S.K, Kyoto, Japan). Metformin order Four slices were obtained from one brain, one each including the SCN, olfactory bulb (OB), CPU/parietal cortex (PC) and substantia nigra (SN). These areas were dissected out in ice-cold Hanks’ balanced salt solution with a surgical knife under a stereoscopic microscope as described elsewhere (Natsubori et al.,

2013a). A dissected tissue was placed on a membrane (Millicell-CM, Millipore, MA, USA; pore size 0.4 μm) in a 35-mm Petri dish, and cultured in air at 37 °C with 1300 μL of DMEM (Invitrogen, CA, USA) supplemented with: HEPES, 10 μm; NaHCO3, 2.7 mm; kanamycin (Gibco, NY, USA), 20 mg/L; apo-transferrin (Sigma, St. Louis, USA), 100 μg/mL; insulin (Sigma), 5 μg/mL; putrescine (Sigma), 100 μm; progesterone (Sigma), 20 nm; sodium selenite (Gibco), 30 nm; and D-Luciferin K salt (Dojindo, Kumamoto, Japan), 0.1 mm. Bioluminescence from each tissue was measured for 1 min at 10-min intervals for 5 successive days with a photomultiplier tube (Lumicycle, Actimetrics or Kronos, Atto, Tokyo, Japan). The discrete brain areas examined were major parts of the brain dopaminergic system. The brain tissue containing the SN

included functionally different structures such as the SN pars compacta, SN pars Selleckchem Saracatinib reticulata and ventral tegmental area. They were not separated in the present study. Twenty-four-hour profiles of spontaneous movement and wheel-running activity in individual rats were analysed in 1-h bins. The individual profiles were averaged for 2 days immediately before the restricted schedule (pre-R) and for the last 2 days (days 12 and 13) of the schedule. When the day corresponded to the estrus in the sexual cycle, the result on that day was replaced by those from the immediately prior proestrus or diestrus day. The group data were

obtained by averaging these individual profiles. Circadian rhythmicity in behavior and its period were evaluated by χ2 periodogram analysis in the range 10–40 h with a significance level of 0.01 (clocklab software, Actimetrics, Evanston, IL, USA). Behavior records in 5-min bins were used for this analysis. The pre-R records of the last 7 days and ad-MAP records of the first 10 days were used for the analyses in the SCN-lesioned rats. The ad-MAP records of the first 5 days were used in the SCN-intact rats. The onset, offset and midpoint of activity Selleck DAPT bands of behavioral rhythms were determined by ClockLab. Time series of bioluminescence data were detrended using a 24-h moving average subtraction method (Yamanaka et al., 2008) and smoothed with a five-point moving average method. A circadian peak was identified when a peak–trough difference in a single circadian range was > 2 SD of the values contained in the range. The circadian peak that appeared within 48 h after the start of culturing was regarded as the first circadian peak. When no clear peak appeared, the data were excluded from further analyses.