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doi:10.1093/brain/awh448
Brain (2005), 128, 1301–1313
Colocalization of corticotropin-releasing hormone
and oestrogen receptor-a in the paraventricular
nucleus of the hypothalamus in mood disorders
Ai-Min Bao,1,2,3 Andon Hestiantoro,3 Eus J. W. Van Someren,3 Dick F. Swaab3 and Jiang-Ning Zhou2
1
Correspondence to: Dr Jiang-Ning Zhou,
Department of Neurobiology, School of Life Science,
University of Science and Technology, China,
Hefei 230032, Anhui, People’s Republic of China
E-mail: [email protected]
Summary
Oestrogens may modulate the activity of the
hypothalamic–pituitary–adrenal (HPA) axis. The present
study was to investigate whether the activity of the HPA
axis in mood disorders might be directly modulated
by oestrogens via oestrogen receptors (ORs) in the
corticotropin-releasing hormone (CRH) neurons of the
human hypothalamic paraventricular nucleus (PVN).
Brains of 13 subjects ranging in age between 45 and
79 years suffering from major depression/major depressive disorder (eight cases) or bipolar disorder (five cases)
and of 13 controls, matched for sex, age, brain weight,
post-mortem delay, fixation time and season and
clock time at death, were studied with double-label
immunocytochemistry. The total number of CRHimmunoreactive (IR) neurons, CRH neurons that colocalized ORa in the neuronal nucleus and the number of
only nuclear ORa-containing neurons in the PVN were
measured using an image analysis system. In addition, the
volume of the PVN delineated on the basis of CRH neurons was determined. It was found that the total number
of CRH-IR neurons in patients with mood disorders was
nearly 1.7 times higher than in controls (P = 0.034).
A novel finding was that the total number of CRH-IR
neurons and the number of CRH-nuclear ORa doublestaining neurons in the PVN were strongly correlated both
in controls and in patients with mood disorders (P < 0.001
and P = 0.022, respectively). The ratio of the CRHnuclear-ORa double-staining neurons to the total CRHIR neurons in patients with mood disorders was similar
to that in the controls (P = 0.448). The volume of the subregion of the PVN that was delineated on the basis of CRH
neurons was significantly larger in patients with mood
disorders than in controls (P = 0.022). Another novel finding was the large population of extra-hypothalamic CRH
neurons that was found in the thalamus. In summary,
oestrogens may directly influence CRH neurons in the
human PVN. The increased numbers of neurons expressing CRH in mood disorders is accompanied by increased
ORa colocalization in the nucleus of these neurons. These
changes seem to be trait- rather than state- related.
Keywords: hypothalamic-pituitary-adrenal axis; corticotropin-releasing hormone; oestrogen receptor-a;
paraventricular nucleus; mood disorders
Abbreviations: BD = bipolar disorder; CRH = corticotropin-releasing hormone; HPA = hypothalamic–pituitary–adrenal;
HPG = hypothalamic–pituitary–gonadal; MD = major depression; MDD = major depressive disorder; OR = oestrogen receptor;
ORE = oestrogen responsive element; PTN = paratenial thalamic nucleus; PVN = paraventricular thalamic nucleus;
TBS = Tris-buffered saline
Received October 1, 2004. Revised December 20, 2004. Accepted January 17, 2005. Advance Access publication
February 10, 2005
Introduction
A number of studies have pointed to the possible involvement
of sex hormones in mood, cognition and a variety of psychiatric disorders. Epidemiological studies have shown that the
lifetime prevalence of major depression is twice as high in
women as in men (Lehtinen and Joukamaa, 1994; Pearlstein
et al., 1997). The prevalence of major depression increases
# The Author (2005). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For Permissions, please email: [email protected]
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Department of Endocrinology, The First Affiliated Hospital
of Anhui Medical University, 2Department of Neurobiology,
School of Life Science, University of Science and
Technology of China, Hefei, Anhui, People’s Republic of
China and 3Netherlands Institute for Brain Research,
Amsterdam, the Netherlands.
1302
A.-M. Bao et al.
drugs attenuate the synthesis of CRH (Fischer et al., 1990;
Nemeroff, 1996; Reus et al., 1997).
The presence of a close functional interaction between
the HPA axis and the hypothalamic–pituitary–gonadal (HPG)
axis has been known for many years. A link of infertile
periods to increased levels of adrenal steroids was observed
in animals (Christian, 1971), while studies in a number of
species have documented inhibitory effects of CRH on
gonadotropin-releasing hormone and luteinizing hormone
secretion (Ferin and Vande Wiele, 1984; Gambacciani
et al., 1986; Olster and Ferin, 1987; Petraglia et al., 1987;
Dudas and Merchenthaler, 2002). Diminished luteinizing
hormone response to gonadotropin-releasing hormone following long-term prednisolone treatment has been found in
women (Sakakura et al., 1975) and, in Cushing’s disease, the
presence of an irregular menstrual cycle was found related
to hypercortisolemia (Lado-Abeal et al., 1998). Moreover,
oestrogen-responsive elements (OREs) are found in the
CRH gene promoter region and oestrogen has been shown
to have modulatory actions on CRH gene expression in
rodents (Vamvakopoulos and Chrousos, 1993).
On the basis of the observations mentioned above, the
present study was aimed at determining whether the activity
of the HPA axis in mood disorders might at least be partly
modulated by oestrogens via ORs and, more specifically,
whether this would affect the CRH neurons in the
human PVN directly or whether it would be mediated by
interneurons.
