终纹床核—下丘脑室旁核GABA能神经环路去抑制调控生命早期应激致成年慢性内脏痛机制研究

This project was continuing our previous exploration of neurobiological mechanism behind the chronic visceral pain. We recently found that the sensitization of CRH neurons in hypothalamic paraventricular nucleus (PVN) participates in the early life stress-induced adult visceral pain. The CRH neurons in PVN are innervated by GABAergic projections originated from BNST to maintain the neural homeostasis. Early life stress is associated with a down-regulation of GAD65 protein expression, activation of glia and up-regulation of proinflammatory factor in adulthood. Accordingly, we hypothesized that early life stress impaired the structure and function of GABAergic neurons in BNST, which led to a disinhibition of GABA innervation on CRH neurons in PVN, causing CRH neuronal hypersensitivity and chronic visceral pain. To validate this hypothesis, we will examine the structure and function of a subgroup of GABAergic neurons in BNST which project to PVN CRH neurons with retrograde tracing and morphological approaches; next, we will employ optogenetic excitation or inhibition of PVN projecting neurons in BNST on PVN CRH neuronal activity and visceral pain performances. In the last, we will explore the glia-neuron crosstalk and neuronal inflammatory mechanism on BNST GABAergic neurons function. This study will enrich our understanding of the neurobiological mechanism of chronic visceral pain, and open a new avenue of the development of therapeutic target against visceral pain.

本课题旨在深入研究慢性内脏痛发病的神经生物学机制。我们发现下丘脑室旁核（PVN）CRH神经元敏化介导生命早期应激致成年慢性内脏痛。PVN内CRH神经元活动受到终纹床核（BNST）GABA投射神经元的抑制性调控以维持稳态。预实验表明生命早期应激可致大鼠成年后BNST GAD65蛋白表达下调，胶质细胞活化和炎性细胞因子增多。据此推测生命早期应激通过炎性细胞因子削弱BNST内GABA能神经元功能，对CRH神经元的抑制减弱，引起神经元敏化和慢性内脏痛。为此我们首先采用束路逆向追踪等方法明确慢性内脏痛大鼠BNST投射到PVN的GABA能神经元结构和功能改变；进而采用光遗传学方法调控BNST→PVN GABA能神经元，观察PVN内CRH神经元活动及内脏痛等改变；最后研究神经炎性反应机制对BNST内GABA神经元功能的调控作用。本研究将丰富慢性内脏痛发病的神经生物学机制，并为临床上开发相关药物提供靶标。

研究背景:大量研究表明生命早期应激（Early life stress，ELS）是内脏痛发生的高危因素，然而其潜在的神经环路机制尚不清楚。我们前期研究表明新生期结直肠扩张（Colorectal distension，CRD）和母婴分离均可引起下丘脑室旁核（Hypothalamic paraventricular nucleus，PVN）中的促肾上腺皮质激素释放激素（Corticotropin releasing hormone，CRH）释放增加，导致皮质激素释放增多，进而介导内脏超敏反应。终纹床核（Bed nucleus of stria terminalis，BNST）可接受边缘系统的信号传入，并将信号传出到PVN，其中腹侧区（anterior ventral，AV）的γ-氨基丁酸（γ-Aminobutyric acid，GABA）能神经元可对PVN中CRH神经元发挥重要作用的调控作用，但终纹床核→下丘脑室旁核GABA能神经环路在慢性内脏痛形成和维持中的作用及机制尚不清楚。.研究目的: 阐明终纹床核→下丘脑室旁核GABA能神经环路调控生命早期应激所致成年慢性内脏痛的机制。.方法：本研究采用大鼠新生期CRD诱导成年慢性内脏痛模型，综合运用电生理学、药理学、行为学测试、分子生物学、化学遗传学和光遗传学的等技术手段。.结果：大鼠新生期 CRD 致成年慢性内脏痛PVN中CRH神经元放电频率升高，自发抑制性突触后电流频率降低；外源性给予GABA和GABAA（muscimol）受体激动剂可以调节PVN中CRH神经元活动介导内脏超敏；BNST→PVN特异性投射的GABA能神经元主要集中在AV区域；内脏超敏状态下，BNST AV→PVN中特异性投射的GABA能神经元兴奋性下降；当损毁AV区GABA能神经元可诱发大鼠内脏痛敏；化学遗传激活BNST AV→PVN环路中特异性投射的GABA能神经元可以缓解内脏痛敏；光遗传激活BNST AV→PVN环路中特异性投射的GABA能神经元可以缓解大鼠的内脏超敏反应，而光抑制则诱发正常大鼠的内脏痛觉过敏。.结论： BNST AV→PVN环路中特异性投射的GABA能神经元去抑制激活PVN中的CRH参与内脏痛敏。