Synapses In Area CA1 Research Articles

A C-terminal peptide from secreted amyloid precursor protein-α enhances long-term potentiation in rats and a transgenic mouse model of Alzheimer's disease

Processing of the amyloid precursor protein by alternative secretases results in ectodomain shedding of either secreted amyloid precursor protein-α (sAPPα) or its counterpart secreted amyloid precursor protein-β (sAPPβ). Although sAPPα contains only 16 additional amino acids at its C-terminus compared to sAPPβ, it displays significantly greater potency in neuroprotection, neurotrophism and enhancement of long-term potentiation (LTP). In the current study, this 16 amino acid peptide sequence (CTα16) was characterised for its ability to replicate the synaptic plasticity-enhancing properties of sAPPα. An N-acetylated version of CTα16 produced concentration-dependent increases in the induction and persistence of LTP at Schaffer collateral/commissural synapses in area CA1 of young adult rat hippocampal slices. A scrambled peptide had no effect. CTα16 significantly enhanced de novo protein synthesis, and correspondingly its enhancement of LTP was blocked by the protein synthesis inhibitor cycloheximide, as well as by the α7-nicotinic receptor blocker α-bungarotoxin. The impaired LTP of 14–16 month old APPswe/PS1dE9 transgenic mice, a mouse model of Alzheimer's disease, was completely restored to the wild-type level by CTα16. These results indicate that the CTα16 peptide fragment of sAPPα mimics the larger protein's functionality with respect to LTP, stimulation of protein synthesis and activation of α7-nAChRs, and thus like sAPPα may have potential as a therapeutic agent against the plasticity and cognitive deficits observed in AD and other neurological disorders.

Activation of Metabotropic Glutamate Receptor 7 Is Required for Induction of Long-Term Potentiation at SC-CA1 Synapses in the Hippocampus.

Of the eight metabotropic glutamate (mGlu) receptor subtypes, only mGlu7 is expressed presynaptically at the Schaffer collateral (SC)-CA1 synapse in the hippocampus in adult animals. Coupled with the inhibitory effects of Group III mGlu receptor agonists on transmission at this synapse, mGlu7 is thought to be the predominant autoreceptor responsible for regulating glutamate release at SC terminals. However, the lack of mGlu7-selective pharmacological tools has hampered direct testing of this hypothesis. We used a novel, selective mGlu7-negative allosteric modulator (NAM), ADX71743, and a newly described Group III mGlu receptor agonist, LSP4-2022, to elucidate the role of mGlu7 in modulating transmission in hippocampal area CA1 in adult C57BL/6J male mice. Interestingly, although mGlu7 agonists inhibit SC-CA1 EPSPs, we found no evidence for activation of mGlu7 by stimulation of SC-CA1 afferents. However, LSP4-2022 also reduced evoked monosynaptic IPSCs in CA1 pyramidal cells and, in contrast to its effect on SC-CA1 EPSPs, ADX71743 reversed the ability of high-frequency stimulation of SC afferents to reduce IPSC amplitudes. Furthermore, blockade of mGlu7 prevented induction of LTP at the SC-CA1 synapse and activation of mGlu7 potentiated submaximal LTP. Together, these data suggest that mGlu7 serves as a heteroreceptor at inhibitory synapses in area CA1 and that the predominant effect of activation of mGlu7 by stimulation of glutamatergic afferents is disinhibition, rather than reduced excitatory transmission. Furthermore, this mGlu7-mediated disinhibition is required for induction of LTP at the SC-CA1 synapse, suggesting that mGlu7 could serve as a novel therapeutic target for treatment of cognitive disorders.