Material and methods
Subjects
Twenty-six autopsied brain samples were studied, of which 13 were
patients clinically followed for mood disorders with an age range
of 45–79 years and 13 were control subjects matched for sex,
age, season and clock time at death, brain weight, post-mortem
delay, fixation time and storage time in paraffin. Brain material
was obtained from the Netherlands Brain Bank (coordinator
Dr R. Ravid) following permission from the patient or next of
kin for a brain autopsy and for the use of the brain material and
clinical data for research purposes.
DSM-IIIR/DSM-IV criteria were used for the diagnosis of major
depression (MD)/major depressive disorder (MDD) or bipolar disorder (BD) at any time during life. The criteria for the presence,
duration and severity of symptoms of either MD/MDD or BD, as
well as the exclusion of other psychiatric and neurological disorders, were systematically scored by two qualified psychiatrists
(Dr W.J.G. Hoogendijk or Dr E. Vermette). Eight patients fulfilled
the criteria for MD/MDD and five fulfilled the criteria for BD. For
detailed information, see Table 1. An overview of the medication in
the past and in the last month before death for patients with mood
disorders and control subjects is also given in Table 1. The medical
records did not reveal any alcohol or other drug abuse among subjects of either group unless stated otherwise (C1, C5). The absence of
neuropathological changes, both in the patients with mood disorders
and in the controls, was confirmed by systematic neuropathological
investigation (van de Nes et al., 1998) by Dr W. Kamphorst (Free
University Amsterdam, The Netherlands).
Downloaded from brain.oxfordjournals.org at Vrije Universiteit - Library on April 8, 2011
during the reproductive years, especially during times of
changes in gonadal hormone levels, e.g. the premenstrual
and postpartum periods and during the transition to the
menopause (Paykel, 1991; Lehtinen and Joukamaa, 1994;
Pearlstein et al., 1997; Young and Korszun, 2002). There
is also some evidence that hormone replacement therapy
could improve and prevent postpartum depression (Sichel
et al., 1995; Gregoire et al., 1996). Moreover, it was
found in males that testosterone levels were lower in severely
depressed patients (Heuser, 2002) and that older men with
lower available testosterone levels were found to be more
frequently depressed (Barrett-Connor et al., 1999).
The effects of oestrogens on the brain are, at least partly,
mediated by oestrogen receptors (ORs) of which two subtypes exist, i.e. ORa and ORb. Both OR subtypes are extensively localized throughout the brain (Pfaff and Keiner, 1973;
Donahue et al., 2000; Osterlund et al., 2000a,b; Kruijver
et al., 2002, 2003), where they may mediate the effects
of oestrogens on emotion, cognition and procreation. It is also
well known that conversion of androgens into oestrogens by
the enzyme aromatase is a key mechanism by which testosterone regulates many physiological and behavioural processes (Balthazart and Ball, 1998). The highest levels of
brain aromatase activity have been found in various limbic
regions, such as the preoptic and hypothalamic regions, which
overlap extensively with brain regions containing oestrogen
receptors (Steimer and Hutchison, 1981; Roselli et al., 1985;
Schumacher and Balthazart, 1987; Vockel et al., 1990).
It is, therefore, logical to hypothesize that the ORs may be
involved in the pathogenesis of mood disorders. However,
although endocrine studies have shown that oestrogens modulate a variety of neurotransmitter systems [e.g. the norepinephrine, dopamine and serotonin system (Clark et al., 1985;
Blaustein et al., 1986)] that have been strongly implicated
in the aetiology of mood disorders (Asberg et al., 1976; van
Praag, 1983; Clark et al., 1985; Blaustein et al., 1986; Pryor and
Sulser, 1991), so far the mechanism and the putative sites of
oestrogen action in the human brain has not been studied in
post-mortem material in relation to mood disorders (Osterlund
and Hurd, 2001). The hypothalamic–pituitary–adrenal (HPA)
axis—the key system in the stress response—has been extensively studied in affective diseases and is considered by many to
be the final common pathway for a number of signs and symptoms of depression (Holsboer and Barden, 1996; Swaab et al.,
2000). The corticotropin-releasing hormone (CRH) neurons in
the hypothalamic paraventricular nucleus (PVN) are the central driving force for the HPA response to stress (Bissette, 1990).
There is a strong increase in the activity of the CRH neurons in
major depression (Raadsheer et al., 1994, 1995) and a CRHoverproducing transgenic mouse model shows symptoms
related to major depression, i.e. an anxious phenotype, that
can be counteracted by a CRH antagonist (Stenzel-Poore
et al., 1994; Muller et al., 2004). Moreover, signs and symptoms of depression can be induced in experimental animals
by intracerebroventricular injection of CRH (Holsboer and
Barden, 1996; Richardson et al., 2000) and antidepressant
Colocalization of CRH and ORa in mood disorders
Human brain material
The hypothalami were dissected and fixed in 0.1 M phosphate
buffered 4% w/v formaldehyde (pH 7.2) for 1–2 months (Table 1).
Tissues were dehydrated in graded ethanol, embedded in paraffin
and serially cut in frontal sections (6 mm) on a Leitz microtome and
stored at room temperature. Every 100th section was stained with
thionine (0.1% w/v thionine in acetate buffer, pH 4) in order to
localize and orient the PVN before immunocytochemical staining.
