Neuroscience 126 (2004) 241–246
CHRONIC CORTISOL SUPPRESSES PITUITARY AND HYPOTHALAMIC
PEPTIDE MESSAGE EXPRESSION IN PIGTAILED MACAQUES
P. SZOT,a,c* C. W. WILKINSON,b,c S. S. WHITE,c
J. B. LEVERENZ,b,c J. L. GREENUP,a
E. A. COLASURDO,b E. R. PESKINDa,c AND
M. A. RASKINDa,c
a
Northwest Network Mental Illness Research, Education and Clinical
Center S-116, Veterans Affairs Puget Sound Health Care System,
Seattle, WA, USA
b
Geriatric Research, Education and Clinical Center, Veterans Affairs
Puget Sound Health Care System, Seattle, WA, USA
c
Department of Psychiatry and Behavioral Sciences, University of
Washington, Seattle, WA 98195, USA
Abstract—The effects of chronic elevations in circulating glu-
cocorticoids on the expression of peptides and peptide re-
ceptors of the hypothalamic–pituitary–adrenal (HPA) axis
have been studied extensively in rodents, but they have not
been examined in primates. To determine the responses of
the HPA axis in primates to elevated cortisol, hypothalamic
and pituitary tissue from normal older pigtailed macaques
(Macaca nemestrina) that had received daily oral administra-
tion of cortisol or placebo for 1 year were studied. Pro-
opiomelanocortin in the anterior pituitary and corticotropin-
releasing factor (CRF) mRNA expression in the hypothalamic
paraventricular nucleus (PVN) were significantly reduced in
cortisol-treated monkeys in comparison with controls. CRF
receptor 1 (CRF-R1) expression in the anterior pituitary and
arginine vasopressin mRNA expression in the PVN were un-
changed by chronic cortisol administration. Sustained eleva-
tion of circulating glucocorticoids results in suppression of
HPA peptide and peptide receptor expression in the PVN and
anterior pituitary similar to those found in rodents. Chronic
therapeutic administration of glucocorticoids in humans may
have unintended consequences for hypothalamic and pitu-
itary function. Published by Elsevier Ltd on behalf of IBRO.
Key words: hypercortisolemia, anterior pituitary, paraven-
tricular nucleus (PVN), corticotropin-releasing factor (CRF),
adrenocorticotropic hormone (ACTH), monkey.
Circulating glucocorticoid concentrations are regulated by
coordinated action at all levels of the hypothalamic–
*Correspondence to: P. Szot, PhD, Veterans Affairs Puget Sound
Health Care System, Mental Illness Research, Education and Clin-
ical Center S-116, 1660 South Columbian Way, Seattle, WA 98108,
USA. Tel: ⫹1-206-277-5052; fax: ⫹1-206-768-5456.
E-mail address: szot@u.washington.edu (P. Szot).
Abbreviations: ACTH, adrenocorticotropic hormone; AVP, arginine
vasopressin; CRF, corticotropin-releasing factor; CRF-R1,
corticotropin-releasing factor receptor subtype 1; HPA, hypothalamic–
pituitary–adrenal; POMC, pro-opiomelanocortin; PVN, paraventricular
nucleus.
0306-4522/04$30.00⫹0.00 Published by Elsevier Ltd on behalf of IBRO.
doi:10.1016/j.neuroscience.2004.03.030
241
pituitary–adrenal (HPA) axis. Glucocorticoid secretion from
the adrenal cortex is controlled by adrenocorticotropic hor-
mone (ACTH), which is secreted by the anterior pituitary.
ACTH secretion is in turn stimulated by hypothalamic
corticotropin-releasing factor (CRF) and arginine vaso-
pressin (AVP) acting synergistically. Activity of the axis is
suppressed by glucocorticoid feedback inhibition acting at
multiple levels: pituitary, hypothalamic, and supra-
hypothalamic. Chronic glucocorticoid treatment disrupts
the auto-regulatory action of the axis and may alter expres-
sion of hypothalamic and hypophyseal peptides and recep-
tors intrinsic to HPA function.
There have been numerous studies performed in ro-
dents examining the effects of chronic glucocorticoid ad-
ministration on the HPA axis. In rodents, elevated circulat-
ing glucocorticoid concentrations decrease expression of
pro-opiomelanocortin (POMC; the biosynthetic precursor
of ACTH; Autelitano et al., 1987; Harbuz et al., 1990;
Young et al., 1995; Zhou et al., 1996) and CRF receptor 1
(CRF-R1), at least transiently, in anterior pituitary (Luo et
al., 1995; Makino et al., 1995; Zhou et al., 1996; Oched-
alski et al., 1998; Aguilera et al., 2001). Glucocorticoids
also down-regulate expression of the hypothalamic ACTH
releasing peptides CRF (Harbuz et al., 1990; Albeck et al.,
1994; Patchev and Almeida, 1996; Swanson and Sim-
mons, 1989) and AVP (Davis et al., 1986; Albeck et al.,
1994; Patchev and Almeida, 1996; Ferrini et al., 1997) in
