Accepted Manuscript Title: Nitric oxide synthase and nitric oxide alterations in chronically stressed rats: a model for nitric oxide in major depressive disorder Author: Shang-Feng Gao Yun-Rong Lu Li-Gen Shi Xue-Yan Wu Bo Sun Xin-Yan Fu Jian-Hong Luo Ai-Min Bao PII: DOI: Reference:
S0306-4530(14)00181-4 http://dx.doi.org/doi:10.1016/j.psyneuen.2014.05.009 PNEC 2699
To appear in: Received date: Revised date: Accepted date:
14-4-2014 13-5-2014 13-5-2014
Please cite this article as: Gao, S.-F., Lu, Y.-R., Shi, L.-G., Wu, X.-Y., Sun, B., Fu, X.-Y., Luo, J.-H., Bao, A.-M.,Nitric oxide synthase and nitric oxide alterations in chronically stressed rats: a model for nitric oxide in major depressive disorder, Psychoneuroendocrinology (2014), http://dx.doi.org/10.1016/j.psyneuen.2014.05.009 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
*Revised Manuscript Click here to view linked References
1
Nitric oxide synthase and nitric oxide alterations in chronically stressed rats: a
2
model for nitric oxide in major depressive disorder
3
Shang-Feng Gao1,2,§, Yun-Rong Lu1,3,§, Li-Gen Shi1, Xue-Yan Wu1, Bo Sun1, Xin-Yan
5
Fu1, Jian-Hong Luo1 and Ai-Min Bao1,*
cr
6
ip t
4
7
1
8
of Health of China; Zhejiang Province Key Laboratory of Neurobiology, Zhejiang
9
University School of Medicine, Hangzhou 310058, Zhejiang, P.R. China.
an
us
Department of Neurobiology, Key Laboratory of Medical Neurobiology of Ministry
10
2
11
Xuzhou 221004, Jiangsu, P.R. China.
12
3
13
Zhejiang University, Hangzhou 310009, Zhejiang, P. R. China.
M
ed
Department of Psychiatry, The Second Affiliated Hospital, Medical School of
ce pt
14
Lab of Neurosurgery, Institute of Nervous System Diseases, Xuzhou Medical College,
§ The authors contributed equally to this work.
16
*Corresponding author:
17
Ai-Min Bao, M.D., Ph.D., Prof of Neurobiology, Department of Neurobiology; Key
18
Laboratory of Medical Neurobiology of Ministry of Health of China; Zhejiang
19
Province Key Laboratory of Neurobiology, Zhejiang University School of Medicine,
20
866 Yu-Hang-Tang Road, Hangzhou 310058, China. Tel: +86 571 88208789; E-mail:
21
[email protected].
22
Running title: Nitric oxide in stress and depression
Ac
15
1
Page 1 of 15
Summary
2
Nitric oxide (NO) and NO synthase-1 (NOS1) are involved in the stress response and
3
in depression. We compared NOS-NO alterations in rats exposed to chronic
4
unpredictable stress (CUS) with alterations in major depressive disorder (MDD) in
5
humans. In the hypothalamus of male CUS rats we determined NOS activity, and in
6
the paraventricular nucleus (PVN) we determined NOS1-immunoreactive (ir) cell
7
densities
8
corticotropin-releasing hormone (CRH), vasopressin (AVP) or oxytocin (OXT). We
9
measured plasma NO levels and cortisol in male medicine-naïve MDD patients and
10
plasma NO and corticosterone (CORT) in CUS rats. In the CUS rat total NOS activity
11
in the hypothalamus (P = 0.018) and NOS1-ir cell density in the PVN were both
12
significantly decreased (P = 0.018), while NOS1 staining was mainly expressed in
13
OXT-ir neurons in this nucleus. Interestingly, plasma NO levels were significantly
14
increased both in male CUS rats (P = 0.001) and in male MDD patients (P < 0.001).
15
Plasma CORT levels were increased in male CUS rats (P = 0.001), while male MDD
16
patients did not show a significant change in cortisol levels. In conclusion, the
17
changes in plasma and hypothalamic NOS-NO of CUS rats and MDD were similar.
18
The male CUS rat model may thus help us with our investigation of the mechanism
19
underlying NOS-NO alterations in depression.
co-localization
of
NOS1
with
stress-related
neuropeptides
Ac
ce pt
ed
M
an
us
and
cr
ip t
1
20 21
KEYWORDS Chronic unpredictable stress; Major depressive disorder; Nitric oxide;
22
Nitric oxide synthase 1; Paraventricular nucleus.
23
2
Page 2 of 15
1. Introduction
2
Stressful life events are crucial precipitants in the development of major depressive
3
disorder (MDD) in people who are vulnerable to this disease, which is characterized
4
by hyperactivity of the hypothalamo-pituitary-adrenal (HPA)-axis (Willner, 2005).
