Light-evoked excitatory synaptic currents of X-type retinal ganglion cells

The excitatory amino acid receptor (EAAR) types involved in the generation of light-evoked excitatory postsynaptic currents (EPSCs) were examined in X-type retinal ganglion cells. Using isolated and sliced preparations of cat and ferret retina, the light-evoked EPSCs of X cells were isolated by adding picrotoxin and strychnine to the bath to remove synaptic inhibition. N-methyl-D-aspartate (NMDA) receptors contribute significantly to the light-evoked EPSCs of ON- and OFF-X cells at many different holding potentials. An NMDA receptor contribution to the EPSCs was observable when retinal synaptic inhibition was either normally present or pharmacologically blocked. NMDA receptors formed 80% of the peak light-evoked EPSC at a holding potential of -40 mV; however, even at -80 mV, 20% of the light-evoked EPSC was NMDA-mediated. An alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) receptor-mediated component to the light-evoked EPSCs predominated at a holding potential of -80 mV. The light-evoked EPSC was blocked by the AMPA receptor-selective antagonist GYKI52466 (50-100 microM). The AMPA receptor-mediated EPSC component had a linear current-voltage relation. AMPA receptors form the main non-NMDA EAAR current on both ON- and OFF- X ganglion cell dendrites. When synaptic transmission was blocked by the addition of Cd(2+) to the Ringer, application of kainate directly to ganglion cells evoked excitatory currents that were strongly blocked by GYKI52466. Experiments using selective EAAR modulators showed the AMPA receptor-selective modulator cyclothiazide potentiated glutamate-evoked currents on X cells, while the kainate receptor-selective modulator concanavalin A (ConA) had no effect on kainate-evoked currents. Whereas the present study confirms the general notion that AMPA EAAR-mediated currents are transient and NMDA receptor-mediated currents are sustained, current-voltage relations of the light-evoked EPSC at different time points showed the contributions of these two receptor types significantly overlap. Both NMDA and AMPA EAARs can transmit transient and sustained visual signals in X ganglion cells, suggesting that much signal shaping occurs presynaptically in bipolar cells.     

Kindling induces transient NMDA receptor-mediated facilitation of high-frequency input in the rat dentate gyrus

To elucidate the gating mechanism of the epileptic dentate gyrus on seizure-like input, we investigated dentate gyrus field potentials and granule cell excitatory postsynaptic potentials (EPSPs) following high-frequency stimulation (10-100 Hz) of the lateral perforant path in an experimental model of temporal lobe epilepsy (i.e., kindled rats). Although control slices showed steady EPSP depression at frequencies greater than 20 Hz, slices taken from animals 48 h after the last seizure presented pronounced EPSP facilitation at 50 and 100 Hz, followed by steady depression. However, 28 days after kindling, the EPSP facilitation was no longer detectable. Using the specific N-methyl-D-aspartate (NMDA) and RS-alpha-amino-3-hydroxy-5-methyl-4-isoxazoleproponic acid (AMPA) receptor antagonists 2-amino-5-phosphonovaleric acid and SYM 2206, we examined the time course of alterations in glutamate receptor-dependent synaptic currents that parallel transient EPSP facilitation. Forty-eight hours after kindling, the fractional AMPA and NMDA receptor-mediated excitatory postsynaptic current (EPSC) components shifted dramatically in favor of the NMDA receptor-mediated response. Four weeks after kindling, however, AMPA and NMDA receptor-mediated EPSCs reverted to control-like values. Although the granule cells of the dentate gyrus contain mRNA-encoding kainate receptors, neither single nor repetitive perforant path stimuli evoked kainate receptor-mediated EPSCs in control or in kindled rats. The enhanced excitability of the kindled dentate gyrus 48 h after the last seizure, as well as the breakdown of its gating function, appear to result from transiently enhanced NMDA receptor activation that provides significantly slower EPSC kinetics than those observed in control slices and in slices from kindled animals with a 28-day seizure-free interval. Therefore, NMDA receptors seem to play a critical role in the acute throughput of seizure activity and in the induction of the kindled state but not in the persistence of enhanced seizure susceptibility.     

Recurrent mossy fiber pathway in rat dentate gyrus: synaptic currents evoked in presence and absence of seizure-induced growth

