Volume 16, Issue 6, Pages 599-605 (March 2006) Synchronization and Maintenance of Timekeeping in Suprachiasmatic Circadian Clock Cells by Neuropeptidergic Signaling  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  Current Biology  Volume 16, Issue 6, Pages 599-605 (March 2006) DOI: 10.1016/j.cub.2006.02.023 Copyright © 2006 Elsevier Ltd Terms and Conditions

Figure 1 Compromised Molecular Circadian Timekeeping in Vipr2−/− SCN Revealed by Bioluminescence Recordings (A) Photomultiplier recordings of total Per1::luciferase bioluminescence (expressed as counts per second) emitted by individual SCN slices reveal high-amplitude circadian activity in wild-type (upper panel) and heterozygous knockout (lower panel, Vipr2+/−) mice (three representative slices per genotype). (B) Circadian gene expression from six representative Vipr2−/− slices was extremely low and apparently nonrhythmic. Rescaling of the ordinate (lower panel) revealed very poorly organized and low-amplitude circadian gene expression. (C) Group data demonstrating that the amplitude of circadian gene expression, monitored by bioluminescence, was severely reduced in Vipr2r−/− SCN slices (mean + SEM). (D) Amplitude of successive circadian luciferase peaks, plotted relative to the initial peak height of each slice (100%), demonstrates that the rhythm of the Vipr2−/− slices (KO) dampened more rapidly than in heterozygotes (HET) (mean + SEM, n = 17 and n = 31). Current Biology 2006 16, 599-605DOI: (10.1016/j.cub.2006.02.023) Copyright © 2006 Elsevier Ltd Terms and Conditions

Figure 2 Desynchronized and Attenuated Cellular Circadian Timekeeping in Vipr2−/− SCN Neurons (A) Phase-contrast and corresponding bioluminescence CCD images captured from representative Vipr2r+/− organotypic slice at 12 hr intervals (48–84 hr of recording) reveal widespread, robust, and tightly synchronized circadian activity across SCN. V denotes third ventricle, and the bar represents 500 μm. See Movie 3. (B) Plots of relative circadian gene expression (grayscale units) from five representative cells in slice (A). Note the well-defined individual cycles and tight synchrony of neurons. (C) Raster plots of bioluminescent circadian gene expression in 25 cells, one cell per line, in slice (A). Neurons are clustered vertically by phase of oscillation; red indicates peak luciferase emission, and green indicates minimal emission. (D) Rayleigh vector plots for cells in (C) confirm tight synchrony. Red arrowheads indicate phases of individual cells, and blue arrow the aggregate phase vector. (E) Phase-contrast and corresponding bioluminescence CCD images captured from representative Vipr2r−/− organotypic slice at 12 hr intervals (54–90 hr of recording). Note few cells with detectable bioluminescence, restricted to the medial SCN. See Movie 4. (F) Enlarged video images from Vipr2−/− slice in (E) at 4 hr intervals show asynchronous Per::luciferase expression, with adjacent individual neurons (green and red arrows) running in antiphase and a third (blue arrows) at an intermediate phase. (G) Plots of relative circadian gene expression (grayscale units) from five representative cells in slice (E). Note the poorly defined and asynchronous circadian activity. (H) Raster plots of bioluminescent circadian gene expression from 29 cells in slice (E); each line represents one cell. Note that cellular circadian oscillation is poorly organized and weakly synchronized, with many circadian phases represented simultaneously. (I) Rayleigh vector plots for cells in (H) confirm statistically borderline cellular synchrony in this Vipr2−/− slice. In six other Vipr2−/− slices, p > 0.05. Current Biology 2006 16, 599-605DOI: (10.1016/j.cub.2006.02.023) Copyright © 2006 Elsevier Ltd Terms and Conditions

Figure 3 Acute Enhancement and Transient Synchronization of Cellular Circadian Gene Expression in Vipr2−/− SCN Slices by Depolarization (A–C) CCD images from Vipr2−/− slice (representative of three independent experiments) recorded before (12–48 hr), during (163–199 hr), and after (238–274 hr) 40 mM K+-induced depolarization, respectively. Note extensive induction of cellular Per::luciferase activity during depolarization. See Movie 5. V denotes third ventricle, and the bar represents 500 μm. (D) Individual bioluminescence recordings (grayscale units expressed as deviation from 24 hr mean) from 24 representative cells from the same slice (C) exhibiting circadian gene expression immediately after removal of 40 mM K+. Note the initial synchrony and then progressive desynchronization. (E) Raster plots of data in (D) and corresponding data from cells in the same slice before (A) and during (B) depolarization. (F) Rayleigh vector plots of data in (E) immediately before depolarization (Pre-K) and then immediately (initial) and 5 days (delayed) after removal of excess K+ (Post-K) reveal transient imposition and then progressive loss of synchrony. Current Biology 2006 16, 599-605DOI: (10.1016/j.cub.2006.02.023) Copyright © 2006 Elsevier Ltd Terms and Conditions

Figure 4 Acute Enhancement and Transient Synchronization of Cellular Circadian Gene Expression in Vipr2−/− SCN Slices by Gastrin-Releasing Peptide (A) CCD images from Vipr2−/− slice (representative of three independent experiments) recorded immediately after addition of GRP (0–18 hr) and 5 days later (120–138 hr). Note the acute induction of cellular Per::luciferase activity 6 hr following GRP. After 5 days, cellular activity is still evident across the slice, especially in the medial SCN, but is asynchronous. V denotes third ventricle, and the bar represents 500 μm. (B) Relative intensity of bioluminescence integrated across SCN (grayscale values expressed as deviation from 24 hr mean) in (A) over 9 days following GRP. Note the progressive dampening of aggregate rhythm. (C) Individual bioluminescence recordings (grayscale values expressed as deviation from 24 hr mean) from 20 cells initially exhibiting circadian gene expression immediately after addition of GRP. Note the progressive desynchronization of the population. (D) Raster plots of data in (C) and corresponding data from (desynchronized) cells in the same slice before GRP addition. (E) Rayleigh vector plots of data in (D) in the 96 hr immediately after GRP (initial) and the final 96 hr of the recording (delayed), by which time cellular synchrony was lost. Current Biology 2006 16, 599-605DOI: (10.1016/j.cub.2006.02.023) Copyright © 2006 Elsevier Ltd Terms and Conditions