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Original Article |

Decreased Glutamic Acid Decarboxylase67 Messenger RNA Expression in a Subset of Prefrontal Cortical γ-Aminobutyric Acid Neurons in Subjects With Schizophrenia FREE

David W. Volk, MS; Mark C. Austin, PhD; Joseph N. Pierri, MS, MD; Allan R. Sampson, PhD; David A. Lewis, MD
[+] Author Affiliations

From the Departments of Neuroscience (Mr Volk and Dr Lewis), Psychiatry (Drs Austin, Pierri, and Lewis), and Statistics (Dr Sampson), University of Pittsburgh, Pittsburgh, Pa.


Arch Gen Psychiatry. 2000;57(3):237-245. doi:10.1001/archpsyc.57.3.237.
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Background  Markers of γ-aminobutyric acid (GABA) neurotransmission seem to be altered in the prefrontal cortex (PFC) of subjects with schizophrenia. We sought to determine whether the expression of the messenger RNA (mRNA) for the synthesizing enzyme of GABA, glutamic acid decarboxylase67 (GAD67), is decreased in the PFC of subjects with schizophrenia, whether this change is present in all or only some GABA neurons, and whether long-term treatment with haloperidol decanoate contributes to altered GAD67 mRNA expression.

Methods  Tissue sections from 10 pairs of subjects with schizophrenia and control subjects and 4 pairs of haloperidol-treated and control monkeys were processed for in situ hybridization histochemical analysis with sulfur-35–labeled oligonucleotide probes for GAD67 mRNA and exposed to nuclear emulsion. Within each layer of PFC area 9, neurons expressing a detectable level of GAD67 mRNA were quantified for cell density and the relative level of mRNA expression per cell (grain density per neuron).

Results  In subjects with schizophrenia, the density of labeled neurons was significantly (P<.05) decreased by 25% to 35% in cortical layers 3 to 5. In contrast, the mean grain density per labeled neuron did not differ across subject groups. Similar analyses in monkeys revealed no effect of long-term haloperidol treatment on either the density of the labeled neurons or the grain density per labeled neuron.

Conclusions  These findings indicate that in subjects with schizophrenia, GAD67 mRNA expression is relatively unaltered in most PFC GABA neurons but is reduced below a detectable level in a subset of GABA neurons. Altered GABA neurotransmission in this subset may contribute to PFC dysfunction in subjects with schizophrenia.

Figures in this Article

CORTICAL markers of γ-aminobutyric acid (GABA) neurotransmission seem to be altered in subjects with schizophrenia. The amount and activity of the synthesizing enzyme for GABA, glutamate decarboxylase (GAD), and the release and uptake of GABA are decreased in subjects with schizophrenia.14 Other studies have found alterations in GABAA receptors, including increased hydrogen-3–muscimol binding,57 an increased density of structures immunoreactive for the α and β2/3 subunits of the GABAA receptor,8 and a shift in the ratio of the messenger RNAs (mRNAs) encoding the splice variants of the γ2 subunit in the prefrontal cortex (PFC) of subjects with schizophrenia.9

Akbarian et al10 found that the number of neurons expressing a detectable level of mRNA for the 67-kd isoform of GAD (glutamic acid decarboxylase67 [GAD67]) was decreased in PFC area 9, located in the superior frontal gyrus, of subjects with schizophrenia. However, the potential confounding factors associated with postmortem studies require that this finding be independently replicated. Furthermore, since the relative level of GAD67 mRNA expression per neuron was not determined by Akbarian and colleagues, it is unclear whether GAD67 mRNA expression was decreased in all GABA neurons such that some were no longer detectable or whether the decrease was primarily limited to a subset of GABA neurons.11 Finally, the long-term pharmacological treatment of schizophrenia necessitates determining if long-term treatment with antipsychotic medications contributes to alterations in GAD67 mRNA expression by examining, for example, monkeys treated with haloperidol in a manner that reflects clinical practice. Consequently, in our study we sought to answer the following questions: (1) Is the density of neurons expressing a detectable level of GAD67 mRNA decreased in PFC area 9 in a new cohort of subjects with schizophrenia? (2) If so, is the relative level of GAD67 mRNA expression decreased in all or only a subset of PFC GABA neurons? (3) Does long-term treatment with haloperidol alter GAD67 mRNA expression in the PFC of monkeys?

