Molecular-Genetic Dissection of Basal Forebrain Circuitry Regulating Arousal

描述（由申请人提供）：基底前脑（BF）是一个高度复杂的大脑区域，涉及广泛的高级神经生物学过程，包括认知、学习、记忆和注意力，几乎所有这些都是在清醒的基础上运行的。BF电路的功能障碍还涉及许多神经精神和神经变性病症的发病机制，例如阿尔茨海默病、帕金森病、精神分裂症和正常衰老的认知障碍。然而，在其最基本的神经生物学背景下，BF（作为“上行网状激活系统”的解剖学组成部分）包含维持行为唤醒和唤醒皮层的关键电路，这是认知和有目的行为的必要条件。然而，值得注意的是，BF调节EEG和神经行为唤醒的机制和底物仍然知之甚少。在理解BF的神经生物学方面的许多困难与其高度的细胞异质性和复杂的解剖组织有关。在这个项目中，我们计划研究在体内的作用，胆碱能，γ-氨基丁酸能和多巴胺能神经元的BF在调节皮层电和行为唤醒。这些细胞群中的每一个都被假设在调节皮层电刺激和神经行为唤醒中发挥重要作用，尽管每个递质系统在这些过程中的各自作用尚未得到解决。我们建议，第一次，使用腺相关病毒（AAV）载体，将含有cre重组酶的腺相关病毒（AAV）载体注射到携带loxP修饰的胆碱乙酰转移酶等位基因的小鼠BF中，研究这三种BF递质系统中每一种的细胞类型特异性病变对EEG和行为觉醒的体内影响。（ChATflox/flox小鼠）、囊泡GABA转运蛋白（Vgat flox/flox小鼠）或囊泡谷氨酸转运蛋白2（Vgat 2 flox/flox小鼠）。总的来说，这些研究将提供关于产生和维持唤醒所必需的底物的重要信息，包括所有三种BF神经递质系统在自由行为、不受限制的动物中对该过程的个体贡献。虽然谷氨酸、GABA或胆碱能神经传递的局灶性消除将潜在地为我们关于这些BF递质系统在EEG和行为唤醒中的长期作用的知识提供显著的进步，但随着时间的推移，可能存在剩余神经递质系统的大量补偿。为了解决这个问题，并提供第二个实验模型系统，用于增加BF和EEG/行为结果中选择性递质中断之间联系的特异性，我们的实验室最近开发了一种含有伊维菌素门控氯离子通道的AAV，该通道允许体内特定神经元亚群的选择性和可逆性沉默。通过将该AAV注射到ChAT-IRES-Cre、Vgat-IRES-Cre和VAT 2-IRES- Cre小鼠的BF中，我们可以分别检查急性和可逆地沉默这些神经元亚型对自由行为动物的皮质EEG和其他神经行为测量的影响。 公共卫生相关性：该计划旨在确定三种神经递质系统的各自体内作用，包括基底前脑，一个高度复杂的大脑区域，涉及广泛的高级神经生物学过程。除了揭示三个基底前脑递质系统在正常功能中的神经生物学作用外，拟议研究的结果可能会对许多神经精神和神经退行性疾病的发病机制提供重要见解，包括阿尔茨海默病，帕金森病，精神分裂症和正常衰老的认知障碍。

DESCRIPTION (provided by applicant): The basal forebrain (BF) is a highly complex brain region that is implicated in a wide range of higher-level neurobiological processes including, cognition, learning, memory and attention, virtually all of which operate on a basis of wakefulness. Dysfunction of BF circuitry is also implicated in the pathogenesis of a host of neuropsychiatric and neurodegenerative conditions such as Alzheimer's disease, Parkinson's disease, schizophrenia and the cognitive impairments of normal aging. In its most fundamental neurobiological context however, the BF (as an anatomical constituent of the "ascending reticular activating system") contains circuitry critical for maintaining behavioral arousal and an aroused cortex, which is the sine qua non for cognition and purposeful behaviors. Remarkably, however, the mechanisms and substrates by which the BF regulates EEG and neurobehavioral arousal remain poorly understood. Much of the difficulty in understanding the neurobiology of the BF is related to its high cellular heterogeneity and complex anatomical organization. In this project we plan to examine the in vivo role of cholinergic, GABAergic and glutamatergic neurons of the BF in the regulation of electrocortical and behavioral arousal. Each of these cell groups has been hypothesized to play an important role in regulating electrocortical and neurobehavioral arousal, although the respective role of each transmitter system in these processes is unresolved. We propose to examine, for the first time, the in vivo effects of cell-type specific lesions of each of these three BF transmitter systems on EEG and behavioral arousal using an adeno-associated viral (AAV) vector containing cre-recombinase injected into the BF of mice harboring loxP-modified alleles of either choline acetyltransferase (ChATflox/flox mice), the vesicular GABA transporter (Vgat flox/flox mice) or the vesicular glutamate transporter 2 (Vglut2 flox/flox mice). Collectively, these studies will provide important information regarding the substrates that are necessary to produce and maintain arousal, including the individual contribution of all three BF neurotransmitter system(s) to this process in a freely behaving, unrestrained animal. While the focal elimination of glutamate, GABA or cholinergic neurotransmission will potentially provide a significant advance in our knowledge regarding the long-term role of these BF transmitter systems in EEG and behavioral arousal, it is possible that there may be substantial compensation by the remaining neurotransmitter systems over time. To address this issue and, also, provide a second experimental model system for increasing the specificity of the linkage between selective transmitter disruption in the BF and EEG/behavioral outcomes, our laboratory has recently developed an AAV containing an ivermectin-gated chloride channel that permits selective and reversible silencing of specific neuronal subpopulations in vivo. By injecting this AAV into the BF of ChAT-IRES-Cre, Vgat-IRES-Cre and Vglut2-IRES- Cre mice we can examine the effects of acutely and reversibly silencing these neuronal subtypes, respectively, on the cortical EEG and other neurobehavioral measures in the freely behaving animal. PUBLIC HEALTH RELEVANCE: This program proposes to determine the respective in vivo role of three neurotransmitter systems comprising the basal forebrain, a highly complex area of the brain that is implicated in a wide-range of higher-level neurobiological processes. In addition to revealing the neurobiological role of the three basal forebrain transmitter systems in normal function, the results from the proposed studies may provide critical insight into the pathogenesis of a host of neuropsychiatric and neurodegenerative disorders, including Alzheimer's disease, Parkinson's disease, schizophrenia and the cognitive impairments of normal aging.