Neuroplasticity with alcohol dependence

描述(由申请人提供)：普遍的观点是，增强中皮质边缘系统中的多巴胺传递是酒精有益特性的基础。这个系统由中脑腹侧被盖区(VTA)中的多巴胺神经元组成，该区域支配伏隔核和其他边缘结构。多巴胺的神经传递由局部回路的GABA中间神经元控制。我们已经在VTA中发现了一个同质的GABA神经元群体，这些神经元被低剂量的乙醇兴奋，但被中到高剂量的乙醇抑制，对慢性乙醇变得耐受，在戒断过程中表现出高度兴奋，并在预期乙醇自我奖赏时加速。这些相关研究得到了我们最近的研究的补充，这些研究证明了VTA GABA神经元在乙醇自我给药中的因果作用。VTA-GABA神经元被广泛认为是DA神经传递的关键调节因子，但它们可能是酒精奖赏和依赖的感觉、皮质和边缘区域汇聚信息的独特和独立的整合者。支持这一建议的核心论点是，反复暴露于酒精会导致GABA(A)受体[GABA(A)R]介导的VTA GABA神经元抑制的适应性变化，并导致伴随酒精依赖而来的中边缘DA动态平衡的失调(Gilpin和Koob，2008；Koob和Le Moal，1997)。根据以前的工作和初步结果，我们假设VTA GABA神经元对慢性乙醇暴露和伴随的依赖的适应是由VTA GABA神经元上GABA(A)R的分子开关导致的，类似于我们在阿片依赖研究中报道的结果(LaViette等人，2004；Vargas-Perez等人，2009)。我们将使用多学科的行为、电生理、分子和新的荧光成像方法来评估短期和长期酒精暴露对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(TrkB)受体在酒精依赖过程中介导GABA(A)R功能转换中的作用。我们的研究将检验以下假设：1)单次酒精暴露(非依赖状态)会增强NMDAR介导的VTA GABA神经元的GLU兴奋，而慢性酒精依赖状态下的戒断会降低GABA(A)R介导的对VTA GABA神经元的抑制；2)慢性酒精暴露会改变GABA(A)R的表达；以及3)慢性酒精暴露会导致GABA(A)R介导的VTA GABA神经元抑制的功能转换，这种抑制是由BDNF TrkB受体激活介导的。为了验证这些假设，我们在GAD GFP小鼠中提出了三个特定的目标，其中GABA神经元可以被明确地识别和表征。我们将集中在机制方法上，以表征VTA GABA神经元中对单一暴露(短期)或多次暴露(长期)做出适应的突触底物，以及BDNF及其高亲和力TrkB受体在介导GABA(A)Rs长期适应中的作用。为了验证这些假说，我们在GAD GFP小鼠中提出了三个特定的目标，其中GABA神经元可以被电生理识别和表征：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、成对脉冲反应、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小时前一次注射乙醇的小鼠(非依赖条件)或在强制液体乙醇饮食程序中饮用乙醇的小鼠(依赖条件)的脑片中的IPSCs和EPSCs来完成：2)我们将使用单细胞定量RTPCR来评估VTA GABA神经元中GABA(A)R亚单位、NMDAR亚单位、酪氨酸羟化酶、Cx36和TrkB受体的转录表达；3)我们将评估BDNF TrkB受体拮抗剂和去除TrkB与siRNA TrkB一起对GABA(A)R介导的抑制性和NMDAR介导的兴奋性突触反应的影响，如目标1所示。此外，我们将使用针对单个VTA GABA神经元的穿孔贴片程序和针对VTA GABA神经元群体的新型Clomeleon荧光成像程序来评估GABA(A)R中的假设开关。这项拟议的研究将为了解GABA(A)Rs在酒精依赖中对VTA GABA神经元的作用提供重要的新见解。VTA GABA神经元表现出与阿片依赖相关的神经适应性反应，其特征是功能从被GABA超极化切换到被去极化。这种转换似乎涉及BDNF，因为它触发了GABA(A)受体功能的长期变化，并在没有慢性阿片类药物的情况下处于依赖状态。我们预计，我们提出的研究将为VTA GABA神经元及其功能连接在酒精消耗中的贡献作用以及BDNF在酒精依赖的VTA GABA神经元上GABA(A)受体功能转换中的作用提供重要的新见解。这项研究的结果可以为选择性作用于GABA(A)受体亚型或BDNFTrkB受体的药物作为治疗酒精依赖的推定治疗剂提供临床前药理学基础。

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.