Seeking rhythmic patterns in microglial cells within the circadian pineal gland from male rats

Microglia, the innate immune cells of the brain, constitute a highly dynamic cell population that displays several functions influenced by the light:dark (L:D) cycle. Within the pineal gland (PG), a key organ of the circadian timing system, microglia actively participate in its development and homeostasis. However, little is known about their rhythmic features in this circadian organ. This study aimed to elucidate morphological and functional phenotypes of pineal microglial cells at two time points of the L:D cycle. We performed immunofluorescence staining and confocal microscopy on paraffin-embedded pineal sections from 3– and 18-month-old Wistar rats, analyzing samples collected at midday (ZT6) and midnight (ZT18). Our results showed that the density and spatial distribution of IBA1 ⁺ microglial cells did not vary between ZT6 and ZT18 in the 3-month-old PG. However, these cells exhibited reduced size and amoeboid-like shapes, along with a reduction in the expression of the phagocytosis inhibitor SIRP alpha, at ZT18 compared to ZT6. Moreover, IBA1 ⁺ cells were immunoreactive for the lysosomal marker CD68 and the autophagic marker LC3B at both ZTs. Additionally, contacts with PAX6 ⁺ cells were detected in the two ZTs analyzed. Interestingly, IBA1 ⁺ cells showed attenuated or abolished rhythmicity in morphological parameters and SIRP alpha expression levels in the 18-month-old PG. Our findings suggest that microglia undergo transitions into alerted states at night, characterized by small, rounded shapes and enhanced phagocytic capacity. This adaptation likely prepares them to respond to potential invading agents and other insults in the PG, which could impact the nocturnal melatonin production.