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We have used immunostaining techniques to reveal a new type of amacrine cell that is immunoreactive for choline acetyltransferase (ChAT), the acetylcholine synthesizing enzyme, in the Ground Squirrel (Spermophilus beecheyi) retina. Cryostat sections and double immunostained wholemount preparations were examined by confocal microscopy. This new ChAT type III cell is distinct in morphology and neurotransmitter content from the well know ‘starburst’ amacrine cells (types I and II) that are so well represented in the ground squirrel retina [J. Comp. Neurol. 365 (1996) 173–216]. The type III cell colocalizes glycine with the acetylcholine and does not appear to be GABAergic or exhibit calcium-binding proteins like the well-known starburst type. As well, type III cells do not occur as a mirror-symmetric pair with normally placed and displaced varieties. The type III cell is probably a small field amacrine type branching broadly in upper sublamina b of the inner plexiform layer, and is most likely A6 of the Ground Squirrel retina [J. Comp. Neurol. 365 (1996) 173–216]. Type III cells are ideally placed in the architecture of the Ground Squirrel retina to influence ON directionally selective ganglion cell types. Supported by Research to Prevent Blindness to HK and DGESIC PB98-0972 to NC.

The histochemistry of reduced nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) and immunoreactivity of neuronal nitric oxide synthase (nNOS-IR) can be demonstrated in various cell types of the vertebrate retina. In this study, we have focused on characterizing the different NADPH-d-positive amacrine cell types in turtle retina. Cryostat sections were examined by confocal laser scanning microscopy for double immunofluorescence with antibodies against nNOS and either GABA or glycine, or by combining histochemistry with immunocytochemistry to obtain triple labeling with NADPH-d, GABA, and glycine. Forty-eight percent of the NADPH-d-labeled amacrine cells colocalized GABA, 52% glycine. Here we show that two morphologically different types of amacrine cell are nNOS/glycine-IR and three types are nNOS/GABA-IR. Antibodies against calretinin, parvalbumin, somatostatin, tyrosine hydroxylase, and choline acetyltransferase did not colocalize with nNOS-IR or NADPH-d-labeled amacrine cells, but 15% of the NOS-labeled amacrine cells showed immunoreactivity against calbindin. Only GABA has been seen to colocalize with NADPH-d in amacrine cells in previous reports in other species. The finding here of glycine colocalizing with NO-containing cells is novel. We suggest that NO, apart from its well known function in gap junction regulation, can also modulate the release of both GABA and glycine in the turtle retina. This study was supported by grant EY04885 to H. Kolb.