SUBJECTS

Brain specimens were obtained during autopsies conducted at the Allegheny County Coroner's Office, Pittsburgh, Pa, after obtaining consent from the next of kin. Ten subjects with schizophrenia, each matched to one control subject for sex, age, and postmortem interval, were used in our study (Table 1). Pairs were completely matched for sex, and the mean (SD) differences within pairs was 5.0 (4.0) years for age and 4.8 (2.8) hours for postmortem interval. Additionally, subject groups did not differ in mean (SD) freezer storage time or postmortem brain pH (Table 1). An independent panel of experienced clinicians arrived at consensus DSM-III-R12 diagnoses for each subject after reviewing medical records and the results of structured interviews conducted with family members of the deceased.13 These interviews also revealed a history of depressive disorder (not otherwise specified) in 1 control subject (case 635), and the presence of alcohol abuse, current at the time of death, in another control subject (case 558); no psychiatric disorders were present in the other control subjects. Four subjects with schizophrenia also had a history of an alcohol and/or other substance abuse disorder (Table 1). Findings from toxicology studies conducted on all subjects were positive for alcohol (46-276 mmol/L) in 3 control subjects; no other drugs of abuse were detected in any subject. Two subjects with schizophrenia (cases 537 and 622) had not been receiving antipsychotic medications for 9.6 and 1.2 months prior to death, respectively (Table 1. The mean (SD) age of subjects with schizophrenia at the onset of illness was 26.4 (10.4) years, and the average duration of illness was 18.8 (8.0) years. The brain specimens used in our study were obtained from a community-based population; consequently, most subjects (7 with schizophrenia and 9 controls) died suddenly outside of a hospital setting.

Table Graphic Jump LocationDemographic Characteristics of Subjects*

Findings from neuropathological examination of each brain revealed abnormalities only in 1 subject (case 622) in whom an infarction limited to the distribution of the inferior branch of the right middle cerebral artery was discovered. However, PFC area 9 appeared to be unaffected. Additionally, thioflavine S staining revealed a few senile plaques in 1 subject (case 685), but clinical and neuropathological criteria for Alzheimer disease were not met.14 All procedures in our study were approved by the University of Pittsburgh's Institutional Review Board for Biomedical Research.

TISSUE PREPARATION AND IN SITU HYBRIDIZATION PROCEDURE

The PFC of the right hemisphere was blocked coronally and immediately frozen and stored at −80°C. Coronal tissue sections (20 µm) containing the superior frontal gyrus were cut and thaw mounted onto slides and stored at −80°C until processed. Every 10th section was stained for Nissl substance, and these sections were used to identify the location of area 9 using cytoarchitectonic criteria.15

A cocktail of 3 oligonucleotide probes (Oligos Etc, Wilsonville, Ore) complementary to bases 808 through 849, 1059 through 1106, and 2657 through 2704 of human GAD67 complementary DNA were used to detect GAD67 mRNA.16 The probe specificity for GAD67 mRNA was previously demonstrated by experiments in which (1) an excess of unlabeled probes eliminated specific hybridization signal, (2) the use of each probe alone revealed similar patterns of cellular localization, and (3) the combination of 2 probes produced an increase in hybridization signal.17 The probes were labeled with 35S-dATP (NEN, Boston, Mass) using terminal deoxynucleotidyl transferase (Bethesda Research Laboratories, Gaithersburg, Md).

All tissue sections from each subject pair were processed together. Eight sections from each subject were processed for in situ hybridization as previously described.18 Briefly, sections were incubated overnight in hybridization buffer containing a cocktail of sulfur 35–labeled oligonucleotide probe (1.5 × 106 disintegrations per minute per section). After a series of stringent washes, sections were coated with photographic emulsion (NTB2; Eastman Kodak, Rochester, NY) and developed after 4 weeks of exposure. Tissue sections were then counterstained with cresyl violet.

