Hippocampal-dependent neural immune interactions regulate heroin withdrawal and future enhanced fear learning

项目摘要 阿片类药物使用障碍是一种全国性的危机，多条证据表明，海洛因的使用导致了 焦虑症的发生率和严重程度，包括创伤后应激障碍(PTSD)。我们的实验室有 建立了一个临床前模型，有效地展示了海洛因戒断后的高反应性 应激源，人类创伤后应激障碍的关键组成部分之一。我们发现戒除不断升级的海洛因 给药后大鼠背侧海马区白介素1β(IL-1β)升高，并阻断背侧海马区IL-1的表达 使用受体拮抗剂(IL-1RA)可预防海洛因戒断，增强恐惧学习。该IL-1β被表达 主要是在星形胶质细胞，海洛因戒断也上调胶质纤维酸性蛋白(GFAP) 表达，星形胶质细胞反应性的标志。这表明星形胶质细胞信号可能与海洛因有关。 戒断和未来增强的恐惧学习，然而，目前尚不清楚是否存在因果联系 在这两者之间。目前的提案是一种有针对性的方法，以检验海洛因戒断的总体假设 通过DH星形胶质细胞信号和星形胶质细胞生理变化增强恐惧学习。AIM 1测试 假设脱氢酶星形胶质细胞介导海洛因戒断诱导的增强恐惧学习。我们已经成功地 将由设计师药物(GI-DREADD)独家激活的GI偶联设计师受体植入我们的 在活体内操纵DH星形胶质细胞信号的研究计划。目标1测试星形胶质细胞GPCR信号是否 能够影响海洛因戒断对增强恐惧学习的长期影响。在《目标1》中，我将成为 接受过化学遗传技术培训，获得了这种尖端工具的广泛知识，并成为 精通使用神经胶质DREADD研究星形胶质细胞信号与行为的关系。结果将 证实海洛因戒断过程中的星形胶质细胞信号转导通路参与了长时程增强的发生。 恐惧一词。目的2验证海洛因戒断改变DH星形胶质细胞形态和星形胶质细胞的假说。 神经元的相互作用。我们已经建立了一种方法来分析单个星形胶质细胞的复杂性 AAV5-GFAP-LCK-GFP构建、高分辨率共聚焦显微镜和高级三维分析 细胞重建。使用这项创新技术，我们将检查海洛因戒断对 星形胶质细胞的形态和共定位与突触后标志物，星形胶质细胞-神经元相互作用的代理。 在目标2中，我将完善和提高我在免疫组化、共聚焦显微镜和高级细胞分析方面的技能。 BitPlane Imaris软件用于量化戒断诱导的星形胶质细胞形态和突触的变化 互动。结果将确定海洛因的使用和戒断直接改变了DH星形胶质细胞的生理 可能会导致恐惧、学习和焦虑的增强。总而言之，这项建议研究了海马体的作用。 星形胶质细胞在海洛因戒断后增强恐惧学习中的作用。这些研究具有重要意义 有助于了解海洛因戒断对星形胶质细胞的影响并提供有价值的学习 培养我在成瘾神经生物学研究生涯中的独立性的经验。

Project Summary Opioid use disorder is a national crisis, and multiple lines of evidence indicate that heroin use leads to increased incidence and severity of anxiety disorders, including post-traumatic stress disorder (PTSD). Our laboratory has established a preclinical model that effectively demonstrates heroin withdrawal-induced hyperreactivity to future stressors, one of the key components of human PTSD. We discovered that withdrawal from escalating heroin administration results in an increase in dorsal hippocampal (DH) interleukin-1β (IL-1β), and blockade of DH IL-1 with receptor antagonist (IL-1RA) prevents heroin withdrawal enhanced fear learning. This IL-1β is expressed predominantly in astrocytes, and heroin withdrawal also upregulates glial fibrillary acidic protein (GFAP) expression, a marker for astrocyte reactivity. This suggests that astrocyte signaling may be involved in heroin withdrawal and future enhanced fear learning, however it is presently unknown whether there is a causal link between the two. The current proposal is a targeted approach to test the overall hypothesis that heroin withdrawal enhances fear learning through DH astrocyte signaling and alterations in astrocyte physiology. Aim 1 tests the hypothesis that DH astrocytes mediate heroin withdrawal-induced enhanced fear learning. We have successfully implemented Gi-coupled designer receptors exclusively activated by designer drugs (Gi-DREADDs) into our research program to manipulate the DH astrocyte signaling in vivo. Aim 1 tests if astroglial GPCR signaling is capable of influencing the long-term effects of heroin withdrawal on enhanced fear learning. In Aim 1, I will be trained in chemogenetic techniques, acquire extensive knowledge of this cutting-edge tool, and become proficient in using glial DREADDs to investigate how astrocyte signaling relates to behavior. Results will establish that DH astroglial signaling during heroin withdrawal mediates changes in the development of long- term fear. Aim 2 tests the hypothesis that heroin withdrawal alters DH astrocyte morphology and astrocyte- neuron interactions. We have established a method to analyze the complexity of individual astrocytes using the AAV5-GFAP-Lck-GFP construct, high-resolution confocal microscopy, and advanced analysis of 3-dimensional cellular reconstructions. Using this innovative technique, we will examine the effect of heroin withdrawal on astrocyte morphology and colocalization with post synaptic markers, a proxy for astrocyte-neuron interactions. In Aim 2, I will refine and enhance my skills in IHC, confocal microscopy, and advanced cellular analysis using the Bitplane Imaris software to quantify withdrawal-induced changes in astrocyte morphology and synaptic interactions. Results will establish that heroin use and withdrawal directly alters DH astrocyte physiology that may lead to enhanced fear learning and anxiety. Collectively, this proposal examines the role of hippocampal astrocytes in mediating enhanced fear learning following heroin withdrawal. These studies make important contributions to understanding the effects of heroin withdrawal on astrocytes and provide a valuable learning experience to foster the independence of my research career in addiction neurobiology.