Immunocytochemistry
(i) deparaffinized and rehydrated using xylene and decreasing
grades of ethanol;
(ii) placed in 0.05 M Tris-HCl buffer (pH 7.6) and microwavetreated (for antigen retrieval) at 90 C for 30 min;
(iii) cooled to room temperature for 30 min and rinsed 33 for
10 min in 0.05 M Tris-buffered saline (TBS) (pH 7.6);
(iv) incubated in milk (elk, campina)-TBS (pH 7.6) for 60 min to
reduce non-specific staining;
(v) incubated overnight at 4 C with rabbit anti-ORa 1:100 and
rat PFU83 1:100 000 diluted in sumi-milk (0.25% gelatin and
0.5 ml Triton X-100 and 5% of milk powder in 100 ml TBS,
pH 7.6);
(vi) rinsed 33 10 min in TBS and incubated with biotinylated
anti-rabbit IgG (Vector Laboratories, Burlingame, CA, USA)
(viii)
(ix)
(x)
(xi)
(xii)
(xiii)
(xiv)
1:200 and anti-rat IgG conjugated with alkaline phosphatase
(K.P.L., Maryland, USA) 1:50 in sumi-milk at room temperature for 60 min;
rinsed 33 10 min in TBS and incubated with ABC elite
kit (1:400, Vector Laboratories) diluted in TBS for
60 min;
rinsed 33 10 min in TBS and incubated with biotinylated
tyramide diluted 1:750 in TBS plus 0.01% hydrogen peroxide
for 15 min to amplify the immunocytochemical reaction of
the ORa antigen (Adams, 1992);
repeat step (vii);
rinsed 33 10 min in TBS and incubated for 5 min in 0.2 M
Tris–HCl buffer pH 8.8;
incubated with a filtered suspension of 2 mg Fast Blue BB
base (Sigma, St Louis, MO, USA) in 10 ml 0.2 M Tris–HCl
buffer (pH 8.8) containing 2 mg Naphtol-AS-AX phosphate
(Sigma) and 3 mg levamisole (Sigma);
rinsed 33 10 min in TBS and incubated for 5 min with 0.2 M
acetic acid buffer pH 5;
incubated with a filtered suspension of 5 mg of 3-amino9-ethylcarbazole (AEC) in 10 ml 0.2 M acetic acid buffer
(pH 5) containing 0.5 ml N,N-dimethylformamide and 5 ml
30% hydrogen peroxide;
rinsed 33 10 min in TBS and coverslipped with Kaisers
glycerin–gelatin (Merck, Darmstadt, Germany).
Since cells showing only ORa and no CRH cytoplasmic staining
generally comprised <10% of all of the cell profiles, we did not
distinguish this as a separate cell population. The total numbers of
the three main classes of cell profiles were determined, i.e. (i) CRH
single or CRH-ORa cytoplasmic double-staining cells, (ii) ORa
nuclear single-staining cells and (iii) cytoplasmic CRH-ORa nuclear
double-staining cells.
Volume measurements
The volume of the PVN delineated by CRH neurons was estimated
unilaterally by measuring the cross-sectional area delineated by
immunoreative CRH neurons in every 100th section using 2.53
objective (Plan-Neofluar) on a Zeiss (Oberkochen, Germany)
Axioskop microscope mounted with a Sony (Tokyo, Japan) black/
white CCD camara (model XC77CE), connected to an IBAS image
analysis system (Kontron-IBAS, Munich, Germany). The total
volume of the PVN delineated by CRH neurons was calculated
according to the Cavalieri principle (Gundersen et al., 1988). A
pilot study had shown that the sampled volume delineated either by
CRH or ORa neurons gave similar results.
Estimation of total neuronal number
Cross-sectional digital images (every 100th section) were made
using a 2.53 objective (Plan-Neofluar) on a Zeiss Axioskop microscope, mounted with a Sony black/white CCD camera (model
XC77CE) that was connected to an IBAS imaging analysis system.
The contour of the entire PVN field occupied by CRH and/or ORaimmunostained neurons was manually outlined by the operator with
the cursor, with a final on-the-monitor magnification of 903 (when
the objective was 2.53); subsequently, the imaging analysis system
overlaid a grid of rectangular fields within the outlined crosssectional area. Each field was equal in size to the area displayed
by the camera at 633 objective (Plan-Apochromat).
Downloaded from brain.oxfordjournals.org at Vrije Universiteit - Library on April 8, 2011
The monoclonal rat anti-CRH antibiody ‘PFU 83’ (IgG2a subclass)
(kindly donated by Professor F.J.H Tilders) was aimed at the
C-terminal part (amino acids 38–39) of rat/human CRH. For
immunocytochemical staining of ORa, the polyclonal rabbit antiORa antibody (MC-20), which recognized the C-terminal epitope
of the ORa (SantaCruz Biotechnology, Santa Cruz, CA, USA;
catalogue no. sc-542) was used. The specificity of these two antibodies has been confirmed previously in our laboratory (Raadsheer
et al., 1993; Ishunina et al., 2000; Kruijver et al., 2002). The staining
was a modification of the method used by Erkut et al. (1995) and
Huitinga et al. (2000), resulting in blue cytoplasmic CRH staining
by alkaline phosphatase, red ORa nuclear or cytoplasmic staining
by peroxidase, and purple CRH-ORa double staining cytoplasm.
We did perform a pilot study of ORb staining according to the
protocol of Ishunina et al. (2000) and looked for CRH-ORb doublestaining in these subjects. However, we found virtually no ORb
immunochemical staining in the PVN of these subjects. This was in
agreement with previous findings that, in contrast to ORa, ORb
and ORb mRNA expression are very low in this hypothalamic area
in humans (Osterlund and Hurd, 2001; Kruijver et al., 2002, 2003).
