ORIGINAL
RESEARCH
Diurnal Corticosterone Presence and Phase Modulate
Clock Gene Expression in the Male Rat Prefrontal
Cortex
Elizabeth R. Woodruff, Lauren E. Chun, Laura R. Hinds, and Robert L. Spencer
Department of Psychology and Neuroscience, University of Colorado Boulder, Boulder, Colorado 80309
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. (Endocrinology 157: 1522–1534, 2016)
O
ptimal organismal function depends on the integrity
of the circadian system (1). Circadian misalignment
is associated with many physiological and psychological
disease states including obesity, diabetes, some forms of
cancer, cardiovascular disease, posttraumatic stress disorder (PTSD), and depression (2– 4). The hypothalamic
suprachiasmatic nucleus (SCN) is an autonomous circadian oscillator that is primarily entrained by the light-dark
cycle and coordinates circadian function throughout the
entire body (5).
Circadian rhythms are generated by the transcriptional/translational feedforward and feedback cycles of
clock gene expression present not only in the SCN, but
also in many other cells throughout the body. This molecular clock is comprised of a positive arm (Bmal1 and
Clock gene products) which induces the transcription of
a negative arm (Period and Cryptochrome genes) whose
protein products inhibit their own production by preventing the actions of Brain and muscle ARNT-like protein (BMAL1) and circadian locomotor output cycles
kaput (CLOCK) proteins (6). REV-ERB␣ and retinoic
acid-like orphan receptor modulate the cycle by inhibiting and inducing Bmal1 transcription, respectively
(7). The completion of this cycle takes approximately 24
hours (6). Although many extra-SCN tissues show oscillations in core clock gene expression, the phase of
ISSN Print 0013-7227 ISSN Online 1945-7170
Printed in USA
Copyright © 2016 by the Endocrine Society
Received October 17, 2015. Accepted February 17, 2016.
First Published Online February 22, 2016
Abbreviations: ACC, anterior cingulate cortex; ADX, adrenalectomized; BMAL, brain and
muscle ARNT-like protein; CLOCK, circadian locomotor output cycles kaput; CORT, corticosterone; FLSD, Fisher’s least significant difference; GR, glucocorticoid receptor; GRE,
glucocorticoid-response element; IL, infralimbic cortex; MDD, major depressive disorder;
nt, nuclear transcript; Per1, Period1; PFC, prefrontal cortex; PL, prelimbic cortex; PTSD,
posttraumatic stress disorder; ROI, region of interest; Rev-erb, NR1D1; SCN, suprachiasmatic nucleus; VO, ventral orbital cortex; ZT, zeitgeber time.
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Endocrinology, April 2016, 157(4):1522–1534
doi: 10.1210/en.2015-1884
doi: 10.1210/en.2015-1884
expression (ie, time of peak and trough expression) varies in a tissue specific manner (8 –10). The SCN is necessary to maintain synchronized rhythmic clock gene
expression throughout the body (11–13), but because it
provides virtually no direct innervation to peripheral
and central tissues outside of the hypothalamus (14), its
mechanism of transduction remains to be established.
In recent years, corticosterone (CORT), the principal
glucocorticoid hormone in rodents, has been found to
serve as a clock gene expression entraining factor in the
liver, kidney, lung, cornea, and salivary gland (8, 15). Glucocorticoid receptors (GRs) are ubiquitously expressed
throughout the body with the noted exception of the SCN
(16). The core clock gene Period1 (Per1) has a functional
glucocorticoid-response element (GRE) in its promoter region (17–19) and is modulated by CORT in both central
(20, 21) and peripheral tissues (8, 16, 22, 23). CORT has
also been shown to directly modulate Per2 (18, 19) and
Rev-erb␣ (24) mRNA expression in some peripheral cell
types. CORT circulation is normally entrained by the SCN
(25) and shows a robust diurnal rhythm with peak levels
occurring around the time of waking (26). CORT therefore is a promising candidate as an entraining factor in
extra-SCN tissues. Interestingly, normal diurnal and
stress-induced CORT release also relies on the presence of
a functional molecular clock within the adrenal gland itself (27, 28), supporting the importance of the interplay
and communication between the circadian and glucocorticoid systems.
Although CORT-dependent clock gene entrainment
has been extensively examined in the periphery, limited
research has examined this process in the brain (20). The
prefrontal cortex (PFC) is a key neural center for regulation of the emotional and physiological response to stress
(29, 30). Many PFC-dependent functions such as attention (31), mood (1, 32), and conditioned fear extinction
learning (33, 34) show diurnal variation. In addition, dendritic branching (which is also CORT-dependent) (35) of
pyramidal cells in the infralimbic subregion of the PFC
exhibits diurnal variation (36). PFC dysfunction is associated with depression and PTSD (37–39). These disorders
are also characterized by impaired circadian function parameters such as sleep, diurnal glucocorticoid circulation,
and PFC clock gene rhythmicity (40, 41). Chronic unpredictable stress can disrupt clock gene oscillation in brain
regions associated with mood regulation (42), and successful treatment of psychiatric disorders is frequently associated with corrected circadian abnormalities (43– 45).
Interestingly, increasing the amplitude of diurnal CORT
circulation in mice has anxiolytic effects (46). To date
there has been no examination of CORT-dependent modulation of clock gene expression in the brain encompassing
press.endocrine.org/journal/endo
1523
both the positive and negative arms of the molecular clock.
