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Original Article |

Density of Glutamic Acid Decarboxylase 67 Messenger RNA–ContainingNeurons That Express the N-Methyl-D-AspartateReceptor Subunit NR2A in the Anterior Cingulate Cortex in Schizophreniaand Bipolar Disorder FREE

Tsung-Ung W. Woo, MD, PhD" TITLE="; John P. Walsh, MS; Francine M. Benes, MD, PhD
[+] Author Affiliations

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 MedicalSchool, Boston, Mass; and the Massachusetts Mental Health Center, Boston (DrWoo).


Arch Gen Psychiatry. 2004;61(7):649-657. doi:10.1001/archpsyc.61.7.649.
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Published online

Background  Disturbances of γ-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 γ-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 NR2A subunit with 35sulfur and the mRNA for the 67-kDa isoform of the γ-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 NR2AmRNA, 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 NR2AmRNA was also decreased by 42% in layer 2. In both disease groups, the expression level of NR2AmRNA in GAD67 mRNA–containing cells was unaltered.

Conclusions  The density of γ-aminobutyric acid interneurons that express the NMDA NR2Asubunit 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.

Figures in this Article

The anterior cingulate cortex (ACCx) (Brodmann's area 24) is a key regionof the large-scale neural network that comprises the limbic system, the dorsolateralprefrontal region, and the motor and premotor cortices. This distributed neuralsystem mediates a wide range of functions, such as affective regulation, motivation,selective attention, separation calls, executive control, and the dynamicorchestration of motor programming. The ACCx is involved in mediating thesefunctions via postulated capabilities, such as error detection and conflictmonitoring.15 Becauseperturbations of many aspects of these functions are commonly seen in schizophreniaand bipolar disorder, it is perhaps not surprising that converging lines ofevidence from postmortem and neuroimaging studies611 haveconsistently demonstrated that the ACCx is structurally and functionally alteredin these disorders.

γ-Aminobutyric acid (GABA) interneurons play an important rolein information processing in the cerebral cortex. Disturbances of these neuronshave been strongly implicated in the pathophysiology of schizophrenia.1219 Increasing,albeit still somewhat limited, evidence7,2023 suggeststhat GABAergic function may also be perturbed in bipolar disorder. In fact,some of the parameters of GABA neurotransmission that have been examined seemto be even more severely altered in subjects with bipolar disorder than inthose with schizophrenia. For example, it has been shown in the ACCx thatthe density of cells with a nonpyramidal shape, putative GABA interneurons,and terminals is decreased in schizophrenia and bipolar disorder, but theextent of this reduction is considerably greater in the latter condition.7,21 In addition, GAD65-immunoreactiveterminals have also been found to be substantially reduced in the ACCx ofbipolar, but not schizophrenic, subjects.24

Converging lines of clinical and preclinical observations2529 stronglysuggest that disturbances of glutamatergic neurotransmission contribute tothe pathophysiology of schizophrenia. Furthermore, it has been postulated,largely based on animal studies, that such disturbances may involve hypofunctioningof N-methyl-D-aspartate (NMDA) receptorson GABA interneurons.30,31 TheNMDA receptor complex is a heteromeric structure composed of different subunits.Among them, the NR2A subunit is abundantly present in the adultcerebral cortex. It has also been implicated in the pathophysiology of schizophrenia.For example, mice lacking the NR2A subunit demonstrated an increasein the release of dopamine in the striatum.32 Behaviorally,these animals exhibited hyperlocomotion, which could be attenuated by treatmentwith antipsychotic agents. Furthermore, NMDA-mediated GABA release in theseanimals was markedly decreased. In this study, as a first step to explorethe question of whether glutamatergic innervation of GABA interneurons viathe NMDA receptor may be altered in schizophrenia and bipolar disorder, weused a double in situ hybridization procedure to simultaneously examine theexpression of messenger RNA (mRNA) for the NR2Asubunit, labeledwith 35sulfur ([35S]), in cells containing GAD67 mRNA, labeled with digoxigenin (DIG), in the ACCx from normal control,schizophrenic, and bipolar subjects.

SUBJECTS

A cohort of 51 human brains obtained from the Harvard Brain Tissue ResourceCenter 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 subjectswas matched to a subject with bipolar disorder and to a normal control subjecton 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 bipolardisorder group and 8:9 for the schizophrenia and normal control groups. Theright hemisphere–left hemisphere ratio was 10:7 for the schizophreniagroup and 8:9 for the bipolar disorder and normal control groups. The mean± SD freezer storage time of brains was not significantly differentamong the normal control (1391 ± 1012 days), schizophrenia (1766 ±958 days), and bipolar disorder (1847 ± 1084 days) groups (F2,48 = 0.97; P = .39). Measurements of tissue pHwere available for 12 of 17 cases in the schizophrenia and bipolar disordergroups 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).

Table Graphic Jump LocationCharacteristics of the 51 Subjects in the Present Study

Psychiatric diagnoses were established using a retrospective reviewof medical records and an extensive family questionnaire that included themedical, psychiatric, and social history of the subjects. For the diagnosisof schizophrenia, the criteria of Feighner et al33 wereused, and the diagnoses of schizoaffective and bipolar disorder were madeaccording to DSM-III-R criteria. Of the 17 schizophrenicsubjects, 3 (cases B2166, B4875, and B4907) had a diagnosis of schizoaffectivedisorder, whereas the remaining cases has a diagnosis of schizophrenia. Threeof the 17 schizophrenic subjects (cases B3146, B4875, and B4256) were nottaking antipsychotic medications at the time of death. In the bipolar disordergroup, 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-equivalentdose) was less than half that of the schizophrenia group (618.3 ± 809.7mg). Some subjects in both disease groups were also taking concomitant psychotropicmedications, such as mood stabilizers, antidepressants, or anxiolytics (Table 1). No subject in the normal controlgroup was receiving any psychotropic agents at the time of death.

TISSUE PREPARATION

Tissue blocks (3 mm thick) from Brodmann's area 24 were removed fromfresh brain specimens at the level of the rostrum of the anterior cingulategyrus between the points at which the gyrus curves above and below the corpuscallosum.7 The blocks were immediately fixedin 0.1% paraformaldehyde in ice-cold 0.1M phosphate buffer (pH 7.4) for 90minutes, immersed in 30% sucrose in the same buffer overnight, and then frozenin Tissue-Tek OCT embedding meduim for frozen tissue (Sakura Finetek USA Inc,Torrance, Calif) on dry ice. Tissue blocks were then sectioned at a thicknessof 10 µm on a cryostat. Two sections per subject and therefore 6 sectionsper matched triplet were used for in situ hybridization. The 6 sections fromeach 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 hybridizationsignals between slides. All mounted sections were stored at –70°Cuntil riboprobe labeling was performed.

