Calcium Circadian Rhythmicity in the Suprachiasmatic Nucleus: Cell Autonomy and Network Modulation

Abstract

Circadian rhythms of mammalian physiology and behavior are coordinated by the suprachiasmatic nucleus (SCN) in the hypothalamus. Within SCN neurons, various aspects of cell physiology exhibit circadian oscillations, including circadian clock gene expression, levels of intracellular Ca²⁺ ([Ca²⁺]_i), and neuronal firing rate. [Ca²⁺]_i oscillates in SCN neurons even in the absence of neuronal firing. To determine the causal relationship between circadian clock gene expression and [Ca²⁺]_i rhythms in the SCN, as well as the SCN neuronal network dependence of [Ca²⁺]_i rhythms, we introduced GCaMP3, a genetically encoded fluorescent Ca²⁺ indicator, into SCN neurons from PER2::LUC knock-in reporter mice. Then, PER2 and [Ca²⁺]_i were imaged in SCN dispersed and organotypic slice cultures. In dispersed cells, PER2 and [Ca²⁺]_i both exhibited cell autonomous circadian rhythms, but [Ca²⁺]_i rhythms were typically weaker than PER2 rhythms. This result matches the predictions of a detailed mathematical model in which clock gene rhythms drive [Ca²⁺]_i rhythms. As predicted by the model, PER2 and [Ca²⁺]_i rhythms were both stronger in SCN slices than in dispersed cells and were weakened by blocking neuronal firing in slices but not in dispersed cells. The phase relationship between [Ca²⁺]_i and PER2 rhythms was more variable in cells within slices than in dispersed cells. Both PER2 and [Ca²⁺]_i rhythms were abolished in SCN cells deficient in the essential clock gene Bmal1. These results suggest that the circadian rhythm of [Ca²⁺]_i in SCN neurons is cell autonomous and dependent on clock gene rhythms, but reinforced and modulated by a synchronized SCN neuronal network.

Keywords: Calcium imaging; PER2; circadian rhythm; luciferase imaging; suprachiasmatic nucleus.

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.