We therefore examined the diurnal expression of 3 core
clock genes (Per1, Per2, and Bmal1) in 4 subregions of the
PFC (anterior cingulate cortex [ACC], prelimbic cortex
[PL], infralimbic cortex [IL], and ventral orbital cortex
[VO]) and the SCN under different chronic CORT conditions. In experiment 1, we compared these expression
profiles in adrenal intact rats, adrenalectomized (ADX)
rats, as well as ADX rats injected with CORT every day for
10 days at the opposite time of day (zeitgeber time [ZT]1)
as the endogenous CORT peak (ZT12) (antiphasic
CORT). In experiment 2, we compared the diurnal expression of the same clock genes in ADX rats, ADX rats
treated with antiphasic CORT, and ADX rats treated with
in-phase CORT (ie, a CORT injection at the time of the
endogenous peak) daily for 13 days. We have previously
found in adrenal intact male and female rats that all 3 clock
genes have a fluctuating daily (“diurnal”) expression pattern in the PFC with peak expression of the positive arm
(Bmal1) approximately antiphasic to the negative arm
(Per1 and Per2) (10). We hypothesized that the normal
phase relationship and robustness of 24-hour clock gene
expression in the PFC depends on the presence of in-phase
diurnal CORT. Consequently, we predicted that the
phase, and possibly even the presence of diurnal PFC clock
gene expression, would be altered by the absence of circulating CORT (ADX) and would be substantially disrupted in ADX rats treated with antiphasic CORT. We
also predicted that clock gene expression in the SCN
would not be affected by CORT status due to the SCN’s
lack of GR expression.
Materials and Methods
Animals
For experiment 1 (N ⫽ 72) and experiment 2 (N ⫽ 80) male
Sprague-Dawley rats (250 –280 g; Harlan Laboratories) were
housed 2 per cage (polycarbonate tubs, 47 ⫻ 23 ⫻ 20 cm) and
given food (Teklad Rodent Diet 8640; Harlan Laboratories) and
either tap water (sham rats) or 0.9% saline (ADX rats) ad libitum. All rats were maintained on a 12-hour light, 12-hour dark
cycle and were given either a 3-day (experiment 1) or 2 week
(experiment 2) acclimation period before surgery. The time of
manipulations and measures for each rat is expressed as ZT, the
time (hours) after light phase onset. Rats were housed in 4 separate sound and light attenuated rooms to ensure proper entrainment to the light-dark cycle. All experiments were conducted in accordance to the guidelines found within the Guide for
the Care and Use of Laboratory Animals (DHHS publication
number [NIH] 80-23, revised 2010, 8th edition) and were approved by the University of Colorado Institutional Animal Care
and Use Committee.
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CORT Regulates PFC Clock Gene mRNA
Endocrinology, April 2016, 157(4):1522–1534
ADX surgery, rats received an ip injection of either vehicle or CORT (2.5 mg/
kg) every day for 13 days at either ZT1
(antiphasic; n ⫽ 24 for CORT, n ⫽ 16 for
vehicle) or ZT11 (in-phase; n ⫽ 24 for
CORT, n ⫽ 16 for vehicle). The day after
13 daily injections, groups of rats within
each CORT status condition were decapitated at 4 different time points through
the day (ZT0, ZT6, ZT12, and ZT18;
Figure 1. Experimental timelines. A, Experiment 1. Starting 2 days after ADX or sham surgery,
n ⫽ 4 or n ⫽ 6 for each ZT with daily
rats were given 10 daily ip injections of either vehicle or CORT (2.5 mg/kg) at ZT1. On the 11th
vehicle or CORT treatment conditions,
day, rats were killed at ZT0, ZT6, ZT12, or ZT18 (n ⫽ 6 per time of death for each CORT status
respectively). For the time points ZT0
condition). B, Experiment 2. Starting 2 days after ADX surgery, rats were given 13 daily ip
and ZT12 rats were decapitated approxinjections of either vehicle or CORT (2.5 mg/kg) at either ZT1 (antiphasic) or ZT11 (in-phase). On
imately 15 minutes before the light tranthe 14th day, rats were killed at ZT0, ZT6, ZT12, or ZT18 (n ⫽ 4 or n ⫽ 6 per time of death for
sition in order to avoid any rapid effects
each daily vehicle or CORT treatment time, respectively).
the light transition may have on clock
gene expression. Rats did not receive an
ADX surgery
injection on the day they were killed in order to avoid any acute
effects of CORT on clock gene expression. Brains were extracted
For experiment 1, rats received either ADX or sham-ADX
and flash-frozen in isopentane chilled to ⫺30°C with dry ice and
surgery. Briefly, rats were anesthetized with halothane during
then stored at ⫺70°C.
surgery, bilateral incisions were made through the dorsal lateral
skin and peritoneal wall near the kidneys, and adrenal glands of
ADX rats were excised. Sham-ADX rats went through the same
CORT assay
procedure but adrenal glands were left in place. For experiment
Plasma CORT levels were measured using an enzyme-linked
2, all rats received ADX surgery as described above.
immunosorbant assay for CORT (Assay Design) in agreement
with the manufacturer’s protocol. Before use, plasma was diluted
Experiment 1 procedure (Figure 1A)
1:50 in assay buffer and incubated at 65°C for 60 minutes in
order to denature corticosteroid binding globulin. Intraassay coThis experiment tested whether diurnal clock gene expression
efficient of variation was 6.3%.
depends on the presence and/or time of day when daily CORT
elevations occur by comparing clock gene expression of sham
rats with ADX rats and ADX rats that received a daily pulse of
In situ hybridization and image analysis
antiphasic CORT. Starting 2 days after surgery, rats received an
Coronal brain slices (12 ␮m) were cut on a Leica Microsysip injection of either vehicle (60% sterile saline, 30% propylene
tems cryostat (model 1850) from the PFC (2.7 mm anterior to
glycol, 10% ethanol 1 mL/kg) or CORT (2.5 mg/kg; Steraloids)
bregma) through the caudal extent of the SCN (1.3 mm posterior
each day at ZT1 for 10 days. We have previously demonstrated
to bregma) (16), thaw mounted on Colorfrost Plus slides (VWR),
that this CORT treatment produces peak plasma CORT levels
and stored at ⫺70°C.
