Automated cell-type-specific electrophysiology for understanding circuit dysregulation in Alzheimer's Disease

项目摘要 预计到2050年，全球将有超过1.5亿人患有痴呆症。阿尔茨海默病（AD） 是痴呆症最常见的形式，占痴呆症病例的70%。一个标志性的病理特征 阿尔茨海默病（AD）的主要症状是进行性神经退行性变， AD患者的认知能力下降。虽然越来越敏感的生化标志物可用于诊断AD 在个体中，在疾病的早期阶段阻止疾病的治疗仍然难以捉摸。AD患者中的证据增加 模型表明，这些生物标志物的显著积累可能在早期回路功能障碍之前。 大量AD患者表现出亚临床癫痫。此外，回路超兴奋性已被 在几种家族性AD小鼠模型中，在斑块形成前也观察到了类似的结果， 散发性AD模型。这些研究的细胞证据表明，电路失调是由于改变了 GABA能中间神经元的回路抑制。特别是，表达小清蛋白（PV）的中间神经元出现 在不同的年龄段，他们的动作电位（AP）放电和潜在的神经传递容易发生变化， 家族性和散发性AD小鼠模型。AD中的神经变性通常被认为是通过良好的- 有意思的是，过度兴奋的回路可能会加速这种病理。是否 PV中间神经元的生理变化首先出现在AD的高脆弱性区域中尚不清楚。我们的中央 假设PV中间神经元将首先在脆弱的脑区域中发展功能障碍性生理缺陷 在早期AD期间，它可能会以Braak式的方式进展到其他大脑区域。评价这一 假设，这将需要从数千个单独的神经元电生理记录，我们将使用 PatcherBot是我们的机器人平台，能够执行高通量，自动化的电生理学， 然而，在这项工作中，我们将增强PatcherBot的机器视觉功能， 荧光成像以特异性靶向脑切片中表达PV的中间神经元。了理论基础和 该建议的可行性显示在初步工作中，演示（1）全自动膜片钳， 使用PatcherBot的荧光靶向中间神经元，（2）全脑范围内引入PV特异性标记， 光遗传学方法在AD小鼠体内，和（3）早期阶段PV放电和神经传递缺陷， 前驱FAD小鼠模型。在这里，我们将在三个主要区域（内嗅）解决我们的假设 皮质、海马、同皮质）和3种相关发育模型（APOE 4、hAPP-KI、5xFAD）中的神经元发育情况。 时间点。这项提案的发现将产生广泛的进展，包括高通量细胞类型， 特定的生理学，有关早期AD认知功能障碍的潜在电路播种的信息。

Project Summary Over 150 million people are projected to be living with dementia worldwide by 2050. Alzheimer’s disease (AD) is the most common form of dementia, responsible for ~70% of dementia cases. A hallmark pathological feature of Alzheimer’s disease (AD) is progressive neurodegeneration, which is thought to initiate and track progressive cognitive decline in AD patients. While increasingly sensitive biochemical markers are available to diagnose AD in individuals, treatments to stall the disease in its early stages remain elusive. Increasing evidence in AD patients models indicates that significant accumulation of these biomarkers may be preceded by early circuit dysfunction. A large plurality of AD patients display subclinical epilepsy. Furthermore, circuit hyperexcitability has been observed before plaque formation in several familial AD mouse models as well, with similar findings in mouse models of sporadic AD. Cellular evidence from these studies suggests that circuit dysregulation is due to altered circuit inhibition from GABAergic interneurons. In particular, parvalbumin-expressing (PV) interneurons appear to be prone to changes in their action potential (AP) firing, and potentially neurotransmission, across distinct familial and sporadic AD mouse models. Neurodegeneration in AD is often thought to progress through well- defined brain regions, and interestingly, hyperexcitable circuits may accelerate this pathology. Whether physiological changes to PV interneurons emerge first in regions of high vulnerability in AD is unclear. Our central hypothesis is that PV interneurons will develop dysfunctional physiological deficits first in vulnerable brain regions during early AD, which may then progress to other brain areas in a Braak-esque fashion. To evaluate this hypothesis, which will require electrophysiological recordings from thousands of individual neurons, we will use the PatcherBot, our robotic platform capable of performing high-throughput, automated electrophysiology of neurons in brain slices; however, in this work, we will augment the PatcherBot’s machine vision capabilities with fluorescence imaging to specifically target PV-expressing interneurons in brain slices. The rationale and feasibility of this proposal are shown in preliminary work, demonstrating (1) fully automated patch clamping of florescent-targeted interneurons using the PatcherBot, (2) brain-wide introduction of PV specific labeling and optogenetic methods in AD mice in vivo, and (3) early-stage PV firing and neurotransmission deficits in a prodromal FAD mouse model. Here, we will address our hypothesis across three major regions (entorhinal cortex, hippocampus, isocortex) in 3 distinct models (APOE4, hAPP-KI, 5xFAD) and 3 relevant developmental timepoints. Findings from this proposal will yield wide-ranging advances, including high-throughput cell-type- specific physiology, to information regarding the potential circuit-seeding of cognitive dysfunction in early AD.