New insights on the role of gephyrin in regulating both phasic and tonic GABAergic inhibition in rat hippocampal neurons in culture.

Gephyrin is a tubulin-binding protein that acts as a scaffold for clustering glycine and GABA(A) receptors at postsynaptic sites. In this study, the role of gephyrin on GABA(A) receptor function was assessed at the post-translational level, using gephyrin-specific single chain antibody fragments (scFv-gephyrin). When expressed in cultured rat hippocampal neurons as a fusion protein containing a nuclear localization signal, scFv-gephyrin were able to remove endogenous gephyrin from GABA(A) receptor clusters. Immunocytochemical experiments revealed a significant reduction in the number of synaptic gamma2-subunit containing GABA(A) receptors and a significant decrease in the density of the GABAergic presynaptic marker vesicular GABA transporter (VGAT). These effects were associated with a slow down of the onset kinetics, a reduction in the amplitude and in the frequency of miniature inhibitory postsynaptic currents (mIPSCs). The quantitative analysis of current responses to ultrafast application of GABA suggested that changes in onset kinetics resulted from modifications in the microscopic gating of GABA(A) receptors and in particular from a reduced entry into the desensitized state. In addition, hampering gephyrin function with scFv-gephyrin induced a significant reduction in GABA(A) receptor-mediated tonic conductance. This effect was probably dependent on the decrease in GABAergic innervation and in GABA release from presynaptic nerve terminals. These results indicate that gephyrin is essential not only for maintaining synaptic GABA(A) receptor clusters in the right position but also for regulating both phasic and tonic inhibition.

Multivesicular release at developing Schaffer collateral-CA1 synapses: an analytic approach to describe experimental data.

We developed and analytically solved a simple and general stochastic model to distinguish the univesicular from the multivesicular mode of glutamate release. The model solution gives analytical mathematical expressions for average values of quantities that can be measured experimentally. Comparison of these quantities with the experimental measures allows one to discriminate the release mode and to determine the most probable values of model parameters. The model has been validated at glutamatergic CA3-CA1 synapses in the hippocampus from newborn (P1-P5 old) rats. Our results strongly support a multivesicular type of release process requiring a variable pool of immediately releasable vesicles. Moreover, computing quantities that are functions of the model parameters, the mean amplitude of the synaptic response to the release of a single vesicle (q) was estimated to be 5-10 pA, in very good agreement with experimental findings. In addition a multivesicular type of release was supported by the following experimental evidences: 1) a high variability of the amplitude of successes, with a coefficient of variation ranging from 0.12 to 0.73; 2) an average potency ratio a2/a1 between the second and first response to a pair of stimuli >1; and 3) changes in the potency of the synaptic response to the first stimulus when the release probability was modified by increasing or decreasing the extracellular calcium concentration. Our results indicate that at Schaffer collateral-CA1 synapses of the neonatal rat hippocampus a single action potential may induce the release of more than one vesicle from the same release site.

Postsynaptic depolarisation enhances transmitter release and causes the appearance of responses at "silent" synapses in rat hippocampus.

Recent data indicate that most "silent" synapses in the hippocampus are "presynaptically silent" due to low transmitter release rather than "postsynaptically silent" due to "latent" receptors of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid type (AMPARs). That synapses bearing only N-methyl-d-aspartate (NMDAR) receptors do exist is suggested by the decreased number of transmission failures during postsynaptic depolarisation and by the presence of NMDA-mediated excitatory postsynaptic currents (EPSCs) in synapses silent at rest. We tested whether these effects could be due to potentiated transmitter release at depolarised postsynaptic potentials rather than removal of Mg(2+) block from NMDARs. Using whole-cell recordings of minimal EPSCs from CA1 and CA3 neurones of hippocampal slices we confirmed decreased incidence of failures at +40 mV as compared with -60 mV. This effect was associated with a gradual increase of EPSC amplitude after switching to +40 mV and with a decrease of paired-pulse facilitation. In initially silent synapses, potentiation of pharmacologically isolated AMPAR-mediated EPSCs was still observed at +40 mV and this persisted after stepping back to -60 mV. All above effects were blocked when the cell was dialysed with the Ca(2+) chelator BAPTA (20 mM). These observations are difficult to reconcile with the "latent AMPAR" hypothesis and suggest an alternative explanation, namely that the reduction in failure rates at positive potentials is due to potentiation of transmitter release following Ca(2+) influx through NMDARs. Our results suggest that silent synapses can be mainly "presynaptically" rather than "postsynaptically silent" and thus increased transmitter release rather than insertion of AMPARs is a major mechanism of early long-term potentiation maintenance.