(⬃50 ␮g/100 mL) within 30 minutes after injection, and those
In situ hybridization for Per1, Per2, and Bmal1 mRNA was
levels return to preinjection levels within 2 hours (47). This maperformed as previously described (49). Briefly, tissue was hynipulation produces a relatively short-lasting pulse of CORT
bridized with 35S-uridine triphosphate-labeled antisense ribolevels within the moderate-to-high physiological range that is
probes in a 50% formamide humidified atmosphere at 54°C for
expected to occupy most GRs in the body (48). All sham rats
16 –18 hours. Slides were then treated with Ribonuclease A
received a vehicle injection (n ⫽ 24), and all ADX rats received
(Sigma) at 37°C for 1 hour, washed in decreasing concentrations
either vehicle (n ⫽ 24) or CORT (n ⫽ 24). The day after 10 daily
of saline citrate solution (2⫻, 1⫻, 0.5⫻, and 0.1⫻), incubated in
injections, groups of rats within each CORT status condition
0.1⫻ saline citrate solution at 65°C for 1 hour, then dehydrated
were decapitated at 4 time points throughout the day (ZT0, ZT6,
through a series of ethanol washes. Dried slides were then exZT12, and ZT18; n ⫽ 6 for each ZT within a CORT status
posed to x-ray film for 2– 4 weeks, after which films were digicondition). For the time points ZT0 and ZT12, rats were decaptized by use of Northern Light lightbox model B95 (Imaging Res,
itated approximately 15 minutes before the light transition in
Inc), a Sony CCD video camera model XC-ST70 fitted with a
order to avoid any rapid effects the light transition may have on
Navitar 7000 zoom lens connected to a LG3– 01 frame grabber
clock gene expression. Rats did not receive an injection on the
(Scion Corp) inside a Dell Dimension 500, and captured with
day they were killed in order to avoid any acute effects of CORT
Scion Image Beta release 4.0.2. The cloned coding portion of
on clock gene expression. Brains were extracted and flash-frozen
each clock gene is as follows: Per1, nuclear transcript (nt) 974 –
in isopentane chilled to ⫺30°C with dry-ice and then stored at
1547, GenBank accession number NM_001034125; Per2, nt
⫺70°C. Trunk blood plasma was collected for CORT assay.
2240 –2869, GenBank accession number NM_031678; and
Bmal1, nt 697–1278, GenBank accession number NM_024362.
Experiment 2 procedure (Figure 1B)
Densitometry measurements from left and right hemispheres on
4 – 6 sections per brain per region of interest (ROI) was perThe second experiment tested whether the altered diurnal
formed by an individual blind to treatment condition. ROIs were
clock gene expression in the PFC of ADX rats observed in expositioned relative to the corpus callosum and longitudinal fisperiment 1 (see Results) is normalized by daily in-phase (ZT11),
sure for PFC subregions and relative to the optic chiasm for the
but not antiphasic (ZT1) CORT injections. Starting 2 days after
doi: 10.1210/en.2015-1884
SCN. ROIs were 12 ⫻ 12 pixels in size and were centered over
the target brain region with reference to a rat brain atlas (50).
Measurements were taken without differentiation between cortical layers, as gene expression was predominantly uniform
throughout. Densitometry measurements (uncalibrated optical
density) were taken using ImageJ software 1.46r (National
Institutes of Health). Optical density values for a given ROI
were averaged across each of the separate tissue sections/
hemisphere measures for each brain to yield a single value for
each brain.
Statistical analysis
Two-way ANOVA was used to examine overall main effects
and interactions of time of death (ZT) and CORT status. Follow-up one-way ANOVA was used to test whether there was a
significant time of death effect for clock gene expression within
each CORT status group. Post hoc pair-wise comparisons used
Fisher’s least significant difference (FLSD). Statistical Package
for the Social Sciences (SPSS v. 21.0, IBM) was used for ANOVA
analysis; P ⬍ .05 was considered significant. There were some
missing data due either to insufficient brain sections for a given
ROI, or variable within brain hybridization signal, accounting
for the variable degrees of freedom for the reported F tests. Data
presented in figures are the mean ⫾ SEM.
Results
Experiment 1. CORT status modulates Per1, Per2,
and Bmal1 mRNA diurnal expression in the PFC
but not in the SCN
Corticosterone
CORT levels in sham rats displayed a strong diurnal
pattern of secretion (ZT effect: F3,23 ⫽ 12.0, P ⬍ .001)
with peak circulation occurring around ZT12 (Figure 2).
CORT levels were below assay detection limits for rats in
both ADX and ADX⫹antiphasic CORT treatment
groups. This is consistent with the very short half-life of
CORT in the rat (ⱕ15 min) (21, 51). Consequently, any
effect that this daily antiphasic CORT treatment had on
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.
press.endocrine.org/journal/endo
1525
clock gene expression levels at the time of death was likely
not due to an acute modulatory effect of concurrent GR
activation by CORT.
Clock gene mRNA
There was a significant main effect of CORT status and
a time of death by CORT status interaction for Per1 and
Per2 mRNA expression in each of the PFC subregions
examined (ACC, PL, IL, and VO) but not in the SCN
(Table 1). Bmal1 mRNA levels appeared to also be affected by CORT status in the PFC, because its expression
showed a significant effect of time of death only in sham
rats (Figure 3 and Table 1). The specific effect of each
CORT status condition on clock gene expression is outlined below.
