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Methods Mol Biol. 2019;2034:191-205. doi: 10.1007/978-1-4939-9658-2_14.

Analysis of Signaling Mechanisms Regulating Microglial Process Movement.

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Department of Immunology, Laboratory of Molecular Genetics, Hellenic Pasteur Institute, Athens, Greece.
Department of Neuroscience, Physiology and Pharmacology, University College London, London, UK.
Département de Physique Nucléaire et Corpusculaire, University of Geneva, Geneva, Switzerland.
CERN, Geneva, Switzerland.
Institute of Neurophysiology, Charité-Universitätsmedizin, Berlin, Germany.


Microglia, the brain's innate immune cells, are extremely motile cells, continuously surveying the central nervous system (CNS) to serve homeostatic functions and to respond to pathological events. In the healthy brain, microglia exhibit a small cell body with long, branched, and highly motile processes, which constantly extend and retract, effectively "patrolling" the brain parenchyma. Over the last decade, methodological advances in microscopy and the availability of genetically encoded reporter mice have allowed us to probe microglial physiology in situ. Beyond their classical immunological roles, unexpected functions of microglia have been revealed, both in the developing and the adult brain: microglia regulate the generation of newborn neurons, control the formation and elimination of synapses, and modulate neuronal activity. Many of these newly ascribed functions depend directly on microglial process movement. Thus, elucidating the mechanisms underlying microglial motility is of great importance to understand their role in brain physiology and pathophysiology. Two-photon imaging of fluorescently labeled microglia, either in vivo or ex vivo in acute brain slices, has emerged as an indispensable tool for investigating microglial movements and their functional consequences. This chapter aims to provide a detailed description of the experimental data acquisition and analysis needed to address these questions, with a special focus on key dynamic and morphological metrics such as surveillance, directed motility, and ramification.


Directed motility; Image processing; Microglia; Ramification; Surveillance; Two-photon imaging

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