GABAA, GABAC and glycine receptor-mediated inhibition differentially affects light-evoked signalling from mouse retinal rod bipolar cells

Rod bipolar cells relay visual signals evoked by dim illumination from the outer to the inner retina. GABAergic and glycinergic amacrine cells contact rod bipolar cell terminals, where they modulate transmitter release and contribute to the receptive field properties of third order neurones. However, it is not known how these distinct inhibitory inputs affect rod bipolar cell output and subsequent retinal processing. To determine whether GABA_A, GABA_C and glycine receptors made different contributions to light-evoked inhibition, we recorded light-evoked inhibitory postsynaptic currents (L-IPSCs) from rod bipolar cells mediated by each pharmacologically isolated receptor. All three receptors contributed to L-IPSCs, but their relative roles differed; GABA_C receptors transferred significantly more charge than GABA_A and glycine receptors. We determined how these distinct inhibitory inputs affected rod bipolar cell output by recording light-evoked excitatory postsynaptic currents (L-EPSCs) from postsynaptic AII and A17 amacrine cells. Consistent with their relative contributions to L-IPSCs, GABA_C receptor activation most effectively reduced the L-EPSCs, while glycine and GABA_A receptor activation reduced the L-EPSCs to a lesser extent. We also found that GABAergic L-IPSCs in rod bipolar cells were limited by GABA_A receptor-mediated inhibition between amacrine cells. We show that GABA_A, GABA_C and glycine receptors mediate functionally distinct inhibition to rod bipolar cells, which differentially modulated light-evoked rod bipolar cell output. Our findings suggest that modulating the relative proportions of these inhibitory inputs could change the characteristics of rod bipolar cell output.     

Propagation of action potentials from the soma to individual dendrite of cultured rat amacrine cells is regulated by local GABA input

Retinal amacrine cells are interneurons that make lateral and vertical connections in the inner plexiform layer of the retina. Amacrine cells do not possess a long axon, and this morphological feature is the origin of their naming. Their dendrites function as both presynaptic and postsynaptic sites. Half of all amacrine cells are GABAergic inhibitory neurons that mediate lateral inhibition, and their light-evoked response consists of graded voltage changes and regenerative action potentials. There is evidence that the amount of neurotransmitter release from presynaptic sites is increased by spike propagation into the dendrite. Thus understanding of how action potentials propagate in dendrites is important to elucidating the extent and strength of lateral inhibition. In the present study, we used the dual whole cell patch-clamp technique on the soma and the dendrite of cultured rat amacrine cells and directly demonstrated that the action potentials propagate into the dendrites. The action potential in the dendrite was TTX sensitive and was affected by the local membrane potential of the dendrite. Propagation of the action potential was suppressed by local application of GABA to the dendrite. Dual dendrite whole cell patch-clamp recordings showed that GABA suppresses the propagation of action potentials in one dendrite of an amacrine cell, while the action potentials propagate in the other dendrites. It is likely that the action potentials in the dendrites are susceptible to various external factors resulting in the nonuniform propagation of the action potential from the soma of an amacrine cell.     

Spike-dependent GABA inputs to bipolar cell axon terminals contribute to lateral inhibition of retinal ganglion cells

The inhibitory surround signal in retinal ganglion cells is usually attributed to lateral horizontal cell signaling in the outer plexiform layer (OPL). However, recent evidence suggests that lateral inhibition at the inner plexiform layer (IPL) also contributes to the ganglion cell receptive field surround. Although amacrine cell input to ganglion cells mediates a component of this lateral inhibition, it is not known if presynaptic inhibition to bipolar cell terminals also contributes to surround signaling. We investigated the role of presynaptic inhibition by recording from bipolar cells in the salamander retinal slice. TTX reduced light-evoked GABAergic inhibitory postsynaptic currents (IPSCs) in bipolar cells, indicating that presynaptic pathways mediate lateral inhibition in the IPL. Photoreceptor and bipolar cell synaptic transmission were unaffected by TTX, indicating that its main effect was in the IPL. To rule out indirect actions of TTX, we bypassed lateral signaling in the outer retina by either electrically stimulating bipolar cells or by puffing kainate (KA) directly onto amacrine cell processes lateral to the recorded cell. In bipolar and ganglion cells, TTX suppressed laterally evoked IPSCs, demonstrating that both pre- and postsynaptic lateral signaling in the IPL depended on action potentials. By contrast, locally evoked IPSCs in both cell types were only weakly suppressed by TTX, indicating that local inhibition was not as dependent on action potentials. Our results show a TTX-sensitive lateral inhibitory input to bipolar cell terminals, which acts in concert with direct lateral inhibition to give rise to the GABAergic surround in ganglion cells.     

