Anterior cingulate cortex has been implicated in affective responses to noxious stimuli contains opioid synthesizing neurons and has high densities of mu and delta opioid receptors. It is not known which neuronal processes bind opioid receptor ligands. The present experiments will employ experimental localization techniques developed by the P.I. for localizing axonal and dendritic muscarinic acetylcholine receptor subtypes. For whole section analysis one of three types of lesions will be made in rat brain followed by assay of mu and delta-selective ligand binding in cryomicrotome sections, coverslip autoradiography and single grain counting in anterior cingulate cortex: 1) full depth or laminar ibotenic acid, 2) selective destruction of cortical projection neurons following injections of the immunotoxin OX7-ss- saporin into the caudate or periaqueductal gray nuclei or into posterior cingulate cortex, 3) deafferentation undercut lesions. A second localization approach will involve prelabeling of caudate, periaqueductal gray or cortical projection neurons with a stable, retrogradely transported fluorescent dye. Cortical neurons will then be enzymatically and mechanically dissociated and assayed for mu-selective binding of beta-funaltrexamine to prelabeled pyramidal neurons and unlabeled pyramidal and multipolar neurons. Another series of experiments will evaluate mu and delta sites in cortical sublaminae and thalamic subnuclei in cryomicrotome sections of rabbit and human brains. As a number of thalamic and cortical connections are likely involved in sensorimotor aspects of responses to noxious stimuli, multiple label connection studies are also proposed to analyze thalamocortical and corticocortical connections of rabbit cingulate cortex. Studies in thalamus will emphasize the lateral magnocellular nucleus because its connections have not been described and it is not known to exist in any other mammal. Studies at the cortical level will emphasize topographic relations among cingulate areas and among cingulate and visual areas. The main goals of these studies are to elucidate a direct means by which opiate and opioid compounds influence cortical neurons which project to autonomic and somatic motor centers, characterize the distribution of opioid receptors on the dendrites and somata of pyramidal projection neurons and multipolar interneurons and describe the connections of a virtually unknown nucleus in rabbit thalamus. Dissociated adult cortical neurons can be used in future studies to evaluate mechanisms of up and down regulation of opioid receptors as well as gating of ionic channels by opioid compounds.
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