Abstract

Decreased function of the anterior cingulate cortex (ACC) is crucially involved in the pathogenesis of depression. A key role of nitric oxide (NO) has also been proposed. We aimed to determine the NO content in the cerebrospinal fluid (CSF) and the expression of NO synthase (NOS) isoforms, that is, NOS1, NOS2, and NOS3 in the ACC in depression. In depressive patients, CSF-NOx levels (the levels of the NO metabolites nitrite and nitrate) were significantly decreased (P = 0.007), indicating a more general decrease of NO production in this disorder. This agreed with a trend toward lower NOS1-mRNA levels (P = 0.083) and a significant decrease of NOS1-immunoreactivity (ir) (P = 0.043) in ACC. In controls, there was a significant positive correlation between ACC-NOS1-ir cell densities and their CSF-NOx levels. Furthermore, both localization of NOS1 in pyramidal neurons that are known to be glutamatergic and co-localization between NOS1 and GABAergic neurons were observed in human ACC. The diminished ACC-NOS1 expression and decreased CSF-NOx levels may be involved in the alterations of ACC activity in depression, possibly by affecting glutamatergic and GABAergic neurotransmission.

Introduction

A crucial involvement of the prefrontal cortex (PFC) in the regulation of mood is apparent in stroke patients, where a significant negative correlation was found between the severity of poststroke depression and the distance between brain injury and frontal pole (Narushima et al. 2003). In addition, a growing number of neuroimaging and postmortem studies have shown both structural and functional abnormalities in PFC subareas in depression, for example, a reduced volume and thickness of gray matter, decreased glucose metabolism and blood flow in the dorsolateral PFC (DLPFC; Drevets, Price, et al. 2008), reduced gray matter volume and decreased metabolic activity in the anterior cingulate cortex (ACC; Drevets et al. 1997), and reduced neuronal and glia cell densities in both DLPFC and ACC (Rajkowska et al. 1999, 2001; Cotter et al. 2001). The altered PFC activities have been proposed to contribute to a number of signs and symptoms of depression, such as cognitive decline, negative self-evaluation, and suicidal tendencies (Roy et al. 2010; Elliott et al. 2011). Transcranial magnetic stimulation or electrical deep brain stimulation applied to the PFC can be effective in the treatment of refractory depressive patients (Drevets, Savitz, et al. 2008; Koenigs and Grafman 2009; Fitzgerald and Daskalakis 2011; Jhanwar et al. 2011). Moreover, successful psychotherapy was found to be accompanied by increased gray matter volume and/or neuronal activity of the prefrontal subareas, particularly of the ACC (Quide et al. 2011). These findings have inspired studies on the neurotransmitter alterations in depression that may be the basis of the changes in PFC activity (Meyer et al. 2006; Gao and Bao 2011). A deficit in gamma aminobutyric acid (GABA) neurotransmission appeared to be present in the PFC in depressive patients, not only judging by the reduced glutamate/glutamine and GABA levels (Hasler et al. 2007; Bhagwagar et al. 2008), but also by the diminished mRNA and protein expression of glutamic acid decarboxylase (GAD), the principal enzyme that catalyzes the conversion of glutamate into GABA (Gao and Bao 2011). In addition, we recently found a significant decrease of GABA receptor beta 2 in the ACC in depression (Zhao et al. 2012). A wealth of evidence indicates that also the glutamatergic system plays a significant role in the pathogenesis of depression. Significant changes have been reported in the PFC in glutamate receptors, glutamate transporters, and glutamine synthetase (Gao and Bao 2011), and a reduced expression in an excitatory postsynaptic density protein (PSD-95; Feyissa et al. 2009; Zhao et al. 2012) in depression.

Recent studies have pointed to a significant role of the gaseous neurotransmitter nitric oxide (NO) in the pathogenesis of depression. NO is synthesized from l-arginine by at least 3 subtypes of NO synthase (NOS), that is, NOS1 (also known as nNOS), NOS2 (or iNOS), and NOS3 (or eNOS; Griffith and Stuehr 1995). NOS1 is present in human brain structures such as the neocortex, hippocampus, and cerebellum (Egberongbe et al. 1994). NOS2 and NOS3 are expressed in the human brain as well. In the human cortex, NOS1 is found in both glutamatergic pyramidal cells and GABAergic interneurons (Sestan and Kostovic 1994; Ohyu and Takashima 1998; Judas et al. 1999; Yew et al. 1999; Kwan et al. 2012). Polymorphisms in the 3 subtypes of NOS confer an increased susceptibility or relapse risk for depressive disorders (Yu et al. 2003; Reif et al. 2006; Galecki et al. 2011), while the genomic locus of NOS1 on chromosome 12q24 was found to be linked with depression (Ewald et al. 1998; Morissette et al. 1999; Fallin et al. 2005; McGuffin et al. 2005). A recent genome-wide association study of mood disorders revealed a single-nucleotide polymorphism located in NOS1 that was associated with general psychological distress (Luciano et al. 2012). The relationship between mood disorder and NOS1 was further supported by a reduced NOS1 expression in the locus coeruleus (Karolewicz et al. 2004) and hypothalamus (Bernstein et al. 1998, 2000, 2002) in depression. In addition, Xing et al. (2002) have shown that constitutive NOS activity that reflects both NOS1 and NOS3 was markedly lower in the DLPFC in depressive patients. The similar findings in different brain regions suggested the possible presence of a more general decrease of NO production in depression. Therefore, we investigated whether the alterations in the brain NOS system in depression were also reflected in the cerebrospinal fluid (CSF). We assessed the NO levels in the postmortem ventricular CSF by measuring the levels of its metabolites, that is, nitrate and nitrite (NOx), since the levels of CSF-NOx were found earlier to reflect changes in the NO content in the brain (Milstien et al. 1994; Tohgi et al. 1998; Rejdak et al. 2008).

Because of the arguments mentioned above, we hypothesized that ACC-NOS expression may be decreased in depression, and that its involvement in depression might be explained by the interactions of NOS–NO with glutamatergic and/or GABAergic neurotransmission. To test this hypothesis, we analyzed the mRNA levels of the 3 NOS subtypes (NOS1, NOS2, and NOS3) and quantified the NOS1-immunoreactivity (ir) in ACC in depressive patients after we had observed meaningful changes in its mRNA levels.

Materials and Methods

Brain Material

Brain material was obtained from the Netherlands Brain Bank (NBB, director Dr I. Huitinga) following permission for a brain autopsy and for the use of the brain material and clinical data for research purposes. The depressive patients had been diagnosed in psychiatric clinics according to Diagnostic and Statistical Manual of Mental Disorders (DSM) criteria to have a major depressive disorder (MDD) or a bipolar disorder (BD) during their lifetime. The diagnosis was confirmed by a board-certified psychiatrist, based on the DSM-IV criteria and the extensive medical records of the NBB. The control subjects had not suffered from either a primary neurological disorder, other psychiatric diseases, or alcohol abuse. 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). The material consisted of ventricular 30 CSF samples (MDD, N = 8; BD, N = 7; control, N = 15), which were taken during autopsy from the anterior horn of the lateral ventricle by a syringe with a long needle and the supernatant was immediately put at −80 °C after centrifugation. In addition, snap-frozen samples of DLPFC (MDD, N = 5; BD, N = 8; control, N = 13) and snap-frozen and paraffin-embedded tissue of ACC (MDD, N = 5; BD, N = 7; control N = 12) were studied. The MDD versus BD subgroup and the combined depression group (MDD + BD) versus the control group were well matched for age, brain weight, clock time, and month of death, postmortem delay (PMD), CSF pH, and fixation time (for ACC paraffin sections). Detailed clinico-pathological information and P-values of parameter matches are given in Supplementary Table 1.