Reciprocal synaptic interactions between rod bipolar cells and amacrine cells in the rat retina.

Reciprocal synaptic transmission between rod bipolar cells and presumed A17 amacrine cells was studied by whole cell voltage-clamp recording of rod bipolar cells in a rat retinal slice preparation. Depolarization of a rod bipolar cell evoked two identifiable types of Ca2+ current, a T-type current that activated at about -70 mV and a current with L-type pharmacology that activated at about -50 mV. Depolarization to greater than or equal to -50 mV also evoked an increase in the frequency of postsynaptic currents (PSCs). The PSCs reversed at approximately ECl (the chloride equilibrium potential), followed changes in ECl, and were blocked by gamma-aminobutyric acidA (GABAA) and GABAC receptor antagonists and thus were identified as GABAergic inhibitory PSCs (IPSCs). Bipolar cells with cut axons displayed the T-type current but lacked an L-type current and depolarization-evoked IPSCs. Thus L-type Ca2+ channels are placed strategically at the axon terminals to mediate transmitter release from rod bipolar cells. The IPSCs were blocked by the non-N-methyl-D-aspartate (non-NMDA) receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione, indicating that non-NMDA receptors mediate the feed-forward bipolar-to-amacrine excitation. The NMDA receptor antagonist 3-((RS)-2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid had no consistent effect on the depolarization-evoked IPSCs, indicating that activation of NMDA receptors is not essential for the feedforward excitation. Tetrodotoxin (a blocker of voltage-gated Na+ channels) reversibly suppressed the reciprocal response in some cells but not in others, indicating that graded potentials are sufficient for transmitter release from A17 amacrine cells, but suggesting that voltage-gated Na+ channels, under some conditions, can contribute to transmitter release.     

Local Gabaergic Modulation of the Activity of Serotoninergic Neurons in the Nucleus Raphe Magnus

Experiments on rat brainstem sections in membrane potential clamping conditions addressed the effects of serotonin and GABA on serotoninergic neurons in the nucleus raphe magnus. Local application of serotonin stimulated inhibitory postsynaptic currents (IPSC) in 45% of the serotoninergic neurons studied. This response was not seen in the presence of the fast sodium channel blocker tetrodotoxin. The GABAA receptor antagonist gabazine blocked IPSC in both serotonin-sensitive and serotonin-insensitive neurons. Application of GABA evoked generation of a membrane current (IGABA), which was completely blocked by gabazine. These results indicate self-regulation of the activity of serotoninergic neurons in the nucleus raphe magnus via a negative feedback circuit involving local GABAergic interneurons.     

Presynaptic inhibition differentially shapes transmission in distinct circuits in the mouse retina

Diverse retinal outputs are mediated by ganglion cells that receive excitatory input from distinct classes of bipolar cells (BCs). These classes of BCs separate visual signals into rod, ON and OFF cone pathways. Although BC signalling is a major determinant of the ganglion cell-mediated retinal output, it is not fully understood how light-evoked, presynaptic inhibition from amacrine cell inputs shapes BC outputs. To determine whether differences in presynaptic inhibition uniquely modulate BC synaptic output to specific ganglion cells, we assessed the inhibitory contributions of GABA_A, GABA_C and glycine receptors across the BC pathways. Here we show that different proportions of GABA_A and GABA_C receptor-mediated inhibition determined the kinetics of GABAergic presynaptic inhibition across different BC classes. Large, slow GABA_C and small, fast GABA_A receptor-mediated inputs to rod BCs prolonged light-evoked inhibitory postsynaptic currents (L-IPSCs), while smaller GABA_C and larger GABA_A receptor-mediated contributions produced briefer L-IPSCs in ON and OFF cone BCs. Glycinergic inhibition also varied across BC class. In the rod-dominant conditions studied here, slow glycinergic inputs dominated L - IPSCs in OFF cone BCs, attributable to inputs from the rod pathway via AII amacrine cells, while rod and ON cone BCs received little and no glycinergic input, respectively. As these large glycinergic inputs come from rod signalling pathways, in cone-dominant conditions L-IPSCs in OFF cone bipolar cells will probably be dominated by GABA_A receptor-mediated input. Thus, unique presynaptic receptor combinations mediate distinct forms of inhibition to selectively modulate BC outputs, enhancing the distinctions among parallel retinal signals.     

