Early in vitro development of voltage- and transmitter-gated currents in GABAergic amacrine cells.

It has been shown in previous studies that a subpopulation of neurons in monolayer cultures prepared from immature embryonic chicken retina acquired a series of functional properties which characterized them as GABAergic amacrine cells after 1 week in vitro. In the present study, we demonstrate that immature precursors of these cells were already identifiable by morphological criteria after 2 days in vitro (DIV). Using the whole cell patch-clamp technique we have studied the time-course of the expression of voltage-dependent and of glutamate and GABA receptor-associated conductances in these identified retinal interneurons developing in vitro. Recordings after 2 DIV revealed a very homogeneous pattern of membrane conductances. In all cells tested, whole cell responses to depolarizing voltage steps consisted solely of a sustained outward potassium current and 100% of the cells responded to the glutamate receptor agonist kainic acid (KA) and to GABA. Fast activating inward sodium currents first appeared after 3 DIV, whereas a transient component of outward potassium currents was not detectable before day 4 in vitro. N-Methyl-D-aspartate (NMDA)-evoked currents were first observed at 3 DIV in the GABAergic neurons. Only 1 day later they were found in all of the GABAergic neurons. Expression of responses to quisqualic acid (QU) started at 3 DIV, but remained restricted to a subpopulation of the GABAergic cells even at later stages (59% at 4 DIV, 63% at 6-9 DIV). Antagonistic effects of QU on KA responses, however, were detectable in all cells tested, independent of the developmental stage and the presence of QU-evoked currents.(ABSTRACT TRUNCATED AT 250 WORDS)

Voltage-gated currents of putative GABAergic amacrine cells in primary cultures and in retinal slice preparations.

To analyze the voltage-dependent ionic conductances of putative GABAergic amacrine cells developing in vitro, whole cell patch clamp recordings were carried out on identified neurons in monolayer cultures from embryonic chick retinae. These recordings were directly compared with those performed on amacrine cells in chick retinal slice preparations. Current responses to depolarizing voltage steps observed in cultured neurons could be separated into at least four different components. A small tetrodotoxin-sensitive sodium inward current was observed in approximately 50% of the cells. The considerably larger outward potassium current consisted of a transient 4-aminopyridine-sensitive component and a sustained component. The latter was reduced in the presence of both tetraethylammonium chloride and Co2+ and thus was probably composed of two conductances. In addition, a Ca(2+)-carried inward current of small amplitude could be identified. Voltage-sensitive currents measured in amacrine cells of retinal slices were very similar. Again, only about half of the cells exhibited sodium currents. Potassium currents contained the above components, but their contributions to the whole cell current seemed to be different. Together with previous findings these results suggest that immature retinal neurons in dissociated cultures undergo a differentiation process similar to that occurring in vivo.

Transmitter-gated currents of GABAergic amacrine-like cells in chick retinal cultures.

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 Mg2+ (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 GABAA 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.

Identification of GABAergic amacrine cell-like neurons developing in chick retinal monolayer cultures.

Purified neuronal monolayer cultures were prepared from embryonic chick retinae. Using immunocytochemistry for GABA and autoradiographic demonstration of [3H]muscimol uptake as markers, approximately 32% of the cells were identified as putative in vitro counterparts of gamma-aminobutyric acidergic (GABAergic) amacrine cells. Whole cell patch clamp recordings revealed close similarities between identified [3H]GABA accumulating neurons in culture and amacrine cells recorded in retinal slices, with respect to voltage-dependent membrane currents and to ion channels gated by exitatory (N-methyl-D-aspartate, kainate, quisqualate, glutamate) and inhibitory (GABA, glycine) amino acid agonists. The results demonstrate that retinal neurons are able to differentiate in dissociated retinal cultures. Such cultures may thus serve as a model system to study development and function of CNS neurons.

Tetanus toxin binding to isolated and cultured rat retinal glial cells.

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.