DESCRIPTION (Applicant's Description): The objective of our research is to understand how the retina of mammals analyzes the visual world and encodes its spatial, temporal and chromatic contrast into a message of action potentials for safe sending to the brain. In the retina, dopamine is the modulator responsible for many of the events that lead to neural adaptation to light. To identify the mechanisms and neural networks that control the release of dopamine, we labeled dopaminergic cells (DA cells) by introducing into the mouse genome the cDNA of human placental alkaline phosphatase linked to a promoter sequence of the gene for tyrosine hydroxylase. Because of the presence of the reporter gene product, we could study the synaptic connections of DA cells, identify their receptors and channels by single-cell RT-PCR and investigate their physiology by patch clamping. We have thus shown that DA cells spontaneously fire action potentials in a rhythmic fashion in absence of synaptic inputs and that this activity cause extrasynaptic release of dopamine. The present application has the following aims: By single-cell RT-PCR we have found that DA cells contain the transcripts for the sodium channels Scn8a and we are planning to establish whether it is responsible for the pacemaker current of DA cells in mice homozygous for a null mutation of its gene. To investigate the light responses of DA cells in the intact retina, we have produced a transgenic line in which these neurons are labeled with the green fluorescent protein (GFP). In these animals, we will study the effects of light stimulation on DA cells responses in the isolated retina maintained in vitro. We will continue our analysis of the receptors and ion channels of DA cells to identify mechanisms and networks that control dopamine release. To this purpose, we will combine the techniques of single-cell RT-PCR, electrophysiology, and immunocytochemistry. We have begun to develop methods for obtaining representative cDNA probes of receptors and channels from single, identified, retinal neurons and test them by hybridization on high density cDNA microarrays. Hopefully, this approach will speed up and enrich our functional studies of DA cells and other types of retinal neurons.