Dual-Color Single-Cell Imaging of the Suprachiasmatic Nucleus Reveals a Circadian Role in Network Synchrony

Abstract

The suprachiasmatic nucleus (SCN) acts as a master pacemaker driving circadian behavior and physiology. Although the SCN is small, it is composed of many cell types, making it difficult to study the roles of particular cells. Here we develop bioluminescent circadian reporter mice that are Cre dependent, allowing the circadian properties of genetically defined populations of cells to be studied in real time. Using a Color-Switch PER2::LUCIFERASE reporter that switches from red PER2::LUCIFERASE to green PER2::LUCIFERASE upon Cre recombination, we assess circadian rhythms in two of the major classes of peptidergic neurons in the SCN: AVP (arginine vasopressin) and VIP (vasoactive intestinal polypeptide). Surprisingly, we find that circadian function in AVP neurons, not VIP neurons, is essential for autonomous network synchrony of the SCN and stability of circadian rhythmicity.

Keywords: Per2 gene; SCN; bioluminescence imaging; circadian rhythms; luciferase; neuronal coupling; neuronal network; suprachiasmatic nucleus.

Figure 1:. Dual-color bioluminescence imaging of VIP-Cre and AVP-Cre SCNs.

(A) Light-path of the dual-color imaging device. (B, C) Example time-lapse images for 48 hr from VIP-Cre and AVP-Cre SCN, respectively. Time interval between frames is three hours. SCN images were positioned with the ventral-dorsal axis vertically, optic chiasma at the bottom. Scalebar represents 100 μm. (D, E) Heat map representations of single-cell real-time imaging of two SCN slices for seven days. Each panel contains 40 single cells of each color in raster plots. The Z-score of detrended and denoised bioluminescence time series were color-coded between −2 to +2 as indicated by the color bar. Time series were ordered by phase with earlier phases placed at the top. (F, G) Rayleigh plots show the phases of single cells on day 6 and phases of green cells are shown relative to the normalized mean phase of red cells. Arrows are the R vector and dashed circles indicate the Rayleigh critical values. Individual data points are scattered as the circular plots. Asterisks indicate significant directional mean phase differences by Watson-Williams test (VIP, p value <0.001; AVP, p value=0.008). Mean +/− SEM period values from multiple slices and experiments (24.85±0.23 hrs, VIP; 24.58±0.15 hrs non-VIP; n=5 for VIP-Cre and 24.05±0.20hrs, AVP; 24.34±0.24, non-AVP; n=6 for AVP-Cre). Asterisks indicate the significance of t-test between green and red (* p ≤0.05; ** p ≤ 0.01; *** p ≤ 0.001). Detailed quantification results are included in Supplemental Data 1.

Figure 2:. Cell-type-specific effects of Bmal loss of function in VIP neurons.

(A, B) Single-cell trajectories from VIP-Cre and VIP-Bmal1^−/− (cKO) SCN through three conditions: pretreatment, 1μM TTX treatment and washout of TTX. (C, D) Heatmap representation of single-cell trajectories from VIP-Cre and VIP-Bmal1^−/− SCN through three conditions. (E, F) Single-cell phase distributions of a VIP SCN (E) and a VIP-Bmal1^−/− SCN (F). Sinusoid curve fitting was performed only on rhythmic cells, and phases on day 4 (midpoint) are presented in Rayleigh plots. Asterisks indicate significant directional mean phase differences by Watson-Williams test and pound signs indicate significant phase homogeneity or variance differences by Watson-Wheeler test. (G-H) Homogeneity of single-cell phase from multiple replicate SCN slices (VIP, n=3 and VIP-Bmal1^−/−, n=4), were evaluated with Mean +/− SEM circular variances. Two-way ANOVA found no effect of genotype across pretreatment, TTX treatment, and washout. One-way repeated measures ANOVA with Tukey’s multiple comparisons was used to assess the effects of pretreatment, TTX treatment, and washout in each cell type (VIP-Cre, non-VIP, VIP-Bmal1^−/−, and non-VIP-Bmal1^−/−). Asterisks and pound signs indicate significant differences (* or ^#, p ≤0.05; ** or ^##, p ≤ 0.01; *** or ^###, p v≤ 0.001). Detailed quantification results are included in Supplemental Data 1.

Figure 3:. Cell-type-specific effects of Bmal loss of function in AVP neurons.

