Temporal expression of seven clock genes in the suprachiasmatic nucleus and the pars tuberalis of the sheep: evidence for an internal coincidence timer

Abstract

The 24-h expression of seven clock genes (Bmal1, Clock, Per1, Per2, Cry1, Cry2, and CK1 epsilon ) was assayed by in situ hybridization in the suprachiasmatic nucleus (SCN) and the pars tuberalis (PT) of the pituitary gland, collected every 4 h throughout 24 h, from female Soay sheep kept under long (16-h light/8-h dark) or short (8-h light/16-h dark) photoperiods. Locomotor activity was diurnal, inversely related to melatonin secretion, and prolactin levels were increased under long days. All clock genes were expressed in the ovine SCN and PT. In the SCN, there was a 24-h rhythm in Clock expression, in parallel with Bmal1, in antiphase with cycles in Per1 and Per2; there was low-amplitude oscillation of Cry1 and Cry2. The waveform of only Per1 and Per2 expression was affected by photoperiod, with extended elevated expression under long days. In the PT, the high-amplitude 24-h cycles in the expression of Bmal1, Clock, Per1, Per2, Cry1, and Cry2, but not CK1 epsilon, were influenced by photoperiod. Per1 and Per2 peaked during the day, whereas Cry1 and Cry2 peaked early in the night. Hence, photoperiod via melatonin had a marked effect on the phase relationship between Per/Cry genes in the PT. This supports the conclusion that an "external coincidence model" best explains the way photoperiod affects the waveform of clock gene expression in the SCN, the central pacemaker, whereas an "internal coincidence model" best explains the way melatonin affects the phasing of clock gene expression in the PT to mediate the photoperiodic control of a summer or winter physiology.

Figure 1

Double-plotted actogram of group-based locomotor activity in Soay sheep. (a) Long-day group (LD 16:8). (b) Short-day group (LD 8:16). The histograms illustrate blood plasma melatonin concentrations collected every 4 h throughout 24 h (ZT 0 = lights on). Horizontal bars indicate time of lights-on (open) and lights-off (filled).

Figure 2

(a) Representative peak (+) and nadir (−) autoradiograms of Bmal1, Clock, Per1, and Cry1 in situ hybridization in coronal sections of the SCN from Soay sheep acclimated to long days (ZT 0 = lights-on). (b) Profiles (24 h) of the expression of Bmal1 and Clock (Top), Per1 and Per2 (Middle), and Cry1 and Cry2 (Bottom) (mean ± SEM) in the SCN in Soay sheep under long days (LD 16:8, Left) and short days (LD 8:16, Right). Data from the repeated ZT 19 samplings are merged into a single time point, and the ZT 3 time point is double-plotted. Horizontal bars indicate time of lights-on (open) and lights-off (filled) (ZT 0 = lights-on). OC, optic chiasm; PeVN, periventricular nucleus.

Figure 3

(a) Representative peak (+) and nadir (−) autoradiograms of Bmal1, Clock, Per1, and Cry1 in situ hybridization in coronal sections of the mediobasal hypothalamus, with the rostral PT attached from Soay sheep acclimated to long days (ZT 0 = lights-on). (b) Profiles (24 h) of the expression of Bmal1 and Clock (Top), Per1 and Per2 (Middle), and Cry1 and Cry2 (Bottom) (mean ± SEM; when error bars are small, they may be contained within the symbol) in the PT in Soay sheep under long days (LD 16:8, Left) and short days (LD 8:16; Right). Data from the repeated ZT 19 samplings are merged into a single time point, and the ZT 3 time point is double-plotted. Horizontal bars indicate time of lights-on (open) and lights-off (filled) (ZT 0 = lights-on). ME, median eminence.

Figure 4

Double plots of 24-h profiles of expression of Per1 (○) and Cry2 (■, mean ± SEM, expressed as % of maximum) in the ovine SCN (a) and PT (b) under long days (LD 16:8) and short days (LD 8:16). Note that in the light-responsive SCN, photoperiod caused a change in waveform in Per1, but not in Cry2, whereas in the melatonin-responsive PT, photoperiod caused a marked change in the phase relationship in peak expression of Per1 and Cry2 (ψ). Horizontal bars indicate time of lights-on (open) and lights-off (filled).