5
Chronic unpredictable stress (CUS) rats are used as a model for this disorder. The
6
question of the present study was whether CUS rats could also be a suitable model to
7
study nitric oxide (NO)-related alterations in depression.
us
cr
ip t
1
NO is a highly diffusible and reactive molecule synthesized from L-arginine by at
9
least 3 subtypes of NO synthase (NOS), i.e. neuronal NOS (NOS1), inducible NOS
10
(NOS2), and endothelial NOS (NOS3) (Griffith and Stuehr, 1995). NOS1 and NOS3
11
are constitutively expressed in vivo and thus known as constitutive NOS (cNOS). We
12
and other research groups have observed decreased NOS1 expression in the
13
hypothalamus, the prefrontal cortex (PFC) and the locus coeruleus, all of which play a
14
key role in stress response and in depression (Bernstein et al., 1998; Karolewicz et al.,
15
2004; Gao et al., 2013). Moreover, a significant decrease of cNOS activity was found
16
in the PFC of depressive patients (Xing et al., 2002). In the hypothalamus,
17
NOS1-immunoreactive (ir) cells have been found to decrease in the PVN (Bernstein
18
et al., 1998), which is the production area for the stress-related neuropeptides
19
corticotropin-releasing hormone (CRH), arginine vasopressin (AVP) and oxytocin
20
(OXT). Whether similar NO-related alterations occur in the CUS rat model is as yet
21
unknown. Furthermore, in previous studies plasma NO levels in depressive patients
22
were found to be either increased (Kim et al., 2006) or decreased (Chrapko et al.,
Ac
ce pt
ed
M
an
8
3
Page 3 of 15
2004), possibly due to the different subtypes of depression or the different treatments
2
the patients received. The issue of plasma NO changes is thus elusive and a
3
comparison with plasma NO levels in CUS rats has not been done. In the present
4
study we determined, in CUS rats, the changes in hypothalamic NOS activity,
5
PVN-NOS1 expression, and plasma NO levels, and we analyzed the co-localization of
6
NOS1 with CRH, AVP or OXT in their PVN. We compared these alterations with
7
changes in plasma NO and cortisol levels in medicine-naïve MDD patients.
8
2. Materials and methods
9
2.1 CUS rats
an
us
cr
ip t
1
Animals were housed in an air-conditioned room at a temperature of 25-27oC, with
11
food and water ad libitum, except when specified otherwise. Prior to the experiments
12
the rats were allowed one week to adapt to their new circumstances. Male Sprague
13
Dawley (SD) rats (n = 32, 280-300g) were randomly divided into a control and a CUS
14
group. CUS was applied according to the literature (Grippo and Johnson, 2009),
15
resulting in the CUS rats showing depression-like behaviors in open field and sucrose
16
preference tests, as well as in body weight (Suppl. Fig. 1). Rats were sacrificed by
17
decapitation between 0900h and 1000h. All procedures were approved by the local
18
animal care committees in accordance with the relevant regulations and laws.
19
2.2 MDD patients and controls
20
Ten male medicine-naïve Chinese Han race MDD patients (age range 26-59 years,
21
mean age = 44.6 years) were recruited by the Department of Psychiatry, the Second
22
Affiliated Hospital of Zhejiang University School of Medicine. All had experienced
Ac
ce pt
ed
M
10
4
Page 4 of 15
life/work stressful events before the depressive episode and all had been diagnosed by
2
qualified psychiatrists as having MDD based upon the criteria of the Diagnostic and
3
Statistical Manual of mental disorders, fourth edition (DSM-IV). The Mini
4
International Neuropsychiatric Interview (Chinese modified version) was used to
5
confirm the DSM-IV diagnosis. The Hamilton Depression Scale (HAMD) was used to
6
evaluate the severity of depression: a score of 35 or above was considered as severe
7
depression. Exclusion criteria were: psychiatric co-morbidity in the form of substance
8
abuse, psychotic or anxiety disorders, mental retardation, chronic physical illness, or
9
abnormal body mass index (BMI ≤ 18 or BMI ≥ 25). In addition, 10 male control
10
subjects (age range 27-58 years, mean age = 44.2 years), who underwent their yearly
11
physical examination in the same hospital, volunteered for this study. The exclusion
12
criteria for the controls were as follows: BMI ≤ 18 or BMI ≥ 25, or medication for
13
chronic illnesses or mental disorders.
ed
M
an
us
cr
ip t
1
The investigation was carried out in accordance with the latest version of the
15
Declaration of Helsinki. All subjects signed informed consent forms and the study
16
was approved by the Medical Ethics Committee of the Second Affiliated Hospital of
17
Zhejiang University.
18
2.3 Plasma NO, cortisol and corticosterone (CORT) measurements
19
Plasma NO levels were determined in rat and human as levels of nitrate and nitrite by
20
means of the Griess reaction method (Zhou et al., 2007). The intra- and inter-assay
21
variations were 7.1% and 10.7%, respectively. Rat plasma CORT was determined
22
using an enzyme-linked immunosorbent assay (DSL, Webster, TX) with intra- and
Ac
ce pt
14
5
Page 5 of 15
inter-assay coefficients of variation of 4.0% and 6.2%, respectively. Human plasma
2
cortisol was measured according to the manufacturer’s protocol (Siemens Healthcare
3
Diagnostics Inc, USA). The intra- and inter-assay variations were 6.1 % and 8.2 %,
4
respectively.
5
2.4 NOS activity measurements
6
The total NOS-activity in the supernatant of rat hypothalami was measured using a
7
commercially available kit (Jiancheng Bioengineering, Nanjing, China). NOS2
8
activity was measured by adding ethylene glycol tetra-acetic acid to chelate free Ca2+
9
from the reaction mixture. cNOS activity was obtained by subtracting NOS2 activity
10
from total NOS activity. NOS activities were expressed in units (U = nanomoles of
11
NO formed in 1 min by 1 mg of protein).
12
2.5 Immunocytochemistry
13
NOS1-staining was performed as described previously (Gao et al., 2013), adding 3%
14
H2O2 in methanol for 30 min to abolish endogenous peroxidase before the sections
15
were incubated with the blocking bufffer. Anti-NOS1 antibody (BD, Lexington, KY)
16
was used 1:100, followed by horseradish peroxidase-conjugated secondary antibody
17
(Zhongshan Goldenbridge Biotechnology, Beijing, China). When the primary
18
antibody was replaced by the blocking buffer no staining was present.