the paraventricular nucleus (PVN) of the hypothalamus.
These functional alterations of the HPA axis in response to
elevated glucocorticoids have been demonstrated repeat-
edly in rodents, but they have not been examined in pri-
mates. Knowledge of the potential existence of similar
effects in primates is clinically relevant because glucocor-
ticoids are commonly prescribed, on a chronic basis, for
many immunological and inflammatory conditions.
We attempted to determine whether chronic cortisol
treatment in primates produces changes in the HPA axis
similar to those found in rodents. Therefore, the effects of
chronic hypercortisolemia in old (18 –29 years) Macaca
nemestrina (pig-tailed macaques) were investigated by
measuring anterior pituitary POMC and CRF-R1 message
expression and PVN CRF and AVP mRNA by in situ
hybridization after 1 year of high-dose oral glucocorticoid
treatment. We hypothesized that the expression of these
peptides and the CRF receptor would be down-regulated
in older macaques receiving cortisol. To our knowledge,
this is the first investigation of the effect of chronic glu-
cocorticoid treatment on HPA-related peptide and receptor
expression in primates.
242 P. Szot et al. / Neuroscience 126 (2004) 241–246
EXPERIMENTAL PROCEDURES
Monkey tissue
Fifteen (five males and 10 females) retired breeder pigtailed ma-
caques (M. nemestrina) in mid- to late life (ages 18 –29 years with
a mean⫾S.E.M. of 23.1⫾1.0 years) received placebo (two males
and six females) or cortisol (three males and four females) orally
for 1 year. Hydrocortisone (cortisol) acetate (3.85–5.78 mg/kg/
day) was administered by mouth twice per day in a highly palat-
able mixture containing peanut butter, molasses, mashed potato
flakes, and ground monkey chow (Leverenz et al., 1999). During
the year of treatment, all animals were individually housed in the
same room so that physical contact between animals was not
possible, but visual and auditory communication was available.
Housing temperature and humidity conditions conformed to the
Animal Welfare Act and Guide for the Care and Use of Laboratory
Animals. All animals were in good general health. Purina monkey
chow was provided during the study on a twice-daily basis, with
fruit supplements. The Washington Regional Primate Research
Center is fully accredited by American Association for the Assess-
ment and Accreditation of Laboratory Animal Care International,
and all procedures were reviewed and approved by the University
of Washington Animal Care and Use Committee. The minimum
number of animals were used for these studies and care was
taken to minimize any suffering. Both plasma (con-
trol⫽318⫾47 ng/ml and treated⫽467⫾61 ng/ml) and CSF (con-
trol⫽13.3⫾1.3 ng/ml and treated⫽40. 8⫾11.0 ng/ml) cortisol were
significantly elevated in cortisol-treated monkeys at the time of
necropsy (Leverenz et al., 1999). After 12 months of treatment,
animals were killed by valium-phenobarbital injections. Brains
were rapidly removed and sectioned into the two hemispheres.
The pituitary was removed and frozen intact. The left portion of the
brain was sectioned into 0.5-cm-thick coronal blocks of the cere-
bral hemispheres and 0.5-cm-thick horizontal blocks of the brain-
stem. The left hemisphere and brainstem blocks were rapidly
frozen between cooled alumina plates in a ⫺70 °C freezer and
stored at that temperature. Blocks including the hypothalamus and
the pituitary were sectioned (20 ␮m) on a cryostat, thaw mounted
onto Fisher Superfrost slides (Fisher Scientific, Houston, TX,
USA) and stored at ⫺70 °C.
Oligonucleotides
PVN sections were atlas matched to the same level for both
treatment groups. The midsection of the PVN was chosen based
on preliminary work, because this region exhibited intense CRF
labeling. Pituitary sections for both treatment groups contained
anterior and posterior regions of the pituitary to demonstrate the
specificity of the oligonucleotide probes for the anterior lobe of the
pituitary. Tissue was prepared as described in detail elsewhere
(Szot et al., 2000). For each oligonucleotide probe studied, tissue
from both treatment groups was processed at the same time.
Oligonucleotide probes (Invitrogen, Carlsbad, CA, USA) comple-
mentary to the first 16 amino acids of the ACTH region of the
POMC sequence (Whitfeld et al., 1982), to the binding region of
