Glutamate-induced suppression of inhibitory synaptic transmission in cultivated hippocampal neurons

In a dissociated culture of rat hippocampal neurons (14 to 24 daysin vitro), modulation effects of glutamate on GABA_A-ergic inhibitory transmission were studied with the use of simultaneous patch-clamp whole-cell recording from monosynaptically connected neuron pairs. In all experiments (n=49), 1.5-min-long or longer extracellular application of 0.5 to 100 μM glutamate suppressed evoked inhibitory postsynaptic currents (IPSC). This suppression usually included fast (seconds) and slow (τ=1.3 min) phases. In 83.7% of the cases studied, IPSC did not return to the control values during the entire subsequent recording period (from 10 to 64 min). When glutamate was applied in the presence of blockers of glutamate ionotropic receptors, DL-APV or CNQX, the fast phase of the effect was removed, while some suppression of inhibitory neuronal responses, although weaker, was preserved (n=19); in most cases (73.3%) this residual suppression was slow and long-lasting. It is concluded that both types of glutamate receptors, ionotropic and metabotropic, are involved in modulation of GABA_A-ergic synaptic transmission. The first above receptor type provides fast and reversible suppression, while the effect provided by the second type is slow and long-lasting.     

Astrocyte activation of presynaptic metabotropic glutamate receptors modulates hippocampal inhibitory synaptic transmission

In the CNS, fine processes of astrocytes often wrap around dendrites, axons and synapses, which provides an interface where neurons and astrocytes might interact. We have reported previously that selective Ca(2+) elevation in astrocytes, by photolysis of caged Ca(2+) by o-nitrophenyl-EGTA (NP-EGTA), causes a kainite receptor-dependent increase in the frequency of spontaneous inhibitory post-synaptic potentials (sIPSCs) in neighboring interneurons in hippocampal slices. However, tetrodotoxin (TTX), which blocks action potentials, reduces the frequency of miniature IPSCs (mIPSCs) in interneurons during Ca(2+) uncaging by an unknown presynaptic mechanism. In this study we investigate the mechanism underlying the presynaptic inhibition. We show that Ca(2+) uncaging in astrocytes is accompanied by a decrease in the amplitude of evoked IPSCs (eIPSCs) in neighboring interneurons. The decreases in eIPSC amplitude and mIPSC frequency are prevented by CPPG, a group II/III metabotropic glutamate receptor (mGluR) antagonist, but not by the AMPA/kainate and NMDA receptor antagonists CNQX/CPP. Application of either the group II mGluR agonist DCG IV or the group III mGluR agonist L-AP4 decreased the amplitude of eIPSCs by a presynaptic mechanism, and both effects are blocked by CPPG. Thus, activation of mGluRs mediates the effects of Ca(2+) uncaging on mIPSCs and eIPSCs. Our results indicate that Ca(2+)-dependent release of glutamate from astrocytes can activate distinct classes of glutamate receptors and differentially modulate inhibitory synaptic transmission in hippocampal interneurons.     

SNAP-29-mediated modulation of synaptic transmission in cultured hippocampal neurons

Identifying the molecules that regulate both the recycling of synaptic vesicles and the SNARE components required for fusion is critical for elucidating the molecular mechanisms underlying synaptic plasticity. SNAP-29 was initially isolated as a syntaxin-binding and ubiquitously expressed protein. Previous studies have suggested that SNAP-29 inhibits SNARE complex disassembly, thereby reducing synaptic transmission in cultured superior cervical ganglion neurons in an activity-dependent manner. However, the role of SNAP-29 in regulating synaptic vesicle recycling and short-term plasticity in the central nervous system remains unclear. In the present study, we examined the effect of SNAP-29 on synaptic transmission in cultured hippocampal neurons by dual patch clamp whole-cell recording, FM dye imaging, and immunocytochemistry. Our results demonstrated that exogenous expression of SNAP-29 in presynaptic neurons significantly decreased the efficiency of synaptic transmission after repetitive firing within a few minutes under low and moderate frequency stimulations (0.1 and 1 Hz). In contrast, SNAP-29 did not affect the density of synapses and basal synaptic transmission. Whereas neurotransmitter release was unaffected during intensive stimulation, recovery after synaptic depression was impaired by SNAP-29. Furthermore, knockdown of SNAP-29 expression in neurons by small interfering RNA increased the efficiency of synaptic transmission during repetitive firing. These findings suggest that SNAP-29 acts as a negative modulator for neurotransmitter release, probably by slowing recycling of the SNARE-based fusion machinery and synaptic vesicle turnover.     