Sham rats
After 10 daily vehicle injections at ZT1, Per1, Per2, and
Bmal1 mRNA expression in adrenal-intact (sham) rats
showed a diurnal pattern (ie, effect of time of death) in
subregions of the PFC and the SCN as determined by oneway ANOVA (Figures 3 and 4; Table 1). Within both the
SCN and some PFC subregions, positive (Bmal1) and negative (Per1 and Per2) regulatory clock genes had peak
mRNA expression levels during opposite phases of the
light-dark cycle (Table 1). Although the time of day for
peak clock gene expression was similar across all PFC
subregions, it varied considerably between the PFC and
SCN, as previously reported (8, 10, 16, 22). Specifically,
we found peak Per1 mRNA occurred at ZT18 in the
PFC and ZT6 in the SCN, peak Per2 occurred around
ZT12 in the PFC and ZT6 in the SCN, and peak Bmal1
mRNA occurred around ZT0 in the PFC and ZT18 in
the SCN.
ADX rats
After 10 daily vehicle injections at ZT1, Per1 and Per2
mRNA expression displayed a diurnal pattern in the PFC
and the SCN of ADX rats as determined by one-way
ANOVA (Figures 3 and 4; Table 1). However, in most PFC
subregions Per1 peak expression was phase advanced
(shift from ZT18 to ZT12 relative to sham rats), and
across all PFC subregions Per2 peak expression was phase
delayed (shift from ZT12 to ZT18) pointing towards a
disruptive effect of ADX on normal PFC clock gene expression phase relationships. A diurnal pattern of Bmal1
mRNA expression was absent across each of the PFC subregions, further supporting disruption in clock gene expression. In contrast, there was no significant effect of
ADX on the diurnal presence or phase of clock gene expression within the SCN.
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Woodruff et al
Table 1.
CORT Regulates PFC Clock Gene mRNA
Endocrinology, April 2016, 157(4):1522–1534
Experiment 1
Peak ZT for CORT Status Groups With
Significant Effect of Time
of Death (P < .05, One-Way ANOVA)
Experiment 1. Rat Clock Gene Expression Two-Way ANOVA Results
Gene
Per1
Per2
Bmal1
ROI
Time of Death
CORT Status
Time of Death ⴛ CORT Status
Sham
ADX
Antiphasic CORT
ACC
PL
IL
VO
SCN
ACC
PL
IL
VO
SCN
ACC
PL
IL
VO
SCN
F3,45 ⫽ 13.6
F3,41 ⫽ 14.2a
F3,39 ⫽ 8.3a
F3,44 ⫽ 17.6a
F3,50 ⫽ 44.6a
F3,55 ⫽ 47.8a
F3,55 ⫽ 35.7a
F3,55 ⫽ 34.3a
F3,55 ⫽ 34.3a
F3,60 ⫽ 193.5a
F3,58 ⫽ 5.4a
F3,53 ⫽ 1.2
F3,54 ⫽ 1.5
F3,58 ⫽ 9.0a
F3,45 ⫽ 41.8a
F2,45 ⫽ 3.3
F2,41 ⫽ 17.1a
F2,39 ⫽ 15.4a
F2,44 ⫽ 6.6a
F2,50 ⫽ 0.67
F2,55 ⫽ 40.7a
F2,55 ⫽ 40.9a
F2,55 ⫽ 43.2a
F2,55 ⫽ 33.1a
F2,60 ⫽ 0.03
F2,58 ⫽ 2.6
F2,53 ⫽ 1.9
F2,54 ⫽ 1.5
F2,58 ⫽ 0.88
F2,45 ⫽ 0.24
F6,45 ⫽ 3.8
F6,41 ⫽ 5.8a
F6,39 ⫽ 4.6a
F6,44 ⫽ 4.6a
F6,50 ⫽ 0.43
F6,55 ⫽ 80.1a
F6,55 ⫽ 70.9a
F6,55 ⫽ 69.6a
F6,55 ⫽ 52.3a
F6,60 ⫽ 0.142
F6,58 ⫽ 2.0
F6,53 ⫽ 1.2
F6,54 ⫽ 1.4
F6,58 ⫽ 1.8
F6,45 ⫽ 1.6
ZT18
ZT18
ZT18
ZT18
ZT6
ZT12
ZT12
ZT12
ZT12
ZT6
ZT0
(P ⫽ .2)
(P ⫽ .3)
ZT0
ZT18
ZT18
ZT12
ZT12
ZT12
ZT6
ZT18
ZT18
ZT18
ZT18
ZT6
(P ⫽ .3)
(P ⫽ .2)
(P ⫽ .2)
(P ⫽ .5)
ZT12
(P ⫽ .05)
(P ⫽ .6)
(P ⫽ .6)
(P ⫽ .2)
ZT6
ZT0
ZT0
ZT0
(P ⫽ .06)
ZT6
(P ⫽ .3)
(P ⫽ .2)
(P ⫽ .4)
(P ⫽ .2)
ZT18
a
a
a
Two- and one-way ANOVA results for clock gene expression in the PFC (ACC, PL, IL, and VO) and SCN. Factors were time of death (ZT0, ZT6,
ZT12, or ZT18) and CORT status (sham, ADX, and ADX⫹antiphasic CORT).
a
P ⬍ .05.
ADXⴙantiphasic CORT rats
After treatment of ADX rats with 10 daily antiphasic
CORT injections at ZT1, a diurnal pattern of Per1 and
Bmal1 mRNA expression was absent across all PFC subregions but was maintained in the SCN as determined by
one-way ANOVA (Figures 3 and 4; Table 1). In the PFC,
Per2 mRNA expression displayed a significant effect of
time of death supporting a diurnal pattern in its expression; however, its diurnal amplitude was substantially
blunted, and the phase of its peak expression was inverted
relative to sham rats (ZT0 as opposed to ZT12) (Figures
3 and 4; Table 1). In contrast, there was no significant
effect of antiphasic CORT treatment in ADX rats on diurnal clock gene expression within the SCN.
