Reciprocal interactions between CA3 network activity and strength of recurrent collateral synapses.

In hippocampal slices, synchronous CA3 network activity induced persistent strengthening of active positive-feedback synapses. This altered network operation by increasing probability of future synchronous network activation. Long-term depression of synaptic strength induced by partial blockade of NMDA receptors during synchronous network activity reversed changes in probability of spontaneous network activation. These results suggest that specific network activity patterns selectively alter strength of active synapses. Stable, reversible alterations in network activity can also be effected by corresponding alterations in synaptic strength. These findings confirm the Hebb memory model at the neural-network level and suggest new therapies for pathological patterns of network activity in epilepsy.     

NMDA receptor subtypes at autaptic synapses of cerebellar granule neurons

We studied the action potential-evoked autaptic N-methyl-d-aspartate receptor-mediated excitatory postsynaptic currents (NMDA-EPSCs) using solitary cerebellar neurons cultured in microislands from wild-type (+/+), NR2A subunit knockout (NR2A-/-), and NR2C subunit knockout (NR2C-/-) mice. The peak amplitude of autaptic NMDA-EPSCs increased for all genotypes between days in vitro 8 (DIV8) and DIV13. Compared with +/+ cells at DIV13, NR2A-/- cells had smaller and NR2C-/- cells had larger NMDA-EPSCs. The decay time of these currents were all unexpectedly fast, except in NR2A-/- neurons, and showed small but significant shortening with development. Comparison of quantal parameters during development indicated an increase in quantal content in all genotypes. The synaptic portion of NMDA receptors measured using MK-801 blockade was roughly 50% in all genotypes at DIV8, and this percentage became slightly larger in NR2A-/- and NR2C-/- neurons at DIV12. The NR2B-selective antagonists Conantokin G and CP101,606 differed in their blocking actions with development, suggesting the presence of both heterodimeric NR1/NR2B and heterotrimeric NR1/NR2A/NR2B receptors. The most striking result we obtained was the significant increase of NMDA-EPSC peak amplitude and charge transfer in NR2C-/- mice. This was mainly the result of an increase in quantal size as estimated from miniature NMDA-EPSCs. The expression of NR2C subunit containing receptors was supported by the decreased Mg(2+) sensitivity of NMDA receptors at DIV13 in +/+ but not in NR2C-/- cells. Thus solitary cerebellar granule neurons provide a novel model to investigate the role of receptor subtypes in the developmental changes of synaptic NMDA receptors.     

Regulation of NMDA receptor trafficking by amyloid-beta

Amyloid-beta peptide is elevated in the brains of patients with Alzheimer disease and is believed to be causative in the disease process. Amyloid-beta reduces glutamatergic transmission and inhibits synaptic plasticity, although the underlying mechanisms are unknown. We found that application of amyloid-beta promoted endocytosis of NMDA receptors in cortical neurons. In addition, neurons from a genetic mouse model of Alzheimer disease expressed reduced amounts of surface NMDA receptors. Reducing amyloid-beta by treating neurons with a gamma-secretase inhibitor restored surface expression of NMDA receptors. Consistent with these data, amyloid-beta application produced a rapid and persistent depression of NMDA-evoked currents in cortical neurons. Amyloid-beta-dependent endocytosis of NMDA receptors required the alpha-7 nicotinic receptor, protein phosphatase 2B (PP2B) and the tyrosine phosphatase STEP. Dephosphorylation of the NMDA receptor subunit NR2B at Tyr1472 correlated with receptor endocytosis. These data indicate a new mechanism by which amyloid-beta can cause synaptic dysfunction and contribute to Alzheimer disease pathology.     

Statistical model relating CA3 burst probability to recovery from burst-induced depression at recurrent collateral synapses.

