The activity of each neuron in the brain is influenced by the actions of many """"""""slow"""""""" neurotransmitters constantly arriving from other neurons, and the total sum of these actions determines the excitability of the neuron. The main objective of this grant proposal is to elucidate the physiological mechanisms by which """"""""slow"""""""" excitatory and """"""""slow"""""""" inhibitory peptide transmitters produce their actions on brain neurons. Cholinergic neurons from the nucleus basalis of Meynert and noradrenergic neurons from the locus coeruleus in culture prepared from the rat brain will be used; the technique for culturing these neurons was first developed in the PI's laboratory in 1985. These neurons are the main source of acetylcholine and noradrenaline in the brain, and in humans these neurons show degeneration in Alzheimer's disease. The projects focus on the signal transduction mechanisms of the following transmitters: substance P, neurotensin, corticotropin-releasing factor, and somatostatin. Some of these transmitters have been reported to be reduced in Alzheimer's disease. Particular emphasis will be placed on how these transmitters modulate the activity of the inward rectifier K+ channel. In many brain neurons, the modulation of the inward rectifier K+channel is responsible for the generation of slow excitatory and slow inhibitory synaptic potentials. The specific projects are: (1) to identify which G protein mediates the signal transduction of the effects of substance P, neurotensin, somatostatin, and corticotropin-releasing factor on the activity of the K+ channels; (2) to investigate the roles of second massagers (phospholipase C and protein kinase C, cyclic AMP and cyclic AMP- dependent protein kinase, and protein phosphatase) in the signal transduction of these transmitters, and (2) to investigate the mechanisms of the interaction of G protein subunits (mutated and non-mutated), protein kinase, and phosphatases with the inward rectifier K+ channels by using inside-out patches. Patch-clamp electrophysiology and immunocytochemistry will be used. In addition, a microinjector will be used to preform intracellular injection of peptides, antibodies, and antisense DNA in order to disrupt the function of a particular mediator of the signal transduction cascades.
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