I4AA-Sensitive chloride current contributes to the center light responses of bipolar cells in the tiger salamander retina

Light-evoked currents in depolarizing and hyperpolarizing bipolar cells (DBCs and HBCs) were recorded under voltage-clamp conditions in living retinal slices of the larval tiger salamander. Responses to illumination at the center of the DBCs' and HBCs' receptive fields were mediated by two postsynaptic currents: DeltaI(C), a glutamate-gated cation current with a reversal potential near 0 mV, and DeltaI(Cl), a chloride current with a reversal potential near -60 mV. In DBCs DeltaI(C) was suppressed by L-2-amino-4-phosphonobutyric acid (L-AP4), and in HBCs it was suppressed by 6,7-dinitroquinoxaline-2,3-dione (DNQX). In both DBCs and HBCs DeltaI(Cl) was suppressed by imidazole-4-acetic acid (I4AA), a GABA receptor agonist and GABA(C) receptor antagonist. In all DBCs and HBCs examined, 10 microM I4AA eliminated DeltaI(Cl) and the light-evoked current became predominately mediated by DeltaI(C). The addition of 20 microM L-AP4 to the DBCs or 50 microM DNQX to HBCs completely abolished DeltaI(C). Focal application of glutamate at the inner plexiform layer elicited chloride currents in bipolar cells by depolarizing amacrine cells that release GABA at synapses on bipolar cell axon terminals, and such glutamate-induced chloride currents in DBCs and HBCs could be reversibly blocked by 10 microM I4AA. These experiments suggest that the light-evoked, I4AA-sensitive chloride currents (DeltaI(Cl)) in DBCs and HBCs are mediated by narrow field GABAergic amacrine cells that activate GABA(C) receptors on bipolar cell axon terminals. Picrotoxin (200 microM) or (1,2,5,6-tetrahydropyridine-4yl) methyphosphinic acid (TPMPA) (2 other GABA(C) receptor antagonists) did not block (but enhanced and broadened) the light-evoked DeltaI(Cl), although they decreased the chloride current induced by puff application of GABA or glutamate. The light response of narrow field amacrine cells were not affected by I4AA, but were substantially enhanced and broadened by picrotoxin. These results suggest that there are at least two types of GABA(C) receptors in bipolar cells: one exhibits stronger I4AA sensitivity than the other, but both can be partially blocked by picrotoxin. The GABA receptors in narrow field amacrine cells are I4AA insensitive and picrotoxin sensitive. The light-evoked DeltaI(Cl) in bipolar cells are mediated by the more strongly I4AA-sensitive GABA(C) receptors. Picrotoxin, although acting as a partial GABA(C) receptor antagonist in bipolar cells, does not suppress DeltaI(Cl) because its presynaptic effects on amacrine cell light responses override its antagonistic postsynaptic actions.     

Presynaptic modulation of GABAergic inhibition by GABA(B) receptors in the rat's inferior colliculus

Whole-cell patch clamp recordings were made from neurons in a brain slice preparation of the inferior colliculus in 11-15-day-old rat pups. Synaptic responses were elicited by applying a current pulse to the lateral lemniscus just below the central nucleus of the inferior colliculus. To examine GABAergic inhibition in the inferior colliculus all excitatory postsynaptic potentials and glycinergic inhibitory postsynaptic potentials were blocked by bath application of their respective antagonists and the contribution of GABA(B) receptors was determined for the remaining inhibitory postsynaptic potentials. For most cells the isolated inhibitory postsynaptic potential was completely blocked by the GABA(A) receptor antagonist, bicuculline, but was unaffected by the GABA(B) receptor antagonist, phaclofen. The GABA(B) receptor agonist, baclofen (10-20 microM), decreased the amplitude of the inhibitory postsynaptic potentials. This effect was completely blocked by phaclofen. Baclofen did not increase the cell membrane conductance or alter the rate of firing produced by depolarization of the cell membrane. In contrast, muscimol, a GABA(A) receptor agonist, greatly increased membrane conductance and lowered the firing rate produced by depolarization. Our results indicate that GABAergic inhibition in the auditory midbrain can be reduced by the activation of GABA(B) receptors and suggest that the effects are presynaptic.     