When neuronal excitability is increased in area CA3 of the hippocampus in vitro, the pyramidal cells generate periodic bursts of action potentials that are synchronized across the network. We have previously provided evidence that synaptic depression at the excitatory recurrent collateral synapses in the CA3 network terminates each population burst so that the next burst cannot begin until these synapses have recovered. These findings raise the possibility that burst timing can be described in terms of the probability of recovery of this population of synapses. Here we demonstrate that when neuronal excitability is changed in the CA3 network, the mean and variance of the interburst interval change in a manner that is consistent with a timing mechanism comprised of a pool of exponentially relaxing pacemakers. The relaxation time constant of these pacemakers is the same as the time constant describing the recovery from activity-dependent depression of recurrent collateral synapses. Recovery was estimated from the rate of spontaneous transmitter release versus time elapsed since the last CA3 burst. Pharmacological and long-term alterations of synaptic strength and network excitability affected CA3 burst timing as predicted by the cumulative binomial distribution if the burst pace-maker consists of a pool of recovering recurrent synapses. These findings indicate that the recovery of a pool of synapses from burst-induced depression is a sufficient explanation for burst timing in the in vitro CA3 neuronal network. These findings also demonstrate how information regarding the nature of a pacemaker can be derived from the temporal pattern of synchronous network activity. This information could also be extracted from less accessible networks such as those generating interictal epileptiform discharges in vivo.     

BK potassium channels control transmitter release at CA3–CA3 synapses in the rat hippocampus

Augmentation is a component of short-term synaptic plasticity with a gradual onset and duration in seconds. To investigate this component at the corticogeniculate synapse, whole cell patch-clamp recordings were obtained from principal cells in a slice preparation of the rat dorsal lateral geniculate nucleus. Trains with 10 stimuli at 25 Hz evoked excitatory postsynaptic currents (EPSCs) that grew in amplitude, primarily from facilitation. Such trains also induced augmentation that decayed exponentially with a time constant τ= 4.6 ± 2.6 s (mean ± standard deviation). When the trains were repeated at 1–10 s intervals, augmentation markedly increased the size of the first EPSCs, leaving late EPSCs unaffected. The magnitude of augmentation was dependent on the number of pulses, pulse rate and intervals between trains. Augmented EPSCs changed proportionally to basal EPSC amplitudes following alterations in extracellular calcium ion concentration. The results indicate that augmentation is determined by residual calcium remaining in the presynaptic terminal after repetitive spikes, competing with fast facilitation. We propose that augmentation serves to maintain a high synaptic strength in the corticogeniculate positive feedback system during attentive visual exploration.     

Protein kinase C modulates NMDA receptor trafficking and gating

Regulation of neuronal N-methyl-D-aspartate receptors (NMDARs) by protein kinases is critical in synaptic transmission. However, the molecular mechanisms underlying protein kinase C (PKC) potentiation of NMDARs are uncertain. Here we demonstrate that PKC increases NMDA channel opening rate and delivers new NMDA channels to the plasma membrane through regulated exocytosis. PKC induced a rapid delivery of functional NMDARs to the cell surface and increased surface NR1 immunofluorescence in Xenopus oocytes expressing NMDARs. PKC potentiation was inhibited by botulinum neurotoxin A and a dominant negative mutant of soluble NSF-associated protein (SNAP-25), suggesting that receptor trafficking occurs via SNARE-dependent exocytosis. In neurons, PKC induced a rapid delivery of functional NMDARs, assessed by electrophysiology, and an increase in NMDAR clusters on the surface of dendrites and dendritic spines, as indicated by immunofluorescence. Thus, PKC regulates NMDAR channel gating and trafficking in recombinant systems and in neurons, mechanisms that may be relevant to synaptic plasticity.     

NMDA receptor-dependent metaplasticity at hippocampal mossy fiber synapses

Hippocampal mossy fiber synapses have been reported to lack NMDA receptor (NMDAR)-dependent long-term potentiation (LTP) of AMPA excitatory postsynaptic currents (EPSCs), unlike conventional glutamatergic synapses. An explanation for this difference may reside in the relatively low number of NMDARs at these synapses. Because mossy fiber synapses display LTP selective for NMDARs, we examined whether this would affect the plasticity rules at mossy fiber-CA3 synapses in mouse hippocampal slices. We found that LTP of NMDARs serves as a metaplastic switch making mossy fiber synapses competent for generating NMDAR-dependent LTP of AMPA EPSCs.     

