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Tsung-Ung W. Woo, MD, PhD; John P. Walsh, MS; Francine M. Benes, MD, PhD

Arch Gen Psychiatry. 2004;61:649-657.

ABSTRACT
Background  Disturbances of -aminobutyric acid interneurons in the cerebral cortex contribute to the pathophysiology of schizophrenia and bipolar disorder. The activity of these neurons is, in turn, modulated by glutamatergic inputs furnished by pyramidal neurons.

Objective  To test the hypothesis that glutamatergic inputs onto -aminobutyric acid interneurons via the N-methyl-D-aspartate (NMDA) receptor are altered in the anterior cingulate cortex in schizophrenia and bipolar disorder.

Design  A double in situ hybridization technique was used to simultaneously label the messenger RNA (mRNA) for the NMDA NR_2A subunit with ³⁵sulfur and the mRNA for the 67-kDa isoform of the -aminobutyric acid synthesizing enzyme glutamic acid decarboxylase (GAD₆₇) with digoxigenin.

Setting  Postmortem human brain studies.

Participants  We studied 17 subjects with schizophrenia, 17 subjects with bipolar disorder, and 17 normal control subjects.

Results  The density of all GAD₆₇ mRNA–containing neurons was decreased by 53% and 28%, in layers 2 and 5, respectively, in subjects with schizophrenia, whereas in subjects with bipolar disorder there was a 35% reduction in layer 2 only. For GAD₆₇ mRNA–containing neurons that co-expressed NR_2AmRNA, their numerical density was decreased by 73% and 52%, in layers 2 and 5, respectively, in subjects with schizophrenia and by 60% in layer 2 in those with bipolar disorder. In the schizophrenia group, the density of the GAD₆₇mRNA–containing neurons that did not co-express NR_2AmRNA was also decreased by 42% in layer 2. In both disease groups, the expression level of NR_2AmRNA in GAD₆₇ mRNA–containing cells was unaltered.

Conclusions  The density of -aminobutyric acid interneurons that express the NMDA NR_2Asubunit appears to be decreased in schizophrenia and bipolar disorder. Future studies will address whether subpopulations of these neurons may be differentially affected in the 2 conditions.

INTRODUCTION

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The anterior cingulate cortex (ACCx) (Brodmann's area 24) is a key region of the large-scale neural network that comprises the limbic system, the dorsolateral prefrontal region, and the motor and premotor cortices. This distributed neural system mediates a wide range of functions, such as affective regulation, motivation, selective attention, separation calls, executive control, and the dynamic orchestration of motor programming. The ACCx is involved in mediating these functions via postulated capabilities, such as error detection and conflict monitoring.^1-5 Because perturbations of many aspects of these functions are commonly seen in schizophrenia and bipolar disorder, it is perhaps not surprising that converging lines of evidence from postmortem and neuroimaging studies^6-11 have consistently demonstrated that the ACCx is structurally and functionally altered in these disorders.

-Aminobutyric acid (GABA) interneurons play an important role in information processing in the cerebral cortex. Disturbances of these neurons have been strongly implicated in the pathophysiology of schizophrenia.^12-19 Increasing, albeit still somewhat limited, evidence^{7, 20-23} suggests that GABAergic function may also be perturbed in bipolar disorder. In fact, some of the parameters of GABA neurotransmission that have been examined seem to be even more severely altered in subjects with bipolar disorder than in those with schizophrenia. For example, it has been shown in the ACCx that the density of cells with a nonpyramidal shape, putative GABA interneurons, and terminals is decreased in schizophrenia and bipolar disorder, but the extent of this reduction is considerably greater in the latter condition.^{7, 21} In addition, GAD₆₅-immunoreactive terminals have also been found to be substantially reduced in the ACCx of bipolar, but not schizophrenic, subjects.²⁴

