Research ArticleNEUROSCIENCE

Differential roles of AVP and VIP signaling in the postnatal changes of neural networks for coherent circadian rhythms in the SCN

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Science Advances  09 Sep 2016:
Vol. 2, no. 9, e1600960
DOI: 10.1126/sciadv.1600960
  • Fig. 1 Tissue-level circadian PER2::LUC rhythms in the cultured SCN slices of WT, Vipr2−/−, Cry1,2−/−, and Cry1,2−/−/Vipr2−/− mice.

    Representative tissue-level PER2::LUC bioluminescence in the neonatal [postnatal day 7 (P7); left] and adult (right) SCN slices of WT, Vipr2−/−, Cry1,2−/−, and Cry1,2−/−/Vipr2−/− mice measured by a PMT. Different colored lines in each panel indicate bioluminescence from the SCN of different mice. The number of days in the culture is indicated on the abscissa with day 0 as the day of harvest of the SCN. The ordinate indicates the intensity of bioluminescence in arbitrary units. The vertical divisions in the panels indicate a local time of 0000.

  • Fig. 2 Pixel-level circadian PER2::LUC rhythms in the cultured SCN slices of WT, Vipr2−/−, Cry1,2−/−, and Cry1,2−/−/Vipr2−/− mice.

    (A) Representative period maps of the circadian PER2::LUC rhythm on the pixel level in the neonatal SCN slices (top) and distributions of the pixel-level circadian periods (bottom). A color key to the right indicates units of the heat map from 10 hours (blue) to 38 hours (white). Scale bar, 200 μm. Superimposed curves in the period distribution of Cry1,2−/−/Vipr2−/− SCN indicate three Gaussian curves fitted to the data. Arrowheads a, b, and c indicate the peaks of a fitted Gaussian curve. (B) Mean circadian periods on the pixel level in the SCN of WT (n = 5), Vipr2−/− (n = 5), and Cry1,2−/− (n = 6) mice (left) and periods of three clusters in the Cry1,2−/−/Vipr2−/− mice (n = 3) (right). (C) Representative period maps of the circadian PER2::LUC rhythm on the pixel level in the adult SCN slices (top) and distributions of pixel-level circadian periods (bottom). See also the legend for (A). (D) Mean circadian periods on the pixel level at the cluster peak of the WT (n = 6) and Vipr2−/− (n = 4) SCN (left) and periods of the three clusters in the Cry1,2−/− (n = 4) (middle) and Cry1,2−/−/Vipr2−/− SCN (n = 4) (right). *P < 0.01, one-way ANOVA with post hoc Tukey-Kramer test.

  • Fig. 3 Pharmacological manipulations of intracellular cAMP or Ca2+ abolish circadian PER2::LUC rhythms in the neonatal SCN of Cry1,2−/− mice.

    (A) Representative PER2::LUC bioluminescence in the neonatal SCN slices of the WT (top) and Cry1,2−/− (bottom) mice treated with a blocker of adenylyl cyclase (MDL-12330A) by exchanging with the culture medium containing each drug. Magenta lines indicate the duration of drug or vehicle treatment. Bioluminescence in the vehicle (black), MDL-12330A (1 μM; pale green), and MDL-12330A (5 μM; dark green) treatment. (B) χ2 periodogram during drug treatment. An oblique line in the periodogram indicates the significance level (P = 0.01). (C) Representative period maps on the pixel level and distributions of period in the WT (left) and Cry1,2−/− (right) SCN treated with 5 μM MDL-12330A. A color key of the heat map is indicated in the right margin from 10 hours (blue) to 38 hours (white). Scale bars, 200 μm. Superimposed curves in the period distribution of Cry1,2−/− indicate three Gaussian curves fitted to the data (far right). Arrowheads a, b, and c indicate the peaks of a fitted Gaussian curve. (D) Representative PER2::LUC bioluminescence in the neonatal SCN slices of the WT (top) and Cry1,2−/− (bottom) mice treated with Ca2+-chelating agent BAPTA-AM [20 μM (orange) or 60 μM (red)]. See also the legend for (A). (E) χ2 periodogram during drug treatment. See also the legend for (B). (F) Representative period maps on the pixel level and distributions of period in a WT (left) and Cry1,2−/− (right) SCN treated with 20 μM BAPTA-AM. See also the legend for (C).

  • Fig. 4 Coculture with the neonatal WT SCN slice rescues the circadian PER2::LUC rhythm in the SCN of Cry1,2−/− and Cry1,2−/−/Vipr2−/− mice.

    (A) Representative bright-field (left) and bioluminescent (middle) images of recipient and graft SCN slices in coculture and their schematic drawing (right). Scale bar, 200 μm. (B) Representative PER2::LUC rhythms in the neonate (P4) cocultured with a neonatal (P7) WT SCN slice are shown using three different colors. The abscissa indicates the days in culture. The day of coculture was indicated by an arrow. (C) Representative bioluminescence in the Cry1,2−/− (blue) and Cry1,2−/−/Vipr2−/− (red) adult SCN cocultured with a neonatal WT SCN slice. (D) Detrended PER2::LUC rhythms are the same as those indicated in (C). (E) Change in the amplitude of circadian PER2::LUC rhythm in the Cry1,2−/− (blue) and Cry1,2−/−/Vipr2−/− (red) adult SCN during the course of coculture with the neonatal WT SCN. The amplitudes are expressed as a ratio of the first amplitude after the coculture. Values are means and SD (n = 7 for both genotypes). *P < 0.05, two-way repeated-measures ANOVA with post hoc Student’s t test. (F) Standardized amplitude of rescued circadian PER2::LUC rhythm on the 12th culture day (6 days after coculture) of the Cry1,2−/− (d) (n = 7) and Cry1,2−/−/Vipr2−/− (t) (n = 7). **P < 0.01, Student’s t test. (G) Periods of rescued circadian PER2::LUC rhythm after coculture with the neonatal WT SCN, evaluated by a χ2 periodogram, showed no significant difference between Cry1,2−/− and Cry1,2−/−/Vipr2−/−. (H) Representative period map (slice A) and period distribution of circadian PER2::LUC rhythm in Cry1,2−/− SCN after coculture on the pixel level (slices A to E). Superimposed curves in the period distribution of slice A indicate three Gaussian curves fitted to the data. Arrowheads a, b, and c indicate the peaks of a Gaussian curve. (I) Representative period map and period distribution of circadian PER2::LUC rhythm in Cry1,2−/−/Vipr2−/− SCN after coculture on the pixel level (slices A to E).

