CRF-PACAP effects on anxiety circuits in mice (Bolshakov)

摘要：项目2(CRF-PACAP对小鼠贫血的影响/Bolshakov) 促肾上腺皮质激素释放因子(CRF)和垂体腺苷环化酶激活多肽 (PACAP)，此前已被认为与人类和实验动物的焦虑调节有关。 人们对其影响的神经回路知之甚少。这两种神经肽都在脑结构中表达。 参与焦虑相关状态的控制--杏仁基底外侧核(BLA)和纹状体床核 终端(BNST)，具体地说，它可能通过引导信息流 调节焦虑相关行为的BLA-BNST回路。我们将通过组合使用 光遗传技术的体外和体内电生理学、小鼠遗传学、病毒示踪 向量和行为测试。在目标1中，我们将探索CRF-和PACAP-之间的相互作用- 介导的信号可能有助于控制焦虑驱动的BLA-BNST电路中的信号流。光遗传学 激活BLA-BNST投射，我们将记录不同BNST亚群中CRF表达的神经元 (ovBNST和adBNST)，并探讨PACAP对兴奋性驱动和抑制性驱动的影响。 记录来自BLA的谷氨酸能投射的神经元及其突触驱动的棘波输出。上一首 研究表明，PACAP可能触发CRF的释放，因此PACAP在BNST的焦虑诱导作用 可能是由PACAP诱导的CRF释放介导的。因此结合了药理学和遗传学的方法， 我们将确定OVBNST中CRFR1和PAC1R受体在CRF-PACAP相互作用中的作用 该控制信号在BLA-BNST电路中流动。因为臂旁核(PBN)是内源性来源 PACAP在BNST和CEL中的作用，在目标2中，我们将结合行为小鼠的光遗传学来探讨CRF-1的作用。 PACAP在BNST和杏仁中央核外侧亚核(CEL)内的相互作用 PACAP能纤维和CRF神经元共同定位，控制焦虑。使用病毒追踪技术，我们 在行为过程中将光基因定位于从PBN到ovBNST和/或细胞内CRF神经元的投射 量化类似焦虑行为的测试。我们将使用CRF-CRE小鼠来去除PAC1R受体 以测试PAC1R在ovBNST和/或cel CRF神经元上特异性激活的可能性 会导致类似焦虑的行为。在目标3中，我们将探索CRF-PACAP相互作用在焦虑中的作用。 增强重复应激的效果。我们假设CRF和PACAP可能协同行动来控制 焦虑驱动的BLA-ovBNST-adBNST投射的信号流及PACAP在ovBNST和/或中的作用 CEL可能是间接的。项目2可能识别可能成为新疗法靶点的神经回路 可以缓解焦虑症核心症状的治疗，因此是该中心的关键联系 增强其他(临床前、临床)元素，并通过这些元素来增强。

SUMMARY: PROJECT 2 (CRF-PACAP EFFECTS ON ANXIETYCIRCUITS IN MICE/BOLSHAKOV) Two neuropeptides, corticotropin-releasing factor (CRF) and pituitary adenylate cyclase-activating polypeptide (PACAP), have been previously implicated in the regulation of anxiety in both humans and laboratory animals. The neurocircuitry of their effects is poorly understood. Both neuropeptides are expressed in brain structures involved in control of anxiety-related states—in the basolateral amygdala (BLA) and the bed nucleus of the stria terminals (BNST), specifically—and could contribute to the production of anxiety by directing information flow in BLA-BNST circuits that regulate anxiety-related behaviors. We will address this possibility by combining the use of optogenetic techniques with ex vivo and in vivo electrophysiology, mouse genetics, tract tracing with viral vectors, and behavioral testing. In Aim 1, we will explore how the interactions between CRF- and PACAP- mediated signaling may contribute to control of signal flow in anxiety-driving BLA-BNST circuits. Optogenetically activating BLA-BNST projections, we will record from CRF-expressing neurons in different BNST subdivisions (ovBNST and adBNST) in brain slices, and explore effects of PACAP on both excitatory and inhibitory drive to recorded neurons in glutamatergic projections from BLA and their synaptically-driven spike output. Previous studies indicate that PACAP may trigger CRF release, and therefore anxiety-inducing effects of PACAP in BNST may be mediated by PACAP-induced CRF release. Thus combining pharmacological and genetic approaches, we will determine the contributions of CRFR1 and PAC1R receptors in ovBNST to the CRF-PACAP interactions that control signal flow in BLA-BNST circuits. Because the parabrachial nucleus (PBn) is the endogenous source of PACAP in BNST and CeL, in Aim 2 we will combine optogenetics in behaving mice to explore the role of CRF- PACAP interactions within BNST and lateral subnucleus of central nucleus of the amygdala (CeL), where PACAPergic fibers and CRF neurons are co-localized, in control of anxiety. Using viral tracing techniques, we will optogenetically target projections from the PBn to CRF neurons in ovBNST and/or CeL during behavioral tests that quantify anxiety-like behaviors. We will use CRF-Cre mice to ablate PAC1R receptors specifically from CRF neurons to test the possibility that activation of PAC1R specifically on ovBNST and/or CeL CRF neurons contributes to anxiety-like behavior. In Aim 3, we will explore the role of CRF-PACAP interactions in the anxiety- enhancing effects of repeated stress. We hypothesize that CRF and PACAP may act in concert to control the signal flow in anxiety-driving BLA-ovBNST-adBNST projections and that the effects of PACAP in ovBNST and/or CeL could be indirect. Project 2 may identify neural circuits that could potentially be targets for novel therapeutic treatments that can alleviate core symptoms of anxiety disorders, and as such is a key nexus of the Center that enhances, and is enhanced by, the other (preclinical, clinical) elements.