DOUBLE IN SITU HYBRIDIZATION
Riboprobe Preparation

Radiolabeled Complementary RNA Probe for NR2A mRNA. The complementary RNA (cRNA) probes for the NR2A subunit(provided by Christine Konradi, PhD) were transcribed in vitro from linearizedcomplementary DNA (cDNA) subclones encoding the rat NMDA NR2A subunit.The specificity of the probe was verified by Northern blot analysis (datanot shown). The probe was derived from a cDNA spanning nucleotides 1185 to2154 (GenBank Accession No. M91561) within the coding region of the subunit.A corresponding sense probe was used as a control. Radiolabeled cRNA probewas prepared by first drying down [35S]UTP (500 µCi/mL ofprobe, PerkinElmer Life and Analytical Sciences Inc, Boston, Mass) in a DNAspeed 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 T3or T7 RNA polymerases (for antisense and sense probes, respectively), and5× transcription buffer were then added. The transcription mixture wassubsequently incubated at 37°C for 1 hour. The cDNA template was digestedby incubating the mixture with R1Q DNase at 37°C for 15 minutes. UnincorporatedNTPs were removed by running the mixture through a push column (NucTrap; Stratagene,La Jolla, Calif). The eluate was collected, and probe concentration was determinedby 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 ngof full-length, linearized human cDNA clones inserted in a bluescript vector(provided by Allan Tobin, PhD, and Niranjula Tillakarantne, PhD, Departmentof Physiological Sciences, University of California at Los Angeles) in thepresence of 0.1M dithiothreitol; 3 U/µL of RNasin; 0.8 U/µL ofT3 and T7 RNA polymerases; 10mM ATP, CTP, and GTP; 6.5mM UTP; and 3.5mM DIG-labeledUTP (Boehringer Mannheim, Indianapolis, Ind). The mixture was incubated at37°C for 1 hour. The cDNA template was digested with RQ1 DNase. Probeconcentration was determined using a standard with known concentrations.

Hybridization

To ensure adequate tissue penetration, the GAD67 probe washydrolyzed to 0.8 kilobase (kb) with an equal volume of sodium bicarbonate–sodiumcarbonate 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 volof 2M sodium acetate in 6.25% glacial acetic acid. Probes were then reconstitutedin a hybridization buffer consisting of 50% formamide, 0.1% yeast transferribonucleic acid, 10% dextran sulfate, 1× Denhardt solution, 0.5M EDTA,0.02% sodium dodecyl sulfate, 4× isotonic sodium chloride solution–sodiumcitrate buffer, 10mM dithiothreitol, and 0.1% single-stranded DNA at a finalconcentration of 0.4 ng probe/µL hybridization buffer. Before hybridization,mounted tissue sections were air-dried and warmed to room temperature. Theywere then postfixed in 4% paraformaldehyde for 10 minutes and incubated in0.1M tetraethylammonium for 5 minutes at room temperature before being dehydratedin a graded series of ethanol. Probes were then added to slides for hybridizationin a prewarmed, humidified dish. Sections were covered with coverslips andincubated at 55°C for 3 hours. At the end of hybridization, coverslipswere soaked off in 4× isotonic sodium chloride solution–sodiumcitrate in the presence of 100 µL of ßMer alcohol. Tissue wasthen 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°Cfor 30 minutes, followed by a high-stringency wash with a solution containing50% formamide, 0.5M sodium chloride/0.05M phosphate buffer, and 100 µLof ßMer at 63°C for 30 minutes. Sections were finally washed overnightin 0.5× isotonic sodium chloride solution–sodium citrate with20mM ßMer alcohol at room temperature.

Visualization of DIG Labeling

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

Emulsion Autoradiography

It was determined that sufficient autoradiographic signal had developedafter 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 developmentin the dark with developer (Kodak D-19; Kodak), slides were counterstainedwith methyl green and coverslipped.

Quantification of GAD67 and NR2A mRNA Expression

All microscopic analyses were conducted under strictly blind conditions.[35S] labeling of NR2A mRNA appeared as clusters ofsilver grains after processing for emulsion autoradiography (Figure 1). After counterstaining with Vector Red, DIG labeling canbe visualized as a red-brown reaction product under a brightfield microscopeor as a fluorescent emission in the red range. Neurons that were single labeledwith DIG (Figure 1A) and those thatwere double labeled with DIG and [35S] (Figure 1B) were identified on images captured on a computer screenusing a microscope (Laborlux; Leica Microsystems, Wetzlar, Germany), whichwas equipped with a solid-state charge-coupled device video camera connectedto an image analysis system (Bioquant Nova; R&M Biometrics, Memphis, Tenn).Using a 100× oil immersion objective lens at a final magnification of×1000, the distributions of single- and double-labeled neurons in a250-µm-wide column extending from the pial surface to the border betweenlayer 6 and the subcortical white matter were obtained for each section. Neighboringsections were stained with cresyl violet for determination of laminar boundaries.Densities of single- and double-labeled neurons for each cortical layer werethen obtained by dividing cell counts by laminar areas. Intrarater reliability,as assessed by counting and recounting profiles in the same column, was establishedto be 93% to 97% before the actual data collection process began.

Place holder to copy figure label and caption
Figure 1.

Photomicrographs showing examplesof digoxigenin single-labeled neurons (arrows) (A) and 35 sulfur/digoxigenindouble-labeled neurons (arrows) (B). Scale bar = 10 µm.

Graphic Jump Location

To quantify the expression level of mRNA for the NR2A subunitin individual GABA cells, the area occupied by each grain cluster was outlinedusing a cursor displayed on the computer monitor. For each cluster, this quantificationwas performed according to the principle of including the largest number ofgrains in the smallest possible area. The cluster area was measured by highlightingthe grains with a thresholding subroutine. This threshold was held constant,and the light intensity was adjusted to ensure that the size of the grainswas neither underrepresented nor overrepresented. This procedure was consistentlyfollowed throughout the entire study. The area covered by autoradiographicgrains in the cluster area was automatically computed based on the thresholdvalue and was represented as a pixel count for NR2A transcriptexpression level. The pixel count was expressed as a function of cluster area(numerical density). By subtracting the background grain density (ie, pixelcount of the area covered by autoradiographic grains per unit area in squaremicrometers in the white matter), the corrected NR2A expressionlevel was obtained. The average NR2A expression level in GABA interneurons(ie, cells positive for GAD67 mRNA) for each cortical layer foreach case was then computed. Intrarater reliability in grain density measurements,which was accessed by repeating the procedures described previously hereinon the same clusters, was determined to be consistently greater than 95% beforethe actual data collection process.