19
2.6 Double immunofluorescent labeling
20
The monoclonal NOS1 antibody and rabbit antibodies against CRH (5Bo) or AVP
21
(Truus) or OXT (O-2-T) (gifts from the Netherlands Institute for Neuroscience,
22
Amsterdam) 1:500 were used for 12–24 h at 4 °C. The specificities of these antibodies
Ac
ce pt
ed
M
an
us
cr
ip t
1
6
Page 6 of 15
had been confirmed previously. Dylight 488 conjugated IgG (1:200) and Dylight 594
2
conjugated IgG (1:600, Jackson lab, West Grove, PA) were added to the sections. The
3
sections were coverslipped and observed under a Leica fluorescence microscope
4
(Germany).
5
2.7 Image analysis
6
The anatomical position of the PVN was determined by Nissl staining of every 5th
7
cryostat section (20 μm). For the quantitative analysis of NOS1-ir signals, PVN
8
sections were chosen from the anterior part (Bregma level -1.4 to 1.6 mm), the middle
9
part (-1.6 to -1.8 mm) and the posterior part (-1.8 to -2.0 mm) (Paxinos and Watson,
10
1986). NOS1-ir cell counting was performed as before (Gao et al., 2013). Cell density
11
was calculated by dividing the number of positive neurons by PVN or supraoptic
12
nucleus (SON) area.
13
2.8 Statistical analyses
14
As the data were found to be not always normally distributed, non-parametric tests
15
were applied. Comparison between two independent samples was done by
16
Mann-Whitney U test and correlations by the Spearman test. P < 0.05 was considered
17
to be significant.
cr
us
an
M
ed
ce pt
Ac
18
ip t
1
19
3. Results
20
3.1 Changes in plasma NO, CORT/cortisol levels in CUS rats and MDD patients
21
Plasma NO levels were significantly increased, both in male CUS rats (P = 0.001,
22
Figure 1 A) and in male MDD patients (P < 0.001, Figure 1 B). Plasma CORT was 7
Page 7 of 15
significantly increased in CUS rats (P = 0.001, Figure 1 C), but no significant change
2
was found in plasma cortisol levels in male MDD patients (P = 0.427). No significant
3
correlations were found between plasma NO and cortisol levels, either in the control
4
(P = 0.160) or in the MDD (P = 0.310) group. There were no significant correlations
5
between plasma NO levels and HAMD scores (P = 0.920) or between plasma cortisol
6
levels and HAMD scores (P = 0.334), nor were any significant correlations found
7
between plasma NO and CORT levels in the control group (P = 0.248) or in the CUS
8
group (P = 0.453).
cr
us
(Insert Figure 1 around here)
an
9
ip t
1
3.2 Alterations in NOS activity and NOS1-ir in CUS rat hypothalamus
11
The total NOS activity and the cNOS activity in the hypothalamus of CUS rats
12
decreased significantly (P = 0.018 and P = 0.048, respectively), while NOS2 activity
13
showed no significant change (Figure 2 A). NOS1-ir was mainly located in the
14
cytoplasm and processes of neuronal cells, with different staining intensity along the
15
PVN (see Suppl. Fig. 2). The NOS1-ir cell density was significantly reduced in the
16
CUS group (P = 0.018, Figure 2 B), mainly due to changes in the anterior (P = 0.018)
17
and middle part (P = 0.068), but not in the posterior PVN (P = 0.201) (Figure 2 C).
18
NOS1-ir cell density showed no significant changes in the SON of the CUS rats (P =
19
0.201). NOS1-ir was found to be mainly co-localized in OXT-ir neurons (Figure 2 D),
20
while no co-localization was observed for NOS1 and CRH or AVP in the rat PVN (see
21
Suppl. Fig. 3).
22
Ac
ce pt
ed
M
10
(Insert Figure 2 around here) 8
Page 8 of 15
1
4. Discussion
3
We found, for the first time, a significant increase of plasma NO levels in the CUS
4
rats, which was in agreement with the increase in plasma NO levels in the
5
medicine-naïve MDD patients. In addition, CUS rats showed significantly decreased
6
hypothalamic NOS activity and NOS1-ir cell densities in the PVN but not in the SON,
7
which was consistent with the finding in human postmortem brain material that had
8
revealed decreased numbers of NOS1-ir neurons in the hypothalamic PVN (Bernstein
9
et al., 1998), but not in the SON (Bernstein et al., 2000) and with the decreased NOS1
10
we found in the PFC of depressive patients (Gao et al., 2013). Moreover, the
11
significantly decreased cNOS activity in the hypothalamus of CUS rats is also in
12
agreement with a decreased cNOS activity in human PFC in depression (Xing et al.,
13
2002). Our findings thus strongly indicate that the CUS rat model could be suitable
14
for further studies on the molecular changes in MDD as far as the NOS-NO system is
15
concerned.
ce pt
ed
M
an
us
cr
ip t
2
NOS1-ir neurons were mostly distributed in the anterior part of the PVN, which
17
agrees with the higher proportion of OXT neurons in the rostral part of the rat PVN
18
(Swaab et al., 1975). Indeed, we observed a co-localization for NOS1 and OXT in the
19
hypothalamic PVN. Since OXT is known to restrain the activity of the HPA axis (Bao
20
et al., 2008) and NO is involved in the regulation of OXT release by the
21
neurohypophyseal system (Cunningham and Sawchenko, 1991), the NOS1-OXT
22
interaction may play a critical role in integrating the different components of the stress
Ac
16
9
Page 9 of 15
1
systems during the stress response and in depression. We did not find significant changes in plasma cortisol levels in MDD. This could
3
be explained by the fact that centrally released CRH may be more important for mood
4
changes than peripherally released CRH. Indeed, increased basal plasma cortisol
5
levels were observed in only about 25% of the MDD patients (Young et al., 2001).
