In-phasic cytosolic-nuclear Ca2+ rhythms in suprachiasmatic nucleus neurons

Abstract

The suprachiasmatic nucleus (SCN) of the hypothalamus is the master circadian clock in mammals. SCN neurons exhibit circadian Ca²⁺ rhythms in the cytosol, which is thought to act as a messenger linking the transcriptional/translational feedback loop (TTFL) and physiological activities. Transcriptional regulation occurs in the nucleus in the TTFL model, and Ca²⁺-dependent kinase regulates the clock gene transcription. However, the Ca²⁺ regulatory mechanisms between cytosol and nucleus as well as the ionic origin of Ca²⁺ rhythms remain unclear. In the present study, we monitored circadian-timescale Ca²⁺ dynamics in the nucleus and cytosol of SCN neurons at the single-cell and network levels. We observed robust nuclear Ca²⁺ rhythm in the same phase as the cytosolic rhythm in single SCN neurons and entire regions. Neuronal firing inhibition reduced the amplitude of both nuclear and cytosolic Ca²⁺ rhythms, whereas blocking of Ca²⁺ release from the endoplasmic reticulum (ER) via ryanodine and inositol 1,4,5-trisphosphate (IP₃) receptors had a minor effect on either Ca²⁺ rhythms. We conclude that the in-phasic circadian Ca²⁺ rhythms in the cytosol and nucleus are mainly driven by Ca²⁺ influx from the extracellular space, likely through the nuclear pore. It also raises the possibility that nuclear Ca²⁺ rhythms directly regulate transcription in situ.

Keywords: SCN; circadian clock; imaging; intracellular Ca2+; nucleus; organelle.

Figure 1

Expression Patterns of the Ca²⁺ Probes in the SCN Slices. (A–B) Expression patterns of the probes of NLS-GCaMP6s (A) and NES-jRGECO1a (B) in the whole SCN (left) and in dorsal and ventral SCN subregions (right). ROIs are indicated by yellow squares in the SCN images. Images are NLS-GCaMP6s or NES-jRGECO1a (left), DAPI (middle), and the merged one (right), respectively. 3 V, third ventricle; OC, optic chiasm.

Figure 2

Dual-color imaging of the Ca²⁺ Dynamics in the nucleus and cytosol of SCN Neurons. (A) Expression patterns of NLS-GCaMP6s (upper) and NES-jRGECO1a (lower) in the SCN. 3 V, third ventricle; OC, optic chiasm. (B) Two-hourly montage images of two representative individual SCN neurons, as indicated by arrowheads in panel (A). The leftmost images show the edge of single SCN neuron. (C) Raw traces of the Ca²⁺ dynamics of the nucleus (green) and the cytosol (magenta) of two representative SCN neurons. (D) Acrophase maps of the Ca²⁺ rhythms of the nucleus (left) and the cytosol (right). The mean acrophase of the entire SCN region was normalized to zero. Color bars indicate the relative time of day (hours). (E) Phase-difference map between the nuclear and cytosolic Ca²⁺ rhythms. (F) Histogram of the pixel distribution of the phase-difference maps (normalized to the total pixels). The frequency (y-axis) represents the value relative to the total number of pixels. Individual SCN data are represented by different colors (n = 5 slices).

Figure 3

Effect of Ca²⁺ release inhibitors from the ER on the nuclear and cytosolic Ca²⁺ Rhythms. (A–D) Representative traces of the Ca²⁺ rhythms in the nucleus (green) and cytosol (magenta). The upper and lower traces in each panel show raw and 24-h detrended data (smoothed with a 3-h moving average), respectively. (A) Ryanodine receptor blocker, dantrolene (10 μM) (n = 5 slices). (B) Ryanodine receptor blocker, high concentration of ryanodine (100 μM) (n = 6 slices). (C) IP₃ receptor blocker, Xestospongin C (10 μM) (n = 6 slices). (D) Sodium channel blocker, TTX (1 μM) (n = 5 slices). (E–F) Summary of blocker effects on the amplitude (E) and trough (F) of the nuclear (green) and cytosolic (magenta) Ca²⁺ rhythms. Data are expressed as percentage of the pretreatment value. One-sample t-test was employed to evaluate the blocker effects. *p < 0.05; **p < 0.01; ***p < 0.001. All data are expressed as mean ± SD.