STATISTICAL ANALYSIS

The numerical densities of single-labeled (GAD67 mRNA only)and double-labeled (GAD67 and NR2A mRNA) neurons andthe amount of mRNA for the NR2A subunit in GAD67 mRNA–containingneurons were compared among groups across layers 2 through 6 using repeated-measuresanalysis of variance (ANOVA), with diagnosis (ie, schizophrenia vs controland bipolar disorder vs control) and layer as main effects. For post hoc analyses,2-tailed paired t tests were used. The Bonferroniprocedure was used to correct for type 1 error as a result of multiple comparisons(layers 2, 3, 5, and 6). Therefore, the α level for significance forall t tests was P = .01(ie, .05 ÷ 4). Layer 1 was not included in the analyses because therewere no GAD67 mRNA–containing neurons with co-expressed NR2A subunit mRNA in this lamina. To evaluate the potential effects ofconfounding variables, such as age, sex, postmortem interval, brain pH, freezerstorage time, hemispheric laterality, and exposure to antipsychotic medications(expressed as the chlorpromazine-equivalent dose), simple Pearson correlationswere obtained for the individual groups and for the control group combinedwith the schizophrenia and bipolar disorder groups. In addition, an analysisof covariance (ANCOVA) was performed to understand how these confounding variablesmight have affected our results. Because none of the conclusions derived fromour findings were affected by the ANCOVA analysis, only results from repeated-measuresANOVAs are reported. Effects of hemispheric laterality on our findings wereevaluated by using 2-tailed unpaired t tests to comparethe measures from the 2 hemispheres within individual groups and when casesfrom the control group were combined with those from the schizophrenia andbipolar disorder groups.

DISTRIBUTION OF GAD67 mRNA– AND GAD67/NR2A mRNA–EXPRESSING NEURONS IN THE ACCx

Neurons that express GAD67 mRNA appeared to be distributedmore or less evenly across all layers in the ACCx, except for layer 1, wherethe density of these neurons was low. In the entire population of GAD67 mRNA–expressing neurons, those that co-expressed NR2A mRNA seemed to be most concentrated in layers 3 to 5, whereas the densityof these neurons seemed to be slightly lower in layers 2 and 6 (Figure 2).

Place holder to copy figure label and caption
Figure 2.

Plots of glutamic acid decarboxylase67 (GAD67) messenger RNA (mRNA)–containing neurons that co-expressedand did not co-express NR2A mRNA from tissue sections from a normalcontrol subject (case B4163) (A) and the matched subject with schizophrenia(case B2774) (B) and the matched subject with bipolar disorder (case B2565)(C).

Graphic Jump Location
DENSITY OF ALL NEURONS THAT EXPRESS GAD67 mRNA

Overall, the repeated-measures ANOVA models revealed a significant diagnosiseffect in the schizophrenia group (F1,32 = 10.19; P = .003). Furthermore, this effect seemed to be layer specific (diagnosis× layer interaction, F = 3.27; P = .03). Thus,the density of GAD67 mRNA–expressing neurons showed the mostprominent change in layer 2 (Figure 3A),with a 53% reduction in the density of these neurons compared with controlsubjects (t = 4.41; P<.001).Besides layer 2, the density of these neurons was also significantly decreasedin layer 5, although the magnitude of reduction (28%) was more modest (t = 2.45; P = .01). The observeddecreases in the density of GAD67 mRNA–expressing neuronsdid not seem to be artifactually related to differences in cortical thicknessbetween the 2 groups because the mean ± SD thickness of the ACCx inthe 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 bipolardisorder, the repeated-measures ANOVA initially did not demonstrate a significantdiagnosis effect (F1,32 = 3.44; P = .07).On closer inspection of the data, it was noticed that the numerical densityof the GAD67 mRNA–expressing cells in layer 2 in a subjectwith bipolar disorder (patient B3817) was 3 SD above the mean density forthat layer. This case and its matched control (case B5122) were, therefore,excluded from all of the numerical density comparisons between controls andsubjects with bipolar disorder reported herein. Case B3817 was the only casein which the mean neuronal density in any layer was beyond 3 SD. After theremoval of these 2 cases, the diagnosis effect in the repeated-measures ANOVAmodel was statistically significant (F1,30 = 4.22; P = .04). When individual layers were examined, the density of GAD67 mRNA–expressing cells in layer 2 was found to be significantlydecreased by 35% in the bipolar disorder group (t =4.12; P<.001). This reduction was smaller in magnitudethan the 53% reduction observed in layer 2 in subjects with schizophrenia(Figure 3A). This apparent differencein the magnitude of reduction in the numerical density of neurons betweenthe 2 subject groups was not statistically significant (t = 1.97, P = .06). Besides the reductionin layer 2, the numerical density of GAD67 mRNA–expressingneurons was essentially unchanged in all other layers in subjects with bipolardisorder. As in the schizophrenia group, there was no statistically significantdifference in mean ± SD cortical thickness between subjects with bipolardisorder (1755.3 ± 318.2 µm) and control subjects (1745.5 ±267.4 µm) (t = 0.097; P =.92).

Place holder to copy figure label and caption
Figure 3.

Mean numerical density of allneurons that express glutamic acid decarboxylase 67 (GAD67) messengerRNA (mRNA) (A), neurons that co-express GAD67 mRNA and NR2A mRNA (B), and neurons that express GAD67 mRNA but notNR2A mRNA (C). Asterisk indicates a statistically significant differenceat P = .01 vs controls. Error bars represent SEM.

Graphic Jump Location
DENSITY OF GAD67 mRNA–CONTAINING NEURONS EXPRESSINGNR2A mRNA

The effect of diagnosis on NR2A-expressing GAD67-positivecells was statistically significant in the schizophrenia (F1,32 =8.97; P = .005) and bipolar disorder (F1,32 = 4.42; P = .04) groups. In the schizophreniagroup, 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 achievestatistical 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 NR2A-positivecells was significantly decreased by 60% in layer 2 (t =2.8; P = .01), but the decreases of 31%, 37%, and29% 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 NOTEXPRESS NR2A mRNA

According to the repeated-measures ANOVA models, the effect of diagnosiswas not statistically significant in subjects with either schizophrenia (F1,32 = 1.38; P = .25) or bipolar disorder (F1,30 = 0.038; P = .85). However, in the schizophreniagroup, there was a significant diagnosis × layer effect (F3,96 = 6.42; P<.001) that seemed to reflectthe 42% decrease in neuronal density in layer 2 (Figure 3C), and this reduction was statistically significant (t = 2.99; P = .005).

EXPRESSION LEVEL OF NR2A mRNA IN CELLS CONTAINING GAD67 mRNA

There were no differences in the density of silver grains in eitherthe schizophrenia (F1,20 = 1.79; P = .20)or the bipolar disorder (F1,24 = 0.21; P =.65) group, suggesting that for GABA interneurons that contained a detectableamount of NR2A mRNA, the level of expression of the transcriptwas unaltered in either disease group (Figure4).

Place holder to copy figure label and caption
Figure 4.

Mean density of silver grainsover glutamic acid decarboxylase 67 (GAD67) messenger RNA (mRNA)–positiveneurons in the anterior cingulate cortex is not different among the 3 studygroups, indicating that the expression level of NR2A mRNA in GAD67 mRNA–positive cells is unchanged in the schizophrenia and bipolardisorder groups. Error bars represent SEM.