6
There was no significant correlation between plasma NO and cortisol levels,
7
indicating that these two systems may function quite independently. In contrast to the
8
significant increase of plasma NO levels, hypothalamic NOS activity and PVN-NOS1
9
expression were decreased in the CUS model and in MDD patients, indicating
10
different sources for the central and the peripheral NO. The peripheral NO might be
11
derived, for example, from the autonomic nervous system (Griffith and Stuehr, 1995).
M
an
us
cr
ip t
2
In conclusion, the changes in plasma NO and hypothalamic PVN-NOS1 expression
13
in the CUS model and in MDD patients are similar, suggesting that the CUS rat is an
14
interesting model for further studies on the underlying mechanisms of alterations in
15
the NOS-NO system in the etiology of depression. The novel finding of
16
co-localization of NOS1 with OXT in the PVN offers an interesting target for further
17
study of the possible role of NO in depression.
Ac
ce pt
ed
12
10
Page 10 of 15
ed
M
an
us
cr
ip t
Bao, A.M., Meynen, G., Swaab, D.F., 2008. The stress system in depression and neurodegeneration: focus on the human hypothalamus. Brain Res Rev 57, 531-53. Bernstein, H.G., Jirikowski, G.F., Heinemann, A., Baumann, B., Hornstein, C., Danos, P., et al., 2000. Low and infrequent expression of nitric oxide synthase/NADPH-diaphorase in neurons of the human supraoptic nucleus: a histochemical study. J Chem Neuroanat 20, 177-83. Bernstein, H.G., Stanarius, A., Baumann, B., Henning, H., Krell, D., Danos, P., et al., 1998. Nitric oxide synthase-containing neurons in the human hypothalamus: reduced number of immunoreactive cells in the paraventricular nucleus of depressive patients and schizophrenics. Neuroscience 83, 867-75. Chrapko, W.E., Jurasz, P., Radomski, M.W., Lara, N., Archer, S.L., Le Melledo, J.M., 2004. Decreased platelet nitric oxide synthase activity and plasma nitric oxide metabolites in major depressive disorder. Biol Psychiatry 56, 129-34. Cunningham, E.T., Jr., Sawchenko, P.E., 1991. Reflex control of magnocellular vasopressin and oxytocin secretion. Trends Neurosci 14, 406-11. Gao, S.F., Qi, X.R., Zhao, J., Balesar, R., Bao, A.M., Swaab, D.F., 2013. Decreased NOS1 Expression in the Anterior Cingulate Cortex in Depression. Cereb Cortex 23, 2956-64. Griffith, O.W., Stuehr, D.J., 1995. Nitric oxide synthases: properties and catalytic mechanism. Annu Rev Physiol 57, 707-36. Grippo, A.J., Johnson, A.K., 2009. Stress, depression and cardiovascular dysregulation: a review of neurobiological mechanisms and the integration of research from preclinical disease models. Stress 12, 1-21. Karolewicz, B., Szebeni, K., Stockmeier, C.A., Konick, L., Overholser, J.C., Jurjus, G., et al., 2004. Low nNOS protein in the locus coeruleus in major depression. J Neurochem 91, 1057-66. Kim, Y.K., Paik, J.W., Lee, S.W., Yoon, D., Han, C., Lee, B.H., 2006. Increased plasma nitric oxide level associated with suicide attempt in depressive patients. Prog Neuropsychopharmacol Biol Psychiatry 30, 1091-6. Paxinos, G., Watson, C., 1986. The Rat Brain in Stereotaxic Coordinates. Academic Press, New York. Swaab, D.F., Pool, C.W., Nijveldt, F., 1975. Immunofluorescence of vasopressin and oxytocin in the rat hypothalamo-neurohypophypopseal system. J Neural Transm 36, 195-215. Willner, P., 2005. Chronic mild stress (CMS) revisited: consistency and behavioural-neurobiological concordance in the effects of CMS. Neuropsychobiology 52, 90-110. Young, E.A., Carlson, N.E., Brown, M.B., 2001. Twenty-four-hour ACTH and cortisol pulsatility in depressed women. Neuropsychopharmacology 25, 267-76. Zhou, Q.G., Hu, Y., Hua, Y., Hu, M., Luo, C.X., Han, X., et al., 2007. Neuronal nitric oxide synthase contributes to chronic stress-induced depression by suppressing hippocampal neurogenesis. J Neurochem 103, 1843-54. Xing, G., Chavko, M., Zhang, L.X., Yang, S., Post, R.M., 2002. Decreased calcium-dependent constitutive nitric oxide synthase (cNOS) activity in prefrontal cortex in schizophrenia
ce pt
2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43
References
Ac
1
11
Page 11 of 15
ce pt
ed
M
an
us
cr
ip t
and depression. Schizophr Res 58, 21-30.