Allosteric interaction of zinc with recombinant alpha(1)beta(2)gamma(2) and alpha(1)beta(2) GABA(A) receptors.

In a recent study we have provided evidence that inhibition of native GABA(A) receptors by zinc depends primarily on the allosteric modulation of receptor gating. Both the kinetics and the sensitivity of the GABA(A) receptor to zinc depend on subunit composition, especially on the presence of the gamma(2) subunit. To analyze the mechanism of action of zinc its effects have been tested on recombinant alpha(1)beta(2)gamma(2) and alpha(1)beta(2) receptors expressed in HEK 293 cells. The currents produced by ultrafast application of GABA have been measured to assess the impact of zinc ions on GABA(A) receptor gating with resolution corresponding to the time scale of synaptic currents. While, as expected, zinc markedly reduced the peak amplitude of alpha(1)beta(2)-mediated currents, its effect on kinetics was significantly different from that observed for alpha(1)beta(2)gamma(2). In particular, unlike alpha(1)beta(2)gamma(2), zinc did not affect the onset of alpha(1)beta(2)-mediated responses. Moreover, zinc increased the extent of desensitisation of alpha(1)beta(2)gamma(2) receptors and reduced desensitisation of alpha(1)beta(2) ones. Quantitative analysis suggests that zinc exerts an allosteric modulation on both alpha(1)beta(2)gamma(2) and alpha(1)beta(2) receptors. Zinc effects on alpha(1)beta(2)gamma(2) were qualitatively similar to those reported for native receptors.

Presynaptic R-type calcium channels contribute to fast excitatory synaptic transmission in the rat hippocampus.

The possibility that R-type calcium channels contribute to fast glutamatergic transmission in the hippocampus has been assessed using low concentrations of NiCl(2) and the peptide toxin SNX 482, a selective antagonist of the pore-forming alpha(1E) subunit of R-type calcium channel. EPSPs or EPSCs were recorded in the whole-cell configuration of the patch-clamp technique mainly from CA3 hippocampal neurons. Effects of both NiCl(2) and SNX 482 were tested on large (composite) EPSCs evoked by mossy and associative-commissural fiber stimulation. NiCl(2) effects were also tested on minimal EPSPs-EPSCs. Both substances reduced the amplitude of EPSPs-EPSCs. This effect was associated with an increase in the number of response failures of minimal EPSPs-EPSCs, an enhancement of the paired-pulse facilitation ratios of both minimal and composite EPSCs, and a reduction of the inverse squared coefficient of variation (CV(-2)). The reduction of CV(-2) was positively correlated with the decrease in EPSC amplitude. The inhibitory effect of NiCl(2) was occluded by SNX 482 but not by omega-conotoxin-MVIIC, a broad-spectrum antagonist thought to interact with N- and P/Q-type calcium channels, supporting a specific action of low concentrations of NiCl(2) on R-type calcium channels. Together, these observations indicate that both NiCl(2) and SNX 482 act at presynaptic sites and block R-type calcium channels with pharmacological properties similar to those encoded by the alpha(1E) gene. These channels are involved in fast glutamatergic transmission at hippocampal synapses.

Regulation of GABA release by nicotinic acetylcholine receptors in the neonatal rat hippocampus.