Graphic Jump Location
POTENTIAL CONFOUNDING VARIABLES

We examined the potential confounding effects of variables such as age,postmortem interval, brain pH, hemispheric laterality, and antipsychotic drugexposure on our findings. None of these factors seem to have affected ourresults (data not shown). Among these variables, pH was particularly importantbecause the integrity of mRNA is known to be sensitive to this variable.3436 There was no statisticallysignificant difference in pH in the 3 study groups. In the statistical analyses,we also found no correlation between pH and any of our measurements eitherin individual diagnostic groups or when subjects from the disease groups andthose from the control group were combined. An ANCOVA incorporating pH asa covariate also did not statistically significantly alter the effect of diagnosison the cell density and grain density measurements. Similar analyses withchlorpromazine-equivalent dose also revealed that exposure to antipsychoticmedications was not statistically significantly correlated with any of ourmeasurements, and neither did it contribute to the observed differences inthe neuronal density measurements in the diagnostic groups. Therefore, thesefindings do not seem to be the result of antipsychotic medication treatmentor any other measurable potential confounds but may in fact reflect the underlyingdisease processes.

Multiple lines of evidence suggest that disturbances of GABA interneuronsrepresent a key feature of the pathophysiology of schizophrenia and bipolardisorder.10,1217,19,3740 Theactivities of GABA interneurons are subject to feedback and feedforward modulationby glutamatergic inputs from pyramidal neurons located locally and in distantcortical or subcortical regions. Together, these mechanisms regulate the flowof information in the cerebral cortex by adjusting the spatial and temporalarchitecture of GABA neurotransmission.4143 Inthis study, we extended previous findings of altered GABAergic neurotransmissionin schizophrenia and bipolar disorder to demonstrate that alteration in glutamatergicinputs onto GABA interneurons via the NMDA receptor may contribute to disturbancesof GABA neurotransmission in both of these conditions. We cannot, however,exclude the possibility that there may be a primary dysregulation in the expressionof the NR2A subunit in a subgroup of GABA neurons.

In situ hybridization labeling with [35S] is more sensitivein detecting transcript signals than nonradioactive DIG-labeled probes.44 In the present study, because DIG was used to labelthe GAD67 transcript, it is possible that we may have underestimatedthe true density of GABA interneurons in all 3 study groups. If the amountof GAD67 transcript per GABA interneuron is equivalent in the 3groups, our conclusions would not have been affected because they were basedon the "relative" changes in neuronal density among the groups. On the otherhand, it is possible that a subpopulation of GABA interneurons may in factexpress a lower level of GAD67 mRNA in subjects with schizophreniaand bipolar disorder and that these cells fell below the detection thresholdfor DIG labeling of GAD67. This scenario could have contributedto the observed reduction in neuronal density in the schizophrenia and bipolardisorder groups compared with controls. Although we cannot rule out thesepossibilities, they seem to be insufficient to account for the magnitude ofneuronal density reduction observed because DIG labeling of GAD67 hasbeen estimated to be only slightly (<7%) less sensitive than similar insitu hybridization labeling with [35S].44 Analternative possibility is that the number of GABA interneurons may be inherentlydifferent in the 2 disease groups and that this was reflected in the differencesin 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 thanin other layers in the ACCx in schizophrenia and bipolar disorder. Thus, therewas 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 inthe density of these neurons in other layers in either subject group, exceptfor a 28% decrease in density in layer 5 in subjects with schizophrenia. Theseobservations are consistent with findings from previous studies45 suggestingthat neural circuits in layer 2 in the ACCx may be a major site of diseasevulnerability in schizophrenia and bipolar disorder. Because neurons in thislayer receive extensive corticocortical projections from other regions ofthe cerebral cortex, such as the prefrontal cortex,46 andthey also receive specific inputs from subcortical and limbic structures,such as the amygdala,47 they may play a criticalrole in integrating diverse streams of information derived from the cognitiveand emotive domains. Therefore, disturbances of information processing inthe neural circuits in this layer could contribute to the multitude of symptomsobserved in schizophrenia and bipolar disorder.

The reduction in the density of neurons that express the mRNA for the67-kDa isoform of GAD may represent a loss of neurons. Alternatively,the amount of GAD67 mRNA in a subpopulation of GABA interneuronsmay be decreased to an experimentally undetectable level. Consideration offindings from previous studies of quantification of nonpyramidal, presumablyGABA interneurons may provide some insights into the possible interpretationsof the current findings. Data from these studies7,48,49 demonstratethat the density of nonpyramidal neurons in layer 2 in the ACCx in bipolardisorder was decreased by 27% to 30%, whereas the magnitude of decrease inschizophrenia was only 16% to 17%. In bipolar disorder, the 35% reductionin the density of GAD67 mRNA–containing neurons in layer2 observed in the present study is similar to the degree of reduction in thedensity of nonpyramidal neurons previously reported.7 Arecent study10 using immunohistochemical techniquesto examine the expression of various calcium-binding proteins, which are differentiallyexpressed by subpopulations of GABA interneurons,5053 hasshown that the density of neurons that express calbindin was decreased by33% and 34% in bipolar disorder and schizophrenia, respectively. In addition,the density of neurons that expressed parvalbumin also seemed to be reducedin 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 ofGAD67mRNA–containing neurons reported in this study. Takentogether, these findings raise the possibility that a subpopulation of GABAinterneurons 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 layer2, which is far greater in magnitude than the 16% to 17% decrease in the densityof nonpyramidal neurons shown in previous cell counting studies7 andthe 34% decrease in the density of calbindin-containing neurons.10 Therefore,although cell loss may still occur to some degree in schizophrenia, it seemsto be insufficient to account for the degree of reduction in the GAD67 mRNA–containing neurons. This conclusion is consistent withthe results of a recent study54 demonstratinga paradoxical decrease in apoptosis markers in the ACCx of subjects with schizophrenia.

Because GABA interneurons are anatomically and functionally heterogeneous,5557 characterizing theidentities of the GABA interneurons that show reduced expression of the NR2A subunit will shed critical light on the nature of neural circuitrydisturbances and their functional consequences in schizophrenia and bipolardisorder. Because subpopulations of GABA interneurons can be characterizedby the presence of calcium-binding proteins and other neuropeptides, suchas cholecystokinin and vasoactive intestinal peptide,5053 futurestudies will examine the co-expression of the NR2A subunit andthese proteins or peptides. The information obtained from these studies willhelp define how glutamatergic modulation of specific GABAergic elements inlayer 2 in the ACCx may be differentially altered in schizophrenia and bipolardisorder. A novel treatment strategy for these conditions could potentiallyinvolve fine-tuning the relative levels of NMDA-mediated glutamatergic activityimpinging on GABA interneurons.

Correspondence: Francine M. Benes, MD, PhD, Program in Structuraland Molecular Neuroscience, McLean Hospital, 115 Mill St, Belmont, MA 02478(benesf@mclean.harvard.edu).

Submitted for publication October 2, 2003; final revision received February11, 2003; accepted February 13, 2004.

This study was supported by grants MH/NS31862, MH00423, and MH42261from the National Institutes of Health, Bethesda, Md.

