Detailed studies of the effects of putative neurotransmitters/modulators and their mechanism of action on postnatal neurons of the mammalian visual system would be aided by studying identified cells in a controlled in vitro environment. For this purpose cultures of rodent retinal ganglion cells, labeled with fluorescent probes, are used in this laboratory.
The specific aims of this proposal are -- 1. To study physiologically and pharmacologically the direct effects of putative neurotransmitters and modulators on solitary mammalian retinal ganglion cells in culture. Ganglion cells, fluorescently labeled by retrograde transport, will be dissociated from the retinas of rodents, maintained in culture, and recorded from with the patch-clamp technique. The detailed effects of various substances and their antagonists on both whole-cell and single-channel currents will be monitored. These agents include classical chemicals such as acetylcholine, GABA, glycine, and glutamate and its analogs, as well as many peptides that are found in the amacrine cells that normally synapse on the ganglion cells. Examples of these peptides are leu-enkephalin, substance P, somatostatin, cholecystokinin,,neurotensin, and vasoactive intestinal peptide (VIP). The analysis of single-channel events activated by these drugs may yield insight into their molecular mechanisms of action and possible differences between retinal and other tissues. 2. To identify the transmitters used at chemical synapses onto ganglion cells. The substances that influence solitary ganglion cells will be tested for their effects on synaptic activity found in the ganglion cells in more dense cultures during whole-cell patch-clamp recording. In this manner an attempt will be made to discern the identity of the physiologically-relevant transmitters and modulators. For example, the effects of antagonists to the various putative transmitters and modulators will be characterized on spontaneous and evoked postsynaptic currents that occur in cultured ganglion cells that have formed synapses with other retinal cells. 3. To quantify plasticity effects of the putative neurotransmitters and modulators and their selective blocking agents by monitoring outgrowth of processes and survival of retinal ganglion cells in culture. Preliminary experiments have suggested that several agents, such as nicotinic agonists and N-methyl-D-aspartate (NMDA), influence the outgrowth of neurites and the survival of cultured retinal ganglion cells. To test the effect on process outgrowth, drugs will be applied directly to the growth cone of a neurite of an isolated retinal ganglion cell while monitoring growth with computer-enhanced video microscopy. Viability of retinal ganglion cells will also be monitored after incubation with putative transmitters. Ionic mechanisms underlying process outgrowth and survival of retinal ganglion cells may be uncovered in these experiments since the ionic basis for the current induced by each substance will have been studied in the patch clamp experiments. The long-term goals of this project are to further the understanding of synaptic communication between neurons in the mammalian visual system and to increase neuronal survival and the potential for process regeneration following injury or ischemic insult.
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