Article Figures & Data
Figures
- Figure 1.
PER2 and [Ca2+]i dynamics of single dispersed SCN neurons. A, Time-lapse images of PER2 and [Ca2+]i imaged simultaneously in a single neuron. Time 0 is 4.3 d after start of imaging. PER2 and [Ca2+]i were reported by bioluminescence intensity of PER2::LUC and fluorescence intensity of GCaMP3, respectively. B, C, Representative patterns of PER2 and [Ca2+]i in single neurons. Shown are relative PER2 expression (black line, left axis) and [Ca2+]i (green line, right axis). Time 0 is start of imaging. Shown are a cell with a clear PER2 rhythm, but no [Ca2+]i rhythm (B) and a cell with rhythmic PER2 and [Ca2+]i (C). Values are calculated by the procedures described in Materials and Methods as well as in Fig. 1-1. Further examples of single cell traces are shown in Fig. 1-2. D, Percentages of cells categorized as having rhythmic PER2 or [Ca2+]i. Black and green portions show proportions of cells with clearly rhythmic PER2 and [Ca2+]i, respectively. Stippled white and light green portions show proportions of cells with nonrhythmic (or very weakly rhythmic) PER2 and [Ca2+]i, respectively. Numbers were rounded to the nearest 1%. E, A Rayleigh histogram showing differences between PER2 and [Ca2+]i rhythm peak time for individual neurons. Negative or positive values indicate that [Ca2+]i peak is leading or lagging PER2 peak, respectively. Length of bars indicates number of cells within each 1-h bin (n = 32 cells in 3 cultures). Arrow indicates a mean vector. F, Relationship between PER2 and [Ca2+]i RI in dispersed cells, with [Ca2+]i RI plotted against PER2 RI of the same cell (black dots). Green dots are exceptional cells categorized as rhythmic for [Ca2+]i but not PER2. The black dotted line is a guide line where PER2 and [Ca2+]i RI are equal. G, Simulation of the relationship between PER and [Ca2+]i amplitude in a mathematical model. [Ca2+]i amplitude is limited by PER amplitude.
- Figure 2.
PER2 and [Ca2+]i dynamics of single neurons in SCN slices. Representative images of PER2 (A) and [Ca2+]i (B) in an SCN slice. Positions of cells selected for data analysis were marked by white squares and a circle. C, Time-lapse images (at 4-h intervals) for the 50 × 50-µm area marked by the white circle in A and B. Time 0 is 3.7 d after start of imaging. D, Representative patterns of PER2 (black lines, left axis) and [Ca2+]i (green lines, right axis) for a single cell within an SCN slice, showing clear PER2 and [Ca2+]i rhythms. Time 0 is start of imaging. E, F, Comparisons of single SCN neurons in dispersed versus slice cultures, showing PER2 and [Ca2+]i rhythmicity (RI) (E) and coherence between PER2 and [Ca2+]i rhythms (F). Values are averages ± SEM, with numbers of cells shown on bars. *, p < 0.05; **, p < 0.01, mixed effect model. G, Rayleigh histogram showing the distribution of differences between PER2 and [Ca2+]i peak times for individual cells in SCN slices. Negative or positive values indicate that [Ca2+]i peak is leading or lagging PER2 peak, respectively. Length of bars indicates number of cells within each 1-h bin (n = 32 cells in three slices). Arrow indicates a mean vector. H–L, Spatiotemporal relationships between PER2 and [Ca2+]i peaks of five SCN slices. H, I, For each of the five slices, cell-like regions with significant rhythmicity in both PER2 and [Ca2+]i are plotted as circles, with peak phases color-coded as in L. PER2 (H) or [Ca2+]i (I) peak times are shown relative to the PER2 peak time of the whole slice, with negative values indicating phase leading of cell-like regions. J, Peak time differences between PER2 and [Ca2+]i are also shown, color-coded as in L. Negative values indicating phase leading of [Ca2+]i peaks relative to PER2 peaks in the same cell-like regions. K, Rayleigh histograms show corresponding distributions of these peak time differences. Numbers of cell-like regions: 267, 238, 149, 331, and 179 in slices 1–5, respectively. Bar length indicates the proportion of cells in each bin. Arrows indicate mean vectors, all of which extend outside the small inner circles marking criterion levels for statistical significance (Rayleigh test, p < 0.01), indicating that the distributions are all significantly different from uniform.
- Figure 3.