CSF-NOx Assay

CSF-NOx levels were measured with the Griess method (Green et al. 1982) by means of an enzymatic colorimetric NO assay according to the manufacturer's protocol (Oxford Biomedical, Oxford, MI, United States of America). Briefly, 85 μL of CSF was incubated in duplicate with nitrate reductase (0.01 U) and nicotinamide adenine diuncleotide hydrogen (2 mM) for 20 min to convert nitrate to nitrite, then an equal volume of Griess reagents was added, and after 5 min the absorbance was measured at 540 nm in a Varioskan Flash Spectral Scanning Multimode Reader (Thermo Scientific, Waltham, MA, United States of America). Linear regression was done by using a set of serial dilutions of standards (0.5–100 μM). The resulting equation was used to calculate the sample concentrations of NOx. The detection limit was 1 μM, the within-run and between-run coefficients of variation were 6.2% and 9.3%, respectively.

Quantification of NOS3-, NOS1-, and NOS2-mRNA in DLPFC and ACC

Tissue Dissection, RNA Extraction, and cDNA Synthesis

Brain tissue was quickly dissected during autopsy and stored at −80 °C until further use. Cryostat sections of 50 μm in thickness were obtained, and the gray matter was separated from the white matter by pre-chilled sterile scalpels. Fifty milligrams of each sample were collected into pre-chilled tubes and immediately put on dry ice. All procedures were conducted at −18 °C. The total RNA was isolated from the collected gray matter samples according to a hybrid protocol of Trizol (Invitrogen Life Technologies, Carlsbad, CA, United States of America) and Qiagen RNeasy Mini Kit (Qiagen, Valencia, CA, United States of America). The RNA isolation methods have been described in detail before (Bossers et al. 2009). RNA yield and purity were determined by a NanoDrop ND-1000 spectrophotometer (Nanodrop Technologies, Wilmington, DE, United States of America). RNA integrity number (RIN) was used to assess RNA quality (scale 1–10, with 1 being the lowest and 10 being the highest RNA quality) and measured by an Agilent 2100 bioanalyzer (Agilent Technologies, Palo Alto, CA, United States of America). The RNA quality of all samples (Supplementary Table 1) was appropriate for real-time quantitative polymerase chain reaction (qPCR) analysis (Fleige and Pfaffl 2006). There was no significant difference in the RIN value, either between the control group and the depression group or between the MDD group and the BD group (Supplementary Table 1). For each sample, 1 μg of total RNA was used for the synthesis of cDNA, the procedure of which has been described before (Bossers et al. 2010).

Primer Design

The primer design strategy and the reference genes' primers have been described in our previous paper (Wang et al. 2008). The primers for NOS1, NOS2 and NOS3 are designed against their common sequences (Table 1). Seven candidate reference genes were tested for both ACC and DLPFC samples. β-Actin, hypoxanthine phosphoribosyltransferase 1, tubulin-α, tubulin-β4, and glyceraldehyde-3-phosphate dehydrogenase were used as reference genes, since these 5 genes showed the most stable expression in ACC and DLPFC as revealed by the geNorm analysis (Vandesompele et al. 2002). There was no significant difference in the used reference genes between depressive patients and matched controls (P ≥ 0.248 for ACC and P ≥ 0.291 for DLPFC). Oligonucleotide primer pairs for the target genes are shown in Table 1.

Table 1

Oligonucleotides of primers

Gene Accession numbers Targeted sequence Predicted size (bp) Primers (5′–3′) 
NOS1 NM_000620.2 3344–3444 101 F-agcagtttgcctcccta 
    R-gttcttgccccatttcc 
NOS2 NM_000625 3875–4007 133 F-gccctttacttgacctcc 
    R-agttccatctttcacccac 
NOS3 NM_000603 1842–1954 113 F-cgagtgaaggcgacaat 
    R-tccatacacaggacccg 
Gene Accession numbers Targeted sequence Predicted size (bp) Primers (5′–3′) 
NOS1 NM_000620.2 3344–3444 101 F-agcagtttgcctcccta 
    R-gttcttgccccatttcc 
NOS2 NM_000625 3875–4007 133 F-gccctttacttgacctcc 
    R-agttccatctttcacccac 
NOS3 NM_000603 1842–1954 113 F-cgagtgaaggcgacaat 
    R-tccatacacaggacccg 

Note: F, forward primer; R, reverse primer; bp, base pair.

Real-Time qPCR

The cDNA template (equivalent to 5 ng of total RNA) was amplified in a final volume of 20 μL using a SYBR Green PCR master mix (Applied Biosystems, CA, United States of America) and a mixture of forward and reverse primers (each 2 pmol/µL) as described before (Wang et al. 2008; Bossers et al. 2009). Data were acquired and processed automatically by the Applied Biosystems 7300 Real-Time PCR System. The specificity of amplification was checked by melting curve analysis and electrophoresis of the products on an 8% polyacrylamide gel. Sterile water and RNA samples—without the addition of reverse transcriptase during cDNA synthesis—served as negative controls. The linearity of each qPCR assay was tested by preparing a series of dilutions of the same stock of cDNA in multiple plates. The expression of target genes was normalized by the geometric mean of the above-mentioned reference genes, and the relative absolute amount of target genes was calculated by 1010 × E−Ct (E = 10−(1/slope)) and used for statistics (Kamphuis et al. 2001).

Quantification of NOS1-ir in ACC

Since NOS1-mRNA levels were found to show a clear trend for decrease in the ACC but not in the DLPFC of the depression group (for details see Results), NOS1-ir distribution and quantification were performed on buffered (pH 7.4) paraformaldehyde-fixed paraffin-embedded 6-µm sections of the ACC from the same subjects.

Antibody Specificity

A polyclonal rabbit anti-NOS1 antibody (catalogue no: Sc-648; Santa Cruz Biotechnology, Santa Cruz, CA, United States of America) was used that was raised against a peptide near the C-terminus of NOS1. All the isoforms were detected in the human ACC in this study, since NOS1-α, NOS1-β, and NOS1-γ share the sequences in the C-terminus (Savidge 2011). This antibody has been used for immunocytochemistry (ICC) and western blot analysis by other research groups (Gingerich and Krukoff 2008; Kang et al. 2009). To confirm the specificity of this antibody for human brain material, we performed a solid phase adsorption test, the procedure of which has been described in our recently published paper (Shan et al. 2011). The anti-NOS1 antibody recognized the corresponding homologous peptide (catalogue no: Sc-648 P; Santa Cruz Biotechnology) on nitrocellulose sheets, while the pre-adsorption with this peptide eliminated the NOS1-ir in the human cortex (Supplementary Fig. 1).

ICC of NOS1-ir

The protocol used for the NOS1 staining has been described in our previous study (Bao and Swaab 2007), with slight modifications. In brief, antigen retrieval was applied for sections with microwave in Tris–HCl buffer (pH 9.0), followed by incubation with tris-buffered saline (TBS) milk [5% milk powder (Elk, Campina, Amersfoort, The Netherlands), pH 7.6] at room temperature (RT) for 60 min to reduce non-specific staining. The sections were incubated with the rabbit anti-NOS1 antibody (1:1000 in 1% TBS milk) overnight at 4 °C. The next day these sections were incubated with biotinylated goat anti-rabbit IgG (Vector Laboratories, Burlingame, CA, United States of America) 1:400, followed by incubation with a streptavidin-biotinylated enzyme complex (Vectastain ABC System, Elite kit, Vector Laboratories, Inc.) 1:800 at RT for 60 min. Finally, the sections were incubated for 10 min in DAB-Ni substrate solution [3′,3′-diaminobenzidine-tetrahydrochloride (0.5 mg/mL); Sigma, Zwijndrecht, The Netherlands] in TBS containing 0.01% hydrogen peroxide and ammonium nickel sulfate (2.2 mg/mL).

