The overall purpose of this proposal is to study the biology and regulation of a brain neuropeptide system--the pro-dynorphin neurons. We have chosen two well identified circuits: the magnocellular-posterior lobe pathway which co-synthesizes dynorphin and vasopressin, and the striato-nigral pathway whereby dynorphin projects to the dopamine rich substantia nigra. These were chosen because of the availability of pharmacological manipulations to alter their level of activity, and because of their possibly contrasting biologies as neurohormonal versus neurotransmitter systems. A number of studies will be aimed at clarifying issues related to the biology of pro-dynorphin in normal animals. These include 1) the unequivocal discrimination between pro-dynorphin and pro-enkephalin neurons, using a combination of immunohistochemistry with multiple antisera and in situ hybridization with messenger-specific cDNA probes. 2) the elucidation of the processing of pro-dynorphin into its final opioid and non-opioid products using in vivo pulse labeling. 3) the description of the anatomical relationships between pro-dynorphin products and multiple opioid receptor subtypes using combined receptor autoradiography-peptide immunohistochemistry. The regulatory cell biology of the two systems in response to changes in activity or demand will be investigated using various treatments, such as salt-loading for the magnocellular system and chronic haloperidol for the striato-nigal system. The response will be investigated at the following levels: 1) the content and ratio of the pro-dynorphin end products in cell body and terminal regions. 2) their biosynthetic rate-incorporation into precursor and conversion to products. 3) release into plasma, if technically possible. 4) changes in mRNA levels using cDNA probes in the contex of dot-blots and in situ hybridization. 5) changes in affinity, number or ratios of multiple opioid receptor subtypes. These studies will increase our understanding of regulatory neuropeptide biology. Further, the pathways under study have been implicated in functions of relevance to mental health: The magnocellular pathway modulates neuroendocrine and stress responses, whereas the striatonigro-striatal loop is critical in mediating some of the effects of antiphyschotic drugs.
