The overall goal of the present proposal is to understand the cellular mechanism(s) by which the gonadal steroid 17 beta-estradiol (E2) modulates opioidergic (beta-endorphin) tone and subsequently the neurosecretion of hypothalamic peptides and amines and activation of reward pathways in the female. We use the guinea pig as a model because the ovulatory cycle mimics the human. Physiological levels of E2 rapidly uncouple mu-opioid and GABAB receptors from K+ channels (IKir) in beta-endorphin (betaEND) neurons via a protein kinase pathway. In addition, chronic opiate uncouples and down-regulates mu-opioid receptors. In Experiments 1, we will test the hypothesis that chronic morphine activates a protein kinase A (PKA) pathway to specifically uncouple mu-opioid receptors in arcuate neurons. We will measure: (a) changes in mu-opioid agonist potency in females treated with morphine using membrane permeable protein kinase inhibitors; (b) the time course of morphine actions; and (c) the effects of morphine on the coupling of the orphanin FQ receptor to IKir. In Experiments 2, we will test the hypothesis that E2 activates a protein kinase C (PKC) pathway to uncouple mu-opioid and GABAB receptors from IKir at the site of the G protein coupling. We will measure: (a) the changes in the potency of mu-opioid and GABAB agonists in ovariectomized females treated acutely with E2 using protein kinase activators and inhibitors; (b) changes in agonist-stimulated 3H-GTPgammaS binding in ARC membranes and autoradiography of 3H-GTPgammaS in ARC slices following E2 treatment; and (c) elucidate the pathway by which longer-term (24 h) E2 uncouples mu-opioid and GABAB receptors. In Experiments 3, we will determine to which effector systems mu-opioid, K-opioid and orphanin FQ receptors are coupled in supraoptic (SON) vasopressin and oxytocin neurons and the effects of chronic morphine. We will: (a) further characterize the inhibition Ih by mu-opioid agonists, the inhibition of a Ca2+ T-current by K-opioid agonists and the specific K+ conductance(s) activated by OFQ; (b) ascertain the effects of chronic morphine on the p-opioid, K-opioid and OFQ responses in SON neurons; (c) measure the changes in mu-opioid, K-opioid and orphanin FQ receptors mRNA expression and receptor binding in the SON with chronic morphine treatment; and (d) characterize the opioid-mediated presynaptic inhibition of excitatory input to SON neurons in morphine-tolerant animals. These results should not only elucidate the mechanisms by which E2 and opioids regulate hypothalamic neurons but also their interaction in altering opioid tone in the female CNS, which will help us understand the gender differences in reward and homeostasis.
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