Converging lines of clinical and preclinical observations^25-29 strongly suggest that disturbances of glutamatergic neurotransmission contribute to the pathophysiology of schizophrenia. Furthermore, it has been postulated, largely based on animal studies, that such disturbances may involve hypofunctioning of N-methyl-D-aspartate (NMDA) receptors on GABA interneurons.^30-31 The NMDA receptor complex is a heteromeric structure composed of different subunits. Among them, the NR_2A subunit is abundantly present in the adult cerebral cortex. It has also been implicated in the pathophysiology of schizophrenia. For example, mice lacking the NR_2A subunit demonstrated an increase in the release of dopamine in the striatum.³² Behaviorally, these animals exhibited hyperlocomotion, which could be attenuated by treatment with antipsychotic agents. Furthermore, NMDA-mediated GABA release in these animals was markedly decreased. In this study, as a first step to explore the question of whether glutamatergic innervation of GABA interneurons via the NMDA receptor may be altered in schizophrenia and bipolar disorder, we used a double in situ hybridization procedure to simultaneously examine the expression of messenger RNA (mRNA) for the NR_2Asubunit, labeled with ³⁵sulfur ([³⁵S]), in cells containing GAD₆₇ mRNA, labeled with digoxigenin (DIG), in the ACCx from normal control, schizophrenic, and bipolar subjects.

METHODS

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SUBJECTS

A cohort of 51 human brains obtained from the Harvard Brain Tissue Resource Center at McLean Hospital was used in this study and included 17 normal controls, 17 subjects with schizophrenia, and 17 subjects with bipolar disorder (Table 1). Each of the schizophrenic subjects was matched to a subject with bipolar disorder and to a normal control subject on the basis of age, postmortem interval, and, whenever possible, sex, hemisphere (ie, right vs left), and pH. The female-male ratio was 7:10 for the bipolar disorder group and 8:9 for the schizophrenia and normal control groups. The right hemisphere–left hemisphere ratio was 10:7 for the schizophrenia group and 8:9 for the bipolar disorder and normal control groups. The mean ± SD freezer storage time of brains was not significantly different among the normal control (1391 ± 1012 days), schizophrenia (1766 ± 958 days), and bipolar disorder (1847 ± 1084 days) groups (F_2,48 = 0.97; P = .39). Measurements of tissue pH were available for 12 of 17 cases in the schizophrenia and bipolar disorder groups and for 15 of 17 cases in the normal control group. The mean ± SD pH was not different among the 3 groups (normal control group: 6.52 ± 0.27; schizophrenia group: 6.54 ± 0.31; bipolar disorder group: 6.45 ± 0.24).

View this table: [in this window] [in a new window] Characteristics of the 51 Subjects in the Present Study

Psychiatric diagnoses were established using a retrospective review of medical records and an extensive family questionnaire that included the medical, psychiatric, and social history of the subjects. For the diagnosis of schizophrenia, the criteria of Feighner et al³³ were used, and the diagnoses of schizoaffective and bipolar disorder were made according to DSM-III-R criteria. Of the 17 schizophrenic subjects, 3 (cases B2166, B4875, and B4907) had a diagnosis of schizoaffective disorder, whereas the remaining cases has a diagnosis of schizophrenia. Three of the 17 schizophrenic subjects (cases B3146, B4875, and B4256) were not taking antipsychotic medications at the time of death. In the bipolar disorder group, 9 subjects were taking antipsychotic medications at the time of death. The dose of antipsychotic drugs that subjects with bipolar disorder (267.4 ± 383.5 mg) were receiving (expressed as chlorpromazine-equivalent dose) was less than half that of the schizophrenia group (618.3 ± 809.7 mg). Some subjects in both disease groups were also taking concomitant psychotropic medications, such as mood stabilizers, antidepressants, or anxiolytics (Table 1). No subject in the normal control group was receiving any psychotropic agents at the time of death.

TISSUE PREPARATION

Tissue blocks (3 mm thick) from Brodmann's area 24 were removed from fresh brain specimens at the level of the rostrum of the anterior cingulate gyrus between the points at which the gyrus curves above and below the corpus callosum.⁷ The blocks were immediately fixed in 0.1% paraformaldehyde in ice-cold 0.1M phosphate buffer (pH 7.4) for 90 minutes, immersed in 30% sucrose in the same buffer overnight, and then frozen in Tissue-Tek OCT embedding meduim for frozen tissue (Sakura Finetek USA Inc, Torrance, Calif) on dry ice. Tissue blocks were then sectioned at a thickness of 10 µm on a cryostat. Two sections per subject and therefore 6 sections per matched triplet were used for in situ hybridization. The 6 sections from each triplet were mounted on 3 slides as follows: (1) normal control + schizophrenia, (2) normal control + bipolar disorder, and (3) schizophrenia + bipolar disorder. This method of mounting sections controls for potential variability of hybridization signals between slides. All mounted sections were stored at –70°C until riboprobe labeling was performed.

