Role of Gi/o-Protein Inhibition of CaV2.2 Channels in Anxiety Behavior

描述（由申请人提供）：杏仁核和海马体功能障碍可引起焦虑症。具有抗焦虑作用的各种神经递质和药物通过激活Gi/o蛋白偶联受体（Gi/oPCR）来调节这些脑区。Gi/oPCR抑制突触前CaV2.2通道，其控制海马和杏仁核中兴奋性和抑制性突触处的递质释放。因此，该项目的假设是Gi/oPCR的激活抑制突触前CaV2.2通道并降低焦虑水平。我的研究计划结合了分子生物学、行为学和与焦虑相关的神经元回路的突触电生理学。该项目的具体目标是： Gi/oPCR与CaV2.2通道的偶联影响海马突触中的神经递质释放。Lipscombe实验室表明，CaV2.2基因中外显子37 a的消除降低了通道对Gi/oPCR抑制的敏感性。为了建立突触作用，我们产生了一只缺乏外显子37 a的小鼠。我们的初始数据显示，与WT相比，37 α缺乏小鼠的内侧穿通路径-齿状回中的配对脉冲比较低，表明通过减少CaV2.2通道的Gi/oPCR抑制产生的神经递质释放增加。在这个目标中，我将进一步分析Gi/oPCR和CaV 2.2之间缺乏偶联对海马神经递质释放的影响。B）确定Gi/oPCR与CaV 2.2通道偶联的破坏是否影响焦虑相关行为。在我们的初步研究中，与野生型相比，e37 a缺乏小鼠在新奇诱导的食欲减退中表现出更短的进食潜伏期，这表明Gi/oPCR对CaV2.2通道的抑制作用的减少降低了焦虑水平。我将扩展和确认这些令人兴奋的结果使用开放领域和高架零迷宫。c）确定在焦虑状态期间，Gi/oPCR对基底外侧杏仁核的GABA能突触的CaV2.2通道的抑制是否上调。先前的报道认为焦虑与基底外侧杏仁核中GABA能传递减少有关。大多数研究都记录了焦虑时突触后的变化，但令人惊讶的是，人们对突触前控制递质释放的情况知之甚少。在这个目标中，我将确定是否CaV2.2依赖GABA释放减少在杏仁核在焦虑状态。d）确定Gi/oPCR激动剂对突触能突触的CaV2.2通道的抑制在抗焦虑药耐受期间是否下调。使用耐受性小鼠模型，我将测量CaV2.2通道对由外源性激动剂介导的Gi/oPCR抑制的敏感性，这些激动剂在天然和药物处理的小鼠中在神经元能传递中。

DESCRIPTION (provided by applicant): Dysfunction of the amygdala and hippocampus can cause anxiety disorders. Various neurotransmitters and drugs with anxiolytic effects modulate these brain areas by activating Gi/o protein coupled receptors (Gi/oPCRs). Gi/oPCRs inhibit presynaptic CaV2.2 channels, which control transmitter release at excitatory and inhibitory synapses in the hippocampus and amygdala. Therefore, the hypothesis of this project is that activation of Gi/oPCRs inhibits presynaptic CaV2.2 channels and reduces anxiety levels. My research plan combines molecular biology, behavior and synaptic electrophysiology of neuronal circuits related to anxiety. The specific aims of this project are: a) To determine if reduction of Gi/oPCR coupling to CaV2.2 channels impacts neurotransmitter release in hippocampal synapses. The Lipscombe lab showed that elimination of exon 37a in the CaV2.2 gene decreases channel sensitivity to Gi/oPCR inhibition. To establish a synaptic role, we generated a mouse that lacks exon 37a. Our initial data showed a lower paired-pulse ratio in the medial perforant path- dentate gyrus of 37a-lacking mice compared to WT, suggesting an increase in neurotransmitter release produced by a reduction of Gi/oPCR inhibition of CaV2.2 channels. In this aim, I will further analyze the effect of the lack of coupling between Gi/oPCRs and CaV2.2 on the neurotransmitter release in hippocampus. b) To determine if the disruption of Gi/oPCR coupling to CaV2.2 channels affect anxiety-related behavior. In our initial studies, e37a-lacking mice exhibited shorter latency times to feed in novelty-induced hypophagia compared to wild-type, suggesting that reduction of Gi/oPCR inhibitory action on CaV2.2 channels lowers anxiety levels. I will extend and confirm these exciting results using open field and elevated zero maze. c) To determine if the inhibition of CaV2.2 channels by Gi/oPCR on gabaergic synapses of basolateral amygdala is upregulated during anxiety states. Previous reports have associated anxiety to a reduction in gabaergic transmission in basolateral amygdala. Most studies have documented changes postsynaptically during anxiety but surprisingly little is known about presynaptic control of transmitter release. In this aim, I will determine whether CaV2.2-dependent GABA release is reduced in the amygdala during anxiety states. d) To determine if the inhibition of CaV2.2 channels by Gi/oPCR agonists on glutamatergic synapses is down regulated during tolerance to anxiolytics. Using mouse models of tolerance, I will measure the sensitivity CaV2.2 channels to Gi/oPCR inhibition mediated by exogenous agonists in na¿ve and drug-treated mice in glutamatergic transmission.