Ac
1 2
12
Page 12 of 15
Figure legends:
3
Figure 1 Changes in plasma NO, CORT levels in MDD patients and CUS rats
4
Plasma NO levels were significantly higher in CUS rats (A, n = 8/group) and MDD
5
patients (B, n = 10/group). Plasma corticosterone (CORT) levels showed significant
6
increases in CUS rats (C, n = 8/group). Data are shown as median, 25th–75th
7
percentiles, and the range. CTR = controls, MDD = major depressive disorder; ** P
S0306-4530(14)00181-4 http://dx.doi.org/doi:10.1016/j.psyneuen.2014.05.009 PNEC 2699
To appear in: Received date: Revised date: Accepted date:
14-4-2014 13-5-2014 13-5-2014
Please cite this article as: Gao, S.-F., Lu, Y.-R., Shi, L.-G., Wu, X.-Y., Sun, B., Fu, X.-Y., Luo, J.-H., Bao, A.-M.,Nitric oxide synthase and nitric oxide alterations in chronically stressed rats: a model for nitric oxide in major depressive disorder, Psychoneuroendocrinology (2014), http://dx.doi.org/10.1016/j.psyneuen.2014.05.009 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
*Revised Manuscript Click here to view linked References
1
Nitric oxide synthase and nitric oxide alterations in chronically stressed rats: a
2
model for nitric oxide in major depressive disorder
3
Shang-Feng Gao1,2,§, Yun-Rong Lu1,3,§, Li-Gen Shi1, Xue-Yan Wu1, Bo Sun1, Xin-Yan
5
Fu1, Jian-Hong Luo1 and Ai-Min Bao1,*
cr
6
ip t
4
7
1
8
of Health of China; Zhejiang Province Key Laboratory of Neurobiology, Zhejiang
9
University School of Medicine, Hangzhou 310058, Zhejiang, P.R. China.
an
us
Department of Neurobiology, Key Laboratory of Medical Neurobiology of Ministry
10
2
11
Xuzhou 221004, Jiangsu, P.R. China.
12
3
13
Zhejiang University, Hangzhou 310009, Zhejiang, P. R. China.
M
ed
Department of Psychiatry, The Second Affiliated Hospital, Medical School of
ce pt
14
Lab of Neurosurgery, Institute of Nervous System Diseases, Xuzhou Medical College,
§ The authors contributed equally to this work.
16
*Corresponding author:
17
Ai-Min Bao, M.D., Ph.D., Prof of Neurobiology, Department of Neurobiology; Key
18
Laboratory of Medical Neurobiology of Ministry of Health of China; Zhejiang
19
Province Key Laboratory of Neurobiology, Zhejiang University School of Medicine,
20
866 Yu-Hang-Tang Road, Hangzhou 310058, China. Tel: +86 571 88208789; E-mail:
21
[email protected].
22
Running title: Nitric oxide in stress and depression
Ac
15
1
Page 1 of 15
Summary
2
Nitric oxide (NO) and NO synthase-1 (NOS1) are involved in the stress response and
3
in depression. We compared NOS-NO alterations in rats exposed to chronic
4
unpredictable stress (CUS) with alterations in major depressive disorder (MDD) in
5
humans. In the hypothalamus of male CUS rats we determined NOS activity, and in
6
the paraventricular nucleus (PVN) we determined NOS1-immunoreactive (ir) cell
7
densities
8
corticotropin-releasing hormone (CRH), vasopressin (AVP) or oxytocin (OXT). We
9
measured plasma NO levels and cortisol in male medicine-naïve MDD patients and
10
plasma NO and corticosterone (CORT) in CUS rats. In the CUS rat total NOS activity
11
in the hypothalamus (P = 0.018) and NOS1-ir cell density in the PVN were both
12
significantly decreased (P = 0.018), while NOS1 staining was mainly expressed in
13
OXT-ir neurons in this nucleus. Interestingly, plasma NO levels were significantly
14
increased both in male CUS rats (P = 0.001) and in male MDD patients (P < 0.001).
15
Plasma CORT levels were increased in male CUS rats (P = 0.001), while male MDD
16
patients did not show a significant change in cortisol levels. In conclusion, the
17
changes in plasma and hypothalamic NOS-NO of CUS rats and MDD were similar.
18
The male CUS rat model may thus help us with our investigation of the mechanism
19
underlying NOS-NO alterations in depression.
co-localization
of
NOS1
with
stress-related
neuropeptides
Ac
ce pt
ed
M
an
us
and
cr
ip t
1
20 21
KEYWORDS Chronic unpredictable stress; Major depressive disorder; Nitric oxide;
22
Nitric oxide synthase 1; Paraventricular nucleus.
23
2
Page 2 of 15
1. Introduction
2
Stressful life events are crucial precipitants in the development of major depressive
3
disorder (MDD) in people who are vulnerable to this disease, which is characterized
4
by hyperactivity of the hypothalamo-pituitary-adrenal (HPA)-axis (Willner, 2005).
5
Chronic unpredictable stress (CUS) rats are used as a model for this disorder. The
6
question of the present study was whether CUS rats could also be a suitable model to
7
study nitric oxide (NO)-related alterations in depression.