DOUBLE IN SITU HYBRIDIZATION

Riboprobe Preparation

Radiolabeled Complementary RNA Probe for NR_2A mRNA. The complementary RNA (cRNA) probes for the NR_2A subunit (provided by Christine Konradi, PhD) were transcribed in vitro from linearized complementary DNA (cDNA) subclones encoding the rat NMDA NR_2A subunit. The specificity of the probe was verified by Northern blot analysis (data not shown). The probe was derived from a cDNA spanning nucleotides 1185 to 2154 (GenBank Accession No. M91561) within the coding region of the subunit. A corresponding sense probe was used as a control. Radiolabeled cRNA probe was prepared by first drying down [³⁵S]UTP (500 µCi/mL of probe, PerkinElmer Life and Analytical Sciences Inc, Boston, Mass) in a DNA speed vac (Savant, Farmingdale, NY); 100 ng/µL of the cDNA template, 0.1M dithiothreitol, 3 U/µL of RNasin, 5mM NTPs, 0.8 U/µL of T3 or T7 RNA polymerases (for antisense and sense probes, respectively), and 5x transcription buffer were then added. The transcription mixture was subsequently incubated at 37°C for 1 hour. The cDNA template was digested by incubating the mixture with R1Q DNase at 37°C for 15 minutes. Unincorporated NTPs were removed by running the mixture through a push column (NucTrap; Stratagene, La Jolla, Calif). The eluate was collected, and probe concentration was determined by scintillation counting. The probe was stored at –20°C until use.

DIG-Labeled GAD₆₇ mRNA Probe. The DIG-UTP–labeled cRNA probes were transcribed using 100 ng of full-length, linearized human cDNA clones inserted in a bluescript vector (provided by Allan Tobin, PhD, and Niranjula Tillakarantne, PhD, Department of Physiological Sciences, University of California at Los Angeles) in the presence of 0.1M dithiothreitol; 3 U/µL of RNasin; 0.8 U/µL of T3 and T7 RNA polymerases; 10mM ATP, CTP, and GTP; 6.5mM UTP; and 3.5mM DIG-labeled UTP (Boehringer Mannheim, Indianapolis, Ind). The mixture was incubated at 37°C for 1 hour. The cDNA template was digested with RQ1 DNase. Probe concentration was determined using a standard with known concentrations.

Hybridization

To ensure adequate tissue penetration, the GAD₆₇ probe was hydrolyzed to 0.8 kilobase (kb) with an equal volume of sodium bicarbonate–sodium carbonate buffer (pH 10.2; 40mM sodium bicarbonate and 60mM sodium carbonate) at 60°C for 6 to 10 minutes. The reaction was stopped by adding 0.08 vol of 2M sodium acetate in 6.25% glacial acetic acid. Probes were then reconstituted in a hybridization buffer consisting of 50% formamide, 0.1% yeast transfer ribonucleic acid, 10% dextran sulfate, 1x Denhardt solution, 0.5M EDTA, 0.02% sodium dodecyl sulfate, 4x isotonic sodium chloride solution–sodium citrate buffer, 10mM dithiothreitol, and 0.1% single-stranded DNA at a final concentration of 0.4 ng probe/µL hybridization buffer. Before hybridization, mounted tissue sections were air-dried and warmed to room temperature. They were then postfixed in 4% paraformaldehyde for 10 minutes and incubated in 0.1M tetraethylammonium for 5 minutes at room temperature before being dehydrated in a graded series of ethanol. Probes were then added to slides for hybridization in a prewarmed, humidified dish. Sections were covered with coverslips and incubated at 55°C for 3 hours. At the end of hybridization, coverslips were soaked off in 4x isotonic sodium chloride solution–sodium citrate in the presence of 100 µL of ßMer alcohol. Tissue was then incubated in 0.5M sodium chloride/0.05M phosphate buffer for 10 minutes, 0.5M sodium chloride with 0.025 mg/mL of ribonuclease (pancreatic) at 37°C for 30 minutes, followed by a high-stringency wash with a solution containing 50% formamide, 0.5M sodium chloride/0.05M phosphate buffer, and 100 µL of ßMer at 63°C for 30 minutes. Sections were finally washed overnight in 0.5x isotonic sodium chloride solution–sodium citrate with 20mM ßMer alcohol at room temperature.

