute to the activity-dependent refinement of neural circuitry during development: (1) long-term potentiation (LTP) and long-term depression (LTD), which involve rapid adjustments in the strengths of individual synapses in response to specific patterns of correlated synaptic activity, and (2) homeostatic synaptic scaling, which entails uniform adjustments in the strength of all synapses on a cell in response to prolonged changes in the cell's electrical activity. Without homeostatic synaptic scaling, neural networks can become unstable and perform suboptimally. Although much is known about the mechanisms underlying LTP and LTD, little is known about the mechanisms responsible for synaptic scaling except that such scaling is due, at least in part, to alterations in receptor content at synapses. Here we show that synaptic scaling in response to prolonged blockade of activity is mediated by the pro-inflammatory cytokine tumour-necrosis factor-alpha (TNF-alpha). Using mixtures of wild-type and TNF-alpha-deficient neurons and glia, we also show that glia are the source of the TNF-alpha that is required for this form of synaptic scaling. We suggest that by modulating TNF-alpha levels, glia actively participate in the homeostatic activity-dependent regulation of synaptic connectivity.     

Arachidonic acid induces a long-term activity-dependent enhancement of synaptic transmission in the hippocampus

Long-term potentiation (LTP) is a widely studied model of the synaptic basis of information storage in the mammalian brain. The induction of LTP is triggered by the postsynaptic entry of calcium through the channel associated with the N-methyl-D-aspartate (NMDA) receptor, whereas its maintenance is mediated, at least in part, by presynaptic mechanisms. To explain how postsynaptic events can lead to an increase in transmitter release, we have postulated the existence of a retrograde messenger to carry information from the postsynaptic side of the synapse to recently active presynaptic terminals. Candidates for a retrograde messenger include arachidonic acid or one of its lipoxygenase metabolites. Here we report that weak activation of the perforant path, when given in the presence of arachidonic acid, leads to a slow-onset persistent increase in synaptic efficacy both in vivo and in vitro. The activity-dependent potentiation thus produced is accompanied by an increase in the release of glutamate, and is non-additive with tetanus-induced LTP. These observations indicate a role for arachidonic acid as a retrograde messenger in the later, but not the initial, stages of LTP.     

Two components of long-term potentiation induced by different patterns of afferent activation

Long-term potentiation (LTP) of excitatory synaptic transmission could be a mechanism underlying memory. Induction of LTP requires Ca2+ influx into postsynaptic neurons through ion channels gated by NMDA (N-methyl-D-aspartate) receptors in hippocampus (area CA1 and dentate gyrus) and neocortex. Here we report that a component of LTP not requiring the activation of NMDA receptors can be induced in area CA1. The component is dependent on tetanus frequency, requires increases in postsynaptic intracellular Ca2+ concentrations, and is suppressed by an antagonist of voltage-dependent Ca2+ channels.     

Opioids block long-term potentiation of inhibitory synapses

Excitatory brain synapses are strengthened or weakened in response to specific patterns of synaptic activation, and these changes in synaptic strength are thought to underlie persistent pathologies such as drug addiction, as well as learning. In contrast, there are few examples of synaptic plasticity of inhibitory GABA (gamma-aminobutyric acid)-releasing synapses. Here we report long-term potentiation of GABA(A)-mediated synaptic transmission (LTP(GABA)) onto dopamine neurons of the rat brain ventral tegmental area, a region required for the development of drug addiction. This novel form of LTP is heterosynaptic, requiring postsynaptic NMDA (N-methyl-d-aspartate) receptor activation at glutamate synapses, but resulting from increased GABA release at neighbouring inhibitory nerve terminals. NMDA receptor activation produces nitric oxide, a retrograde signal released from the postsynaptic dopamine neuron. Nitric oxide initiates LTP(GABA) by activating guanylate cyclase in GABA-releasing nerve terminals. Exposure to morphine both in vitro and in vivo prevents LTP(GABA). Whereas brief treatment with morphine in vitro blocks LTP(GABA) by inhibiting presynaptic glutamate release, in vivo exposure to morphine persistently interrupts signalling from nitric oxide to guanylate cyclase. These neuroadaptations to opioid drugs might contribute to early stages of addiction, and may potentially be exploited therapeutically using drugs targeting GABA(A) receptors.