Rapid recruitment of NMDA receptor transport packets to nascent synapses

Although many of the molecules involved in synaptogenesis have been identified, the sequence and kinetics of synapse assembly in the central nervous system (CNS) remain largely unknown. We used simultaneous time-lapse imaging of fluorescent glutamate receptor subunits and presynaptic proteins in rat cortical neurons in vitro to determine the dynamics and time course of N-methyl-D-aspartate receptor (NMDAR) recruitment to nascent synapses. We found that both NMDA and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) subunits are present in mobile transport packets in neurons before and during synaptogenesis. NMDAR transport packets are more mobile than AMPAR subunits, moving along microtubules at about 4 microm/min, and are recruited to sites of axodendritic contact within minutes. Whereas NMDAR recruitment to new synapses can be either concurrent with or independent of the protein PSD-95, AMPARs are recruited with a slower time course. Thus, glutamatergic synapses can form rapidly by the sequential delivery of modular transport packets containing glutamate receptors.     

NMDA receptor-mediated control of protein synthesis at developing synapses

We demonstrate a rapid and complex effect of N-methyl-d-aspartate receptor (NMDAR) activation on synaptic protein synthesis in the superior colliculi of young rats. Within minutes of receptor activation, translation of alpha Ca2+/calmodulin dependent kinase II (alphaCamK II) was increased, whereas total protein synthesis was reduced. NMDAR activation also increased phosphorylation of eukaryotic elongation factor 2 (eEF2), a process known to inhibit protein translation by reducing peptide chain elongation. Low doses of cycloheximide, which reduce elongation rate independently of eEF2 phosphorylation, decreased overall protein synthesis but increased alphaCaMK II synthesis. These observations suggest that regulation of peptide elongation via eEF2 phosphorylation can link NMDAR activation to local increases in the synthesis of specific proteins during activity-dependent synaptic change.     

Properties of quantal transmission at CA1 synapses

We have used Monte Carlo simulations to understand the generation of quantal responses at the single active zones of CA1 synapses. We constructed a model of AMPA channel activation that accounts for the responses to controlled glutamate application and a model of glutamate diffusion in the synaptic cleft. With no further adjustments to these models, we simulated the response to the release of glutamate from a single vesicle. The predicted response closely matches the rise time of observed responses, which recent measurements show is much faster (<100 micros) than previously thought. The simulations show that initial channel opening is driven by a brief (<100 micros) glutamate spike near the site of vesicle fusion, producing a hotspot of channel activation (diameter: approximately 250 nm) smaller than many synapses. Quantal size therefore depends more strongly on the density of channels than their number, a finding that has important implications for measuring synaptic strength. Recent measurements allow estimation of AMPA receptor density at CA1 synapses. Using this value, our simulations correctly predicts a quantal amplitude of approximately 10 pA. We have also analyzed the properties of excitatory postsynaptic currents (EPSCs) generated by the multivesicular release that can occur during evoked responses. We find that summation is nearly linear and that the existence of multiple narrow peaks in amplitude histograms can be accounted for. It has been unclear how to reconcile the existence of these narrow peaks, which indicate that the variation of quantal amplitude is small (CV < 0.2) with the highly variable amplitude of miniature EPSCs (mEPSCs; CV approximately 0.6). According to one theory, mEPSC variability arises from variation in vesicle glutamate content. However, both our modeling results and recent experimental results indicate that this view cannot account for the observed rise time/amplitude correlation of mEPSCs. In contrast, this correlation and the high mEPSC variability can be accounted for if some mEPSCs are generated by two or more vesicles released with small temporal jitter. We conclude that a broad range of results can be accounted for by simple principles: quantal amplitude (approximately 10 pA) is stereotyped, some mEPSCs are multivesicular at moderate and large synapses, and evoked responses are generated by quasi-linear summation of multiple quanta.     