An adjacent section to each NOS1-stained section was stained with 0.1% w/v thionine in acetate buffer (pH 4) for 2 min in order to localize the different layers in the ACC.

Image Analysis

All the images were collected under a Zeiss Axioskop microscope (Zeiss, Jena, Germany) with neofluar objectives (Zeiss) and a motorized XYZ stage, black-and-white camera (Sony, Minato, Japan). Cell counting and density analysis were both performed with the software Image Pro 6.3 (Mediacybernetics, Bethesda, United States of America) plus home-developed macros, with the identities of all the subjects unknown to the investigator.

The method of determining cell densities of NOS1-ir neuronal profiles in the ACC was similar to what was described in our earlier work (Fronczek et al. 2007; Bossers et al. 2009). In brief, images were collected at low magnification (×10), the intact gray matter and the layers II–III in particular were manually outlined based upon the adjacent thionine-stained section (Fig. 2). Randomly selected fields were counted under a ×40 microscope objective, covering in total 20% of the manually outlined areas. To prevent double counting, only positively stained cell profiles containing a nucleolus were counted (Bao et al. 2005). The estimated cell densities per cubic millimeter were calculated by dividing the number of identified structures by the measured area, and by correcting for section thickness (6 μm) in order to rule out the confounding effect of possible changes in gray matter volume in the ACC in depression (Drevets et al. 1997).

The methods of determining the NOS1-positive area and the optical density (OD) of NOS1 signal were as follows: A black-and-white camera was fitted on the microscope with a ×20 objective in front. The light was adjusted to get the same penetration for unstained control areas for each section. The collected images were transformed into OD images by use of a standard transformation curve. The collected images included an intact ACC gyrus, in which the gray matter and layers II–III were delineated based upon the adjacent thionine-stained section (Fig. 2). OD values >6 times the background value were considered a positive signal, a level that was determined by a pilot study for the threshold value. The average size of NOS1-ir cells was approached by dividing the NOS1-positive area by the cell numbers in layers II–III. The average OD was calculated by multiplying the percentage of the NOS1-positive area by the OD of NOS1 signal in the ACC.

Immunofluorescence and Confocal Laser Scanning Microscopy

Antigen retrieval and TBS milk blocking were performed as described in the “ICC of NOS1-ir” section, then sections were incubated with monoclonal anti-NOS1 antibody (1:50, BD Transduction Laboratories, Lexington, KY, United States of America) and rabbit polyclonal GAD65/67 antibody (1:500, Chemicon Int. Temecula, CA, United States of America). Primary antibody incubation for 2h at RT was followed by overnight incubation at 4 °C. NOS1 was visualized in red by SA-Alexa594 (1:200, Invitrogen) using a biotinylated anti-mouse antibody (1:400, Vector Laboratories Inc.). GAD65/67 was detected in green by anti-rabbit-Alexa488 (1:400, Invitrogen). A fluorochrome-conjugated antibody was incubated for 3 h at RT. The sections were observed using a Zeiss 510 confocal laser scanning microscope equipped with lasers emitting at 488 and 543 nm.

Statistical Analysis

As not all data were distributed normally, the differences between groups were evaluated by the Mann–Whitney U-test. The differences in clock time or month of death (circular parameters) between groups were tested with the Mardia–Watson–Wheeler test. A correlation was examined with the Spearman test. Tests were 2-tailed and values of P ≤ 0.05 were considered to be significant.

Results

CSF-NOx Levels

CSF-NOx levels showed a significant 53% decrease in depressive patients (10.4 ± 2.4 μmol/L) compared with the matched controls (15.2 ± 1.4 μmol/L; P = 0.007, Fig. 1). No significant difference was found in the CSF-NOx levels between BD (10.4 ± 2.1 μmol/L) and MDD patients (10.4 ± 4.3 μmol/L; P = 0.189). There was no significant correlation between CSF-NOx levels and PMD either in the control subjects (P = 0.277) or in the depressive patients (P = 0.602).

Figure 1.

Changes of CSF-NOx levels in depression. Boxplot showing the median, 25th–75th percentiles, and the range of the nitrate and nitrite (NOx) levels in the postmortem CSF of depressive patients (open symbols) and controls (solid circles). CSF-NOx levels showed a significant 53% decrease in depressive patients. Triangles: MDD; empty circles: BD. Double asterisks indicates P < 0.01.

Figure 1.

Changes of CSF-NOx levels in depression. Boxplot showing the median, 25th–75th percentiles, and the range of the nitrate and nitrite (NOx) levels in the postmortem CSF of depressive patients (open symbols) and controls (solid circles). CSF-NOx levels showed a significant 53% decrease in depressive patients. Triangles: MDD; empty circles: BD. Double asterisks indicates P < 0.01.

Expression of NOS3-, NOS1-, and NOS2-mRNA in ACC and DLPFC

There was a trend toward the decrease of NOS1-mRNA in the ACC in the depression group (P = 0.083, Table 2) compared with the matched controls. This observation was followed up on the protein level. No significant changes of NOS2- or NOS3-mRNA levels in ACC, or NOS1-, NOS2-, NOS3-mRNA levels in DLPFC, were found in the depression group (P ≥ 0.249, Table 2). No significant difference was found in the mRNA levels of the 3 NOS subtypes between MDD and BD patients, either in ACC or in DLPFC (Table 2).

Table 2

NOS1-, NOS2-, and NOS3-mRNA–relative levels presented as mean ± standard error of the mean (×10−6)

 NOS1 in ACC NOS2 in ACC NOS3 in ACC NOS1 in DLPFC NOS2 in DLPFC NOS3 in DLPFC 
Control 1575 ± 133 759 ± 122 390 ± 64 981 ± 110 1997 ± 327 179 ± 26 
Depression 1355 ± 188 896 ± 159 449 ± 104 959 ± 99 2003 ± 370 257 ± 50 
P-value 0.083 0.729 0.954 0.939 0.939 0.249 
MDD 1077 ± 118 811 ± 329 348 ± 113 926 ± 101 1707 ± 298 206 ± 47 
BD 1553 ± 298 957 ± 163 522 ± 161 980 ± 153 2188 ± 580 288 ± 76 
P-value 0.149 0.432 0.530 0.943 0.833 0.833 
 NOS1 in ACC NOS2 in ACC NOS3 in ACC NOS1 in DLPFC NOS2 in DLPFC NOS3 in DLPFC 
Control 1575 ± 133 759 ± 122 390 ± 64 981 ± 110 1997 ± 327 179 ± 26 
Depression 1355 ± 188 896 ± 159 449 ± 104 959 ± 99 2003 ± 370 257 ± 50 
P-value 0.083 0.729 0.954 0.939 0.939 0.249 
MDD 1077 ± 118 811 ± 329 348 ± 113 926 ± 101 1707 ± 298 206 ± 47 
BD 1553 ± 298 957 ± 163 522 ± 161 980 ± 153 2188 ± 580 288 ± 76 
P-value 0.149 0.432 0.530 0.943 0.833 0.833 

Note: ACC, anterior cingulate cortex; DLPFC, dorsolateral prefrontal cortex; MDD, major depressive disorder; BD, bipolar disorder.

