Contributions of GABAA receptors and GABAC receptors to acetylcholine release and directional selectivity in the rabbit retina.

GABA is a major inhibitory neurotransmitter in the mammalian retina and it acts at many different sites via a variety of postsynaptic receptors. These include GABAA receptors and bicuculline-resistant GABAC receptors. The release of acetylcholine (ACh) is inhibited by GABA and strongly potentiated by GABA antagonists. In addition, GABA appears to mediate the null inhibition which is responsible for the mechanism of directional selectivity in certain ganglion cells. We have used these two well-known examples of GABA inhibition to compare three GABA antagonists and assess the contributions of GABAA and GABAC receptors. All three GABA antagonists stimulated ACh release by as much as ten-fold. By this measure, the ED50s for SR-95531, bicuculline, and picrotoxin were 0.8, 7.0, and 14 microM, respectively. Muscimol, a potent GABAA agonist, blocked the effects of SR-95531 and bicuculline, but not picrotoxin. This indicates that SR-95531 and bicuculline are competitive antagonists at the GABAA receptor, while picrotoxin blocks GABAA responses by acting at a different, nonreceptor site such as the chloride channel. In the presence of a saturating dose of SR-95531 to completely block GABAA receptors, picrotoxin caused a further increase in the release of ACh. This indicates that picrotoxin potentiates ACh release by a mechanism in addition to the block of GABAA responses, possibly by also blocking GABAC receptors, which have been associated with bipolar cells. All three GABA antagonists abolished directionally selective responses from ON/OFF directional-selective (DS) ganglion cells. In this system, the ED50S for SR-95531, bicuculline, and picrotoxin were 0.7 microM, 8 microM, and 94.6 microM, respectively. The results with SR-95531 and bicuculline indicate that GABAA receptors mediate the inhibition responsible for directional selectivity. The addition of picrotoxin to a high dose of SR-95531 caused no further increase in firing rate. The comparatively high dose required for picrotoxin also suggests that GABAC receptors do not contribute to directional selectivity. This in turn suggests that feedforward GABAA inhibition, as opposed to feedback at bipolar terminals, is responsible for the null inhibition underlying directional selectivity.

Pharmacology of directionally selective ganglion cells in the rabbit retina.

In this report we describe extracellular recordings made from ON and ON-OFF directionally selective (DS) ganglion cells in the rabbit retina during perfusion with agonists and antagonists to acetylcholine (ACh), glutamate, and gamma-aminobutyric acid (GABA). Nicotinic ACh agonists strongly excited DS ganglion cell in a dose-dependent manner. Dose-response curves showed a wide range of potencies, with (+/-)-exo-2-(6-chloro-3pyridinyl)-7-azabicyclo[2.2.1] heptane dihydrochloride (epibatidine) > > > nicotine > 1,1-dimethyl-4-phenylpiperazinium iodide = carbachol. In addition, the mixed cholinergic agonist carbachol produced a small excitation, mediated by muscarinic receptors, that could be blocked by atropine. The specific nicotinic antagonists hexamethonium bromide (100 microM), dihydro-beta-erythroidine (50 microM), mecamylamine (50 microM), and tubocurarine (50 microM) blocked the responses to nicotinic agonists. In addition, nicotinic antagonists reduced the light-driven input to DS ganglion cells by approximately 50%. However, attenuated responses were still DS. We deduce that cholinergic input is not required for directional selectivity. These experiments reveal the importance of bipolar cell input mediated by glutamate. N-methyl-D-aspartic acid (NMDA) excited DS ganglion cells, but NMDA antagonists did not abolish directional selectivity. However, a combined cholinergic and NMDA blockade reduced the responses of DS ganglion cells by > 90%. This indicates that most of the noncholinergic excitatory input appears to be mediated by NMDA receptors, with a small residual made up by alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/kainate (KA) receptors. Responses to AMPA and KA were highly variable and often evoked a mixture of excitation and inhibition due to the release of ACh and GABA. Under cholinergic blockade AMPA/KA elicited a strong GABA-mediated inhibition in DS ganglion cells. AMPA/KA antagonists, such as 2,3-dihydroxy-6-nitro-7-sulfamoylbenzo (F)quinoxaline dione and GYKI-53655, promoted null responses and abolished directional selectivity due to the blockade of GABA release. We conclude that GABA release, mediated by non-NMDA glutamate receptors, is an essential part of the mechanism of directional selectivity. The source of the GABA is unknown, but may arise from starburst amacrine cells.