Up-regulation of GLT-1 severely impairs LTD at mossy fibre–CA3 synapses

Glutamate transporters are responsible for clearing synaptically released glutamate from the extracellular space. By this action, they maintain low levels of ambient glutamate, thus preventing excitotoxic damage, and contribute to shaping synaptic currents. We show that up-regulation of the glutamate transporter GLT-1 by ceftriaxone severely impaired mGluR-dependent long-term depression (LTD), induced at rat mossy fibre (MF)–CA3 synapses by repetitive stimulation of afferent fibres. This effect involved GLT-1, since LTD was rescued by the selective GLT-1 antagonist dihydrokainate (DHK). DHK per se produced a modest decrease in fEPSP amplitude that rapidly regained control levels after DHK wash out. Moreover, the degree of fEPSP inhibition induced by the low-affinity glutamate receptor antagonist γ-DGG was similar during basal synaptic transmission but not during LTD, indicating that in ceftriaxone-treated rats LTD induction did not alter synaptic glutamate transient concentration. Furthermore, ceftriaxone-induced GLT-1 up-regulation significantly reduced the magnitude of LTP at MF–CA3 synapses but not at Schaffer collateral–CA1 synapses. Postembedding immunogold studies in rats showed an increased density of gold particles coding for GLT-1a in astrocytic processes and in mossy fibre terminals; in the latter, gold particles were located near and within the active zones. In both CEF-treated and untreated GLT-1 KO mice used for verifying the specificity of immunostaining, the density of gold particles in MF terminals was comparable to background levels. The enhanced expression of GLT-1 at release sites may prevent activation of presynaptic receptors, thus revealing a novel mechanism by which GLT-1 regulates synaptic plasticity in the hippocampus.     

Mossy fibre synaptic NMDA receptors trigger non-hebbian long-term potentiation at entorhino-CA3 synapses in the rat

Hippocampal CA3 pyramidal cells receive two independent afferents from the enthorinal cortex, i.e. a direct input via the temporoammonic pathway (TA, perforant path) and an indirect input via the mossy fibres (MF) of dentate granule cells. In spite of past suggestions that the TA is assigned an important role in exciting the pyramidal cells, little is known about their physiological properties. By surgically making an incision through the sulcus hippocampi and a small part of the dentate molecular layer, we succeeded in isolating TA-mediated monosynaptic responses in CA3 stratum lacunosum-moleculare. The TA-CA3 synaptic transmission was completely blocked by a combination of d,l -2-amino-5-phosphonopentanoic acid (AP5) and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), NMDA and non-NMDA receptor antagonists, respectively, and displayed paired-pulse facilitation and NMDA receptor-dependent long-term potentiation, which are all typical of glutamatergic synapses. We next addressed the heterosynaptic interaction between TA-CA3 and MF-CA3 synapses. The TA-CA3 transmission was partially attenuated by single-pulse MF pre-stimulation at inter-pulse intervals of up to 70 ms. However, surprisingly, burst stimulation of the MF alone induced long-lasting facilitation of TA-CA3 synaptic efficacy. This non-Hebbian form of synaptic plasticity was efficiently prevented by local application of AP5 into the MF synapse-rich area. Therefore, MF-activated NMDA receptors are responsible for the heterosynaptic modification of TA-CA3 transmission, and thereby, the history of MF activity may be etched into TA-CA3 synaptic strength. Our findings predict a novel form of spatiotemporal information processing in the hippocampus, i.e. a use-dependent intersynaptic memory transfer.     