QUANTIFICATION OF GAD67 mRNA EXPRESSION

Slides were coded to conceal subject number and diagnosis from the investigator (D.W.V.). Four tissue sections from each subject were randomly chosen for analysis. Using a Microcomputer Imaging Device (MCID; Imaging Research Inc, London, Ontario), three 140 × 140-µm sampling frames were randomly placed in each cortical layer (Figure 1). The bright-field image (objective ×40) of the sampling frame was digitized, and the outline of each Nissl-stained soma with at least 2 overlying silver grains was traced on the computer monitor at a final magnification of ×1510.19 In a dark-field image of the same sampling frame, the number of grains over each encircled cell was then counted by the Microcomputer Imaging Device system. More than 10,000 cells were sampled in our study, ranging from 310 to 649 cells per subject. The mean (SD) coefficient of error for cell counts in each subject, averaged across layers, was identical in the schizophrenic and control groups (0.14 [0.05]) and did not differ in any layer between the groups (paired t test, t9<2.4, P>.24). For background measurements, the number of grains in 3 sampling frames randomly placed in the subjacent white matter was determined. The thickness of the gray matter in area 9 was determined by measuring the distance from the pial surface to the layer 6–white matter border at 3 locations on 2 Nissl-stained sections located immediately rostral and caudal to the tissue sections processed for in situ hybridization.

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Figure 1.

Nissl-stained coronal tissue section from prefrontal cortex area 9 showing the approximate placement of sampling frames (140 µm × 140 µm) in each cortical layer as well as in the subjacent white matter (WM) (scale bar, 200 µm).

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HALOPERIDOL-TREATED MONKEYS

To mimic the clinical treatment of subjects with schizophrenia, 4 male cynomolgus (Macaca fascicularis) monkeys were treated with haloperidol and benztropine mesylate for 9 to 12 months as previously described.20,21 Mean (SD) trough serum haloperidol levels (11 [3] nmol/L) were within the reported therapeutic range for the treatment of schizophrenia.22 Each haloperidol-treated animal was matched to one control animal for sex, age (using bone dating and/or an assessment of developmental stage), and weight (haloperidol-treated animals, 3.8 [1.1] kg; control animals, 4.0 [1.0] kg). Both animals in each matched pair were euthanized at the same time, and following a 45-minute postmortem interval, coronal tissue blocks were frozen and stored at −80°C.

Tissue processing procedures were identical to those described previously in this article with a few minor modifications in section thickness and exposure time. The mean (SD) coefficient of error for neuron counts in each subject averaged across layers was 0.07 (0.01) in both subject groups and did not differ in any layer between the groups (paired t test, t3<1.4; P>.26).

DATA ANALYSIS

To measure the relative level of GAD67 mRNA expression per neuron, grain density per neuron (grains per 100 µm2 somal area) was determined for each sampled cell. Grain density per neuron was then corrected for nonspecific background labeling by subtracting the average background level of grains in the subjacent white matter of the same tissue section. To identify a threshold of grain density per neuron that would exclude nonspecifically labeled cells from analysis, histograms of grain density per neuron for all sampled neurons per layer from the controls and subjects with schizophrenia were constructed. These histograms revealed a distribution that appeared bimodal in each layer, representing the modes of nonspecifically and specifically labeled neuron populations.23 Similar histograms including only neurons with a grain density greater than the background ×5 showed a distribution that appeared normal and unimodal in both the schizophrenic and control groups. Therefore, a threshold of the background ×5 provided a cutoff at the point of rarity in the distribution of all cells that permitted the identification of specifically labeled neurons. Thus, only neurons with a grain density greater than the background ×5 of the individual tissue section were included in the data analysis and are subsequently referred to as GAD67 mRNA-positive (+) neurons. The mean neuron density, mean grain density per neuron, and mean cross-sectional somal area of all GAD67 mRNA+ neurons were then determined for every layer of every subject.

