The role of BNST CGRP in stress-enhanced anxiety behavior and HPA-axis function

描述（由申请人提供）：暴露于引起恐惧的压力源的个体患多种精神疾病的风险增加，包括创伤后应激障碍、重度抑郁症和广泛性焦虑症。这些使人衰弱的疾病是当今美国面临的最严重的公共卫生问题之一。了解驱动恐惧应激源反应的中枢神经药理学和分子机制将使我们能够确定可能导致压力相关疾病发展的失调点，并确定开发有效治疗方案的目标。终纹床核是最近才出现的应激调节回路的重要组成部分，它含有密集的终端，可以释放神经肽降钙素基因相关肽（CGRP）。最近，行为学证据表明BNST内的CGRP信号与焦虑有关，并指出BNST CGRP可能调节对压力的行为和神经内分泌反应。因此，本研究的第一个目标是验证BNST内CGRP信号增强焦虑样行为和下丘脑-垂体-肾上腺（HPA）轴对压力的反应这一假设。我将通过以下方法来操纵BNST的CGRP信号传导：1)通过颅内插管向BNST输注CGRP拮抗剂1CGRP8-37； 2)通过慢病毒介导的RAMP1的过表达来致敏BNST的CGRP受体，RAMP1是CGRP受体亚基，赋予CGRP的药理学特异性，并影响CGRP受体的糖基化和转运到细胞表面。在操纵BNST信号后，将大鼠暴露于引起恐惧的应激源（三甲噻唑啉气味）。我们将测量恐惧和焦虑样行为（声惊吓和防御性冻结），神经内分泌反应（血清皮质酮和促肾上腺皮质激素），CGRP mRNA表达的变化，以及CGRP受体成分RAMP1和降钙素受体样受体（CRLR）。此外，由于BNST内的CGRP终末在含有应激相关神经肽促肾上腺皮质激素释放因子（CRF）的神经元周围形成组织周围筐，本研究的另一个目标是确定CGRP信号是否影响BNST中含有CRF的神经元的激活，从而影响焦虑行为和HPA轴的活性。为了验证BNST CGRP增强BNST CRF神经元激活从而增加应激相关靶结构中CRFr1受体信号传导的假设，研究人员将用系统性CRFr1拮抗剂（GSK876008）治疗大鼠，或用慢病毒介导的CRF siRNA感染大鼠，以降低BNST内CRF的表达，然后将其注入BNST内CGRP并进行上述测试。这些研究结果为BNST CGRP调节行为和神经内分泌应激反应以及影响应激相关神经递质信号通路提供了第一个证据。本研究也可能为应激相关精神障碍的治疗提供新的治疗靶点。

DESCRIPTION (provided by applicant): Individuals exposed to fear-evoking stressors are at increased risk for a number of psychiatric illnesses including post-traumatic stress disorder, major depressive disorder, and generalized anxiety disorder. These debilitating conditions are among the most significant public health problems facing the United States today. Understanding the central neuropharmacological and molecular mechanisms that drive responses to fear- evoking stressors will enable us to identify points of dysregulation that may contribute to development of stress-related disorders and also identify targets for the development of effective treatment options. The bed nucleus of the stria terminalis, which has recently emerged as an important component of stress regulation circuitry, contains dense terminals that release the neuropeptide calcitonin gene-related peptide (CGRP). Very recently, behavioral evidence has implicated CGRP signaling within the BNST in anxiety and also points to the possibility that BNST CGRP may modulate behavioral and neuroendocrine responses to stress. The first goal of this present research, then, is to test the hypothesis that CGRP signaling within the BNST enhances anxiety-like behavioral and hypothalamic-pituitary-adrenal (HPA) axis responses to stress. I will manipulate BNST CGRP signaling using 1) infusion of a CGRP antagonist, 1CGRP8-37 into the BNST via intracranial cannulae and 2) BNST CGRP receptor sensitization by lentiviral-mediated over-expression of RAMP1, the CGRP receptor subunit that confers pharmacological specificity for CGRP and influences glycosylation and trafficking of the CGRP receptor to the cell surface. Following manipulation of BNST signaling, rats will be exposed to a fear-evoking stressor (trimethlythiazoline odor). We will measure fear- and anxiety-like behavior (acoustic startle and defensive freezing), neuroendocrine responses (serum corticosterone and adrenocorticotropin hormone), and changes in mRNA expression of CGRP, and the CGRP receptor components RAMP1 and calcitonin receptor-like receptor (CRLR). Also, because CGRP terminals within the BNST form perisomatic baskets around neurons containing the stress-related neuropeptide corticotropin- releasing factor (CRF), another goal of this research is to determine if CGRP signaling affects activation of BNST CRF-containing neurons to influence anxiety behavior and HPA axis activity. In order to test the hypothesis that BNST CGRP enhances BNST CRF neuron activation to increase CRFr1 receptor signaling in stress-related target structures, rats will be treated with a systemic CRFr1 antagonist (GSK876008) or infected with lentiviral-mediated CRF siRNA to knock down CRF expression within the BNST and then they will be infused with intra-BNST CGRP and tested as described above. The results of these studies could provide the first pieces of evidence to demonstrate BNST CGRP modulation of behavioral and neuroendocrine stress responses and influence on stress-related neurotransmitter signaling pathways. This research may also present a novel therapeutic target in the treatment of stress-related psychiatric disorders. PUBLIC HEALTH RELEVANCE: Individuals exposed to fear-evoking stressors are at increased risk for a number of psychiatric illnesses including post-traumatic stress disorder, major depressive disorder, and generalized anxiety disorder. The bed nucleus of the stria terminalis (BNST), which contains numerous calcitonin gene-related peptide (CGRP) immunopositive terminals, has recently been identified as a pivotal relay of cortical information to neural structures mediating behavioral and neuroendocrine responses to stress. The proposed research will investigate the role of CGRP signaling within the BNST in fear- and anxiety-like behavioral and neuroendocrine stress responses, and may present a novel therapeutic target in the treatment of stress-related psychopathologies.