Pinealectomy does not affect diurnal PER2 expression in the rat limbic forebrain

A role for the pineal hormone, melatonin, in the regulation of the rhythmic expression of circadian clock genes is suggested by the finding that surgical removal of the pineal gland abolishes the rhythm of expression of clock genes such as Per1 in several neural and endocrine tissues in rodents, including the caudate-putamen (CP) and nucleus accumbens, the hypophyseal pars tuberalis and adrenal cortex. Pinealectomy has no effect on clock gene rhythms in the suprachiasmatic nucleus (SCN), the master circadian clock, as well as in the eyes and heart, indicating that the effect of melatonin on clock gene rhythms is tissue specific. To further study the role of melatonin in the regulation of the rhythm of clock genes, we assessed in rats the effect of pinealectomy on the rhythm of expression of the clock protein, PER2, in a number of key limbic forebrain structures, the oval nucleus of the bed nucleus of the stria terminalis (BNST-OV), the central nucleus of the amygdala (CEA) and the hippocampus (HIPP). Despite previous evidence showing that these regions are sensitive to melatonin, pinealectomy had no effect on the daily rhythm of expression of PER2 within these structures, further supporting the view that the role of endogenous melatonin in the regulation of clock gene expression is tissue specific

Comprehensive Mapping of Regional Expression of the Clock Protein PERIOD2 in Rat Forebrain across the 24-h Day

In mammals, a light-entrainable clock located in the suprachiasmatic nucleus (SCN) regulates circadian rhythms by synchronizing oscillators throughout the brain and body. Notably, the nature of the relation between the SCN clock and subordinate oscillators in the rest of the brain is not well defined. We performed a high temporal resolution analysis of the expression of the circadian clock protein PERIOD2 (PER2) in the rat forebrain to characterize the distribution, amplitude and phase of PER2 rhythms across different regions. Eighty-four LEW/Crl male rats were entrained to a 12-h: 12-h light/dark cycle, and subsequently perfused every 30 min across the 24-h day for a total of 48 time-points. PER2 expression was assessed with immunohistochemistry and analyzed using automated cell counts. We report the presence of PER2 expression in 20 forebrain areas important for a wide range of motivated and appetitive behaviors including the SCN, bed nucleus, and several regions of the amygdala, hippocampus, striatum, and cortex. Eighteen areas displayed significant PER2 rhythms, which peaked at different times of day. Our data demonstrate a previously uncharacterized regional distribution of rhythms of a clock protein expression in the brain that provides a sound basis for future studies of circadian clock function in animal models of disease

The expression of the clock protein PER2 in the limbic forebrain is modulated by the estrous cycle

Daily behavioral and physiological rhythms are linked to circadian oscillations of clock genes in the brain and periphery that are synchronized by the master clock in the suprachiasmatic nucleus. In addition, there are a number of inputs that can influence circadian oscillations in clock gene expression in a tissue-specific manner. Here we identify an influence on the circadian oscillation of the clock protein PER2, endogenous changes in ovarian steroids, within two nuclei of the limbic forebrain: the oval nucleus of the bed nucleus of the stria terminalis and central nucleus of the amygdala. We show that the daily rhythm of PER2 expression within these nuclei but not in the suprachiasmatic nucleus, dentate gyrus, or basolateral amygdala is blunted in the metestrus and diestrus phases of the estrus cycle. The blunting of the PER2 rhythm at these phases of the cycle is abolished by ovariectomy and restored by phasic estrogen replacement suggesting that fluctuations in estrogen levels or their sequelae are necessary to produce these effects. The finding that fluctuations in ovarian hormones have area-specific effects on clock gene expression in the brain introduces a new level of organizational complexity in the control of circadian rhythms of behavior and physiology

Restricted access to food, but not sucrose, saccharine, or salt, synchronizes the expression of Period2 protein in the limbic forebrain

Restricted feeding schedules (RF) in which daily access to food is limited to a few hours each day can entrain the rhythms of expression of circadian clock genes in the brain and periphery in rodents. The critical factors mediating the effect of RF on rhythms of clock gene expression are unknown. Previously, we demonstrated that daytime RF shifts the phase of expression of the clock protein, Period2 (PER2) in the oval nucleus of the bed nucleus of the stria terminalis in rats kept on a 12-h light/dark cycle, and restored the rhythm of PER2 expression in rats housed in constant light. We now report that RF also modifies the rhythms of PER2 expression in the central and basolateral nuclei of the amygdala and in the dentate gyrus, such that all three areas become synchronized, peaking 12 h after the time of food presentation. Daily limited access to sucrose or saccharine in freely fed rats or scheduled access to saline in sodium-deprived rats had no effect on these PER2 rhythms. Thus, it would appear that the rhythms of PER2 in limbic forebrain structures are sensitive to signals that arise from the alleviation of a negative metabolic state associated with scheduled feeding and that access to rewarding substances in the absence of food deprivation or metabolic challenges, per se, is not sufficient to alter the rhythms of PER2 expression in these regions

Hippocampus.

<p>Representative photomicrographs of PER2-ir cells in the dentate gyrus (<b>A–B</b>), CA1 (<b>D–E</b>), and CA3 (<b>G–H</b>) at ZT1 and ZT13, scale bar 100 µm. Mean number of PER2-immunoreactive cells in the DG (<b>C</b>), CA1 (<b>F</b>), and CA3 (<b>I</b>) of each individual rat (diamonds) across 48 <i>zeitgeber</i> times fitted with a 24-h sine wave. R² = Goodness of fit value for given region. <i>n</i> = 82 for DG, <i>n</i> = 84 for CA1 and CA3 (C: outliers = red diamonds).</p

Clustered phases of peak PER2 expression.

<p>Twenty-four hour circular diagram displaying peak PER2 expression in all 18 rhythmic regions analyzed. Numbers around the ‘clock’ are in ZT time. : SCN, □: amygdala, ⋄: hippocampus, ○: striatum, : cortex.</p

PER2 in the SCN.

<p>Representative photomicrographs of PER2-ir cells in the SCN every hour across the 24-h day. <i>Zeitgeber</i> (ZT) time 0 denotes lights on, ZT12 lights off. Scale bar 100 µm.</p

Suprachiasmatic nucleus.

<p>Graphs illustrating the temporal organization (a–c), phase relationship (d, e) and relative amplitudes (f) of <i>Per2</i>, <i>Bmal1</i> and <i>Dbp</i> mRNA rhythms in the SCN. The double plotted graphs show relative mRNA levels across 24 <i>zeitgeber</i> times fitted with a 24-h sine wave for <i>Per2</i> (a), <i>Bmal1</i> (b), and <i>Dbp</i> (c). Each point represents means ± SEM of mRNA data from 6–7 rats sampled every 30 min and grouped in consecutive 2-h intervals. R² = Goodness of fit value for sine wave. <i>n</i> = 73 for all.</p

Blocking experiment.

<p>Representative photomicrographs of the piriform cortex (<b>A, B</b>), striatum (<b>C, D</b>), and hippocampus (<b>E, F</b>), stained for PER2 without (<b>A, C, E</b>) and with (<b>B, D, C</b>) the PER2 peptide. Scale bar 100 µm for the piriform and striatum; 200 µm for the hippocampus.</p