Localization of photoperiod responsive circadian oscillators in the mouse suprachiasmatic nucleus

The circadian pacemaker in the suprachiasmatic nucleus (SCN) yields photoperiodic response to transfer seasonal information to physiology and behavior. To identify the precise location involved in photoperiodic response in the SCN, we analyzed circadian Period1 and PERIOD2 rhythms in horizontally sectioned SCN of mice exposed to a long or short day. Statistical analyses of bioluminescence images with respective luciferase reporters on pixel level enabled us to identify the distinct localization of three oscillating regions; a large open-ring-shape region, the region at the posterior end and a sharply demarcated oval region at the center of the SCN. The first two regions are the respective sites for the so-called evening and morning oscillators, and the third region is possibly a site for mediating photic signals to the former oscillators. In these regions, there are two classes of oscillating cells in which Per1 and Per2 could play differential roles in photoperiodic responses.


Supporting Information
Materials and Methods Animals. Transgenic mice carrying a Period1-luciferase reporter (Per1-luc mice) 1 and knock-in mice carrying a PER2::LUC reporter (Per2 Luc mice) 2 were bred and raised in our animal facility under 12:12 light dark cycles (LD 12:12) with lights on at 06:00 h and off at 18:00 h. The light intensity during the light phase was about 100 lux. Room temperature was 22 ± 2 °C , and humidity was 60 ± 10 %. Chow (Oriental Yeast Co., Tokyo, Japan) and tap water were available at all times. Weaning was done at the postnatal day 21 and only male mice were used for the experiments. At age 2-5 months old, mice were transferred to individual cages in light-tight boxes where light intensity was approximately 300 lux in the light phase.
In the experiment of bioluminescence imaging in two photoperiods, 20 mice were used (Per1-luc mice, n = 10, Per2 Luc mice, n = 10). They were randomly assigned to the long or short day group (each n = 5). The number of animals was determined by a principle of the minimum number for the parametric evaluation of significance level, taking the general variance of circadian parameters into account. The photoperiod was changed to either LD 18:6 (long day, light-on at 3:00 and light-off at 21:00) or LD 6:18 (short day, light-on at 9:00 and light-off at 15:00) as described previously 1 . The mice were exposed to either of the photoperiods for 3-5 weeks before the brain sampling for SCN slice culture. In the experiment of exposure to constant darkness (DD), Per1-luc mice (n = 15) were kept in LD 18:6 for 3 weeks, and randomly assigned to the long day or constant darkness (DD) group. The Per1-luc mice in the DD (n = 7) were subjected to SCN slice culture on the fourth day in DD. The mice kept in LD 18:6 (n = 8) were used as the control.
In the experiments of immunohistochemical analyses, Per1-luc mice (n = 4) were used for staining AVP, VIP and GRP. To visualize the retinal projection to the SCN, Per1-luc mice (n = 5) were used for an anterograde tracer injection. The mice were kept in LD18:6 for 3 weeks before subjected to the experiments. In the experiment of fluorescence in situ hybridization (FISH), C57BL/6J mice (n = 12) were purchased from a local breeder and kept in our animal quarters for exposing to LD18:6 for 3 weeks before hybridization.
SCN slice culture and bioluminescence recording. Mice were decapitated and enucleated after cervical dislocation without anesthesia between 11:00 and 15:00 h. For mice in DD the above procedures were performed under infrared light (< 1 lux). The brain was removed and chilled in ice cold Hanks' balanced salt solution, which was followed by serial slicing of the brain tissue on a horizontal plane in 100 μm-thick with a microslicer (Dosaka EM, Kyoto, Japan). The brain slice was obtained from ca. 300 μm above the bottom of the optic chiasm, which contains the anterior tip to posterior end of the bilateral SCN. The brain slice was cultured on a membrane (Millicell-CM membrane, Millipore) with 1.3 ml of DMEM containing 0.2 mM D-luciferin K and 5 % culture supplements as previously reported 3 . Bioluminescence images were obtained using one of the following CCD cameras: ImagEM (Hamamatsu Photonics, Hamamatsu, Japan) cooled down to -80 °C, iXon3 (Andor, Belfast, UK) down to -80 °C and ORCA II (Hamamatsu Photonics) down to -60 °C. Bioluminescence was recorded every hour for at least 3 consecutive days. At the end of bioluminescence recording the brain slices were fixed with 4% paraformaldehyde in 0.1 M phosphate buffer (PFA) and subjected to immunohistochemical analysis as described below.