NOS1-ir in ACC

On the basis of the thionine staining in adjacent sections (Fig. 2A,H), NOS1-ir appeared to be mainly present in the layer II of the human ACC (Fig. 2B,I). In the control subjects, NOS1-ir displayed light to medium staining in the perikarya and the proximal parts of the major dendrites of small- or medium-sized pyramidal neurons in layers II–III (Fig. 2C,D), while it was also present in some scattered fibers (Fig. 2E,F) and in some large multipolar neurons with an intense and Golgi-like staining (Fig. 2G) in layers V–VI. In depressive patients, fewer and smaller NOS1-ir neurons were found in layer II (Fig. 2I, see also Fig. 3), while the morphology of the NOS1-ir neurons was unchanged. NOS1 staining in depressive patients was again present in the proximal parts of major dendrites and in the perikarya of layers II–III pyramidal neurons (Fig. 2J,K), scattered fibers (Fig. 2L) and large multipolar neurons (Fig. 2M,N) in layers V–VI. In addition, the co-expression of NOS1 and GAD65/67 was present in neurons, particularly in the depressive patients (Supplementary Fig. 2).

Figure 2.

Representative microphotographs of NOS1-ir in the ACC. The laminar structure is visible in the adjacent thionine-stained sections (A and H). In the control subject (AG, NBB #97-143), the majority of NOS1-immunoreactivity (ir) was observed to distribute in the layer II of the ACC (B). Note that in the ACC, the NOS1 staining is present in small- and medium-sized pyramidal neurons in layers II–III (C and D), in scattered fibers (E and F), and large multipolar neurons (G) in layers V–VI. In the depressive patient (HN, NBB #06-011), the number and size of NOS1-ir neurons are reduced in layer II (I), the morphology of NOS1-ir neurons in all the layers is comparable with the control subject, that is, in proximal parts of major dendrites and in the perikarya of layers II–III pyramidal neurons (J and K), scattered fibers (L), and large multipolar neurons (M and N) in layers V–VI. Scale bars: A, B, H, and I, 100 mm; CG and JN, 20 mm.

Figure 2.

Representative microphotographs of NOS1-ir in the ACC. The laminar structure is visible in the adjacent thionine-stained sections (A and H). In the control subject (AG, NBB #97-143), the majority of NOS1-immunoreactivity (ir) was observed to distribute in the layer II of the ACC (B). Note that in the ACC, the NOS1 staining is present in small- and medium-sized pyramidal neurons in layers II–III (C and D), in scattered fibers (E and F), and large multipolar neurons (G) in layers V–VI. In the depressive patient (HN, NBB #06-011), the number and size of NOS1-ir neurons are reduced in layer II (I), the morphology of NOS1-ir neurons in all the layers is comparable with the control subject, that is, in proximal parts of major dendrites and in the perikarya of layers II–III pyramidal neurons (J and K), scattered fibers (L), and large multipolar neurons (M and N) in layers V–VI. Scale bars: A, B, H, and I, 100 mm; CG and JN, 20 mm.

Figure 3.

Alterations of NOS1-ir in the ACC in depression. Boxplot showing the median, 25th–75th percentiles, and the range of the cell density (A), the approximate size of NOS1-positive cells (B), and the average OD (C) of NOS1-ir in the ACC of depressive patients (open symbols) and controls (solid circles). There was a significant 49% reduction of NOS1-ir cell density in layers II–III and a trend towards a lower NOS1-ir cell density in the entire gray matter of ACC in depressive patients (A). The approximation of NOS1-ir cell size showed a significant decrease (60%) in layers II–III (B). The average OD of NOS1-ir showed a significant reduction in the entire gray matter (45%) and a trend towards a reduction in layers II–III of the ACC in depression (C). There was a significantly positive correlation between CSF-NOx levels and NOS1-ir cell densities in the gray matter of ACC in the control subjects (rho = 0.667, P = 0.050, N = 9) (D). Triangles: MDD, empty circles: BD. Asterisk indicates P < 0.05.

Figure 3.

Alterations of NOS1-ir in the ACC in depression. Boxplot showing the median, 25th–75th percentiles, and the range of the cell density (A), the approximate size of NOS1-positive cells (B), and the average OD (C) of NOS1-ir in the ACC of depressive patients (open symbols) and controls (solid circles). There was a significant 49% reduction of NOS1-ir cell density in layers II–III and a trend towards a lower NOS1-ir cell density in the entire gray matter of ACC in depressive patients (A). The approximation of NOS1-ir cell size showed a significant decrease (60%) in layers II–III (B). The average OD of NOS1-ir showed a significant reduction in the entire gray matter (45%) and a trend towards a reduction in layers II–III of the ACC in depression (C). There was a significantly positive correlation between CSF-NOx levels and NOS1-ir cell densities in the gray matter of ACC in the control subjects (rho = 0.667, P = 0.050, N = 9) (D). Triangles: MDD, empty circles: BD. Asterisk indicates P < 0.05.

A significant 49% reduction in NOS1-ir cell density was observed in the ACC layers II–III (P = 0.043), and a trend toward lower NOS1-ir cell density in the entire gray matter of ACC (P = 0.083) was found in the depression group (Fig. 3A). The approximation of NOS1-ir cell size showed a significant 60% decrease in the layers II–III (P = 0.015, Fig. 3B). The average OD of NOS1-ir showed a significant 45% reduction in the gray matter and a trend toward a reduction in the layers II–III of ACC in depression (Fig. 3C, P = 0.011 and 0.057, respectively). Furthermore, CSF-NOx levels were found to be positively correlated with NOS1-ir cell density in the gray matter of the ACC in the control group (rho = 0.667, P = 0.050, N = 9, Fig. 3D), but not in the depression group (rho = −0.179, P = 0.702, N = 7).

Throughout the entire gray matter (P ≥ 0.149) and in the layers II–III (P ≥ 0.639) of ACC, no significant difference could be detected in the NOS1-ir cell density, the approximation of NOS1-ir cell size, or the average OD of NOS1-ir between BD and MDD patients (Supplementary Table 2).

Possible Confounding Factors

As shown in Supplementary Table 1, BD–MDD and depression–control are well matched between the compared groups for age, PMD, CSF pH, brain weight, clock time, and month of death. There was thus no indication of the effect of these possible confounders on our data. The relationship of CSF-NOx, NOS1-mRNA, and NOS1-ir measurements with suicide, sex, and mood state at time of death is given in SupplementaryResult 1. The possible influence of antidepressant treatment on NOS–NO measurements is analyzed in the Discussion section.

Discussion

The present study shows a significantly reduced NO concentration in the ventricular CSF and a decreased NOS1 expression in the ACC (but not in the DLPFC) in depressive patients. In addition, we observed a significant positive correlation between the NOS1-ir cell densities in the ACC and the CSF-NOx levels in the control subjects but not in the depressive patients, which supports the presence of a globally altered brain NOS–NO system in depression. No differences were found between the 2 types of depression: BD and MDD.

The significantly decreased CSF-NOx levels we found in depression will not only be due to the decreased NOS1 expression in ACC, but also to the reduced NOS expression or activity that was reported in other brain areas in depression (Xing et al. 2002; Karolewicz et al. 2004). The postmortem CSF-NOx levels of control subjects measured in the present study were within the same range of NOx levels reported for the lumbar puncture CSF of living subjects (Tohgi et al. 1998; de Bustos et al. 1999; Ramirez et al. 2004; Boll et al. 2008). In accordance with the NOS/NO studies in other human brain regions (Blum-Degen et al. 1999; Yao et al. 2004), parameters as PMD, CSF pH, or age showed no correlation with CSF-NOx levels in the control group, indicating that NO metabolites in postmortem CSF were independent of these possible confounders for which we also carefully matched. The lower levels of NO metabolites in the ventricular CSF we found in depression provide thus further evidence for a more general brain NO deficiency in this disorder.

