Glycinergic synaptic inputs to bipolar cells in the salamander retina

Glycine activated strychnine-sensitive chloride conductances at both the dendrites and the axonal telodendria of most bipolar cells in the salamander retina. The chloride equilibrium potential of bipolar cells was found to be negative to -50 mV, indicating that glycinergic synapses on bipolar cells are inhibitory. Some bipolar cells exhibited discrete, strychnine-sensitive, chloride-mediated inhibitory postsynaptic currents (IPSCs). These were elicited by focal application of glutamate at the inner plexiform layer (IPL). Glycinergic synapses were localized using simultaneous focal application of calcium to retinal slices bathed in calcium-free media. Both dendritic and telodendritic glycinergic IPSCs were observed. The decay of the telodendritic IPSCs was well fitted by a single exponential with a time constant of 17.7 ± 8.7 ms. Similar kinetics were observed for dendritic IPSCs in some cells, but in one class of on-centre bipolar cell the decay of the dendritic IPSCs was better fitted by a sum of two exponentials with time constants 9.9 ± 4.3 and 51.3 ± 24.3 ms. The dendritic IPSCs were best driven by application of glutamate at the distal IPL (the off sublamina), while the telodendritic IPSCs were driven best by application near the telodendria. These results suggest that bipolar cell dendrites receive inhibitory glycinergic inputs from interplexiform cells that are excited by off-centre bipolar cells, whereas bipolar cell telodendria receive glycinergic amacrine cell inputs that are antagonistic to the photoreceptor inputs. Both inputs could be elicited in the presence of tetrodotoxin (TTX), but the dendritic IPSCs were sometimes abolished by TTX, suggesting that sodium-dependent spikes play an important role in the transmission of interplexiform cell signals to the outer plexiform layer. Bipolar cells are non-spiking neurons involved in the integration of signals at both synaptic layers of the retina (Werblin & Dowling, 1969). At the outer plexiform layer (OPL), excitatory conductances on bipolar cell dendrites are modulated by photoreceptor synapses. Light hyperpolarizes photoreceptors and causes a reduction of transmitter release at these synapses. On-centre bipolar cells are depolarized by illumination of their receptive field centres, because the photoreceptor transmitter, glutamate, deactivates excitatory dendritic conductances in these cells (Slaughter & Miller, 1981; Attwell, Mobbs, Tessier-Lavigne & Wilson, 1987). Off-centre bipolar cells are hyperpolarized by illumination of their receptive field centres, because for these cells glutamate activates excitatory dendritic conductances (Slaughter & Miller, 1983; Maple, Werblin & Wu, 1994). Bipolar cells are the first cells in the visual system to exhibit centre-surround receptive field organization (Werblin & Dowling, 1969). The surround antagonism of their receptive fields is thought to result primarily from presynaptic inhibition of photoreceptors by GABAergic horizontal cells (Wu, 1992), although horizontal cell inputs to bipolar cell dendrites may also play a role (Yang & Wu, 1991; Hare & Owen, 1992). In addition, bipolar cell dendrites receive synapses from interplexiform cells (Smiley & Yazulla, 1990), but little is known about the role of these inputs in the generation of bipolar cell receptive fields. At the inner plexiform layer (IPL) bipolar cell axonal processes make excitatory glutamatergic synapses on ganglion cells (the output neurons of the retina), on amacrine cells (responsible for lateral inhibition at the IPL), and on interplexiform cells (Maguire, Lukasiewicz & Werblin, 1990; Mittman, Taylor & Copenhagen, 1990, Dixon & Copenhagen, 1992; Marc, Murray & Basinger, 1995). In the salamander retina, bipolar cell axons give rise to many fine telodendria, which ramify laterally in IPL. The bipolar cell telodendria and associated output synapses are segregated in the IPL according to bipolar cell type. Off- centre bipolar cells have telodendria that ramify and synapse on amacrine and ganglion cell dendrites in the distal IPL (sublamina A), while on-centre bipolar cell telodendria ramify and synapse on amacrine and ganglion cell dendrites in the proximal IPL (sublamina B) (Famiglietti & Kolb, 1976; Famiglietti, Kaneko & Tachibana, 1977; Nelson, Famiglietti & Kolb, 1978; Maple & Wu, 1996). Bipolar cells also receive inhibitory synaptic inputs from amacrine cells (Wong-Riley, 1974; Marc, 1992; Maple & Wu, 1996). Much of this inhibition is probably GABAergic, since in the salamander retina a large fraction of amacrine cells accumulate GABA (Wu, 1991) and GABA activates large chloride conductances localized at the telodendria of bipolar cells (Lukasiewicz & Werblin, 1994; Maple & Wu, 1996). In contrast to the considerable body of research that has been devoted to GABAergic circuits in the retina, relatively little attention has been given to the role of glycinergic circuitry. Immunocytochemical studies have shown that in the salamander retina, glycine is accumulated by subpopulations of amacrine cells and interplexiform cells, but not by horizontal cells (Wu, 1991). It is possible, therefore, that bipolar cells might receive synaptic inputs from glycinergic interplexiform cells at the OPL and/or glycinergic amacrine cells at the IPL. This article presents a systematic study of glycinergic inputs to retinal bipolar cells and provides the first direct electrophysiological evidence for glycinergic synapses from both amacrine cells and interplexiform cells. It begins with an examination of the spatial distribution of glycine-activated chloride conductances on bipolar cells. This is followed by a description of strychnine-sensititive inhibitory postsynaptic currents (IPSCs) observed in bipolar cells. A method is demonstrated for localizing the sites of these synaptic inputs through the focal application of calcium to a retinal slice. Based on the results of experiments combining focal stimulation of the retina with focal application of calcium, we present a detailed model for the circuitry of glycinergic synaptic inputs to one class of on-centre bipolar cell in the salamander retina.