Paradoxical reduction of synaptic inhibition by vigabatrin.

GABAergic inhibition, a primary target for pharmacological modulation of excitability in the CNS, can be altered by multiple mechanisms including alteration of GABA metabolism. Gamma-vinyl GABA (vigabatrin, GVG) is an irreversible inhibitor of the GABA catabolic enzyme GABA transaminase, thus its anticonvulsant properties are thought to result from an elevation of brain GABA levels. We examined the effects of GVG on GABAergic synaptic transmission in hippocampal slices. GVG unexpectedly reduced miniature and evoked inhibitory postsynaptic currents (IPSCs) in dentate granule cells. The reduction in synaptic events was accompanied by an increase in tonic GABA(A) receptor-mediated current. These effects developed slowly and persisted following wash out of GVG. The GVG pretreatment reduced sucrose-evoked GABA release as well as postsynaptic sensitivity to exogenous GABA, indicating that both pre- and postsynaptic mechanisms contributed to the reduction in synaptic currents. These results suggest that tonic rather than phasic increases in GABA underlie the anticonvulsant properties of GVG, and that mechanisms that elevate brain neurotransmitter levels do not necessarily correlate with enhanced synaptic release.

Slow desensitization regulates the availability of synaptic GABA(A) receptors.

At central synapses, a large and fast spike of neurotransmitter efficiently activates postsynaptic receptors. However, low concentrations of transmitter can escape the cleft and activate presynaptic and postsynaptic receptors. We report here that low concentrations of GABA reduce IPSCs in hippocampal neurons by preferentially desensitizing rather than opening GABA(A) channels. GABA transporter blockade also caused desensitization by locally elevating GABA to approximately 1 microm. Recovery of the IPSC required several seconds, mimicking recovery of the channel from slow desensitization. These results indicate that low levels of GABA can regulate the amplitude of IPSCs by producing a slow form of receptor desensitization. Accumulation of channels in this absorbing state allows GABA(A) receptors to detect even a few molecules of GABA in the synaptic cleft.

Glutamate transporters contribute to the time course of synaptic transmission in cerebellar granule cells.

Transporters are thought to assist in the termination of synaptic transmission at some synapses by removing neurotransmitter from the synapse. To investigate the role of glutamate transport in shaping the time course of excitatory transmission at the mossy fiber-granule cell synapse, the effects of transport impairment were studied using whole-cell voltage- and current-clamp recordings in slices of rat cerebellum. Impairment of transport by L-trans-pyrrolidine-2,4-dicarboxylate (PDC) produced a prolongation of the decay of the AMPA receptor-mediated current after a repetitive stimulus, as well as prolongation of single stimulus-evoked EPSCs when AMPA receptor desensitization was blocked. PDC also produced a prolongation of both single and repetitive-evoked NMDA receptor-mediated EPSCs. Enzymatic degradation of extracellular glutamate did not reverse the PDC-induced prolongation of AMPA receptor-mediated current after a repetitive stimulus, suggesting that transporter binding sites participate in limiting glutamate spillover. In current-clamp recordings, PDC dramatically increased the total area of the EPSP and the burst duration evoked by single and repetitive stimuli. These data indicate that glutamate transporters play a significant role in sculpting the time course of synaptic transmission at granule cell synapses, most likely by limiting the extent of glutamate spillover. The contribution of transporters is particularly striking during repetitive stimulus trains at physiologically relevant frequencies. Hence, the structural arrangement of the glomerulus may enhance the contribution of transporters to information processing by limiting the extent of glutamate spillover between adjacent synapses.

Prolonged physiological entrapment of glutamate in the synaptic cleft of cerebellar unipolar brush cells.

