Proteomic reconstructive microscopy of healthy and diseased dendrites

描述（由申请人提供）：在过去的几十年里，我们对离子通道的多样性及其在神经元电信号中的作用的理解呈指数增长。这些进步有助于理解和治疗一些脑部疾病，但许多其他常见脑部疾病的潜在机制仍有待发现。事实上，在研究的范围内，许多这些情况都与突触或通道异常有关，包括在青春期出现的，随着年龄的增长，或作为经验的结果（例如，吸毒-当然，创伤后应激障碍）。因此，加深我们对离子通道、突触、树突和脑部疾病之间联系的理解是基础和临床神经科学研究的一个基本目标。这种努力的一个关键瓶颈是，薄树突仍然无法使用膜片钳生理学，而膜片钳生理学是我们对树突和神经元整合的大部分理解的基础。因此，我们对神经元和树突功能的理解主要是基于大直径树突记录和计算模型的推断结合。为了规避这些限制并填补关键的知识空白，该项目将使用或接近超微结构分辨率技术确定海马CA1锥体神经元整个完整树突的几个关键配体和电压门控离子通道的表达模式。为了实现这一目标，提出的实验将分别结合场发射扫描电子显微镜（FESEM）和阵列断层扫描（AT）与免疫金或免疫荧光通道检测。这种方法将填补我们对小鼠和人类单树突蛋白质组的知识的有限空白。而且，如果成功，所提出的方法有可能彻底改变我们对离子通道在树突/神经元功能中的作用的理解，因为在整个大脑的单个神经元中，运输网络和表达水平可能在树突内不同。最后，为了验证这种方法的潜在临床/翻译相关性，在两种不同的阿尔茨海默病小鼠模型中，随着年龄的增长出现的通道病变将在药理学上急剧逆转，然后用FESEM和AT进行探测，以确定表达是否确实恢复正常，或者这种治疗是否诱导了功能性而不是恢复性的通道病变逆转。

DESCRIPTION (provided by applicant): Over the last several decades, our understanding of the diversity of ion channels and their role in electrical signaling in neurons has increased exponentially. Such advances have helped understand and treat some brain disorders, but the mechanisms underlying many other common brain disorders remain to be discovered. In- deed, to the extent examined, many of these conditions are associated with synaptic or channelopathic abnormalities, including ones that emerge in adolescence, with aging, or as a result of experience (e.g., drug expo- sure, post-traumatic stress disorder). Deepening our understanding of the links among ion channels, synapses, dendrites, and brain disease is therefore a fundamental goal of both basic and clinical neuroscience research. One critical bottleneck to such an endeavor is that thin dendrites remain inaccessible to patch-clamp physiology, the technique responsible for much of our understanding with regard to dendritic and neuronal integration. Consequently, our understanding of neuronal and dendritic function is based primarily on a combination of inferences from large-diameter dendritic recordings and computational models. To circumvent such limitations and fill crucial knowledge gaps, this project will determine the expression patterns of several key ligand- and voltage-gated ion channels for entire, complete dendrites from hippocampal CA1 pyramidal neurons using at or near ultrastructural resolution techniques. To accomplish this, the proposed experiments will combine field emission scanning electron microscopy (FESEM) and array tomography (AT) with immunogold or immunoflourescence channel detection, respectively. Such an approach will fill limiting gaps in our knowledge of the single-dendrite proteome in both mice and humans. And, if successful, the proposed approach has the poten- tial to revolutionize our understanding of the role of ion channels in dendritic/neuronal function because trafficking networks and expression levels are likely to differ intradendritically in single neurons throughout the brain. Finally, to validate the potential clinical/translational relevance of such an approach, a channelopathy that emerges with age in two different mouse models of Alzheimer's disease will be acutely reversed pharmacologically, and then probed with FESEM and AT to determine whether expression is indeed rendered normal again, or whether such a treatment induces a functional, but not a restorative, reversal of the channelopathy.