Paus  T Primate anterior cingulate cortex: where motor control, drive and cognitioninterface. Nat Rev Neurosci. 2001;2417- 424
PubMed Link to Article
Carter  CSBotvinick  MMCohen  JD The contribution of the anterior cingulate cortex to executive processesin cognition. Rev Neurosci. 1999;1049- 57
PubMed
Carter  CSBraver  TSBarch  DMBotvinick  MMNoll  DCohen  JD Anterior cingulate cortex, error detection, and the online monitoringof performance. Science. 1998;280747- 749
PubMed Link to Article
Falkenstein  MHohnsbein  JHoormann  JBlanke  L Effects of crossmodal divided attention on late ERP components, II:error processing in choice reaction tasks. Electroencephalogr Clin Neurophysiol. 1991;78447- 455
PubMed Link to Article
Gehring  WJKnight  RT Prefrontal-cingulate interactions in action monitoring. Nat Neurosci. 2000;3516- 520
PubMed Link to Article
Benes  FM Alterations of neural circuitry within layer II of anterior cingulatecortex in schizophrenia. J Psychiatr Res. 1999;33511- 512
PubMed Link to Article
Benes  FMVincent  SLTodtenkopf  M The density of pyramidal and nonpyramidal neurons in anterior cingulatecortex of schizophrenic and bipolar subjects. Biol Psychiatry. 2001;50395- 406
PubMed Link to Article
Carter  CSMacDonald  AW  IIIRoss  LLStenger  VA Anterior cingulate cortex activity and impaired self-monitoring ofperformance in patients with schizophrenia: an event-related fMRI study. Am J Psychiatry. 2001;1581423- 1428
PubMed Link to Article
Eastwood  SLHarrison  PJ Synaptic pathology in the anterior cingulate cortex in schizophreniaand mood disorders: a review and a Western blot study of synaptophysin, GAP-43and the complexins. Brain Res Bull. 2001;55569- 578
PubMed Link to Article
Cotter  DLandau  SBeasley  C  et al.  The density and spatial distribution of GABAergic neurons, labelledusing calcium binding proteins, in the anterior cingulate cortex in majordepressive disorder, bipolar disorder, and schizophrenia. Biol Psychiatry. 2002;51377- 386
PubMed Link to Article
Chana  GLandau  SBeasley  CEverall  IPCotter  D Two-dimensional assessment of cytoarchitecture in the anterior cingulatecortex in major depressive disorder, bipolar disorder, and schizophrenia:evidence for decreased neuronal somal size and increased neuronal density. Biol Psychiatry. 2003;531086- 1098
PubMed Link to Article
Akbarian  SKim  JJPotkin  SG  et al.  Gene expression for glutamic acid decarboxylase is reduced withoutloss of neurons in prefrontal cortex of schizophrenics. Arch Gen Psychiatry. 1995;52258- 266
PubMed Link to Article
Volk  DWAustin  MCPierri  JNSampson  ARLewis  DA Decreased glutamic acid decarboxylase67 messenger RNA expression ina subset of prefrontal cortical γ-aminobutyric acid neurons in subjectswith schizophrenia. Arch Gen Psychiatry. 2000;57237- 245
PubMed Link to Article
Volk  DAustin  MPierri  JSampson  ALewis  D GABA transporter-1 mRNA in the prefrontal cortex in schizophrenia:decreased expression in a subset of neurons. Am J Psychiatry. 2001;158256- 265
PubMed Link to Article
Benes  FMVincent  SLAlsterberg  GBird  EDSanGiovanni  JP Increased GABAA receptor binding in superficial layers of cingulatecortex in schizophrenics. J Neurosci. 1992;12924- 929
PubMed
Benes  FMBerretta  S GABAergic interneurons: implications for understanding schizophreniaand bipolar disorder. Neuropsychopharmacology. 2001;251- 27
PubMed Link to Article
Costa  EDavis  JGrayson  DRGuidotti  APappas  GDPesold  C Dendritic spine hypoplasticity and downregulation of reelin and GABAergictone in schizophrenia vulnerability. Neurobiol Dis. 2001;8723- 742
PubMed Link to Article
Lewis  DAPierri  JNVolk  DWMelchitzky  DSWoo  TU Altered GABA neurotransmission and prefrontal cortical dysfunctionin schizophrenia. Biol Psychiatry. 1999;46616- 626
PubMed Link to Article
Woo  TUWWhitehead  REMelchitzky  DSLewis  DA A subclass of prefrontal γ-aminobutyric acid axon terminals areselectively altered in schizophrenia. Proc Natl Acad Sci U S A. 1998;955341- 5346
PubMed Link to Article
Benes  FMKwok  EWVincent  SLTodtenkopf  MS A reduction of nonpyramidal cells in sector CA2 of schizophrenics andmanic depressives. Biol Psychiatry. 1998;4488- 97
PubMed Link to Article
Benes  FMTodtenkopf  MSLogiotatos  PWilliams  M Glutamate decarboxylase(65)-immunoreactive terminals in cingulate andprefrontal cortices of schizophrenic and bipolar brain. J Chem Neuroanat. 2000;20259- 269
PubMed Link to Article
Guidotti  AAuta  JDavis  JM  et al.  Decrease in reelin and glutamic acid decarboxylase67 (GAD67) expressionin schizophrenia and bipolar disorder: a postmortem brain study. Arch Gen Psychiatry. 2000;571061- 1069
PubMed Link to Article
Kromkamp  MUylings  HBSmidt  MPHellemons  AJBurbach  JPKahn  RS Decreased thalamic expression of the homeobox gene DLX1 in psychosis. Arch Gen Psychiatry. 2003;60869- 874
PubMed Link to Article
Todtenkopf  MSBenes  FM Distribution of glutamate decarboxylase65 immunoreactive puncta onpyramidal and nonpyramidal neurons in hippocampus of schizophrenic brain. Synapse. 1998;29323- 332
PubMed Link to Article
Tamminga  CA Schizophrenia and glutamatergic transmission. Crit Rev Neurobiol. 1998;1221- 36
PubMed Link to Article
Coyle  JT The glutamatergic dysfunction hypothesis for schizophrenia. Harv Rev Psychiatry. 1996;3241- 253
PubMed Link to Article
Goff  DCCoyle  JT The emerging role of glutamate in the pathophysiology and treatmentof schizophrenia. Am J Psychiatry. 2001;1581367- 1377
PubMed Link to Article
Carlsson  AWaters  NHolm-Waters  STedroff  JNilsson  MCarlsson  ML Interactions between monoamines, glutamate, and GABA in schizophrenia:new evidence. Annu Rev Pharmacol Toxicol. 2001;41237- 260
PubMed Link to Article
Javitt  DCZukin  SR Recent advances in the phencyclidine model of schizophrenia. Am J Psychiatry. 1991;1481301- 1308
PubMed
Olney  JWFarber  NB Glutamate receptor dysfunction and schizophrenia. Arch Gen Psychiatry. 1995;52998- 1007
PubMed Link to Article
Olney  JWNewcomer  JWFarber  NB NMDA receptor hypofunction model of schizophrenia. J Psychiatr Res. 1999;33523- 533
PubMed Link to Article
Miyamoto  YYamada  KNoda  YMori  HMishina  MNabeshima  T Hyperfunction of dopaminergic and serotonergic neuronal systems inmice lacking the NMDA receptor ϵ1 subunit. J Neurosci. 2001;21750- 757
PubMed
Feighner  JPRobins  EGuze  SBWoodruff  RA  JrWinokur  GMunoz  R Diagnostic criteria for use in psychiatric research. Arch Gen Psychiatry. 1972;2657- 63
PubMed Link to Article
Kingsbury  AEFoster  OJNisbet  AP  et al.  Tissue pH as an indicator of mRNA preservation in human post-mortembrain. Brain Res Mol Brain Res. 1995;28311- 318
PubMed Link to Article
Harrison  PJHeath  PREastwood  SLBurnet  PWMcDonald  BPearson  RC The relative importance of premortem acidosis and postmortem intervalfor human brain gene expression studies: selective mRNA vulnerability andcomparison with their encoded proteins. Neurosci Lett. 1995;200151- 154
PubMed Link to Article
Eastwood  SLKerwin  RWHarrison  PJ Immunoautoradiographic evidence for a loss of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate–preferring non–N-methyl-d-aspartateglutamate receptors within the medial temporal lobe in schizophrenia. Biol Psychiatry. 1997;41636- 643
PubMed Link to Article
Benes  FM Altered glutamatergic and GABAergic mechanisms in the cingulate cortexof the schizophrenic brain. Arch Gen Psychiatry. 1995;521015- 1024
PubMed Link to Article
Lewis  DA GABAergic local circuit neurons and prefrontal cortical dysfunctionin schizophrenia. Brain Res Brain Res Rev. 2000;31270- 276
PubMed Link to Article
Reynolds  GPBeasley  CL GABAergic neuronal subtypes in the human frontal cortex: developmentand deficits in schizophrenia. J Chem Neuroanat. 2001;2295- 100
PubMed Link to Article
Heckers  SStone  DWalsh  JShick  JKoul  PBenes  FM Differential hippocampal expression of glutamic acid decarboxylase65 and 67 messenger RNA in bipolar disorder and schizophrenia. Arch Gen Psychiatry. 2002;59521- 529
PubMed Link to Article
Constantinidis  CWilliams  GVGoldman-Rakic  PS A role for inhibition in shaping the temporal flow of information inprefrontal cortex. Nat Neurosci. 2002;5175- 180
PubMed Link to Article
Constantinidis  CFranowicz  MNGoldman-Rakic  PS Coding specificity in cortical microcircuits: a multiple-electrodeanalysis of primate prefrontal cortex. J Neurosci. 2001;213646- 3655
PubMed
Rao  SGWilliams  GVGoldman-Rakic  PS Isodirectional tuning of adjacent interneurons and pyramidal cellsduring working memory: evidence for microcolumnar organization in PFC. J Neurophysiol. 1999;811903- 1916
PubMed
Stone  DJWalsh  JBenes  FM Localization of cells preferentially expressing GAD(67) with negligibleGAD(65) transcripts in the rat hippocampus: a double in situ hybridizationstudy. Brain Res Mol Brain Res. 1999;71201- 209
PubMed Link to Article
Benes  FM Emerging principles of altered neural circuitry in schizophrenia. Brain Res Brain Res Rev. 2000;31251- 269
PubMed Link to Article
Barbas  HPandya  DN Architecture and intrinsic connections of the prefrontal cortex inthe rhesus monkey. J Comp Neurol. 1989;286353- 375
PubMed Link to Article
Barbas  HDe  Olmos J Projections from the amygdala to basoventral and mediodorsal prefrontalregions in the rhesus monkey. J Comp Neurol. 1990;300549- 571
PubMed Link to Article
Benes  FMDavidson  JBird  ED Quantitative cytoarchitectural studies of the cerebral cortex of schizophrenics. Arch Gen Psychiatry. 1986;4331- 35
PubMed Link to Article
Benes  FMMcSparren  JBird  EDSanGiovanni  JPVincent  SL Deficits in small interneurons in prefrontal and cingulate corticesof schizophrenic and schizoaffective patients. Arch Gen Psychiatry. 1991;48996- 1001
PubMed Link to Article
DeFelipe  J Types of neurons, synaptic connections and chemical characteristicsof cells immunoreactive for calbindin-D28K, parvalbumin and calretinin inthe neocortex. J Chem Neuroanat. 1997;141- 19
PubMed Link to Article
Conde  FLund  JSJacobowitz  DMBaimbridge  KGLewis  DA Local circuit neurons immunoreactive for calretinin, calbindin D-28kor parvalbumin in monkey prefrontal cortex: distribution and morphology. J Comp Neurol. 1994;34195- 116
PubMed Link to Article
Gabbott  PLBacon  SJ Local circuit neurons in the medial prefrontal cortex (areas 24a,b,c,25 and 32) in the monkey, II: quantitative areal and laminar distributions. J Comp Neurol. 1996;364609- 636
PubMed Link to Article
Kawaguchi  YKondo  S Parvalbumin, somatostatin and cholecystokinin as chemical markers forspecific GABAergic interneuron types in the rat frontal cortex. J Neurocytol. 2002;31277- 287
PubMed Link to Article
Benes  FMWalsh  JBhattacharyya  SSheth  ABerretta  S DNA fragmentation decreased in schizophrenia but not bipolar disorder. Arch Gen Psychiatry. 2003;60359- 364
PubMed Link to Article
Kawaguchi  YKubota  Y GABAergic cell subtypes and their synaptic connections in rat frontalcortex. Cereb Cortex. 1997;7476- 486
PubMed Link to Article
Somogyi  PTamas  GLujan  RBuhl  EH Salient features of synaptic organisation in the cerebral cortex. Brain Res Brain Res Rev. 1998;26113- 135
PubMed Link to Article
Gupta  AWang  YMarkram  H Organizing principles for a diversity of GABAergic interneurons andsynapses in the neocortex. Science. 2000;287273- 278
PubMed Link to Article

