Day/night variations of microglial morphology in the mouse lateral hypothalamus

Microglia are glial cells derived from mesenchymal progenitors (i.e., of the myeloid lineage). They provide the first line of defense against immune challenges in the central nervous system (CNS) and can be regarded as resident brain macrophages. They represent 10-15% of brain cells and are distributed throughout the CNS. Although several different microglial phenotypes have been identified through the years, in this study, we take into consideration a more classical definition with two significant phenotypes: i) the ramified “resting” (also known as “surveillant”) phenotype, in which microglia constantly scan the surrounding microenvironment through their highly motile processes, which are thin and extensively ramified; ii) the “activated” microglia, in response to various kinds of insults, in which cells can reach different degrees of activation (pro-inflammatory or anti-inflammatory) with either “hypertrophic” or “amoeboid” phenotype (the latter with phagocytic functions). The shift between ramified and activated phenotypes is critical for brain homeostasis and defense processes. While microglial activation in various pathological conditions has been extensively investigated, microglia plasticity in normal, unchallenged conditions remains largely unexplored. This is of critical importance since, in basal conditions, transient microglia-synapse interactions play a role in synaptic activity regulation. The hypothalamus is the diencephalic brain region that controls many physiological functions (such as body temperature, food and water intake, sleep-wake cycle, and circadian rhythms). The neurons of the lateral hypothalamus containing the orexin/hypocretin peptides play a crucial role in many of these functions, especially energy homeostasis and wake stability. Orexinergic neurons (about 6700 in the rat and about 50000-80000 in humans) widely project to many CNS structures. On this basis, the present study investigated microglia in the lateral hypothalamus, with particular reference to day/night changes in morphology. Adult male heterozygous CX3CR1-GFP transgenic mice, in which microglial cells were tagged with a green fluorescent protein (GFP), were sacrificed at two time points (early night and early day). Using immunofluorescence, confocal microscopy was used to visualize both fluorescent microglia and orexinergic neurons. The ramification patterns of GFP-labeled microglia were evaluated quantitatively using dedicated software (IMARIS). Animals showed cyclic changes in microglial cell morphology, with more ramified processes at night (when mice are predominantly awake) than during daytime (the period of sleep predominance). For the first time, the findings point out diurnal plastic changes of microglia in the lateral hypothalamus, which could be related to the functional properties of the local neuronal circuitry, including synaptic plasticity phenomena. Such cyclic variations suggest that during wakefulness, microglia could play significant roles in maintaining homeostasis and surveilling the numerous synaptic contacts reached by the highly ramified distal processes. On the other hand, the reduced ramification pattern observed during the sleep period might suggest a shift towards a scavenging and, possibly, pruning role of microglia.