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) 是最常见的痴呆症形式，约占痴呆病例的70%。一个标志性的病理特征 阿尔茨海默病(AD)是一种进行性神经变性，被认为是启动和跟踪进行性的 阿尔茨海默病患者认知功能下降。虽然越来越敏感的生化标记物可用于诊断AD 对于个人来说，在疾病的早期阶段拖延疾病的治疗仍然难以捉摸。AD患者中越来越多的证据 模型表明，这些生物标志物的显著积累可能先于早期回路功能障碍。 大量AD患者表现为亚临床癫痫。此外，电路的超兴奋性已经被 在几个家族性AD小鼠模型中也观察到斑块形成之前的情况，在小鼠中也有类似的发现 散发性AD的模型。来自这些研究的细胞证据表明，电路失调是由于改变 GABA能中间神经元的环路抑制。特别是，出现了表达小白蛋白的中间神经元。 容易改变他们的动作电位(AP)放电，以及潜在的神经传递，跨不同 家族性和散发性AD小鼠模型。阿尔茨海默病的神经变性通常被认为是通过良好的- 确定的大脑区域，有趣的是，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的高易损区，目前尚不清楚。我们的中央 假设PV中间神经元将首先在脆弱的脑区出现功能失调的生理缺陷。 在AD早期，它可能会以布拉克式的方式进展到其他大脑区域。要评估这一点 假设，这将需要数千个单个神经元的电生理记录，我们将使用 Patcher Bot，我们的机器人平台，能够执行高通量、自动化的电生理 脑片中的神经元；然而，在这项工作中，我们将通过以下方式增强Patcher Bot的机器视觉能力 荧光成像以特异性靶向脑片中表达PV的中间神经元。其基本原理和 初步工作证明了该方案的可行性，论证了(1)全自动膜片钳技术。 使用Patcher Bot的荧光靶向中间神经元，(2)全脑引入PV特异性标记和 在活体AD小鼠中的光遗传学方法，以及(3)早期PV放电和神经传递缺陷 前驱FAD小鼠模型。在这里，我们将讨论我们在三个主要区域(内嗅区)的假设 3个不同的模型(APOE4、HAPP-KI、5xFAD)和3个相关发育 时间点。这项提案的发现将产生广泛的进步，包括高通量细胞类型- 具体的生理学，到有关早期AD认知功能障碍的潜在回路播种的信息。

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.