A common feature of temporal lobe epilepsy and of animal models of epilepsy is the growth of hippocampal mossy fibers into the dentate molecular layer, where at least some of them innervate granule cells. Because the mossy fibers are axons of granule cells, the recurrent mossy fiber pathway provides monosynaptic excitatory feedback to these neurons that could facilitate seizure discharge. We used the pilocarpine model of temporal lobe epilepsy to study the synaptic responses evoked by activating this pathway. Whole cell patch-clamp recording demonstrated that antidromic stimulation of the mossy fibers evoked an excitatory postsynaptic current (EPSC) in approximately 74% of granule cells from rats that had survived >10 wk after pilocarpine-induced status epilepticus. Recurrent mossy fiber growth was demonstrated with the Timm stain in all instances. In contrast, antidromic stimulation of the mossy fibers evoked an EPSC in only 5% of granule cells studied 4-6 days after status epilepticus, before recurrent mossy fiber growth became detectable. Notably, antidromic mossy fiber stimulation also evoked an EPSC in many granule cells from control rats. Clusters of mossy fiber-like Timm staining normally were present in the inner third of the dentate molecular layer at the level of the hippocampal formation from which slices were prepared, and several considerations suggested that the recorded EPSCs depended mainly on activation of recurrent mossy fibers rather than associational fibers. In both status epilepticus and control groups, the antidromically evoked EPSC was glutamatergic and involved the activation of both AMPA/kainate and N-methyl-D-aspartate (NMDA) receptors. EPSCs recorded in granule cells from rats with recurrent mossy fiber growth differed in three respects from those recorded in control granule cells: they were much more frequently evoked, a number of them were unusually large, and the NMDA component of the response was generally much more prominent. In contrast to the antidromically evoked EPSC, the EPSC evoked by stimulation of the perforant path appeared to be unaffected by a prior episode of status epilepticus. These results support the hypothesis that recurrent mossy fiber growth and synapse formation increases the excitatory drive to dentate granule cells and thus facilitates repetitive synchronous discharge. Activation of NMDA receptors in the recurrent pathway may contribute to seizure propagation under depolarizing conditions. Mossy fiber-granule cell synapses also are present in normal rats, where they may contribute to repetitive granule cell discharge in regions of the dentate gyrus where their numbers are significant.     

Excitatory synapse in the rat hippocampus in tissue culture and effects of aniracetam.

Excitatory synaptic connections between rat hippocampal neurons were established in tissue culture. The electrophysiological and pharmacological properties of these synapses were studied with the use of the tight-seal whole-cell recording technique. The excitatory postsynaptic current (EPSC) in a dissociated CA1 neuron evoked by stimulation of an explant from the CA3/CA4 region of the hippocampus had two distinct components in Mg(2+)-free medium. The fast component was abolished by the non-NMDA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) (2 microM), whereas the slow component was abolished by the N-methyl-D-aspartate (NMDA) receptor antagonist D-2-amino-5-phosphonovalerate (D-APV) (50 microM). In solution containing 1 mM Mg2+, the peak amplitude of the fast component was almost linearly related to the membrane potential. In contrast, the conductance change underlying the slow component of the EPSC was voltage-dependent with a region of negative-slope conductance in the range of -80 to -20 mV. A nootropic drug, aniracetam, increased both the amplitude and duration of the fast component of the EPSC in a concentration-dependent manner in the range of 0.1-5 mM, whereas it had no potentiating effect on the slow component. Aniracetam (0.1-5 mM) similarly increased current responses of the postsynaptic neuron to alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA). Current responses to quisqualate and glutamate in the presence of D-APV were also potentiated by aniracetam. However, neither NMDA- nor kainate-induced current was potentiated by 1 mM aniracetam.     

Postsynaptic currents in deep cerebellar nuclei

Postsynaptic currents were studied by whole cell recordings in visually identified large neurons of the deep cerebellar nuclei (DCN) in slices of 4- to 11-day-old mice. Spontaneous postsynaptic currents were abolished by the GABA(A) receptor antagonist bicuculline and had a single-exponential decay with a mean time constant of 13.6 +/- 3.2 (SD) ms. Excitatory postsynaptic currents (EPSCs) were evoked in 48/56 neurons recorded. The addition of AMPA and N-methyl-D-aspartate (NMDA) receptor antagonists together completely abolished all synaptic responses. In 1 mM [Mg(2+)](o) and at a holding potential of -60 mV, the peak amplitude of the NMDA component of the EPSC (NMDA-EPSC) was 83.2 +/- 21.2% of the AMPA component (AMPA-EPSC). This indicates that in DCN neurons, at a physiological [Mg(2+)](o) and at the resting membrane potential, NMDA receptors contribute to the synaptic signal. AMPA-EPSCs had a linear current-voltage relationship with a reversal potential of +2.3 +/- 0.4 mV and a single-exponential decay with a voltage-dependent time constant that at -60 mV was 7.1 +/- 3.3 ms. In 10 microM glycine and 1 mM [Mg(2+)](o), the I-V relationship of NMDA-EPSCs had a reversal potential of -0.5 +/- 3.3 mV and a maximal inward current at -33.4 +/- 5.8 mV. The apparent dissociation constant (K(D)) of Mg(2+) for the NMDA receptor-channel at -60 mV, measured by varying [Mg(2+)](o), was 135.5 +/- 55.3 microM, and when measured by fitting the I-V curves with a theoretical function, it was 169.9 +/- 119.5 microM. Thus in the DCN, NMDA receptors have a sensitivity to Mg(2+) that corresponds to subunits that are weakly blocked by this ion (epsilon 3 and epsilon 4) of which the DCN express epsilon 4. NMDA-EPSCs had a double-exponential decay with voltage-dependent time constants that at -60 mV were 20.2 +/- 8.9 and 136.4 +/- 62.8 ms. At positive voltages, the time constants were slower and their contributions were about equal, while in the negative slope conductance region of the I-V curve, the faster time constant became predominant, conferring faster kinetics to the EPSC. The weak sensitivity to Mg(2+) of NMDA receptors, together with a relatively fast kinetics, provide DCN neurons with strong excitatory inputs in which fast dynamic signals are relatively well preserved.     