For each section, the values of the 3 dependent variables (neuron density, grain density per neuron, and somal size) were averaged across the 3 sampling frames for each cortical layer, with the value of each sampling frame weighted by the number of observations within that frame. Thus, in every layer of each subject, 4 section averages were obtained for each dependent variable. These 4 averages were treated as repeated measures with a compound symmetric covariance structure24 because the values were possibly correlated and were also exchangeable within a given subject. Pair effect was included to reflect the matching of subjects with schizophrenia with controls for sex, age, and postmortem interval. Postmortem brain pH was included as a covariate because it may reflect the integrity of some mRNA species.25 Thus, the effect of diagnostic group on each of the 3 dependent variables in each layer was examined using a multivariant analysis of covariance model with the 4 section averages having a compound symmetric covariance matrix, with pair as a blocking effect and brain pH as a covariate.

For all measures, the Holm simultaneous inference procedure26 was used to identify which of the 6 layers showed a significant diagnostic group effect for that variable. The Holm procedure maintains the overall family-wise error rate at the .05 level and tests individual laminar diagnostic effects at certain prescribed significance levels. Specifically, the P values, Pi*, for diagnostic group effect are ordered from smallest (i* = 1) to largest (i* = 6) among the 6 layers. The layer corresponding to Pi* is declared to have a significant diagnostic effect at the family-wise .05 level if Pi*≤.05/[(N + 1) − i*], where N is the number of comparisons. For example, the laminar level corresponding to the smallest P value (i* = 1) is declared to have a significant diagnostic effect if that P value ≤.05/[(6 + 1) − 1] = .0083. To maintain consistency throughout the text, the prescribed significance level, and consequently the quoted P value, for each laminar diagnostic group have been adjusted to correspond with the family-wise error rate of .05 (ie, Pi* × [(N + 1) − i*]). For the haloperidol-treated monkeys, 2-tailed paired t tests and the Holm correction were used to determine the effect of treatment group on each of the 3 dependent variables in each cortical layer.

SPECIFICITY OF OLIGONUCLEOTIDE PROBES FOR GAD67 mRNA

The specificity of the oligonucleotide probes for GAD67 mRNA was confirmed by the morphological characteristics and laminar distribution of labeled neurons. Specific hybridization signal, or the clustering of silver grains over Nissl-stained cell bodies, was clearly present for small- and medium-sized neurons but was noticeably absent for both pyramidal neurons and glial cells (Figure 2). Additionally, the relative laminar densities of GAD67 mRNA+ neurons, greatest in layers 2 and 4 and lowest in layer 6 (Figure 3), matched the reported laminar distribution of GAD67 mRNA expression in human PFC.10 These observations, in concert with previously reported data,17 demonstrate the specificity of these oligonucleotide probes for GAD67 mRNA.

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Figure 2.

Prefrontal cortex area 9 from a control subject (case 567) processed by in situ hybridization for glutamic acid decarboxylase67 messenger RNA and then counterstained with cresyl violet. Note the accumulation of silver grains over small- and medium-sized neurons (open arrows) but not over the larger pyramidal neurons (large arrows) or the smaller glial cells (small arrows) (scale bar, 50 µm).

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Figure 3.

Prefrontal cortex area 9 of a control subject (case 558) showing the laminar distribution of grain clusters, representing glutamic acid decarboxylase67 messenger RNA+ neurons. Note that the density of labeled neurons appears greatest in layers 2 and 4, intermediate in layers 3 and 5, and lowest in layers 1 and 6. WM indicates white matter (scale bar, 300 µm).

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GAD67 mRNA EXPRESSION IN HUMAN PFC

As shown in Figure 4, the density of grain clusters, representing GAD67 mRNA+ neurons, appear to be decreased in subjects with schizophrenia compared with matched controls. This qualitative impression was confirmed by quantitative analyses, which revealed that the mean density of GAD67 mRNA+ neurons was decreased by 25% to 35% in layers 1 through 5 in subjects with schizophrenia (Figure 5, top). Statistical analysis of GAD67 mRNA+ neuron density in each layer revealed a significant effect of diagnosis in layers 3 through 5 (superficial layer 3, F1,8 = 10.64, P = .046; layer 3-4 border, F1,8 = 12.51, P = .046; and layer 5, F1,8 = 11.82, P = .044), a trend in layers 1 and 2 (layer 1, F1,8 = 5.34, P = .099; and layer 2, F1,8 = 8.66, P = .056), and no effect in layer 6 (F1,8 = 0.79, P = .40). Furthermore, in each of layers 1 through 5, at least 8 of 10 subject pairs showed a decrease in GAD67 mRNA+ neuron density in the subject with schizophrenia (Figure 6).