Contribution of voltage-gated sodium channels to the b-wave of the mammalian flash electroretinogram

Voltage-gated sodium channels (Na(v) channels) in retinal neurons are known to contribute to the mammalian flash electroretinogram (ERG) via activity of third-order retinal neurons, i.e. amacrine and ganglion cells. This study investigated the effects of tetrodotoxin (TTX) blockade of Na(v) channels on the b-wave, an ERG wave that originates mainly from activity of second-order retinal neurons. ERGs were recorded from anaesthetized Brown Norway rats in response to brief full-field flashes presented over a range of stimulus energies, under dark-adapted conditions and in the presence of steady mesopic and photopic backgrounds. Recordings were made before and after intravitreal injection of TTX (approximately 3 microm) alone, 3-6 weeks after optic nerve transection (ONTx) to induce ganglion cell degeneration, or in combination with an ionotropic glutamate receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 200 microm) to block light-evoked activity of inner retinal, horizontal and OFF bipolar cells, or with the glutamate agonist N-methyl-D-aspartate (NMDA, 100-200 microm) to reduce light-evoked inner retinal activity. TTX reduced ERG amplitudes measured at fixed times corresponding to b-wave time to peak. Effects of TTX were seen under all background conditions, but were greatest for mesopic backgrounds. In dark-adapted retina, b-wave amplitudes were reduced only when very low stimulus energies affecting the inner retina, or very high stimulus energies were used. Loss of ganglion cells following ONTx did not affect b-wave amplitudes, and injection of TTX in eyes with ONTx reduced b-wave amplitudes by the same amount for each background condition as occurred when ganglion cells were intact, thereby eliminating a ganglion cell role in the TTX effects. Isolation of cone-driven responses by presenting test flashes after cessation of a rod-saturating conditioning flash indicated that the TTX effects were primarily on cone circuits contributing to the mixed rod-cone ERG. NMDA significantly reduced only the additional effects of TTX on the mixed rod-cone ERG observed under mesopic conditions, implicating inner retinal involvement in those effects. After pharmacological blockade with CNQX, TTX still reduced b-wave amplitudes in cone-isolated ERGs indicating Na(v) channels in ON cone bipolar cells themselves augment b-wave amplitude and sensitivity. This augmentation was largest under dark-adapted conditions, and decreased with increasing background illumination, indicating effects of background illumination on Na(v) channel function. These findings indicate that activation of Na(v) channels in ON cone bipolar cells affects the b-wave of the rat ERG and must be considered when analysing results of ERG studies of retinal function.     

Acetylcholine and norepinephrine mediate GABAergic but not glycinergic transmission enhancement by melittin in adult rat substantia gelatinosa neurons

GABAergic and glycinergic inhibitory synaptic transmissions in substantia gelatinosa (SG; lamina II of Rexed) neurons of the spinal dorsal horn play an important role in regulating nociceptive transmission from the periphery. It has not yet been well known whether each of the inhibitory transmissions plays a distinct role in the regulation. We report an involvement of neurotransmitters in GABAergic but not glycinergic transmission enhancement produced by the PLA(2) activator melittin, where the whole-cell patch-clamp technique is applied to the SG neurons of adult rat spinal cord slices. Glycinergic but not GABAergic spontaneous inhibitory postsynaptic current (sIPSC) was increased in frequency and amplitude by melittin in the presence of nicotinic, muscarinic acetylcholine, and α(1)-adrenergic receptor antagonists (mecamylamine, atropine, and WB-4101, respectively). GABAergic transmission enhancement produced by melittin was unaffected by the 5-hydroxytryptamine 3 receptor and P2X receptor antagonists (ICS-205,930 and pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid, respectively). Nicotinic and muscarinic acetylcholine receptor agonists [(-)-nicotine and carbamoylcholine, respectively] and norepinephrine, as well as melittin, increased GABAergic sIPSC frequency and amplitude. A repeated application of (-)-nicotine, carbamoylcholine, and norepinephrine, but not melittin, at an interval of 30 min produced a similar transmission enhancement. These results indicate that melittin produces the release of acetylcholine and norepinephrine, which activate (nicotinic and muscarinic) acetylcholine and α(1)-adrenergic receptors, respectively, resulting in GABAergic but not glycinergic transmission enhancement in SG neurons. The desensitization of a system leading to the acetylcholine and norepinephrine release is slow in recovery. This distinction in modulation between GABAergic and glycinergic transmissions may play a role in regulating nociceptive transmission.     