1. The whole-cell configuration of the patch-clamp technique was used to study the modulation of giant depolarizing potentials (GDPs) by nicotinic acetylcholine receptors (nAChRs) in CA3 hippocampal neurons in slices from postnatal day (P) 2-6 rats. 2. Bath application of nicotine increased GDP frequency in a concentration-dependent manner. For example, nicotine (0.5-1 microM) enhanced GDP frequency from 0.05 +/- 0.04 to 0.17 +/- 0.04 Hz. This effect was prevented by the broad-spectrum nicotinic receptor antagonist dihydro-beta-erythtroidine (DHbetaE, 50 microM) and partially antagonized by methyllycaconitine (MLA, 50 nM) a competitive antagonist of alpha7 nAChRs. GDP frequency was also enhanced by AR-17779 (100 microM), a selective agonist of alpha7 nAChRs. 3. The GABA(A) receptor antagonist bicuculline (10 microM) and the non-NMDA glutamate receptor antagonist DNQX (20 microM) blocked GDPs and prevented the effects of nicotine on GDPs. In the presence of DNQX, nicotine increased GABA-mediated synaptic noise, indicating that this drug may have a direct effect on GABAergic interneurons. 4. Bath application of edrophonium (20 microM), a cholinesterase inhibitor, in the presence of atropine (1 microM), increased GDP frequency, indicating that nAChRs can be activated by ACh released from the septo-hippocampal fibres. This effect was prevented by DHbetaE (50 microM). 5. In the majority of neurons tested, MLA (50 nM) and DHbetaE (50 microM) reduced the frequency of GDPs with different efficacy: a reduction of 98 +/- 11 and 61 +/- 29 % was observed with DHbetaE and MLA, respectively. In a subset of cells (40 % in the case of MLA and 17 % in the case of DHbetaE) these drugs induced a twofold increase in GDP frequency. 6. It is suggested that, during development, nAChRs modulate the release of GABA, assessed as GDPs, through distinct nAChRs. The rise of intracellular calcium via nAChRs would further strengthen GABA-mediated oscillatory activity. This can be crucial for consolidation of synaptic contacts and for the fine-tuning of the developing hippocampus.

Generating diversity at GABAergic synapses.

GABA-mediated transmission is characterized by high variability of synaptic responses. Major contributors to this variability are: presynaptic factors, including release probability and number of release sites; factors that determine synaptic GABA transients in the cleft, including diffusion and the actions of GABA transporters; and postsynaptic factors, including GABA(A) receptors subtypes, their location and number, their modulation by endogenous and exogenous factors, and their interactions with postsynaptic-anchoring proteins.

Early expression of GABA(A) receptor delta subunit in the neonatal rat hippocampus.

The cDNA library screening strategy was used to identify the genes encoding for GABA(A) receptor subunits in the rat hippocampus during development. With this technique, genes encoding eleven GABA(A) receptor subunits were identified. The alpha5 subunit was by far the most highly expressed, followed by the gamma2, alpha2 and alpha4 subunits respectively. The expression of the beta2, alpha1, gamma1, beta1 and beta3 subunits was moderate, although that of the alpha3 and delta subunits was weak. In situ hybridization experiments, using digoxigenin-labeled cRNA probes, confirmed that the delta subunit was expressed in the neonatal as well as in the adult hippocampus, and is likely to form functional receptors in association with other subunits of the GABA(A) receptor. When the more sensitive RT-PCR approach was used, the gamma3 subunit was also detected, suggesting that this subunit is present in the hippocampus during development but at low levels of expression. The insertion of the delta subunit into functional GABA(A) receptors may enhance the efficacy of GABA in the immediate postnatal period when this amino acid is still exerting a depolarizing and excitatory action.

Differences in amplitude-voltage relations between minimal and composite mossy fibre responses of rat CA3 hippocampal neurons support the existence of intrasynaptic ephaptic feedback in large synapses.

