Neuroplasticity with alcohol dependence

描述（由申请人提供）：普遍的观点是，中皮质边缘系统中多巴胺传递的增强是酒精的奖励特性的基础。该系统由中脑腹侧被盖区（VTA）中的多巴胺神经元组成，其支配丘脑核和其他边缘系统结构。多巴胺的神经传递是由GABA中间神经元控制的。我们已经确定了一个均匀的GABA神经元的VTA的人口是由低剂量的乙醇兴奋，但抑制中到高剂量的乙醇，成为耐受慢性乙醇，表现出hyperexcitabilityduring撤退，并加快预期乙醇自我奖励。这些相关的研究补充了我们最近的研究表明，在乙醇自我管理的VTA GABA神经元的因果关系的作用。VTA GABA神经元被广泛认为是DA神经传递的关键调节者，但它们可能是来自感觉、皮层和边缘系统区域的会聚信息的独特和独立的整合者，从而促进酒精奖励和依赖。该提议的核心论点是，反复暴露于酒精导致GABA（A）受体[GABA（A）R]介导的腹侧被盖区GABA神经元抑制的适应性变化，并导致伴随酒精依赖的中脑边缘DA稳态失调（Gilpin和Koob，2008; Koob和Le Moal，1997）。基于先前的工作和初步结果，我们假设腹侧被盖区GABA神经元对慢性乙醇暴露的适应和伴随的依赖性是由腹侧被盖区GABA神经元上GABA（A）Rs的分子开关引起的，类似于我们对阿片依赖的研究中所报道的（Laviolette等人，2004; Vargas-Perez等人，2009年）。我们将采用多学科的行为，电生理，分子和新的荧光成像方法来评估短期和长期乙醇暴露对GABA（A）R介导的抑制和谷氨酸（GLU）NMDAR介导的兴奋和受体表达的适应性影响，以及脑源性神经营养因子（BDNF）酪氨酸激酶B（Trk B）受体在介导GABA功能转换中的作用（A）酒精依赖期间的RS。我们的研究将检验以下假设：1）从单一乙醇暴露中退出（非依赖性条件）将增强NMDAR介导的GLU兴奋VTA GABA神经元，而从慢性乙醇中戒断（依赖性条件）将减少GABA（A）R介导的对VTA GABA神经元的抑制：2）从慢性乙醇暴露中退出将改变GABA（A）Rs的表达;和3）从长期暴露于乙醇中的戒断将导致GABA（A）R介导的对VTA GABA神经元的抑制的功能转换，所述抑制由BDNF TrkB受体激活介导。为了验证这些假设，我们提出了GAD GFP小鼠的三个特定目标，其中GABA神经元可以被明确识别和表征。我们将专注于机械方法，以表征VTA GABA神经元中的突触底物，其适应于单次暴露（短期）或多次暴露于乙醇（长期），以及BDNF及其高亲和力TrkB受体在介导GABA（A）Rs的长期适应中的作用。为了验证这些假设，我们提出了在GAD GFP小鼠中的三个具体目标，其中GABA神经元可以被识别和电生理学表征：1）我们将使用膜片钳电生理学来评估自发和诱发的抑制性和兴奋性突触传递、成对脉冲响应、总电荷转移、AMPAR/NMDAR比率和AMPA整流指数。这些研究将通过记录24小时前给予小鼠的单次注射乙醇后退出期间脑切片的IPSC和EPSC来完成（非依赖性条件）或在强制液体乙醇饮食程序中消耗乙醇的小鼠中（依赖条件）; 2）对GABA（A）R亚单位、NMDAR亚单位、酪氨酸羟化酶、Cx 36、用单细胞定量RTPCR检测VTA GABA神经元中TrkB受体转录本的表达;和3）我们将评估BDNF TrkB受体拮抗剂和用siRNA TrkB耗竭TrkB对GABA（A）R介导的抑制和NMDAR-1的影响。介导的兴奋性突触反应，如在Aim 1中。此外，我们将评估假设开关GABA（A）R使用穿孔补丁程序的个别腹侧被盖区GABA神经元和使用新的Clomeleon荧光成像程序的人口的腹侧被盖区GABA神经元。这些研究为探讨GABA（A）受体在酒依赖中对腹侧被盖区GABA神经元的作用提供了新的思路。VTA GABA神经元表现出与阿片依赖相关的神经适应性反应，其特征在于功能从GABA超极化到去极化的转换。这种开关似乎涉及BDNF，因为它触发GABA（A）受体功能的长期变化和无慢性阿片类药物的依赖状态。我们预计，我们提出的研究将提供重要的新的见解VTA GABA神经元的贡献作用和它们的功能连接在乙醇消耗和BDNF介导的功能开关的GABA（A）受体对VTA GABA神经元与酒精依赖的作用。这项研究的结果可以提供一个临床前药理学原理，考虑药物选择性作用于GABA（A）受体亚型或BDNF TrkB受体作为治疗酒精依赖的假定治疗药物。

DESCRIPTION (provided by applicant): The prevailing view is that enhancement of dopamine transmission in the mesocorticolimbic system underlies the rewarding properties of alcohol. This system consists of dopamine neurons in the midbrain ventral tegmental area (VTA) that innervate the nucleus accumbens and other limbic structures. Dopamine neurotransmission is controlled by local-circuit GABA interneurons. We have identified a homogeneous population of GABA neurons in the VTA that are excited by low-dose ethanol, but inhibited by moderate to high-dose ethanol, become tolerant to chronic ethanol, evince hyperexcitabililty during withdrawal, and accelerate in anticipation of ethanol self-reward. These correlative studies are complemented by our recent studies demonstrating a causal role for VTA GABA neurons in ethanol self-administration. VTA GABA neurons are widely accepted to be critical regulators of DA neurotransmission, but they may serve as unique and independent integrators of convergent information from sensory, cortical and limbic areas subserving alcohol reward and dependence. The core thesis underlying this proposal is that repeated exposure to alcohol causes adaptive changes in GABA(A) receptor [GABA(A)R]-mediated inhibition of VTA GABA neurons and contributes to the dysregulation of mesolimbic DA homeostasis that accompanies alcohol dependence (Gilpin and Koob, 2008; Koob and Le Moal, 1997). Based on previous work and preliminary results, we hypothesize that adaptation of VTA GABA neurons to chronic ethanol exposure and accompanying dependence results from a molecular switch in GABA(A)Rs on VTA GABA neurons, similar to what has been reported in our studies of opiate dependence (Laviolette et al., 2004; Vargas-Perez et al., 2009). We will employ multidisciplinary behavioral, electrophysiological, molecular and novel