The high ORa/b ratio seems to be a species-specific phenomena,
since ORb is abundantly present in the PVN in rat and mouse, while
ORa is absent or barely expressed (Simonian and Herbison, 1997;
Alves et al., 1998; Hrabovszky et al., 1998). However, ORb is also
barely present in CRH neurons of the PVN in mouse (Nomura et al.,
2002).
The double staining for CRH and ORa was performed on every
100th section throughout the complete PVN region, stretched
and mounted on SuperFrost/Plus slides (Menzel, Braunschweig,
Germany). The rostral-caudal length of the PVN was defined as the
distance between the most rostral and most caudal section that contained three or more CRH or ORa positive cells. Both criteria gave
similar results. Run-effects were counteracted by a design in which
sections from equal numbers of depressive patient(s) and control(s)
were stained in the same run while the investigator was blind to the
identities of the subjects. Staining was performed as follows:
(vii)
1303
M
F
F
M
M
M
M
M
M
F
D4
D5
D6
D7
D8
D9 b
D10 b
D11 b
D12 b
D13 b
Median
Range
Mean 6 SD
F
M
D2
D3
71/44
31/46
67.9 6 9.2/
44.9 6 13.0
73/28
75/40
68/32
70/35
74/74
79/ND
68/48
72/53
72/54
71/53
61/50
55/40
Mood disorder patients
D1
M
45/32
Age at
death/age
of onset
(years)
1320
555
1309.5 6 167.7
1260
1123
1204
1490
1444
1530
1424
1116
1287
975
1424
1320
1427
Brain
weight
(gram)
12:00
58:55
18:54 6 17:28
5:15
4:00
5:55
4:50
62:55
21:10
12:00
28:25
22:00
16:15
40:20
4:54
7:00
Post-mortem
delay (hours:
minutes)
38
26
38.7 6 7.6
36
38
33
43
35
28
30
35
39
38
42
52
54
Fixation
time
(days)
56.5 6 4.2
20
23
53
32
100
101
4
12
87
105
98
92
7
Storage
time in
paraffin
(months)
23:48 6 4:40*
9:30
20:45
23:15
2:45
17:05
17:50
2:30–9:45
4:20
19:00
16:15
4:40
7:45
2:30
Clock
time at
death
0.4 6 2.2*
1
1
10
10
3
9
2
4
1
2
10
11
6
Month
of
death
diabetes, pneumonia,
infarction right
hemisphere
cerebral ischaemia,
pneumonia
mesothelioma,
pneumonia
heart failure, septic
shock, pyelonephritis
Strangulation (suicide)
suicide
subdural haematoma
following fall
cardiac ischaemia
cardiac arrest, ileus
due to intestinal
haemorrhage
cachexia, dehydration
CVA left, acute
abdomen secondary
to perforation either
stomach or intestines
brain haemorrhage
(pons)
heart failure, urosepsis
Clinicopathological
information
Yes
Yes
Yes
No
Yes
Yes
No
No
No
Yes
No
Yes
No
Suicide
attempt
Yes
Probably
yes
SSRI, BRO
PHT
None
BZD
BZD
ZUC, SSRI, BZD
SSRI
MAOI
Li, TCA
Li
SSRI, MAOI
BZD, Tol, TCA,
SSRI, Haldol, Mo
MAP, BZD,
TCA, PHT
TCA
BZD, MAOI, TCA
MAP
MIA, TCA
None
None
Li
None
Li
Mo, Li
SSRI,
NSAID
4
1
ND
4
2
9
3
4
8
3
4
1
Yes
Yes
No
Manic
Yes
Yes
No
Yes
Yes
Yes
No
SSRI
1
SSRI
Died
during
depressive
episode?
Medication
taken
in the
last month
Medication
taken in
the past
No. of
episodes
Downloaded from brain.oxfordjournals.org at Vrije Universiteit - Library on April 8, 2011
Sex
Table 1 Brain material of patients with mood disorders and control subjects
1304
A.-M. Bao et al.