Visualization of DIG Labeling

After incubation in blocking buffer (100mM Tris hydrochloride, 150mM sodium chloride [pH 7.5], 3% normal goat serum, and 0.3% Triton X-100), sections were incubated overnight at 4°C in buffer containing 1:200 dilution of alkaline phosphatase–conjugated sheep -DIG antibody (Roche Diagnostics, Indianapolis). Sections were then incubated in an alkaline phosphatase substrate (Vector Red; Vector Laboratories, Burlingame, Calif), at room temperature for 40 minutes in complete darkness.

Emulsion Autoradiography

It was determined that sufficient autoradiographic signal had developed after the slides were apposed to x-ray film (Kodak BioMax MS; Kodak, Rochester, NY) for 10 days. The slides were then dipped in emulsion (Kodak NTB-2; Kodak), air-dried, and stored at 4°C in darkness for 5 weeks. After development in the dark with developer (Kodak D-19; Kodak), slides were counterstained with methyl green and coverslipped.

Quantification of GAD₆₇ and NR_2A mRNA Expression

All microscopic analyses were conducted under strictly blind conditions. [³⁵S] labeling of NR_2A mRNA appeared as clusters of silver grains after processing for emulsion autoradiography (Figure 1). After counterstaining with Vector Red, DIG labeling can be visualized as a red-brown reaction product under a brightfield microscope or as a fluorescent emission in the red range. Neurons that were single labeled with DIG (Figure 1A) and those that were double labeled with DIG and [³⁵S] (Figure 1B) were identified on images captured on a computer screen using a microscope (Laborlux; Leica Microsystems, Wetzlar, Germany), which was equipped with a solid-state charge-coupled device video camera connected to an image analysis system (Bioquant Nova; R&M Biometrics, Memphis, Tenn). Using a 100x oil immersion objective lens at a final magnification of x1000, the distributions of single- and double-labeled neurons in a 250-µm-wide column extending from the pial surface to the border between layer 6 and the subcortical white matter were obtained for each section. Neighboring sections were stained with cresyl violet for determination of laminar boundaries. Densities of single- and double-labeled neurons for each cortical layer were then obtained by dividing cell counts by laminar areas. Intrarater reliability, as assessed by counting and recounting profiles in the same column, was established to be 93% to 97% before the actual data collection process began.

View larger version (223K): [in this window] [in a new window] Figure 1. Photomicrographs showing examples of digoxigenin single-labeled neurons (arrows) (A) and 35 sulfur/digoxigenin double-labeled neurons (arrows) (B). Scale bar = 10 µm.

To quantify the expression level of mRNA for the NR_2A subunit in individual GABA cells, the area occupied by each grain cluster was outlined using a cursor displayed on the computer monitor. For each cluster, this quantification was performed according to the principle of including the largest number of grains in the smallest possible area. The cluster area was measured by highlighting the grains with a thresholding subroutine. This threshold was held constant, and the light intensity was adjusted to ensure that the size of the grains was neither underrepresented nor overrepresented. This procedure was consistently followed throughout the entire study. The area covered by autoradiographic grains in the cluster area was automatically computed based on the threshold value and was represented as a pixel count for NR_2A transcript expression level. The pixel count was expressed as a function of cluster area (numerical density). By subtracting the background grain density (ie, pixel count of the area covered by autoradiographic grains per unit area in square micrometers in the white matter), the corrected NR_2A expression level was obtained. The average NR_2A expression level in GABA interneurons (ie, cells positive for GAD₆₇ mRNA) for each cortical layer for each case was then computed. Intrarater reliability in grain density measurements, which was accessed by repeating the procedures described previously herein on the same clusters, was determined to be consistently greater than 95% before the actual data collection process.

