Melanopsin (Opsin 4), a photopigment found in a small subset of retinal ganglion cells projecting to the suprachiasmatic nucleus and other brain areas, is implicated in nonvisual responses to environmental light such as the pupillary light reflex, seasonal adaptations in physiology, photic inhibition of nocturnal melatonin release, and modulation of sleep, alertness and activity. Because melanopsin containing ganglion cells are few in number and scattered throughout the retina, they are difficult to study. To address these limitations, we engineered a mouse line in which the Enhanced Green Fluorescent Protein (EGFP) is expressed under the control of the mouse melanopsin promoter employing BAC (bacterial artificial chromosome) transgenesis. We will perform two types of studies in these mice: (1) single cell electrophysiological recordings of EGFP positive neurons in whole mount retinas to characterize their functional properties during development, (2) electrophysiological recordings of acutely isolated EGFP positive neurons to characterize their intrinsic properties. Taking advantage of our ability to selectively target the melanopsin expressing neurons we will also engineer an additional mouse line: Opn4tTA line, in which expression of a transcriptional activator will be regulated both reversibly and quantitatively by exposing the transgenic animals to varying concentrations of doxycycline (Dox). The Opn4 tTA line will be used to allow the conditional expression of tetanus toxin light chain, a molecule that inhibits synaptic release or conditional overexpression of the inward rectifying potassium channel, Kir2.1, to diminish the excitability of melanopsin expressing neurons. These mouse lines will provide important tools to the neuroscience and vision research community to address the mechanisms of signal transduction in melanopsin expressing cells, their physiological properties, and their roles in vivo. Within the retina of the vertebrate eye there are photoreceptors that capture light to regulate non visual processes such as day night rhythms and the narrowing and widening of the pupil. To capture light, these special cells called intrinsically photoreceptive retinal ganglion cells, require pigments called melanopsins, similar to the opsins that the rod and cone cells use for turning light into vision. While cones and rods have been extensively studied, much less is known about the melanopsin expressing ganglion cells, although they appear to function more like the photoreceptors found in invertebrate eyes. This study will generate novel mouse models that will allow the study of these cells in vivo and in vitro. Determining how the intrinsically photosensitive ganglion cells work may allow the treatment of disorders such as sleep disorders and seasonal depression. ? ? ?
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