Circadian rhythmicity is the overt manifestation of an innate timekeeping mechanism, i.e., a """"""""circadian clock."""""""" Recognition of the clinical and practical importance of the human circadian timing system is increasing, and disordered clock function may underlie the symptoms of a number of neuropsychiatric illnesses. This research plan proposes an investigation into the basic neurobiology of a putative circadian pacemaker in rodents, the suprachiasmatic nuclei (SCN) in the anterior hypothalamus. An experimental approach is presented that permits continuous infusion of reversible pharmacological probes into the SCN of unanesthetized and unrestrained rodents for 14 days. This design allows systematic assessment and distinction of the three components of a functioning circadian system in vivo: an input pathway for entrainment to the environmental light-dark cycle, a pacemaker that actually generates the circadian oscillation, and an output pathway for expression of overt, measurable rhythms. In preliminary studies using this approach, tetrodotoxin (TTX) was infused into the SCN of rats. TTX blocked the function of both input and output pathways without disturbing the internal timekeeping mechanism of the pacemaker. Na+-dependent action potentials are needed for the entrainment and expression of overt circadian rhythms but are not required for the pacemaker to keep time. The first set of proposed experiments more fully characterizes the effects of chronic TTX infusion into the SCN. Topographical specificity is delineated, an electrophysiological assay is developed, the behavioral paradigm is refined and extended, and another output (the circadian rhythm of arginine vasopressin in cerebrospinal fluid) and other inputs (those mediated by carbachol or neuropeptide Y) are assayed. These studies help to validate and extend the experimental approach as a generally useful tool for further pharmacological studies. The second set of experiments tests the general utility and applicability of the experimental approach as an in vivo assay system for pharmacological probes other than TTX. To put this strategy to the test, two protocols are proposed to determine whether voltage-dependent Ca2+ channels or membrane potential can be implicated in the internal mechanism of the pacemaker in the SCN. The third set of experiments begins to define the processes responsible for the circadian rhythm of SCN energy metabolism. The effects of TTX on SCN glucose utilization (measured by the 14C-labeled deoxyglucose technique) are assessed.
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