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. 2017 Mar 22;93(6):1420–1435.e5. doi: 10.1016/j.neuron.2017.02.030

Figure 1.

Figure 1

Circadian Landscape of Interlocked, Anti-phasic Oscillations in SCN Neurons and Astrocytes

(A–E) Photomicrographs and representative traces of SCN slices transduced with AAVs encoding Syn-RCaMP1h reporting neuronal [Ca2+]i paired with (A) Syn-ArcLightD reporting membrane voltage; (B) gfaABC1D-GCaMP3 reporting astrocytic [Ca2+]i; (C) gfaABC1D-LCK::GCaMP6 detecting [Ca2+]i in astrocytic microdomains; and (D and E) iGluSnFR driven by Syn or GFAP, respectively, to detect [Glu]e localized on neuronal or astrocytic cell membranes. ArclightD y axis inverted for presentation purposes. SCN area is outlined (white dashed lines) for presentation purposes.

(F) Circadian landscape of reporters in (A)–(E), plotted relative to phase of RCaMP1h (n depicted on graph; each dot is a biological replicate). PER2::LUC is used to register circadian time (CT12). Photomicrographs are false colors LUT ΔF/F.

(G) Fluorescent traces and ratios showing GFAP-iGluSnFR fluorescence upon MSO treatment, compared with co-expressed GFAP-hM3DGq::mCherry, used as an internal control. n = 3 for both reporters.

(H) Representative traces of circadian oscillations of PER2::LUC in SCN slices treated with MSO or vehicle, in serum-free conditions.

(I and J) Bar graphs showing amplitude ratio (I) and relative amplitude error (RAE) ratio (J) in SCN slices treated with MSO or vehicle.

(K) Representative traces of PER2::LUC oscillations of SCN slices treated with astrocytically (blue line) or neuronally (red line) restricted flex-taCasp3-TEV to specifically ablate those populations.

(L) Mean ± SEM iGluSnFR traces from astrocytically ablated or neuronally ablated SCN slices. Scale bars, 100 μM. Measurement windows in gray.

(M) Bar graphs showing variations of iGluSnFR fluorescent intensity ratios in astrocytically or neuronally ablated SCN slices.

All bar graphs are mean ± SEM; n experimental replicates depicted on bars. Statistical tests are as follows: (I and J) one-way ANOVA, Bonferroni corrected; (M) two-tailed paired t test. ∗p < 0.05; ∗∗∗∗p < 0.0001. See also Figures S1–S4 and Movie S1.