Confocal Imaging and 3D Reconstruction to Determine How Genetic Perturbations Impact Presynaptic Morphology at the Mouse Calyx of Held.
Confocal Imaging and 3D Reconstruction to Determine How Genetic Perturbations Impact Presynaptic Morphology at the Mouse Calyx of Held.
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DOI：
10.21769/bioprotoc.4799
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发表时间：
2023-09-05
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影响因子：
0.8
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神经元通过突触进行通信，突触是由一个神经元的突触前末端和另一个神经元的突触后末端组成的专门结构。随着人们逐渐认识到调节突触功能的分子突变是许多神经系统疾病的基础，阐明调节突触功能的分子机制以理解突触强度、可塑性、调制和病理学是至关重要的，这些最终影响神经元回路输出和行为。Held突触前萼是听脑干中一个巨大的神经元突触前末梢，由于其可接近性和选择性分子微扰的可能性，是研究突触前蛋白在调节突触功能中作用的理想模型。在本协议中，我们描述了使用共聚焦成像和三维重建的萼举行，以评估分子扰动后的总体形态的变化。使用病毒载体传递在不同的发育时间点进行分子扰动，我们提供了一种快速和具有成本效益的方法来研究突触前蛋白如何在神经元回路的整个生命周期中调节总形态，如表面积和突触体积。 突触前末梢的共聚焦成像和3D重建。与病毒介导的mEGFP表达一起使用，以实现突触前区室的有效、细胞类型特异性标记。该方案是用Held的萼开发的，但适用于多个哺乳动物和无脊椎动物物种的各种神经元的突触前和突触后隔室。
Neurons communicate via synapses—specialized structures that consist of a presynaptic terminal of one neuron and a postsynaptic terminal of another. As knowledge is emerging that mutations in molecules that regulate synaptic function underpin many neurological disorders, it is crucial to elucidate the molecular mechanisms regulating synaptic function to understand synaptic strength, plasticity, modulation, and pathology, which ultimately impact neuronal circuit output and behavior. The presynaptic calyx of Held is a large glutamatergic presynaptic terminal in the auditory brainstem, which due to its accessibility and the possibility to selectively perform molecular perturbations on it, is an ideal model to study the role of presynaptic proteins in regulating synaptic function. In this protocol, we describe the use of confocal imaging and three-dimensional reconstruction of the calyx of Held to assess alterations in gross morphology following molecular perturbation. Using viral-vector delivery to perform molecular perturbations at distinct developmental time points, we provide a fast and cost-effective method to investigate how presynaptic proteins regulate gross morphology such as surface area and synapse volume throughout the lifetime of a neuronal circuit. Key features Confocal imaging and 3D reconstruction of presynaptic terminals. Used with a virus-mediated expression of mEGFP to achieve efficient, cell-type specific labeling of the presynaptic compartment. Protocol was developed with the calyx of Held but is suitable for pre- and postsynaptic compartments of various neurons across multiple mammalian and invertebrate species.