HCN channel activity-dependent modulation of inhibitory synaptic transmission in the rat basolateral amygdala

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are expressed in the central nervous system and play a regulatory role in neuronal excitability. In the present study, we examined a physiological role of HCN channels in the rat basolateral amygdala (BLA). In vitro electrophysiological studies showed that ZD7288 decreased spontaneous inhibitory postsynaptic current (sIPSC) without changing miniature IPSC (mIPSC). HCN channel blockade also attenuated feedback inhibitions in BLA principal neurons. However, blockade of HCN channel had little effects on spontaneous excitatory postsynaptic current (sEPSC) and mEPSC. Therefore, HCN channel appeared to decrease BLA excitability by increasing the action potential-dependent inhibitory control over the BLA principal neurons. Anxiety is reported to be influenced by neuronal excitability in the BLA and inhibitory synaptic transmission is thought to play a pivotal role in regulating overall excitability of the amygdala. As expected, blockade of HCN channels by targeted injection of ZD7288 to the BLA increased anxiety-like behavior under elevated plus maze test. Our results suggest that HCN channel activity can modulate the GABAergic synaptic transmission in the BLA, which in turn control the amygdala-related emotional behaviors such as anxiety.     

Bi-directional modulation of fast inhibitory synaptic transmission by leptin

The hormone leptin has widespread actions in the CNS. Indeed, leptin markedly influences hippocampal excitatory synaptic transmission and synaptic plasticity. However, the effects of leptin on fast inhibitory synaptic transmission in the hippocampus have not been evaluated. Here, we show that leptin modulates GABA_A receptor-mediated synaptic transmission onto hippocampal CA1 pyramidal cells. Leptin promotes a rapid and reversible increase in the amplitude of evoked GABA_A receptor-mediated inhibitory synaptic currents (IPSCs); an effect that was paralleled by increases in the frequency and amplitude of miniature IPSCs, but with no change in paired pulse ratio or coefficient of variation, suggesting a post-synaptic expression mechanism. Following washout of leptin, a persistent depression (inhibitory long-lasting depression) of evoked IPSCs was observed. Whole-cell dialysis or bath application of inhibitors of phosphoinositide 3 (PI 3)-kinase or Akt prevented leptin-induced enhancement of IPSCs indicating involvement of a post-synaptic PI 3-kinase/Akt-dependent pathway. In contrast, blockade of PI 3-kinase or Akt activity failed to alter the ability of leptin to induce inhibitory long-lasting depression, suggesting that this process is independent of PI 3-kinase/Akt. In conclusion these data indicate that the hormone leptin bi-directionally modulates GABA_A receptor-mediated synaptic transmission in the hippocampus. These findings have important implications for the role of this hormone in regulating hippocampal pyramidal neuron excitability.     

Calcium and endocannabinoids in the modulation of inhibitory synaptic transmission

Synapses in the central nervous system can be highly plastic devices, being able to modify their efficacy in relaying information in response to several factors. Calcium ions are often fundamental in triggering synaptic plasticity. Here, we will shortly review the effects induced by postsynaptic increases of calcium concentration at GABAergic and glycinergic synapses. Both postsynaptic and presynaptic mechanisms mediating changes in synaptic strength will be examined. Particular attention will be devoted to phenomena of retrograde signaling and, specifically, to the recently discovered role, played by the endocannabinoid system in retrograde synaptic modulation.     