M
M
F
M
M
M
M
F
M
M
M
F
C2
C3
C4
C5
C6
C7
C8
C9
C10
C11
C12
C13
70
40
66.5 6 14.0
78
83
78
78
77
74
70
69
61
63
47
44
43
1317
463
1322.8 6 123.4
1310
1102
1442
1160
1235
1317
1454
1352
1296
1250
1368
1565
1345
9:00
89:20
17:08 6 23:04
4:20
7:45
8:25
4:00
2:40
8:00
9:00
19:15
17:45
10:20
29:13
10:00
92:00
43
149
54.3 6 40.1
43
34
24
39
47
60
28
ND
119
32
68
149
63
55
84
56.5 6 30.0
79
42
77
59
34
33
95
28
55
35
112
56
30
9:53 6 4:45*
16:15
21:00
12:15
12:15
8:30
13:00
8:00
23:00–3:30
23:15
5:00
11:47
7:00
21:00
10.5 6 2.2*
9
2
7
10
11
11
2
8
12
1
12
7
7
pancreatic cancer
acute myocardial
infarction
cardiac arythmia
myocardiac infarction,
(alcohol abuse)
acute myocardiac
infarction
hypercholesterolaemia,
acute myocardial
infarction
acute heart failure
pneumonia, myocardial
infarction, (alcohol
abuse)
pneumonia, sudden
death during sleep
(delirium)
Pneumonia, lung
carcinoma, renal failure
heart failure,
myocardial infarction
septic shock,
metastasized pancreas
carcinoma
pulmonary carcinoma
Downloaded from brain.oxfordjournals.org at Vrije Universiteit - Library on April 8, 2011
None
Statine
None
Antibiotics
bB, Hal, BZD#
ND
Sympaticomemetics
bB, antibiotics
PG, prednisone
5 mg
Ca-antagonists,
furosimide
Mo, BZD, fentanyl
Vit. B, digoxin,
nitrate
None
Statine
None
None
None
ND
Sympaticomemetics
None
bB, PG
Mo, BZD, fentanyl
Vit. B, digoxin,
nitrate
Prednisone, digoxin
Antibiotics
None
#Temazepam was started 3 days and Haloperidol 2 days before death for a state of delirium. *Circular mean and SD of the clock time at death or the month of death. Neither the clock time or month of death differed significantly between the groups as tested
with the Mardia-Watson-Wheeler test. bB = beta-blocker; BRO = bromperidol; BZD = benzodiazepine; F = female; Hal = haloperidol; Li = lithium; MAOI = monoamine oxidase inhibitor; M = male; MAP = maprotiline; MIA = mianserin; Mo = morphine;
ND = no data; None = no medication; NSAID = non-steroidal anti-inflammatory drugs; PG = prostaglandin synthetase inhibitor; PHT = phenothiazine; SSRI = selective serotonin reuptake inhibitor; TCA = tricyclic antidepressant; Tol = Tolvon;
ZUC = zuclopenthixol.
Median
Range
Mean 6 SD
F
Controls
C1
Colocalization of CRH and ORa in mood disorders
1305
1306
A.-M. Bao et al.
Statistical analysis
Differences among the groups were statistically evaluated by the
nonparametric Mann–Whitney U-test, since the data were discrete
and not normally distributed. The association between the number of
total CRH cells and the number of CRH-ORa nuclear-colocalizing
cells and the association between the duration of mood disorders
with the volume of PVN and CRH data were examined using
Spearman’s correlation coefficient. Differences in clock time of
death and month of death (circular parameters) between controls
and patients with mood disorders were tested with the Mardia–
Watson–Wheeler test (Batschelet, 1981). Tests were two-tailed.
Values of P < 0.05 were considered to be significant.
Results
The sex, age (z = 0.103, P = 0.920), season and clock time
at death (F = 2, x2 = 0.553, P = 0.766; and F = 2, x2 = 3.320,
P = 0.190; respectively), brain weight (z = 0.026, P = 1.000),
post-mortem delay (z = 0.0.359, P = 0.724), fixation time
(z = 0.821, P = 0.418) and storage time in paraffin
(z = 0.333, P = 0.762) were well matched for the mood
disorders and control group. The intensity of the staining, the
distribution of immunocytochemical reaction product and the
appearance of the immunoreactive neurons in patients with
mood disorders were similar to the results of the control cases,
except that there were more CRH neurons present in some
of the patients with mood disorders. Scattered CRH neurons
were found in the PVN (our main area of interest), but also in
the hypothalamic infundibular (= arcuate) nucleus. CRH fibres
were found in the area of the suprachiasmatic nucleus.
A surprisingly large population of CRH neurons was found
in the thalamus, i.e. in the paraventricular thalamic nucleus
(PVN) and paratenial thalamic nucleus (PTN), extending to
the anteromedial thalamic nucleus and the anterior principle
nucleus (for an example, see Fig. 1A). Clear CRH-staining in
the thalamus was present, both in controls and in patients
with mood disorders. No obvious characteristic differences
were found between the CRH neurons in the thalamus and
those in the PVN (Fig. 2), except that the staining intensity of
the CRH neurons in the thalamus was generally weaker than
those in the PVN.
The total population of CRH neurons (including the only
CRH-containing and CRH-ORa nuclear staining neurons)
was significantly larger than in controls (Fig. 3). The median
of the total number of CRH neurons (i.e. 15 298 in the
patients with mood disorders) was 1.7 times higher than
that found in the controls (z = 2.128, P = 0.034) (Fig. 3A).
The median of the total number of CRH-ORa nuclear doublestaining neurons was 5594, which was 1.2 times higher than
found in the controls—a trend that did not reach significance
(z = 1.769, P = 0.081). The median proportion of the number of CRH-ORa nuclear double-staining neurons of the total
number of CRH neurons in patients with mood disorders was
Fig. 1 A large population of CRH neurons in the thalamic anterior principal nucleus: a frontal section (6 mm) of the human thalamus
of a control subject (C11) (A,B). (B) represents a 12.53 higher magnification of (A) and shows a cytoplasmic CRH (blue)–ORa (red)
nuclear double-staining neuron.
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For analysis, 10% of the rectangular fields (each field covering at
least 10% of the outlined area) for the ORa nuclear single-staining,
60% for the CRH single-staining or for the CRH-ORa cytoplasmic
double-staining was selected by a random systematic sampling
procedure. A pilot study had shown that these counting percentages
yielded a good estimation of the total numbers of cell profiles.
