The cellular basis of motor learning in the cerebellum has been attributed mostly to long-term depression (LTD) at excitatory parallel fiber (PF)-Purkinje cell (PC) synapses. LTD is induced when PFs are activated in conjunction with a climbing fiber (CF), the other excitatory input to PCs. Recently, by using whole-cell patch-clamp recording from PCs in cerebellar slices, a new form of synaptic plasticity was discovered. Stimulation of excitatory CFs induced a long-lasting (usually longer than 30 min) “rebound potentiation (RP)” of γ-amino-butyric acid A (GABAa)-receptor mediated inhibitory postsynaptic, currents (IPSCs). As in LTD, induction of RP requires transient elevation of intracellular calcium concentration ([Ca2+]1) due to activation of voltage-gated Ca2+ channels. Activity of inhibitory synapses seems also to be necessary for RP to occur. RP is mainly due to up-regulation of postsynaptic GABAa receptor function, since PC response to bath-applied exogenous GABA is also potentiated with a time course similar to RP. The difference in the time scale between the Ca2+ transients (10–30 sec) and the durations of RP (>30 min) strongly suggests that some intracellular biochemical machinery is involved. Pharmacological evidence suggests that protein kinases are involved in RP of inhibitory synapses and LTD of excitatory PF synapses. Besides the well-described LTD, RP could be a cellular mechanism that plays an important role in motor learning.

Bipolar cells (BCs) are critical relay neurons in the retina that are organized into parallel signaling pathways. The three main signaling pathways in the mammalian retina are the rod, ON cone, and OFF cone BCs. Rod BCs mediate incrementing dim light signals from rods, and ON cone and OFF cone BCs mediate incrementing and decrementing brighter light signals from cones, respectively. The outputs of BCs are shaped by inhibitory inputs from GABAergic and glycinergic amacrine cells in the inner plexiform layer, mediated by three distinct types of inhibitory receptors: GABAA, GABAC, and glycine receptors. The three main BC pathways receive distinct forms of inhibition from these three receptors that shape their light-evoked inhibitory signals. Rod BC inhibition is dominated by slow GABAC receptor inhibition, while OFF cone BCs are dominated by glycinergic inhibition. The inhibitory inputs to BCs are also shaped by serial inhibitory connections between GABAergic amacrine cells that limit the spatial profile of BC inhibition. We discuss our recent studies on how inhibitory inputs to BCs are shaped by receptor expression, receptor properties, and neurotransmitter release properties and how these affect the output of BCs.

This paper describes experiments on GABA-activated whole-cell membrane currents in bipolar cells freshly isolated from the adult rat retina. The main goal was to determine whether bipolar cell responses to GABA could be resolved in terms of mediation by the GABAA receptor, the GABAB receptor, or both. Bipolar cells were isolated by gentle enzymatic dissociation and identified by their distinct morphology. GABA agonists and antagonists were applied focally by pressure and the resultant currents were recorded under whole-cell voltage clamp. In all bipolar cells tested, GABA (0.1–100 μM) induced a monophasic response associated with a conductance increase (IGABA). The shift in reversal potential for IGABA as a function of pipet [CI] paralleled that predicted based on the Nernst equation for Cl−. IGABA was mimicked by muscimol (5–20 μM) and antagonized by bicuculline (20–100 μM). Baclofen (0.1–1.0 mM) produced no apparent conductance change. “Hot spots” of sensitivity to GABA which might be associated with regions of synaptic contact were not found; both the soma and processes of all bipolar cells were responsive to focally applied GABA. Furthermore, all bipolar cells tested responded to glycine.

In conclusion, we have established the presence of GABAA receptors on rat retinal bipolar cells. Our data suggest further that these cells lack GABAB receptors. Finally, our observation that bipolar cells in the rat retina are relatively homogeneous in terms of their sensitivity to GABA and glycine lead us to postulate that the functional significance of the presence of receptors and their distribution on a neuron may be dictated more by the topography of the presynaptic inputs than by its inherent chemosensitivity.

A large population of amacrine cells in the retina are thought to use GABA as an inhibitory neurotransmitter in their synaptic interactions within the inner plexiform layer. However, little is known about their synaptic targets; the neurons that express the receptors for GABA have not been clearly identified. Recently, the GABAA receptor has been isolated and antibodies have been raised against it. These antibodies have proven useful for the immunocytochemical localization of the receptor, and two brief reports describing the distribution of GABAA receptor immunoreactivity in the retina have appeared (Richards et al., 1987; Mariani et al., 1987). We used a monoclonal antibody (62–3G1) against the GABAA receptor to study the retina of the New World primate Saimiri sciureus.