We did not find a significant change of NOS1-, NOS2-, or NOS3-mRNA levels in the DLPFC in depression, which is in agreement with previous observations of unaltered NOS1 or NOS3 expression at the protein level (Xing et al. 2002) and stable NOS1-mRNA expression in the DLPFC of depressive patients (Silberberg et al. 2010). It should be noted that the mRNA levels of NOS1 in ACC were only 14% lower in the depressed group, while the difference in protein in ICC was about 50%. Our novel finding of a decreased NOS1 expression in the ACC in depression suggests that this cortical area is more vulnerable than the DLPFC concerning the molecular pathological changes of NOS1 in depression. A special role of the ACC in the pathogenesis of depression is also indicated by the observation that deep brain stimulation of this area can ameliorate depressive symptoms in treatment-resistant depressive patients (Drevets, Savitz, et al. 2008).

NOS1 has been shown to be localized in the human cortex in both pyramidal and nonpyramidal neurons by ICC and/or NADPH-diaphorase histochemistry (Sestan and Kostovic 1994; Ohyu and Takashima 1998; Judas et al. 1999; Yew et al. 1999; Kwan et al. 2012). Our NOS1-ICC study in the ACC is fully in agreement with previous findings in other brain regions (Ohyu and Takashima 1998; Yew et al. 1999). In layers II–III, the small- and medium-sized pyramidal NOS1-ir neurons with light-to-medium reactivity in perikarya and initial dendritic segments have been defined as type II neurons that are glutamatergic and exclusively present in the primate and human brain (Judas et al. 1999; Kwan et al. 2012). In layers V–VI and the border between white- and gray matter, the large multipolar NOS1-ir neurons with an intense and Golgi-like staining have been defined as type I neurons that are GABAergic interneurons and usually co-localize with neuropeptide Y and somatostatin (Yan et al. 1996; Judas et al. 1999).

The decreased activity of the brain NO system in depression may cause the alterations of NO-mediated fundamental functions such as neuroprotection, cerebral blood flow regulation, and synaptic plasticity, which may in turn underlie the structural and functional abnormalities of the PFC found in depression (Swaab et al. 2000; Drevets, Price, et al. 2008). NO-mediated neuronal activity may affect not only the glutamatergic neurotransmission in the PFC (Resstel et al. 2008; Roenker et al. 2012), but also the GABAergic neurotransmission in hippocampus, striatum, and basal forebrain (Segovia et al. 1994; Casamenti et al. 1999). This latter mechanism may also take place in the ACC, as we found: 1) Co-localization in neurons of GAD65/67 (a marker of GABAergic neurons) and NOS1 and 2) a significant correlation in mRNA levels between NOS1 and GAD67 (rho = −0.664, P = 0.018, N = 12) in the depression group. In addition, 3) we found in a pilot study a positive correlation between CSF-GABA and the here reported CSF-NOx levels (rho = 0.426, P = 0.043, N = 23, unpublished data). Another possible mechanism is that the observed deficiency in the NOS–NO system in depression may result from a diminished activity in the glutamatergic system. Glutamate, through activation of N-methyl-d-aspartate (NMDA) receptors, is capable of enhancing NO synthesis by activating NOS1. The NOS1-expressing pyramidal neurons in ACC are known to be glutamatergic, suggesting the existence of an NMDA–NO transduction pathway that has indeed been suggested to be involved in the pathophysiology of depression. In this disorder, decreased glutamate levels were found in both ACC and CSF (Mirza et al. 2004; Frye et al. 2007), while our data in the ACC of the same patients as in the present study showed a significantly reduced expression of PSD-95, a scaffolding protein between the NMDA receptor and NOS1 (Zhao et al. 2012). Co-staining of NOS1 with pyramidal neuron- and interneuron markers in both controls and depressed patients may provide in the future valuable insights in the possibility of a cell type-dependent loss of NOS1 in depression. Since NOS1-containing neurons may be selectively spared from apoptosis in neurodegenerative diseases (Hyman et al. 1992; Mufson and Brandabur 1994), the decreased NOS1 expression that we found in depression may increase the risk for apoptosis and eventually cell loss, as was indeed found in the ACC in depression (Cotter et al. 2001).

The decreased ACC-NOS1 expression and CSF-NO levels in depression are unlikely to be the result of the chronic administration of antidepressants, since it has been found that antidepressants may enhance, rather than attenuate, NO production in vitro (Chapleau and Abboud 1994; Miller and Hoffman 1994), while chronic therapy with imipramine or citalopram or lithium was found not to change NOS activity in the rat cortex (Corasaniti et al. 1995; Jopek et al. 1999). Thus, had antidepressants or mood stabilizers interfered with our measurements, this would rather have led to an underestimation of the observed differences between controls and depressive patients in the brain's NO system. We also analyzed the possible effects of medication in the last 3 months (Supplementary Table 1) on our data. Consistent with the studies mentioned above, MDD patients not taking antidepressants and BD patients on mood stabilizers not taking antidepressants did indeed have lower ACC-NOS1 expression and CSF-NOx levels compared with BD and MDD patients taking antidepressants, although the reduction did not reach statistical significance, probably due to the small sample size. It should also be noted that, for the depression group, we recruited both MDD and BD patients, as a series of previous studies on the NOS1-ir alteration in the human brain material (Bernstein et al. 1998, 2000, 2002), and some genetic association studies with respect to NOS1 and depression (Ewald et al. 1998; Morissette et al. 1999; Fallin et al. 2005; McGuffin et al. 2005) did not yield differences between these 2 subtypes of patients, nor did we find significant differences between MDD and BD for both the ACC-NOS1 expression and the ventricular CSF-NOx levels.

Conclusion

Here, we report for the first time significantly decreased CSF-NOx levels and a reduction of NOS1 expression in the ACC in depression. There was also a significant positive correlation between ACC-NOS1-ir cell densities and CSF-NOx levels in the control subjects. In addition, we found a co-localization between NOS1-ir and GABAergic neurons in ACC. Our data together with the literature suggest that the NOS–NO system may participate in PFC activity regulation possibly by influencing GABAergic and glutamatergic neurotransmission, and thus be involved in the pathogenesis of depression.

Supplementary Material

Supplementary material can be found at: http://www.cercor.oxfordjournals.org/.

Funding

This work was supported by Nature Science Foundation of China (31271130 and 30970928 to A.-M.B.); Nature Science Foundation of Zhejiang Province (Y12H090054 to A.-M.B.); Science and Technology Program of Zhejiang Province (2009C34020 to A.-M.B.); China Scholarship Council for State Scholarship Fund (2010632112 to S.-F.G.); and China Exchange Program of the Royal Netherlands Academy of Arts and Sciences (08CDP012 to A.-M.B. and D.F.S.).

Notes

We thank Mrs. W.T.P. Verweij for correcting the English, Mr. Joop J. van Heerikhuize for image analysis, and Mr. Michiel Kooreman, Mr. Bart Fisser, Ms. Arja Sluiter, and Mr. Ling Shan for their technical assistance. Brain material was provided by the Netherlands Brain Bank (Director Dr I. Huitinga). Conflict of Interest: None declared.