(A, B) Single-cell trajectories from AVP-Cre and AVP-Bmal1^−/− (cKO) SCN through three conditions: pretreatment, 1μM TTX treatment and washout of TTX. (C, D) Heatmap representation of single-cell trajectories from AVP-Cre and an AVP-Bmal1^−/− SCN through three conditions. (E, F) Single-cell phase distributions of an AVP SCN (E) and an AVP-Bmal1^−/− SCN, (F). Sinusoid curve fitting was performed only on rhythmic cells, and phases on day 4 (midpoint) are presented in Rayleigh plots. Asterisks indicate significant directional mean phase differences by Watson-Williams test and pound signs indicate significant phase homogeneity or variance differences by Watson-Wheeler test. (G-H) Homogeneity of single-cell phase from multiple replicate SCN slices (AVP, n=5 and AVP-Bmal1^−/−, n=4), were evaluated with Mean +/− SEM circular variances. Two-way ANOVA revealed a main effect of genotype, and asterisks indicate the significant Holm-Sidak’s multiple comparisons between AVP-Cre and AVP-Bmal1^−/− across pretreatment, TTX treatment, and washout. One-way repeated measures ANOVA with Tukey’s multiple comparisons was used to assess the effects of pretreatment, TTX treatment and washout in each cell type (AVP-Cre, non-AVP, AVP-Bmal1^−/−, and non-AVP Bmal1^−/−). Asterisks and pound signs indicate significant differences (* or ^#, p ≤0.05; ** or ^##, p ≤ 0.01; *** or ^###, p ≤ 0.001). Detailed quantification results are included in Supplemental Data 1.

Figure 4:. Comparison of the effects of loss of Bmal1 on Coupling in VIP and AVP neurons in the SCN.

(A, B) Pairwise MIC score plots of neurons in the VIP SCN versus the VIP-Bmal1^−/− (cKO) SCN, during pretreatment, TTX treatment, and washout. As annotated in D, horizontal and vertical dashed lines split the plot into four parts, including the lower-left part showing the pair-wise MIC value among green cells, lower-right part is MIC values of green-red pairs and higher-right part is red-red pairs. Correlation network was constructed from pairwise MIC analysis: A connection line was drawn when the MIC value of the cell pair is larger than 0.8. Right-left SCN associations are not shown. (C) Mean +/− SEM MIC scores are shown to compare three types of associations between VIP-Cre and VIP-Bmal1^−/− SCNs. The associations were categorized as green-green (or KO-KO), red-red (or Cre-Cre) and green-red (or KO-Cre). Each mean MIC value from a replicate is represented by a point in the overlaid scatter plot (VIP, n=3; VIP-Bmal1^−/−, n=4). Higher asterisks represent the significance of genotype factor in two-way ANOVA, and lower asterisks represent significance of Holm-Sidak’s multiple comparisons. (E, F) Pairwise MIC score plots of neurons in the AVP SCN versus the AVP-Bmal1^−/− SCN, during pretreatment, TTX treatment, and washout. (G ) Mean +/− SEM of MIC scores are shown to compare three types of associations (as in C) between AVP-Cre and AVP-Bmal1^−/− SCNs (AVP, n=5; AVP-Bmal1^−/−, n=4). Asterisks indiciate significant differences (* p ≤0.05; ** p ≤ 0.01; *** p ≤ 0.001). Detailed quantification results are included in Supplemental Data 1.

Figure 5:. Immunofluorescent staining of key components of VIP and AVP signaling in VIP-Bmal1^−/−, AVP-Bmal1^−/− and VIP/AVP-Bmal1^−/− SCNs.

(A - D) Immunofluorescent images from 50μm thick SCN slices from wildtype control, VIP-Bmal1^−/− (cKO), AVP-Bmal1^−/−(cKO) and VIP/AVP-Bmal1^−/− (cKO) SCNs, stained with AVP, VIP, AVP receptor V1a and VIP receptor VPAC2 antibodies. Green signals from the neuropeptides or receptors are merged with the blue staining of nuclear DNA. Scale bars represent 100μm. (E-H) Mean +/− SEM plots of fluorescence intensity normalized to wildtype, VIP-Cre and AVP-Cre controls (one of each) are shown as the relative abundance. Each replicate is shown as a point in the overlaid scatter plots (all n=3). Asterisks indicate the significance of Holm-Sidak’s comparisons to controls and ANOVA adjusted p-value (* p ≤0.05; ** p ≤ 0.01; *** p ≤ 0.001).

Figure 6:. A stochastic circadian computation model to explain experimental observations.

(A) Computational model as the outcome of parameterization of the circadian circuits based on approximated ratios of SCN neurons (53 AVP cells, 27 VIP cells, and 170 non-AVP/non-VIP cells). The AVP:VIP coupling strength was set to 2.5:1. (B) Single-cell trajectories of each cell type. The signals represent relative expression level. Three models representing a wildtype, a VIP-Bmal1^−/− (cKO) or an AVP-Bmal1^−/− (cKO) SCN are shown. (C) Pairwise MIC scores represent six inter- or intra-class connections and correlations. X axis and Y axis represent the cell classes, and the pairing pattern of the classes is divided by dashed lines. (D) Proposed mechanism of the differential roles of VIP and AVP neurons in the SCN. Entrainment occurs under daylight and switches to free-running under constant darkness. Green color and minus signs indicate the excision of Bmal1 and ablation of autonomous circadian rhythms, while red color and plus signs indicate intact cells.