Synaptic Tagging and Capture: Differential Role of Distinct Calcium/Calmodulin Kinases in Protein Synthesis-Dependent Long-Term Potentiation

Weakly tetanized synapses in area CA1 of the hippocampus that ordinarily display long-term potentiation lasting approximately 3 h (called early-LTP) will maintain a longer-lasting change in efficacy (late-LTP) if the weak tetanization occurs shortly before or after strong tetanization of an independent, but convergent, set of synapses in CA1. The synaptic tagging and capture hypothesis explains this heterosynaptic influence on persistence in terms of a distinction between local mechanisms of synaptic tagging and cell-wide mechanisms responsible for the synthesis, distribution, and capture of plasticity-related proteins (PRPs). We now present evidence that distinct CaM kinase (CaMK) pathways serve a dissociable role in these mechanisms. Using a hippocampal brain-slice preparation that permits stable long-term recordings in vitro for >10 h and using hippocampal cultures to validate the differential drug effects on distinct CaMK pathways, we show that tag setting is blocked by the CaMK inhibitor KN-93 (2-[N-(2-hydroxyethyl)]-N-(4-methoxybenzenesulfonyl)amino-N-(4-chlorocinnamyl)-N-methylbenzylamine) that, at low concentration, is more selective for CaMKII. In contrast, the CaMK kinase inhibitor STO-609 [7H-benzimidazo(2,1-a)benz(de)isoquinoline-7-one-3-carboxylic acid] specifically limits the synthesis and/or availability of PRPs. Analytically powerful three-pathway protocols using sequential strong and weak tetanization in varying orders and test stimulation over long periods of time after LTP induction enable a pharmacological dissociation of these distinct roles of the CaMK pathways in late-LTP and so provide a novel framework for the molecular mechanisms by which synaptic potentiation, and possibly memories, become stabilized.

Activation of kainate receptors controls the number of functional glutamatergic synapses in the area CA1 of rat hippocampus

The expression and functions of kainate-type glutamate receptors (KARs) in the hippocampus are developmentally regulated. In particular, presynaptic KARs depressing glutamate release are tonically activated during early postnatal development, and this activity is down-regulated in parallel with maturation of the synaptic circuitry. In order to understand the physiological relevance of the tonic KAR-mediated signalling, we have here studied the effect of long-term pharmacological activation of KARs on glutamatergic synaptic connectivity in hippocampal slice cultures where presynaptic KARs are expressed but not endogenously activated. Prolonged (16-20 h) activation of the GluR5 subunit-containing KARs using the agonist ATPA (1 microM) caused a specific and enduring increase in the number of glutamatergic synapses in area CA1, evidenced as an increase in the frequency of action potential-independent spontaneous EPSCs (mEPSCs) and in immunostaining against synaptic marker proteins. The long-term ATPA treatment had no detectable effect on GABAergic transmission or on glutamate release probability. Further, the effect of ATPA on synaptic density was independent of action potential firing and dependent on protein kinase C. A critical role of endogenous KAR activity in synaptic development was revealed by chronic treatment of the cultures with the selective GluR5 antagonist LY382884, which caused a significant impairment of glutamatergic transmission to CA1 pyramidal neurons. Together, these data suggest a role for the GluR5 subunit-containing KARs in the formation and/or stabilization of functional glutamatergic synapses in area CA1.

Regulation of Histone Acetylation during Memory Formation in the Hippocampus

Formation of long term memory begins with the activation of many disparate signaling pathways that ultimately impinge on the cellular mechanisms regulating gene expression. We investigated whether mechanisms regulating chromatin structure were activated during the early stages of long term memory formation in the hippocampus. Specifically, we investigated hippocampal histone acetylation during the initial stages of consolidation of long term association memories in a contextual fear conditioning paradigm. Acetylation of histone H3 in area CA1 of the hippocampus was regulated in contextual fear conditioning, an effect dependent on activation of N-methyl-D-aspartic acid (NMDA) receptors and ERK, and blocked using a behavioral latent inhibition paradigm. Activation of NMDA receptors in area CA1 in vitro increased acetylation of histone H3, and this effect was blocked by inhibition of ERK signaling. Moreover, activation of ERK in area CA1 in vitro through either the protein kinase C or protein kinase A pathways, biochemical events known to be involved in long term memory formation, also increased histone H3 acetylation. Furthermore, we observed that elevating levels of histone acetylation through the use of the histone deacetylase inhibitors trichostatin A or sodium butyrate enhanced induction of long term potentiation at Schaffer-collateral synapses in area CA1 of the hippocampus, a candidate mechanism contributing to long term memory formation in vivo. In concert with our findings in vitro, injection of animals with sodium butyrate prior to contextual fear conditioning enhanced formation of long term memory. These results indicate that histone-associated heterochromatin undergoes changes in structure during the formation of long term memory. Mimicking memory-associated changes in heterochromatin enhances a cellular process thought to underlie long term memory formation, hippocampal long term potentiation, and memory formation itself.