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Figure 4.

Layer 3 of prefrontal cortex area 9 showing glutamic acid decarboxylase67 messenger RNA expression in a matched pair of a control (top) and a subject with schizophrenia (bottom) (Table 1, pair 1). Note the apparent decrease in number of grain clusters in the subject with schizophrenia (scale bar, 150 µm).

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Figure 5.

Bar graphs showing (top) the mean (SD) density of glutamic acid decarboxylase67 (GAD67) messenger RNA+ (mRNA+) neurons and (bottom) the mean grain density per GAD67 mRNA+ neuron in each prefrontal cortex area 9 layer of the schizophrenic and control groups. 3s indicates superficial layer 3.

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Figure 6.

Comparison of glutamic acid decarboxylase67 (GAD67) messenger RNA+ (mRNA+) neuron density within each matched pair of subjects with schizophrenia and control subjects for superficial layer 3, layer 3-4 border, and layer 5. Eight of 10 pairs in superficial layer 3 and layer 5 and 9 of 10 pairs in layer 3-4 border show a decrease in GAD67 mRNA+ neuron density in the subject with schizophrenia. Closed circles indicate pairs in which the value for the control subject is greater than the value for the matched subject with schizophrenia; open triangles, pairs in which the value for the control subject is less than the value for the matched subject with schizophrenia.

Graphic Jump Location

In contrast to these differences in the density of labeled neurons, the mean grain density per GAD67 mRNA+ neuron, a relative measure of GAD67 mRNA expression per neuron, did not differ (F1,8<4.1, P>.45) in any layer between subjects with schizophrenia and control subjects (Figure 5, bottom). The mean cross-sectional somal area of GAD67 mRNA+ neurons also did not differ (F1,8<4.1, P>.43) in any layer between subjects with schizophrenia and control subjects. Furthermore, the mean (SD) thickness of the cortical gray matter for the subjects with schizophrenia (2.73 [0.33] mm) and control subjects (3.02 [0.47] mm) also did not differ (t9 = −1.65, P = .13).

GAD67 mRNA EXPRESSION IN THE PFC OF MONKEYS

In the monkey tissue, silver grains were also selectively clustered over small- and medium-sized neurons, with the density of GAD67 mRNA+ neurons greatest in layers 2 and 4. This laminar distribution matched that of GABA-immunoreactive neurons in the PFC of cynomolgus monkeys.27 However, in contrast to the observations in humans, neither neuron density nor grain density of the GAD67 mRNA+ neurons differed significantly (t3<3.47, P>.24) in any layer between haloperidol-treated monkeys and controls (Figure 7).

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Figure 7.

Bar graphs showing (top) the mean (SD) density of glutamic acid decarboxylase67 (GAD67) messenger RNA+ (mRNA+) neurons and (bottom) the mean grain density per GAD67 mRNA+ neuron in each layer of the prefrontal cortex in haloperidol-treated monkeys and control monkeys. No significant differences were observed for either measure in any layer. 3s indicates superficial layer 3.

Graphic Jump Location

We found that the density of GAD67 mRNA+ neurons was significantly reduced in layers 3 through 5, with a trend (P = .06) toward a reduction in layer 2 of PFC area 9 in a new cohort of subjects with schizophrenia. In contrast, grain density per GAD67 mRNA+ neuron, a relative measure of the cellular level of GAD67 mRNA expression, did not differ between subject groups. Together, these observations suggest that in the PFC of subjects with schizophrenia, GAD67 mRNA expression is relatively unaltered in most GABA neurons but is reduced below a detectable level in a subset of GABA neurons. In addition, the results from the study of haloperidol-treated monkeys suggest that decreased GAD67 mRNA expression in the PFC of subjects with schizophrenia is not a consequence of long-term treatment with haloperidol.