Effect of chronic stress on synaptic currents in rat hippocampal dentate gyrus neurons

We investigated the effect of chronic stress on synaptic responses of rat dentate granule cells to perforant path stimulation. Rats were subjected for 3 wk to unpredictable stressors twice daily or to control handling. One day after the last stressor, hippocampal slices were prepared and synaptic responses were determined with whole-cell recording. At that time, adrenal weight was found to be increased and thymus weight as well as gain in body weight were decreased in the stressed versus control animals, indicative of corticosterone hypersecretion during the stress period. In slices from rats with basal corticosteroid levels (at the circadian trough, under rest), no effect of prior stress exposure was observed on synaptic responses. However, synaptic responses of dentate granule cells from chronically stressed and control rats were differently affected by in vitro activation of glucocorticoid receptors, i.e., 1-4 h after administration of 100 nM corticosterone for 20 min. Thus the maximal response to synaptic activation of dentate cells at holding potential of -70 mV [when N-methyl-D-aspartate (NMDA) receptors are blocked by magnesium] was significantly enhanced after corticosterone administration in chronically stressed but not in control animals. In accordance, the amplitude of alpha-amino-3-hydroxy-5-methylisolazole-4-propionic acid (AMPA) but not of NMDA receptor-mediated currents was increased by corticosterone in stressed rats, over the entire voltage range. Corticosterone treatment also decreased the time to peak of AMPA currents, but this effect did not depend on prior stress exposure. The data indicate that following chronic stress exposure synaptic excitation of dentate granule cells may be enhanced when corticosterone levels rise. This enhanced synaptic flow could contribute to enhanced excitation of projection areas of the dentate gyrus, most notably the CA3 hippocampal region.     

Mu opioid receptor-mediated modulation of synaptic currents in dentate granule cells of rat hippocampus.

1. The effect of a selective mu opioid agonist, [N-MePhe3-D-Pro4]morphiceptin (PL017), on synaptic transmission in the dentate gyrus was examined in hippocampal slices. Synaptic currents were evoked by stimulation of the outer molecular layer and recorded from granule cells using whole-cell voltage-clamp techniques. 2. Monosynaptic inhibitory postsynaptic currents (IPSCs) were evoked in the presence of D(-)-2-amino-5-phosphonovaleric acid (D-APV), and N-methyl-D-aspartate (NMDA) receptor antagonist, and 6,7-dinitroquinoxaline-2,3-dione (DNQX), a non-NMDA type of glutamate receptor antagonist. The IPSCs consisted of a gamma-aminobutyric acid (GABA)A receptor-mediated early component and a GABAB receptor-mediated late component. 3. Bath application of PL017 (0.3-3 microM) induced a dose-dependent reduction in the amplitude of both early IPSCs (21-56%) and late IPSCs (43-81%). These effects could be reversed by the opiate antagonist naloxone (1 microM) or prevented by the selective mu antagonist beta-funaltrexamine hydrochloride (10 microM). 4. NMDA receptor-mediated excitatory postsynaptic currents (EPSCs) were revealed in the presence of DNQX and the GABAA antagonist bicuculline methiodide. PL017 (3 microM) caused a 35% reduction in the amplitude of NMDA EPSCs. NMDA receptor-mediated population EPSPs recorded extracellularly were also inhibited by 3 microM PL017 to a similar degree. 5. Non-NMDA receptor-mediated EPSCs were demonstrated in the presence of D-APV and bicuculline methiodide. The amplitude of non-NMDA EPSCs was not affected by PL017.(ABSTRACT TRUNCATED AT 250 WORDS)     

Zinc-induced augmentation of excitatory synaptic currents and glutamate receptor responses in hippocampal CA3 neurons