Experiment 2. Diurnal pattern of Per1, Per2, and
Bmal1 mRNA in the PFC of ADX rats is normalized
by daily in-phase CORT treatment but not daily
antiphasic CORT treatment; CORT status has no
effect on clock gene mRNA in the SCN
There was no significant difference in clock gene expression in any brain area between ADX rats given a daily
vehicle injection at ZT1 or ZT11 (two-way ANOVA, P ⬎
.05) (Supplemental Figure 1). Accordingly, data from vehicle-treated rats were pooled for subsequent statistical
analyses and graphical presentation (ADX⫹vehicle
group: total of 32 rats, with n ⫽ 8 for each ZT of death).
As in the first experiment, there was an overall significant main effect of CORT status for Per1 and Per2
mRNA expression in each of the PFC subregions examined (ACC, PL, IL, and VO), but not in the SCN (two-way
ANOVA) (Table 2). There was also a significant time of
death by CORT status interaction for each of the clock
genes throughout the PFC, and there was a time of death
by CORT status interaction for Per1 and Bmal1 mRNA
expression in the SCN. However, post hoc tests and visual
inspection of the graphs indicate that the interaction
within the SCN, in contrast to the PFC, was due to small
group differences at a single time point that did not reflect
a CORT status dependent difference in clock gene expression pattern, amplitude, or phase relationship (Figures 5
and 6). The specific effect of each CORT status condition
on clock gene expression is outlined below.
ADXⴙvehicle rats
The phase relationship for the peak of each clock gene
mRNA in the SCN corresponded to that of all rats in
experiment 1, with the exception that in this experiment
we found Bmal1 mRNA peak expression at ZT12 instead
of ZT18 (Figure 5 and Table 2). The positive (Bmal1) and
negative (Per1 and Per2) arms of the molecular clock still
displayed peak expression levels during opposite phases of
the light-dark cycle. Similar to the first experiment, in the
PFC of ADX rats Per2 mRNA expression was diurnal in
nature but phase delayed relative to the sham rats in experiment 1 (Figure 5 and Table 2). In this second experiment, Per1 expression in the VO subregion of the PFC of
ADX rats had a significant diurnal pattern with peak expression levels at ZT18 (Figure 5 and Table 2). There was
also a significant time of death effect for Per1 mRNA levels
in the IL, and a near significant trend in the ACC and PL;
however, in each of those PFC subregions, there appeared
to be 2 peaks of expression at ZT6 and ZT18 (Figure 5 and
Table 2). For this experiment there was also a significant
time of death effect for Bmal1 expression in all subregions
of the PFC with a peak at ZT18 (Figures 3 and 5). Sup-
doi: 10.1210/en.2015-1884
press.endocrine.org/journal/endo
1527
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).
portive of a disruptive nature of ADX on PFC clock gene
expression, as in the first experiment, ADX⫹vehicletreated rats lacked peak expression of the positive (Bmal1)
and negative (Per1 and Per2) arms of the molecular clock
during opposite phases of the light-dark cycle.
ADXⴙin-phase CORT rats
After 13 daily CORT (2.5 mg/kg) injections at ZT11,
Per1, Per2, and Bmal1 mRNA expression displayed a diurnal pattern in the PFC and the SCN as determined by
one-way ANOVA (Figures 5 and 6; Table 2). Within each
subregion of the PFC, clock gene expression patterns were
largely normalized in these ADX rats. That is, peak expression for the positive (Bmal1) and negative (Per1 and
Per2) arms of the molecular clock occurred at opposite
phases of the light-dark cycle with the same diurnal timing
as observed in adrenal intact rats (Figure 3).
ADXⴙantiphasic CORT rats
After 13 daily CORT (2.5 mg/kg) injections at ZT1,
Per1, Per2, and Bmal1 mRNA exhibited a diurnal expression pattern in the SCN but not in the PFC for Per1 and
Bmal1 expression (Figures 5 and 6; Table 2). Consistent
with the first experiment, Per2 mRNA expression in the
PFC did exhibit a significant time of death effect, but it had
a somewhat blunted diurnal peak amplitude and its peak
expression was phase-inverted relative to Per2 mRNA expression in in-phase CORT-treated rats (ZT12) (Figure 5
1528
Woodruff et al
CORT Regulates PFC Clock Gene mRNA
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.
and Table 2) or sham rats in experiment 1 (ZT12) (Figure
3 and Table 1). Consequently, as in the first experiment,
antiphasic CORT treatment of ADX rats substantially disrupted the normal diurnal expression of both the positive
and negative arms of PFC clock gene expression.
Discussion
In these experiments, we demonstrated that diurnal clock
gene expression in the PFC was strongly modulated by the
presence and phase of daily fluctuations in circulating
CORT. The diurnal variations in Per1, Per2, and Bmal1
mRNA expression that we observed in the SCN are consistent with the well-established rhythmic expression of
Endocrinology, April 2016, 157(4):1522–1534
these clock genes that constitutes a molecular clock (5, 6).
The diurnal variation of clock gene expression in PFC
subregions of adrenal-intact rats suggests that under normal conditions there may also be an operational molecular
clock here as well. Interestingly, CORT manipulations
substantially affected both the presence and timing of diurnal clock gene expression patterns in the PFC. In contrast, clock gene expression in the SCN was unaffected by
CORT manipulations, likely due to the absence of GR
expression in the SCN (16, 18, 19). To aid in the discussion
of these data we have provided summary figures for each
treatment group (Figure 7 and Supplemental Figure 2) that
show whether there was a significant diurnal variation in
clock gene expression and if so, the observed peak time of
that expression. Comparison of the time of peak diurnal
expression of these clock genes provides some indication
of the phase relationships that would be expected in the
case of sustained rhythmic expression.