the CRF-R1 sequence (Ross et al., 1994), to nucleotides 511–558
of the CRF sequence (Shibahara et al., 1983) and the first 16
amino acids of the neurophysin II portion of the rat AVP sequence
(Land et al., 1982) were utilized. Preliminary work with each of the
oligonucleotide probes indicated specific labeling to regions that
have been previously documented (Pepe et al., 1994; Austin et al.,
1995; Liebsch et al., 1995). Each oligonucleotide probe was 3⬘-
end-labeled with [33P]dATP (Perkin Elmer, Boston, MA, USA)
using terminal deoxyribonucleotidyl transferase (Life Technolo-
gies, Gaithersburg, MD, USA) and then purified on NEN-Sorb
columns. The hybridization buffer for POMC assay contained
1.35⫻106 cpm/50 ␮l, for CRF-R1, 0.65⫻106 cpm/50 ␮l, for CRF,
0.80⫻106 cpm/50 ␮l, and for AVP, 0.55⫻106 cpm/50 ␮l. Slides
hybridized with each of the [33P] oligonucleotide probes were
coated with NTB2 Nuclear Track Emulsion (undiluted) and stored
at ⫺20 °C for 1 day for POMC, 2 days for AVP, 7 days for CRF-R1,
and 2 weeks for CRF. The slides were developed in Kodak D-19
developer (diluted 1:1 with water) at 17 °C, rinsed in water, and
fixed in Kodak general fixer. The slides were stained with Cresyl
Violet acetate, dehydrated (70, 95 and 100% alcohol), allowed to
air dry, and mounted with coverslips.
Quantification of mRNA in anterior pituitary
To quantify POMC and CRF-R1 mRNA expression in the anterior
pituitary in the monkey tissue, three consecutive sections from
each animal were processed, hybridized, and washed in the same
session. The density of POMC and CRF-R1 grains/cell was de-
termined by analyzing the quantity of silver grains over a cell body
at a threshold value approximately three-fold higher than back-
ground under dark-field illumination with a 20⫻ objective. Grain
intensity (i.e. grains/cell) was measured as pixels using the Micro-
Computer Imaging Device (MCID; Imaging Research Inc., St.
Catherines, Ontario, Canada). The data are represented as the
mean⫾S.E.M. of the number of grains localized over cell bodies
(grains/cell) in the anterior pituitary from three sections. Data were
analyzed with unpaired Student’s t-test using StatView (SAS In-
stitute, Inc., Cary, NC, USA) with the significance level at P⬍0.05.
Simple linear regressions were performed between mRNA mea-
surements and age, urinary, plasma and CSF cortisol and plasma
ACTH using StatView, with significance at P⬍0.05.
Quantification of mRNA in hypothalamic PVN
CRF and AVP mRNA expression in the monkey PVN tissue was
quantified in the same way as POMC and CRF-R1 mRNA in the
anterior pituitary tissue, with three consecutive sections from each
animal processed, hybridized, and washed in the same session.
Since an alteration in mRNA expression in neurons can result in
either changes in the number of cells expressing the mRNA or the
amount of mRNA expressed per cell (grains/cell), both of these
measurements were performed in the PVN. The number of cells
that achieved the threshold criteria described above was recorded
and totaled unilaterally for the PVN and expressed as the number
of positively labeled cells. Unilateral readings were made in the
PVN (the other half of forebrain was fixed with paraformaldehyde
for other studies). An average of three readings was used to
determine a single value of grains/cell and number of positively
labeled cells for each animal. Measurement and analysis of grain
density (grains/cell) were as described for pituitary tissue.
RESULTS
Anterior pituitary
Expression of POMC and CRF-R1 mRNA was observed in
the anterior but not the posterior pituitary. Levels of POMC
or CRF-R1 mRNA in the anterior pituitary of control- and
cortisol-treated monkeys are shown in Fig. 1 as grains/cell.
Levels of POMC mRNA expression in the anterior pituitary
of control-treated monkeys demonstrated a significant
negative correlation with plasma ACTH levels (r⫽⫺0.776;
P⫽0.02). POMC and CRF-R1 mRNA expression were not
significantly correlated with age-within the narrow age
range of these animals -or to urinary, plasma or CSF
cortisol concentrations. POMC mRNA expression per cell
(grains/cell) in cortisol-treated monkeys was significantly
reduced in comparison with control animals (Fig. 1A; dark-
field photomicrographs of POMC mRNA expression per
cell [grains/cell] in control- [1C] and cortisol-treated [1D]
 P. Szot et al. / Neuroscience 126 (2004) 241–246 243