The pupillary response to light in the turtle.

When intense adapting lights are turned off, the pupil of the turtle, Pseudemys scripta elegans, enlarges. The recovery functions for pupillary dilation have different time constants that are defined by red- and green-sensitive cones and rods as they are affected by prior light adaptation and time in the dark. Pupillary area related to dilation responds over at least a three- to four-fold range. Following white-light adaptation, the course of pupil dilation in the dark shows a three-legged curve of differing time constants. With spectral-light adaptations, the contributions of separate classes of photoreceptors can be isolated. Red- and green-sensitive cones contribute shorter time constants of 3.31 and 3.65 min to prior white-light adaptation--4.81 and 4.18 min to prior spectral-light adaptations. Rods contribute a much longer time constant of 6.69 min to prior white-light adaptation--7.60 min to prior spectral-light adaptation. The ratios are in keeping with the flash sensitivities of photoreceptors in this same animal, as well as with psychophysical visual threshold mechanisms of color sensitivity.

Effect of ON pathway blockade on directional selectivity in the rabbit retina.

1. In this report we describe extracellular recordings made from directionally selective (DS) ganglion cells in the rabbit retina during perfusion with 2-amino-4-phosphonobutyric acid (APB) to block ON channels through the retina. 2. Application of 100 microM APB selectively and reversibly abolished the responses of ON ganglion cells in the rabbit retina. In addition, 100 microM APB completely and reversibly blocked ON component responses of ON-OFF DS ganglion cells to both stationary and moving stimuli. These results are consistent with the idea that APB blocks ON pathways through the retina. 3. Under ON pathway blockade with APB, OFF component responses of ON-OFF DS ganglion cells remained DS. DS OFF responses retained the same preferred direction as the pre-APB ON-OFF responses and could be driven using either normal or reversed contrast stimuli. 4. Extracellular responses of ON DS ganglion cells were completely blocked by APB. Under APB, these cells showed no response to stationary or moving stimuli. 5. Application of the gamma-aminobutyric acid-A (GABAA) antagonist 2-(3-Carboxypropyl)-3-amino-6-(4-methoxyphenyl)pyridazinium bromide (SR95531) reversibly abolished directional selectivity of ON DS and ON-OFF DS ganglion cells in the rabbit retina. This finding is consistent with previous data for picrotoxin. 6. Application of SR95531 during ON channel blockade by APB caused OFF component responses of ON-OFF DS ganglion cells to lose their directional selectivity. Under these conditions, OFF responses to movement in the preferred and null directions became virtually identical. 7. These results indicate that simultaneous ON and OFF layer input is not required to generate directional responses in ON-OFF DS ganglion cells. In addition, it appears that a GABAA-dependent mechanism for directional selectivity may operate independently in the two separate dendritic layers of the ON-OFF DS ganglion cell.

Functional morphologies of retinal ganglion cells in the turtle.

Retinal ganglion cells in the turtle, Pseudemys scripta elegans, were examined by intracellular recording with a protocol of stationary and moving lights. Responses were apportioned among OFF, ON, and ON-OFF categories, and directional selectivity. Cells were injected with Neurobiotin, then later conjugated with avidin-horseradish peroxidase in standard procedure. Morphological analysis of the stained cells included measurements of soma and dendritic field sizes, dendritic stratification, number of cell processes, dendritic branchings, and dendritic symmetry ratios. ON and ON-OFF cells are at least bistratified, sometimes tristratified, in both sublaminae A and B whether directionally selective or not. OFF cells, in contrast, are monostratified, or at least confined to sublamina A. Morphological parameters of somal and dendritic field areas, branch point densities, and dendritic field asymmetries do not predict directional selectivity. Membrane polarization accompanying moving stimulation is discussed in terms of shunting inhibition and recording site.