Silence Analysis of AMPA Receptor Mutated at the CaM-Kinase II Phosphorylation Site

Direct phosphorylation of the GluR1 subunit of postsynaptic AMPA receptors by Ca 2+/calmodulin-dependent protein kinase II (CaM-KII) is believed to be one of the major contributors to the enhanced strength of glutamatergic synapses in CA1 area of hippocampus during long-term potentiation. The molecular mechanism of AMPA receptor regulation by CaM-KII is examined here by a novel approach, silence analysis, which is independent of previously used variance analysis. I show that three fundamental channel properties—single-channel conductance, channel open probability, and the number of functional channels—can be measured in an alternative way, by analyzing the probability of channels to be simultaneously closed (silent). Validity of the approach was confirmed by modeling, and silence analysis was applied then to the GluR1 AMPA receptor mutated at S831, the site phosphorylated by CaM-KII during long-term potentiation. Silence analysis indicates that a negative charge at S831 is a critical determinant for the enhanced channel function as a charge carrier. Silence and variance analyses, when applied to the same sets of data, were in agreement on the receptor regulation upon mutations. These results provide independent evidences for the mechanism of AMPA receptor regulation by CaM-KII and further strengthens the idea how calcium-dependent phosphorylation of AMPA receptors can contribute to the plasticity at central glutamatergic synapses.

Quantal variability at glutamatergic synapses in area CA1 of the rat neonatal hippocampus.

Quantal variability was determined at glutamatergic synapses in the neonatal (postnatal days 1-7) rat hippocampal slice preparation. Synaptic AMPA and NMDA quantal EPSCs were recorded from CA1 pyramidal neurones using the whole-cell, or perforated, patch-clamp technique. Release was evoked by minimal stimulation using brief trains (10 impulses, 50 Hz), and various tests ascertained that this stimulation activated single release sites releasing single vesicles. Both AMPA and NMDA quantal responses at a given release site varied considerably in magnitude, the coefficient of variation (CV) among the synapses averaging 0.39 and 0.30, respectively. This variability differed among the synapses, from 0.2 to 0.7, and 0.10 to 0.50, respectively, and CV values of AMPA responses co-varied with those of the NMDA responses. Both for AMPA and NMDA, low CV values were associated with a Gaussian distribution of EPSC peak values, whereas synapses with high CV values displayed distributions skewed towards lower values. Analysis of successive NMDA responses during a train revealed a considerable degree of non-saturation of NMDA receptors by a single vesicle. The results are compatible with a quantal variability based, to a large extent, on non-saturated AMPA and NMDA responses fluctuating as a function of the amount of transmitter released from each vesicle.

Vesicle release probability and pre-primed pool at glutamatergic synapses in area CA1 of the rat neonatal hippocampus.

Factors determining the release probability were examined using whole-cell patch-clamp recording and minimal stimulation (10 impulses, 50 Hz) of individual glutamatergic synapses, containing single release sites, on pyramidal neurones in the CA1 region of hippocampal slices from neonatal rats. Release probability following the first action potential in the burst (P1) varied among the synapses from 0 to 0.87 (mean +/- s.d.; 0.35 +/- 0.28, n = 52) and the average release during the burst (burst pool) varied from 0.4 to 4.1 events (1.7 +/- 0.85, n = 52). Heterogeneity in P1 did not co-vary with that of the burst pool. By selecting burst trials during which only one release event occurred, the vesicle release probability (Pves) at a release site could be determined. It was found to vary considerably among the synapses, from 0.04 to 0.94 (0.43 +/- 0.28, n = 43). This heterogeneity correlated significantly with that of P1 such that more than half of the variation of P1 could be explained by a variation in Pves. The average number of vesicles directly available for release at the onset of the burst (the pre-primed pool) was estimated as the cumulative release up to that point in the burst where a second release event did not produce higher initial release probability than that found in trials where only one vesicle was released. The average pre-primed pool varied among the synapses from 0.4 to 2.1 (1.03 +/- 0.42, n = 43). It co-varied significantly with that of P1 such that it could explain the remaining variation in P1. The difference between the burst pool and the pre-primed pool suggests the presence of a fast (< 100 ms), activity-dependent priming of vesicles. Some synapses (9/52) did not show any initial release (P1 = 0), but release occurred later during the burst ('low frequency mute synapses'). Their behaviour was explained by an absence of a pre-primed pool.

