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

描述(申请人提供)：杏仁核和海马体功能障碍可导致焦虑症。各种神经递质和具有抗焦虑作用的药物通过激活Gi/o蛋白偶联受体(Gi/oPCRs)来调节这些脑区。GI/oPCRs抑制突触前Cav2.2通道，该通道控制海马区和杏仁核兴奋性突触和抑制性突触的递质释放。因此，该项目的假设是GI/oPCRs的激活抑制了突触前Cav2.2通道，降低了焦虑水平。我的研究计划结合了与焦虑相关的神经回路的分子生物学、行为学和突触电生理学。该项目的具体目标是：a)确定是否减少 Gi/oPCR偶联到Cav2.2通道影响海马突触中神经递质的释放。Lipscombe实验室表明，Cav2.2基因37a外显子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，我们培育了一只缺少外显子37a的小鼠。我们的初步数据显示，与WT相比，37A缺乏小鼠的内侧穿透路径-齿状回中的成对脉冲比率较低，这表明Cav2.2通道的Gi/oPCR抑制减少所产生的神经递质释放增加。为此，我将进一步分析GI/oPCRs和Cav2.2之间缺乏偶联对海马区神经递质释放的影响。B)确定Gi/oPCR连接到Cav2.2通道的中断是否影响与焦虑相关的行为。在我们最初的研究中，与野生型相比，缺乏e37a的小鼠在新奇诱导的低吞噬中表现出更短的进食潜伏期，这表明减少对Cav2.2通道的Gi/oPCR抑制作用可以降低焦虑水平。我将使用开阔的场地和高架的零迷宫来扩展和证实这些令人兴奋的结果。C)确定焦虑状态下，GI/oPCR对杏仁基底外侧核GABA能突触的抑制作用是否上调。以前的报道将焦虑与杏仁基底外侧核GABA能传递的减少联系在一起。大多数研究都记录了焦虑过程中突触后的变化，但令人惊讶的是，对突触前对递质释放的控制知之甚少。在这个目标中，我将确定在焦虑状态下杏仁核中依赖Cav2.2的GABA释放是否减少。D)确定在抗焦虑药物耐受过程中，Gi/oPCR激动剂对谷氨酸能突触的抑制作用是否下调。利用小鼠耐受模型，我将在NA和药物治疗的小鼠中测量Cav2.2通道对外源性激动剂介导的Gi/oPCR抑制谷氨酸能传递的敏感性。

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.