Adrenal-intact rats had diurnal expression patterns of
Per1, Per2, and Bmal1 mRNA in both the PFC and SCN
(Figure 7 and Supplemental Figure 2). The phase of peak
clock gene mRNA that we observed in the SCN is similar
to other reports of SCN clock gene expression in the rodent (8 –10, 16, 24, 49). Sham rats displayed a different
phase relationship for the expression of each clock gene
within the PFC compared with the SCN, and this PFCspecific clock gene expression profile is in close agreement
with our recent report of diurnal clock gene expression in
the PFC of male and female rats (10). Importantly, there
was an antiphasic relationship between the time of day for
peak expression of a positive (Bmal1) and negative (Per1/
Per2) arm of the molecular clock in both the SCN and PFC
of sham rats, as would be expected for an operational
intrinsic molecular clock (Figure 7).
ADX alters PFC diurnal clock gene expression
Although there was no significant difference in SCN
clock gene expression between ADX and sham rats, PFC
clock gene expression profiles were significantly altered by
ADX. In the first experiment, Per1 and Per2 mRNA expression continued to exhibit a diurnal pattern in the PFC
of ADX rats, but the apparent phase of that expression
was altered with a delay of peak Per2 mRNA and an advance of peak Per1 mRNA relative to the expression patterns in sham rats. In the second experiment, ADX rats
with vehicle treatment again displayed a phase delay in
PFC Per2 mRNA expression relative to sham rats, and
PFC Per1 mRNA expression lacked a reliable diurnal variation across the PFC subregions. ADX also affected PFC
Bmal1 expression patterns in both experiments. The discrepancy between experiments in the specific effects of
ADX on PFC clock gene expression patterns may be re-
doi: 10.1210/en.2015-1884
Table 2.
press.endocrine.org/journal/endo
Experiment 2
Peak ZT for CORT Status Groups With Significant
Effect of Time of Death (P < .05, One-Way ANOVA)
Experiment 2. Rat Clock Gene Expression Two-Way ANOVA Results
Gene
Per1
Per2
Bmal1
1529
ROI
Time of Death
CORT Status
Time of Death ⴛ CORT Status
ADX ⴙ In-Phase CORT
ADX ⴙ Veh
ADX ⴙ Antiphasic CORT
ACC
PL
IL
VO
SCN
ACC
PL
IL
VO
SCN
ACC
PL
IL
VO
SCN
F3,52 ⫽ 5.8
F3,58 ⫽ 7.8a
F3,53 ⫽ 8.5a
F3,58 ⫽ 7.9a
F3,67 ⫽ 165.2a
F3,53 ⫽ 6.1a
F3,55 ⫽ 1.5
F3,56 ⫽ 1.4
F3,54 ⫽ 9.9a
F3.67 ⫽ 194.4a
F3,61 ⫽ 4.5a
F3,60 ⫽ 2.3a
F3,62 ⫽ 2.4
F3,61 ⫽ 8.7a
F3,67 ⫽ 52.8a
F2,52 ⫽ 3.6
F2,58 ⫽ 5.1a
F2,53 ⫽ 13.3a
F2,58 ⫽ 3.3a
F2,67 ⫽ 2.0
F2,53 ⫽ 7.2a
F2,55 ⫽ 4.5a
F2,56 ⫽ 4.5a
F2,54 ⫽ 3.5a
F2,67 ⫽ 0.04
F2,61 ⫽ 0.11
F2,60 ⫽ 0.28
F2,62 ⫽ 0.29
F2,61 ⫽ 0.49
F2,67 ⫽ 1.3
F6,52 ⫽ 4.7
F6,58 ⫽ 5.3a
F6,53 ⫽ 6.6a
F6,58 ⫽ 5.5a
F6,67 ⫽ 2.9a
F6,53 ⫽ 6.5a
F6,55 ⫽ 2.8a
F6,56 ⫽ 5.3a
F6,54 ⫽ 6.3a
F6,67 ⫽ 1.1
F6,61 ⫽ 4.2a
F6,60 ⫽ 4.3a
F6,62 ⫽ 5.5a
F6,61 ⫽ 7.9a
F6,67 ⫽ 2.8a
ZT12
ZT18
ZT18
ZT12
ZT6
ZT12
(P ⫽ .07)
ZT12
ZT12
ZT6
ZT0
ZT0
ZT0
ZT0
ZT12
(P ⫽ .06)
(P ⫽ .05)
ZT6/18
ZT18
ZT6
ZT18
ZT18
ZT18
ZT18
ZT6
ZT18
ZT18
ZT18
ZT18
ZT12
(P ⫽.5)
(P ⫽ .7)
(P ⫽ .4)
(P ⫽ .7)
ZT6
(P ⫽ .3)
(P ⫽ .3)
ZT0
ZT0
ZT6
(P ⫽ .4)
(P ⫽ .5)
(P ⫽ .1)
(P ⫽ .4)
ZT12
a
a
a
Two-way ANOVA and one-way ANOVA results for clock gene expression in the PFC (ACC, PL, IL, and VO) and SCN. Factors were time of death
(ZT0, ZT6, ZT12, or ZT18) and CORT status (ADX⫹vehicle, ADX⫹in-phase CORT, and ADX⫹antiphasic.
a
P ⬍ .05.
lated to the longer duration of ADX in the second experiment (13 d) compared with the first experiment (10 d).
CORT is known to stabilize circadian rhythms (8, 26, 52,
53), therefore the extended absence of endogenous CORT
in experiment 2 may have been enough to further alter the
clock gene expression patterns in the PFC. ADX has been
found in other studies to disrupt diurnal clock gene expression in some but not all peripheral tissues (8, 20, 22)
and to blunt diurnal PER2 immunoreactivity in subregions of the extended amygdala (20).