A B 400 A B

Cell Number
200 Control 200 120

Grains/cell
Control
Treated 150 Treated 100
80
300 100 60
Grains/cell

50
40
*
200 0 * 20
0
100

* 100

0 0

Fig. 2. (A) Number of positively labeled neurons and (B) grains/cell of

CRF mRNA expression in grains/cell of CRF mRNA expression in the
hypothalamic PVN of monkeys treated with either placebo or cortisol
for 1 year. Dark-field images of CRF mRNA labeling in the PVN of
placebo (control; C) and cortisol-treated (D) monkey. * Indicates sig-
nificant difference from control treated group, P⬍0.05. Scale
bar⫽300 ␮m.

control-treated monkeys are shown in Fig. 3. AVP mRNA

Fig. 1. Amount of POMC (A) and CRF-R1 (B) mRNA expression
(grains/cell) in the anterior pituitary of monkeys treated with either
placebo or cortisol (control) for 1 year. Dark-field images of POMC
mRNA labeling in the anterior pituitary of placebo (control; C) and
cortisol-treated (D) monkey. * Indicates significant difference from
control treated group, P⬍0.05. Scale bar⫽200 ␮m.
monkeys). CRF-R1 mRNA expression in the anterior pitu-
itary was not statistically different between treatment
groups (Fig. 1B).
PVN
The cell number and grains/cell of CRF expression in the
PVN of control- and cortisol-treated monkeys are shown in
Fig. 2. CRF mRNA expression in the PVN was negatively
correlated with urinary (r⫽⫺0.724; P⬍0.01), plasma
(r⫽⫺0.648; P⬍0.05) and CSF (r⫽⫺0.558; P⬍0.05) corti-
sol and positively correlated with plasma ACTH concen-
trations (r⫽0.616; P⬍0.05) when both groups (control and
cortisol-treated) were combined, but analysis of each
group separately did not result in a significant correlation
with any parameter. AVP mRNA expression did not exhibit
significant correlations with age, urinary, plasma or CSF
expression in cortisol-treated animals did not differ signif-
icantly from that of control animals, in either number of
positively labeled neurons or grains/cell.
DISCUSSION
These studies comprise the first demonstration in a pri-
mate of the effects of 1-year, high-dose cortisol treatment
on hypothalamic and hypophyseal expression of peptides
and peptide receptors that are fundamental components of
the regulation of the HPA axis. Chronic cortisol treatment
resulted in marked decreases in anterior pituitary POMC
and hypothalamic PVN CRF mRNA without significantly
affecting pituitary CRF-R1 or PVN AVP expression. The
decrease in CRF mRNA expression was negatively corre-
lated with urinary, plasma and CSF cortisol levels, indicat-
ing suppression of CRF mRNA expression by elevated
A B
300 200
Control
Treated
250
Cell Number

