Clifford B. Saper - US grants

A considerable body of evidence indicates that behavioral and emotional factors may play an important role in cardiovascular disease, but the central nervous system mechanisms underlying this influence are poorly understood. The goal of the proposed project is to investigate the neuroanatomical, physiological and biochemical organization of the parabrachial nucleus, a cell group which appears to be a crucial link between the forebrain and brainstem areas involved in cardiovascular control. In the first series of experiments, the subnuclear organization of the afferent connections of the parabrachial nucleus will be studied in the rat, using antero-grade and retrograde axonal transport methods. In the second series of experiments, the neurotransmitter specificity of parabrachial subnuclei and their connections will be studied using immunohistochemical staining for enkephalin, somatostatin, cholecystokinin, corticotropin releasing factor, neurotensin and substance P and catecholamine synthetic enzymes (tyrosine hydroxylase and phenylethanolamine N-methyltransferase). In the third series of experiments, the potential neurotransmitters in specific parabrachial connections will be studied in the rat, using combined retrograde fluorescent tracers and immunofluorescence with the same antisera used in series two. In the fourth series of experiments, the responses of single parabrachial neurons in the rat to cardiovascular and other visceral stimuli will be recorded; when a cardiovascular-responsive neuron is found, a small iontophoretic deposit of either an anterograde or retrograde tracer will be made. The area will then be stimulated electrically, and cardiovascular and other visceral responses recorded. Finally, the correlation of pathways labeled in these brains with various potential neurotransmitters will be studied, by combining the tracer methods with immunohistochemistry. In the fifth series of experiments, similar though abbreviated studies will be done in cats and monkeys. Finally, the cytoarchitectonic and immunohistochemical organization of the parabrachial nucleus in the human brain will be studied. These experiments should give us some insight into the mechanisms of forebrain influence on cardiovascular control, in a variety of mammalian species up to, and including, man. The results may, eventually, allow the design of rational therapies at the central nervous system level for myocardial ischemia and cardiac arrhythmias.

Accumulating evidence suggests that the neuromodulator adenosine (AD) may be an important mediator of sleep. This proposal outlines experiments to identify the neural mechanisms through which AD produced sleep. The ventrolateral pre-optic are (VLPO) may promote sleep by causing widespread inhibition of the cholinergic and monoaminergic arousal systems. We hypothesize that AD produced sleep by indirectly activating VLPO neurons. Specifically, we propose that AD inhibits neurons that tonically inhibit the VLPO during wakefulness; as AD accumulates during wakefulness, these neurons become less active, disinhibiting the VLPO. We will use in situ hybridization histochemistry to determine the distribution and neurochemical phenotype of neurons expressing AD receptors in the rat and human hypothalamus and basal forebrain. We will combine these receptor studies with injections of a retrograde tracer to identify which neurons expressing AD acts to produce or inhibit sleep by then will determine where in the hypothalamus or basal forebrain AD acts to produce or inhibit sleep by microinjecting AD agonists into specific hypothalamic regions and recording sleep/wake behavior. TO determine whether endogenous AD in the HYPO/BF influences sleep, we also will microinject drugs which increase or decrease the local concentration of AD and study their effect on sleep. Finally, we will study the electrophysiologic effects of AD agonists and antagonists on VLPO neurons using an in vitro slice preparation. This technique also will be used to record the response to AD agonists and antagonists of neurons that project to the VLPO. These experiments will help determine the biochemical source of AD, the anatomic sites at which AD induces sleep and the electrophysiologic mechanism through which AD produces sleep.

The Neuroanatomy Core will provide both light and electron microscopic services to the Projects, including tissue staining, sectioning and imaging. Ten aims in three projects will utilize this Core facility. Projects 2( Rosenberg) and 3 (Saper) will use the Core for in situ hybridization histochemistry to localize mRNA for phosphodiesterases, adenosine receptors and glutamic acid decarboxylase in rat brain, and the latter also in human brain. Light microscopic immunocytochemical services will be used by Project 2, 3, and 4 to localize cholinergic neurons, as well as immunocytochemistry for neurons containing enkephalin, in some cases combined with retrograde tract tracing or in situ hybridization methods. Electron microscopic immunocytochemistry will be used by Project 4 to examine the chemical specificity of afferents to hypoglossal motor neurons. The centralization of these services in one laboratory will give the laboratories that do relative little neuroanatomy (Rosenberg and Greene) access to the technical proficiency of the Core laboratory and staff and will allow economy of scale in order and using reagents, isotopes and photographic emulsion.