Potentiation of GABAergic synaptic transmission in the rostral nucleus of the solitary tract

Whole-cell recordings were made from neurons in the rostral nucleus of the solitary tract in horizontal brainstem slices. Monosynaptic GABAA receptor-mediated inhibitory postsynaptic potentials were evoked by single stimulus shocks or by high-frequency tetanic stimulation in the presence of glutamate receptor blockers. While single stimulus-evoked inhibitory postsynaptic potentials had variable amplitudes, tetanic stimulation-induced, hyperpolarizing postsynaptic potentials were of a more constant amplitude. Furthermore, tetanic stimulation resulted in potentiation of the amplitude of single stimulus shock-evoked inhibitory postsynaptic potentials. Of 55 neurons that were tested, potentiation lasted over 30 min for 11, 10-30 min for 13, less than 10 min for 23 and no potentiation occurred in eight. Tetanic stimulation did not result in potentiation of the tetanic stimulus-evoked hyperpolarizing postsynaptic potentials. Both the single stimulus shock- and tetanic stimulus-evoked potentials had similar inhibition concentration-response curves to the GABAA antagonist, bicuculline methiodide (EC50 = 0.75 and 0.83, respectively), indicating that they were mediated by the same postsynaptic receptors. By comparing the effect of bicuculline methiodide on the amplitude of the single stimulus shock-evoked inhibitory postsynaptic potentials and the tetanic stimulus-evoked hyperpolarizing potentials, we concluded that a single stimulus shock does not activate all postsynaptic GABAA receptors. However, tetanic stimulation results in activation of all postsynaptic GABAA receptors and induces long-lasting changes in the presynaptic GABAergic neuron. These long-lasting changes of the presynaptic neuron facilitate the release of GABA during single stimulus shock and, as a consequence, more postsynaptic receptors are activated during single stimulus shock-evoked synaptic transmission. This conclusion is supported by the results of experiments in which the extracellular Ca2+ concentration was manipulated to change the amount of neurotransmitter released from the presynaptic GABAergic terminals. The single stimulus shock-evoked inhibitory postsynaptic potentials were sensitive to the extracellular Ca2+ concentration, whereas tetanic stimulus-evoked inhibitory post-synaptic potentials were essentially insensitive to extracellular Ca2+ concentration. The relationship between the single stimulus shock-evoked inhibitory postsynaptic potential amplitude and extracellular Ca2+ concentration indicates that, in control physiological saline containing 2.5 mM Ca2+, a single stimulus shock activates less than half the postsynaptic GABA receptors. The phenomenon of long-lasting potentiation of inhibitory transmission within the rostral nucleus of the solitary tract may be important in the processing of gustatory information and play a role in taste-guided behaviors.     

Glycinergic inputs cause the pause of pontine omnipause neurons during saccades

Pontine omnipause neurons (OPNs) are inhibitory neurons projecting to saccade-related premotor burst neurons. OPNs exhibit sustained discharge during fixations and cease firing before and during saccades. The pause in OPN discharge releases the burst neurons from tonic inhibition, resulting in generation of saccadic eye movements. OPNs are thought to receive two major inhibitory inputs during saccades: an early component that determines the pause onset and a late component that controls the pause duration. Although there is evidence that numerous glycinergic and GABAergic terminals contact OPNs, their physiological roles remain unclear. To reveal functions of glycinergic and GABAergic inputs, we investigated effects of iontophoretic application of strychnine, a glycine receptor antagonist, and bicuculline, a GABAA receptor antagonist, on discharge patterns of OPNs in alert cats. Application of strychnine reduced the ratio of pause duration to saccade duration. Analysis of the timing of pause relative to saccades showed that pause onset was delayed and pause end was advanced. These effects were observed for saccades in all directions. Application of bicuculline, in contrast, had no effect on the OPN pause duration or timing. Both strychnine and bicuculline increased tonic firing rate during intersaccadic intervals. These results suggest that glycinergic, but not GABAergic, afferents convey inhibitory signals that determine the onset as well as duration of pause in OPN activity during saccades.     

