NEUROSTEROIDS AND ETHANOL INTERACTIONS

本提案的目的是探索内源性3 α-羟基-5 α-葡聚糖-20-酮的潜在作用 (3a，5a-THP）在乙醇的电生理和行为作用中的作用，包括乙醇的发育， 宽容我们最近发现，给大鼠注射乙醇可使血浆和大脑中的 有效的GABAA受体神经活性类固醇的水平。3a、5a-THP。相关的 浓度的乙醇对大脑皮层3a，5a-THP水平的影响具有时间和剂量依赖性， 足以增强GABAA受体功能。此外，酒精睡眠与酒精睡眠之间有很强的相关性， 5a-THP的时间和大脑皮层水平。与此相反，急性脑损伤并没有改变脑内3a，5a-THP的水平。 因此，在乙醇依赖性大鼠中乙醇挑战，耐受性可能发展到乙醇对 3 α，5 α-THP诱导。3a，5a-THP水平的乙醇诱导的丧失可能是对乙醇耐受的基础。 乙醇的药理作用。因此，我们建议检验3a，5cc-THP 介导乙醇在体内的药理学作用。 第一个目标是研究3a，5a-THP在行为和神经生理学效应中的作用， 乙醇3a，5cc-THP的形成将通过预处理类固醇生物合成抑制剂和 乙醇对神经元放电率的影响，GABAA受体介导的自发神经元活动的抑制， 将测量中毒、空中翻正反射和癫痫发作阈值。3a，5a-THP的作用也将是 在缺乏3a-羟基类固醇脱氢酶（3a，5a- THP形成。第二个目标将确定3a，5c-THP是否在对HLA-THP的耐受性的发展中起作用。 使用基因敲除小鼠和类固醇生物合成抑制剂。第三个目标将侧重于机制 3 α，5 α-THP的积累。将进行研究，以确定乙醇是否 直接改变3 α，5 α-THP生物合成酶的活性。初步结果表明，乙醇可能 增加3 α，5 α-THP生物合成，而高剂量乙醇可释放或暴露3 α，5 α-THP的"储存"。的 将测量乙醇对从培养的星形胶质细胞释放3 α，5 α-THP的影响。这些研究将解决 可能与电生理作用相关的快速时间点（秒至分钟）乙醇的影响 乙醇。这些研究可能阐明了乙醇在中枢神经系统中作用的新机制，并解释了为什么 乙醇对GABA能神经传递的直接作用不能充分解释， GABA.4受体。这项研究的结果将扩大我们对神经类固醇潜在作用的认识 在酒精作用和酒精耐受性方面的作用，并可能确定参与酒精中毒病因学的新因素。

The objective of this proposal is to explore the potential role of endogenous 3a-hydroxy-5a-pregnan-20-one (3a,5a-THP) in the electrophysiological and behavioral actions of ethanol, including the development of ethanpl tolerance. We recently discovered that ethanol administration to rats produces an elevation in plasma and brain levels of the potent GABAA receptor neuroactive steroid. 3a,5a-THP. to pharmacologically relevant concentrations. The effects of ethanol on 3a,5a-THP levels in cerebral cortex are time and dose dependent and sufficient to potentiate GABAA receptor function. Moreover, there is a strong correlation between ethanol sleep time and cerebral cortical levels of 3a.5a-THP. In contrast, brain levels of 3a,5a-THP are not altered by acute ethanol challenge in ethanol dependent rats, therefore, tolerance may develop to the effect of ethanol on the induction of 3a,5a-THP. The loss of ethanol induction of 3a,5a-THP levels may underlie tolerance to the pharmacological effects of ethanol. Therefore, we propose to test the overall hypothesis that 3a,5cc-THP mediates pharmacological effects of ethanol in vivo. The first goal is to investigate the role of 3a,5a-THP in the behavioral and neurophysiological effects of ethanol. 3a,5cc-THP formation will be prevented by pretreatment steroid biosynthesis inhibitors and the effects of ethanol on neuronal firing rates, GABAA receptor-mediated inhibition of spontaneous neuronal activity, intoxication, aerial righting reflex and seizure thresholds will be measured. The role of 3a,5a-THP will also be investigated in conditional knock-out mice that lack 3a-hydroxysteroid dehydrogenase - the final step in 3a,5a- THP formation. The second aim will determine if 3a,5oc-THP plays a role in the development of tolerance to ethanol using both knock out mice and steroid biosynthesis inhibitors. The third aim will focus on the mechanisms of 3a,5cc-THP accumulation following ethanol administration. Studies will be conducted to determine if ethanol directly alters the activity of the 3a,5cc-THP biosynthetic enzymes. Preliminary results suggest that ethanol may increase 3a,5a-THP biosynthesis, while high dose ethanol may release or uncover a "store" of 3a,5oc-THP. The effect of ethanol on 3oc,5a-THP release from cultured astrocytes will be measured. These studies will address the effects of ethanol at rapid time points (seconds to minutes) that may be relevant to the electrophysiological actions of ethanol. These studies may elucidate a new mechanism of ethanol action in the CNS and explain why the effects of ethanol on GABAergic neurotransmission could not be adequately explained by the direct action of ethanol at GABA.4 receptors. The results of this investigation will extend our knowledge of the potential role of neurosteroids in ethanol action and ethanol tolerance and may identify new factors involved in the etiology of alcoholism.