L-type Ca currents at CA1 synapses,but not CA3 or dentate granule neuron synapses,are increased in 3xTgAD mice in an age-dependent manner

Abnormal neuronal excitability and impaired synaptic plasticity might occur before the degeneration and death of neurons in Alzheimer's disease (AD). To elucidate potential biophysical alterations underlying aberrant neuronal network activity in AD, we performed whole-cell patch clamp analyses of L-type (nifedipine-sensitive) Ca²⁺ currents (L-VGCC), 4–aminopyridine-sensitive K⁺ currents, and AMPA (2-amino-3-(3-hydroxy-5-methyl-isoxazol-4-yl)propanoic acid) and NMDA (N-methyl-D-aspartate) currents in CA1, CA3, and dentate granule neurons in hippocampal slices from young, middle-age, and old 3xTgAD mice and age-matched wild type mice. 3xTgAD mice develop progressive widespread accumulation of amyloid β-peptide, and selective hyperphosphorylated tau pathology in hippocampal CA1 neurons, which are associated with cognitive deficits, but independent of overt neuronal degeneration. An age-related elevation of L-type Ca²⁺ channel current density occurred in CA1 neurons in 3xTgAD mice, but not in wild type mice, with the magnitude being significantly greater in older 3xTgAD mice. The NMDA current was also significantly elevated in CA1 neurons of old 3xTgAD mice compared with in old wild type mice. There were no differences in the amplitude of K⁺ or AMPA currents in CA1 neurons of 3xTgAD mice compared with wild type mice at any age. There were no significant differences in Ca²⁺, K⁺, AMPA, or NMDA currents in CA3 and dentate neurons from 3xTgAD mice compared with wild type mice at any age. Our results reveal an age-related increase of L-VGCC density in CA1 neurons, but not in CA3 or dentate granule neurons, of 3xTgAD mice. These findings suggest a potential contribution of altered L-VGCC to the selective vulnerability of CA1 neurons to tau pathology in the 3xTgAD mice and to their degeneration in AD patients.     

Shift in induction mechanisms underlies an age-dependent increase in DHPG-induced synaptic depression at CA3 CA1 synapses

Several forms of log-term synaptic plasticity have been identified and the mechanisms for induction and expression of synaptic modifications change over development and maturation. The present study examines age-related changes in the induction of group I metabotropic receptor selective agonist (R,S)-3,5-dihydroxyphenylglycine (DHPG) induced long-term synaptic depression (DHPG-LTD) at CA3-CA1 synapses. The results demonstrate that the magnitude of DHPG-LTD is enhanced in male aged Fischer 344 rats compared with young adults. The role of mGluR1 in the induction of DHPG-LTD was increased with advanced age and, in contrast to young adults, induction involved a significant contribution of NMDA receptors and L-type Ca(2+) channels. Moreover, the protein tyrosine phosphatase inhibitor sodium orthovanadate significantly attenuated DHPG-LTD only in young adults. The expression of DHPG-LTD in aged animals was dependent on protein synthesis and the enhanced expression was associated with an increase in paired-pulse facilitation. The results provide evidence that DHPG-LTD is one of the few forms of synaptic plasticity that increases with advanced age and suggest that DHPG-LTD may contribute to age-related changes in hippocampal function.     

Burst firing induces postsynaptic LTD at developing mossy fibre–CA3 pyramid synapses

Synaptic development is an activity-dependent process utilizing coordinated network activity to drive synaptogenesis and subsequent refinement of immature connections. Hippocampal CA3 pyramidal neurons (PYRs) exhibit intense burst firing (BF) early in development, concomitant with the period of mossy fibre (MF) development. However, whether developing MF–PYR synapses utilize PYR BF to promote MF synapse maturation remains unknown. Recently, we demonstrated that transient tonic depolarization of postsynaptic PYRs induces a persistent postsynaptic form of long-term depression (depolarization-induced long-term depression, DiLTD) at immature MF–PYR synapses. DiLTD induction is NMDAR independent but does require postsynaptic Ca²⁺ influx through L-type voltage gated Ca²⁺ channels (L-VGCCs), and is expressed as a reduction in AMPAR function through the loss of GluR2-lacking AMPARs present at immature MF–PYR synapses. Here we examined whether more physiologically relevant phasic L-VGCC activation by PYR action potential (AP) BF activity patterns can trigger DiLTD. Using combined electrophysiological and Ca²⁺ imaging approaches we demonstrate that PYR BF effectively drives L-VGCC activation and that brief periods of repetitive PYR BF, produced by direct current injection or intrinsic network activity induces NMDAR-independent LTD by promoting Ca²⁺ influx through the activated L-VGCCs. This BF induced LTD, just like DiLTD, is specific for developing MF–PYR synapses, is PICK1 dependent, and is expressed postsynaptically. Our results demonstrate that DiLTD can be induced by phasic L-VGCC activation driven by PYR BF, suggesting the engagement of natural PYR network activity patterns for MF synapse maturation.     