Mutant presenilin 1 alters synaptic transmission in cultured hippocampal neurons

Mutations in presenilins are the major cause of familial Alzheimer disease, but the precise pathogenic mechanism by which presenilin (PS) mutations cause synaptic dysfunction leading to memory loss and neurodegeneration remains unclear. Using autaptic hippocampal cultures from transgenic mice expressing human PS1 with the A246E mutation, we demonstrate that mutant PS1 significantly depressed the amplitude of evoked alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid and N-methyl-D-aspartate receptor-mediated synaptic currents. Analysis of the spontaneous miniature synaptic activity revealed a lower frequency of miniature currents but normal miniature amplitude. Both alterations could be rescued by the application of a gamma-secretase blocker. On the other hand, the application of synthetic soluble Abeta42 in wild-type neurons induced the PS1 mutant phenotype on synaptic strength. Together, these findings strongly suggest that the expression of mutant PS1 in cultured neurons depresses synaptic transmission by causing a physical reduction in the number of synapses. This hypothesis is consistent with morphometic and semiquantitative immunohistochemical analysis, revealing a decrease in synaptophysin-positive puncta in PS1 mutant hippocampal neurons.     

CLC-3 channels modulate excitatory synaptic transmission in hippocampal neurons

It is well established that ligand-gated chloride flux across the plasma membrane modulates neuronal excitability. We find that a voltage-dependent Cl(-) conductance increases neuronal excitability in immature rodents as well, enhancing the time course of NMDA receptor-mediated miniature excitatory postsynaptic potentials (mEPSPs). This Cl(-) conductance is activated by CaMKII, is electrophysiologically identical to the CaMKII-activated CLC-3 conductance in nonneuronal cells, and is absent in clc-3(-/-) mice. Systematically decreasing [Cl(-)](i) to mimic postnatal [Cl(-)](i) regulation progressively decreases the amplitude and decay time constant of spontaneous mEPSPs. This Cl(-)-dependent change in synaptic strength is absent in clc-3(-/-) mice. Using surface biotinylation, immunohistochemistry, electron microscopy, and coimmunoprecipitation studies, we find that CLC-3 channels are localized on the plasma membrane, at postsynaptic sites, and in association with NMDA receptors. This is the first demonstration that a voltage-dependent chloride conductance modulates neuronal excitability. By increasing postsynaptic potentials in a Cl(-) dependent fashion, CLC-3 channels regulate neuronal excitability postsynaptically in immature neurons.     

Reversine inhibits spontaneous synaptic transmission in cultured rat hippocampal neurons

The dedifferentiation agent "reversine" [2-(4-morpholinoanilino)-N6-cyclohexyladenine 2], which can induce myogenic lineage-committed cells to become multipotent mesenchymal progenitor cells, was discovered by Shuibing Chen et al. in 2003. But its effects on neurons were unknown. Using patch-clamp technique, we found that reversine inhibits spontaneous synaptic transmission in cultured rat hippocampal neurons without influencing the dynamics function of potassium, sodium and calcium channels. This result suggests that reversine may also act as a dedifferentiation agent in neurons, and inhibiting the synaptic transmission maybe the early step of neuronal dedifferentiation.     

Wnt-7a modulates the synaptic vesicle cycle and synaptic transmission in hippocampal neurons

Wnt signaling is essential for neuronal development and the maintenance of the developing nervous system. Recent studies indicated that Wnt signaling modulates long term potentiation in adult hippocampal slices. We report here that different Wnt ligands are present in hippocampal neurons of rat embryo and adult rat, including Wnt-4, -5a, -7a, and -11. Wnt-7a acts as a canonical Wnt ligand in rat hippocampal neurons, stimulates clustering of presynaptic proteins, and induces recycling and exocytosis of synaptic vesicles as studied by FM dyes. Wnt-3a has a moderate effect on recycling of synaptic vesicles, and no effect of Wnt-1 and Wnt-5a was detected. Electrophysiological analysis on adult rat hippocampal slices indicates that Wnt-7a, but not Wnt-5a, increases neurotransmitter release in CA3-CA1 synapses by decreasing paired pulse facilitation and increasing the miniature excitatory post-synaptic currents frequency. These results indicate that the presynaptic function of rat hippocampal neurons is modulated by the canonical Wnt signaling.     

