In Vivo Imaging of the Structural Plasticity of Cortical Neurons After Stroke

: The comprehension of the finest mechanisms underlying experience-dependent plasticity requires the investigation of neurons and synaptic terminals in the intact brain over prolonged periods of time. Longitudinal two-photon imaging together with the expression of fluorescent proteins enables high-resolution imaging of dendritic spines and axonal varicosities of cortical neurons in vivo. Importantly, the study of the mechanisms of structural reorganization is relevant for a deeper understanding of the pathophysiological mechanisms of neurological diseases such as stroke and for the development of new therapeutic approaches. This protocol describes the principal steps for in vivo investigation of neuronal plasticity both in healthy conditions and after an ischemic lesion. First, we give a description of the surgery to perform a stable cranial window that allows optical access to the mouse brain cortex. Then we explain how to perform longitudinal two-photon imaging of dendrites, axonal branches, and synaptic terminals in the mouse brain cortex in vivo, in order to investigate the plasticity of synaptic terminals and orientation of neuronal processes. Finally, we describe how to induce an ischemic lesion in a target region of the mouse brain cortex through a cranial window by applying the photothrombotic stroke model.