Modelling Huntington’s disease

Huntington’s disease (HD) is one of a group of inherited neurodegenerative disorders caused by a polyglutamine (CAG)-repeat expansion. The pathology of these disorders is associated with the presence of neuronal intranuclear inclusions, which contain the polyglutamine- repeat-containing protein that is specific to the disease, along with ubiquitin. Sathasivam et al.1xFormation of polyglutamine inclusions in non-CNS tissue. Sathasivam, K. et al. Hum. Mol. Genet. 1999; 8: 813–822Crossref | PubMedSee all References1 report the formation of nuclear inclusions (NIs) in non-neuronal tissue in previously described transgenic mice that express exon 1 of the human HD gene that contains a highly expanded CAG repeat. In this model, NIs were found in several tissues, including cardiac and skeletal muscle, liver, kidney, adrenal gland and pancreas. They report a correlation between the presence of NIs and a progressive decrease in organ size. It is not known whether similar non-neuronal NIs occur in human HD patients, and as such the relevance of this aspect of the model is unclear. Despite this, the presence of NIs in tissues such as skeletal muscle provides a good system to study NIs and to investigate ways to prevent their formation.Although the association of NIs with HD is well documented, their role in the pathology of the disease remains unproven, and an unknown property of the expanded polyglutamine repeat that is independent of NI formation might underlie certain aspects of the disease. A new mouse model, described by Shelbourne et al.2xA Huntington’s disease CAG expansion at the murine Hdh locus is unstable and associated with behavioural abnormalities in mice. Shelbourne, P.F. et al. Hum. Mol. Genet. 1999; 8: 763–774Crossref | PubMedSee all References2, provides a valuable tool to study this possibility. This mouse has a stretch of 72–80 CAG repeats inserted into the endogenous Hdh gene. This ‘knockin’ mutation is similar to the human HD mutation as the mice express full-length mutant huntingtin in a wild-type genomic context. From a genetic viewpoint, this model appears to closely mirror the human HD mutation. As in humans, the expanded CAG repeat is unstable and a parent-of-origin effect on the repeat length is observed, with expansions tending to be associated with paternal transmission and contractions being associated with maternal transmission. The brains of the mutant mice are histologically normal with no evidence of NI formation. However, a reduction in brain size is seen in the mutant strains. The mutant mice also displayed chronic aggressive behaviour; human HD patients often exhibit symptoms such as irritability and aggression and ∼50% of patients present with psychiatric symptoms. These mice might therefore provide a model for the psychiatric disorder in HD.The knockin model was used to study the possible basis of cognitive impairment in HD (Ref. 3xImpaired synaptic plasticity in mice carrying the Huntington’s disease mutation. Usdin, M.T. et al. Hum. Mol. Genet. 1999; 8: 839–846Crossref | PubMedSee all ReferencesRef. 3). Long-term potentiation (LTP) is the sustained increase in synaptic strength following a high-frequency conditioning stimulus. LTP provides a model for a mechanism that underlies some forms of learning and memory. LTP was significantly reduced in the stratum radiatum of area CA1 of hippocampal slices from the mutant mice. Post-tetanic potentiation and paired-pulse facilitation were also shown to be impaired, suggesting that the presynaptic terminals might be less able to sustain neurotransmitter release with repetitive stimulation. This was tested by monitoring the opening of N-methyl-d-aspartate (NMDA) channels by glutamate release. Glutamatergic synapses in area CA1 were shown to transmit normally in response to single stimuli but were impaired in their ability to release neurotransmitter at moderate levels of stimulation. The authors propose that the polyglutamine expansion might interfere with neurotransmitter release at the presynaptic terminals. This hypothesis is consistent with previous reports of wild-type HD protein being associated with microtubules and synaptic vesicles.This new mouse model provides evidence that some of the symptoms of HD might be independent of NI formation and might precede neurodegeneration. The two mouse models provide insights into distinct aspects of HD; taken together they should complement each other to facilitate research into the molecular basis and treatment of this disease.