STATISTICAL ANALYSIS

The numerical densities of single-labeled (GAD₆₇ mRNA only) and double-labeled (GAD₆₇ and NR_2A mRNA) neurons and the amount of mRNA for the NR_2A subunit in GAD₆₇ mRNA–containing neurons were compared among groups across layers 2 through 6 using repeated-measures analysis of variance (ANOVA), with diagnosis (ie, schizophrenia vs control and bipolar disorder vs control) and layer as main effects. For post hoc analyses, 2-tailed paired t tests were used. The Bonferroni procedure was used to correct for type 1 error as a result of multiple comparisons (layers 2, 3, 5, and 6). Therefore, the level for significance for all t tests was P = .01 (ie, .05 ÷ 4). Layer 1 was not included in the analyses because there were no GAD₆₇ mRNA–containing neurons with co-expressed NR_2A subunit mRNA in this lamina. To evaluate the potential effects of confounding variables, such as age, sex, postmortem interval, brain pH, freezer storage time, hemispheric laterality, and exposure to antipsychotic medications (expressed as the chlorpromazine-equivalent dose), simple Pearson correlations were obtained for the individual groups and for the control group combined with the schizophrenia and bipolar disorder groups. In addition, an analysis of covariance (ANCOVA) was performed to understand how these confounding variables might have affected our results. Because none of the conclusions derived from our findings were affected by the ANCOVA analysis, only results from repeated-measures ANOVAs are reported. Effects of hemispheric laterality on our findings were evaluated by using 2-tailed unpaired t tests to compare the measures from the 2 hemispheres within individual groups and when cases from the control group were combined with those from the schizophrenia and bipolar disorder groups.

RESULTS

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DISTRIBUTION OF GAD₆₇ mRNA– AND GAD₆₇/NR_2A mRNA–EXPRESSING NEURONS IN THE ACCx

Neurons that express GAD₆₇ mRNA appeared to be distributed more or less evenly across all layers in the ACCx, except for layer 1, where the density of these neurons was low. In the entire population of GAD₆₇ mRNA–expressing neurons, those that co-expressed NR_2A mRNA seemed to be most concentrated in layers 3 to 5, whereas the density of these neurons seemed to be slightly lower in layers 2 and 6 (Figure 2).

View larger version (35K): [in this window] [in a new window] Figure 2. Plots of glutamic acid decarboxylase 67 (GAD67) messenger RNA (mRNA)–containing neurons that co-expressed and did not co-express NR2A mRNA from tissue sections from a normal control subject (case B4163) (A) and the matched subject with schizophrenia (case B2774) (B) and the matched subject with bipolar disorder (case B2565) (C).

DENSITY OF ALL NEURONS THAT EXPRESS GAD₆₇ mRNA

Overall, the repeated-measures ANOVA models revealed a significant diagnosis effect in the schizophrenia group (F_1,32 = 10.19; P = .003). Furthermore, this effect seemed to be layer specific (diagnosis x layer interaction, F = 3.27; P = .03). Thus, the density of GAD₆₇ mRNA–expressing neurons showed the most prominent change in layer 2 (Figure 3A), with a 53% reduction in the density of these neurons compared with control subjects (t = 4.41; P<.001). Besides layer 2, the density of these neurons was also significantly decreased in layer 5, although the magnitude of reduction (28%) was more modest (t = 2.45; P = .01). The observed decreases in the density of GAD₆₇ mRNA–expressing neurons did not seem to be artifactually related to differences in cortical thickness between the 2 groups because the mean ± SD thickness of the ACCx in the schizophrenia (1753.45 ± 269.1 µm) and control (1745.5 ± 267.4 µm) groups was not significantly different (t = 0.009; P = .93). In subjects with bipolar disorder, the repeated-measures ANOVA initially did not demonstrate a significant diagnosis effect (F_1,32 = 3.44; P = .07). On closer inspection of the data, it was noticed that the numerical density of the GAD₆₇ mRNA–expressing cells in layer 2 in a subject with bipolar disorder (patient B3817) was 3 SD above the mean density for that layer. This case and its matched control (case B5122) were, therefore, excluded from all of the numerical density comparisons between controls and subjects with bipolar disorder reported herein. Case B3817 was the only case in which the mean neuronal density in any layer was beyond 3 SD. After the removal of these 2 cases, the diagnosis effect in the repeated-measures ANOVA model was statistically significant (F_1,30 = 4.22; P = .04). When individual layers were examined, the density of GAD₆₇ mRNA–expressing cells in layer 2 was found to be significantly decreased by 35% in the bipolar disorder group (t = 4.12; P<.001). This reduction was smaller in magnitude than the 53% reduction observed in layer 2 in subjects with schizophrenia (Figure 3A). This apparent difference in the magnitude of reduction in the numerical density of neurons between the 2 subject groups was not statistically significant (t = 1.97, P = .06). Besides the reduction in layer 2, the numerical density of GAD₆₇ mRNA–expressing neurons was essentially unchanged in all other layers in subjects with bipolar disorder. As in the schizophrenia group, there was no statistically significant difference in mean ± SD cortical thickness between subjects with bipolar disorder (1755.3 ± 318.2 µm) and control subjects (1745.5 ± 267.4 µm) (t = 0.097; P = .92).

