Basal forebrain hypothalamic networks supporting wakefulness

 描述（由申请人提供）：基底前脑（BF）在维持清醒和激活脑电图（EEG）方面发挥关键作用。然而，BF调节行为和EEG唤醒的机制和底物仍然知之甚少。对文献的回顾表明，尽管存在以下事实，但绝大多数关于BF回路的研究都集中在生理和病理生理背景下的胆碱能BF系统上：1）胆碱能BF系统的损伤在EEG或行为唤醒中产生有限的改变;和2）BF还含有至少另外两种神经元群-GABA能和谷氨酸能神经元，与胆碱能神经元混合在一起。虽然已经认识到其他BF神经递质系统可以在控制睡眠和觉醒中与胆碱能神经元相互作用或并行工作，但GABA能BF神经元，特别是它们的网络输出对这些过程的贡献仍然很大程度上未被探索。我们最近发现，选择性激活BF GABA能神经元，而不是胆碱能或谷氨酸能神经元，意外地产生持续的觉醒。在这个建议中，我们试图确定在体外（细胞）和体内（系统）的机制和基板BF GABA能神经元支持觉醒。我们推测，BF GABA能群体在行为觉醒中的影响是通过激活外侧下丘脑，特别是通过食欲素/下丘脑泌素神经元的去抑制来介导的。我们的模型，这直接来自我们的初步数据，预测BF GABA能神经元调节觉醒通过两个输出：一个分支的电路抑制睡眠调节神经元在视前下丘脑，而第二个分支通过本地GABA能神经元抑制食欲素神经元。我们进一步提出，这些网络对稳态睡眠压力敏感，腺苷可能以位点和受体依赖的方式作用于这些不同的节点。因此，目前的建议，旨在确定，首先在体外，然后在体内，突触后的BF GABA能输入的目标是必要的，促进觉醒。为了测试我们的假设，我们将首先在特定目标1和2中采用体外回路映射范例，其组合了基于光遗传学和AAV的通道视紫红质2（ChR 2）辅助回路映射（CRACM）和逆行微球，以确定这些推定的皮层下回路如何支持觉醒。然后我们将确定腺苷如何调节这些回路。最后，在具体目标3中，我们是否将cre激活的病毒载体系统离散注射到BF中，并将cre激活的毒素注射到这些BF神经元的三个传出（突触后）靶点之一中？这样做将使我们能够激活行为小鼠中的BF GABA能神经元，其中BF输入的特定突触神经元靶点已经被破坏。反过来，这将揭示哪些BF GABA能传出输出对于支持行为动物的觉醒是必要的。我们希望这项合作工作的结果能够为支持清醒的大脑回路提供重要而新颖的见解。

 DESCRIPTION (provided by applicant): The basal forebrain (BF) is critically involved in maintaining wakefulness and an activated electroencephalogram (EEG). The mechanisms and substrates by which the BF regulates behavioral and EEG wake remains however poorly understood. A review of the literature reveals that the vast majority of studies on BF circuitry have focused on the cholinergic BF system in physiological and pathophysiological contexts, despite the facts that 1) lesions of the cholinergic BF system produce limited changes in EEG or behavioral wake; and 2) the BF also contains at least other two neuronal populations - GABAergic and glutamatergic neurons - that roughly intermingle with the cholinergic neurons. While it has been appreciated for some time that other BF neurotransmitter systems may interact or work in parallel with cholinergic neurons in the control of sleep and wake, the contribution of GABAergic BF neurons, and in particular their network output, to these processes remains largely unexplored. We recently found that selective activation of BF GABAergic, but not cholinergic or glutamatergic, neurons unexpectedly produces sustained wakefulness. In this proposal we seek to determine the in vitro (cellular) and in vivo (system) mechanisms and substrates by which BF GABAergic neurons support wakefulness. We hypothesize that the influence of BF GABAergic population in behavioral arousal is mediated by activation of the lateral hypothalamus and specifically through disinhibition of orexin/hypocretin neurons. Our model, which derives directly from our preliminary data, predicts that BF GABAergic neurons regulate wakefulness through two outputs: one branch of the circuit inhibits sleep regulatory neurons in the preoptic hypothalamus whereas the second branch disinhibits orexin neurons through local GABAergic neurons. We further propose that these networks are sensitive to homeostatic sleep pressure and that adenosine might act on these different nodes, in a site- and receptor- dependent manner. The current proposal thus seeks to identify, first in vitro and then in vivo, the postsynaptic targets of BF GABAergic input that are necessary for promoting wakefulness. To test our hypotheses we will first employ in Specific Aims 1 and 2 an in vitro circuit mapping paradigm that combines optogenetic- and AAV- based channelrhodopsin2 (ChR2)-assisted circuit mapping (CRACM) and retrograde microspheres to determine how these putative subcortical circuits support wakefulness. We will then determine how adenosine modulates these circuits. Finally in Specific Aim 3 will we place discrete injections of a cre-enabled viral vector system into the BF and a cre-enabled toxin into one of three efferent (post-synaptic) targets of these BF neurons? Doing so will allow us to activate BF GABAergic neurons in behaving mice in which specific synaptic neuronal targets of this BF input have been disrupted. In turn this will reveal which BF GABAergic efferent outputs are necessary for supporting wakefulness in behaving animals. We expect the results from this collaborative work to provide important and novel insights into the brain circuitry supporting wakefulness.