Basal Forebrain Cellular Mechanisms of Cortical Activation

描述(由申请人提供)： 清醒状态和睡眠状态如何增强信息处理能力？这个问题的答案涉及大脑通过神经元振荡同步神经元组装活动的能力，从而使显著的信息片段以连贯的感知结合在一起，并加强相关神经元之间的突触连接，这最初是由Donald Hebb提出的。这种神经元振荡的频率、区域分布和幅度在睡眠-觉醒周期中各不相同。特别是，伽马振荡(30-80赫兹或更宽，以40赫兹为中心)是清醒和快速眼动睡眠期间脑电(EEG)的一个显著特征。这些振荡被认为对大脑功能，如注意力、知觉和记忆的形成是必不可少的。我们小组和其他人的研究表明，精神分裂症患者的前额叶和初级感觉(听觉和视觉)皮质存在伽马异常。此外，伽马缺乏是睡眠障碍、昏迷和阿尔茨海默病的一个显著特征，这些疾病在退伍军人和军人中很常见。因此，伽马振荡的调节代表了治疗这些疾病症状的一个有希望的治疗靶点。这一建议侧重于基底前脑(BF)神经元向大脑皮层的投射对皮质激活和伽马振荡的调制，因为在最近的一项研究中，BF区域的广泛损伤显示皮质激活/伽马活动显著减少，导致昏迷状态。虽然这样的损伤表明了BF的重要性，但它们并不能告诉我们是哪种特定的BF细胞 类型对皮质激活很重要，BF如何受到其他大脑区域和神经递质的影响，以及哪些回路和神经递质将是最佳治疗靶点。在前一个资助周期开发的方法的基础上，遵循我们实验室使用多水平方法的实力和记录，拟议的实验使用集成的分子、体外和体内(系统)方法，以提供对所研究的神经回路的最佳理解。在第一系列实验中，新型的光遗传光激活离子通道将被插入特定的BF亚群(含有小白蛋白的胆碱能和GABA能神经元)，以研究激活或抑制这些特定神经细胞类型对皮质激活/伽马活性的影响。多导睡眠记录仪将确定这些手法对睡眠和清醒的影响。大脑皮层局部场电位(LFP)记录将用于精确确定精神分裂症患者中受睡眠剥夺和表现异常影响的三个不同皮质区域的局部伽马振荡。此外，Fos免疫组织化学将用于提供光刺激这些eBF亚群后皮质激活的空间和细胞类型特异性分析。在我们成功地在脑干中使用小干扰RNA(SiRNA)之后，同样的技术将被用于敲除BF中的增食欲素受体，并揭示它们在睡眠-觉醒控制中的作用，提供可逆性，而不会像结构性敲除一样，潜在地混淆发育补偿。选择性毒素MUP75-Saporin将被用来研究胆碱能BF神经元在觉醒调节中的作用以及食欲素的作用。在最后一系列实验中，我们将使用GAD67-GFP敲入小鼠，这是一种在上一个赠款周期中得到验证的新型遗传工具，以识别皮质投射的BF GABA 体外培养的神经元。利用膜片钳记录，我们将揭示胆碱能和食欲素能化合物对它们的调制，并确定激活的受体和离子通道。综上所述，我们建议使用最先进的方法来识别调节觉醒和皮质伽马活动的BF投射到皮质的细胞和分子成分。因此，我们为针对性治疗奠定了基础，以提高在影响退伍军人群体的条件下的警觉性、注意力和执行功能，如精神分裂症、阿尔茨海默病和睡眠障碍。 公共卫生相关性： 最近的证据表明，在退伍军人中流行的几种疾病，包括睡眠障碍、精神分裂症和昏迷，皮质激活和伽马节律都是异常的。提出的研究将为针对性治疗奠定基础，以提高患有这些疾病的患者的警觉性、注意力和执行功能。这项研究的重点是特定的皮质投射的基底前脑神经元如何调节觉醒、皮质激活和伽马频率范围(30-120赫兹)的脑电振荡。拟议的实验将采用多水平、最先进的新方法，包括光遗传学、siRNA和体外药理学程序，以确定神经递质受体和效应系统，这些受体和效应系统可以作为未来药物治疗或遗传治疗的靶点，以调节皮质激活和伽马活性。

DESCRIPTION (provided by applicant): How do the states of wakefulness and sleep enhance the processing of information? The answer to this question involves the ability of the brain to synchronize the activities of assemblies of neurons by means of neuronal oscillations so that salient pieces of information are bound together in coherent percepts and synaptic connections between related neurons are strengthened, as originally proposed by Donald Hebb. The frequency, regional distribution and amplitude of such neuronal oscillations vary across the sleep-wake cycle. In particular, gamma oscillations (30-80 Hz or broader, centered on 40 Hz) are a prominent feature of the electroencephalogram (EEG) during waking and REM sleep. These oscillations are thought to be essential for brain functions such as attention, perception, and memory formation. Work from our group and from others has demonstrated gamma abnormalities in prefrontal and primary sensory (auditory and visual) cortices in schizophrenic patients. Furthermore, gamma deficits are a prominent feature of sleep disorders, coma, and Alzheimer's disease, other conditions prevalent in veterans and military personnel. Modulation of gamma oscillations thus represents a promising therapeutic target to treat symptoms of these disorders. This proposal focuses on the modulation of cortical activation and gamma oscillations by the basal forebrain (BF) neuronal projections to the cerebral cortex, since, in a recent study, extensive lesions of the BF region revealed dramatic reductions in cortical activation/gamma activity leading to a coma-like state. While such lesions point to the importance of BF, they do not tell us which specific BF cell types are important for cortical activation, how BF is influenced by other brain regions and neurotransmitters, and which circuits and