Computer simulations and electrophysiological experiments have been performed to test the hypothesis on the existence of an ephaptic interaction in purely chemical synapses. According to this hypothesis, the excitatory postsynaptic current would depolarize the presynaptic release site and further increase transmitter release, thus creating an intrasynaptic positive feedback. For synapses with the ephaptic feedback, computer simulations predicted non-linear amplitude-voltage relations and voltage dependence of paired-pulse facilitation. The deviation from linearity depended on the strength of the feedback determined by the value of the synaptic cleft resistance. The simulations showed that, in the presence of the intrasynaptic feedback, recruitment of imperfectly clamped synapses and synapses with linear amplitude-voltage relations tended to reduce the non-linearity and voltage dependence of paired-pulse facilitation. Therefore, the simulations predicted that the intrasynaptic feedback would particularly affect small excitatory postsynaptic currents induced by activation of electrotonically close synapses with long synaptic clefts. In electrophysiological experiments performed on hippocampal slices, the whole-cell configuration of the patch-clamp technique was used to record excitatory postsynaptic currents evoked in CA3 pyramidal cells by activation of large mossy fibre synapses. In accordance with the simulation results, minimal excitatory postsynaptic currents exhibited "supralinear" amplitude-voltage relations at hyperpolarized membrane potentials, decreases in the failure rate and voltage-dependent paired-pulse facilitation. Composite excitatory postsynaptic currents evoked by activation of a large amount of presynaptic fibres typically bear linear amplitude-voltage relationships and voltage-independent paired-pulse facilitation. These data are consistent with the hypothesis on a strong ephaptic feedback in large mossy fibre synapses. The feedback would provide a mechanism whereby signals from large synapses would be amplified. The ephaptic feedback would be more effective on synapses activated in isolation or together with electrotonically remote inputs. During synchronous activation of a large number of neighbouring inputs, suppression of the positive intrasynaptic feedback would prevent abnormal boosting of potent signals.

Postsynaptic hyperpolarization increases the strength of AMPA-mediated synaptic transmission at large synapses between mossy fibers and CA3 pyramidal cells.

In chemical synapses information flow is polarized. However, the postsynaptic cells can affect transmitter release via retrograde chemical signaling. Here we explored the hypothesis that, in large synapses, having large synaptic cleft resistance, transmitter release can be enhanced by electrical (ephaptic) signaling due to depolarization of the presynaptic release site induced by the excitatory postsynaptic current itself. The hypothesis predicts that, in such synapses, postsynaptic hyperpolarization would increase response amplitudes "supralinearly", i.e. stronger than predicted from the driving force shift. We found supralinear increases in the amplitude of minimal excitatory postsynaptic potential (EPSP) during hyperpolarization of CA3 pyramidal neurons. Failure rate, paired-pulse facilitation, coefficient of variation of the EPSP amplitude and EPSP quantal content were also modified. The effects were especially strong on mossy fiber EPSPs (MF-EPSPs) mediated by the activation of large synapses and identified pharmacologically or by their kinetics. The effects were weaker on commissural fiber EPSPs mediated by smaller and more remote synapses. Even spontaneous membrane potential fluctuations were associated with supralinear MF-EPSP increases and failure rate reduction. The results suggest the existence of a novel mechanism for retrograde control of synaptic efficacy from postsynaptic membrane potential and are consistent with the ephaptic feedback hypothesis.

Silent synapses in the developing hippocampus: lack of functional AMPA receptors or low probability of glutamate release?

At early developmental stages, silent synapses have been commonly found in different brain areas. These synapses are called silent because they do not respond at rest but are functional at positive membrane potentials. A widely accepted interpretation is that N-methyl-d-aspartate (NMDA) but not alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors are functionally expressed on the subsynaptic membrane. Here we show that, in both CA3 and CA1 hippocampal regions, AMPA-mediated synaptic responses can be detected already at early stages of postnatal development. However, some synapses appear silent because of a very low probability of glutamate release. They can be converted into functional ones by factors that enhance release probability such as paired-pulse stimulation, increasing the temperature or cyclothiazide (CTZ), a drug that blocks AMPA receptor desensitization and increases transmitter release. Conversely, conducting synapses can be switched off by increasing the frequency of stimulation. Although we cannot exclude that "latent AMPA receptors" can become functional after activity-dependent processes, our results clearly indicate that, in the neonatal hippocampus, a proportion of glutamatergic synaptic connections are presynaptically rather than postsynaptically silent.

'Specific' oligonucleotides often recognize more than one gene: the limits of in situ hybridization applied to GABA receptors.

