Neuronal Circuitry in Sleep

Shiromani, Priyattam

Abstract

This is an application for a K02 Scientist Research Award. The applicant's research on the neurobiology of sleep has progressed from single-cell recording of pontine neurons to in-vivo microinjection to anatomical networking related to REM sleep, and now intracellular molecular changes associated with sleep. Data from these studies have begun to shape a first-order map of sleep-wake control. To date the PI has concentrated on determining a circuit diagram of sleep-wake control because then investigators would know where to look for molecular changes. The PI proposes to continue his studies to define the neural circuitry, but also extend these studies to include the intracellular molecular events. Thus, it will be possible to understand sleep at both the network and single cell level. The proposed career plan consists of neuroanatomy studies under the guidance of Dr. Clifford B. Saper. The PI will utilize anterograde and retrograde tracers in conjunction with double and single labelling immunohistochemistry to identify the neural circuitry underlying sleep-wakefulness. The applicant has never used anterograde tracers (because of unfamiliarity with microiontophoretic injection and labelling procedures) in his previous studies, so the training is necessary. Newer retrograde tracers are now available which the PI will learn to use. Training will also involve cell counting and tracing procedures, morphometric analysis, and computer digitization and plotting techniques. The PI will also learn in-situ hybridization (ISH) and other molecular procedures (northern blot analysis, PCR, gel shift assays). These procedures will be used because in studying sleep, where non-REM and REM sleep last for only a few minutes, it is necessary to monitor markers that are rapidly induced. Protein markers (such as c-Fos) require the animal to remain in a state for at least 30-60 min so that sufficient levels of the protein are produced to be visualized using immunohistochemical procedures. The applicant has never used ISH or other molecular procedures. However, clearly the studies are going in that direction. The PI will be trained in the molecular studies by Dr. William Schwartz.