Functionall characterization of nicotinic receptors in amacrine cells of rat retina

FERNANDA GUMILAR; LUIS POLITI; CECILIA BOUZAT

Bahía Blanca

Workshop; Workshop: Neuronal Communication: From Structure to physiology; 2008

Sociedad Argentina de Investigación en Neurociéncias

Amacrine cells are a heterogeneous class of interneurons that modulate the transfer of the light signals through the retina. The cholinergic systems present in these neurons are involved in theses modulation through, in part, of the neuronal nicotinic receptors activation. However, the characterization of amacrine cells AChRs and their possible roles during the retina development have not been established. In this study, we have identified functionally subtypes of neuronal AChRs in amacrine cells in different postnatal days and determined their effects on the neurite outgrowth during activation or blocking of these receptors. We have recorded macroscopic currents in amacrine cells at different times of development in vitro. We have identified the neurons by their morphology and by microscopic and immunocytochemical techniques using the monoclonal antibody HPC-1. We have used confocal microscopy and the Ca2+-sensitive fluorescent indicator Fluo-3/AM to reveal Ca2+ influx. We have demonstrated that highest levels of a7 AChRs were founded on 8-9 days in development in vitro because co-cultures were stained with rhodamine-labelled a-bungarotoxin. Amacrine cells have been examined with whole-cell recording technique. Two different responses were observed only after of day 8. A slow current decays with a td = 111 +/- 15 ms, and the fast current decays with td = 11 +/- 2 ms. The last one could be attribute to functional a7-AChR, because this receptor is quickly desensitized. Since a7-AChR is permeable to calcium ions, its functionality was assessed by measuring [Ca2+]i oscillations with Fluo3-AM. We have evaluated the effects of nicotine y/or a-Bgt on calcium signaling. We have not observed spontaneous calcium signals in the amacrine cells. When cells were exposed to ACh or nicotine, we have observed Ca2+ influx change as an increase in the fluorescence intensity. a-Bgt inhibits this signal indicating functional a7 AChRs. Nicotine significant increase in neuritic length was observed in cultured amacrine cells. The neurite outgrowth effect of nicotine was abolished by co treatment with a-Bgt. In conclusion, amacrine cells have at least two subtypes of nicotinic receptors suggesting a functional role of these receptors during the synaptogenesis in the retina.