Circadian modulation of calcium levels in cells in the suprachiasmatic nucleus

Abstract

There is reason to believe that resting free calcium concentration [Ca2+]i in neurons in the suprachiasmatic nucleus (SCN) may vary with the circadian cycle. In order to start to examine this hypothesis, optical techniques were utilized to estimate resting Ca2+ levels in SCN cells in a rat brain slice preparation. [Ca2+]i measured from the soma was significantly higher in the day than in the night. Animals from a reversed light-dark cycle were used to confirm that the phase of the rhythm was determined by the prior light-dark cycle. The rhythm in Ca2+ levels continued to be expressed in tissue collected from animals maintained in constant darkness, thus confirming the endogenous nature of this variation. Interestingly, the rhythm in Ca2+ levels was not observed when animals were housed in constant light. Finally, the rhythm in Ca2+ levels was prevented when slices were exposed to tetrodotoxin (TTX), a blocker of voltage-sensitive sodium channels. Similar results were obtained with the voltage-sensitive Ca2+ channel blocker methoxyverapamil. These observations suggest a critical role for membrane events in driving the observed rhythm in Ca2+. Conceptually, this rhythm can be thought of as an output of the circadian oscillator. Because [Ca2+]i is known to play a critical role in many cellular processes, the presence of this rhythm is likely to have many implications for the cell biology of SCN neurons.

FIG. 1

Neurons in SCN brain slices visualized by IR DIC video microscopy. Left: image of SCNunder lower power magnification. Scale bar, 100 μM. 3V, third ventricle; OC, optic chiasm. Right: higher power view of same slice. Scale bar, 10 μm. This technology allows a clear view of soma and, in some cases, processes of SCNcells. In the present study, this technology was used primarily to identify cells in the SCNregion for further analysis and to exclude cells from the surrounding hypothalamic regions. Tissue from 14-day-old rat.

FIG. 2

SCNcells in brain slice loaded with the Ca²⁺ indicator dye fura2. Left: SCNcells in brain slice preparation bulk loaded with the fura2 AM. Right: SCNcell loaded with fura2 salt through the patch pipette. Cells were excited at 380 nm and images were over-exposed to aid in visualization of the filled cells. Rats were 14 days old.

FIG. 3

Diurnal rhythm of resting Ca²⁺ levels in cells in the SCN. In these experiments, resting Ca²⁺ levels were estimated in SCNneurons in brain slices from animals during their day and compared with data obtained from brain slices from animals during their night. Animals were killed at either ZT 0 for the day group or ZT 12 for the night group. Each cell is sampled only once. Top panels: histograms showing that Ca²⁺ levels in SCNcells peak during the day. [Ca²⁺]_i was estimated to be 135 ± 6 nM (n = 238) for the day group and 62 ± 3 nM (n = 180) for the night group (P < 0.001). When the phase of the light-dark cycle to which the animals were exposed was reversed, so did the resulting rhythm. This result demonstrates that the observed daily variation is determined by the phase of the LD cycle and not some other unknown variable. Middle panel: histograms illustrating the daily variation in the distribution of Ca²⁺ levels. Bottom panel: histograms illustrating average Ca²⁺ levels as a function of time from which the data were collected.

FIG. 4

Circadian rhythm of resting Ca²⁺ levels in cells in the SCN. In these experiments animals were maintained in constant conditions and resting Ca²⁺ levels were estimated in SCNneurons in brain slices from animals during their subjective day and compared with data obtained from brain slices from animals during their subjective night. Left panel: when animals were maintained in constant dark, the rhythm in Ca²⁺ levels in SCNcells continues to be observed with phase determined by prior LD cycle. Right panel: when animals were placed in bright constant light for 10-14 days, the rhythm in Ca²⁺ levels was no longer observed. The observations that the rhythm continues in constant darkness, but not in constant light, strongly suggest that the rhythm in [Ca²⁺]_i is circadian.

FIG. 5

Daily rhythm in Ca²⁺ levels was blocked by application of ion channel inhibitors. In these experiments, resting Ca²⁺ levels were estimated in SCN neurons in brain slices from animals during their day and compared with data obtained from brain slices from animals during their night. Left panel: in the presence of TTX, the rhythm in Ca²⁺ was not observed (day, 81 ± 2 nM, n = 131; night, 78 ± 4 nM, n = 69). Right panel: in the presence of methoxyverapamil, the rhythm in Ca²⁺ was not observed (day, 76 ± 4 nM, n = 33; night, 68 ± 3 nM, n = 45). In both cases, it was the higher day levels that were significantly inhibited (P < 0.001) by application of these inhibitors, while the night levels were not significantly impacted. The net result was the loss of the rhythm in the presence of these ion channel inhibitors.