The goal of this research is to furnish an understanding of the complex neural substrates underlying the regulation of the circadian oscillator located in the hypothalamic suprachiasmatic nucleus (SCN), which functions as our biological clock. Increased knowledge of the functional organization of the hypothalamus and the circadian timing system is relevant to major public health concerns such as sleep disorders, sleep disruption (as in jet lag or shift work), and serious affective disorders. Photic information essential for the daily resetting of the SCN circadian clock is conveyed directly to the SCN from the retina via the retinohypothalamic tract. The SCN also receives a dense serotonergic innervation arising from the midbrain raphe and additional afferents from the intergeniculate leaflet (IGL). In recent years, multiple serotonin (5-HT) receptor subtypes have been described and grouped into several families: 5-HT1 - 5-HT7. In this proposal, we will test the hypotheses that 5-HT1B receptors are located on the terminals of IGL afferents in the SCN and that 5-HT7 receptors are present on SCN neurons and on GABAergic terminals in the SCN. Moreover, we will test the hypothesis that 5-HT1B receptors are not located on a subset of retinal ganglion cells that send axonal branches to the SCN and IGL. Electron microscopic immunocytochemical techniques will be used to test the hypotheses that 5-HT1B receptors are presynaptic on IGL terminals in the SCN and that 5-HT7 receptors are located on neurons and presynaptically on GABA terminals in the SCN. In situ hybridization will be used to test the hypothesis that IGL neurons express 5-HT1B receptor mRNA. Using whole-cell patch-clamp techniques, we will test the hypothesis that 5-HT7 receptor agonists inhibit optic nerve stimulation-evoked EPSCs in SCN neurons in vitro in wild-type and in 5-HTlA receptor knock-out mice and that these same agonists inhibit GABA release in the SCN via a presynaptic mechanism. Behavioral pharmacological analyses of circadian wheel-running activity will be used to complement the in vitro analysis. The use of behavioral, pharmacological, morphological, and electrophysiological approaches will provide an integrated analysis of 5-HT receptor subtype function in the SCN and on afferents to the SCN that regulate clock function.
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