Diurnal Spatial Rearrangement of Microglial Processes through the Rhythmic Expression of P2Y12 Receptors

All efforts were made to minimize animal suffering and to reduce the number of animals used.Eight-week-old male C57BL/6 mice and Clock-mutant (Clock/Clock) mice, which have a point mutation in exon 19 of the Clock gene (Jackson Laboratory, Bar Harbor, ME, USA), on an ICR background were purchased from Charles River Japan, Inc. (Kanagawa, Japan).The mice were housed under either normal light/dark (LD) condition (light on at 07:00 and light off at 19:00) or constant darkness (DD) in a temperature-controlled room (24 ± 1°C) with a humidity of 60 ± 10%, and were provided food and water ad libitum.They were adapted to the LD for two weeks before the experiments.Under the LD condition, the ZT0 (Zeitgeber time) was designated as lights on and ZT12 as lights off.For mice housed under the DD condition for one day, the circadian time (CT) was used instead of ZT, and CT0 was characterized as the beginning of the subjective light phase, and CT12 was defined as the beginning of the subjective dark phase.All the preparations were performed under the red light.All animals were treated in accordance with the guidelines stipulated by the animal care and use committee of Kyushu University.