1. Intracellular and extracellular recordings were obtained from ganglion cells in the rabbit retina. The effect of N-methyl-DL-aspartate (NMDLA) and N-methyl-D-aspartate (NMDA) antagonists were studied with the use of a perfusion method for drug application. 2. NMDLA excited all ganglion cell types and caused a characteristic burst firing pattern, which is not typical of physiological responses in the retina. When synaptic transmission was blocked with cobalt, NMDLA still excited ganglion cells, indicating a direct action. 3. A comparison of DL-2-amino-5-phosphonopentanoate (DL-AP-5) and DL-2-amino-7-phosphonoheptanoate (DL-AP-7) revealed that DL-AP-7 was a more specific NMDA antagonist. DL-AP-5 partially blocked the b-wave of the electroretinogram (ERG), an action typical of L-2-amino-4-phosphonobutyrate (L-APB), which specifically blocks on channels in the retina. 4. DL-AP-7 reversibly blocked the action of NMDLA on all ganglion cell types, but the effects of kainate (KA) and carbachol were unchanged. AP-7 was stereospecific and pharmacologically specific, with action typical of a competitive NMDA antagonist in the rabbit retina. 5. DL-AP-7 did not block light responses driven by center or surround stimulation for ON or OFF ganglion cells. Directional selectively was unchanged by DL-AP-7. However, most ganglion cells showed a reduction, typically 20-30%, in the number of action potentials produced by light stimulation. 6. In contrast to a previous report, we found no evidence that DL-AP-7 specifically inhibited sustained ON ganglion cells. The inhibition of sustained ON responses by DL-AP-5, previously attributed to NMDA antagonism, is probably because of the weak APB activity of L-AP-5. 7. We conclude that NMDA receptors do not mediate the major light-driven input to ganglion cells in the rabbit retina. By exclusion, transmission from bipolar cells to ganglion cells appears to be carried mostly by KA or quisqualate (QQ) receptors. However, because NMDA antagonists reduced the number of action potentials produced by light stimulation, it is likely that NMDA receptors carry a portion of the signal transmission to ganglion cells. The presence of NMDA receptors on third-order neurons is consistent with the release of glutamate from presynaptic neurons such as bipolar cells.

Using the in vivo rabbit eyecup, we have studied the light-evoked release of acetylcholine (ACh) which is presumed to indicate the activity of cholinergic amacrine cells. Gamma-Aminobutyric acid (GABA) inhibited the light-evoked release of ACh (IC50 congruent to 1 mM), but the GABA antagonists bicuculline (5 micro M) and picrotoxin (20 micro M) potentiated the light-evoked release and markedly increased the resting release of ACh. This bicuculline/picrotoxin-evoked release was calcium dependent and the effects of bicuculline, but not picrotoxin, were blocked by muscimol, a potent GABA agonist. Muscimol also inhibited the light-evoked release of ACh (IC50 less than 1 micro M) and was at least 1000 times more potent than GABA. Nipecotic acid (1 mM), a GABA transport blocker, also inhibited the light-evoked release of ACh, but the effect was slow in onset and recovery was prompt. We conclude that the cholinergic amacrine cells of rabbit retina are inhibited by GABA. The relatively weak action of GABA, compared to muscimol, may be due to the presence of avid GABA transport systems. We ascribe the excitatory effects of bicuculline and picrotoxin to the antagonism of endogenous GABA, suggesting that the cholinergic cells are influenced by a tonic release of GABA. This is consistent with the effects of nipecotic acid. Although we are unable to specify the synaptic arrangements involved, we suggest that the most likely interaction is directly between GABA amacrine cells and the cholinergic amacrine cells and/or their presumed bipolar cell inputs.

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The patterns of glutamate, gamma-aminobutyric acid (GABA), and glycine distribution in the zebrafish retina were determined using immunocytochemical localization of antisera at the light-microscope level. The observed GABA immunoreactivity (GABA-IR) patterns were further characterized using antibodies to both isoforms of glutamic acid decarboxylase (GAD65 and GAD67), the synthetic enzyme for GABA. Glutamate-IR was observed in all retinal layers with photoreceptors, bipolar cells, and ganglion cells prominently labeled. Bipolar cells displayed the most intense glutamate-IR and bipolar cell axon terminals were clearly identified as puncta arranged in layers throughout the inner plexiform layer (IPL). These findings suggest the presence of multiple subtypes of presumed OFF- and ON-bipolar cells, including some ON-bipolar cells characterized by a single, large (9 microm X 6 microm) axon terminal. GABA-, GAD-, and glycine-IR were most intense in the inner retina. In general, the observed labeling patterns for GABA, GAD65, and GAD67 were similar. GABA- and GAD-IR were observed in a population of amacrine cells, a few cells in the ganglion cell layer, throughout the IPL, and in horizontal cells. In the IPL, both GABA- and GAD-IR structures were organized into two broad bands. Glycine-IR was observed in amacrine cells, interplexiform cells, and in both plexiform layers. Glycine-positive terminals were identified throughout the IPL, with a prominent band in sublamina 3 corresponding to an immunonegative region observed in sections stained for GAD and GABA. Our results show the distribution of neurons in the zebrafish retina that use glutamate, GABA, or glycine as their neurotransmitter. The observed distribution of neurotransmitters in the inner retina is consistent with previous studies of other vertebrates and suggests that the advantages of zebrafish for developmental studies may be exploited for retinal studies.

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