BETA ENDORPHIN NEURONS AND THE CONTROL OF HOMEOSTASIS

描述(摘自申请人的摘要)： 本提案的总体目标是了解细胞 β-内啡肽(BetaEND)神经元控制下丘脑的机制 神经分泌细胞活动，以及 最终，下丘脑多肽和胺的神经分泌在 女性。我们将使用豚鼠作为模型，因为它的排卵周期 模拟人类和生理水平的E2快速解偶联阿片类药物 和来自K+通道(LKIR)的GABAβ受体通过一种蛋白在β神经元中表达 蛋白水解酶途径。在实验1中，我们将检验这样的假设：雌激素会激活 解偶联Mu阿片受体和GABAβ受体的蛋白激酶C(PKC)途径 来自弓状(ARC)神经元的1KIR。我们将衡量：(A) 阿片类药物和GABAβ受体激动剂对去卵巢女性的治疗效果 使用PKC激活剂和抑制物急性使用E2；(B)阐明该途径 通过较长时间(24小时)的E2解偶联Mu-阿片和GABAβ受体； (C)G蛋白信号转导调节蛋白(RGS)蛋白表达的变化 用原位杂交和核糖核酸酶保护试验检测E2 治疗。在实验2中，我们将确定哪个效应器系统 视上(SON)催产素中偶联MU-阿片受体和孤啡肽FQ受体 和加压素神经元以及长期E2治疗的效果。我们会：(A) 进一步研究Mu-阿片激动剂对促黄体生成素的抑制作用 K-阿片激动剂抑制的钙电导(S)； 被OFQ激活的特定K+电导(S)； SON神经元的Mu阿片、k阿片和OFQ反应；(C)表征 雌二醇组动物SON神经元的阿片类突触输入； 受体放射自显影。在实验3中，我们将使用一种菌株 基因靶向BetaEND缺陷(Beta末端敲除，KO)小鼠以确定 Mu-阿片受体偶联作用减弱是由于缺乏 内源性阿片类药物。我们将：(A)研究Mu-阿片受体的变化 弓状神经元与Lkir的偶联；(B)测量突触前 阿片类药物抑制兴奋性传入ARC神经元的作用，(C)测量 雌二醇的作用。在Mu-阿片受体与IkIR偶联后； 最后(D)测量一种GABAβ激动剂的效力和疗效以避免 KO小鼠体内的IkIR。这些结果将阐明细胞机制，通过 雌二醇2改变了阿片受体与其效应系统的偶联， 最终决定女性中枢神经系统中的类阿片音调。此外，结果将是 帮助我们理解神经元在动态平衡控制中的作用。

DESCRIPTION (from applicant's abstract): The overall goal of the present proposal is to understand the cellular mechanisms by which beta-endorphin (betaEND) neurons control hypothalamic neurosecrelory cell activity, and Ultimately the neurosecretion of hypothalamic peptides and amines in the female. We will use the guinea pig as a model because its ovulalory cycle mimics the Human, and physiologicaal levels of E2 rapidly uncouple mu-opioid and GABAbeta receptors from K+ channels (lKir) in betaEND neurons via a protein kinase pathway. In Experiments 1, we will test the hypothesis that E2 activates a protein kinase C (PKC) pathway to uncouple mu-opioid and GABAbeta receptors from lKir in arcuate (ARC) neurons. We will measure: (a) the changes in the potency of mu-opioid and GABAbeta agonists in ovaricetomized females treated acutely with E2 using PKC activators and inhibitors; (b) elucidate the pathway by which longer-term (24 h) E2 uncouples mu-opioid and GABAbeta receptors; and (c) changes in expression of regulators of G-protein signaling (RGS) proteins using in situ hybridization and ribonuclease protection assay following E2 treatment. In Experiments 2, we will determine to which effector systems mu-opioid and orphanin FQ receptors are coupled in supraoptic (SON) oxytocin and vasopressin neurons and the effects of long term E2 treatment. We will: (a) further characterize the inhibition of lh by mu-opioid agonists and ascertain the specific Ca2+ conductance (s) inhibited by K-opioid agonists; and the specific K+ conductance(s) activated by OFQ; (b) ascertain the effects of E 2 on the mu-opioid, k-opioid and OFQ responses in SON neurons; (c) characterize the opioid synaptic synaptic input to SON neurons in E2-treated animals; and receptor autoradiography. In Experiments 3, we will use a strain of gene-targeted betaEND deficient (beta END Knockout, KO) mice to ascertain if there is a decrease in mu-opioid receptor coupling induced by the absence of an endogenous opioid. We will: (a) investigate the changes in mu-opioid receptor coupling to lkir in arcuate neurons; (b) measure changes in the presynaptic actions of mu-opioids to inhibit excitatory input to ARC neurons, (C) measure the effects of E2. to after the coupling of mu-opioid receptors to lkir; and finally (d) to measure the potency and efficacy of a GABAbeta agonist to aviate lkir in KO mice. These results will elucidate the cellular mechanisms by which E2 alters the coupling of the mu-opioid receptor to its effector system, which ultimately determinesmu-opioid tone in the female CNS. Also the results will help us understand the role betaEND neurons in the control of homeostasis.