Akbarian et al10 previously reported a 30% to 50% decrease in GAD67 mRNA+ neuron density in layers 1 through 5 of PFC area 9 from the left hemisphere. In the present study, GAD67 mRNA+ neuron density was also decreased by 25% to 35% in these same layers of area 9 from the right PFC, suggesting a common, bilateral decrease in GAD67 mRNA+ neuron density in the PFC of subjects with schizophrenia. In addition, decreased GAD protein has been reported in the temporal cortex of subjects with schizophrenia.4 Together, these observations suggest that decreased GAD67 mRNA expression in the association regions of the neocortex may be a frequent feature of schizophrenia.

Comparisons with other studies suggest that the decrease in the density of GAD67 mRNA+ neurons was not due to a decrease in the number of PFC neurons in subjects with schizophrenia. Our finding of a decreased density of GAD67 mRNA+ neurons is strikingly similar in magnitude and laminar distribution to the previous report by Akbarian et al,10 who also found that the density of small, round, presumably GABA cells was unchanged. Consistent with this observation, most previous studies have reported either no change or an increase in neuron density,10,15,2831 or no change in total neuron number32 in the PFC of subjects with schizophrenia. In addition, in the same cortical region of the same subjects used in the present study, we found no change in the density of neurons expressing synaptophysin mRNA,33 which is found in virtually every cortical neuron, providing direct evidence that the present cohort of subjects with schizophrenia does not have a reduced number of neurons.

Our results suggest that a subset of GABA neurons does not express a detectable level of GAD67 mRNA in subjects with schizophrenia. The axon terminals of the chandelier subclass of GABA neurons have been reported to be selectively altered in subjects with schizophrenia,21,34 and we found that the density of GAD67 mRNA+ neurons was decreased in layers 2 through 5, the primary location of chandelier neurons.35 However, the magnitude of the decrease in GAD67 mRNA+ neuron density suggests that other subpopulations of GABA neurons may be affected as well.

The typical long-term exposure of subjects with schizophrenia to antipsychotic medications requires determining whether pharmacotherapy may contribute to the altered expression of GAD67 mRNA in the PFC. Although long-term treatment with haloperidol and other dopamine D2-like receptor antagonists can reportedly affect GAD67 mRNA expression in the rat basal ganglia,3639 it was unclear whether long-term use of antipsychotic medications could also affect GAD67 mRNA expression in the PFC, where the density of D2-like receptors is much lower.40,41 In our study, long-term treatment with haloperidol did not affect GAD67 mRNA expression in the PFC of monkeys. Consistent with this observation, the 2 subjects with schizophrenia (cases 537 and 622) who were not receiving antipsychotic medications at the time of death had densities of GAD67 mRNA+ neurons less than that of their matched controls. In addition, the 4 subjects who had received atypical antipsychotic agents (Table 1) showed a similar decrease in GAD67 mRNA+ neuron density compared with those who had been treated only with typical antipsychotics.

Although stereological approaches to determine absolute cell number are advantageous in many situations, they remain problematic in studies of individual cortical regions lacking clearly delineated boundaries and in studies using in situ hybridization.42 Thus, a 2-dimensional sampling technique was used to make relative comparisons of differences in neuron density and, importantly, was not confounded by cross-subject differences in somal size. In addition, the modest reduction of cortical gray matter in subjects with schizophrenia observed in many other studies4347 would be expected to elevate neuronal density. Consistent with these reports, we found a 10% (but nonsignificant) decrease in cortical gray matter in subjects with schizophrenia. Thus, the observed 25% to 35% decrease in the relative density of GAD67 mRNA+ neurons may actually be an underestimate of the differences between subjects with schizophrenia and control subjects.

A threshold of grain density per neuron (>5× the background) was identified to exclude nonspecifically labeled cells from analysis. The use of a lower threshold (>3× the background) revealed similar differences in neuron density and grain density per GAD67 mRNA+ neuron. Thus, the level of GAD67 mRNA expression in a subset of GABA neurons appears to be so low that these neurons are still not detectable even when a less stringent threshold for specific labeling is used.