Dendritic spine changes associated with hippocampal long-term synaptic plasticity.

Long-term enhancement of synaptic efficacy in the hippocampus is an important model for studying the cellular mechanisms of neuronal plasticity, circuit reorganization, and even learning and memory. Although these long-lasting functional changes are easy to induce, it has been very difficult to demonstrate that they are accompanied or even caused by morphological changes on the subcellular level. Here we combined a local superfusion technique with two-photon imaging, which allowed us to scrutinize specific regions of the postsynaptic dendrite where we knew that the synaptic changes had to occur. We show that after induction of long-lasting (but not short-lasting) functional enhancement of synapses in area CA1, new spines appear on the postsynaptic dendrite, whereas in control regions on the same dendrite or in slices where long-term potentiation was blocked, no significant spine growth occurred.

Multiple presynaptic metabotropic glutamate receptors modulate excitatory and inhibitory synaptic transmission in hippocampal area CA1

The metabotropic glutamate receptors (mGluRs) have many important roles in regulation of neuronal excitability and synaptic transmission. In hippocampal area CA1, activation of mGluRs can reduce both excitatory and inhibitory synaptic transmission. The conventional view is that the presynaptic effects are mediated by L-2-amino-4-phosphonobutyric acid (L-AP4)-sensitive, or group III mGluRs (mGluR4, mGluR6, mGluR7, mGluR8). However, some studies suggest that other mGluR subtypes may also be involved in regulation of excitatory and inhibitory synaptic transmission in area CA1. We have found that two pharmacologically distinct presynaptic receptors are involved in the depression of excitatory transmission at the Schaffer collateral--CA1 synapse. Consistent with previous studies, one receptor subtype is an L-AP4-sensitive receptor that is pharmacologically similar to mGluR4 or mGluR7. However, we have found that a second mGluR subtype, which is pharmacologically similar to mGluR1 and mGluR5 (group I mGluRs), can also reduce excitatory synaptic transmission in area CA1. Analysis of effects of agonists of these two receptors on miniature EPSCs and paired-pulse facilitation suggest that both receptors are localized presynaptically. It is also shown that the mGluR that reduces transmission at inhibitory synapses in area CA1 is presynaptically localized, is insensitive to L-AP4, and is sensitive to agonists selective for mGluR1 and mGluR5.

Transient protein kinase C activation primes long-term depression and suppresses long-term potentiation of synaptic transmission in hippocampus.

Activity-dependent long-lasting plasticity in hippocampus and neocortex includes long-term potentiation (LTP) and long-term depression (LTD) of synaptic strength. Recent studies have confirmed theoretical predictions that the sensitivity of LTP- and LTD-inducing mechanisms is dynamically regulated by previous synaptic history. In particular, prior induction of either repeated short-term potentiations or LTP lowers the threshold for induction of LTD and raises the threshold for LTP. In the current study, transient activation of protein kinase C with phorbol 12,13-diacetate was able to substitute for synaptic activity in priming synapses to exhibit enhanced homosynaptic LTD and to suppress the induction of LTP at Schaffer collateral synapses in area CA1 of hippocampal slices. This priming lasted 30 min, but not 3 hr, following phorbol 12,13-diacetate bath application. These data suggest that a protein kinase C-sensitive phosphorylation site may be an activity-sensitive target mediating the rapid expression of LTP and LTD.

Potassium-induced long-term potentiation in area CA1 of the hippocampus involves phospholipase activation.