References

Bao
AM
Hestiantoro
A
Van Someren
EJ
Swaab
DF
Zhou
JN
Colocalization of corticotropin-releasing hormone and oestrogen receptor-alpha in the paraventricular nucleus of the hypothalamus in mood disorders
Brain
 , 
2005
, vol. 
128
 (pg. 
1301
-
1313
)
Bao
AM
Swaab
DF
Gender difference in age-related number of corticotropin-releasing hormone-expressing neurons in the human hypothalamic paraventricular nucleus and the role of sex hormones
Neuroendocrinology
 , 
2007
, vol. 
85
 (pg. 
27
-
36
)
Bernstein
HG
Heinemann
A
Krell
D
Mawrin
C
Bielau
H
Danos
P
Diekmann
S
Keilhoff
G
Bogerts
B
Baumann
B
Further immunohistochemical evidence for impaired NO signaling in the hypothalamus of depressed patients
Ann N Y Acad Sci
 , 
2002
, vol. 
973
 (pg. 
91
-
93
)
Bernstein
HG
Jirikowski
GF
Heinemann
A
Baumann
B
Hornstein
C
Danos
P
Diekmann
S
Sauer
H
Keilhoff
G
Bogerts
B
Low and infrequent expression of nitric oxide synthase/NADPH-diaphorase in neurons of the human supraoptic nucleus: a histochemical study
J Chem Neuroanat
 , 
2000
, vol. 
20
 (pg. 
177
-
183
)
Bernstein
HG
Stanarius
A
Baumann
B
Henning
H
Krell
D
Danos
P
Falkai
P
Bogerts
B
Nitric oxide synthase-containing neurons in the human hypothalamus: reduced number of immunoreactive cells in the paraventricular nucleus of depressive patients and schizophrenics
Neuroscience
 , 
1998
, vol. 
83
 (pg. 
867
-
875
)
Bhagwagar
Z
Wylezinska
M
Jezzard
P
Evans
J
Boorman
E
M Matthews
P
J Cowen
P
Low GABA concentrations in occipital cortex and anterior cingulate cortex in medication-free, recovered depressed patients
Int J Neuropsychopharmacol
 , 
2008
, vol. 
11
 (pg. 
255
-
260
)
Blum-Degen
D
Heinemann
T
Lan
J
Pedersen
V
Leblhuber
F
Paulus
W
Riederer
P
Gerlach
M
Characterization and regional distribution of nitric oxide synthase in the human brain during normal ageing
Brain Res
 , 
1999
, vol. 
834
 (pg. 
128
-
135
)
Boll
MC
Alcaraz-Zubeldia
M
Montes
S
Rios
C
Free copper, ferroxidase and SOD1 activities, lipid peroxidation and NO(x) content in the CSF. A different marker profile in four neurodegenerative diseases
Neurochem Res
 , 
2008
, vol. 
33
 (pg. 
1717
-
1723
)
Bossers
K
Meerhoff
G
Balesar
R
van Dongen
JW
Kruse
CG
Swaab
DF
Verhaagen
J
Analysis of gene expression in Parkinson's disease: possible involvement of neurotrophic support and axon guidance in dopaminergic cell death
Brain Pathol
 , 
2009
, vol. 
19
 (pg. 
91
-
107
)
Bossers
K
Wirz
KT
Meerhoff
GF
Essing
AH
van Dongen
JW
Houba
P
Kruse
CG
Verhaagen
J
Swaab
DF
Concerted changes in transcripts in the prefrontal cortex precede neuropathology in Alzheimer's disease
Brain
 , 
2010
, vol. 
133
 (pg. 
3699
-
3723
)
Casamenti
F
Prosperi
C
Scali
C
Giovannelli
L
Colivicchi
MA
Faussone-Pellegrini
MS
Pepeu
G
Interleukin-1beta activates forebrain glial cells and increases nitric oxide production and cortical glutamate and GABA release in vivo: implications for Alzheimer's disease
Neuroscience
 , 
1999
, vol. 
91
 (pg. 
831
-
842
)
Chapleau
MW
Abboud
FM
Modulation of baroreceptor activity by ionic and paracrine mechanisms: an overview
Braz J Med Biol Res
 , 
1994
, vol. 
27
 (pg. 
1001
-
1015
)
Corasaniti
MT
Paoletti
AM
Palma
E
Granato
T
Navarra
M
Nistico
G
Systemic administration of pramiracetam increases nitric oxide synthase activity in the cerebral cortex of the rat
Funct Neurol
 , 
1995
, vol. 
10
 (pg. 
151
-
155
)
Cotter
D
Mackay
D
Landau
S
Kerwin
R
Everall
I
Reduced glial cell density and neuronal size in the anterior cingulate cortex in major depressive disorder
Arch Gen Psychiatry
 , 
2001
, vol. 
58
 (pg. 
545
-
553
)
de Bustos
F
Navarro
JA
de Andres
C
Molina
JA
Jimenez-Jimenez
FJ
Orti-Pareja
M
Gasalla
T
Tallon-Barranco
A
Martinez-Salio
A
Arenas
J
Cerebrospinal fluid nitrate levels in patients with multiple sclerosis
Eur Neurol
 , 
1999
, vol. 
41
 (pg. 
44
-
47
)
Drevets
WC
Price
JL
Furey
ML
Brain structural and functional abnormalities in mood disorders: implications for neurocircuitry models of depression
Brain Struct Funct
 , 
2008
, vol. 
213
 (pg. 
93
-
118
)
Drevets
WC
Price
JL
Simpson
JR
Jr
Todd
RD
Reich
T
Vannier
M
Raichle
ME
Subgenual prefrontal cortex abnormalities in mood disorders
Nature
 , 
1997
, vol. 
386
 (pg. 
824
-
827
)
Drevets
WC
Savitz
J
Trimble
M
The subgenual anterior cingulate cortex in mood disorders
CNS Spectr
 , 
2008
, vol. 
13
 (pg. 
663
-
681
)
Egberongbe
YI
Gentleman
SM
Falkai
P
Bogerts
B
Polak
JM
Roberts
GW
The distribution of nitric oxide synthase immunoreactivity in the human brain
Neuroscience
 , 
1994
, vol. 
59
 (pg. 
561
-
578
)
Elliott
R
Zahn
R
Deakin
JF
Anderson
IM
Affective cognition and its disruption in mood disorders
Neuropsychopharmacology
 , 
2011
, vol. 
36
 (pg. 
153
-
182
)
Ewald
H
Degn
B
Mors
O
Kruse
TA
Significant linkage between bipolar affective disorder and chromosome 12q24
Psychiatr Genet
 , 
1998
, vol. 
8
 (pg. 
131
-
140
)
Fallin
MD
Lasseter
VK
Avramopoulos
D
Nicodemus
KK
Wolyniec
PS
McGrath
JA
Steel
G
Nestadt
G
Liang
KY
Huganir
RL
Bipolar I disorder and schizophrenia: a 440-single-nucleotide polymorphism screen of 64 candidate genes among Ashkenazi Jewish case-parent trios
Am J Hum Genet
 , 
2005
, vol. 
77
 (pg. 
918
-
936
)
Feyissa
AM
Chandran
A
Stockmeier
CA
Karolewicz
B
Reduced levels of NR2A and NR2B subunits of NMDA receptor and PSD-95 in the prefrontal cortex in major depression
Prog Neuropsychopharmacol Biol Psychiatry
 , 
2009
, vol. 
33
 (pg. 
70
-
75
)
Fitzgerald
PB
Daskalakis
ZJ
A practical guide to the use of repetitive transcranial magnetic stimulation in the treatment of depression
Brain Stimul
 , 
2011
, vol. 
5
 (pg. 
287
-
296
)
Fleige
S
Pfaffl
MW
RNA integrity and the effect on the real-time qRT-PCR performance
Mol Aspects Med
 , 
2006
, vol. 
27
 (pg. 
126
-
139
)
Fronczek
R
Overeem
S
Lee
SY
Hegeman
IM
van Pelt
J
van Duinen
SG
Lammers
GJ
Swaab
DF
Hypocretin (orexin) loss in Parkinson's disease
Brain
 , 
2007
, vol. 
130
 (pg. 
1577
-
1585
)
Frye
MA
Tsai
GE
Huggins
T
Coyle
JT
Post
RM
Low cerebrospinal fluid glutamate and glycine in refractory affective disorder
Biol Psychiatry
 , 
2007
, vol. 
61
 (pg. 
162
-
166
)
Galecki
P
Maes
M
Florkowski
A
Lewinski
A
Galecka
E
Bienkiewicz
M
Szemraj
J
Association between inducible and neuronal nitric oxide synthase polymorphisms and recurrent depressive disorder
J Affect Disord
 , 
2011
, vol. 
129
 (pg. 
175
-
182
)
Gao
SF
Bao
AM
Corticotropin-releasing hormone, glutamate, and gamma-aminobutyric acid in depression
Neuroscientist
 , 
2011
, vol. 
17
 (pg. 
124
-
144
)
Gingerich
S
Krukoff
TL
Activation of ERbeta increases levels of phosphorylated nNOS and NO production through a Src/PI3K/Akt-dependent pathway in hypothalamic neurons
Neuropharmacology
 , 
2008
, vol. 
55
 (pg. 
878
-
885
)
Green
LC
Wagner
DA
Glogowski
J
Skipper
PL
Wishnok
JS
Tannenbaum
SR
Analysis of nitrate, nitrite, and [15N]nitrate in biological fluids
Anal Biochem
 , 
1982
, vol. 
126
 (pg. 
131
-
138
)
Griffith
OW
Stuehr
DJ
Nitric oxide synthases: properties and catalytic mechanism