Labeled somata were found in the inner nuclear layer (INL) and ganglion cell layer (GCL). The staining was confined to what appeared to be the cell's plasmalemma and small cytoplasmic granules. Most of the labeled neurons in the INL had small somata (5–7 μm in diameter) located at the vitreal edge of the layer. They arborized in two laminae (approximately 2 and 4) of inner plexiform layer (IPL). Ventral to the optic disc (2.5 mm) they comprised 29% of the cells present. A few of the labeled neurons appeared to be interplexiform cells or flat bipolar cells, with labeled processes that extended into both the IPL and the inner half of the outer plexiform layer. In the GCL, the labeled somata were among the largest present (13–20 μm in diameter), and 2.5 mm ventral to the optic disc they made up 15% of the cells present. Experiments in which immunoreactive somata were retrogradely labeled following the injection of fluorescent tracers into the optic tract provided a conclusive demonstration that some of the immunoreactive somata were ganglion cells. The antibody often labeled their axons in the optic fiber layer. This suggests that the GABAA receptors are transported anterogradely to the retinal terminal fields. The dendrites of the immunoreactive ganglion cells extended into the 2 laminae of labeled processes in the IPL, and their primary dendritic arbors were, at any given eccentricity, quite similar in appearance. This homogeneity suggests that they comprise a particular subset of the ganglion cells.

Sections simultaneously labeled with the monoclonal antibody against the GABAA receptor and antisera against either L-glutamic acid decarboxylase (GAD) or GABA revealed that the GAD/GABA was distributed much more widely in the IPL than the GABAA receptor. This variance may reflect either the presence of other, perhaps GABAB, receptors in the IPL or GABA in portions of the IPL where it is ineffective as a neurotransmitter.

Gamma-aminobutyric acid (GABA) is an inhibitory neurotransmitter used by many neurons of the mammalian retina. To identify the synaptic targets of these cells, we undertook an immunohistochemical study with a monoclonal antibody that recognizes the GABAA receptors (62−3G1, generously donated by A. de Blas). This antibody labels the somata of at least one group of amacrine cells in the inner nuclear layer. It also labels two groups of somata in the ganglion cell layer: one small and the other much larger. The small cells are likely to be displaced amacrine cells based on their size, although some could be gamma ganglion cells. The much larger receptor-positive cells are clearly ganglion cells, based both on their size and the antibody labeling of the initial portion of their axon. In the peripheral retina, the size of these large somata suggests that many are beta ganglion cells. However, at any point across the retina the density of these cells never exceeded 50% of the density of beta cells as a whole.

The antibody also labels a dense plexus of processes that extends throughout the inner plexiform layer (IPL), with a marked concentration in the inner third of the layer. This is the portion of the IPL in which the rod bipolar cells terminate. It is difficult to recognize processes of individual cells in the IPL, so retinae were dissociated. The rod bipolar cells were identified by protein kinase C immunoreactivity (Negishi et al., 1988; Karschin & Wässle, 1990). They were not labeled by the GABAA receptor antibody. This is surprising in light of tight-seal, whole cell voltage-clamp recordings that have shown that the rod bipolars express functional GABAA receptors. One possible explanation is that the antibody recognizes only a subset of the GABAA receptors.

Dry autoradiography was used to determine the distribution of GABAA binding sites in tiger salamander retina. High-affinity binding of [3H]-flunitrazepam ([3H]-FNZ) was used to localize benzodiazepine receptors (BZR) and [3H]-muscimol was used to localize the GABAA recognition site. Specific [3H]-FNZ binding was present only in the inner retina, primarily in the inner plexiform layer (IPL). Co-incubation with GABA enhanced [3H]-FNZ binding by 20–50%. [3H]-muscimol binding was found throughout the IPL and in the outer plexiform layer (OPL). Mouse monoclonal antibodies 62–3G1 and BD-17, that recognize the GABAAβ2, β3 polypeptides, and BD-24, that recognizes the GABAA α1, polypeptide, did not label either the OPL or IPL, despite numerous variations in the fixation and immunoprocessing methods. GABAA receptor location, as revealed by [3H]-muscimol binding, matches the distribution of presumed GABAergic terminals in the OPL and IPL. We suggest that there are at least two subtypes of GABAA receptor in the tiger salamander retina: one type is present only in the inner retina, primarily in the IPL and is functionally coupled to BZRs; the other type is located in the OPL and is not coupled to the BZRs. Furthermore, GABAA receptors in the tiger salamander retina appear to be of a different epitope than GABAA receptors in numerous other preparations that are recognized by mAbs 62–3G1, BD-17, and BD-24.

Amphibian oocytes meiotic arrest is released under the stimulus of progesterone; this hormone interacts with the oocyte surface and starts a cascade of events leading to the activation of a cytoplasmic maturation promoting factor (MPF) that induces germinal vesicle breakdown (GVBD), chromosome condensation and extrusion of the first polar body.