Zinc is found throughout the CNS at synapses co-localized with glutamate in presynaptic terminals. In particular, dentate granule cells' (DGC) mossy fiber (MF) axons contain especially high concentrations of zinc co-localized with glutamate within vesicles. To study possible physiological roles of zinc, visualized slice-patch techniques were used to voltage-clamp rat CA3 pyramidal neurons, and miniature excitatory postsynaptic currents (mEPSCs) were isolated. Bath-applied zinc (200 microM) enhanced median mEPSC peak amplitudes to 153.0% of controls, without affecting mEPSC kinetics. To characterize this augmentation further, rapid agonist application was performed on perisomatic outside-out patches to coapply zinc with glutamate extremely rapidly for brief (1 ms) durations, thereby emulating release kinetics of these substances at excitatory synapses. When zinc was coapplied with glutamate, zinc augmented peak glutamate currents (mean +/- SE, 116.6 +/- 2.8% and 143.8 +/- 9.8% of controls at 50 and 200 microM zinc, respectively). This zinc-induced potentiation was concentration dependent, and pharmacological isolation of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor-mediated currents (AMPAR currents) gave results similar to those observed with glutamate application (mean, 115.0 +/- 5.4% and 132.5 +/- 9.1% of controls at 50 and 200 microM zinc, respectively). Inclusion of the AMPAR desensitization blocker cyclothiazide in the control solution, however, abolished zinc-induced augmentation of glutamate-evoked currents, suggesting that zinc may potentiate AMPAR currents by inhibiting AMPAR desensitization. Based on the results of the present study, we hypothesize that zinc is a powerful modulator of both excitatory synaptic transmission and glutamate-evoked currents at physiologically relevant concentrations. This modulatory role played by zinc may be a significant factor in enhancing excitatory neurotransmission and could significantly regulate function at the mossy fiber-CA3 synapse.     

Postnatal development of excitatory postsynaptic currents in nucleus accumbens medium spiny neurons

We have recorded excitatory postsynaptic currents (EPSCs) evoked by local electrical stimulation in 243 nucleus accumbens (nAcb) neurons in vitro during postnatal development from the day of birth (postnatal day 0; P0) to P27 and in young adults rats (P59-P71). An EPSC sensitive to glutamatergic antagonists was found in all neurons. In the majority of cases (189/243), the EPSC had two distinct components: an early one sensitive to 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and a late one that was sensitive to d-2-amino-5-phosphonovaleric acid (APV) showing that early and late components of the EPSC were mediated by AMPA/kainate (KA) and N-methyl-d-aspartate (NMDA) receptors respectively. During the first four postnatal days, the amplitudes of both the AMPA/KA and NMDA components of the EPSC were relatively small and then began to increase until the end of the second postnatal week. Whereas the amplitude of the early component appeared to stabilize from that point on, the late component began to decrease and became virtually undetectable in preparations from animals older than 3 weeks unless the AMPA/KA response was blocked with CNQX. In addition, the ratio between the amplitude of the NMDA and AMPA/KA receptor-mediated components of the EPSC followed a developmental pattern parallel to that of the NMDA receptor component showing an increase during the first two postnatal weeks followed by a decrease. Together, these results show that, during postnatal development, there is a period when NMDA receptor-mediated EPSC are preeminent and that time frame might represent a period during which the development of the nAcb might be sensitive to environmental manipulation.     

Shunting of excitatory input to dentate gyrus granule cells by a depolarizing GABAA receptor-mediated postsynaptic conductance.

1. Stimulation of the perforant path in the outer molecular layer of the adult rat dentate gyrus produced a depolarizing post-synaptic potential (DPSP) in granule cells when recorded using whole-cell techniques in the standard hippocampal slice preparation at 34 degrees C. The postsynaptic currents (PSCs) contributing to the DPSP were analyzed using specific receptor antagonists in current- and voltage-clamp recordings. 2. The DPSP reversal potential was dependent on the intracellular chloride concentration, and the amplitude of the DPSP was increased 55% after perfusion of the gamma-aminobutyric acid-A (GABAA) receptor antagonist bicuculline methiodide (BMI). The GABAA receptor-mediated PSC reversed at -66 mV, which was 19 mV positive to the resting membrane potential (-85 mV) but hyperpolarized relative to action potential threshold. At -35 mV, the GABAA PSC had a latency to peak of 12.9 ms after the stimulus and decayed monoexponentially with an average time constant of 23.4 ms. 3. The component of the PSC blocked by the Quis/AMPA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) had a latency to peak of 7.1 ms and decayed monoexponentially with a time constant of 9.9 ms at -35 mV. The N-methyl-D-aspartate (NMDA) receptor-mediated PSC, which was blocked by D-amino-5-phosphonovaleric acid (D-AP5), had a waveform that was similar to the GABAA PSC: the latency to peak was 16 ms and the decay was monoexponential with a time constant of 24.5 ms at -35 mV. 4. The ratio of the peak PSCs mediated by GABAA, Quis/AMPA, and NMDA receptors measured at -35 mV with cesium gluconate electrode solutions was 1:0.2:0.1. This ratio was essentially constant over the range of stimulus intensities that produced compound PSC amplitudes of 80-400 pA. 5. Measured at its reversal potential, the GABAA receptor-mediated postsynaptic conductance (GGABA-A) decreased the peak DPSP amplitude by 35%, shunted 50% of the charge transferred to the soma by the excitatory PSC, and completely inhibited the NMDA receptor-mediated component of the DPSP. 6. Simultaneous stimulation of presynaptic fibers from both the perforant path and interneurons results in a large depolarizing GGABA-A that inhibits the granule cell by shunting the excitatory PSCs. As predicted by models of shunting, the similar kinetics of the GABAA and NMDA PSCs leads to particularly effective inhibition of the NMDA PSC. The more rapid Quis/AMPA PSC is less affected by the GGABA-A, so that granule cell excitation under these conditions is primarily due to Quis/AMPA receptor activation.     

