A circadian rhythm in the expression of PERIOD2 protein reveals a novel SCN-controlled oscillator in the oval nucleus of the bed nucleus of the stria terminalis

Abstract

Circadian rhythms in mammals are regulated not only globally by the master clock in the suprachiasmatic nucleus (SCN), but also locally by widely distributed populations of clock cells in the brain and periphery that control tissue-specific rhythmic outputs. Here we show that the oval nucleus of the bed nucleus of the stria terminalis (BNST-OV) exhibits a robust circadian rhythm in expression of the Period2 (PER2) clock protein. PER2 expression is rhythmic in the BNST-OV in rats housed under a light/dark cycle or in constant darkness, in blind rats, and in mice, and is in perfect synchrony with the PER2 rhythm of the SCN. Constant light or bilateral SCN lesions abolish the rhythm of PER2 in the BNST-OV. Large abrupt shifts in the light schedule transiently uncouple the BNST-OV rhythm from that of the SCN. Re-entrainment of the PER2 rhythm is faster in the SCN than in the BNST-OV, and it is faster after a delay than an advance shift. Bilateral adrenalectomy blunts the PER2 rhythm in the BNST-OV. Thus, the BNST-OV contains circadian clock cells that normally oscillate in synchrony with the SCN, but these cells appear to require both input from the SCN and circulating glucocorticoids to maintain their circadian oscillation. Taken together with what is known about the functional organization of the connections of the BNST-OV with systems of the brain involved in stress and motivational processes, these findings place BNST-OV oscillators in a position to influence specific physiological and behavioral rhythms downstream from the SCN clock.

Figure 1.

Expression of PER2 immunoreactivity in the BNST-OV. Top, Photomicrograph of a coronal brain section containing the BNST from a rat killed at ZT12. PER2 immunoreactivity is concentrated in the BNST-OV (marked by the black arrows). Expression in all other subdivisions of the BNST is exceedingly low. Scale bar, 200 μm. Bottom, High magnification of BNST-OV showing PER2 immunostaining. Scale bar, 100 μm. aco, Anterior commissure; av, anteroventral BNST; fu, fusiform nucleus of the BNST; al, anterolateral BNST; ju, juxtacapsular nucleus of the BNST; cc, anterolateral BNST central core; int, internal capsule; MS, medial septal nucleus; Lsv, ventral lateral septal nucleus; Lsi, intermediate part of the lateral septal nucleus; VL, lateral ventricle.

Figure 2.

Synchronous rhythmic expression of PER2 in the BNST-OV and SCN. a, Photomicrographs showing examples of PER2 immunostaining in the BNST-OV and SCN from rats housed under a 12 hr LD cycle and killed at different ZTs (ZT0, lights on; ZT12, lights off). Scale bar, 200μm. b, Mean ± SEM number of PER2-immunoreactive (Per2-IR Cells) nuclei in the SCN and BNST-OV of rats killed at different ZTs (n = 4 per time point). c, Mean ± SEM number of PER2-immunoreactive nuclei in the SCN and BNST-OV of rats that were housed in constant darkness and killed at different CTs (n = 4 per time point). d, Mean ± SEM number of PER2-immunoreactive nuclei in the SCN and BNST-OV of adult rats that were enucleated during the neonatal period and killed at CT0 or CT12 (n = 4 per time point).

Figure 3.

Synchronous expression of PER2 in the BNST-OV and SCN of the mouse. The photomicrographs show examples of PER2 immunostaining in the SCN and BNST-OV in mice housed under a 12 hr LD cycle and killed at ZT0 or ZT12. Arrows point to the BNST-OV. Scale bars: SCN, 100 μm; BNST-OV, 200 μm. The bar graphs show mean ± SEM number of PER2-immunoreactive nuclei in the SCN and BNST-OV as a function of ZT (n = 3 per time point).

Figure 4.

Bilateral SCN lesions that disrupt circadian activity rhythms abolish PER2 oscillations in the BNST-OV. a, Representative double-plotted actograms of wheel-running activity in an SCN-lesioned arrhythmic rat (left) and in a lesioned rat that remained rhythmic (right). The rats were housed under a 12 hr LD cycle. The vertical marks indicate periods of activity of at least 10 wheel revolutions per 10 min. Successive days are plotted from top to bottom. b, Photomicrographs showing the largest extent of the SCN lesions (arrows) of rats whose actograms are shown in a. Scale bar, 200μm. c, Photomicrographs showing examples of PER2 immunostaining in the BNST-OV from SCN-lesioned, arrhythmic rats (left) and from lesioned, rhythmic rats (right) as a function of ZT. Magnification, 20×. d, Mean ± SEM number of PER2-immunoreactive nuclei in the BNST-OV of SCN-lesioned arrhythmic rats (left) and of lesioned, rhythmic rats (right) as a function of ZT. The numbers in brackets indicate the number of rats per time point.

Figure 5.

Constant-light housing disrupts circadian activity rhythms and abolishes PER2 rhythms in the SCN and BNST-OV. a, Representative double-plotted actograms showing wheel-running activity in two rats housed in LL. b, Photomicrographs showing examples of PER2 immunoreactivity in the SCN and BNST-OV of LL-housed rats killed at four equally spaced times around the clock. Scale bar, 200μm. c, Mean ± SEM number of PER2-immunoreactive nuclei in the SCN (top) and BNST-OV (bottom) of LL-housed rats (n = 3–4 per time point).

Figure 6.

PER2 expression in the BNST-OV after unilateral SCN lesion. a, Photomicrographs showing examples of unilateral SCN lesions (arrows) in rats killed at ZT0 or ZT12. PER2 expression in the intact SCN is low at ZT0 and high at ZT12. Scale bar, 100 μm. b, Photomicrographs showing examples of PER2 immunoreactivity in the BNST-OV ipsilateral and contralateral to the lesioned SCN. Magnification, 20×. c, Mean ± SEM number of PER2-immunoreactive nuclei in the ipsilateral and contralateral BNST-OV as a function of ZT. Asterisks indicate significant differences from the ipsilateral side (p < 0.05; n = 5–7 per time point).

Figure 7.

Uncoupling between PER2 expression in the SCN and BNST-OV after a shift in the light cycle. a, Schematic representation of the experimental design. The rats were entrained to a 12 hr LD cycle. The onset of light was then advanced or delayed by 8 hr, and rats were killed 2, 4, 6, or 8 d later at the new ZT12. Representative actograms of rats from the advance and delay groups killed 8 d after the shift are shown. b, Mean ± SEM number of PER2-immunoreactive nuclei in the SCN and BNST-OV of rats killed at ZT12 without a shift (day 0) or 2, 4, 6, or 8 d after an advance shift at the new ZT12. c, Mean ± SEM number of PER2-immunoreactive nuclei in the SCN and BNST-OV of rats killed at ZT12 without a shift (day 0) or 2, 4, 6, or 8 d after a delay shift at the new ZT12. Asterisks indicate significant difference from day 0 for each brain area.

Figure 8.

Effect of adrenalectomy on PER2 expression in the SCN and BNST-OV. Mean ± SEM number of PER2-immunoreactive nuclei in the SCN (top) and BNST-OV (bottom) of adrenalectomized and sham-operated rats as a function of ZT (n = 5–7 per group per time point). Adrenalectomy had no effect on the rhythm of PER2 expression in the SCN, whereas it completely blunted the rhythm in the BNST-OV.