Imaging of Hippocampal Activity Across Sleep/Wake and Disease States

描述（由申请人提供）：脑电图功率在θ频率范围（6- 9hz），特别是在清醒和快速眼动睡眠期间，被认为主要是由于海马锥体细胞的同步放电。迄今为止，支持这一假设的研究依赖于细胞外单位记录，其中监测的细胞数量相对较少，单个细胞的活动只能在短时间内进行跟踪。在这里，我们将通过监测数百个个体可识别细胞的活动，在数周到数月的时间内，提供一个前所未有的跨唤醒状态的海马体神经活动的网络水平视图。我们将把脑电图和肌电图活动的遥测记录与一项令人兴奋的新技术结合起来，当与基因编码的荧光钙指示器（例如，GCaMP5和6）结合使用时，可以在未麻醉的自由活动的动物的局部大脑区域同时成像数百个神经元的活动。Inscopix nVista高清成像系统由一个微型（<2 g）荧光显微镜组成，该显微镜可以在小鼠颅骨上携带，软件可以在高达0.5 mm2的视场上进行高速成像（20-100 Hz），显微内窥镜可以在皮层下大脑结构中进行微米级分辨率的成像。由于在海马体及其层流组织中进行的丰富的行为研究历史，该区域是理想的神经结构，可以利用该技术获得跨唤醒状态的神经活动的新见解。首先，我们将确定CA1内部局部网络在唤醒状态下的活动，以及在稳态睡眠压力可能不同的情况下的活动，例如在“亮灯”时期的早期与黑暗时期的早期，以及对睡眠剥夺和随后的恢复性睡眠的反应。同时测量数百个细胞的活动与脑电图将使我们不仅能够跟踪单个细胞在睡眠和清醒状态下的活动，而且还可以评估局部网络内关于脑电图频率和特定脑电图事件的同步性。在确定了野生型小鼠海马网络的参数后，我们将在亨廷顿病（HD）小鼠模型中确定CA1局部网络的活性。我们最近对R6/2 HD小鼠模型的睡眠和清醒以及脑电图的变化进行了表征，并发现随着疾病的进展，θ波范围的功率显著增加，θ波峰值频率（TPF）减慢。利用theta功率和TPF作为生物标志物，我们将利用Inscopix技术的力量，对已识别的神经元进行长期记录，以确定R6/2小鼠的海马体网络如何随着疾病进展而重组。在记录自由行为小鼠脑电图的同时观察数百个细胞的钙动态，可能会对海马在HD行为状态中的作用和海马发生的网络变化有新的认识。

DESCRIPTION (provided by applicant): EEG power in the theta frequency range (6-9 Hz), particularly during waking and REM sleep, is believed to primarily be due to synchronous firing of hippocampal pyramidal cells. To date, studies supporting this hypothesis have relied on extracellular unit recordings where a relatively small number of cells are monitored and the activity of individual cells can only be followed for short periods of time. Here, we will provide n unprecedented network level view of neural activity in the hippocampus across arousal states by monitoring the activity of hundreds of individually identifiable cells over weeks to months. We will combine telemetric recording of EEG and EMG activity with an exciting new technology that, when used in conjunction with genetically-encoded fluorescent calcium indicators (e.g., GCaMP5 and 6), enables imaging the activity of hundreds of neurons simultaneously from a local brain region in unanesthetized, freely-moving animals. The Inscopix nVista HD imaging system is comprised of a miniature (<2 g) fluorescence microscope that can be borne on the skull of a mouse, software that enables high-speed imaging (20-100 Hz) over a field of view up to ~0.5 mm2, and microendoscopes that allow imaging with micron-scale resolution in subcortical brain structures. Because of the rich history of behavioral studies conducted in the hippocampus and its laminar organization, this region is the ideal neural structure in which to utilize this technology to obtain novel insights into neural activity across arousal states. First,we will determine the activity of localized networks within CA1 across arousal states and during conditions when homeostatic sleep pressure can be expected to differ, such as early in the "lights on" period vs. early in the dark period and in response to sleep deprivation and subsequent recovery sleep. Concurrent measurement of the activity of hundreds of cells in conjunction with the EEG will enable us to not only follow the activity of individual cells across sleep and wakefulness, but also to evaluate synchrony within the local network with respect to EEG frequencies and specific EEG events. Having defined the parameters of the hippocampal network in wildtype mice, we will then determine the activity of local networks within CA1 in a mouse model of Huntington's disease (HD). We have recently characterized changes in sleep and wakefulness and in the EEG of the R6/2 mouse model of HD and found both tremendously increased power in the theta range and slowing of the theta peak frequency (TPF) as disease progresses. Using theta power and TPF as biomarkers, we will exploit the power of the Inscopix technology to enable long-term recordings of identified neurons to determine how the hippocampal network is reorganized as disease progresses in R6/2 mice. The ability to observe calcium dynamics of hundreds of cells while simultaneously recording EEG in freely-behaving mice may lead to new insights into the role of the hippocampus in behavioral states and the network changes that occur in the hippocampus in HD.