Effects of TTX on SCN neurons in slice and dispersed cultures. Representative patterns of PER2 and [Ca2+]i for a cell in an SCN slice (A) and a dispersed cell (B). Each plot shows relative levels of PER2 expression (black lines, left axis) and [Ca2+]i (green lines, right axis) for a single SCN neuron. Time 0 is start of imaging. Black bar indicates duration of TTX application. In SCN slices, PER2 expression and rhythmicity decreased during TTX in all cells, and [Ca2+]i rhythmicity also decreased significantly on average, whereas in dispersed cells TTX had no significant effects. Shown here, A is a cell in a slice for which the PER2 rhythm damped substantially and the [Ca2+]i rhythm damped more modestly during TTX, and B is a dispersed cell in which TTX had no discernible effect on either rhythm. C–H, Bar graphs of RI (C, F), PER2 expression (D, G), and coherence between PER2 and [Ca2+]i rhythms (E, H) before, during, and after TTX application, for cells in slices and dispersed cells, respectively. All values shown are averages ± SEM, with numbers of cells shown on bars. **, p < 0.01; ns, not significant (mixed effect model compared to before TTX application). Effects of ryanodine are shown in Fig. 3-1. I, J, Simulations of PER expression (black lines, left axis) and [Ca2+]i (green lines, right axis) for a single cell in a multicellular model (I) and a single-cell model (J). Black bars indicate duration of simulated TTX application. In the multicellular model, PER and [Ca2+]i levels and rhythmicity decreased during TTX, whereas in the single-cell model, TTX had no effect.
- Figure 4.
A, PER2 and [Ca2+]i patterns of a representative cell in a Bmal1–/– SCN slice culture. Relative levels of PER2 (black lines, left axis) and [Ca2+]i (green lines, right axis) are shown. Time 0 is start of imaging. B, Comparison of average RI values for PER2 rhythms (black bars) and [Ca2+]i rhythms (green bars) for cells in WT and Bmal1–/– SCN slices. C, Coherence between PER2 and [Ca2+]i rhythms. **, p < 0.01, t test. All values shown are averages ± SEM, with numbers of cells shown on bars.
Extended Data
Figure 1-1
Normalization of FL intensity of cells using background. (A) Example of a whole field of view of [Ca2+]i reported by GCaMP3. Note the considerable variation across the image in intensity of background areas devoid of cells (arrow heads 1–6). (B) Examples of fluctuation of background intensity over time, showing intensities of background (BG) areas indicated by arrow heads in A. CCD camera and software had to be reset at ∼5.7 d after start of imaging. (C-D) Linear correlation between intensity of BGn and coefficient An (C) or constant Bn (D) (for details, see Materials and Methods). An and Bn values for background regions (blue diamonds) could be used to extrapolate corresponding values for cell-containing regions of higher intensities (Acell, Bcell, magenta diamond), and from these to calculate an expected background intensity value for each cell. (E-F) Patterns of estimated background (blue) and raw FL intensity (black) for two representative cells, one non-rhythmic (E, cell1) and the other rhythmic (F, cell2). (G) Ratios of raw FL intensity to expected BG for cell1 (black) and cell2 (green). (H) Ratios shown in G after detrending by subtracting a 24 h running average. Download Figure 1-1, EPS file.
Figure 1-2
Additional plots of PER2 (black lines, left axis) and [Ca2+]i (green lines, right axis) for SCN cells exhibiting various patterns of [Ca2+]i. Shown at left are cells in dispersed cultures (A-E), including a cell with a sinusoidal [Ca2+]i rhythm (A), a cell with a [Ca2+]i rhythm showing a secondary peak (B), an initially non-rhythmic cell with spontaneous recovery of both PER2 and [Ca2+]i rhythms (C), and cells in which the [Ca2+]i rhythm became weaker (D) or stronger (E) during TTX. Shown at right are cells in SCN slice cultures (F-J), including a cell with a sinusoidal [Ca2+]i rhythm (F), a cell with a [Ca2+]i rhythm showing a secondary peak (G), a cell with an unusually phased [Ca2+]i rhythm peaking after PER2 (H), a cell in which TTX had no discernible effect on the [Ca2+]i rhythm (I), and a cell in which the [Ca2+]i rhythm was weaker during TTX (J). Download Figure 1-2, EPS file.
Figure 3-1
Effects of ryanodine on PER2 and [Ca2+]i rhythm in dispersed SCN cells. (A) PER2 and [Ca2+]i patterns of a representative cell in a dispersed cell culture. Relative levels of PER2 (black lines, left axis) and [Ca2+]i (green lines, right axis) are shown. Time 0 is start of imaging. (B) Comparison of average RI values for PER2 rhythms (black bars) and [Ca2+]i rhythms (green bars) for cells before and during 100 μM ryanodine application. n.s. p > 0.05, mixed effect model. Download Figure 3-1, EPS file.
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