Nociceptin/orphanin FQ (N/OFQ) inhibits excitatory and inhibitory synaptic signaling in the suprachiasmatic nucleus (SCN)

Environmental synchronization of the endogenous mammalian circadian rhythm involves glutamatergic and GABAergic neurotransmission within the hypothalamic suprachiasmatic nucleus (SCN). The neuropeptide nociceptin/orphanin FQ (N/OFQ) inhibits light-induced phase shifts, evokes K(+)-currents and reduces the intracellular Ca(2+) concentration in SCN neurons. Since these effects are consistent with a modulatory role for N/OFQ on synaptic transmission in the SCN, we examined the effects of N/OFQ on evoked and spontaneous excitatory and inhibitory currents in the SCN. N/OFQ produced a consistent concentration-dependent inhibition of glutamate-mediated excitatory postsynaptic currents (EPSC) evoked by optic nerve stimulation. N/OFQ did not alter the amplitude of currents induced by application of (RS)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) or N-methyl-d-aspartate (NMDA) nor the amplitude of miniature EPSC (mEPSC) consistent with a lack of N/OFQ effect on postsynaptic AMPA or NMDA receptors. N/OFQ significantly reduced the mEPSC frequency. The inhibitory actions of N/OFQ were blocked by omega-conotoxin GVIA, an N-type Ca(2+)channel antagonist and partially blocked by omega-agatoxin TK, a P/Q type Ca(2+) channel blocker. These data indicate that N/OFQ reduces evoked EPSC, in part, by inhibiting the activity of N- and P/Q-type Ca(2+) channels. In addition, N/OFQ produced a consistent reduction in baseline Ca(2+) levels in presynaptic retinohypothalamic tract terminals. N/OFQ also inhibited evoked GABA(A) receptor-mediated inhibitory postsynaptic currents (IPSC) in a concentration dependent manner. However, N/OFQ had no effect on currents activated by muscimol application or on the amplitude of miniature IPSC (mIPSC) and significantly reduced the mIPSC frequency consistent with an inhibition of GABA release downstream from Ca(2+) entry. Finally, N/OFQ inhibited the paired-pulse depression observed in SCN GABAergic synapses consistent with a presynaptic mechanism of action. Together these results suggest a widespread modulatory role for N/OFQ on the synaptic transmission in the SCN.     

A voltage-clamp study of the effects of Joro spider toxin and zinc on excitatory synaptic transmission in CA1 pyramidal cells of the guinea pig hippocampal slice.

Using the single-electrode voltage-clamp technique, we have examined the effects of a non-N-methyl-D-aspartate (NMDA) antagonist. Joro spider toxin (JSTX), and of an NMDA antagonist, zinc, on excitatory postsynaptic currents (EPSCs) evoked by stimulation of stratum radiatum in CA1 pyramidal cells of the guinea-pig hippocampal slice. Pressure application of a synthesized JSTX (JSTX-3) at 10-200 microM greatly reduced the EPSCs (14/19 cells). The block by JSTX-3 was observed in pyramidal cells where the EPSCs showed linear peak current-voltage (I-V) relations in the control. EPSCs remaining after JSTX-3 application showed non-linear peak I-V relationships (10/14 cells), and were blocked by puff application of the selective NMDA receptor antagonist DL-2-amino-5-phosphonovalerate (APV) at 200 microM (6/10 cells). In the presence of JSTX-3, the decay time constant of the EPSC was increased and was less affected by membrane potential. JSTX-3 had no detectable effects on EPSCs apparently mediated solely by NMDA receptor. These observations suggest that JSTX-3 blocks excitatory synaptic transmission mainly by suppressing non-NMDA-receptor-mediated EPSCs, and that the JSTX-3-insensitive component is mediated at least in part by NMDA receptors in the hippocampal slice. Zinc (100-200 microM) reversibly attenuated EPSCs (6/9 cells) and appeared to block a slower component of the EPSCs, suggesting that mainly NMDA receptor-mediated currents were affected.     