Figures

Place holder to copy figure label and caption
Figure 1.

Photomicrographs showing examplesof digoxigenin single-labeled neurons (arrows) (A) and 35 sulfur/digoxigenindouble-labeled neurons (arrows) (B). Scale bar = 10 µm.

Graphic Jump Location
Place holder to copy figure label and caption
Figure 2.

Plots of glutamic acid decarboxylase67 (GAD67) messenger RNA (mRNA)–containing neurons that co-expressedand did not co-express NR2A mRNA from tissue sections from a normalcontrol subject (case B4163) (A) and the matched subject with schizophrenia(case B2774) (B) and the matched subject with bipolar disorder (case B2565)(C).

Graphic Jump Location
Place holder to copy figure label and caption
Figure 3.

Mean numerical density of allneurons that express glutamic acid decarboxylase 67 (GAD67) messengerRNA (mRNA) (A), neurons that co-express GAD67 mRNA and NR2A mRNA (B), and neurons that express GAD67 mRNA but notNR2A mRNA (C). Asterisk indicates a statistically significant differenceat P = .01 vs controls. Error bars represent SEM.

Graphic Jump Location
Place holder to copy figure label and caption
Figure 4.

Mean density of silver grainsover glutamic acid decarboxylase 67 (GAD67) messenger RNA (mRNA)–positiveneurons in the anterior cingulate cortex is not different among the 3 studygroups, indicating that the expression level of NR2A mRNA in GAD67 mRNA–positive cells is unchanged in the schizophrenia and bipolardisorder groups. Error bars represent SEM.

Graphic Jump Location

Tables

Table Graphic Jump LocationCharacteristics of the 51 Subjects in the Present Study