As exquisite probes for gene sequences, oligonucleotides are one of the most powerful tools of recombinant molecular biology. In studying the GABA receptor subunits in the neonatal hippocampus we have used oligonucleotide probes in in situ hybridization and cloning techniques. The oligonucleotides used and assumed to be specific for the target gene, actually recognized more than one gene, leading to surprising and contradictory results. In particular, we found that a GABA(A)-rho specific oligonucleotide recognized an abundant, previously unknown, transcription factor in both in situ and library screening, while oligos 'specific' for GABA(A) subunits were able to recognize 30 additional unrelated genes in library screening. This suggests that positive results obtained with oligonucleotides should be interpreted with caution unless confirmed by identical results with oligonucleotides from different parts of the same gene, or cDNA library screening excludes the presence of other hybridizing species.

GABA- and glutamate-mediated network activity in the hippocampus of neonatal and juvenile rats revealed by fast calcium imaging.

In the rat hippocampus, during the first postnatal week, network activity is characterized by GABA-driven giant depolarizing potentials (GDPs) associated with calcium signals that are readily blocked when the GABAA antagonist bicuculline is applied to the bath. Towards the end of the first postnatal week, in concomitance with the shift of GABA responses from the depolarizing to the hyperpolarizing direction, functional glutamatergic connections start appearing. At this developmental stage, application of bicuculline blocks GABAA-mediated inhibition and induces the appearance of interictal epileptiform discharges. In the present experiments, we have used a high spatio-temporal resolution imaging system to compare, on a time scale of tens of ms, the onset and propagation of fast calcium transients generated within a GABAergic or glutamatergic network. We found that, during the first postnatal week, calcium signals associated to evoked GDPs arise from the activation of a local circuitry of neurons spanning the stratum radiatum and the pyramidal layer. Similar activation patterns were elicited by focal application of GABA in the presence of kynurenic acid, a broad spectrum ionotropic glutamatergic antagonist, and were blocked by bicuculline. During the second postnatal week, in the presence of bicuculline, calcium signals associated with interictal discharges evoked by stimulation of glutamatergic fibres propagated along the well-defined three-synaptic pathway from the dentate gyrus to the CA1 hippocampal area.

Zinc inhibits miniature GABAergic currents by allosteric modulation of GABAA receptor gating.

Zinc is abundantly present in the CNS, and after nerve stimulation is thought to be released in sufficient quantity to modulate the synaptic transmission. Although it is known that this divalent cation inhibits the GABAergic synaptic currents, the underlying mechanisms were not fully elucidated. Here we report that zinc reduced the amplitude, slowed the rise time, and accelerated the decay of mIPSCs in cultured hippocampal neurons. The analysis of current responses to rapid GABA applications and model simulations indicated that these effects on mIPSCs are caused by zinc modulation of GABA(A) receptor gating. In particular, zinc slowed the onset of GABA-evoked currents by decreasing both the binding (k(on)) and the transition rate from closed to open state (beta(2)). Moreover, slower onset and recovery from desensitization as well as an increased unbinding rate (k(off)) were shown to underlie the accelerated deactivation kinetics in the presence of zinc. The nonequilibrium conditions of GABA(A) receptor activation were found to strongly affect zinc modulation of this receptor. In particular, an extremely fast clearance of synaptic GABA is implicated to be responsible for a stronger zinc effect on mIPSCs than on current responses to exogenous GABA. Finally, the analysis of currents evoked by GABA coapplied with zinc indicated that the interaction between zinc and GABA(A) receptors was too slow to explain zinc effects in terms of competitive antagonism. In conclusion, our results provide evidence that inhibition of mIPSCs by zinc is attributable to the allosteric modulation of GABA(A) receptor gating.

Long-term synaptic changes induced by intracellular tetanization of CA3 pyramidal neurons in hippocampal slices from juvenile rats.

