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Cell-autonomous clock of astrocytes drives circadian behavior in mammals

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Science  11 Jan 2019:
Vol. 363, Issue 6423, pp. 187-192
DOI: 10.1126/science.aat4104
  • Fig. 1 An astrocytic clockwork can autonomously drive circadian clock gene expression in the SCN.

    (A) Experimental design to restrict expression of Cry1-Flex-Luc to neurons or astrocytes by AAVs cotransduced with Syn-mCherry::Cre or GFAP-mCherry::Cre. (B) Stills from live-image recordings of SCN slices cotransduced with Cry1-Flex-Luc, alongside Syn-mCherry::Cre or GFAP-mCherry::Cre, showing circadian variation of the bioluminescent Cry1-Luc signal, phase-aligned to Syn-GCaMP3. Signals are false lookup table colors. (C) Representative detrended traces of neuronally or astrocytically restricted Cry1-Flex-Luc circadian oscillations, phase-aligned to Syn-GCaMP3. A.U., arbitrary units. (D and E) Period and RAE values of Cry1-Luc oscillations, restricted to neurons or astrocytes. Data are means ± SEM, n = 5 per group. (F) Waveform traces of neuronal and astrocytic Cry1-Flex-Luc expression phase-aligned to Syn-GCaMP3. Data are means ± SEM, n = 5 for each experimental group. The asterisk indicates that the circadian phase is based on previous data (3, 9). (G) Representative Per2::Luc traces from SCN slices of Cry1/2-null pups sequentially transduced with Cry1-Flex-Cry1::EGFP and then either Syn-mCherry::Cre or GFAP-mCherry::Cre AAVs to restore Cry1 expression in neurons or astrocytes, respectively. Insets show amplitudes of Per2::Luc in the early (inset 1) and late (inset 2) stages of neuronally and astrocytically restricted Cry1 expression. (H) Period values after neuronally or astrocytically restricted Cry1 expression in the late phases of the treatment. Data are means ± SEM, n = 4. Statistical test was an unpaired two-tailed t test. *P < 0.05. Scale bars, 50 μm.

  • Fig. 2 Genetic complementation of Cry1 in SCN astrocytes initiates and sustains robust circadian patterns of locomotor activity in circadian-incompetent Cry1/2-null mice.

    (A) Experimental design of in vivo expression of Flex-Cry1::EGFP restricted to SCN astrocytes or neurons by Syn- or GFAP-driven Cre, respectively. (B and C) Representative actograms and wavelet analyses of Cry1/2-null mice targeted with Cry1-Flex-Cry1::EGFP together with AAVs expressing GFAP-EGFP (control) (B) or Cre (C). Rhythmicity in LD1 and -2 is due to a masking effect of the light-dark cycle. (D and E) Representative confocal tiled microphotographs of SCN sections from control and Cre-treated mice evaluated post hoc to assess effective targeting of the SCN. Histograms represent colocalization of fluorescence signals from mCherry::Cre and Cry1::EGFP in Cre-treated mice (insets). Total number of cells counted: GFAP-Cre, N(DAPI+) = 5491, n = 5 targeted mice; Syn-Cre, N(DAPI+) = 6037, n = 5 targeted mice. DAPI, 4′,6-diamidino-2-phenylindole. (F) Periods of circadian activity rhythms of control and Cre-treated mice before (DD1) and after (DD2) stereotaxic surgery. (G) Correlation analysis of number of Cry1::EGFP+ astrocytes or neurons and behavioral period (Syn-mCherry-Cre: r = 1, n = 5, P = 0.02; GFAP-mCherry-Cre: r = −0.70, n = 10, P = 0.03, two-way Spearman test). (H) Locomotor activity plotted across the circadian day (means ± SEM). Group sizes were n(GFAP-EGFP) = 7, n(GFAP-Cre) = 10, and n(Syn-Cre) = 5. The statistical test was a two-way repeated measures analysis of variance (RM-ANOVA) with Bonferroni correction. **P < 0.01; ***P < 0.001; §§P < 0.01 (ad hoc unpaired two-tailed t test with Sidak-Bonferroni correction). Scale bars, 50 μm.

  • Fig. 3 Temporal dynamics of circadian bioluminescence rhythms of single cells initiated in Cry1/2-null SCN explants after neuronally or astrocytically restricted expression of Cry1.