To prevent double counting, only neurons containing a nucleolus
(2 mm diameter) were counted. This counting procedure, which
was judged to be the best for the thin (6 mm) section used, is based
on the principle that nucleoli can be considered as hard particles
that will not be sectioned by a microtome knife but, instead, are pushed
either in or out of the paraffin when hit the knife (Jones, 1937;
Cammermeyer, 1967; Koningsmark, 1970; Braendgaard and
Gundersen, 1986; Huitinga et al., 2000; Chung et al., 2002). No
double nucleoli were observed, confirming the observation of
Huitinga et al. (2000) for the PVN. All visible neurons with a nucleus
containing a nucleolus, within the exclusion lines, were counted using
a 633 objective with a final on-the-monitor magnification of 24003.
By dividing the total number of determined cell profiles by the sampled
volume, an estimate of the cellular density was calculated for each
PVN area and the total number of the cell profiles was calculated by
multiplying this volume with the cellular density. The measurements
were made without knowledge of the identity of the sections.
A test of the precision of the method for estimating CRH neurons
was performed in eight subjects whose sections were stained and
counted in two separate runs by one investigator (I. Huitinga; data
from Huitinga et al., 2000), who was blind to the subjects’ identities.
This test revealed highly significantly correlated values (r = 0.905,
P = 0.002).
Colocalization of CRH and ORa in mood disorders
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Fig. 2 Frontal section of the PVN in a control (C12) (A,B) and a patient with mood disorder (D10) (C,D) stained for CRH (blue) and ORa
(red). (B) and (D) represent a 43 higher magnification of (A) and (C). The arrows, solid and hollow arrowheads in (A,B) and (C,D) indicate
the same place in the preparation to facilitate comparison. Both sections show the central part (mid-level) of the PVN and contain the largest
number of stained neurons. It is clear by comparing (A) with (C) and (B) with (D), that the number of stained neurons is markedly increased
in this patient of mood disorder. III: the third ventricle. The arrow points to an ORa nuclear single-staining cell; the solid arrowhead points to
a cytoplasmic CRH-ORa nuclear double-staining cell and the hollow arrowhead points to a CRH single-staining cell.
similar to that in the controls (48% verus 37%) (z = 0.795,
P = 0.448). Significant correlations between the number of
CRH cells and the number of CRH-ORa nuclear doublestaining neurons in the PVN were found both in control
and mood disorders groups (r = 0.852, P < 0.001 and
r = 0.626, P = 0.022, respectively) (Fig. 4). The same holds
for the two combined groups (r = 0.801, P < 0.001). The total
number of ORa single nuclear staining neurons in patients
with mood disorders (56 018) was not significantly higher
than that in controls (44 433) (z = 1.308, P = 0.204).
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A.-M. Bao et al.
Fig. 4 Linear regression between total number of CRH neurons (x-axis) and the number of CRH-ORa nuclear double-staining neurons
in the PVN of controls (dashed line: r = 0.852, P < 0.001) and patients with mood disorders (solid line: r = 0.626, P = 0.024). Values of
controls (closed circles) and patients with mood disorders (triangles) are delineated by a minimum convex polygon (dashed for
controls and solid for patients with mood disorders).
The median of the PVN volume delineated on the basis
of CRH neurons in mood disorders (20.88 mm3) was found
to be significantly larger than that in the controls (13.74 mm3)
(z = 2.282, P = 0.022) (Fig. 3B). This significance did not
depend on the highest value in the mood disorders group
(D1, who was depressive 12 years earlier, refused therapy).
When this value was left out, the difference between the
groups remained significant (z = 2.067, P = 0.040).
No significant differences were found between MD/MDD
(n = 8) and BD subjects (n = 5) (P > 0.127). Similar values
were obtained in patients that died during a depressed state
(n = 9) or not (n = 3) (P > 0.260) or in a manic state (one
patient) in any one of the parameters mentioned above, i.e.
the total population of CRH neurons, the total number of
CRH-ORa nuclear double-staining neurons, the proportion
of the number of CRH-ORa nuclear double-staining neurons
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Fig. 3 The total number of CRH-IR neurons in the PVN (A) and the volume of the PVN as delineated by the presence of CRH neurons
(B) of controls (n = 13) and patients with mood disorders (n = 13). Note that the total number of CRH neurons and the PVN volume in mood
disorders were significantly larger than in the controls (A: z = 2.128, P = 0.034; B: z = 2.282, P = 0.022). Horizontal line indicates
the median value.
Colocalization of CRH and ORa in mood disorders
Discussion
The present study demonstrates for the first time that ORa
is co-localized with CRH in 40% of total CRH neurons in
the human PVN and that increased numbers of CRH neurons
in mood disorders are accompanied by a similar increase in
nuclear ORa staining in these neurons. Oestrogens are thus
capable of influencing CRH neurons directly, as is clear from
the colocalization of ORa in these neurons. This finding
agrees fully with the presence of OREs in the CRH promoter
region (Torpy et al., 1997). The increased total number of
CRH neurons in mood disorders confirmed earlier studies by
our group on the protein and mRNA level (Raadsheer et al.,
1994, 1995). Previous studies of our group did not find a
difference in the PVN volume delineated by a conventional
staining (Raadsheer et al., 1994) nor in the PVN volume as
stained for oxytocin or vasopressin neurons (Purba et al.,
1996), which agrees with the finding of Bernstein et al.
