BETA ENDORPHIN NEURONS AND THE CONTROL OF HOMEOSTASIS

描述（来自申请人摘要）： 本提案的总体目标是了解蜂窝网络中的 β-内啡肽（β-END）神经元控制下丘脑的机制 神经分泌细胞活性，和 最后，下丘脑肽和胺的神经分泌， 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，生理水平的E2迅速解偶联μ-阿片样物质 和GABA β受体通过蛋白质从β END神经元中的K+通道（IKir） 激酶途径在实验1中，我们将测试E2激活的假设， 使μ-阿片样物质和GABA β受体解偶联的蛋白激酶C（PKC）途径 从弓状（ARC）神经元中的IKir。我们将测量：（a） μ-阿片样物质和GABA β激动剂在接受治疗的卵巢切除雌性中的效力 使用PKC激活剂和抑制剂与E2急性接触;（B）阐明途径 通过其长期（24小时）E2解偶联μ-阿片样物质和GABA β受体;和 (c)G蛋白信号调节因子（RGS）蛋白表达的变化 使用E2后的原位杂交和核糖核酸酶保护试验 治疗在实验2中，我们将确定哪些效应器系统 μ-阿片样物质和FQ受体在视上（SON）催产素中偶联 和血管加压素神经元以及长期E2治疗的影响。我们将：（a） 进一步表征μ-阿片激动剂对LH的抑制作用 K-阿片受体激动剂抑制的比Ca 2+电导; （B）确定E2的作用 对SON神经元中的μ-阿片样物质、k-阿片样物质和OFQ反应的影响;（c）表征 在E2处理的动物中，向SON神经元的阿片样物质突触突触输入;以及 受体放射自显影在实验3中，我们将使用 基因靶向的β END缺陷（β END敲除，KO）小鼠，以确定 由于缺乏一种受体， 内源性阿片样物质我们将：（a）研究μ-阿片受体的变化 与弓状神经元中的lkir偶联;（B）测量突触前神经元中的变化， μ-阿片样物质抑制对ARC神经元的兴奋性输入的作用，（C）测量 E2的影响。在μ-阿片样物质受体与Lkir偶联之后;和 最后（d）测量GABA β激动剂的效力和功效， KO小鼠中的IKir。这些结果将阐明细胞机制， E2改变μ-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μ阿片张力。结果也将 帮助我们理解β END神经元在体内平衡控制中的作用。

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.