The aim of this work was to determine whether the activation of a GABAA receptor is able to induce GVBD in fully grown denuded oocytes of Bufo arenarum and to analyse its possible participation in progesterone-induced maturation. We also evaluated the role of purines and phospholipids in the maturation process induced by a GABAA receptor agonist such as muscimol.

Our results indicated that the activation of the GABAA receptor by muscimol induces maturation in a dose- and time-dependent manner and that this activation is a genuine maturation that enables oocytes to form pronuclei. Assays with a receptor antagonist, picrotoxine, showed that the maturation induced by muscimol was inhibited. Treatment with picrotoxine, however, shows that the participation of GABAA receptor in progesterone-induced maturation is not significant.

In addition, our results indicate that high intracellular levels of purines obtained by the use of db-AMPc and theophylline or the inhibition of the phosphatidylinositol 4,5-bisphosphate (PIP2 hydrolysis by neomycin and PIP2 turn over by LiCl, respectively, inhibited the maturation induced by muscimol. Treatment with H-7 indicated, however, that PKC activation is not necessary for GVBD induced by the GABAA receptor agonist. Results suggest that the transduction pathway used by the GABAA receptor to induce maturation is different from those used by progesterone.

The synaptic terminals of mammalian rod bipolar cells are the targets of multiple presynaptic inhibitory inputs arriving from glycinergic and GABAergic amacrine cells. To investigate the contribution of these different inhibitory receptor types, we have applied the patch-clamp technique in acutely isolated slices of the adult mouse retina. By using the whole-cell configuration, we measured and analyzed the spontaneous postsynaptic currents (PSCs) in rod bipolar cells. The spontaneous synaptic activity of rod bipolar cells was very low. However, when amacrine cells were depolarized by AMPA or kainate, the PSC frequency in rod bipolar cells increased significantly. These PSCs comprised several types that could be distinguished by pharmacological and kinetic criteria. Strychnine-sensitive, glycinergic PSCs were characterized by a mean peak amplitude of −43.5 pA and a weighted decay time constant (τw) of 10.9 ms. PSCs that persisted in the presence of strychnine, but were completely inhibited by bicuculline, were mediated by GABAARs. They had a mean peak amplitude of −20.0 pA and a significantly faster τw of 5.8 ms. Few PSCs remained in the presence of strychnine and bicuculline, suggesting that they were mediated by GABACRs. These PSCs were characterized by much smaller amplitudes (−6.2 pA) and a significantly slower decay kinetics (τw = 51.0 ms). We conclude that rod bipolar cells express at least three types of functionally different inhibitory receptors, namely GABAARs, GABACRs, and GlyRs that may ultimately regulate the Ca2+ influx into rod bipolar cell terminals, thereby modulating their glutamate release.

Background and objective The γ1-aminobutyric acidA receptor (GABAAR) is a target for anaesthetic agents. We investigated the interactions of sevoflurane with a recombinant GABAAR. Emphasis was on the mechanism of block, as relevant open-channel block by a volatile anaesthetic would possibly explain prolonged GABAergic postsynaptic currents.

Methods The effect of sevoflurane on GABA–induced currents through recombinant α1β1γ1 GABAAR channels was studied (patch clamp; HEK293 cells). GABA 0.01mM or 1mM was applied alone or together with sevoflurane (0.05mM to 5mM).

Results Currents elicited by GABA 0.01mM were increased by low sevoflurane concentrations to 183% and decreased by high sevoflurane concentrations (> 1mM) to 34% (P < 0.05). Ten- to 90%-rise times of the currents were reduced by sevoflurane concentration dependently. At GABA (1mM), peak currents and 10–90%-rise times decreased with increasing sevoflurane concentrations. A transient current increase was induced by discontinuation of GABA and sevoflurane. Such rebound currents indicate a reversal of an openchannel block by sevoflurane.

Conclusions Sevoflurane (a) increases the apparent affinity of GABA to the GABAAR, as suggested by the decreased current rise times. This explains the enhancement of the currents induced by low GABA concentrations (0.01mM). Additionally, sevoflurane (b) induces a picrotoxin-like open-channel block at the GABAAR. The reversal of the open-channel block elicits a delayed GABA response. These findings indicate at least two different sites of action of sevoflurane at this receptor that are both important for an enhanced GABAergic synaptic transmission.