GABA-Mediated inhibition between amacrine cells in the goldfish retina

Retinal amacrine cells have abundant dendro-dendritic synapses between neighboring amacrine cells. Therefore an amacrine cell has both presynaptic and postsynaptic aspects. To understand these synaptic interactions in the amacrine cell, we recorded from amacrine cells in the goldfish retinal slice preparation with perforated- and whole cell-patch clamp techniques. As the presynaptic element, 19% of the cells recorded (15 of 78 cells) showed spontaneous tetrodotoxin (TTX)-sensitive action potentials. As the postsynaptic element, all amacrine cells (n = 9) were found to have GABA-evoked responses and, under perforated patch clamp, 50 microM GABA hyperpolarized amacrine cells by activating a Cl(-) conductance. Bicuculline-sensitive spontaneous postsynaptic currents, carried by Cl(-), were observed in 82% of the cells (64 of 78 cells). Since the source of GABA in the inner plexiform layer is amacrine cells alone, these events are likely to be inhibitory postsynaptic currents (IPSCs) caused by GABA spontaneously released from neighboring amacrine cells. IPSCs were classified into three groups. Large amplitude IPSCs were suppressed by TTX (1 microM), indicating that presynaptic action potentials triggered GABA release. Medium amplitude IPSCs were also TTX sensitive. Small amplitude IPSCs were TTX insensitive (miniature IPSCs; n = 26). All of spike-induced, medium amplitude, and miniature IPSCs were Ca(2+) dependent and blocked by Co(2+). Blocking of glutamatergic inputs by DL-2-amino-phosphonoheptanoate (AP7; 30 microM) and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 2 microM) had almost no effect on spontaneous GABA release from presynaptic amacrine cells. We suggest that these dendro-dendrotic inhibitory networks contribute to receptive field spatiotemporal properties.     

Electrophysiological and pharmacological analysis of synaptic inputs to pulmonary rapidly adapting receptor relay neurons in the rat

The information from pulmonary rapidly adapting stretch receptors (RARs) to the central nervous system (CNS) is relayed in the nucleus tractus solitarii (NTS). The second-order neurons in the NTS referred to as RAR cells have recently been shown to receive rhythmic inputs from the central respiratory system in addition to the main inputs from RAR afferents. The present study analyzed these synaptic inputs by intracellular recordings from RAR cells, and by extracellular recordings combined with local applications of neuroactive drugs to RAR cells, in Nembutal-anesthetized, paralyzed, and artificially ventilated rats. The intracellular analysis identified both excitatory postsynaptic potentials (EPSPs) elicited presumably by RAR afferents and inhibitory postsynaptic potentials (IPSPs) synchronous with central inspiratory activity. This inhibitory input, called I suppression, was the origin of respiratory modulation of RAR cell firing, and its time course suggested that some unidentified inspiratory neurons with an augmenting firing pattern were the source of the inhibition. The pharmacological analysis suggested the types of neurotransmitters used in these synaptic events. First, glutamate was shown to be the primary neurotransmitter at the synapse between RAR afferents and RAR cells. Iontophoretic applications of the non-NMDA glutamate antagonist, CNQX, abolished RAR cell firing almost completely in response to lung inflation and deflation and to electrical stimulation of the vagus nerve. Second, glycinergic inputs which inhibited RAR cells in the inspiratory phase were revealed by applications of the glycine antagonist, strychnine. That is, the I suppression was greatly diminished by applications of strychnine. Third, although applications of the GABA_A receptor antagonist, bicuculline, had little effect on I suppression, bicuculline markedly increased the baseline firing of RAR cells. These results imply that the information path from RARs to the CNS is regulated at the level of RAR cells by phasically-acting glycinergic inhibition in the inspiratory phase and tonically-acting GABAergic inhibition; the results also provide new insights into the neuronal mechanisms of RAR-induced reflexes. Received: 3 February 1999 / Accepted: 12 May 1999     

Kainate receptor-mediated synaptic currents in mudpuppy inner retinal neurons reduced by D-O-phosphoserine

