The presence of immunocytochemically detectable membrane receptors for tetanus toxin, supposedly composed of higher gangliosides, is widely accepted as a marker of neuronal cells. We now demonstrate that Müller cells, a unique glial cell type of the vertebrate retina, possess specific tetanus toxin (TT)-binding sites. Single cell suspensions were prepared from adult rat retina by a gentle dissociation method, and the Müller cells, unequivocally identified by their morphology, could be immunocytochemically double-labeled by antisera to vimentin and to TT. The expression of complex gangliosides by identified Müller cells was also demonstrated by immunofluorescence labeling with the monoclonal antibody A2B5. Using the double-immunolabeling method for the identification of Müller cells we show that specific tetanus toxin binding is acquired by these cells during postnatal maturation both in vivo and in vitro. In vivo the percentage of tetanus toxin-positive Müller cells increases from 0% in 4-day-old animals to 10% on postnatal day 8, reaching the adult level of about 95-100% around day 30. In retinal monolayer cultures prepared from newborn rats, the majority (65%) of vimentin-positive non-neuronal cells became TT-positive during a 2-week culture period, indicating that this population of non-neuronal cells represents differentiating Müller cells. Again, comparable results were obtained with A2B5, supporting the conclusion that Müllerian glia expresses surface molecules, which are normally regarded as neuronal markers.
A subpopulation of cells developing in dissociated neuronal cultures prepared from 8-day-old embryonic chick retinae can be identified as putative in vitro counterparts of GABAergic amacrine cells by immunocytochemical and autoradiographic markers and by their electrophysiological responses to transmitter agonists. In the present study, transmitter-gated conductances expressed by these neurons were examined using the whole-cell patch-clamp technique. At negative holding potentials, the excitatory amino acid agonists N-methyl-D-aspartate (NMDA), kainate quisqualate, and glutamate induced inward currents with reversal potentials close to 0 mV in most of the cells selected for recording. NMDA-evoked responses were selectively blocked by the noncompetitive inhibitor MK 801 and by Mg 2+ (in a voltage-dependent manner) and were potentiated in the presence of submicromolar concentrations of glycine. Glutamate apparently interacted with both NMDA and non-NMDA type receptors. All cells tested responded to the inhibitory transmitters GABA and glycine. Both inhibitory agonists could be shown to activate chloride conductances. Responses to GABA and glycine were specifically inhibited in the presence of bicuculline and strychnine, respectively. Thus, GABAergic neurons in retinal cultures express at least two different excitatory amino acid receptors -NMDA and non-NMDA -and two different inhibitory amino acid receptors -the GABA A and the glycine receptor. The results demonstrate the ability of the cultured neurons to develop an apparently mature phenotype and contribute to the understanding of the functional properties of GABAergic amacrine cells in the vertebrate retina.
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