Supplemental Data Synchronization and Maintenance of Timekeeping in Suprachiasmatic Circadian Clock Cells by Neuropeptidergic Signaling

• Main Authors: Elizabeth S. Maywood, Akhilesh B. Reddy, Gabriel K. Y. Wong, John S. O’neill, John A. O’brien, Douglas G. Mcmahon, Anthony J. Harmar, Hitoshi Okamura, Michael H. Hastings
• Other Authors: The Pennsylvania State University CiteSeerX Archives
• Format: Text
• Language: English
• Subjects: Straume; Orca
• Online Access: http://citeseerx.ist.psu.edu/viewdoc/summary?doi=10.1.1.325.7909; http://download.cell.com/current-biology/mmcs/journals/0960-9822/piis0960982206011730.mmc1.pdf

and the University of Cambridge. Per1::luciferase [S1] and Per1::dsGFP transgenic mice [S2] and Vipr22/2 mutants [S3] and crosses between these lines were produced in local colonies. For organotypic slice culture, brains were removed from pups 5–10 days old and sectioned at 300 um with a McIlwain ‘‘Tissue Chopper.’’ Slices were sorted and trimmed to contain principally SCN tissue and placed onto a Millipore membrane insert (PICMORG) for culture at 37ºC in 5 % CO2 as described previously [S4]. For long-term recordings, slices were transferred to 1.1 ml HEPES buffered medium with 100 uM beetle luciferin (Promega) [S5] in a glass-bottomed Petri dish sealed with a coverslip and vacuum grease. Total bioluminescence was recorded with Hamamatsu photomultiplier tube assemblies housed in a light-tight 37ºC incubator. For bioluminescence recording of cells, slices were mounted on a Zeiss Axiovert microscope with heated stage and imaged with 1 hr integration via a Hamamatsu Orca 2 CCD camera cooled to 265ºC. Fluorescence recordings were made as described previously [S5]. Post-hoc analyses, including determination of integrated intensity of bioluminescence and fluorescence in regions of interest were made with IP Lab software (Scanalytics, Fairfax Virginia). Photomultiplier recordings were expressed as counts per second integrated over 6 min sample bins. CCD and fluorescence recordings were expressed as relative pixel intensity (grayscale). Rhythmicity was assessed with Brass software package kindly provided by A. Millar, University of Edinburgh, and M. Straume, University of Virginia. Rayleigh tests of synchrony were conducted in Oriana Software with data from R20 individual cells per SCN (except for one Vipr22/2 SCN in which only 15 cells could be detected after exposure to K +). Raster plots were produced with EPCLUST online software