Grains/cell

cortisol or with plasma ACTH. CRF mRNA expression in 150 p<0.20

the PVN of cortisol-treated monkeys was significantly re- 200
duced compared with controls, both in terms of the number
of positively labeled neurons (Fig. 2A) and the amount of 150 100
CRF expressed per cell (grains/cell; Fig. 2B; dark-field
p<0.10
photomicrographs of CRF expression in control- [2C] and 100
cortisol-treated [2D] monkeys).
50
AVP mRNA expression was found only in the magno- 50
cellular region of the PVN, the region in which AVP peptide
concentration is highest in monkeys (Kawata and Sano, 0 0
1982). AVP mRNA levels were undetectable in the parvi-
cellular region of the PVN. The number of positively la- Fig. 3. (A) Number of positively labeled neurons and (B) grains/cell of
beled neurons and grains/cell of AVP mRNA expression in AVP mRNA expression in the hypothalamic PVN of monkeys treated
the magnocellular region of the PVN of cortisol- and with either placebo or cortisol for 1 year.
244 P. Szot et al. / Neuroscience 126 (2004) 241–246
cortisol levels. Since cortisol also suppresses ACTH se-
cretion (Leverenz et al., 1999), CRF mRNA is positively
correlated with plasma ACTH levels. Plasma ACTH con-
centrations in the control group exhibited a significant neg-
ative correlation with POMC mRNA expression, reflecting
the increasing inhibition of POMC mRNA expression with
increases in endogenous ACTH and cortisol. There was no
significant correlation with POMC mRNA in the cortisol
treated monkeys because of complete feedback inhibition
of ACTH in this group. Four of the seven monkeys in the
cortisol-treated group had unmeasurable plasma ACTH.
The majority of studies in rats have also reported a
significant reduction of POMC mRNA in the anterior pitu-
itary and of CRF mRNA in the PVN (Davis et al., 1986;
Autelitano et al., 1987; Swanson and Simmons, 1989;
Harbuz et al., 1990; Albeck et al., 1994; Young et al., 1995;
Zhou et al., 1996; Patchev and Almeida, 1996), although
Konakchieva et al. (1998) found increases in rat hypotha-
lamic CRF and AVP expression after 5 days of i.p. injec-
tions of very high doses of dexamethasone. In the single
human study published to date, chronic glucocorticoid ad-
ministration of varying duration (2–18 days) resulted in a
significant decrease in the number of CRF immunohisto-
chemically labeled neurons in the hypothalamus (PVN;
Erkut et al., 1998). Therefore, the down-regulation of
POMC and CRF mRNA in the pituitary and hypothalamus,
respectively, is a consistent effect of chronic glucocorticoid
administration in rodents and non-human primates despite
differences in route of administration, type of glucocorti-
coid, dose, nature of the control group, or duration of
treatment.
Regulation of CRF-R1 has been shown to be complex
and exerted at multiple levels including translational and
post-translational modification (Aguilera et al., 2001; Xu et
al., 2001). Glucocorticoid administration to rats generally
has been shown to result in sustained decreases in
CRF-R1 number and binding sites in the anterior pituitary
(Childs et al., 1986; Schwartz et al., 1986; Hauger et al.,
1987), but CRF-R1 mRNA has been found to exhibit only
a transient reduction (Ochedalski et al., 1998; Iredale and
Duman, 1997). In addition, pituitary CRF-R1 mRNA levels
are not affected by long-term adrenalectomy and thus are
not under tonic inhibition by glucocorticoids (Rabadan-
Diehl et al., 1997). Therefore, the lack of effect of chronic
glucocorticoid treatment on CRF-R1 mRNA in macaque
anterior pituitary coincides with findings in rats.
The lack of an effect on AVP mRNA expression in the
hypothalamic PVN in the monkeys after 1 year of cortisol
treatment is not completely consistent with rodent data,
although there are major differences between the rodent
studies and the one performed here. AVP is expressed in
parvicellular and magnocellular neurons of the PVN with
the majority of AVP being localized to magnocellular neu-
rons (Kawata and Sano, 1982; Ma and Aguilera, 1999).
However, AVP expression in magnocellular PVN neurons
is not generally considered to be under regulatory control
by cortisol feedback. The levels of AVP mRNA expression