Modulation of desensitization at glutamate receptors in isolated crucian carp horizontal cells by concanavalin A, cyclothiazide, aniracetam and PEPA

In horizontal cells freshly dissociated from crucian carp (Carassius auratus) retina, we examined the effects of modulators of glutamate receptor desensitization, concanavalin A, cyclothiazide, aniracetam and 4-[2-(phenylsulfonylamino)ethylthio]-2,6-difluoro-phenoxyacetam ide (PEPA), on responses to rapid application of glutamate and kainate, using whole-cell voltage-clamp techniques. Incubation of concanavalin A suppressed the peak response but weakly potentiated the equilibrium response of horizontal cells to glutamate. Cyclothiazide blocked glutamate-induced desensitization in a dose-dependent manner, which resulted in a steady increase of the equilibrium current. The concentration of cyclothiazide causing a half-maximal potentiation for the equilibrium response was 85 microM. Furthermore, cyclothiazide shifted the dose-response relationship of the equilibrium current to the right, but slightly suppressed the kainate-induced sustained current. These effects of concanavalin A and cyclothiazide are consistent with the supposition that glutamate receptors of carp horizontal cells may be an alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA)-preferring subtype. In order to further characterize the AMPA receptors of horizontal cells, modulation by aniracetam and PEPA of glutamate- and kainate-induced currents was studied. Aniracetam, a preferential modulator of flop variants of AMPA receptors, considerably blocked desensitization of glutamate-induced currents, but only slightly potentiated kainate-induced currents. It was further found that PEPA, a flop-preferring allosteric modulator of AMPA receptor desensitization, slightly suppressed the peak current, while it dramatically potentiated the equilibrium current induced by glutamate in a dose-dependent manner. PEPA was much potent than aniracetam at these receptors and showed the effect on glutamate-induced desensitization even at a concentration as low as 3 microM. PEPA also potentiated non-desensitizing currents induced by kainate, but with much less extent. These modulatory effects of concanavalin A, cyclothiazide, aniracetam and PEPA on AMPA receptors in carp horizontal cells were rather similar to those obtained at AMPA receptors assembled from flop variants expressed in Xenopus oocyte and HEK cell. Consequently, we speculate that the AMPA receptor on carp horizontal cells may predominantly carry the flop splice variants.     

Modulation of excitatory synaptic transmission by GABA(C) receptor-mediated feedback in the mouse inner retina.

In many vertebrate CNS synapses, the neurotransmitter glutamate activates postsynaptic non-N-methyl-D-aspartate (NMDA) and NMDA receptors. Since their biophysical properties are quite different, the time course of excitatory postsynaptic currents (EPSCs) depends largely on the relative contribution of their activation. To investigate whether the activation of the two receptor subtypes is affected by the synaptic interaction in the inner plexiform layer (IPL) of the mouse retina, we analyzed the properties of the light-evoked responses of ON-cone bipolar cells and ON-transient amacrine cells in a retinal slice preparation. ON-transient amacrine cells were whole cell voltage-clamped, and the glutamatergic synaptic input from bipolar cells was isolated by a cocktail of pharmacological agents (bicuculline, strychnine, curare, and atropine). Direct puff application of NMDA revealed the presence of functional NMDA receptors. However, the light-evoked EPSC was not significantly affected by D(-)-2-amino-5-phosphonopentanoic acid (D-AP5), but suppressed by 2,3-dioxo-6-nitro-1,2,3,4-tetrahydrobenzo[f]quinoxaline-7-sulfonamide (NBQX) or 1-(4-aminophenyl)-4-methyl-7,8-methylenedioxy-5H-2,3-benzodiazepine hydrochloride (GYKI 52466). These results indicate that the light-evoked EPSC is mediated mainly by AMPA receptors under this condition. Since bipolar cells have GABA(C) receptors at their terminals, it has been suggested that bipolar cells receive feedback inhibition from amacrine cells. Application of (1,2,5,6-tetrahydropyridin-4-yl)methylphosphinic acid (TPMPA), a specific blocker of GABA(C) receptors, suppressed both the GABA-induced current and the light-evoked feedback inhibition observed in ON-cone bipolar cells and enhanced the light-evoked EPSC of ON-transient amacrine cells. In the presence of TPMPA, the light-evoked EPSC of amacrine cells was composed of AMPA and NMDA receptor-mediated components. Our results suggest that photoresponses of ON-transient amacrine cells in the mouse retina are modified by the activation of presynaptic GABA(C) receptors, which may control the extent of glutamate spillover.     

Effects of corticosteroids on synaptic transmission in rat dorsolateral septal nucleus

The effects of corticosteroids on synaptic transmission in the rat dorsolateral septal nucleus (DLSN) were examined, in vitro, by using intracellular and voltage-clamp recording methods. Prednisolone (100 microM) increased the amplitude of excitatory postsynaptic potential (EPSP) and depressed both fast and slow inhibitory postsynaptic potentials (IPSP). Under voltage-clamp conditions, prednisolone (100 microM) increased the amplitude of excitatory postsynaptic current (EPSC) and depressed the fast and slow inhibitory postsynaptic currents (IPSCs). Corticosterone (100 microM) mimicked the effects of prednisolone on the postsynaptic currents (PSCs). To examine the direct effects of prednisolone on the EPSC and slow IPSC, the fast IPSC was blocked by bicuculline (20 microM). Under these experimental conditions, prednisolone (100 microM) did not alter the isolated EPSC but depressed slow IPSC by 22 +/- 3% (n = 10). The fast IPSC was isolated by pretreatment with kynurenic acid and CGP55845A, where the EPSC and slow IPSC were blocked. Prednisolone (100 microM) depressed the isolated fast IPSC in DLSN neurons. Prednisolone (100 microM) did not change either the inward current produced by glutamate or the outward current produced by gamma-aminobutyric acid (GABA). The results suggest that corticosteroids facilitate excitatory synaptic transmission in the DLSN by reducing the release of GABA from the presynaptic nerve terminals of interneurons.     