Calcium channel structural determinants of synaptic transmission between identified invertebrate neurons

We report here that unlike what was suggested for many vertebrate neurons, synaptic transmission in Lymnaea stagnalis occurs independent of a physical interaction between presynaptic calcium channels and a functional complement of SNARE proteins. Instead, synaptic transmission in Lymnaea requires the expression of a C-terminal splice variant of the Lymnaea homolog to mammalian N- and P/Q-type calcium channels. We show that the alternately spliced region physically interacts with the scaffolding proteins Mint1 and CASK, and that synaptic transmission is abolished following RNA interference knockdown of CASK or after the injection of peptide sequences designed to disrupt the calcium channel-Mint1 interactions. Our data suggest that Mint1 and CASK may serve to localize the non-L-type channels at the active zone and that synaptic transmission in invertebrate neurons utilizes a mechanism for optimizing calcium entry, which occurs independently of a physical association between calcium channels and SNARE proteins.     

Synaptic localization and presynaptic function of calcium channel beta 4-subunits in cultured hippocampal neurons

Neurotransmitter release is triggered by the influx of Ca(2+) into the presynaptic terminal through voltage gated Ca(2+)-channels. The shape of the presynaptic Ca(2+) signal largely determines the amount of released quanta and thus the size of the synaptic response. Ca(2+)-channel function is modulated in particular by the auxiliary beta-subunits that interact intracellularly with the pore-forming alpha(1)-subunit. Using retrovirus-mediated gene transfer in cultured hippocampal neurons, we demonstrate that functional GFP-beta(4) constructs colocalize with the synaptic vesicle marker synaptobrevin II and endogenous P/Q-type channels, indicating that beta(4)-subunits are localized to synaptic sites. Costaining with the dendritic marker MAP2 revealed that the beta(4)-subunit is transported to dendrites as well as axons. The nonconserved amino- and carboxyl-termini of the beta(4)-subunit were found to target the protein to the synapse. Physiological measurements in autaptic hippocampal neurons infected with green fluorescent protein (GFP)-beta(4) revealed an increase in both excitatory post-synaptic current amplitude and paired pulse facilitation ratio, whereas the GFP-beta(4) mutant, GFP-beta(4)(Delta50-407), which demonstrated a cytosolic localization pattern, did not alter these synaptic properties. In summary, our data suggest a pre-synaptic function of the Ca(2+)-channel beta(4)-subunit in synaptic transmission.     

Astrocyte-induced modulation of synaptic transmission

The idea that astrocytes simply provide structural and trophic support to neurons has been challenged by recent evidence demonstrating that astrocytes exhibit a form of excitability and communication based on intracellular Ca2+ variations and intercellular Ca2+ waves, which can be initiated by neuronal activity. These astrocyte Ca2+ variations have now been shown to induce glutamate-dependent Ca2+ elevations and slow inward currents in neurons. More recently, it has been demonstrated that synaptic transmission between cultured hippocampal neurons can be directly modulated by astrocytes. We have reported that astrocyte stimulation can increase the frequency of miniature synaptic currents. Furthermore, we also have demonstrated that an elevation in the intracellular Ca2+ in astrocytes induces a reduction in both excitatory and inhibitory evoked synaptic transmission through the activation of selective presynaptic metabotropic glutamate receptors.     

Calcium-induced modulation of synaptic transmission

Calcium (Ca²⁺) is a second messenger regulating a wide variety of intracellular processes. Using GABA-and glycinergic synapses as examples, this review analyzes two functions of this unique ion: postsynaptic Ca²⁺-dependent modulation of receptor-operated channels and Ca²⁺-induced retrograde regulation of neurotransmitter release from the presynaptic terminals. Phosphorylation, rapid Ca²⁺-induced modulation via intermediate Ca²⁺-binding proteins, and changes in the number of functional receptors represent the main pathways of short-and long-term plasticity of postsynaptic receptor-operated channel machinery. Retrograde signaling is an example of synaptic modulation triggered by stimulation of postsynaptic cells and mediated via regulation of presynaptic neurotransmitter release. This mechanism provides postsynaptic neurons with efficient tools to control the presynaptic afferents in an activity-dependent mode. Elevation of intracellular Ca²⁺ in a postsynaptic neuron triggers the synthesis of endocannabinoids (derivatives of arachidonic acid). Their retrograde diffusion through the synaptic cleft and consequent activation of presynaptic G-protein coupled to CB1 receptors inhibits the release of neurotransmitter. These mechanisms of double modulation, which include control over the function of postsynaptic ion channels and retrograde suppression of the release machinery, play an important role in Ca²⁺-dependent control of the main excitatory and inhibitory synaptic pathways in the mammalian nervous system.     