The cellular mechanism underlying the genesis of the long-lasting alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-receptor-mediated excitatory postsynaptic currents (EPSCs) at the mossy fiber (MF)-unipolar brush cell (UBC) synapse in rat vestibular cerebellum was examined with the use of whole cell and excised patch-clamp recording methods in thin cerebellar slices. Activation of MFs evokes an all-or-none biphasic AMPA-receptor-mediated synaptic current with a late component that peaks at 100-800 ms, which has been proposed to originate from an entrapment of glutamate in the MF-UBC synaptic cleft and is generated by the steady-state activation of AMPA receptors. Bath application of cyclothiazide, which blocks desensitization of AMPA receptors, produced a dose-dependent enhancement of the amplitude of the synaptic current (median effective dose 30 microM) and slowing of the rise time of the fast EPSC. N-methyl-D-aspartate-receptor-mediated EPSCs in UBCs were not potentiated in amplitude or time course by cyclothiazide (100 microM). The dose-response relations for the steady-state current evoked by glutamate acting at AMPA receptors in excised outside-out patches from UBC and granule somatic membranes was biphasic, peaking at 50 microM and declining to 50-70% of this value at 1 mM glutamate. When glutamate was slowly washed from patches to simulate the gradual decline of glutamate in the synapse, a late hump in the transmembrane current was observed in patches from both cell types. The delivery of a second MF stimulus at the peak of the slow EPSC evoked a fast EPSC of reduced amplitude followed by an undershoot of the subsequent slow current, consistent with the hypothesis that the peak of the slow EPSC reflects the peak of the biphasic steady-state dose-response curve. Estimates of receptor occupancy and glutamate concentration derived from the ratio of fast EPSC amplitudes, and the amplitude and polarity of the initial steady-state current in paired-pulse experiments, predict a slow decline of glutamate with a time constant of 800 ms, declining to ineffective concentrations at 5.4 s. Manipulation of cleft glutamate concentration by lowered extracellular calcium or delivery of brief stimulus trains abolished the slow EPSC and restored the undershoot to paired stimuli, respectively, in a manner consistent with a prolonged lifetime of glutamate in the cleft. The slow component of the EPSC was prolonged in duration by the glutamate reuptake inhibitor L-trans-pyrrolidine-2,4-dicarboxylate, suggesting that glutamate transport contributes to the time course of the synaptic current in UBCs. The data support the notion that the MF-UBC synapse represents an ultrastructural specialization to effectively entrap glutamate for unusually prolonged periods of time following release from MF terminals. The properties of the postsynaptic receptors and constraints on diffusional escape of glutamate imposed by synaptic ultrastructure and glutamate transporters act in concert to sculpt the time course of the resulting slow EPSC. This in turn drives a long-lasting train of action potentials in response to single presynaptic stimuli.

Metabotropic glutamate receptor mediated long-term depression in developing hippocampus.

The effects of bath application of the metabotropic glutamate receptor (mGluR) agonist 1S,3R-1-aminocyclopentane-1,3-dicarboxylic acid (ACPD, 10 microM) were studied at the Schaffer collateral-CA1 synapse in hippocampal slices from rats of 8-33 days postnatal age. In immature animals (8-12 days) ACPD induced a biphasic response characterized by an acute decrease in field EPSP slope (approximately 50-60% of baseline) in the presence of the agonist, followed by long-term depression (LTD, approximately 75-80% of baseline) after washout. In animals older than 20 days, ACPD induced a slow onset potentiation or minimal change. Both the acute depression and LTD were blocked by the mGluR antagonist alpha-methyl-4-carboxyphenyl glycine (MCPG). ACPD-induced LTD was blocked by the N-methyl-D-aspartate receptor (NMDAR) antagonists D(-)-2-amino-5 phosphopentanoic acid (AP5) and dizocilpine maleate (MK-801), and by ethanol. Glutamic pyruvic transaminase, an enzyme that selectively metabolizes endogenous extracellular glutamate, also blocked LTD suggesting that the requisite NMDA currents were tonically activated by extracellular rather than synaptically released glutamate. ACPD-induced LTD was blocked by staurosporine, indicating a requirement for serinethreonine kinase activation, and was unaffected by the L-type voltage sensitive calcium channel blocker nitrendipine and the A1 adenosine receptor antagonist 8-cyclopentyl-1,3-dimethylxanthine (CPT). Because mGluR-mediated LTD was observed only in immature CA1, mGluRs may play a role in hippocampal development, perhaps by contributing to synapse pruning in a temporally restricted fashion.

A role for adenosine A2 receptors in the induction of long-term potentiation in the CA1 region of rat hippocampus.