1. The effects of D-O-phosphoserine (DOS) were examined on proximal neurons in the superfused mudpuppy retinal-eyecup preparation by measuring their synaptically evoked whole-cell currents with the use of patch-clamp electrodes. 2. DOS reduced the light-evoked excitatory postsynaptic potentials (EPSPs) of amacrine and ganglion cells. This suppression was present even though the center responses of both ON- and OFF-bipolar cells were unaffected by DOS. 3. When recordings were done under voltage-clamp conditions. DOS diminished the magnitude of light-evoked synaptic currents associated with a reduction in synaptic conductance. 4. To determine which acidic amino acid receptor mediated the network-selective action of DOS, various glutamate agonists were tested against this excitatory amino acid receptor (EAAR) antagonist. DOS blocked the depolarizing effects of kainate (KA), but not those of N-methyl-D-aspartate (NMDA) or quisqualate (QQ). Thus DOS was a selective KA antagonist, and KA receptors appear to be the dominant EAAR subtype that mediates synaptic inputs into the inner retina of the mudpuppy.     

GABAB Receptor- and Metabotropic Glutamate Receptor-Dependent Cooperative Long-Term Potentiation of Rat Hippocampal GABAA Synaptic Transmission

Repetitive stimulation of Schaffer collaterals induces activity-dependent changes in the strength of polysynaptic inhibitory postsynaptic potentials (IPSPs) in hippocampal CA1 pyramidal neurons that are dependent on stimulation parameters. In the present study, we investigated the effects of two stimulation patterns, theta-burst stimulation (TBS) and 100 Hz tetani, on pharmacologically isolated monosynaptic GABAergic responses in adult CA1 pyramidal cells. Tetanization with 100 Hz trains transiently depressed both early and late IPSPs, whereas TBS induced long-term potentiation (LTP) of early IPSPs that lasted at least 30 min. Mechanisms mediating this TBS-induced potentiation were examined using whole-cell recordings. The paired-pulse ratio of monosynaptic inhibitory postsynaptic currents (IPSCs) was not affected during LTP, suggesting that presynaptic changes in GABA release are not involved in the potentiation. Bath application of the GABA_B receptor antagonist CGP55845 or the group I/II metabotropic glutamate receptor antagonist E4-CPG inhibited IPSC potentiation. Preventing postsynaptic G-protein activation or Ca²⁺ rise by postsynaptic injection of GDP-β-S or BAPTA, respectively, abolished LTP, indicating a G-protein- and Ca²⁺-dependent induction in this LTP. Finally during paired-recordings, activation of individual interneurons by intracellular TBS elicited solely short-term increases in average unitary IPSCs in pyramidal cells. These results indicate that a stimulation paradigm mimicking the endogenous theta rhythm activates cooperative postsynaptic mechanisms dependent on GABA_BR, mGluR, G-proteins and intracellular Ca²⁺, which lead to a sustained potentiation of GABA_A synaptic transmission in pyramidal cells. GABAergic synapses may therefore contribute to functional synaptic plasticity in adult hippocampus.     

Roles of aspartate and glutamate in synaptic transmission in rabbit retina. II. Inner plexiform layer

Intracellular recordings were obtained from amacrine and ganglion cells in the superfused, isolated retina-eyecup of the rabbit. The putative neurotransmitters aspartate, glutamate, and several of their analogues were added to the superfusate while the membrane potential and light-responsiveness of the retinal neurons were monitored. Both L-aspartate and L-glutamate displayed excitatory actions on the activity of the vast majority of amacrine and ganglion cells studied. However, these agents occasionally appeared to inhibit the responses of the inner retinal neurons by producing hyperpolarization of the membrane potential and blockage of the light-evoked responses. In either case, the effects of aspartate and glutamate were indistinguishable. The glutamate analogues kainate and quisqualate produced strong excitatory effects on the responses of amacrine and ganglion cells at concentrations some 200-fold less than those needed to obtain similar effects with aspartate or glutamate. The aspartate analogue, n-methyl DL-aspartate (NMDLA), also produced strong excitatory effects but was approximately three times less potent than kainate or quisqualate. On one occasion, we encountered a ganglion cell that was depolarized by kainate, but hyperpolarized by NMDLA. The glutamate antagonist alpha-methyl glutamate and the aspartate antagonist alpha-amino adipate effectively blocked the responses of amacrine and ganglion cells. However, on any one cell, one antagonist was always clearly more potent than the other. We examined the actions of the glutamate analogue 2-amino-4-phosphonobutyrate (APB) on the responses of inner retinal neurons and found that it selectively abolished all "on" activity in the inner retina. Together with our finding that APB selectively abolishes on-bipolar cell responses (see Ref. 6), these data support the hypothesis that on-bipolar cells subserve the "on" activity of amacrine and ganglion cells. Our data suggest that aspartate and glutamate are excitatory transmitters in the inner retina, possibly being released from bipolar cell axon terminals in the inner plexiform layer.     

