Bruce R. Maple

Amino acid neurotransmitters in the retina : a functional overview

Physiological and pharmacological mechanisms of glutamatergic, GABAergic and glycinergic synapses in the tiger salamander retina were studied. We used immunocytochemical and autoradiographic methods to study localizations of these neurotransmitters and their uptake transporters; and electrophysiological methods (intracellular, extracellular and whole cell patch electrode recordings) to study the light responses, miniature postsynaptic currents and neurotransmitter-induced postsynaptic currents in various retinal neurons. Our results are consistent with the following scheme: Glutamate is used by the photoreceptor and bipolar cell output synapses and the release of glutamate is largely mediated by calcium-dependent vesicular processes. The postsynaptic glutamate receptors in DBCs are L-AP4 receptors, in HBCs, HCs and ganglion cells are the kainate/AMPA and NMDA receptors. Subpopulations of HCs make GABAergic synapses on cones and gate chloride condunctance through GABAA receptors. GABAergic HCs do not make feedforward synapses on bipolar cell dendrites and the neurotransmitter identity of the HCs making feedforward synapses is unknown. Subpopulations of amacrine cells make GABAergic synapses on bipolar cell synaptic terminals, other amacrine cells and ganglion cells and GABA gates chloride conductances in theses cells. Glycinergic amacrine cells make synapses on bipolar cell synaptic terminals, other amacrine cells and ganglion cells and glycine opens postsynaptic chloride channels. Glycinergic interplexiform cells make synapses on bipolar cells in the outer retina and glycine released from these cells open chloride channels in bipolar cell dendrites.

Glycinergic synaptic inputs to bipolar cells in the salamander retina

1 Glycine activated strychnine‐sensitive chloride conductances at both the dendrites and the axonal telodendria of most bipolar cells in the salamander retina. 2 The chloride equilibrium potential of bipolar cells was found to be negative to ‐50 mV, indicating that glycinergic synapses on bipolar cells are inhibitory. 3 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. 4 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. 5 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. 6 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.

Miniature excitatory postsynaptic currents in bipolar cells of the tiger salamander retina

The synapse between photoreceptor and bipolar cell is important for at least three reasons: (1) it is the first synapse in the visual pathway; (2) it is the best-known tonic chemical synapse; and (3) it has perhaps the most complex and highly organized synaptic morphology in the entire brain. Yet little is known about how neurotransmitter is released from this synapse. We present in this report evidence which suggests that the release of photoreceptor neurotransmitter, presumably glutamate, is probably mediated by clusters of synaptic vesicles which give rise to discrete miniature excitatory postsynaptic currents (MEPSCs) in bipolar cells. The MEPSCs are Ca(2+)-, osmotic- and CNQX-sensitive, and they share the same reversal potential (near -3 mV) as the glutamate-induced postsynaptic current. The frequency of MEPSCs increases upon presynaptic depolarization, and the mean peak conductance is about 54 pS. MEPSCs exhibit wide variations of amplitudes and durations, probably resulting from random variations of number of synaptic vesicles and the degree of synchronization in individual release clusters.

Postsynaptic responses of horizontal cells in the tiger salamander retina are mediated by AMPA-preferring receptors

