Primary afferent terminals in the dorsal horn (DH) of the spinal cord release besides glutamate (GLU) and aspartate (ASP) also peptides that may be involved in the slow excitatory synaptic transmission and the central processing of pain information. However, the sites and the molecular mechanisms by which the peptide signals produce cellular responses have yet to be elucidated. The co-existence of tachykinins and GLU, has been reported in small primary sensory neurons. Chemical signal transfer via such neurons presents new aspects and complexities of presynaptic (synaptic efficacy) and postsynaptic (membrane excitability) regulation which have not previously been considered and may have important implications for the performance of the somatosensory system. This proposal focuses on the functional postsynaptic aspects of the co-release of tachykinins and excitatory amino acids (EAAs) in the rat spinal DH and especially on the analysis of their direct excitatory interactions and underlying cellular mechanisms. We propose to examine the hypothesis that modulation of the responses of rat spinal DH neurons to EAA (glutamate, aspartate, N-methyl- D-aspartate, alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid, kainate and quisqualate) by tachykinins (e.g. substance P, neurokinin A) and opioid peptides (acting at mu, delta and kappa receptors) occurs and might involve the distinct subtypes of glutamate and the peptide receptors. Possible involvement of G proteins, changes in the concentration of intracellular Ca2+ and second messenger mechanisms in the regulation of the chemosensitivity of DH neurons to NMDA and tachykinins will also be investigated. Whole-cell voltage-clamp and nystatin- perforated patch-clamp techniques and monitoring of the levels of intracellular free Ca2+ concentration using fura-2 based microfluorimetry in freshly isolated DH neurons will be used to obtain information about the molecular mechanisms underlying the interactions between NMDA and tachykinins. Delineating the cellular mechanisms of tachykinin actions on DH neurons is an important step toward understanding anatomical and neurochemical organization of the spinal DH. Such knowledge may provide the possibility of selective pharmacological manipulation of sensory perception mechanisms with important therapeutic implications, especially for pain.
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