Previous studies have shown that potassium-induced long-term potentiation (LTP) of the Schaffer collateral/commissural synapses in area CA1 of the hippocampus shares common properties with tetanus-induced LTP. In the present investigation, we performed electrophysiological and binding experiments on CA1 hippocampal slices to evaluate the location and nature of the changes underlying potassium-induced LTP. Paired-pulse facilitation, which represents an index of transmitter release, was markedly reduced by potassium-induced LTP. In addition, KCl-induced LTP was associated with an increase in 3H-AMPA ([3H]-amino-3-hydroxy-5-methylisoxazole-4-propionate) binding to CA1 synaptic membranes when measured 40 min after high-potassium exposure; however, no changes were detected in binding of an antagonist ([3H]-6-cyano-7-nitroquinoxaline-2,3-dione; 3H-CNQX) to AMPA receptors in slices expressing KCl-induced LTP. Administration of the phospholipase A2 (PLA2) inhibitor bromophenacyl bromide (BPB) prior to potassium application prevented LTP formation as well as the changes in paired-pulse facilitation and 3H-AMPA binding that characterized this type of potentiation. Taken together, these data indicate that potassium-induced LTP may be related to modifications in both pre- and postsynaptic properties and confirm the hypothesis that PLA2 activation is an important mechanism in long-term changes of synaptic operation.

Activation of NMDA receptors in hippocampal area CA1 by low and high frequency orthodromic stimulation and their contribution to induction of long-term potentiation.

N-methyl-D-aspartate (NMDA) receptors are important in many instances of synaptic plasticity. In hippocampal area CA1, long-term potentiation (LTP) can be induced by both NMDA receptor-dependent and -independent mechanisms. Using intracellular recordings and single-electrode voltage clamp, we isolated and characterized NMDA receptor-mediated synaptic responses. NMDA receptor-mediated responses evoked by low frequency orthodromic stimulation were inhibited in a dose-dependent manner by the competitive antagonist D,L-2-amino-5-phosphonovaleric acid (APV). High frequency (tetanic) stimulation, which facilitates synaptic release of glutamate, failed to overcome the blockade of NMDA receptors by APV. Using extracellular recordings of field potentials, we studied the contribution of NMDA receptors to LTP induced by different patterns of tetanic stimulation. LTP was inhibited in a dose-dependent manner by APV, but was more sensitive to APV than were NMDA receptor-mediated synaptic responses. This most likely reflects a threshold for NMDA receptor activation in LTP induction. A component of LTP that resisted blockade by APV was induced by high (200 Hz), but not low (25 Hz), frequency tetanization. This NMDA receptor-independent component of LTP persisted for > 4 hours and accounted for approximately half the potentiation induced by 200 Hz tetanization. Procedures necessary to induce LTP at the Schaffer collateral/commissural synapses in area CA1 by both NMDA receptor-dependent and -independent mechanisms are now well characterized. Using the same neuronal population, it will be possible to ask if processes involved in the maintenance of LTP are shared even when LTP is induced through two different mechanisms.

Membrane structure at synaptic junctions in area CA1 of the rat hippocampus

In tissue from area CA1 of the rat hippocampus prepared for electron microscopic study by thin-sectioning, asymmetric synaptic junctions were found on dendritic spines, spiny dendritic shafts, and non-spiny dendritic shafts. In freeze-fractured preparations, aggregates of large particles were found on the extracellular half of the postsynaptic membrane at each of these synaptic junctions. Particle aggregate areas were measured and particle packing densities were computed at dendritic spine synapses and dendritic shaft synapses in area CA1, and compared to similar measures of particle aggregates on dendritic spines of cerebellar Purkinje cells. All of these CA1 and cerebellar synapses are excitatory and are thought to use glutamate as a neurotransmitter. There was a tendency for the dispersion of particles within individual aggregates to be less uniform in larger aggregates in both area CA1 and cerebellar cortex. Distinct particle-free zones could be distinguished in the center of particle aggregates on large “mushroom-shaped” spines in area CA1. There was no statistically significant difference between the particle densities at CA1 dendritic spines (2848 ± 863particles/μm 2) and CA1 dendritic shafts (2707 ± 718particles/μm 2). However, the average density of particles at cerebellar dendritic spine synapses (3614 ± 1081particles/μm 2) was significantly greater than at dendritic spine or shaft synapses found in area CA1. Symmetric synaptic junctions were observed on the CA1 pyramidal cell somas and dendritic shafts in thin-sectioned preparations. These synapses typically exert an inhibitory action mediated by gammaaminobutyric acid. In freeze-fracture preparations, large varicosities were found apposed to the pyramidal somal and dendric membranes, but there were no specializations of particle distribution on either the extracellular or the cytoplasmic half of the fractured postsynaptic membranes. This finding parallels observations from freeze-fracture preparations of other GABAergic synapses in the central nervous system.