The postsynaptic responses of sign-preserving second-order retinal neurons (horizontal cells (HCs) and off-bipolar cells) are mediated by CNQX-sensitive AMPA/KA glutamate receptors. In this study we used receptor-specific allosteric regulators of desensitization and selected antagonists to determine the glutamate receptor subtypes in tiger salamander horizontal cells. Two approaches were employed in this study. The first was to measure postsynaptic currents induced by exogenously applied glutamate under voltage clamp conditions in living retinal slices; and the second was to record voltage responses controlled by endogenous glutamate released from photoreceptors in whole retinas. Application of 100 microM cyclothiazide (a specific AMPA receptor desensitization blocker) enhanced the glutamate-induced current by about 5 fold. In contrast, 300 microgram ml-1 Co nA (a specific kainate receptor desensitization blocker), had no effect. GYKI 52466 (a specific AMPA receptor antagonist) at 30 microM almost completely suppressed the glutamate-induced inward current in HCs. Cyclothiazide at 100 microM depolarized the HC dark membrane potential by about 5 mV and reduced the amplitudes of the voltage responses to dim lights, but enhanced the voltage responses to bright lights. Cyclothiazide had no effect on either the dark potential or the light responses of rods and cones. Con A at 300 microgram ml-1 had no effect on either the dark potential or the light responses of the HC. GYKI 52466 (30 microM) hyperpolarized the HC dark membrane potential by about 55 mV and almost completely suppressed the light responses. We conclude from these results that the postsynaptic glutamate- and light-induced responses in the tiger salamander retinal horizontal cells are mediated by AMPA-preferring, and not kainate-preferring glutamate receptors. The functional roles of AMPA receptors and their desensitization kinetics in visual information processing are discussed.

Synaptic inputs mediating bipolar cell responses in the tiger salamander retina

Postsynaptic receptors in bipolar cells were studied by focal application of glutamate and GABA to the outer and inner plexiform layers (OPL and IPL) under visual guidance in living retinal slices of the tiger salamander. Two different types of conductance change could be elicited in bipolar cells by applying glutamate to the OPL. In off-center cells, which had axon telodendria ramifying in the distal 55% of the IPL, glutamate elicited a conductance increase associated with a reversal potential near -5 mV. In on-center cells, which had telodendria stratified in the proximal 45% of the IPL, glutamate caused a conductance decrease associated with a reversal potential near -11 mV. These observations suggest that glutamate gates relatively nonspecific cation channels at synapses between photoreceptors and bipolar cell dendrites. Application of glutamate to the IPL elicited no conductance change in Co2+ Ringers solution, but in normal Ringers it generated a conductance increase associated with a reversal potential near the chloride equilibrium potential (ECl). These findings are consistent with the notion that glutamate receptors exist in GABAergic and/or glycinergic amacrine cells, and that glutamate in the IPL depolarizes these cells, causing GABA and/or glycine release and the opening of chloride channels in bipolar cell axon terminals. In Co2+ Ringers, application of GABA at the OPL elicited no conductance changes in bipolar cells, suggesting that GABA receptors do not exist on bipolar cell dendrites. Applied at the IPL, GABA elicited large conductance increases associated with a reversal potential near ECl. Implications of these results for the functional circuitry of the tiger salamander retina are discussed.

Glutamate receptors differ in rod- and cone-dominated off-center bipolar cells.

The axon morphology of off-center bipolar cells in salamander retinal slices was correlated with light responses and dendritic glutamate responses of these cells under voltage clamp. Cells with telodendria ramifying exclusively in the distal one-quarter of the inner plexiform layer were rod dominated, whereas cells with more centrally ramifying telodendria were cone dominated. The glutamate responses of the centrally ramifying cells displayed more apparent desensitization and a smaller underlying single channel conductance (1.2+/-0.3 pS) than did the distally ramifying cells (2.8+/-0.4 pS), although the responses for both classes of cells were strongly enhanced by cyclothiazide. These results suggest that different subtypes of AMPA receptors are present on rod- and cone-dominated cells, and that these may differ with respect to desensitization kinetics.

Characterization of displaced bipolar cells in the tiger salamander retina

In immunocytochemical studies of the tiger salamander retina, 17% of neurons in the outer nuclear layer did not label for recoverin, a photoreceptor marker. Lucifer yellow injection showed a population of cells in the ONL to be displaced bipolar cells, with axon terminals that stratified exclusively in the OFF sublamina of the inner plexiform layer (IPL), and predominately within the cone-dominated region of the OFF sublamina. Glutamate generated a dendritic cationic conductance increase in all displaced bipolar cells tested, and typical cone-dominated bipolar cell light responses were observed among displaced cells that stratified in the central IPL. We conclude that displaced bipolar cells in the tiger salamander retina are entirely OFF-center cells, and predominately cone-dominated cells.