Analysis of Neuronal Morphology by Two-Photon Microscopy

During the development of mammalian brains, pyramidal neurons in the cerebral cortex form highly organized six layers with different functions. These neurons undergo developmental processes such as axon extension, dendrite outgrowth, and synapse formation. A proper integration of the neuronal connectivity through dynamic changes of dendritic branches and spines is required for learning and memory. Disruption of these crucial developmental processes is associated with many neurodevelopmental and neurodegenerative disorders. To investigate the complex dendritic architecture, several useful staining tools and genetic methods to label neurons have been well established. Monitoring the dynamics of dendritic spine in a single neuron is still a challenging task. Here, we provide a methodology that combines in vivo two-photon brain imaging and in utero electroporation, which sparsely labels cortical neurons with fluorescent proteins. This protocol may help elucidate the dynamics of microstructure and neural complexity in living rodents under normal and disease conditions.