Molecular-Genetic Dissection of Basal Forebrain Circuitry Regulating Arousal

描述(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，几乎所有这些过程都是在清醒的基础上进行的。BF回路功能障碍也与一系列神经精神疾病和神经退行性疾病的发病有关，如阿尔茨海默病、帕金森氏病、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“上升网状激活系统”的解剖组成部分)包含维持行为觉醒的关键回路和被唤醒的皮质，这是认知和有目的行为的必要条件。然而，值得注意的是，BF调节脑电和神经行为唤醒的机制和底物仍然知之甚少。了解BF神经生物学的困难很大程度上与其高度的细胞异质性和复杂的解剖结构有关。在这个项目中，我们计划研究BF的胆碱能、GABA能和谷氨酸能神经元在调节大脑皮层和行为觉醒中的作用。这些细胞群中的每一个都被假设在调节大脑皮层和神经行为觉醒方面发挥重要作用，尽管每个递质系统在这些过程中各自的作用尚未解决。我们首次使用含有重组酶的腺相关病毒(AAV)载体注射到携带loxP修饰等位基因的小鼠的BF中，在体内首次检测了这三种BF递质系统的细胞类型特异性损伤对脑电和行为唤醒的影响。这些小鼠的等位基因包括胆碱乙酰转移酶(ChATflx/Flox小鼠)、囊泡性GABA转运体(Vgat Flox/Flox鼠)或囊泡性谷氨酸转运体2(V2 Glu/Flox鼠)。总而言之，这些研究将提供有关产生和维持唤醒所必需的底物的重要信息，包括在行为自由、不受约束的动物中，所有三种BF神经递质系统(S)对这一过程的单独贡献。虽然谷氨酸、GABA或胆碱能神经传递的局部消除可能会显著提高我们对这些BF递质系统在脑电和行为觉醒中的长期作用的了解，但随着时间的推移，剩余的神经递质系统可能会有实质性的补偿。为了解决这个问题，并提供第二个实验模型系统，以提高BF中选择性递质干扰与EEG/行为结果之间联系的特异性，我们实验室最近开发了一种包含伊维菌素门控氯离子通道的AAV，它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Vlos2-IRES-Cre小鼠的BF中，我们可以检测分别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脑电和其他神经行为指标的影响。 公共卫生相关性：该计划建议确定三个神经递质系统在体内各自的作用，包括基底前脑，这是大脑的一个高度复杂的区域，涉及广泛的更高水平的神经生物学过程。除了揭示三个基础前脑递质系统在正常功能中的神经生物学作用外，拟议中的研究结果还可能为一系列神经精神和神经退行性疾病的发病机制提供关键的见解，包括阿尔茨海默病、帕金森氏病、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.