Chemokine CCL2 modulation of neuronal excitability and synaptic transmission in rat hippocampal slices

In addition to its well-characterized effects in immune system, chemokine CC motif ligand 2 (CCL2, formerly known as monocyte chemoattractant protein-1) is believed to play an important role in brain physiological and pathological processes. It has been shown that CCL2 and its cognate receptor chemokine CC motif receptor 2 are constitutively expressed in several brain regions including the hippocampus, and the expression is up-regulated under pathological conditions. Whereas most investigations have so far focused on its involvement in CNS pathology, few studies have examined the effects of CCL2 on neuronal and synaptic physiology. In this study, we tested the effects of CCL2 on neuronal excitability and excitatory synaptic transmission in the CA1 region of rat hippocampal slices using whole-cell patch clamp techniques. Bath application of CCL2 depolarized membrane potential and increased spike firing in CA1 neuronal cells. Bath application of CCL2 also produced an increase of excitatory post-synaptic currents recorded in Schaffer-collateral fibers to CA1 synapses. Quantal analysis revealed that CCL2 increased the frequency of spontaneous excitatory post-synaptic current occurrence and mean quantal content. Taken together, our data indicate that CCL2 enhances neuronal excitability and synaptic transmission via pre-synaptic mechanisms. These results support the emerging concept that chemokines function as neuromodulators in the CNS.     

Serotoninergic modulation of GABAergic synaptic transmission in developing rat CA3 pyramidal neurons

Serotoninergic modulation of GABAergic mIPSCs was investigated in immature (postnatal 12–16-days old) rat CA3 pyramidal neurons using a conventional whole-cell patch clamp technique. Serotonin or 5-hydroxytryptamine (5-HT) (10 μmol/L) transiently and explosively increased mIPSC frequency with a small increase in the current amplitude. However, 5-HT did not affect the GABA-induced postsynaptic currents, indicating that 5-HT acts presynaptically to facilitate the probability of spontaneous GABA release. The 5-HT action on GABAergic mIPSC frequency was completely blocked by 100 nmol/L MDL72222, a selective 5-HT₃ receptor antagonist, and mimicked by mCPBG, a selective 5-HT₃ receptor agonist. The 5-HT action on GABAergic mIPSC frequency was completely occluded either in the presence of 200 μmol/L Cd²⁺ or in the Na⁺-free external solution, suggesting that the 5-HT₃ receptor-mediated facilitation of mIPSC frequency requires a Ca²⁺influx passing through voltage-dependent Ca²⁺channels from the extracellular space, and that presynaptic 5-HT₃ receptors are less permeable to Ca²⁺. The 5-HT action on mIPSC frequency in the absence or presence of extracellular Na⁺ gradually increased with postnatal development. Such a developmental change in the 5-HT₃ receptor-mediated facilitation of GABAergic transmission would play important roles in the regulation of excitability as well as development in CA3 pyramidal neurons.     

丙泊酚拮抗戊四氮对大鼠海马CA1区动作电位和突触传递的影响

目的：观察戊四氮对大鼠海马CA1区动作电位（action potential,AP）和兴奋性突触后电流（excitatory postsynaptic current,EPSC）的影响和丙泊酚的拮抗作用。方法：断头法分离Wistar大鼠海马半脑,切片机切出400μm厚度的海马脑片,全细胞电流钳记录CA1区锥体神经元动作电位发放情况,全细胞电压钳记录电刺激Schaeffer侧支／联合纤维诱发的CA1区锥体神经元EPSC的变化。结果：戊四氮使动作电位发放频率增加,EPSC值降低;丙泊酚拮抗戊四氮的作用,使动作电位发放减少甚至消失,EPSC值上升至加入丙泊酚前的2倍左右。结论：丙泊酚拮抗戊四氮对动作电位和EPSC的作用,所以临床上可用于抗癫痫治疗。