Annu Rev Physiol
 , 
1995
, vol. 
57
 (pg. 
707
-
736
)
Hasler
G
van der Veen
JW
Tumonis
T
Meyers
N
Shen
J
Drevets
WC
Reduced prefrontal glutamate/glutamine and gamma-aminobutyric acid levels in major depression determined using proton magnetic resonance spectroscopy
Arch Gen Psychiatry
 , 
2007
, vol. 
64
 (pg. 
193
-
200
)
Hyman
BT
Marzloff
K
Wenniger
JJ
Dawson
TM
Bredt
DS
Snyder
SH
Relative sparing of nitric oxide synthase-containing neurons in the hippocampal formation in Alzheimer's disease
Ann Neurol
 , 
1992
, vol. 
32
 (pg. 
818
-
820
)
Jhanwar
VG
Bishnoi
RJ
Jhanwar
MR
Utility of repetitive transcranial stimulation as an augmenting treatment method in treatment-resistant depression
Indian J Psychol Med
 , 
2011
, vol. 
33
 (pg. 
92
-
96
)
Jopek
R
Kata
M
Nowak
G
The activity of rat brain nitric oxide synthase following chronic antidepressant treatment
Acta Pol Pharm
 , 
1999
, vol. 
56
 (pg. 
307
-
310
)
Judas
M
Sestan
N
Kostovic
I
Nitrinergic neurons in the developing and adult human telencephalon: transient and permanent patterns of expression in comparison to other mammals
Microsc Res Tech
 , 
1999
, vol. 
45
 (pg. 
401
-
419
)
Kamphuis
W
Schneemann
A
van Beek
LM
Smit
AB
Hoyng
PF
Koya
E
Prostanoid receptor gene expression profile in human trabecular meshwork: a quantitative real-time PCR approach
Invest Ophthalmol Vis Sci
 , 
2001
, vol. 
42
 (pg. 
3209
-
3215
)
Kang
YM
He
RL
Yang
LM
Qin
DN
Guggilam
A
Elks
C
Yan
N
Guo
Z
Francis
J
Brain tumour necrosis factor-alpha modulates neurotransmitters in hypothalamic paraventricular nucleus in heart failure
Cardiovasc Res
 , 
2009
, vol. 
83
 (pg. 
737
-
746
)
Karolewicz
B
Szebeni
K
Stockmeier
CA
Konick
L
Overholser
JC
Jurjus
G
Roth
BL
Ordway
GA
Low nNOS protein in the locus coeruleus in major depression
J Neurochem
 , 
2004
, vol. 
91
 (pg. 
1057
-
1066
)
Koenigs
M
Grafman
J
The functional neuroanatomy of depression: distinct roles for ventromedial and dorsolateral prefrontal cortex
Behav Brain Res
 , 
2009
, vol. 
201
 (pg. 
239
-
243
)
Kwan
KY
Lam
MM
Johnson
MB
Dube
U
Shim
S
Rasin
MR
Sousa
AM
Fertuzinhos
S
Chen
JG
Arellano
JI
Species-dependent posttranscriptional regulation of NOS1 by FMRP in the developing cerebral cortex
Cell
 , 
2012
, vol. 
149
 (pg. 
899
-
911
)
Luciano
M
Huffman
JE
Arias-Vasquez
A
Vinkhuyzen
AA
Middeldorp
CM
Giegling
I
Payton
A
Davies
G
Zgaga
L
Janzing
J
Genome-wide association uncovers shared genetic effects among personality traits and mood states
Am J Med Genet B Neuropsychiatr Genet
 , 
2012
, vol. 
159B
 (pg. 
684
-
695
)
McGuffin
P
Knight
J
Breen
G
Brewster
S
Boyd
PR
Craddock
N
Gill
M
Korszun
A
Maier
W
Middleton
L
Whole genome linkage scan of recurrent depressive disorder from the depression network study
Hum Mol Genet
 , 
2005
, vol. 
14
 (pg. 
3337
-
3345
)
Meyer
JH
Ginovart
N
Boovariwala
A
Sagrati
S
Hussey
D
Garcia
A
Young
T
Praschak-Rieder
N
Wilson
AA
Houle
S
Elevated monoamine oxidase a levels in the brain: an explanation for the monoamine imbalance of major depression
Arch Gen Psychiatry
 , 
2006
, vol. 
63
 (pg. 
1209
-
1216
)
Miller
KJ
Hoffman
BJ
Adenosine A3 receptors regulate serotonin transport via nitric oxide and cGMP
J Biol Chem
 , 
1994
, vol. 
269
 (pg. 
27351
-
27356
)
Milstien
S
Sakai
N
Brew
BJ
Krieger
C
Vickers
JH
Saito
K
Heyes
MP
Cerebrospinal fluid nitrite/nitrate levels in neurologic diseases
J Neurochem
 , 
1994
, vol. 
63
 (pg. 
1178
-
1180
)
Mirza
Y
Tang
J
Russell
A
Banerjee
SP
Bhandari
R
Ivey
J
Rose
M
Moore
GJ
Rosenberg
DR
Reduced anterior cingulate cortex glutamatergic concentrations in childhood major depression
J Am Acad Child Adolesc Psychiatry
 , 
2004
, vol. 
43
 (pg. 
341
-
348
)
Morissette
J
Villeneuve
A
Bordeleau
L
Rochette
D
Laberge
C
Gagne
B
Laprise
C
Bouchard
G
Plante
M
Gobeil
L
Genome-wide search for linkage of bipolar affective disorders in a very large pedigree derived from a homogeneous population in quebec points to a locus of major effect on chromosome 12q23-q24
Am J Med Genet
 , 
1999
, vol. 
88
 (pg. 
567
-
587
)
Mufson
EJ
Brandabur
MM
Sparing of NADPH-diaphorase striatal neurons in Parkinson's and Alzheimer's diseases
Neuroreport
 , 
1994
, vol. 
5
 (pg. 
705
-
708
)
Narushima
K
Kosier
JT
Robinson
RG
A reappraisal of poststroke depression, intra- and inter-hemispheric lesion location using meta-analysis
J Neuropsychiatry Clin Neurosci
 , 
2003
, vol. 
15
 (pg. 
422
-
430
)
Ohyu
J
Takashima
S
Developmental characteristics of neuronal nitric oxide synthase (nNOS) immunoreactive neurons in fetal to adolescent human brains
Brain Res Dev Brain Res
 , 
1998
, vol. 
110
 (pg. 
193
-
202
)
Quide
Y
Witteveen
AB
El-Hage
W
Veltman
DJ
Olff
M
Differences between effects of psychological versus pharmacological treatments on functional and morphological brain alterations in anxiety disorders and major depressive disorder: a systematic review
Neurosci Biobehav Rev
 , 
2011
, vol. 
36
 (pg. 
626
-
644
)
Rajkowska
G
Halaris
A
Selemon
LD
Reductions in neuronal and glial density characterize the dorsolateral prefrontal cortex in bipolar disorder
Biol Psychiatry
 , 
2001
, vol. 
49
 (pg. 
741
-
752
)
Rajkowska
G
Miguel-Hidalgo
JJ
Wei
J
Dilley
G
Pittman
SD
Meltzer
HY
Overholser
JC
Roth
BL
Stockmeier
CA
Morphometric evidence for neuronal and glial prefrontal cell pathology in major depression
Biol Psychiatry
 , 
1999
, vol. 
45
 (pg. 
1085
-
1098
)
Ramirez
J
Garnica
R
Boll
MC
Montes
S
Rios
C
Low concentration of nitrite and nitrate in the cerebrospinal fluid from schizophrenic patients: a pilot study
Schizophr Res
 , 
2004
, vol. 
68
 (pg. 
357
-
361
)
Reif
A
Strobel
A
Jacob
CP
Herterich
S
Freitag
CM
Topner
T
Mossner
R
Fritzen
S
Schmitt
A
Lesch
KP
A NOS-III haplotype that includes functional polymorphisms is associated with bipolar disorder
Int J Neuropsychopharmacol
 , 
2006
, vol. 
9
 (pg. 
13
-
20
)
Rejdak
K
Petzold
A
Stelmasiak
Z
Giovannoni
G
Cerebrospinal fluid brain specific proteins in relation to nitric oxide metabolites during relapse of multiple sclerosis
Mult Scler
 , 
2008
, vol. 
14
 (pg. 
59
-
66
)
Resstel
LB
Correa
FM
Guimaraes
FS
The expression of contextual fear conditioning involves activation of an NMDA receptor-nitric oxide pathway in the medial prefrontal cortex
Cereb Cortex
 , 
2008
, vol. 
18
 (pg. 
2027
-
2035
)
Roenker
NL
Gudelsky
GA
Ahlbrand
R
Horn
PS
Richtand
NM
Evidence for involvement of nitric oxide and GABA(B) receptors in MK-801-stimulated release of glutamate in rat prefrontal cortex
Neuropharmacology
 , 
2012
, vol. 
63
 (pg. 
575
-
581
)
Roy
M
Harvey
PO
Berlim
MT
Mamdani
F
Beaulieu
MM
Turecki
G
Lepage
M
Medial prefrontal cortex activity during memory encoding of pictures and its relation to symptomatic improvement after citalopram treatment in patients with major depression
J Psychiatry Neurosci
 , 
2010
, vol. 
35
 (pg. 
152
-
162
)
Savidge
TC
S-nitrosothiol signals in the enteric nervous system: lessons learnt from big brother
Front Neurosci
 , 
2011
, vol. 
5
 pg. 
31
 