  • Fig. 5 AVP in the graft SCN is responsible for the rescue of circadian PER2::LUC rhythms in the adult Cry1,2−/− and Cry1,2−/−/Vipr2−/− SCN.

    (A and B) Representative circadian PER2::LUC rhythms rescued in the arrhythmic adult SCN of Cry1,2−/− (A) and Cry1,2−/−/Vipr2−/− (B) mice by coculture of a neonatal WT SCN before and after treatment of AVP receptor (V1a and V1b) antagonists (blue and red) or vehicle (black). An arrow indicates the day of coculture, and a horizontal magenta line indicates the days under treatment. Coculture was done without exchanging culture medium, whereas the drug treatment was done by exchanging into a new medium containing drug or vehicle. The histogram shown on the right side demonstrates the mean ratios of circadian amplitude on the third day after drug treatment to that immediately before the treatment (Cry1,2−/−, n = 6; Cry1,2−/−/Vipr2−/−, n = 4; each for vehicle and antagonists). **P < 0.01, Student’s t test, significant difference between antagonists and vehicle treatments. (C and D) Representative period maps and period distributions of the rescued circadian PER2::LUC rhythm (left), after AVP antagonist treatment (middle) and washout of the antagonists (right) in the adult SCN of Cry1,2−/− (C) and Cry1,2−/−/Vipr2−/− (D) mice by coculture with a neonatal WT SCN on the pixel level. Superimposed curves in the period distribution indicate three Gaussian curves fitted to the data. Scale bars, 200 μm. (E) Schematic drawing of the coculture of the SCN slices and representative circadian PER2::LUC rhythm of the adult SCN slice of Cry1,2−/−/Vipr2−/− mice (orange) cocultured with the SCN from the heterozygote (top) and homozygote (bottom) of Avp knockout mice (green). Recipient adult SCN exhibits PER2::LUC bioluminescence (orange) and graft neonatal (P4 + 3 days culture) SCN exhibits Avp-ELuc bioluminescence (green), which were calculated after alternatively monitoring total and filtered bioluminescence in the same cocultured SCN slices. The abscissa indicates days in coculture. (F) χ2 periodograms for the circadian Avp-ELuc rhythms of graft SCN (left) and PER2::LUC rhythms of recipient SCN (right) in culture. An oblique line in the periodogram indicates the level of significance (P < 0.01).

  • Fig. 6 Cry1,2−/− mice show attenuated circadian rhythms in Avp expression in the cultured SCN.

    (A) Representative Avp-ELuc rhythms in the neonatal and adult SCN slices of WT (black) and Cry1,2−/− (red) mice. (B) Avp expression levels in the SCN of WT (black) and Cry1,2−/− (red) mice. Mean intensity of bioluminescence between 24 and 48 hours after culture was calculated as the Avp expression level in each SCN. Values are means and SD (n = 3 to 5). *P < 0.05; **P < 0.01, Student’s t test, significant difference between the WT and Cry1,2−/− mice.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/2/9/e1600960/DC1

    fig. S1. Circadian rhythms in locomotor activity in the WT, Vipr2−/−, Cry1,2−/−, and Cry1,2−/−/Vipr2−/− mice.

    fig. S2. Circadian rhythms in PER2::LUC in the cultured SCN of Cry1,2−/−/Vipr2−/− mice.

    fig. S3. Circadian PER2::LUC rhythms in the cultured SCN slice of Cry1,2−/− mice applied with MDL-12330A or BAPTA-AM of different doses.

    fig. S4. Rescue of the circadian PER2::LUC rhythm in the arrhythmic adult SCN slice of Cry1,2−/− or Cry1,2−/−/Vipr2−/− mice by coculture with a neonatal WT SCN.

    fig. S5. AVP receptor antagonists modify rescued circadian PER2::LUC rhythms in the arrhythmic adult Cry1,2−/− or Cry1,2−/−/Vipr2−/− SCN.

    fig. S6. A differential cell cluster model for the mouse SCN neural network.

  • Supplementary Materials

    This PDF file includes:

    • fig. S1. Circadian rhythms in locomotor activity in theWT, Vipr2−/−, Cry1,2−/−, and Cry1,2−/−/Vipr2−/− mice.
      fig. S2. Circadian rhythms in PER2::LUC in the cultured SCN of Cry1,2−/−/Vipr2−/− mice.
      fig. S3. Circadian PER2::LUC rhythms in the cultured SCN slice of Cry1,2−/− mice applied with MDL-12330A or BAPTA-AM of different doses.
      fig. S4. Rescue of the circadian PER2::LUC rhythm in the arrhythmic adult SCN slice of Cry1,2−/− or Cry1,2−/−/Vipr2−/− mice by coculture with a neonatal WT SCN.
      fig. S5. AVP receptor antagonists modify rescued circadian PER2::LUC rhythms in the arrhythmic adult Cry1,2−/− or Cry1,2−/−/Vipr2−/− SCN.
      fig. S6. A differential cell cluster model for the mouse SCN neural network.

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