Background

Microglia are the immunocompetent cells of the brain. They have been classically described as defenders of the CNS under pathological conditions. Indeed, they are able to detect pathological stimuli or brain insults and rapidly switch to an activated phenotype migrating toward the site of injury (Hanisch and Kettenmann, 2007). However, in the last years this interpretation has been challenged. Through time-lapse imaging techniques, it has been demonstrated that, in physiological conditions, microglia engage minimal overlapping territories and continuously scan brain microenvironment, extending and retracting their thin processes (Davalos et al., 2005; Nimmerjahn et al., 2005). This dynamic reorganization of microglial processes may be considered a housekeeping function by which microglia monitor the environment, physically interact with other cells, remove tissue debris and apoptotic cells, and sense neuronal activity and structural alterations, in order to preserve and organize neuronal networks (reviewed in Hristovska and Pascual, 2016).
The CX3CR1-GFP mouse model (Jung et al., 2000) provides an exceptional tool for the visualization in vivo and ex vivo of microglia morphology, including the distal processes and fine protrusions. The heterozygous Cx3cr1 mouse model has been previously used in several studies in order to highlight the dynamic behavior of microglial cells in different brain regions (Lee et al., 2008; Wake et al., 2009; Tremblay et al., 2010). However, it should be also considered that different studies have highlighted a slightly different microglial function in heterozygous mice compared to wild type (Lee et al., 2010; Rogers et al., 2011; Sellner et al., 2016).

The current protocol allows investigating the scanning ability of microglial cells, providing a deep analysis of the fine movement of microglial processes at rest. Using this approach, we highlighted an impairment in microglia patrolling ability of mice lacking the CX3CR1, in which neuron-microglia crosstalk mediated by the fractalkine/CX3CR1 signaling is disrupted (Basilico et al., 2019).

Moreover, this protocol can be easily adapted to study the acute effect of drugs, possibly applied through the perfusion system, on microglial processes rearrangement.