Minimal excitatory postsynaptic potentials were evoked in CA3 pyramidal neurons by activation of the mossy fibres in hippocampal slices from seven- to 16-day-old rats. Conditioning intracellular depolarizing pulses were delivered as 50- or 100-Hz bursts. A statistically significant depression and potentiation was induced in four and five of 13 cases, respectively. The initial state of the synapses influenced the effect: the amplitude changes correlated with the pretetanic paired-pulse facilitation ratio. Afferent (mossy fibre) tetanization produced a significant depression in four of six inputs, and no significant changes in two inputs. Quantal content decreased or increased following induction of the depression or potentiation, respectively, whereas no significant changes in quantal size were observed. Compatible with presynaptic maintenance mechanisms of both depression and potentiation, changes in the mean quantal content were associated with modifications in the paired-pulse facilitation ratios, coefficient of variation of response amplitudes and number of response failures. Cases were encountered when apparently "presynaptically silent" synapses were converted into functional synapses during potentiation or when effective synapses became "presynaptically silent" when depression was induced, suggesting respective changes in the probability of transmitter release. It is concluded that, in juvenile rats, it is possible to induce lasting potentiation at the mossy fibre-CA3 synapses by purely postsynaptic stimulation, while afferent tetanization is accompanied by long-lasting depression. The data support the existence not only of a presynaptically induced, but also a postsynaptically induced form of long-term potentiation in the mossy fibre-CA3 synapse. Despite a postsynaptic induction mechanism, maintenance of both potentiation and depression is likely to occur presynaptically.

Facilitation of miniature GABAergic currents by chlorpromazine in cultured rat hippocampal cells.

The whole cell configuration of the patch clamp technique was used to study the effects of chlorpromazine (CPZ), a widely used antipsychotic drug, on miniature GABAA-mediated synaptic currents (mIPSCs) in hippocampal cells in culture. CPZ (10-30 microM) induced a clear dose-dependent increase of mIPSCs frequency that was associated with a decrease in amplitude and with an acceleration of their decay kinetics. When applied in a calcium-free medium, CPZ was less effective in enhancing mIPSCs frequency, suggesting that this effect was partially calcium dependent. While a low (10 microM) CPZ concentration induced a 2-fold increase in the total charge transfer a higher (30 microM) dose of this drug produced no changes, indicating that the presynaptic effect was counterbalanced by the postsynaptic one.

Low expression of the ClC-2 chloride channel during postnatal development: a mechanism for the paradoxical depolarizing action of GABA and glycine in the hippocampus.

In early postnatal development, during the period of synapse formation, gamma-aminobutyric acid (GABA) and glycine, the main inhibitory transmitters in the adult brain, paradoxically excite and depolarize neuronal membranes by an outward flux of chloride. The mechanisms of chloride homeostasis are not fully understood. It is known that in adult neurons intracellular chloride accumulation is prevented by a particular type of chloride channel, the ClC-2. This channel strongly rectifies in the inward direction at potentials negative to ECl thus ensuring chloride efflux. We have tested the hypothesis that in the developing hippocampus, a differential expression or regulation of ClC-2 channels may contribute to the depolarizing action of GABA and glycine. We have cloned a truncated form of ClC-2 (ClC-2nh) from the neonatal hippocampus which lacks the 157 bp corresponding to exon 2. In situ hybridization experiments show that ClC-2nh is the predominant form of ClC-2 mRNA in the neonatal brain. ClC-2nh mRNA is unable to encode a full-length protein due to a frameshift, consequently it does not induce any currents upon injection into Xenopus oocytes. Low expression of the full-length ClC-2 channel, could alter chloride homeostasis, lead to accumulation of [Cl-]i and thereby contribute to the depolarizing action of GABA and glycine during early development.

Muscarinic receptor modulation of GABA-mediated giant depolarizing potentials in the neonatal rat hippocampus.

1. The whole-cell patch clamp technique was used to study the role of muscarinic receptors in regulating the frequency of giant depolarizing potentials (GDPs) in CA3 hippocampal neurones in slices from postnatal (P) P1-P8 rats. 2. Atropine (1 microM) reduced the frequency of GDPs by 64.2 +/- 2.9 %. The acetylcholinesterase inhibitor edrophonium (20 microM) increased the frequency of GDPs in a developmentally regulated way. This effect was antagonized by the M1 muscarinic receptor antagonist pirenzepine. 3. In the presence of edrophonium, tetanic stimulation of cholinergic fibres induced either an enhancement of GDP frequency (179 +/- 79 %) or a membrane depolarization (27 +/- 16 mV) associated with an increase in synaptic noise. These effects were prevented by atropine. 4. Application of carbachol (3 microM) produced an increase in GDP frequency that at P5-P6 was associated with a membrane depolarization and an increase in synaptic noise. These effects were prevented by atropine, pirenzepine (3 microM) and bicuculline (10 microM). 5. In the presence of pirenzepine, carbachol reduced GDP frequency by 50 +/- 4 %. Conversely, in the presence of methoctramine (3 microM), carbachol enhanced GDP frequency by 117 +/- 4 %. 6. It is concluded that endogenous acetylcholine, through the activation of M1 receptors, enhances the release of gamma-aminobutyric acid (GABA), in a developmentally regulated way. On the other hand, carbachol exerts both an up- and downregulation of GABA release through the activation of M1 and M2 receptors, respectively.