(1998). To our knowledge, our study is the first to find a
significantly larger volume of the sub-region of the PVN,
which is delineated on the basis of CRH neurons, in patients
with mood disorders than in controls. This indicates that,
during activation, more cells in the periphery that do not stain
in controls are ‘recruited’ to produce CRH. Since in mood
disorders some 7000 more cells express CRH and the median
value of single ORa nuclear expressing neurons goes up
from 44 433 to 56 018 (see Results), it seems that cells
expressing neither CRH nor ORa in controls are recruited
in mood disorders. The observed larger volume of the CRH
sub-region of the PVN in patients with mood disorders is
consistent with the presence of a permanent hyperactivity
of the HPA axis. As has been described before, depressive
illness is presumed to be the result of an interaction between
the effects of environmental stress and genetic and developmental predisposition (Holsboer and Barden, 1996; Swaab
et al., 2000). The set point of the HPA-axis activity is not
only programmed by genotype, but can be changed to another
level by early life events (De Kloet et al., 1997; Swaab et al.,
2000; Heim and Nemeroff, 2001). Stressful life events
during development may predispose individuals to adultonset depression by a permanent hyperactivity and hyperresponsiveness of the HPA axis (Levitan et al., 1998;
De Bellis et al., 1999; Weiss et al., 1999; Swaab et al.,
2000). Interestingly, prenatal oestrogen administration may
also increase the risk of affective disorders, as appeared from
a study on individuals who, during foetal life, were exposed
to diethylstilbestrol, a synthetic non-steroidal oestrogen
(Meyer-Bahlburg, 1987). Since the HPA axis is considered
to be the ‘final common pathway’ in the stress responsive and
depressive symptomatology (Holsboer and Barden, 1996;
Swaab et al., 2000), this observation (Meyer-Bahlburg,
1987) and our results raise the possibility that oestrogens
are involved in prenatal programming of the level of activity
of CRH neurons, thus influencing the increased risk of mood
disorders. This hypothesis should be further investigated.
Oestrogen receptors, like many of the other members of
the nuclear receptor superfamily, act as ligand-activated
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of the total number of CRH neurons, correlations between the
number of CRH cells and the number of CRH-ORa nuclear
double-staining neurons in the PVN, the total number of ORa
single nuclear staining neurons and the median of the PVN
volume delineated on the basis of CRH neurons. There was
no difference between the patients with mood disorders with
a history of suicide attempt and those without such a history
(n = 7 and n = 6, respectively; P > 0.138) in all these parameters. In addition, we did not find differences in any one
of these parameters between females and males, either within
the control group (n = 4 and n = 9, respectively; P > 0.148),
or in the mood disorders group (n = 4 and n = 9, respectively;
P > 0.199). It should be noted, however, that the clinical data
in these sub-groups of patients were not perfectly matched.
Some trends of differences may exist in the statistics mentioned above that did not achieve significant levels due to
the small sample size in each sub-group.
When we checked the clinical pathological data of the
two patients with mood disorders with the largest numbers of
CRH-IR neurons, no other common characteristics presented
besides the common diagnosis of MD/MDD. There were no
significant correlations between: (i) the total number of CRH
neurons, ORa nuclear single staining neurons, or the volume
of the PVN delineated on the basis of CRH neurons; and
(ii) the post-mortem delay, fixation time or storage time in
paraffin (P > 0.289) in the control group, nor did these
parameters have significant correlations with the duration
of the mood disorders (from <1 year to 45 years) (P > 0.114)
in the mood disorders group. A significantly negative
correlation was found in the mood disorders group between
the number of single ORa nuclear staining neurons and the
post-mortem delay (r = 0.775, P = 0.002). However, use of
ANOVA (analysis of variance) controlling for differences in
post-mortem delay between the groups showed that there was
no significant difference in the number of single ORa nuclear
staining neurons between the depressive and the control
subjects (P = 0.078). No significant correlations were found
between age and the number of immunostained cell profiles
in either the control or the mood disorders group (P > 0.627
and P > 0.589, respectively). It was noticed that control
subject C1, a 43-year-old female with liver cirrhosis due
to alcohol abuse, had a much higher total number of CRH
(21 410), CRH-ORa nuclear double-staining neurons (10 749)
and volume of the PVN delineated by the presence of CRH
neurons (19.15 mm3) than the rest of the controls. Activation
of the HPA axis is, indeed, a well-known characteristic of
alcoholism (see Discussion). The significances found
above—comparison of the total number of CRH neurons
with the volume of PVN delineated on the basis of CRH
neurons between the patients with mood disorders and
controls—were reinforced (z = 2.339, P = 0.019 and
z = 2.393, P = 0.016, respectively) and the trend in the
number of CRH-ORa nuclear double-staining neurons
between the patients with mood disorders and controls
reached statistical significance when the subject with alcohol
abuse was left out of the analysis (z = 2.067, P = 0.040).
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A.-M. Bao et al.
similar activities of the HPA axis is also consistent with
previous studies (Raadsheer et al., 1995; Zhou et al., 2001).
It was not surprising to find an extremely high total number
of CRH neurons in control subject C1, since it is known
that chronic alcohol abuse is associated with activation of
the HPA axis and resistance to dexamethasone (Wand, 1999;
Nurnberger et al., 2001).
Some limitations of the present post-mortem study should
be mentioned. There were not enough data available on the
CSF pH as an indicator for agonal changes (Swaab, 2003).