    (A) Stills from live-image recordings of Per2::Luc expression from Cry1/2-null SCN slices, showing circadian variation of the bioluminescent signal in the early (upper rows) and late (lower rows) stages of neuronal or astrocytic Cry1 expression. Co-detected mCherry and EGFP are shown to compare spatial distribution and temporal dynamics of mCherry::Cre and Cry1::EGFP expression. (B) Representative single-cell (colored lines) and mean (black lines) traces of Per2::Luc oscillations after Cre-mediated expression of Cry1 in either neurons or astrocytes within SCN slices. (C and D) Period and RAE after neuronal or astrocytic expression of Cry1 in an individual SCN and across multiple explants. Traces for aggregate data are means ± SEM. Group size is n = 3 for each group. The statistical test was a two-way RM-ANOVA with Bonferroni correction. (E) Stills from live-image recordings showing circadian variations of Per2::Luc and Syn-RCaMP1h in Cry1/2-null SCN slices transduced with GFAP-mCherry::Cre or Cry1-Flex-Cry1::EGFP. (F) Representative traces of data presented in (E). (G) Period quantification of Per2::Luc and Syn-RCaMP1h in Cry1/2-null SCN expressing Cry1 only in astrocytes. Data are means ± SEM, n = 4. (H and I) Mean traces ± SEM (H) and Rayleigh plots (I) showing waveforms and phase differences of Per2::Luc and Syn-RCaMP1h oscillations in GFAP-mCherry::Cre or Cry1-Flex-Cry1::EGFP SCN slices and wild-type SCN. (J and K) Representative spatial phase map of Syn-RCaMP1h signal (J) and quantification of the dorsal-to-ventral phase relationship (K) in SCN expressing astrocytic Cry1 in comparison to wild type. Phase data were normalized to dorsal values. Values are means ± SEM, and group sizes are plotted. **P < 0.01; ***P < 0.001; ****P < 0.0001. Statistical tests included a paired two-tailed t test (G) and unpaired ANOVA (K). Scale bars, 50 μm.

  • Fig. 4 Astrocytically released glutamate mediates astrocytic control of circuit-level circadian time-keeping in Cry1/2-null SCN expressing GFAP-restricted Cry1.

    (A) Confocal tiled microphotographs of adult SCN showing colocalization of GFAP-EGFP and Cx43, detected by polyclonal antiserum (results are representative of findings with three independent brains). (B) Representative Per2::Luc PMT traces and group data (mean + SEM), showing dose-response effects of TAT-Gap19 on the amplitude ratio (with drug/before drug) and period in wild-type SCN slices. The statistical test for the amplitude ratio was an unpaired ANOVA with Bonferroni correction. Analysis for period employed a two-way RM-ANOVA with Bonferroni correction [n = 3 for each group, except vehicle (Veh), n = 4]. (C and D) Representative Per2::Luc PMT trace (C) and paired scatter plot of RAE and amplitude (D) of Cry1/2-null SCN slices transduced with GFAP-mCherry::Cre and Cry1-Flex-Cry1::EGFP and treated with TAT-Gap19 (50 μM). The statistical test was a paired one-tailed t test, n = 5. (E and F) Representative iGluSnFR traces (E) and paired scatter plot of RAE (F) of Cry1/2-null SCN slices before and after GFAP-mCherry::Cre and Cry1-Flex-Cry1::EGFP transduction and treatment with TAT-Gap19 (50 μM). The statistical test was an RM-ANOVA with Bonferroni correction, n = 4. (G) Representative Per2::Luc PMT traces of Cry2-null and Cry1/2-null SCN slices transduced with GFAP-mCherry::Cre and Cry1-Flex-Cry1::EGFP and treated with DQP-1105 (50 μM) or vehicle, with subsequent washout. (H) Group data (means + SEM) of bioluminescence baseline traces represented in (G) before, in the presence of, and after removal of DQP-1105. The statistical test was a two-way RM-ANOVA with Bonferroni correction. (I) Group data (means + SEM) showing peak-trough differences in the presence of DQP-1105 of traces represented in (G). (J) Group data (means + SEM) showing amplitude ratio (after drug/with drug) of data presented in (G). The statistical test for (H) was a two-way RM-ANOVA with Bonferroni correction. The statistical test for (I) and (J) was an unpaired ANOVA with Bonferroni correction (n = 4 for Cry2-null and Veh groups; n = 3 for DQP-1105 treatment in Cry1/2-null group). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001 (n = 4). Scale bars, 20 μm.

Supplementary Materials

  • Cell-autonomous clock of astrocytes drives circadian behavior in mammals

    Marco Brancaccio, Mathew D. Edwards, Andrew P. Patton, Nicola J. Smyllie, Johanna E. Chesham, Elizabeth S. Maywood, Michael H. Hastings

    Materials/Methods, Supplementary Text, Tables, Figures, and/or References

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    • Materials and Methods
    • Figs. S1 to S4
    • Captions for Movies S1 and S2
    • References

    Images, Video, and Other Media

    Movie S1
    Progressive establishment of Per2::Luc oscillation in Cry1/2-null SCN slices following astrocytically restricted expression of Cry1. Per2::Luc signal from Cry1/2-null SCN slices on astrocytic Cry1 expression (single SCN). The slice had previously been transduced with Cry1-Flex-Cry1::EGFP AAV. The recording starts immediately after super-transduction with GFAP-mCherry::Cre AAVs triggering astrocytic specific expression of Cry1. V is third ventricle. Time is hours (h), as indicated in the movie.
    Movie S2
    Astrocytically restricted expression of Cry1 fully initiates circadian neuronal activity and spatio-temporal circuit organisation of the SCN in Cry1/2-null mice. Syn-RCaMP1 detecting neuronal intracellular calcium signal from Cry1/2-null SCN slices transduced with Cry1-Flex-Cry1::EGFP/ GFAP-mCherry::Cre AAVs to express Cry1 solely in astrocytes. The movie is obtained by subtracting 25 hour moving average to filter noise. 3V is third ventricle. Time is hours (h), as indicated in the movie.

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