Although reductions in neurotransmission have been reported in response to agonist-mediated adenosine A1 receptor activation, the implications of A2 receptor activation on synaptic transmission have not been well explored. We examined the role adenosine A2 receptors play in the efficacy of neurotransmission between the Schaffer collateral-CA1 pathway in the rat transverse hippocampal slice. A2 receptor blockade in the presence of complete A1 receptor inhibition led to a reversible reduction of the field excitatory post-synaptic potential (EPSP) slope in response to low-frequency test pulses (0.033 Hz) indicating that A2 receptors can enhance synaptic transmission. A2 receptor blockade by the A2 antagonist, DMPX (3,7-dimethyl-1-propargylxanthine) prevented the induction of tetanus-induced long-term potentiation (LTP) of the EPSP. In contrast, no such effect on LTP induction was observed during A1 receptor blockade. We also examined the effects of DMPX on the induction of LTP during continued A1 receptor blockade with CPT. Under this condition, LTP was significantly reduced when compared to LTP induced in the presence of CPT alone. A similar result was found using the highly polar A2 antagonist 8-SPT (8-(p-sulfophenyl)theophylline) suggesting that the effects of DMPX on LTP were not due to a direct action on an intracellular intermediate. DMPX had no effect on LTP expression if applied 45 min following the tetanus indicating that A2 receptors play no significant role in the maintenance phase of LTP. Selective A2a receptor activation did not alter the field EPSP. Similarly, selective blockade of the A2a receptor did not interfere with tetanus-induced LTP. Increases in neuronal firing rates can result in elevations in the concentration of extracellular adenosine. Together, these results suggest that the A2 receptors may play an important role in the induction although not the maintenance of hippocampal LTP and that the effect is likely to be mediated by the A2b receptor.

Impaired cerebellar synaptic plasticity and motor performance in mice lacking the mGluR4 subtype of metabotropic glutamate receptor.

The application of the glutamate analog L-2-amino-4-phosphonobutyric acid (L-AP4) to neurons produces a suppression of synaptic transmission. Although L-AP4 is a selective ligand at a subset of metabotropic glutamate receptors (mGluRs), the precise physiological role of the L-AP4-activated mGluRs remains primarily unknown. To provide a better understanding of the function of L-AP4 receptors, we have generated and studied knockout (KO) mice lacking the mGluR4 subtype of mGluR that displays high affinity for L-AP4. The mGluR4 mutant mice displayed normal spontaneous motor activity and were unimpaired on the bar cross test, indicating that disruption of the mGluR4 gene did not cause gross motor abnormalities, impairments of novelty-induced exploratory behaviors, or alterations in fine motor coordination. However, the mutant mice were deficient on the rotating rod motor-learning test, suggesting that mGluR4 KO mice may have an impaired ability to learn complex motor tasks. Patch-clamp and extracellular field recordings from Purkinje cells in cerebellar slices demonstrated that L-AP4 had no effect on synaptic responses in the mutant mice, whereas in the wild-type mice 100 microM L-AP4 produced a 23% depression of synaptic responses with an EC50 of 2.5 microM. An analysis of presynaptic short-term synaptic plasticity at the parallel fiber-->Purkinje cell synapse demonstrated that paired-pulse facilitation and post-tetanic potentiation were impaired in the mutant mice. In contrast, long-term depression (LTD) was not impaired. These results indicate that an important function of mGluR4 is to provide a presynaptic mechanism for maintaining synaptic efficacy during repetitive activation. The data also suggest that the presence of mGluR4 at the parallel fiber-->Purkinje cell synapse is required for maintaining normal motor function.

AP5 blocks LTP in developing rat dentate gyrus and unmasks LTD.

The hippocampal dentate gyrus undergoes active neuronogenesis as well as growth and regression of neuronal elements and connections during the early postnatal period. In some brain regions, most notably in the visual system, both activity-dependent synaptic plasticity and NMDA receptor activation are candidate mechanisms by which neuronal architecture may be refined during brain maturation. To investigate whether similar mechanisms might obtain in developing dentate, we studied the effects of tetanic stimulation before and after NMDA receptor blockade in hippocampal slices from rats at 7-33 days. Field potentials were recorded in the suprapyramidal granule cell layer in response to stimulation of the medial perforant path. Robust long-term potentiation (LTP) of population spike amplitude (approximately 200% of baseline) was produced by a single tetanus (100 Hz, 2 s, 200 microseconds) at all ages studied. Application of 10 microM AP5 depressed population spike amplitude only in the younger slices (approximately 81% of baseline at 8-15 days; approximately 86% of baseline at 16-24 days), suggesting that the NMDA receptor-mediated component of normal synaptic transmission is higher in early development and decreases with maturation. AP5 prevented or significantly diminished LTP at all ages, establishing the NMDA dependence of LTP induction in the medial perforant path throughout development. AP5 also unmasked tetanus-induced homosynaptic long-term depression (62-75% of baseline) in the younger slices (8-24 days). Thus, prominent NMDA receptor-mediated activity and the capacity for bidirectional synaptic plasticity are characteristic of immature dentate. These processes may influence dentate morphogenesis by contributing to the growth, regression, and stabilization of neuronal elements.