It should be noted that in the second experiment peak
SCN Bmal1 mRNA occurred at ZT12 instead of ZT18 as
seen in the first experiment. Although this may reflect an
effect of ADX on molecular clock operation within the
SCN, we believe that is not the case for the following
reasons. First, we did not see this effect of ADX in the first
experiment. Second, in contrast to clock gene expression
in the PFC, neither in-phase nor antiphasic CORT treatment had an effect on SCN clock gene expression in ADX
rats. Third, other studies report no effect of ADX on SCN
clock gene expression (8, 20, 31, 54). Instead, we suspect
that this difference in Bmal1 mRNA peak times reflects a
cohort difference in timing that may appear more pronounced than it was due to the 6-hour interval between
sampling times. Other studies with greater temporal resolution typically report a Bmal1 mRNA acrophase in the
rat SCN around ZT16 –ZT18 (9, 10, 49).
In-phase diurnal CORT normalizes PFC diurnal
clock gene expression
Daily in-phase CORT pulses were sufficient to normalize the diurnal pattern in PFC clock gene expression of
ADX rats (Figure 7 and Supplemental Figure 2). Similar
in-phase glucocorticoid treatment (glucocorticoid in
drinking water) of ADX rats has previously been shown to
normalize ex vivo Per1 gene promoter rhythmic activity in
some peripheral tissues (8) and normalize diurnal PER2
expression in select extended amygdala subregions of the
brain (54). In contrast, treatment of ADX rats with constant glucocorticoid levels was not sufficient to restore
normal brain PER2 expression patterns (54). Our study is
the first to demonstrate a regulatory influence of CORT
treatment on clock gene expression in the PFC. Importantly, our study also demonstrated that this influence was
evident for both positive (Bmal1) and negative (Per1 and
Per2) transcriptional regulatory components of the core
molecular clock. We are the first to systematically test and
report the extent of in-phase CORT normalization of negative and positive clock gene expression in extra-SCN tissue and to directly compare this with antiphasic CORT
treatment of ADX rats. This result, combined with the
general antiphasic relationship between Bmal1 mRNA
(peak at onset of the light phase) and Per1/Per2 mRNA
(peak during the early to midportion of the dark phase) in
subregions of the PFC suggests that there is not only 24hour rhythmic clock gene expression present within the
PFC, but also intrinsic oscillatory control of that expression. Support for rhythmic and generally oscillatory clock
gene expression in the PFC has also been observed in humans (41, 55). However, the clock gene expression phase
relationship profile in the human PFC was opposite to
what we have found in the rat, ie, the negative components
of the clock tended to reach peak expression during the
light phase and the positive components during the dark
phase. This species difference may be related to the dif-
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Woodruff et al
CORT Regulates PFC Clock Gene mRNA
Endocrinology, April 2016, 157(4):1522–1534
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).
ferent awake/sleep activity phase profile of diurnal humans compared with nocturnal rats. If such is the case,
then clock gene expression in the PFC of both species may
depend more on entraining influences that align with activity patterns which are known to influence endogenous
CORT circulation rather than light-dark cycle phase (56).
Antiphasic diurnal CORT substantially disrupts PFC
diurnal clock gene expression
In contrast to the normalizing influence of daily inphase CORT, antiphasic CORT, matched in dose and
delivery procedure, produced a dramatic disruption of PFC clock gene
expression in ADX rats (Figure 7 and
Supplemental Figure 2). In both of
our experiments, antiphasic CORTtreated rats lacked a significant diurnal variation in PFC Per1 and Bmal1
mRNA. This effect was especially
pronounced for Per1 mRNA. Diurnal expression of Per2 mRNA was
substantially blunted in most PFC
subregions, at least for the specific
sample times used in the study. These
results are similar to what was observed in the liver of adrenal-intact
mice after daily antiphasic, but
not in-phase Prednisolone treatment (15). Interestingly, despite the
blunted diurnal amplitude of PFC
Per2 mRNA expression, we saw a
distinct 12-hour phase shift of Per2
mRNA peak expression relative to
its profile in sham rats or ADX rats
with in-phase CORT treatment. A
similar phase inversion of PER2 diurnal immunoreactivity in the extended amygdala was observed after
daily antiphasic CORT injections of
ADX rats (20). Despite the dramatic
effect of antiphasic CORT treatment
on clock gene expression in the PFC,
it had no significant effect on clock
gene expression in the SCN.
Possible mechanisms of
glucocorticoid modulation of
the PFC molecular clock
In our study, CORT was a strong
modulatory factor for PFC diurnal
Per1 mRNA levels, a clock gene with
a well-characterized functional GRE
(17–19). Glucocorticoids rapidly induce Per1 expression
in a variety of cell lines and tissues (16, 17, 21). CORT
manipulations in our study also effectively modulated
PFC expression patterns of Per2 and Bmal1 mRNA. These
clock genes are also associated with candidate GREs; however, there is much less evidence that these GREs are functional across a range of tissues and conditions (18, 19, 57).
Thus, the entraining influence of CORT on PFC rhythmic
clock gene expression may be due to the activation of GR
within the PFC (which expresses a high level of GR) (58)
by daily CORT surges/pulses, and subsequent induction
doi: 10.1210/en.2015-1884
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.
of Per1 gene expression. In hepatocytes, the expression of
GR is necessary for glucocorticoid modulation of the
phase of liver rhythmic clock gene expression (16). Recent
studies show direct protein-protein interactions between
GR and several clock gene proteins, including CLOCK,
BMAL1, and CRYPTOCHROME1, each of which modulates GR-dependent transcriptional activity (19, 59, 60).
Glucocorticoids have also been shown to down-regulate
Rev-erb␣ expression in both rat and human liver tissue (8,
15, 24). REV-ERB␣ is a negative controller of Bmal1 transcription, and the expression of Rev-erb␣ is suppressed by
PERIOD proteins (7).
We also cannot rule out the possibility that CORT indirectly affects PFC clock gene expression by acting at
other brain regions. For example, normal CORT secretion
is necessary for diurnal fluctuations in tryptophan hy-
press.endocrine.org/journal/endo
1531
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.
droxylase 2 expression in the dorsal raphe nuclei (61).
