The primary aim of this proposal is to study ionic and molecular mechanisms of action of several neuropeptides and the functional role of putative second messenger systems in peptide actions, and in transmission and regulation of sensory information at afferent synapses in the dorsal horn of the immature rat spinal cord. Substance P (SP), neurokinin A (NKA) and calcitonin gene-related peptide (CGRP), have been found to excite dorsal horn neurons, although the ionic and molecular mechanisms by which the peptide signals produce cellular responses have not been completely understood. In addition, the cellular mechanisms of the slow excitatory synaptic potentials have not been adequately tested. Therefore, we propose to utilize intracellular recording methodology in the rat dorsal horn slice preparation, and the isolated dorsal horn neurons, to learn more about possible ionic and molecular mechanisms underlying the peptide actions and the fast and slow excitatory transmission in the dorsal horn of the spinal cord. The following topics will be investigated: 1. Voltage-clamp analysis of the slow excitatory transmission in the rat spinal dorsal horn slice preparation: possible mediation by tachykinins (SP, NKA). 2. Possible molecular mechanisms underlying the SP, NKA and CGRP actions and the fast and slow excitatory synaptic transmission in the rat spinal dorsal horn. The three second messenger systems that may be linked to peptide receptors and which will be examined for their role in the control of neuronal excitability in the rat spinal dorsal horn, are as follows: 1) The adenylate cyclase and cyclic AMP-dependent protein kinase system, 2) The guanylate cyclase and cyclic GMP-dependent protein kinase system, and 3) The inositol triphosphate/diacylglycerol-protein kinase C system. 3. Mechanisms of modulation of calcium conductances by neuropeptides (SP, NKA, and CGRP) in acutely isolated rat dorsal horn neurons using patch-clamp technique.
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