in parvicellular PVN neurons are low under physiological
conditions, but are significantly increased following adre-
nalectomy in rodents. Glucocorticoid administration subse-
quent to adrenalectomy reduces AVP expression to basal
levels (Davis et al., 1986; Albeck et al., 1994; Ferrini et al.,
1997; Ma and Aguilera, 1999; Tanimura and Watts, 2000).
In our study, AVP mRNA levels were undetectable in ma-
caque parvicellular PVN neurons, and this extremely low
parvicellular expression is reflected by low protein levels
(Kawata and Sano, 1982). The lack of an effect on AVP
mRNA expression in the hypothalamic PVN in monkeys
after 1 year of cortisol treatment may be the result of a
“floor” effect in parvicellular neurons and the relative insen-
sitivity of magnocellular neurons to glucocorticoid
feedback.
However, there is some evidence to indicate that the
magnocellular neurons of the PVN do respond to glucocor-
ticoids under some conditions. Adrenalectomy followed by
glucocorticoid replacement may not be an appropriate
means of determining glucocorticoid regulation because
removal of the adrenals also removes the source of min-
eralocorticoids. The consequent alterations of plasma os-
molality can have a profound effect on magnocellular AVP
neurons that may mask any effect of glucocorticoids on
these neurons. Electrophysiologically, magnocellular AVP
neurons in the PVN are inhibited by the administration of
glucocorticoids (Saphier and Feldman, 1988). The expres-
sion of glucocorticoid receptors also increases in magno-
cellular neurons during chronic hyperosmolality (Berghorn
et al., 1995). Adrenocortical insufficiency results in in-
creased levels of plasma AVP, the source of which is
magnocellular PVN neurons. Glucocorticoid administration
reduces the plasma AVP concentrations (Ahmed et al.,
1967). Erkut et al. (1998) found a significant decrease in
total PVN AVP expression in postmortem tissue of sub-
jects exposed to short term glucocorticoid treatment (2–18
days) but did not differentiate between parvicellular and
magnocelluar neurons of the PVN. We have been unable
to detect AVP mRNA in postmortem human parvicellular
PVN (unpublished data), and in light of the extremely low
expression of AVP in parvicellular neurons, the changes
observed by Erkut et al. (1998) can be surmised to have
occurred in the magnocellular PVN. It is possible that
short-term glucocorticoid administration reduces AVP ex-
pression in the magnocellular neurons of the primate PVN,
but that compensatory AVP expression may occur when
glucocorticoid treatment is prolonged as in the current
study.
Overall, the changes in HPA-related peptide and re-
ceptor expression in the hypothalamus and anterior pitu-
itary of macaques to prolonged glucocorticoids are very
similar to the changes that have been documented in
rodents. Significant suppression of pituitary POMC and
hypothalamic CRF expression are consistent and predom-
inant responses to chronic glucocorticoid administration.
Suppression of pituitary CRF-R1 and PVN AVP expression
may be transitory and/or regulated primarily by transla-
tional or post-translational mechanisms. Chronic therapeu-
tic administration of high levels of glucocorticoids in hu-
mans is highly likely to result in prolonged suppression of
pituitary POMC and hypothalamic CRF suppression. This
 P. Szot et al. / Neuroscience 126 (2004) 241–246
prolonged suppression may not only alter HPA functional
dynamics, but may also affect CRF-dependent CNS me-
diation of anxiety and/or POMC-dependent peripheral en-
dorphin responses to pain.
Acknowledgements—These studies were supported by the De-
partment of Veterans Affairs Research and Development Service,
Northwest Network Mental Illness Research, Education and Clin-
ical Center (MIRECC) and Geriatric Research, Education and
Clinical Center (GRECC), VA Puget Sound Health Care System;
the National Alliance for Research on Schizophrenia and Depres-
sion and the University of Washington Alzheimer’s Disease Re-
search Center (AGO5136); the Geriatric Academic program
(AGO0503); and the Alhadeff Alzheimer’s Research Fund.
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(Accepted 18 March 2004)