References

Paus  T Primate anterior cingulate cortex: where motor control, drive and cognitioninterface. Nat Rev Neurosci. 2001;2417- 424
PubMed Link to Article
Carter  CSBotvinick  MMCohen  JD The contribution of the anterior cingulate cortex to executive processesin cognition. Rev Neurosci. 1999;1049- 57
PubMed
Carter  CSBraver  TSBarch  DMBotvinick  MMNoll  DCohen  JD Anterior cingulate cortex, error detection, and the online monitoringof performance. Science. 1998;280747- 749
PubMed Link to Article
Falkenstein  MHohnsbein  JHoormann  JBlanke  L Effects of crossmodal divided attention on late ERP components, II:error processing in choice reaction tasks. Electroencephalogr Clin Neurophysiol. 1991;78447- 455
PubMed Link to Article
Gehring  WJKnight  RT Prefrontal-cingulate interactions in action monitoring. Nat Neurosci. 2000;3516- 520
PubMed Link to Article
Benes  FM Alterations of neural circuitry within layer II of anterior cingulatecortex in schizophrenia. J Psychiatr Res. 1999;33511- 512
PubMed Link to Article
Benes  FMVincent  SLTodtenkopf  M The density of pyramidal and nonpyramidal neurons in anterior cingulatecortex of schizophrenic and bipolar subjects. Biol Psychiatry. 2001;50395- 406
PubMed Link to Article
Carter  CSMacDonald  AW  IIIRoss  LLStenger  VA Anterior cingulate cortex activity and impaired self-monitoring ofperformance in patients with schizophrenia: an event-related fMRI study. Am J Psychiatry. 2001;1581423- 1428
PubMed Link to Article
Eastwood  SLHarrison  PJ Synaptic pathology in the anterior cingulate cortex in schizophreniaand mood disorders: a review and a Western blot study of synaptophysin, GAP-43and the complexins. Brain Res Bull. 2001;55569- 578
PubMed Link to Article
Cotter  DLandau  SBeasley  C  et al.  The density and spatial distribution of GABAergic neurons, labelledusing calcium binding proteins, in the anterior cingulate cortex in majordepressive disorder, bipolar disorder, and schizophrenia. Biol Psychiatry. 2002;51377- 386
PubMed Link to Article
Chana  GLandau  SBeasley  CEverall  IPCotter  D Two-dimensional assessment of cytoarchitecture in the anterior cingulatecortex in major depressive disorder, bipolar disorder, and schizophrenia:evidence for decreased neuronal somal size and increased neuronal density. Biol Psychiatry. 2003;531086- 1098
PubMed Link to Article
Akbarian  SKim  JJPotkin  SG  et al.  Gene expression for glutamic acid decarboxylase is reduced withoutloss of neurons in prefrontal cortex of schizophrenics. Arch Gen Psychiatry. 1995;52258- 266
PubMed Link to Article
Volk  DWAustin  MCPierri  JNSampson  ARLewis  DA Decreased glutamic acid decarboxylase67 messenger RNA expression ina subset of prefrontal cortical γ-aminobutyric acid neurons in subjectswith schizophrenia. Arch Gen Psychiatry. 2000;57237- 245
PubMed Link to Article
Volk  DAustin  MPierri  JSampson  ALewis  D GABA transporter-1 mRNA in the prefrontal cortex in schizophrenia:decreased expression in a subset of neurons. Am J Psychiatry. 2001;158256- 265
PubMed Link to Article
Benes  FMVincent  SLAlsterberg  GBird  EDSanGiovanni  JP Increased GABAA receptor binding in superficial layers of cingulatecortex in schizophrenics. J Neurosci. 1992;12924- 929
PubMed
Benes  FMBerretta  S GABAergic interneurons: implications for understanding schizophreniaand bipolar disorder. Neuropsychopharmacology. 2001;251- 27
PubMed Link to Article
Costa  EDavis  JGrayson  DRGuidotti  APappas  GDPesold  C Dendritic spine hypoplasticity and downregulation of reelin and GABAergictone in schizophrenia vulnerability. Neurobiol Dis. 2001;8723- 742
PubMed Link to Article
Lewis  DAPierri  JNVolk  DWMelchitzky  DSWoo  TU Altered GABA neurotransmission and prefrontal cortical dysfunctionin schizophrenia. Biol Psychiatry. 1999;46616- 626
PubMed Link to Article
Woo  TUWWhitehead  REMelchitzky  DSLewis  DA A subclass of prefrontal γ-aminobutyric acid axon terminals areselectively altered in schizophrenia. Proc Natl Acad Sci U S A. 1998;955341- 5346
PubMed Link to Article
Benes  FMKwok  EWVincent  SLTodtenkopf  MS A reduction of nonpyramidal cells in sector CA2 of schizophrenics andmanic depressives. Biol Psychiatry. 1998;4488- 97
PubMed Link to Article
Benes  FMTodtenkopf  MSLogiotatos  PWilliams  M Glutamate decarboxylase(65)-immunoreactive terminals in cingulate andprefrontal cortices of schizophrenic and bipolar brain. J Chem Neuroanat. 2000;20259- 269
PubMed Link to Article
Guidotti  AAuta  JDavis  JM  et al.  Decrease in reelin and glutamic acid decarboxylase67 (GAD67) expressionin schizophrenia and bipolar disorder: a postmortem brain study. Arch Gen Psychiatry. 2000;571061- 1069
PubMed Link to Article
Kromkamp  MUylings  HBSmidt  MPHellemons  AJBurbach  JPKahn  RS Decreased thalamic expression of the homeobox gene DLX1 in psychosis. Arch Gen Psychiatry. 2003;60869- 874
PubMed Link to Article
Todtenkopf  MSBenes  FM Distribution of glutamate decarboxylase65 immunoreactive puncta onpyramidal and nonpyramidal neurons in hippocampus of schizophrenic brain. Synapse. 1998;29323- 332
PubMed Link to Article
Tamminga  CA Schizophrenia and glutamatergic transmission. Crit Rev Neurobiol. 1998;1221- 36
PubMed Link to Article
Coyle  JT The glutamatergic dysfunction hypothesis for schizophrenia. Harv Rev Psychiatry. 1996;3241- 253
PubMed Link to Article
Goff  DCCoyle  JT The emerging role of glutamate in the pathophysiology and treatmentof schizophrenia. Am J Psychiatry. 2001;1581367- 1377
PubMed Link to Article
Carlsson  AWaters  NHolm-Waters  STedroff  JNilsson  MCarlsson  ML Interactions between monoamines, glutamate, and GABA in schizophrenia:new evidence. Annu Rev Pharmacol Toxicol. 2001;41237- 260