Modulation of LTP at rat hippocampal CA3-CA1 synapses by direct current stimulation

Transcranial direct current stimulation (tDCS) can produce a lasting polarity-specific modulation of cortical excitability in the brain, and it is increasingly used in experimental and clinical settings. Recent studies suggest that the after-effects of tDCS are related to molecular mechanisms of activity-dependent synaptic plasticity. Here we investigated the effect of DCS on the induction of one of the most studied N-methyl-d-aspartate receptor-dependent forms of long-term potentiation (LTP) of synaptic activity at CA3-CA1 synapses in the hippocampus. We show that DCS applied to rat brain slices determines a modulation of LTP that is increased by anodal and reduced by cathodal DCS. Immediate early genes, such as c-fos and zif268 (egr1/NGFI-A/krox24), are rapidly induced following neuronal activation, and a specific role of zif268 in the induction and maintenance of LTP has been demonstrated. We found that both anodal and cathodal DCS produce a marked subregion-specific increase in the expression of zif268 protein in the cornus ammonis (CA) region, whereas the same protocols of stimulation produce a less pronounced increase in c-fos protein expression in the CA and in dentate gyrus regions of the hippocampus. Brain-derived neurotrophic factor expression was also investigated, and it was found to be reduced in cathodal-stimulated slices. The present data demonstrate that it is possible to modulate LTP by using DCS and provide the rationale for the use of DCS in neurological diseases to promote the adaptive and suppress the maladaptive forms of brain plasticity.     

Forskolin-induced LTP in the CA1 hippocampal region is NMDA receptor dependent

Chemically induced long-term potentiation (cLTP) could potentially work by directly stimulating the biochemical machinery that underlies synaptic plasticity, bypassing the need for synaptic activation. Previous reports suggested that agents that raise cAMP concentration might have this capability. We examined the cLTP induced in acute slices by application of Sp-cAMPS or a combination of the adenylyl cyclase activator, forskolin, and the phosphodiesterase inhibitor, rolipram. Under our conditions, cLTP was induced but only if inhibition was reduced. We found that this form of cLTP was blocked by a N-methyl-d-aspartate receptor (NMDAR) antagonist and required the low-frequency test stimulation typically used to monitor the strength of synapses. Interestingly, similar LTP could be induced by lowering the Mg(2+) concentration of the ACSF during forskolin/rolipram or Sp-cAMPS application or even by just lowering Mg(2+) concentration alone. This LTP was also NMDAR dependent and required only a few ( approximately 5) low-frequency stimuli for its induction. The finding that even low-frequency synaptic stimulation was sufficient for LTP induction indicates that a highly sensitized plasticity state was generated. The fact that some stimulation was required means that potentiation is probably restricted to the stimulated axons, limiting the usefulness of this form of cLTP. However, when similar experiments were conducted using slice cultures, potentiation occurred without test stimuli, probably because the CA3-CA1 connections are extensive and because presynaptic spontaneous activity is sufficient to fulfill the activity requirement. As in acute slices, the potentiation was blocked by an NMDAR antagonist. Our general conclusion is that the induction of LTP caused by elevating cAMP requires presynaptic activity and NMDA channel opening. The method of inducing cLTP in slice cultures will be useful when it is desirable to produce NMDAR-dependent LTP in a large fraction of synapses.