fluorescent imaging approaches to evaluate the adaptive effects of short-term and long-term ethanol exposure on GABA(A)R-mediated inhibition and glutamate (GLU) NMDAR-mediated excitation, and receptor expression, as well as the role of brain-derived neurotrophic factor (BDNF) tyrosine kinase B (TrkB) receptors in mediating the functional switch of GABA(A)Rs during ethanol dependence. Our studies will test the following hypotheses: 1) Withdrawal from a single exposure to ethanol (non-dependent condition) will enhance NMDAR-mediated GLU excitation of VTA GABA neurons, while withdrawal from chronic ethanol (dependent condition) will reduce GABA(A)R-mediated inhibition of VTA GABA neurons; 2) Withdrawal from chronic ethanol exposure will modify the expression of GABA(A)Rs; and 3) Withdrawal from chronic exposure to ethanol will result in a functional switch in GABA(A)R-mediated inhibition of VTA GABA neurons that is mediated by BDNF TrkB receptor activation. To test these hypotheses, we propose three Specific Aims in GAD GFP mice, wherein GABA neurons can be identified and characterized unambiguously. We will focus on mechanistic approaches in order to characterize the synaptic substrates in VTA GABA neurons that adapt in response to a single exposure (short-term) or multiple exposures to ethanol (long-term), and the role of BDNF and its high- affinity TrkB receptor in mediating the long-term adaptation of GABA(A)Rs. To test these hypotheses, we propose three Specific Aims in GAD GFP mice, wherein GABA neurons can be identified and characterized electrophysiologically: 1) We will evaluate spontaneous and evoked inhibitory and excitatory synaptic transmission, paired-pulse responses, total charge transfer, AMPAR/NMDAR ratio and AMPA rectification index using patch clamp electrophysiology. These studies will be accomplished by recording IPSCs and EPSCs from brain slices during withdrawal from a single injection of ethanol administered to mice 24 hrs previous (non-dependent condition) or in mice consuming ethanol in the forced liquid ethanol diet procedure (dependent condition); 2) We will evaluate the GABA(A)R subunit, NMDAR subunit, tyrosine hydroxylase, Cx36, and TrkB receptor transcript expression in VTA GABA neurons using single-cell quantitative RTPCR; and 3) We will evaluate the effects of BDNF TrkB receptor antagonists and TrkB depletion with siRNA TrkB on GABA(A)R-mediated inhibitory and NMDAR-mediated excitatory synaptic responses as in Aim 1. In addition, we will evaluate the hypothetical switch in GABA(A)R using the perforated patch procedure for individual VTA GABA neurons and using the novel Clomeleon fluorescent imaging procedure for populations of VTA GABA neurons. The proposed studies will provide important new insights into the role of GABA(A)Rs on VTA GABA neurons in alcohol dependence. VTA GABA neurons evince neuroadaptive responses in association with opiate dependence, characterized by a switch in functionality from being hyperpolarized by GABA to being depolarized. This switch appears to involve BDNF, as it triggers long-term changes in the functionality of GABA(A) receptors and a state of dependence without chronic opioids. We anticipate that the studies we propose will provide important new insights into the contributory role of VTA GABA neurons and their functional connectivity in ethanol consumption and the role of BDNF in mediating a switch in the functionality of GABA(A) receptors on VTA GABA neurons with alcohol dependence. Results from this study could provide a preclinical pharmacologic rationale for considering drugs that act selectively on GABA(A) receptor subtypes or on BDNF TrkB receptors as putative therapeutic agents for the treatment of alcohol dependence.