PubMed Link to Article
Javitt  DCZukin  SR Recent advances in the phencyclidine model of schizophrenia. Am J Psychiatry. 1991;1481301- 1308
PubMed
Olney  JWFarber  NB Glutamate receptor dysfunction and schizophrenia. Arch Gen Psychiatry. 1995;52998- 1007
PubMed Link to Article
Olney  JWNewcomer  JWFarber  NB NMDA receptor hypofunction model of schizophrenia. J Psychiatr Res. 1999;33523- 533
PubMed Link to Article
Miyamoto  YYamada  KNoda  YMori  HMishina  MNabeshima  T Hyperfunction of dopaminergic and serotonergic neuronal systems inmice lacking the NMDA receptor ϵ1 subunit. J Neurosci. 2001;21750- 757
PubMed
Feighner  JPRobins  EGuze  SBWoodruff  RA  JrWinokur  GMunoz  R Diagnostic criteria for use in psychiatric research. Arch Gen Psychiatry. 1972;2657- 63
PubMed Link to Article
Kingsbury  AEFoster  OJNisbet  AP  et al.  Tissue pH as an indicator of mRNA preservation in human post-mortembrain. Brain Res Mol Brain Res. 1995;28311- 318
PubMed Link to Article
Harrison  PJHeath  PREastwood  SLBurnet  PWMcDonald  BPearson  RC The relative importance of premortem acidosis and postmortem intervalfor human brain gene expression studies: selective mRNA vulnerability andcomparison with their encoded proteins. Neurosci Lett. 1995;200151- 154
PubMed Link to Article
Eastwood  SLKerwin  RWHarrison  PJ Immunoautoradiographic evidence for a loss of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate–preferring non–N-methyl-d-aspartateglutamate receptors within the medial temporal lobe in schizophrenia. Biol Psychiatry. 1997;41636- 643
PubMed Link to Article
Benes  FM Altered glutamatergic and GABAergic mechanisms in the cingulate cortexof the schizophrenic brain. Arch Gen Psychiatry. 1995;521015- 1024
PubMed Link to Article
Lewis  DA GABAergic local circuit neurons and prefrontal cortical dysfunctionin schizophrenia. Brain Res Brain Res Rev. 2000;31270- 276
PubMed Link to Article
Reynolds  GPBeasley  CL GABAergic neuronal subtypes in the human frontal cortex: developmentand deficits in schizophrenia. J Chem Neuroanat. 2001;2295- 100
PubMed Link to Article
Heckers  SStone  DWalsh  JShick  JKoul  PBenes  FM Differential hippocampal expression of glutamic acid decarboxylase65 and 67 messenger RNA in bipolar disorder and schizophrenia. Arch Gen Psychiatry. 2002;59521- 529
PubMed Link to Article
Constantinidis  CWilliams  GVGoldman-Rakic  PS A role for inhibition in shaping the temporal flow of information inprefrontal cortex. Nat Neurosci. 2002;5175- 180
PubMed Link to Article
Constantinidis  CFranowicz  MNGoldman-Rakic  PS Coding specificity in cortical microcircuits: a multiple-electrodeanalysis of primate prefrontal cortex. J Neurosci. 2001;213646- 3655
PubMed
Rao  SGWilliams  GVGoldman-Rakic  PS Isodirectional tuning of adjacent interneurons and pyramidal cellsduring working memory: evidence for microcolumnar organization in PFC. J Neurophysiol. 1999;811903- 1916
PubMed
Stone  DJWalsh  JBenes  FM Localization of cells preferentially expressing GAD(67) with negligibleGAD(65) transcripts in the rat hippocampus: a double in situ hybridizationstudy. Brain Res Mol Brain Res. 1999;71201- 209
PubMed Link to Article
Benes  FM Emerging principles of altered neural circuitry in schizophrenia. Brain Res Brain Res Rev. 2000;31251- 269
PubMed Link to Article
Barbas  HPandya  DN Architecture and intrinsic connections of the prefrontal cortex inthe rhesus monkey. J Comp Neurol. 1989;286353- 375
PubMed Link to Article
Barbas  HDe  Olmos J Projections from the amygdala to basoventral and mediodorsal prefrontalregions in the rhesus monkey. J Comp Neurol. 1990;300549- 571
PubMed Link to Article
Benes  FMDavidson  JBird  ED Quantitative cytoarchitectural studies of the cerebral cortex of schizophrenics. Arch Gen Psychiatry. 1986;4331- 35
PubMed Link to Article
Benes  FMMcSparren  JBird  EDSanGiovanni  JPVincent  SL Deficits in small interneurons in prefrontal and cingulate corticesof schizophrenic and schizoaffective patients. Arch Gen Psychiatry. 1991;48996- 1001
PubMed Link to Article
DeFelipe  J Types of neurons, synaptic connections and chemical characteristicsof cells immunoreactive for calbindin-D28K, parvalbumin and calretinin inthe neocortex. J Chem Neuroanat. 1997;141- 19
PubMed Link to Article
Conde  FLund  JSJacobowitz  DMBaimbridge  KGLewis  DA Local circuit neurons immunoreactive for calretinin, calbindin D-28kor parvalbumin in monkey prefrontal cortex: distribution and morphology. J Comp Neurol. 1994;34195- 116
PubMed Link to Article
Gabbott  PLBacon  SJ Local circuit neurons in the medial prefrontal cortex (areas 24a,b,c,25 and 32) in the monkey, II: quantitative areal and laminar distributions. J Comp Neurol. 1996;364609- 636
PubMed Link to Article
Kawaguchi  YKondo  S Parvalbumin, somatostatin and cholecystokinin as chemical markers forspecific GABAergic interneuron types in the rat frontal cortex. J Neurocytol. 2002;31277- 287
PubMed Link to Article
Benes  FMWalsh  JBhattacharyya  SSheth  ABerretta  S DNA fragmentation decreased in schizophrenia but not bipolar disorder. Arch Gen Psychiatry. 2003;60359- 364
PubMed Link to Article
Kawaguchi  YKubota  Y GABAergic cell subtypes and their synaptic connections in rat frontalcortex. Cereb Cortex. 1997;7476- 486
PubMed Link to Article
Somogyi  PTamas  GLujan  RBuhl  EH Salient features of synaptic organisation in the cerebral cortex. Brain Res Brain Res Rev. 1998;26113- 135
PubMed Link to Article
Gupta  AWang  YMarkram  H Organizing principles for a diversity of GABAergic interneurons andsynapses in the neocortex. Science. 2000;287273- 278
PubMed Link to Article

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