Alterations in microglial morphology concentrate in the habitual sleeping period of the mouse

In nocturnal animals, waking appears during the dark period while maximal non-rapid-eye-movement sleep (NREMS) with electroencephalographic slow-wave-activity (SWA) takes place at the beginning of the light period. Vigilance states associate with variable levels of neuronal activity: waking with high-frequency activity patterns while during NREMS, SWA influences neuronal activity in many brain areas. On a glial level, sleep deprivation modifies microglial morphology, but only few studies have investigated microglia through the physiological sleep-wake cycle. To quantify microglial morphology (territory, volume, ramification) throughout the 24 h light-dark cycle, we collected brain samples from inbred C57BL male mice (n = 51) every 3 h and applied a 3D-reconstruction method for microglial cells on the acquired confocal microscopy images. As microglia express regional heterogeneity and are influenced by local neuronal activity, we chose to investigate three interconnected and functionally well-characterized brain areas: the somatosensory cortex (SC), the dorsal hippocampus (HC), and the basal forebrain (BF). To temporally associate microglial morphology with vigilance stages, we performed a 24 h polysomnography in a separate group of animals (n = 6). In line with previous findings, microglia displayed de-ramification in the 12 h light- and hyper-ramification in the 12 h dark period. Notably, we found that the decrease in microglial features was most prominent within the early hours of the light period, co-occurring with maximal sleep SWA. By the end of the light period, all features reached maximum levels and remained steadily elevated throughout the dark period with minor regional differences. We propose that vigilance-stage specific neuronal activity, and SWA, could modify microglial morphology.