To understand brain stem mechanisms important for control of the desynchronized phase of sleep (D), we shall investigate the physiology and anatomy of the pontine reticular formation (RF) core as related to sleep, and aspects of connectivity and physiology of the Ch5 (pedunculopontine) and Ch6 (laterodorsal) cholinergic neurons as they relate to sleep and connections with pontobulbar reticular formation. A guiding hypothesis is that an essential element of the occurrence of D is the membrane potential (MP) depolarization and increased excitability observed in the population of medial pontine reticular formation (mPRF) neurons and other, densely connected RF neurons, and that cholinergic input may be important for initiating these events. Chronic intracellular recording experiments in the naturally sleeping cat will examine whether mPRF reticulo-reticular projection neurons have, compared with reticulo-spinal neurons, an earlier, D-anticipatory onset of MP depolarization, thus implying functional differentiation of RF cellular function and a special role in state-related changes for these neurons. Chronic intracellular and extracellular recordings will determine if cholinergic neurons in the Ch5-Ch6 groups have a time course of discharge activity that is compatible with initiation of D- anticipatory events in mPRF. Intracellular HRP combined with acetylcholinesterase (AChE) or choline acetyltransferase (ChAT) labeling will identify recorded neurons. Anatomical- physiological studies in acute cats will examine the morphology and histochemical nature of neurons responsible for connectivity within pontobulbar RF (PBRF), between PBRF and Ch 5-6, and the rostral and spinal cord projections of PBRF. Extracellular and intracellular HRP injection techniques combined with ChAT/AChE staining will be used. Using mPRF intracellular recordings in the pontine RF slice, a novel in vitro preparation developed by us, we propose to: perform initial identification and characterization of the mPRF-mPRF neurotransmitter(s); identify intrinsic voltage- dependent currents important for mediation of D state-related changes; and characterize the effects of cholinergic agonists. Better understanding of D mechanisms will aid development of more rational treatment of disorders with D sleep pathology, including depression and narcolepsy.
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