However, it is unlikely that a difference in the activity of the
PVN CRH neurons is related to agonal state, since a previous
study by our group found that agonal differences do not
significantly affect the activity of the HPA axis as indicated
by CSF cortisol levels (Erkut et al., 2004). In addition, the
mood disorders group was made up of a heterogeneous
patient sample, i.e. patients diagnosed with MD/MDD or
BD who had died during a depressed state or not, or in a
manic state. Although no significant differences were found
between these sub-groups, the numbers of each sub-group are
too small to allow definite conclusions. Some trends do exist
in these sub-analyses, suggesting that, possibly, no significant
differences were achieved due to the small sample size in
each sub-group. Moreover, the patients’ data were not
perfectly matched in these sub-analyses. The increased
expression of CRH and ORa in mood disorders seems to
be related to trait rather than state, as no differences were
found between those who died during a depressive episode
or when not in a depressed state. Future studies with larger
post-mortem samples of particular diagnostic entity are thus
needed for the various aspects mentioned above.
The large CRH neuron population we found in the human
thalamus opens a new topic for further study. The exact
nature of these CRH-containing neurons and whether their
function changes in physiological or pathological conditions
is not known. The question f whether these neurons may
contribute—as an extrahypothalamic source—to the CRH
levels in the CSF also deserves further study. These CSFCRH levels probably do not reflect the activity of the PVN,
but rather reflect fluctuations in extrahypothalamic CRH
(Mitchell, 1998; Arborelius et al., 1999; Boyer, 2000;
Vythilingam et al., 2000), e.g. from the neocortex, limbic
and brainstem regions and from the thalamus (this study).
The present study could not answer the question how ORa
is upregulated in the PVN in mood disorders. Since a similar
pattern of upregulation was found in males and females,
the role of circulating levels of oestrogens in the regulation
of ORs in CRH neurons is doubtful. Whether a higher
local production of oestrogens—produced via the enzyme
aromatase from adrenal or brain-derived androgens—may
act as ligand and upregulate ORa in the brain (Kroboth
et al., 1999) and/or whether the upregulation of ORa is
modulated by neurotransmitters such as norepinephrine,
dopamine, serotonin—all of which are involved in the central
stress responses and mood disorders (Asberg et al., 1976;
Blaustein et al., 1986)—deserves further study in human.
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transcription factors. The human CRH gene contains five
perfect half-palindromic OREs and estradiol may enhance the
HPA-axis activity by stimulation of CRH gene transcription
(Torpy et al., 1997). Our finding of the close relationship
of CRH with ORa nuclear double-staining in the PVN is
consistent with these data. Upregulation of ORa seems to
accompany activation of CRH neurons in mood disorders.
The similar ratios of the amount of CRH with ORa nuclear
double-staining neurons to the total number of CRH neurons
in patients with mood disorders (48%) and control subjects
(37%) suggest that oestrogens may contribute to CRH activation via OR. However, the exact mechanism of the action
of oestrogens on CRH neurons should be a topic for further
study.
The finding that there virtually no ORb immunochemical
staining in the PVN of these subjects was in agreement with
previous reports showing that, in contrast to ORa, ORb and
ORb mRNA expression are very low in this hypothalamic
area in human (Osterlund and Hurd, 2001; Kruijver
et al., 2002, 2003) and that the high ORa/b ratio seems to
be a species- and area-specific phenomena (Simonian
and Herbison, 1997; Alves et al., 1998; Hrabovszky et al.,
1998; Nomura et al., 2002; Kruijver et al., 2003). Indeed, it
was previously shown that there are antagonistic effects
mediated by ORa and ORb in HeLa cells in vitro, where
ORa mediated the activation and ORb the inhibition of
transcription (Paech et al., 1997). Previous studies by our
group have proposed that ORb mediates inhibition and that
ORa mediates stimulation—also of AVP cells in human
supraoptic nucleus—by oestrogens acting on the genomic
level (Ishunina and Swaab, 1999; Ishunina et al., 1999, 2000).
It would certainly be of interest to investigate whether ORa
mediates activation of CRH neurons in the human PVN.
The increased activity of the PVN CRH neurons in the
patients with mood disorders is unlikely to be the result of
the duration of mood disorders or the chronic administration
of antidepressants (Table 1), since it has been found that
both the chronicity of the disease and antidepressants may
attenuate rather than enhance the activity of CRH neurons
(Fischer et al., 1990; Vingerhoets et al., 1996; Nemeroff,
1997; Oldehinkel et al., 2001). Thus, if antidepressants
interfered with our measurements, this would have led to
an underestimation of the observed difference between controls and patients with mood disorders in the state of activity
of their CRH-expressing neurons in the PVN. Moreover,
no significant differences were found between subjects taking
lithium or mood stabilizing drugs and subjects taking antidepressants or other psychoactive drugs. However, we did
not include this point in Results because the statistical
analyses were doubtful due to the small sub-samples and
the overlap in the use of these drugs among these patients.
A proper comparison between the effects of those drugs on
the HPA and/or the HPG axes deserves further study. No
relationship was found between the total number of CRH
neurons and the duration of mood disorders in the present
study. The finding that MD/MDD and BD patients showed
Colocalization of CRH and ORa in mood disorders
Acknowledgements
We wish to thank the Netherlands Brain Bank (coordinator
Dr R. Ravid) at the Netherlands Institute for Brain Research
for providing us with brain material. We also wish to thank:
Mr B. Fisser for his technical assistance; Mr J.J. van
Heerikhuize and Dr C.W. Pool for their assistance with the
image analysis system; Mrs W.T.P. Verweij for correcting the
English; and Professor H.B.M. Uylings for his comments,
especially on the anatomy of the thalamus. This investigation was supported by Nature Science Foundation of
China (30470537), Project of Chinese Academy of Science
(KSCX2-SW-217), KNAW 02CDP019 and 03CDP001.
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