A preparation of isolated presynaptic terminals of rat retinal rod bipolar cells was developed. Patch-clamp recordings were performed on the isolated terminal to determine the type(s) of voltage-activated Ca2+ channels and the contribution of GABAA and GABAC receptor-mediated currents localized in the terminal region. Both low-voltage-activated (LVA) and high-voltage-activated (HVA) Ca2+ currents, with properties similar to those found in intact cell recordings, were observed in the isolated terminal recordings. Consistent with previous studies, the HVA Ca2+ currents are L-type since the currents were blocked by low micromolar concentrations of nimodipine and potentiated by BayK 8644. Also, both GABAA and GABAC receptor-mediated currents were observed in the isolated terminal. The current density of GABAC receptors in the terminal was more than three times higher than that in the soma. In contrast, the current density of GABAA currents between the terminal and the soma was not significantly different. Assessed by 100 μM GABA, the contributions of GABAA and GABAC receptors to the total GABA-mediated currents at the terminal were comparable. This study directly demonstrates the localization of LVA Ca2+ channels at the axon terminal of mammalian rod bipolar cells, suggesting that LVA Ca2+ channels may play a role in bipolar cell transmitter release. Results of this study also support the notion that both types of ionotropic GABA receptors regulate synaptic transmission in mammalian rod bipolar cells. In addition, this study reports for the first time the feasibility of direct patch-clamp recordings of isolated axon terminals of mammalian retinal bipolar cells. The isolated presynaptic terminal preparation of mammalian retinal bipolar cells could be a valuable system for the study of transmitter release in the central nervous system (CNS).

Gamma aminobutyric acid (GABA) has been established as an important developmental signal in a number of regions of the central nervous system (CNS), including retina. Our previous studies have shown that GABAergic horizontal cells act as the initial synaptic target for developing cone photoreceptors in neonatal rabbit retina. Since intraocular injections of the GABAA receptor antagonists, picrotoxin or bicuculline, disrupt cone synaptogenesis in vivo, GABA released from horizontal cells may provide a necessary signal for cone axon growth and/or synapse formation. In the current report, we have used cultured retinal explants to examine the effects of GABAA receptor antagonists on other aspects of developing cones. These include the distribution pattern of cone cell bodies across the outer surface of the retina and the expression of GABAA receptors within both cone cell bodies and axonal processes. Peanut agglutinin (PNA), a plant lectin that specifically labels cone plasma membrane and extracellular matrix, was used to monitor cone development, and a GABAA receptor antibody against the β2/3 subunits of the protein was used to label GABAA receptors. Results showed that cones maintained in the explant culture express GABAA receptors in a temporal and spatial pattern similar to that observed in vivo, namely a low expression of receptors on cone cell bodies at postnatal day 1 (P1), peaking around P3 and diminishing by P7. Neonatal retinal explants exposed to the GABAA receptor antagonists, bicuculline (10 μM) or SR95531 (5 μM), for 24 h in culture showed disruption of the normal distribution of cone cell bodies. When GABA (100 μM) was added along with either antagonist, cone cell bodies appeared normal. Neither bicuculline nor SR95531 alone had any effect on the general morphology of other retinal layers, suggesting that these GABAA receptor antagonists at the concentrations used were not acting as nonspecific disruption agents. The effects of GABA antagonists were confined to the first week after birth with no disruption seen in P9 or adult explant cultures. These data provide a direct demonstration of the necessity for GABAergic input to cones during active synaptogenesis. As we have previously shown, GABAA receptor activation causes a substantial increase in intracellular calcium concentrations in cones and thereby could provide a mechanism by which GABA regulates cone maturation.

The physiological function of GABA transporters and GABA receptors in retinal horizontal cells (HCs) under dark- and light-adapted conditions were studied by whole-cell voltage clamp and intracellular recording techniques in retinal slices and whole-mounted isolated retinas of the larval tiger salamander. Puff application of GABA in picrotoxin elicited a NO-711 (a potent GABA transporter blocker)-sensitive inward current that did not exhibit a reversal potential in the physiological range, consistent with the idea that these HCs contain electrogenic GABA transporters. Application of GABA in NO-711 elicited a chloride current in HCs; about half of the current was suppressed by bicuculline or I4AA (a GABAC receptor antagonist), and the remaining half was suppressed by bicuculline + I4AA or picrotoxin. In whole-mount retinas, NO-711, bicuculline, I4AA, or picrotoxin hyperpolarized the HCs and enhanced the light responses under dark-adapted conditions, and blocked the time-dependent recovery of HC membrane potential and light responses during background illumination. Based on the parallel conductance model, GABA released in darkness mediates a chloride conductance about three times greater than the leak conductance or the glutamate-gated cation conductance. About half of this chloride conductance is mediated by GABAA receptors, and the other half is mediated by GABAC receptors. These results suggest that GABA released from HCs through the NO-711-sensitive GABA transporters activates GABAA and GABAC receptors, resulting in chloride conductance increase which leads to a HC depolarization and reduction of the light response. Additionally, GABA transporters also mediate GABA release in background light that is responsible for the recovery of HC membrane potential and light responses.