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.

The GABAC receptor subtypes on bipolar cells of rats and cold-blooded vertebrates differ in their pharmacological properties and probably have different molecular compositions. With the exception of the rat, native GABAC receptors in mammals had not been studied. In ferret, whole-cell, voltage-clamp recordings were made from bipolar cells in the retinal slice preparation to determine which subtype of GABAC receptor predominated. Puff-evoked GABA currents in bipolar cells were partially reduced by the GABAA receptor antagonist bicuculline, indicating that both GABAA and GABAC receptors mediated the responses. By contrast, GABA currents of ganglion cells were always completely blocked by bicuculline, indicating that GABAA receptors predominated on these cells. Small-amplitude GABA currents of bipolar cells evoked by short-duration puffs were less sensitive to bicuculline than large-amplitude currents evoked by long-duration puffs. This indicates that GABAc receptors mediated proportionately more of the small-amplitude, puff-evoked responses and GABAA receptors mediated more of the large-amplitude, puff-evoked responses. In bipolar cells, the bicuculline-resistant component of the GABA current was entirely blocked by 3-APMPA (3-aminopropyl-(methyl)phosphonic acid), a GABAC receptor antagonist. Picrotoxin, which is relatively ineffective at rat GABAC receptors, completely blocked GABA currents in ferret bipolar cells, indicating that GABAC receptors on ferret bipolar cells resemble those in lower vertebrates rather than those in the rat retina. These results suggest that there may be a diversity of GABAc receptor subtypes on mammalian bipolar cells.

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.

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).

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.