Tryptophan hydroxylase 2 is the rate-limiting enzyme for
serotonin synthesis, and the dorsal raphe nucleus provides
a strong serotonergic input to the PFC (62).
Although our results demonstrate that diurnal fluctuations in circulating CORT had a strong influence on PFC
diurnal clock gene expression, we do not believe that the
results point to CORT as the sole entrainment factor of
PFC clock gene expression. In ADX⫹vehicle rats we still
observed a diurnal pattern of PFC clock gene mRNA lev-
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Woodruff et al
CORT Regulates PFC Clock Gene mRNA
els, albeit a pattern that was altered relative to those observed in adrenal-intact rats. In addition, although treatment of ADX rats with in-phase CORT pulses normalized
PFC clock gene expression, treatment with antiphasic
CORT pulses inverted the phase only of PFC Per2 expression, and that diurnal expression amplitude was substantially blunted. If CORT is the sole entrainer for PFC clock
gene expression, then one would expect that Per1 and
Bmal1 expression would have been inverted as well. It is
therefore likely that the SCN has some influence on PFC
clock gene expression that is independent of circadian
CORT rhythms. The SCN does not directly innervate the
PFC, but it provides strong indirect input to the PFC IL via
a relay through the paraventricular nucleus of the thalamus (14, 63). SCN-dependent input could then be exchanged throughout the subregions of the PFC which
communicate extensively with each other (64). Consequently, the PFC may receive circadian entrainment input
from multiple sources including the master clock in the
SCN and diurnal CORT circulation. In instances when
these inputs are not tightly coupled to each other, a “competition” for entrainment may occur and lead to a complete loss of normal circadian rhythmicity in the PFC, as
appears to be the case with antiphasic CORT treatment
in our study. On the other hand, the presence of daily
in-phase CORT pulses combined with an indirect SCN
influence may lead to robust rhythmic and oscillatory expression of Per1, Per2, and Bmal1 mRNA in the PFC.
Regardless of the process by which CORT modulates
clock gene expression in the PFC, that process appears to
be similar across PFC subregions. We observed very similar CORT treatment effects on the extent of diurnal clock
gene expression patterns and peak expression timing in
each of the 4 subregions that we examined. Although the
functionality and connectivity of these various subregions
of the PFC are similar in some regards, they also exhibit
distinct differences (64 – 66). Perhaps CORT provides a
similar modulatory influence in clock gene expression
across PFC subregions by regulation of the indirect SCN
input to the PFC. Alternatively, CORT may directly affect
clock gene expression in each PFC subregion via the abundant presence of GR found throughout the PFC (58). The
similarity across PFC subregions in diurnal clock gene expression profiles and their sensitivity to CORT suggest
that this CORT modulatory influence is important for a
shared and coordinated aspect of PFC subregion function.
Glucocorticoid circadian physiology and
implications for PFC-dependent function
The PFC modulates many cognitive (29, 67) and physiological functions (68), and its operation and dendritic
architecture are strongly affected by time of day and stress
Endocrinology, April 2016, 157(4):1522–1534
(36). It is noteworthy that many PFC-dependent behaviors
show circadian variation (31–33). Mood and anxiety disorders are characterized by both abnormal PFC functioning (37, 38) and abnormal diurnal CORT circulation (4,
69). Intriguingly, Li et al (41) found reduced diurnal patterns of clock gene mRNA in the postmortem PFC of major depressive disorder (MDD) patients. Successful treatment of MDD is associated with normalization of both
diurnal CORT circulation (70) and peripheral blood cell
clock gene expression (69). Chen et al (55) have similarly
found altered diurnal clock gene expression in the postmortem PFC of older humans compared with younger
humans, potentially related to age-associated alterations
in cognition and mood. Karatsoreos et al (71) have shown
that a rodent model of circadian disruption produces sequelae of behaviors resembling those that occur after PFC
lesion (decreased cognitive flexibility and altered emotionality). In addition, McClung and coworkers have shown
that both mutation and knock down of Clock (a second
component of the positive arm) lead to disruption in mood
related behavior in mice (72, 73). That research group has
also found that chronic stress leads to similar behavioral
disruption as well as altered clock gene expression in several relevant brain areas (42).
Our results have important implications for some of the
consequences of situations in which normal diurnal
CORT circulation is no longer present, as can occur with
hypothalamic adrenal pituitary axis dysregulation or
pharmacological treatment with glucocorticoids. Disrupted clock gene oscillatory expression due to abnormal
CORT circulation may be a mechanism that causes or
exacerbates MDD, PTSD, and other PFC-related psychiatric disorders. This will be an important consideration in
the future when optimizing treatment strategies for these
disorders. It was because of these clinically relevant considerations that the primary aim of this study was to examine the effect that varying chronic patterns of diurnal
CORT circulation have on clock gene expression in the
PFC in the presence of a fully functioning master clock and
a normal environmental light-dark cycle. It would be interesting, however, to examine the effects of various patterns of CORT circulation on PFC clock gene expression
in the absence of the master clock (eg, SCN lesion) or under
free running SCN conditions (eg, under constant darkness). Those studies will be useful in determining the mechanistic relationship between the SCN and CORT in control of PFC clock gene expression and circadian function.
Acknowledgments
We thank Sara Fardi and Nicolas Varra for technical assistance.
doi: 10.1210/en.2015-1884
Address all correspondence and requests for reprints to:
Elizabeth R. Woodruff, Department of Psychology and Neuroscience, University of Colorado Boulder, UCB 345, Boulder, CO
80309. E-mail: [email protected].
This work was supported the National Institutes of Health
Grant MH075968 and the National Science Foundation Grant
IOS-1456706.
Disclosure Summary: The authors have nothing to disclose.
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