Segovia
G
Porras
A
Mora
F
Effects of a nitric oxide donor on glutamate and GABA release in striatum and hippocampus of the conscious rat
Neuroreport
 , 
1994
, vol. 
5
 (pg. 
1937
-
1940
)
Sestan
N
Kostovic
I
Histochemical localization of nitric oxide synthase in the CNS
Trends Neurosci
 , 
1994
, vol. 
17
 (pg. 
105
-
106
)
Shan
L
Bossers
K
Luchetti
S
Balesar
R
Lethbridge
N
Chazot
PL
Bao
AM
Swaab
DF
Alterations in the histaminergic system in the substantia nigra and striatum of Parkinson's patients: a postmortem study
Neurobiol Aging
 , 
2011
, vol. 
33
 (pg. 
1488.e1
-
1488.e13
)
Silberberg
G
Ben-Shachar
D
Navon
R
Genetic analysis of nitric oxide synthase 1 variants in schizophrenia and bipolar disorder
Am J Med Genet B Neuropsychiatr Genet
 , 
2010
, vol. 
153B
 (pg. 
1318
-
1328
)
Swaab
DF
Fliers
E
Hoogendijk
WJ
Veltman
DJ
Zhou
JN
Interaction of prefrontal cortical and hypothalamic systems in the pathogenesis of depression
Prog Brain Res
 , 
2000
, vol. 
126
 (pg. 
369
-
396
)
Tohgi
H
Abe
T
Yamazaki
K
Murata
T
Isobe
C
Ishizaki
E
The cerebrospinal fluid oxidized NO metabolites, nitrite and nitrate, in Alzheimer's disease and vascular dementia of Binswanger type and multiple small infarct type
J Neural Transm
 , 
1998
, vol. 
105
 (pg. 
1283
-
1291
)
van de Nes
JA
Kamphorst
W
Ravid
R
Swaab
DF
Comparison of beta-protein/A4 deposits and Alz-50-stained cytoskeletal changes in the hypothalamus and adjoining areas of Alzheimer's disease patients: amorphic plaques and cytoskeletal changes occur independently
Acta Neuropathol
 , 
1998
, vol. 
96
 (pg. 
129
-
138
)
Vandesompele
J
De Preter
K
Pattyn
F
Poppe
B
Van Roy
N
De Paepe
A
Speleman
F
Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes
Genome Biol
 , 
2002
, vol. 
3
  
RESEARCH0034.1?12
Wang
SS
Kamphuis
W
Huitinga
I
Zhou
JN
Swaab
DF
Gene expression analysis in the human hypothalamus in depression by laser microdissection and real-time PCR: the presence of multiple receptor imbalances
Mol Psychiatry
 , 
2008
, vol. 
13
 (pg. 
786
-
799
741
Xing
G
Chavko
M
Zhang
LX
Yang
S
Post
RM
Decreased calcium-dependent constitutive nitric oxide synthase (cNOS) activity in prefrontal cortex in schizophrenia and depression
Schizophr Res
 , 
2002
, vol. 
58
 (pg. 
21
-
30
)
Yan
XX
Jen
LS
Garey
LJ
NADPH-diaphorase-positive neurons in primate cerebral cortex colocalize with GABA and calcium-binding proteins
Cereb Cortex
 , 
1996
, vol. 
6
 (pg. 
524
-
529
)
Yao
JK
Leonard
S
Reddy
RD
Increased nitric oxide radicals in postmortem brain from patients with schizophrenia
Schizophr Bull
 , 
2004
, vol. 
30
 (pg. 
923
-
934
)
Yew
DT
Wong
HW
Li
WP
Lai
HW
Yu
WH
Nitric oxide synthase neurons in different areas of normal aged and Alzheimer's brains
Neuroscience
 , 
1999
, vol. 
89
 (pg. 
675
-
686
)
Yu
YW
Chen
TJ
Wang
YC
Liou
YJ
Hong
CJ
Tsai
SJ
Association analysis for neuronal nitric oxide synthase gene polymorphism with major depression and fluoxetine response
Neuropsychobiology
 , 
2003
, vol. 
47
 (pg. 
137
-
140
)
Zhao
J
Bao
AM
Qi
XR
Kamphuis
W
Luchetti
S
Lou
JS
Swaab
DF
Gene expression of GABA and glutamate pathway markers in the prefrontal cortex of non-suicidal elderly depressed patients
J Affect Disord
 , 
2012
, vol. 
138
 (pg. 
494
-
502
)