us
cr
ip t
1
NO is a highly diffusible and reactive molecule synthesized from L-arginine by at
9
least 3 subtypes of NO synthase (NOS), i.e. neuronal NOS (NOS1), inducible NOS
10
(NOS2), and endothelial NOS (NOS3) (Griffith and Stuehr, 1995). NOS1 and NOS3
11
are constitutively expressed in vivo and thus known as constitutive NOS (cNOS). We
12
and other research groups have observed decreased NOS1 expression in the
13
hypothalamus, the prefrontal cortex (PFC) and the locus coeruleus, all of which play a
14
key role in stress response and in depression (Bernstein et al., 1998; Karolewicz et al.,
15
2004; Gao et al., 2013). Moreover, a significant decrease of cNOS activity was found
16
in the PFC of depressive patients (Xing et al., 2002). In the hypothalamus,
17
NOS1-immunoreactive (ir) cells have been found to decrease in the PVN (Bernstein
18
et al., 1998), which is the production area for the stress-related neuropeptides
19
corticotropin-releasing hormone (CRH), arginine vasopressin (AVP) and oxytocin
20
(OXT). Whether similar NO-related alterations occur in the CUS rat model is as yet
21
unknown. Furthermore, in previous studies plasma NO levels in depressive patients
22
were found to be either increased (Kim et al., 2006) or decreased (Chrapko et al.,
Ac
ce pt
ed
M
an
8
3
Page 3 of 15
2004), possibly due to the different subtypes of depression or the different treatments
2
the patients received. The issue of plasma NO changes is thus elusive and a
3
comparison with plasma NO levels in CUS rats has not been done. In the present
4
study we determined, in CUS rats, the changes in hypothalamic NOS activity,
5
PVN-NOS1 expression, and plasma NO levels, and we analyzed the co-localization of
6
NOS1 with CRH, AVP or OXT in their PVN. We compared these alterations with
7
changes in plasma NO and cortisol levels in medicine-naïve MDD patients.
8
2. Materials and methods
9
2.1 CUS rats
an
us
cr
ip t
1
Animals were housed in an air-conditioned room at a temperature of 25-27oC, with
11
food and water ad libitum, except when specified otherwise. Prior to the experiments
12
the rats were allowed one week to adapt to their new circumstances. Male Sprague
13
Dawley (SD) rats (n = 32, 280-300g) were randomly divided into a control and a CUS
14
group. CUS was applied according to the literature (Grippo and Johnson, 2009),
15
resulting in the CUS rats showing depression-like behaviors in open field and sucrose
16
preference tests, as well as in body weight (Suppl. Fig. 1). Rats were sacrificed by
17
decapitation between 0900h and 1000h. All procedures were approved by the local
18
animal care committees in accordance with the relevant regulations and laws.
19
2.2 MDD patients and controls
20
Ten male medicine-naïve Chinese Han race MDD patients (age range 26-59 years,
21
mean age = 44.6 years) were recruited by the Department of Psychiatry, the Second
22
Affiliated Hospital of Zhejiang University School of Medicine. All had experienced
Ac
ce pt
ed
M
10
4
Page 4 of 15
life/work stressful events before the depressive episode and all had been diagnosed by
2
qualified psychiatrists as having MDD based upon the criteria of the Diagnostic and
3
Statistical Manual of mental disorders, fourth edition (DSM-IV). The Mini
4
International Neuropsychiatric Interview (Chinese modified version) was used to
5
confirm the DSM-IV diagnosis. The Hamilton Depression Scale (HAMD) was used to
6
evaluate the severity of depression: a score of 35 or above was considered as severe
7
depression. Exclusion criteria were: psychiatric co-morbidity in the form of substance
8
abuse, psychotic or anxiety disorders, mental retardation, chronic physical illness, or
9
abnormal body mass index (BMI ≤ 18 or BMI ≥ 25). In addition, 10 male control
10
subjects (age range 27-58 years, mean age = 44.2 years), who underwent their yearly
11
physical examination in the same hospital, volunteered for this study. The exclusion
12
criteria for the controls were as follows: BMI ≤ 18 or BMI ≥ 25, or medication for
13
chronic illnesses or mental disorders.
ed
M
an
us
cr
ip t
1
The investigation was carried out in accordance with the latest version of the
15
Declaration of Helsinki. All subjects signed informed consent forms and the study
16
was approved by the Medical Ethics Committee of the Second Affiliated Hospital of
17
Zhejiang University.
18
2.3 Plasma NO, cortisol and corticosterone (CORT) measurements
19
Plasma NO levels were determined in rat and human as levels of nitrate and nitrite by
20
means of the Griess reaction method (Zhou et al., 2007). The intra- and inter-assay
21
variations were 7.1% and 10.7%, respectively. Rat plasma CORT was determined
22
using an enzyme-linked immunosorbent assay (DSL, Webster, TX) with intra- and
Ac
ce pt
14
5
Page 5 of 15
inter-assay coefficients of variation of 4.0% and 6.2%, respectively. Human plasma
2
cortisol was measured according to the manufacturer’s protocol (Siemens Healthcare
3
Diagnostics Inc, USA). The intra- and inter-assay variations were 6.1 % and 8.2 %,
4
respectively.
5
2.4 NOS activity measurements
6
The total NOS-activity in the supernatant of rat hypothalami was measured using a
7
commercially available kit (Jiancheng Bioengineering, Nanjing, China). NOS2
8
activity was measured by adding ethylene glycol tetra-acetic acid to chelate free Ca2+
9
from the reaction mixture. cNOS activity was obtained by subtracting NOS2 activity
10
from total NOS activity. NOS activities were expressed in units (U = nanomoles of
11
NO formed in 1 min by 1 mg of protein).
12
2.5 Immunocytochemistry
13
NOS1-staining was performed as described previously (Gao et al., 2013), adding 3%
14
H2O2 in methanol for 30 min to abolish endogenous peroxidase before the sections
15
were incubated with the blocking bufffer. Anti-NOS1 antibody (BD, Lexington, KY)
16
was used 1:100, followed by horseradish peroxidase-conjugated secondary antibody
17
(Zhongshan Goldenbridge Biotechnology, Beijing, China). When the primary
18
antibody was replaced by the blocking buffer no staining was present.