View larger version (100K): [in this window] [in a new window] Figure 3. Mean numerical density of all neurons that express glutamic acid decarboxylase 67 (GAD67) messenger RNA (mRNA) (A), neurons that co-express GAD67 mRNA and NR2A mRNA (B), and neurons that express GAD67 mRNA but not NR2A mRNA (C). Asterisk indicates a statistically significant difference at P = .01 vs controls. Error bars represent SEM.

DENSITY OF GAD₆₇ mRNA–CONTAINING NEURONS EXPRESSING NR_2A mRNA

The effect of diagnosis on NR_2A-expressing GAD₆₇-positive cells was statistically significant in the schizophrenia (F_1,32 = 8.97; P = .005) and bipolar disorder (F_1,32 = 4.42; P = .04) groups. In the schizophrenia group, neuronal density was significantly decreased by 73% (t = 3.08; P = .007) and 52% (t = 2.95; P = .009) in layers 2 and 5, respectively, whereas 37% and 40% reductions in layers 3 and 6, respectively, did not achieve statistical significance under the stringent Bonferroni correction (t = 2.62; P = .02 and t = 2.27; P = .04, respectively) (Figure 3B). In the bipolar disorder group, the numerical density of GAD₆₇-positive and NR_2A-positive cells was significantly decreased by 60% in layer 2 (t = 2.8; P = .01), but the decreases of 31%, 37%, and 29% in layers 3, 5, and 6, respectively, did not achieve statistical significance (t = 1.63; P = .12, t = 2.24; P = .04, and t = 1.54; P = .14, respectively) (Figure 3B).

DENSITY OF GAD₆₇ mRNA–CONTAINING NEURONS THAT DO NOT EXPRESS NR_2A mRNA

According to the repeated-measures ANOVA models, the effect of diagnosis was not statistically significant in subjects with either schizophrenia (F_1,32 = 1.38; P = .25) or bipolar disorder (F_1,30 = 0.038; P = .85). However, in the schizophrenia group, there was a significant diagnosis x layer effect (F_3,96 = 6.42; P<.001) that seemed to reflect the 42% decrease in neuronal density in layer 2 (Figure 3C), and this reduction was statistically significant (t = 2.99; P = .005).

EXPRESSION LEVEL OF NR_2A mRNA IN CELLS CONTAINING GAD₆₇ mRNA

There were no differences in the density of silver grains in either the schizophrenia (F_1,20 = 1.79; P = .20) or the bipolar disorder (F_1,24 = 0.21; P = .65) group, suggesting that for GABA interneurons that contained a detectable amount of NR_2A mRNA, the level of expression of the transcript was unaltered in either disease group (Figure 4).

View larger version (38K): [in this window] [in a new window] Figure 4. Mean density of silver grains over glutamic acid decarboxylase 67 (GAD67) messenger RNA (mRNA)–positive neurons in the anterior cingulate cortex is not different among the 3 study groups, indicating that the expression level of NR2A mRNA in GAD67 mRNA–positive cells is unchanged in the schizophrenia and bipolar disorder groups. Error bars represent SEM.