Data analyses of bioluminescence images. The day of slice preparation for culture was designated as day 0 in culture. Bioluminescence obtain from 0:00 h on day 1 in culture was subjected to the analyses of circadian rhythm parameters, unless otherwise mentioned. The local time was used for plotting the circadian rhythms.
For the ROI-level analyses, the peak phase of circadian rhythms were determined as described previously 1 . The amplitude of circadian rhythm was defined as a difference between the maximum and minimum level of the bioluminescence rhythm. The horizontal plane of an SCN slice was divided into the anterior and posterior portions which contain approximately 60 and 40 % of the SCN, respectively. The temporal distribution of the peak phases in each ROI was examined in one hour bins and was fitted to a single or double Gaussian curve to obtain the median (μ) and variance (σ) of distribution 4 . The median and variance were calculated for each SCN slice of mice exposed to either LD18:6 or LD6:18. The mean median and variance were obtained for the circadian Per1-luc and PER2::LUC rhythms under different photoperiods (n = 5).
For pixel level analyses (pixel size, 3.7 × 3.7 μm) a time series of bioluminescence of each pixel was fitted to a cosine curve, as described previously 5 .
Briefly, the background level of bioluminescence was subtracted from the original data.
The background level was defined as the mean bioluminescence intensity of 9 pixels at the darkest corner of each image plus 5 SD of the mean. An acrophase and an amplitude (a double value of mathematical amplitude) of a best fitted cosine curve were used as the circadian peak phase and rhythm amplitude, respectively. Goodness of fitting was statistically evaluated by percent rhythm 6 at a significance level of P < 0.01. The circadian peak phase and amplitude were illustrated in pseudocolor.
For statistical comparison of the circadian rhythm parameters on pixel-level, the shape of SCN slice was normalized for each slice to obtain the mean bioluminescence image. The shape of SCN image in each slice was geometrically transformed into the shape of template SCN slice which was selected beforehand. Eight reference points were placed at the margin of the bioluminescence (Fig. 4a). Those  Double labeled fluorescent in situ hybridization. C57BL/6J mice (n = 12) were exposed to LD18:6 for three weeks. The brains were sampled at the mid-dark (0:00 h) or mid-light (12:00 h), frozen in crashed dry ice, and stored at -80 °C until further treatment. Frozen brain was sectioned serially (20 μm) by a cryostat (CM1900; Leica Microsystems), mounted on silane-coated glass slides and fixed as reported previously 11 . The sections were double-labeled by FISH with fluorescein-, and digoxigenin (DIG)-labeled cRNA probes 11 for Per1 (390-1643; GenBank accession number AB030818) and Per2 (1428-2953; NM011066) mRNAs, respectively. FISH was performed as described elsewhere 11 . Finally, sections were stained with NeuroTrace 640/660 Nissl stain (Invitrogen). One brain slice was missed in the course of preparation. Statistical analyses. Statistical analyses were performed using Excel Tokei 2012 (SSRI, Japan). A one-way ANOVA with a post-hoc Tukey-Kramer test was used to evaluate differences in the same condition. A two-way ANOVA with a post-hoc Tukey-Kramer test was used to evaluate difference among different conditions. Bartlett's test was performed prior to ANOVA to confirm the variance among groups were equal. Two-sided Student's t-test was used to evaluate difference between two groups in the pixel level analysis.