Diurnal Corticosterone Presence and Phase Modulate Clock Gene Expression in the Male Rat Prefrontal Cortex

Abstract

Mood disorders are associated with dysregulation of prefrontal cortex (PFC) function, circadian rhythms, and diurnal glucocorticoid (corticosterone [CORT]) circulation. Entrainment of clock gene expression in some peripheral tissues depends on CORT. In this study, we characterized over the course of the day the mRNA expression pattern of the core clock genes Per1, Per2, and Bmal1 in the male rat PFC and suprachiasmatic nucleus (SCN) under different diurnal CORT conditions. In experiment 1, rats were left adrenal-intact (sham) or were adrenalectomized (ADX) followed by 10 daily antiphasic (opposite time of day of the endogenous CORT peak) ip injections of either vehicle or 2.5 mg/kg CORT. In experiment 2, all rats received ADX surgery followed by 13 daily injections of vehicle or CORT either antiphasic or in-phase with the endogenous CORT peak. In sham rats clock gene mRNA levels displayed a diurnal pattern of expression in the PFC and the SCN, but the phase differed between the 2 structures. ADX substantially altered clock gene expression patterns in the PFC. This alteration was normalized by in-phase CORT treatment, whereas antiphasic CORT treatment appears to have eliminated a diurnal pattern (Per1 and Bmal1) or dampened/inverted its phase (Per2). There was very little effect of CORT condition on clock gene expression in the SCN. These experiments suggest that an important component of glucocorticoid circadian physiology entails CORT regulation of the molecular clock in the PFC. Consequently, they also point to a possible mechanism that contributes to PFC disrupted function in disorders associated with abnormal CORT circulation.

Figure 1.

Experimental timelines. A, Experiment 1. Starting 2 days after ADX or sham surgery, rats were given 10 daily ip injections of either vehicle or CORT (2.5 mg/kg) at ZT1. On the 11th day, rats were killed at ZT0, ZT6, ZT12, or ZT18 (n = 6 per time of death for each CORT status condition). B, Experiment 2. Starting 2 days after ADX surgery, rats were given 13 daily ip injections of either vehicle or CORT (2.5 mg/kg) at either ZT1 (antiphasic) or ZT11 (in-phase). On the 14th day, rats were killed at ZT0, ZT6, ZT12, or ZT18 (n = 4 or n = 6 per time of death for each daily vehicle or CORT treatment time, respectively).

Figure 2.

Experiment 1. Diurnal CORT profile in sham-ADX rats. There was a prominent diurnal CORT peak at ZT12 in trunk blood from sham-ADX rats (n = 6). Plasma CORT levels were undetectable in trunk blood of ADX and ADX+antiphasic CORT-treated rats. The black bar above the x-axis denotes dark phase.

Figure 3.

Experiment 1. Effect of ADX and daily antiphasic CORT treatment on clock gene expression in PFC and SCN. In sham rats, Per1, Per2, and Bmal1 mRNA expression exhibited a diurnal pattern in the PFC and SCN, but the phase of peak expression differed between the PFC and SCN. ADX shifted the time of peak Per1 and Per2 mRNA expression and disrupted Bmal1 mRNA diurnal expression pattern in the PFC. Ten days of antiphasic CORT disrupted Per1, Per2, and Bmal1 mRNA diurnal expression. There was no effect of CORT status in the SCN. The black bar above the x-axis denotes dark phase. #, sham vs ADX difference at that ZT, P < .05; *, sham vs antiphasic CORT difference at that ZT P < .05 (FLSD, n = 4–6). Brain ROIs used for analyses are depicted on brain atlas images adapted from Paxinos and Watson (50).

Figure 4.

Experiment 1. Representative autoradiogram images of Per1, Per2, and Bmal1 mRNA (in situ hybridization) in the PFC (coronal section) and SCN (ventral portion of coronal section) of sham and ADX rats across 4 times of day (ZT0, ZT6, ZT12, and ZT18). Note the different phase relationships of peak clock gene expression in the PFC of sham vs ADX rats but similar phase relationships of both treatment groups in the SCN.

Figure 5.

Experiment 2. Effect of daily in-phase or antiphasic CORT treatment on clock gene expression in PFC and SCN of ADX rats. All rats were ADX and treated for 13 days with either vehicle or CORT at either ZT11 (in-phase CORT) or ZT1 (antiphasic CORT). In vehicle-treated rats (regardless of the time of daily injection) (Supplemental Figure 1) Per1, Per2, and Bmal1 mRNA in PFC subregions either lacked a significant diurnal expression pattern or the time of peak expression was shifted relative to sham rats in experiment 1, whereas 13 days of in-phase CORT treatment normalized PFC diurnal clock gene expression patterns compared with sham rats (Figure 3). Thirteen days of antiphasic CORT substantially disrupted Per1, Per2, and Bmal1 diurnal expression, and in the IL and VO, it inverted the diurnal expression profile of Per2. There were only minor effects of CORT status in the SCN. The black bar above the x-axis denotes dark phase. #, in-phase vs vehicle difference at that ZT, P < .05; *, in-phase vs antiphasic CORT difference at that ZT, P < .05 (FLSD, n = 4–8).

Figure 6.

Experiment 2. Representative autoradiogram images of Per1, Per2, and Bmal1 mRNA (in situ hybridization) in the PFC (coronal section) and SCN (ventral portion of coronal section) of in-phase CORT and antiphasic CORT-treated rats across 4 times of day (ZT0, ZT6, ZT12, and ZT18). Note the absent or phase shifted relationships of diurnal clock gene expression in the PFC of antiphasic CORT rats compared with in-phase CORT rats but similar phase relationships of both treatment groups in the SCN.

Figure 7.

Phase comparisons of peak Per1, Per2, and Bmal1 mRNA diurnal expression within the PFC (most typical timing across all subregions is depicted) (Supplemental Figure 2) and SCN of each treatment group in experiments 1 and 2. Circles are oriented as clock faces with ZT labels, and shading of the dark phase. Time of peak expression is shown only where a significant effect of time of death was observed (Tables 1 and 2). Note in the SCN the lack of effect of CORT manipulations on clock gene expression phase relationships, but in the PFC the dramatic effect relative to sham rats of all manipulations except daily in-phase CORT treatment of ADX rats.