Light-evoked oscillatory discharges in retinal ganglion cells are generated by rhythmic synaptic inputs

In the visual system, optimal light stimulation sometimes generates gamma-range (ca. 20 approximately 80 Hz) synchronous oscillatory spike discharges. This phenomenon is assumed to be related to perceptual integration. Applying a planar multi-electrode array to the isolated frog retina, Ishikane et al. demonstrated that dimming detectors, off-sustained type ganglion cells, generate synchronous oscillatory spike discharges in response to diffuse dimming illumination. In the present study, applying the whole cell current-clamp technique to the isolated frog retina, we examined how light-evoked oscillatory spike discharges were generated in dimming detectors. Light-evoked oscillatory ( approximately 30 Hz) spike discharges were triggered by rhythmic ( approximately 30 Hz) fluctuations superimposed on a depolarizing plateau potential. When a suprathreshold steady depolarizing current was injected into a dimming detector, only a few spikes were evoked at the stimulus onset. However, repetitive spikes were triggered by a gamma-range sinusoidal current superimposed on the steady depolarizing current. Thus the light-evoked rhythmic fluctuations are likely to be generated presynaptically. The light-evoked rhythmic fluctuations were suppressed not by intracellular application of N-(2,6-dimethyl-phenylcarbamoylmethyl)triethylammonium bromide (QX-314), a Na(+) channel blocker, to the whole cell clamped dimming detector but by bath-application of tetrodotoxin to the retina. The light-evoked rhythmic fluctuations were suppressed by a GABA(A) receptor antagonist but potentiated by a GABA(C) receptor antagonist, whereas these fluctuations were little affected by a glycine receptor antagonist. Because amacrine cells are spiking neurons and because GABA is one of the main transmitters released from amacrine cells, amacrine cells may participate in generating rhythmically fluctuated synaptic input to dimming detectors.     

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.     

Glycinergic inputs to cardiac vagal neurons in the nucleus ambiguus are inhibited by nociceptin and mu-selective opioids

Most parasympathetic regulation of heart rate originates from preganglionic cardiac vagal neurons within the nucleus ambiguus. Little is known regarding the modulation of glycinergic transmission to these neurons. However, the presence of mu-opioid receptors and opioid-receptor-like (ORL1) receptors within the ambiguus, together with the presence of endogenous ligands for both receptor types in the same area, suggests opioids may modulate synaptic transmission to cardiac vagal neurons. This study therefore examined the effects of endomorphin-1 and endomorphin-2 (the mu-selective endogenous peptides), DAMGO (a synthetic, mu-selective agonist), and nociceptin (the ORL1-selective endogenous peptide) on spontaneous glycinergic inhibitory postsynaptic currents (IPSCs) in rat cardiac parasympathetic neurons. All four of the opioids used in this study decreased spontaneous IPSCs. At concentrations of 100 microM, the amplitude of the IPSCs was reduced significantly by nociceptin (-56.6%), DAMGO (-46.5%), endomorphin-1 (-45.1%), and endomorphin-2 (-26%). IPSC frequency was also significantly reduced by nociceptin (-61.1%), DAMGO (-69.9%), and endomorphin-1 (-40.8%) but not endomorphin-2. Lower concentrations of nociceptin and DAMGO (10-30 microM) also effectively decreased IPSC amplitude and frequency. The inhibitory effects of DAMGO were blocked by d-Phe-Cys-Tyr-d-Trp-Orn-Thr-Pen-Thr-NH2 (C-TOP; 10 microM), a selective mu-receptor antagonist. Neither nociceptin nor DAMGO inhibited the postsynaptic responses evoked by exogenous application of glycine or affected TTX-insensitive glycinergic mini-IPSCs. These results indicate that mu-selective opioids and nociceptin act on preceding neurons to decrease glycinergic inputs to cardiac vagal neurons in the nucleus ambiguus. The resulting decrease in glycinergic transmission would increase parasympathetic activity to the heart and may be a mechanism by which opioids induce bradycardia.