POTENTIAL CONFOUNDING VARIABLES

We examined the potential confounding effects of variables such as age, postmortem interval, brain pH, hemispheric laterality, and antipsychotic drug exposure on our findings. None of these factors seem to have affected our results (data not shown). Among these variables, pH was particularly important because the integrity of mRNA is known to be sensitive to this variable.^34-36 There was no statistically significant difference in pH in the 3 study groups. In the statistical analyses, we also found no correlation between pH and any of our measurements either in individual diagnostic groups or when subjects from the disease groups and those from the control group were combined. An ANCOVA incorporating pH as a covariate also did not statistically significantly alter the effect of diagnosis on the cell density and grain density measurements. Similar analyses with chlorpromazine-equivalent dose also revealed that exposure to antipsychotic medications was not statistically significantly correlated with any of our measurements, and neither did it contribute to the observed differences in the neuronal density measurements in the diagnostic groups. Therefore, these findings do not seem to be the result of antipsychotic medication treatment or any other measurable potential confounds but may in fact reflect the underlying disease processes.

COMMENT

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Multiple lines of evidence suggest that disturbances of GABA interneurons represent a key feature of the pathophysiology of schizophrenia and bipolar disorder.^{10, 12-17,19, 37-40} The activities of GABA interneurons are subject to feedback and feedforward modulation by glutamatergic inputs from pyramidal neurons located locally and in distant cortical or subcortical regions. Together, these mechanisms regulate the flow of information in the cerebral cortex by adjusting the spatial and temporal architecture of GABA neurotransmission.^41-43 In this study, we extended previous findings of altered GABAergic neurotransmission in schizophrenia and bipolar disorder to demonstrate that alteration in glutamatergic inputs onto GABA interneurons via the NMDA receptor may contribute to disturbances of GABA neurotransmission in both of these conditions. We cannot, however, exclude the possibility that there may be a primary dysregulation in the expression of the NR_2A subunit in a subgroup of GABA neurons.

In situ hybridization labeling with [³⁵S] is more sensitive in detecting transcript signals than nonradioactive DIG-labeled probes.⁴⁴ In the present study, because DIG was used to label the GAD₆₇ transcript, it is possible that we may have underestimated the true density of GABA interneurons in all 3 study groups. If the amount of GAD₆₇ transcript per GABA interneuron is equivalent in the 3 groups, our conclusions would not have been affected because they were based on the "relative" changes in neuronal density among the groups. On the other hand, it is possible that a subpopulation of GABA interneurons may in fact express a lower level of GAD₆₇ mRNA in subjects with schizophrenia and bipolar disorder and that these cells fell below the detection threshold for DIG labeling of GAD₆₇. This scenario could have contributed to the observed reduction in neuronal density in the schizophrenia and bipolar disorder groups compared with controls. Although we cannot rule out these possibilities, they seem to be insufficient to account for the magnitude of neuronal density reduction observed because DIG labeling of GAD₆₇ has been estimated to be only slightly (<7%) less sensitive than similar in situ hybridization labeling with [³⁵S].⁴⁴ An alternative possibility is that the number of GABA interneurons may be inherently different in the 2 disease groups and that this was reflected in the differences in neuronal density observed in this study (see the following paragraphs).

Our findings indicate that the decrease in the density of GAD₆₇ mRNA–expressing neurons may be more prominent in layer 2 than in other layers in the ACCx in schizophrenia and bipolar disorder. Thus, there was a 53% and a 35% reduction in the density of neurons that express GAD₆₇ mRNA in layer 2 in the schizophrenia and bipolar disorder groups, respectively, whereas there was no statistically significant reduction in the density of these neurons in other layers in either subject group, except for a 28% decrease in density in layer 5 in subjects with schizophrenia. These observations are consistent with findings from previous studies⁴⁵ suggesting that neural circuits in layer 2 in the ACCx may be a major site of disease vulnerability in schizophrenia and bipolar disorder. Because neurons in this layer receive extensive corticocortical projections from other regions of the cerebral cortex, such as the prefrontal cortex,⁴⁶ and they also receive specific inputs from subcortical and limbic structures, such as the amygdala,⁴⁷ they may play a critical role in integrating diverse streams of information derived from the cognitive and emotive domains. Therefore, disturbances of information processing in the neural circuits in this layer could contribute to the multitude of symptoms observed in schizophrenia and bipolar disorder.

