Activity and connectivity of hippocampal newborn neurons underlie alcohol withdrawal-associated syndromes

慢性酒精暴露(AE)后戒酒(AW)会产生一系列症状。其中， 全身性强直阵挛发作是最严重、最危险的症状。严重程度和易感性 复发和长期酗酒突出表明迫切需要了解其潜在的机制 酒精依赖与AW，以开发干预和治疗AW相关的新治疗策略 癫痫等症状。在这个应用程序中，我们将测试活动和连接性这一新的假设 新生齿状颗粒细胞(DGC)是AW相关癫痫发作的基础。DGC是主要的 不断产生的兴奋性神经元，整合到海马神经回路中，并改变 海马神经发生与癫痫发作有关。我们之前的研究揭示了 新生海马区DGC在AW相关惊厥表达中的作用声发射减弱的脊柱形成 AW可增加新生大鼠海马区DGC的突触连接。我们的狂犬病病毒介导的逆行 示踪研究发现，兴奋性和非兴奋性的新生DGC神经元连接性改变 AW惊厥期间的抑制性神经元。此外，我们对DREADD(Designer Receptor)的功能研究 仅由特制药物激活)方法证明了海马新生神经元的活动 在AW相关癫痫的表达中起重要作用。这些观察提供了理论上的 我们假说的基础是改变了新生海马区DC的神经元连接和活性 破坏兴奋性和抑制性(E/I)信号的平衡，最终导致AW相关的癫痫发作。因此， 这项提议的中心目标是使用新的映射方法、成像工具以及细胞和分子 了解负责海马神经回路的活动和连接性的方法 与AW相关的癫痫发作。在目标1中，我们将确定AW是否改变了神经元和功能的连接 狂犬病病毒和麦胚凝集素(WGA)介导的DGC逆行和顺行示踪 方法分别进行了实验研究。我们还将使用多个DREADD并评估新生神经的基本作用 在AW相关癫痫发作中，新生海马区DGC和输入神经元之间形成回路。目标2，使用 CA2成像和各种磁共振成像(MRI)模式，允许我们进行纵向研究， 我们将确定与AW相关的海马区和全局神经回路的活性和连接性 癫痫发作。在目标3中，我们将识别和验证可能存在于改变的突触和神经元的转录组。 海马区新生DGC对声发射和声发射的反应。单细胞RNA测序技术的应用 将允许我们不仅将差异表达的基因登记到细胞类型特定的方式，而且还可以确定 在AE和AW期间区分病理性新生儿DGC和正常DGC的转录本。总而言之， 我们的提案将剖析新生海马体的分子、细胞和神经回路机制。 DGC是AW相关癫痫发作的基础。

Alcohol withdrawal (AW) after chronic alcohol exposure (AE) produces a series of symptoms. Among them, generalized tonic-clonic seizures are the most severe and dangerous symptom. The severity and susceptibility to relapse, and perpetuation of alcohol abuse underscore the urgent need to understand mechanisms underlying alcohol dependence and AW in order to develop new therapeutic strategies to intervene and treat AW-associated syndromes such as seizures. In this application, we will test the novel hypothesis that activity and connectivity of hippocampal newborn dentate granule cells (DGCs) underlie AW-associated seizures. DGCs are principal excitatory neurons that are continuously produced and integrate into hippocampal neural circuits, and altered hippocampal neurogenesis has been implicated in seizures. Our previous studies have revealed the essential roles of hippocampal newborn DGCs in the expressions of AW-associated seizures. AE reduced spine formation while AW increased synaptic connectivity of hippocampal newborn DGCs. Our rabies virus-mediated retrograde tracing study discovered altered neuronal connectivity of hippocampal newborn DGCs with both excitatory and inhibitory neurons during AW seizures. Moreover, our functional study with a DREADD (Designer Receptors Exclusively Activated by Designer Drugs) method demonstrated that activity of hippocampal newborn neurons plays an essential role in the expression of AW-associated seizures. These observations provided the theoretical foundation for our hypothesis that altered neuronal connectivity and activity of hippocampal newborn DGCs disrupts the balance of excitatory and inhibitory (E/I) signals, ultimately leading to AW-associated seizures. Thus, the central goal of this proposal is to use novel mapping methods, imaging tools, and cellular and molecular approaches in order to understand activity and connectivity of hippocampal neural circuits that are responsible for AW-associated seizures. In Aim 1, we will determine whether AW alters neuronal and functional connectivity of DGCs by using a rabies virus- and wheat germ agglutinin (WGA)-mediated retrograde and anterograde tracing methods, respectively. We will also use multiple DREADDs and assess the essential role of de novo neural circuits formed between hippocampal newborn DGCs and input neurons in AW-associated seizures. Aim 2, using Ca2+ imaging and various magnetic resonance imaging (MRI) modalities that allows us for longitudinal studies, we will determine activity and connectivity of hippocampal and global neural circuits underlying AW-associated seizures. In Aim 3, we will identify and validate transcriptome that may underlie altered synaptic and neuronal connectivity of hippocampal newborn DGCs in response to AE and AW. The usage of single cell RNA sequencing will allow us to not only register differentially expressed genes to cell type-specific manner, but determine transcriptomes that distinguish pathological newborn DGCs from normal DGCs during AE and AW. Altogether, our proposal will dissect the molecular, cellular, and neural circuitry mechanisms by which hippocampal newborn DGCs underlie AW-associated seizures.