Presynaptic and postsynaptic NMDA receptors mediate distinct effects of brain-derived neurotrophic factor on synaptic transmission

In addition to its effects on neuronal survival and differentiation, brain-derived neurotrophic factor (BDNF) plays an important role in modulating synaptic transmission and plasticity in many brain areas, most notably the neocortex and hippocampus. These effects may underlie a role for BDNF in learning and memory as well as developmental plasticity. Consistent with localization of the tropomyosin-related kinase B receptor to both sides of the synapse, BDNF appears to have pre- and postsynaptic effects, but the underlying cellular mechanisms are unclear and it is not known whether pre- and postsynaptic modulations by BDNF occur simultaneously. To address these issues, we recorded dual-component (alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid [AMPA] and N-methyl-D-aspartate [NMDA]) miniature excitatory postsynaptic currents (mEPSCs) from cortical and hippocampal pyramidal neurons and dentate gyrus granule cells from acute brain slices. BDNF had no effect on the fast component of mEPSC decay or on the peak amplitude, suggesting that BDNF did not modulate postsynaptic AMPA receptors, although BDNF rapidly modulated NMDA receptors, as seen by an enhancement of the slow component of mEPSC decay that was prevented by blocking postsynaptic NMDA receptors. At the same time, BDNF acted presynaptically to enhance mEPSC frequency. Surprisingly, the effect on frequency was also NMDA receptor dependent, but required activation of presynaptic, not postsynaptic, NMDA receptors. BDNF also enhanced action potential-dependent glutamate release via presynaptic NMDA receptors, an effect that was unmasked when voltage-gated calcium channels were partially inhibited. Our results indicate that BDNF acutely modulates presynaptic release and postsynaptic responsiveness through simultaneous effects on pre- and postsynaptic NMDA receptors.     

Corticostriatal paired-pulse potentiation produced by voltage-dependent activation of NMDA receptors and L-type Ca(2+) channels.

AMPA and N-methyl-D-aspartate (NMDA) receptor-mediated synaptic responses expressed differential paired-pulse plasticity when examined in the same cell using intracellular or whole cell voltage-clamp recordings. Electrical stimulation of corticostriatal afferents in brain slices bathed in artificial cerebrospinal fluid containing bicuculline produces excitatory postsynaptic potentials and excitatory postsynaptic currents (EPSCs) mediated primarily by AMPA receptors. Cell-to-cell variation existed in AMPA receptor paired-pulse plasticity, but within-cell plasticity was stable over a range of stimulation intensities. Addition of 6-cyano-7-nitroquinoxalene-2,3-dione blocked most of the synaptic response leaving behind a small AP-5-sensitive component. Increasing the stimulation intensity produced large, long-lasting NMDA receptor-mediated responses. In contrast to AMPA receptor-mediated responses, NMDA receptor responses consistently showed an increase in paired-pulse potentiation with increasing stimulation intensity. This relationship was restricted to interstimulus intervals shorter than 100 ms. Paired-pulse potentiation of NMDA receptor responses was voltage-dependent and reduced by removal of extracellular Mg(2+). Block of postsynaptic L-type Ca(2+) channels with nifedipine produced a voltage-dependent reduction of NMDA receptor excitatory postsynaptic currents (EPSCs) and a voltage-dependent reduction of NMDA receptor paired-pulse potentiation. These data indicate depolarization during the first NMDA receptor response causes facilitation of the second by removing voltage-dependent block of NMDA receptors by Mg(2+) and by activating voltage-dependent Ca(2+) channels.     

Excitatory amino acid antagonists inhibit synaptic responses in the guinea pig hypothalamic paraventricular nucleus.