neurotransmitters would be optimal treatment targets. Building on the methodologies developed in the previous grant cycle and following our laboratory's strength and track record of using a multilevel approach, the proposed experiments use integrated molecular, in vitro, and in vivo (systems) methods in mice to provide optimal understanding of the neural circuits studied. In the first series of experiments novel 'optogenetic light- activated ion channels will be inserted into specific BF subpopulations (cholinergic and GABAergic neurons containing parvalbumin) to study the effect of activating or inhibiting these specific neuronal cell types on cortical activation/gamma activity. Polysomnographic recordings will determine the effect of these manipulations on sleep and wakefulness. Cortical local field potential (LFP) recordings will be used to gain a precise determination of local gamma oscillations in three different cortical regions affected by sleep deprivation and exhibiting abnormalities in schizophrenia. In addition, Fos immunohistochemistry will be used to provide a spatial and cell-type specific analysis of cortical activation following light stimulation of theseBF subpopulations. Following our successful use of small interfering RNA (siRNA) in the brainstem, the same technique will be used to knockdown orexin receptors in the BF and reveal their role in sleep-wake control, providing reversibility without the potential confound of developmental compensation often seen with constitutive knockouts. The selective toxin mu p75-saporin will be used to investigate the role of cholinergic BF neurons in the regulation of wakefulness, and the effect of orexins. In the last series of experiments we will use GAD67-GFP knock-in mice, a novel genetic tool validated in the previous grant cycle, to identify cortically projecting BF GABA neurons in vitro. Using patch-clamp recordings we will reveal their modulation by cholinergic and orexinergic compounds and determine the receptors and ion channels activated. In summary, we propose to use state-of-the-art methods to identify the cellular and molecular components of the BF projections to the cortex modulating wakefulness and cortical gamma activity. We thereby lay the groundwork for targeted therapies to improve alertness, attention, and executive function in conditions that affect the Veteran population such as schizophrenia, Alzheimer's disease and sleep disorders. PUBLIC HEALTH RELEVANCE: Recent evidence suggests cortical activation and gamma rhythms are abnormal in several diseases prevalent in veterans and military personnel, including sleep disorders, schizophrenia, and coma. The research proposed will lay the ground work for targeted therapies to improve alertness, attention, and executive function in patients suffering from these conditions. The research focuses on how specific cortically projecting basal forebrain neurons modulate wakefulness, cortical activation and electroencephalographic oscillations in the gamma frequency range (30-120 Hz). The experiments proposed will employ multilevel, state of the art, novel approaches including optogenetic, siRNA, and in vitro pharmacological procedures to identify the neurotransmitter receptors and effector systems that can be targeted for future pharmacological, or genetic treatments, to modulate cortical activation and gamma activity.