19
2.6 Double immunofluorescent labeling
20
The monoclonal NOS1 antibody and rabbit antibodies against CRH (5Bo) or AVP
21
(Truus) or OXT (O-2-T) (gifts from the Netherlands Institute for Neuroscience,
22
Amsterdam) 1:500 were used for 12–24 h at 4 °C. The specificities of these antibodies
Ac
ce pt
ed
M
an
us
cr
ip t
1
6
Page 6 of 15
had been confirmed previously. Dylight 488 conjugated IgG (1:200) and Dylight 594
2
conjugated IgG (1:600, Jackson lab, West Grove, PA) were added to the sections. The
3
sections were coverslipped and observed under a Leica fluorescence microscope
4
(Germany).
5
2.7 Image analysis
6
The anatomical position of the PVN was determined by Nissl staining of every 5th
7
cryostat section (20 μm). For the quantitative analysis of NOS1-ir signals, PVN
8
sections were chosen from the anterior part (Bregma level -1.4 to 1.6 mm), the middle
9
part (-1.6 to -1.8 mm) and the posterior part (-1.8 to -2.0 mm) (Paxinos and Watson,
10
1986). NOS1-ir cell counting was performed as before (Gao et al., 2013). Cell density
11
was calculated by dividing the number of positive neurons by PVN or supraoptic
12
nucleus (SON) area.
13
2.8 Statistical analyses
14
As the data were found to be not always normally distributed, non-parametric tests
15
were applied. Comparison between two independent samples was done by
16
Mann-Whitney U test and correlations by the Spearman test. P < 0.05 was considered
17
to be significant.
cr
us
an
M
ed
ce pt
Ac
18
ip t
1
19
3. Results
20
3.1 Changes in plasma NO, CORT/cortisol levels in CUS rats and MDD patients
21
Plasma NO levels were significantly increased, both in male CUS rats (P = 0.001,
22
Figure 1 A) and in male MDD patients (P < 0.001, Figure 1 B). Plasma CORT was 7
Page 7 of 15
significantly increased in CUS rats (P = 0.001, Figure 1 C), but no significant change
2
was found in plasma cortisol levels in male MDD patients (P = 0.427). No significant
3
correlations were found between plasma NO and cortisol levels, either in the control
4
(P = 0.160) or in the MDD (P = 0.310) group. There were no significant correlations
5
between plasma NO levels and HAMD scores (P = 0.920) or between plasma cortisol
6
levels and HAMD scores (P = 0.334), nor were any significant correlations found
7
between plasma NO and CORT levels in the control group (P = 0.248) or in the CUS
8
group (P = 0.453).
cr
us
(Insert Figure 1 around here)
an
9
ip t
1
3.2 Alterations in NOS activity and NOS1-ir in CUS rat hypothalamus
11
The total NOS activity and the cNOS activity in the hypothalamus of CUS rats
12
decreased significantly (P = 0.018 and P = 0.048, respectively), while NOS2 activity
13
showed no significant change (Figure 2 A). NOS1-ir was mainly located in the
14
cytoplasm and processes of neuronal cells, with different staining intensity along the
15
PVN (see Suppl. Fig. 2). The NOS1-ir cell density was significantly reduced in the
16
CUS group (P = 0.018, Figure 2 B), mainly due to changes in the anterior (P = 0.018)
17
and middle part (P = 0.068), but not in the posterior PVN (P = 0.201) (Figure 2 C).
18
NOS1-ir cell density showed no significant changes in the SON of the CUS rats (P =
19
0.201). NOS1-ir was found to be mainly co-localized in OXT-ir neurons (Figure 2 D),
20
while no co-localization was observed for NOS1 and CRH or AVP in the rat PVN (see
21
Suppl. Fig. 3).
22
Ac
ce pt
ed
M
10
(Insert Figure 2 around here) 8
Page 8 of 15
1
4. Discussion
3
We found, for the first time, a significant increase of plasma NO levels in the CUS
4
rats, which was in agreement with the increase in plasma NO levels in the
5
medicine-naïve MDD patients. In addition, CUS rats showed significantly decreased
6
hypothalamic NOS activity and NOS1-ir cell densities in the PVN but not in the SON,
7
which was consistent with the finding in human postmortem brain material that had
8
revealed decreased numbers of NOS1-ir neurons in the hypothalamic PVN (Bernstein
9
et al., 1998), but not in the SON (Bernstein et al., 2000) and with the decreased NOS1
10
we found in the PFC of depressive patients (Gao et al., 2013). Moreover, the
11
significantly decreased cNOS activity in the hypothalamus of CUS rats is also in
12
agreement with a decreased cNOS activity in human PFC in depression (Xing et al.,
13
2002). Our findings thus strongly indicate that the CUS rat model could be suitable
14
for further studies on the molecular changes in MDD as far as the NOS-NO system is
15
concerned.
ce pt
ed
M
an
us
cr
ip t
2
NOS1-ir neurons were mostly distributed in the anterior part of the PVN, which
17
agrees with the higher proportion of OXT neurons in the rostral part of the rat PVN
18
(Swaab et al., 1975). Indeed, we observed a co-localization for NOS1 and OXT in the
19
hypothalamic PVN. Since OXT is known to restrain the activity of the HPA axis (Bao
20
et al., 2008) and NO is involved in the regulation of OXT release by the
21
neurohypophyseal system (Cunningham and Sawchenko, 1991), the NOS1-OXT
22
interaction may play a critical role in integrating the different components of the stress
Ac
16
9
Page 9 of 15
1
systems during the stress response and in depression. We did not find significant changes in plasma cortisol levels in MDD. This could
3
be explained by the fact that centrally released CRH may be more important for mood
4
changes than peripherally released CRH. Indeed, increased basal plasma cortisol
5
levels were observed in only about 25% of the MDD patients (Young et al., 2001).
