Srivastava Deepak P, Woolfrey Kevin M, Penzes Peter
Department of Physiology, Northwestern University Feinberg School of Medicine, USA.
J Vis Exp. 2011 Jul 13(53):e2794. doi: 10.3791/2794.
Dendritic spines are the sites of the majority of excitatory connections within the brain, and form the post-synaptic compartment of synapses. These structures are rich in actin and have been shown to be highly dynamic. In response to classical Hebbian plasticity as well as neuromodulatory signals, dendritic spines can change shape and number, which is thought to be critical for the refinement of neural circuits and the processing and storage of information within the brain. Within dendritic spines, a complex network of proteins link extracellular signals with the actin cyctoskeleton allowing for control of dendritic spine morphology and number. Neuropathological studies have demonstrated that a number of disease states, ranging from schizophrenia to autism spectrum disorders, display abnormal dendritic spine morphology or numbers. Moreover, recent genetic studies have identified mutations in numerous genes that encode synaptic proteins, leading to suggestions that these proteins may contribute to aberrant spine plasticity that, in part, underlie the pathophysiology of these disorders. In order to study the potential role of these proteins in controlling dendritic spine morphologies/number, the use of cultured cortical neurons offers several advantages. Firstly, this system allows for high-resolution imaging of dendritic spines in fixed cells as well as time-lapse imaging of live cells. Secondly, this in vitro system allows for easy manipulation of protein function by expression of mutant proteins, knockdown by shRNA constructs, or pharmacological treatments. These techniques allow researchers to begin to dissect the role of disease-associated proteins and to predict how mutations of these proteins may function in vivo.
树突棘是大脑中大多数兴奋性连接的部位，构成突触的突触后部分。这些结构富含肌动蛋白，并且已被证明具有高度的动态性。响应经典的赫布可塑性以及神经调节信号，树突棘可以改变形状和数量，这被认为对于神经回路的细化以及大脑内信息的处理和存储至关重要。在树突棘内部，一个复杂的蛋白质网络将细胞外信号与肌动蛋白细胞骨架连接起来，从而控制树突棘的形态和数量。神经病理学研究表明，从精神分裂症到自闭症谱系障碍等多种疾病状态都表现出异常的树突棘形态或数量。此外，最近的遗传学研究已经在许多编码突触蛋白的基因中发现了突变，这表明这些蛋白质可能导致异常的棘突可塑性，而这在一定程度上是这些疾病病理生理学的基础。为了研究这些蛋白质在控制树突棘形态/数量方面的潜在作用，使用培养的皮质神经元具有几个优点。首先，该系统允许对固定细胞中的树突棘进行高分辨率成像以及对活细胞进行延时成像。其次，这个体外系统允许通过突变蛋白的表达、shRNA构建体的敲低或药物处理来轻松操纵蛋白质功能。这些技术使研究人员能够开始剖析疾病相关蛋白质的作用，并预测这些蛋白质的突变在体内可能如何发挥作用。
