Studies in the continuing project period will be based primarily on a critical finding, made over the last year with a congenic strain of rats derived from the Spontaneously Hypertensive Rat (SHR) and Brown Norway parentage. This strain contains a 3 lcM replacement in chromosome 8 into the SHR background, and quite fortuitously this region contains a cluster of nicotinic acetylcholine receptor genes that encode receptor subunits expressed in the CNS (alpha3, beta4 and alpha7). These animals show diminished blood pressure (systolic and diastolic) relative to their SHR counterparts. More importantly, intrathecal administration of nicotinic agonists shows markedly diminished pressor and nociceptive responses. For sometime we have felt that the SHR trait was one of enhanced excitability, and as the animal ages, blood pressure becomes one manifestation of this. If we consider more widespread excitability responses, loci of amplification evident in the presynaptic control of neurotransmitter release would be a logical starting point. Recent work has also documented that most nicotinic receptors appear to lie presynaptically in spinal and supraspinal regions. We have chosen to study the spinal system, not because we believe it to be an area that globally controls hypertension, but rather pressor responses to nicotinic agonists injected into segmental cord areas can be monitored in the conscious animal. In turn, measurements of target receptor number, their subtypes, cellular responses (ie: release of neurotransmitters detected in microdialysates), and the cellular localization of neurotransmitter and receptor subtype in various lamina and cell types in the cord have been and will be measured. Accordingly, we propose to more fully characterize the pressor response in conscious SHR and SHR-Lx rats, and then begin to localize the gene(s) responsible for the different pressor response by delimiting the region around the nicotinic receptor gene cluster. If the response remains associated with the AChR gene substitution, we then will attempt to identify which of the three genes are involved, through a characterization of the coding and critical noncoding regions. Other regions showing quantitative trait loci will also be investigated. Since the window of the response to spinal nicotinic agonists is augmented, we will characterize the localization of receptors and transmitters in this region through the use of immunocytochemistry and confocal microscopy. In addition, relatively specific cell ablation is possible in the spinal cord through the use of capsaicin and a Substance P-saporin conjuate, and by comparing altered pressor responses of treated and untreated animals, we should be able to map spinal pathways and receptor subtypes involved in the altered responses.
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