1. The effects of specific excitatory amino acid (EAA) antagonists on evoked excitatory synaptic responses were studied in the hypothalamic paraventricular nucleus (PVN) of the guinea pig, by the use of the in vitro slice preparation. Intracellular recordings were obtained from paraventricular neurons, and excitatory postsynaptic potentials (EPSPs) and currents (EPSCs) were induced by perifornical electrical stimulation. To reduce the influence of a potential gamma-aminobutyric acidA (GABAA) inhibitory component on the synaptic responses, all experiments were performed in the presence of 50 microM picrotoxin. 2. Of 20 cells tested, 13 had electrophysiological characteristics similar to magnocellular neuropeptidergic cells (MNCs) and 7 displayed low-threshold Ca2+ spikes (LTSs). No difference was detected in the effect of the antagonists on the synaptic responses of cells with or without LTS potentials. 3. The broad-spectrum EAA antagonist kynurenic acid decreased the amplitude of the EPSPs and EPSCs in a dose-dependent manner: the mean decrease was 5% for 100 microM, 43% for 300 microM, and 70% for 1 mM. 4. The quisqualate/kainate-receptor-selective antagonist 6-cyano-2,3-dihydroxy-7-nitroquinoxaline (CNQX) induced a dose-dependent decrease of the EPSPs and EPSCs: 1 microM had no detectable effect, 3 and 10 microM caused 30 and 70% decreases, respectively, and 30 microM blocked the response almost completely. This effect was not accompanied by a change in resting membrane potential or input resistance and was slowly reversible. 5. The N-methyl-D-aspartate (NMDA)-receptor-selective antagonist DL-2-amino-5-phosphonopentanoic acid (AP5), applied at 30 and 300 microM, reduced slightly the amplitude of the decay phase of the EPSP but did not significantly affect the peak amplitude. In some cells, the current-voltage relationship of the decay phase of the EPSC revealed a region of negative slope conductance between -70 and -40 mV. 6. These results suggest that 1) glutamate or a related EAA is responsible for the fast excitatory input to magnocellular and parvocellular neurons in the PVN and probably also for cells around PVN, 2) a quisqualate/kainate receptor type is responsible for the rising phase and peak amplitude of the synaptic current, and 3) an NMDA receptor contributes to the late part of the synaptic response.     

Modulation of AMPA and NMDA responses in rat spinal dorsal horn neurons by trans-1-aminocyclopentane-1,3-dicarboxylic acid.

In freshly isolated spinal dorsal horn (DH) neurons (laminae I-IV) of the young rat the effects of 25-100 microM of (+/-)-trans-1-aminocyclopentane-1,3-dicarboxylic acid (trans-ACPD), 1S,3R-ACPD and 1R,3S-ACPD, a metabotropic glutamate receptor (mGluR) agonist, on inward currents induced by glutamate (Glu), alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), N-methyl-D-aspartate (NMDA) and kainate were studied under whole-cell voltage-clamp conditions. When the cells were clamped to -60 mV, the racemic mixture and both stereo isomers of trans-ACPD increase the responses elicited by Glu, AMPA, and NMDA, but little those of kainate. In addition, quisqualate (10-50 microM), in the presence of CNQX (5-20 microM) or NBQX (5 microM), potentiated NMDA-induced currents. The enhancing effect lasted 10-75 min, depending upon both dose and length of application. In a smaller proportion of dorsal horn neurons, the enhancing effect was preceded by a transient depression of the responses to Glu, AMPA, and NMDA. 2-Amino-3-phosphonopropionic acid (L-AP3), a putative antagonist of mGluR exerted little effect on responses to AMPA itself, but reduced or prevented the enhancing effect of 1S,3R-ACPD. It is concluded that activation of a metabotropic glutamate receptor by trans-ACPD, and its two enantiomers, may mediate the enhancement of AMPA and NMDA responses in acutely isolated rat spinal dorsal horn neurons. These results are consistent with the possibility that the activation of metabotropic glutamate receptor may contribute to the regulation of the strength of excitatory amino-mediated primary afferent neurotransmission, including nociception.     

17beta-estradiol enhances NMDA receptor-mediated EPSPs and long-term potentiation

Gonadal steroid hormones influence CNS functioning through a variety of different mechanisms. To test the hypothesis that estrogen modulates synaptic plasticity in the hippocampus, in vitro hippocampal slices from 2-mo-old Sprague-Dawley male rats were used to determine the effect of 17beta-estradiol on both N-methyl-D-aspartate (NMDA) receptor-mediated excitatory postsynaptic potentials (EPSPs) through intracellular recordings and long-term potentiation (LTP) through extracellular recordings. Intracellular EPSPs and extracellular field EPSPs (fEPSPs) were recorded from CA1 pyramidal cells by stimulating Schaffer collateral fibers. In intracellular experiments, slices were perfused with medium containing bicuculline (5 microM) and low Mg2+ (0.1 mM) to enhance the NMDA receptor-mediated currents and 6, 7-dinitroquinoxaline-2,3-dione (DNQX) (10 microM) to block the alpha-amino-3-hydroxy-5-methyl-4-isoxazoleproprianate (AMPA) receptor-mediated component. The effects of 17beta-estradiol on NMDA receptor-mediated activity were excitatory; concentrations >10 nM induced seizure activity, and lower concentrations (1 nM) markedly increased the amplitude of NMDA-mediated EPSPs (both the first and second responses increased during paired pulse stimulation by 180 and 197%, respectively). In extracellular experiments, slices perfused with 17beta-estradiol (100 pM) exhibited a pronounced, persisting, and significant enhancement of LTP of both the fEPSP slope (192%) and fEPSP amplitude (177%) compared with control slices (fEPSP slope = 155%; fEPSP amplitude = 156%) 30 min after high-frequency stimulation. These data demonstrate that estrogen enhances NMDA receptor-mediated currents and promotes an enhancement of LTP magnitude.