Glutamate controls the induction of GABA-mediated giant depolarizing potentials through AMPA receptors in neonatal rat hippocampal slices.

Glutamate controls the induction of GABA-mediated giant depolarizing potentials through AMPA receptors in neonatal rat hippocampal slices. Giant depolarizing potentials (GDPs) are generated by the interplay of the depolarizing action of GABA and glutamate. In this study, single and dual whole cell recordings (in current-clamp configuration) were performed from CA3 pyramidal cells in hippocampal slices obtained from postnatal (P) days P1- to P6-old rats to evaluate the role of ionotropic glutamate receptors in GDP generation. Superfusion of 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) (10-40 microM) completely blocked GDPs. However, in the presence of CNQX, it was still possible to re-induce the appearance of GDPs with GABA (20 microM) or (RS)-alpha-amino-3-hydroxy-5-methyl-4-isoxadepropionate (AMPA) (5 microM). This effect was prevented by the more potent and selective AMPA receptor antagonist GYKI 53655 (50-100 microM). In the presence of GYKI 53655, both kainic or domoic acid (0.1-1 microM) were unable to induce GDPs. In contrast, bath application of D-(-)-2-amino-5-phosphonopentanoic acid (50 microM) or (+)-3-(2carboxy-piperazin-4-yl)-propyl-L-phosphonic acid (20 microM) produced only a 37 +/- 9% (SE) and 36 +/- 11% reduction in GDPs frequency, respectively. Cyclothiazide, a selective blocker of AMPA receptor desensitization, increased GDP frequency by 76 +/- 14%. Experiments were also performed with an intracellular solution containing KF to block GABAA receptor-mediated responses. In these conditions, a glutamatergic component of GDP was revealed. GDPs could still be recorded synchronous with those detected simultaneously with KCl-filled electrodes, although their amplitude was smaller. Similar results were found in pair recordings obtained from minislices containing only a small portion of the CA3 area. These data suggest that GDP generation requires activation of AMPA receptors by local release of glutamate from recurrent collaterals.

Chlorpromazine inhibits miniature GABAergic currents by reducing the binding and by increasing the unbinding rate of GABAA receptors.

Recent studies have emphasized that nonequilibrium conditions of postsynaptic GABAA receptor (GABAAR) activation is a key factor in shaping the time course of IPSCs (Puia et al., 1994; Jones and Westbrook, 1995). Such nonequilibrium, resulting from extremely fast agonist time course, may affect the interaction between pharmacological agents and postsynaptic GABAARs. In the present study we found that chlorpromazine (CPZ), a widely used antipsychotic drug known to interfere with several ligand and voltage-gated channels, reduces the amplitude and accelerates the decay of miniature IPSCs (mIPSCs). A good qualitative reproduction of the effects of CPZ on mIPSCs was obtained when mIPSCs were mimicked by responses to ultrafast GABA applications to excised patches. Our experimental data and model simulations indicate that CPZ affects mIPSCs by decreasing the binding (kon) and by increasing the unbinding (koff) rates of GABAARs. Because of reduction of kon by CPZ, the binding reaction becomes rate-limiting, and agonist exposure of GABAARs during mIPSC is too short to activate the receptors to the same extent as in control conditions. The increase in unbinding rate is implicated as the mechanism underlying the acceleration of mIPSC decaying phase. The effect of CPZ on GABAAR binding rate, resulting in slower onset of GABA-evoked currents, provides a tool to estimate the speed of synaptic clearance of GABA. Moreover, the onset kinetics of recorded responses allowed the estimate the peak synaptic GABA concentration.