6
There was no significant correlation between plasma NO and cortisol levels,
7
indicating that these two systems may function quite independently. In contrast to the
8
significant increase of plasma NO levels, hypothalamic NOS activity and PVN-NOS1
9
expression were decreased in the CUS model and in MDD patients, indicating
10
different sources for the central and the peripheral NO. The peripheral NO might be
11
derived, for example, from the autonomic nervous system (Griffith and Stuehr, 1995).
M
an
us
cr
ip t
2
In conclusion, the changes in plasma NO and hypothalamic PVN-NOS1 expression
13
in the CUS model and in MDD patients are similar, suggesting that the CUS rat is an
14
interesting model for further studies on the underlying mechanisms of alterations in
15
the NOS-NO system in the etiology of depression. The novel finding of
16
co-localization of NOS1 with OXT in the PVN offers an interesting target for further
17
study of the possible role of NO in depression.
Ac
ce pt
ed
12
10
Page 10 of 15
ed
M
an
us
cr
ip t
Bao, A.M., Meynen, G., Swaab, D.F., 2008. The stress system in depression and neurodegeneration: focus on the human hypothalamus. Brain Res Rev 57, 531-53. Bernstein, H.G., Jirikowski, G.F., Heinemann, A., Baumann, B., Hornstein, C., Danos, P., et al., 2000. Low and infrequent expression of nitric oxide synthase/NADPH-diaphorase in neurons of the human supraoptic nucleus: a histochemical study. J Chem Neuroanat 20, 177-83. Bernstein, H.G., Stanarius, A., Baumann, B., Henning, H., Krell, D., Danos, P., et al., 1998. Nitric oxide synthase-containing neurons in the human hypothalamus: reduced number of immunoreactive cells in the paraventricular nucleus of depressive patients and schizophrenics. Neuroscience 83, 867-75. Chrapko, W.E., Jurasz, P., Radomski, M.W., Lara, N., Archer, S.L., Le Melledo, J.M., 2004. Decreased platelet nitric oxide synthase activity and plasma nitric oxide metabolites in major depressive disorder. Biol Psychiatry 56, 129-34. Cunningham, E.T., Jr., Sawchenko, P.E., 1991. Reflex control of magnocellular vasopressin and oxytocin secretion. Trends Neurosci 14, 406-11. Gao, S.F., Qi, X.R., Zhao, J., Balesar, R., Bao, A.M., Swaab, D.F., 2013. Decreased NOS1 Expression in the Anterior Cingulate Cortex in Depression. Cereb Cortex 23, 2956-64. Griffith, O.W., Stuehr, D.J., 1995. Nitric oxide synthases: properties and catalytic mechanism. Annu Rev Physiol 57, 707-36. Grippo, A.J., Johnson, A.K., 2009. Stress, depression and cardiovascular dysregulation: a review of neurobiological mechanisms and the integration of research from preclinical disease models. Stress 12, 1-21. Karolewicz, B., Szebeni, K., Stockmeier, C.A., Konick, L., Overholser, J.C., Jurjus, G., et al., 2004. Low nNOS protein in the locus coeruleus in major depression. J Neurochem 91, 1057-66. Kim, Y.K., Paik, J.W., Lee, S.W., Yoon, D., Han, C., Lee, B.H., 2006. Increased plasma nitric oxide level associated with suicide attempt in depressive patients. Prog Neuropsychopharmacol Biol Psychiatry 30, 1091-6. Paxinos, G., Watson, C., 1986. The Rat Brain in Stereotaxic Coordinates. Academic Press, New York. Swaab, D.F., Pool, C.W., Nijveldt, F., 1975. Immunofluorescence of vasopressin and oxytocin in the rat hypothalamo-neurohypophypopseal system. J Neural Transm 36, 195-215. Willner, P., 2005. Chronic mild stress (CMS) revisited: consistency and behavioural-neurobiological concordance in the effects of CMS. Neuropsychobiology 52, 90-110. Young, E.A., Carlson, N.E., Brown, M.B., 2001. Twenty-four-hour ACTH and cortisol pulsatility in depressed women. Neuropsychopharmacology 25, 267-76. Zhou, Q.G., Hu, Y., Hua, Y., Hu, M., Luo, C.X., Han, X., et al., 2007. Neuronal nitric oxide synthase contributes to chronic stress-induced depression by suppressing hippocampal neurogenesis. J Neurochem 103, 1843-54. Xing, G., Chavko, M., Zhang, L.X., Yang, S., Post, R.M., 2002. Decreased calcium-dependent constitutive nitric oxide synthase (cNOS) activity in prefrontal cortex in schizophrenia
ce pt
2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43
References
Ac
1
11
Page 11 of 15
ce pt
ed
M
an
us
cr
ip t
and depression. Schizophr Res 58, 21-30.
Ac
1 2
12
Page 12 of 15
Figure legends:
3
Figure 1 Changes in plasma NO, CORT levels in MDD patients and CUS rats
4
Plasma NO levels were significantly higher in CUS rats (A, n = 8/group) and MDD
5
patients (B, n = 10/group). Plasma corticosterone (CORT) levels showed significant
6
increases in CUS rats (C, n = 8/group). Data are shown as median, 25th–75th
7
percentiles, and the range. CTR = controls, MDD = major depressive disorder; ** P
