Modeling excitotoxic ischemic brain injury of cerebellar purkinje neurons by intravital and in vitro multi-photon laser scanning microscopy

The analysis of cell morphology and connectivity of nerve cells is of particular interest for the study of various forms of cellular damage in the central nervous system (CNS) caused, for example, by stroke or neurodegenerative pathologies. Several experimental models have been established over the years to study structural changes under these conditions using both in vitro and in vivo approaches. In this context, the use of transgenic reporter mice has revolutionized study of neuronal cytoarchitecture in living and fixed tissue. In order to choose the right system for studying a particular aspect of pathological changes in the CNS, it is essential to understand the benefits and limitations of these models. Here, we provide a detailed comparison between in vitro and intravital analysis of structural changes of neuronal networks using the example of altered complexity of the dendritic arbor of Purkinje neurons in response to excitotoxicity and ischemic brain injury. A particular focus of this chapter is the discussion of technical considerations for carrying out multi-photon laser scanning microscopy (MP-LSM) analysis of Purkinje neuron morphology in the brain of living mice and in slice cultures of cerebellar tissue. This includes aspects for optimizing experimental conditions such as multi-photon excitation, fluorescence emission detection, and factors impacting on the level of spatial resolution. While this chapter focuses on excitotoxic damage, it also serves as a guide for experimental designs studying neuronal damage in the CNS caused by other means.