The reduction in the density of neurons that express the mRNA for the 67-kDaisoform of GAD may represent a loss of neurons. Alternatively, the amount of GAD₆₇ mRNA in a subpopulation of GABA interneurons may be decreased to an experimentally undetectable level. Consideration of findings from previous studies of quantification of nonpyramidal, presumably GABA interneurons may provide some insights into the possible interpretations of the current findings. Data from these studies^{7, 48-49} demonstrate that the density of nonpyramidal neurons in layer 2 in the ACCx in bipolar disorder was decreased by 27% to 30%, whereas the magnitude of decrease in schizophrenia was only 16% to 17%. In bipolar disorder, the 35% reduction in the density of GAD₆₇ mRNA–containing neurons in layer 2 observed in the present study is similar to the degree of reduction in the density of nonpyramidal neurons previously reported.⁷ A recent study¹⁰ using immunohistochemical techniques to examine the expression of various calcium-binding proteins, which are differentially expressed by subpopulations of GABA interneurons,^50-53 has shown that the density of neurons that express calbindin was decreased by 33% and 34% in bipolar disorder and schizophrenia, respectively. In addition, the density of neurons that expressed parvalbumin also seemed to be reduced in the 2 disorders, although the differences did not achieve statistical significance. Furthermore, the magnitude of reduction in the densities of the calbindin- and parvalbumin-expressing neurons seems to be similar in bipolar disorder, and it is quantitatively almost identical to the decrease in the density of GAD₆₇mRNA–containing neurons reported in this study. Taken together, these findings raise the possibility that a subpopulation of GABA interneurons in layer 2, especially those that contain calbindin or parvalbumin, may indeed be lost in bipolar disorder. In schizophrenia, we observed a 53% reduction in the density of GAD₆₇ mRNA-containing neurons in layer 2, which is far greater in magnitude than the 16% to 17% decrease in the density of nonpyramidal neurons shown in previous cell counting studies⁷ and the 34% decrease in the density of calbindin-containing neurons.¹⁰ Therefore, although cell loss may still occur to some degree in schizophrenia, it seems to be insufficient to account for the degree of reduction in the GAD₆₇ mRNA–containing neurons. This conclusion is consistent with the results of a recent study⁵⁴ demonstrating a paradoxical decrease in apoptosis markers in the ACCx of subjects with schizophrenia.

Because GABA interneurons are anatomically and functionally heterogeneous,^55-57 characterizing the identities of the GABA interneurons that show reduced expression of the NR_2A subunit will shed critical light on the nature of neural circuitry disturbances and their functional consequences in schizophrenia and bipolar disorder. Because subpopulations of GABA interneurons can be characterized by the presence of calcium-binding proteins and other neuropeptides, such as cholecystokinin and vasoactive intestinal peptide,^50-53 future studies will examine the co-expression of the NR_2A subunit and these proteins or peptides. The information obtained from these studies will help define how glutamatergic modulation of specific GABAergic elements in layer 2 in the ACCx may be differentially altered in schizophrenia and bipolar disorder. A novel treatment strategy for these conditions could potentially involve fine-tuning the relative levels of NMDA-mediated glutamatergic activity impinging on GABA interneurons.

AUTHOR INFORMATION

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Correspondence: Francine M. Benes, MD, PhD, Program in Structural and Molecular Neuroscience, McLean Hospital, 115 Mill St, Belmont, MA 02478 (benesf{at}mclean.harvard.edu).

Submitted for publication October 2, 2003; final revision received February 11, 2003; accepted February 13, 2004.

This study was supported by grants MH/NS31862, MH00423, and MH42261 from the National Institutes of Health, Bethesda, Md.

From the Program in Structural and Molecular Neuroscience, McLean Hospital, Belmont, Mass (Drs Woo and Benes and Mr Walsh); the Department of Psychiatry (Drs Woo and Benes) and the Program in Neuroscience (Dr Benes), Harvard Medical School, Boston, Mass; and the Massachusetts Mental Health Center, Boston (Dr Woo).

REFERENCES

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