Premortem agonal state events may affect postmortem levels of some mRNA species.25 Brain pH, reportedly an inverse correlate of agonal state,25 did not differ between the subject groups in our study. Additionally, GAD67 mRNA was not generally degraded in the subjects with schizophrenia because most GAD67 mRNA+ neurons in subjects in the schizophrenic group had normal levels of GAD67 mRNA expression. Furthermore, a similar analysis of the cellular levels of synaptophysin mRNA revealed no differences between the same subjects with schizophrenia and control subjects examined in the present study.33

Four subjects with schizophrenia in our study met criteria for a substance abuse disorder (Table 1), but the available data suggest that these comorbid conditions did not contribute to the decreased density of GAD67 mRNA+ neurons. First, the only 2 subjects with schizophrenia who, compared with their matched control subject, showed a similar or increased density of GAD67 mRNA+ neurons in several layers both met criteria for alcohol abuse (Table 1, pairs 6 and 9). Second, the only control subject (case 558) with alcohol abuse had a higher GAD67 mRNA+ neuron density than the matched subject with schizophrenia. Finally, the 3 control subjects with positive plasma alcohol levels (46-276 mmol/L) still had a higher density of GAD67 mRNA+ neurons than their matched subjects with schizophrenia.

Our study provides further insight into the potential pathophysiological mechanisms underlying altered PFC GABA neurotransmission in subjects with schizophrenia. The alteration in GAD67 mRNA expression may reflect an intrinsic defect in a subset of PFC GABA neurons. For example, in PFC area 9 in subjects with schizophrenia, the decreased density of chandelier neuron axon terminals immunoreactive for the GABA membrane transporter21,34 suggests that the uptake of GABA is impaired at these axon terminals. As a consequence, inhibitory GABA activity may be increased at postsynaptic sites.48 Mice lacking the dopamine transporter exhibit evidence of excessive dopamine activity and show a 90% decrease in the level of tyrosine hydroxylase, the rate-limiting enzyme for dopamine synthesis.49,50 If the same relationships hold true for the GABA system, then GAD67 mRNA expression may be down-regulated in chandelier neurons as a compensatory response to excessive GABA activity.

Alternatively, an abnormality in afferents to the PFC may result in a reduced level of GAD67 mRNA expression in the PFC. For example, several studies of schizophrenia have found decreased neuron number in and/or volume of the mediodorsal nucleus of the thalamus,5154 a major source of excitatory input to the PFC. In addition, reports of fewer dendritic spines55,56 and axon terminals57 in PFC layers 3 and 4, the principal termination zone of projections from the thalamus, are consistent with a decrease in these afferents in subjects with schizophrenia. Monocular deprivation studies in monkeys indicate that a loss of thalamic input produces decreased GAD67 mRNA expression in layer 4 and adjacent layers of visual cortex.58,59 Thus, the decreased expression of GAD67 mRNA observed in our study may reflect a down-regulation of inhibition in the PFC to compensate for a decrease in excitatory drive from the mediodorsal nucleus of the thalamus. Further studies are needed to discriminate between these or other possible mechanisms underlying decreased GAD67 mRNA expression in a subset of GABA neurons in subjects with schizophrenia.

Accepted for publication November 6, 1999.

This work was supported by grants MH43784, MH00519, and MH45156 from the National Institutes of Health, Bethesda, Md (Dr Lewis).

We thank Sandra O'Donnell, BS, for technical assistance, Mary Brady, BS, for photographic assistance, and Sungyoung Auh, MS, for statistical consultation.

Corresponding author: David A. Lewis, MD, University of Pittsburgh, 3811 O'Hara St, W1650 BST, Pittsburgh, PA 15213 (e-mail: lewisda@msx.upmc.edu).

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Pierri  JNChaudry  ASWoo  T-ULewis  DA Alterations in chandelier neuron axon terminals in the prefrontal cortex of schizophrenic subjects. Am J Psychiatry. 1999;1561709- 1719
Janicak  PGDavis  JMPreskorn  SHAyd  FJ Principles and Practice of Psychopharmacotherapy.  Baltimore, Md Williams & Wilkins1993;
Gerfen  CRMcGinty  JFYoung  WS Dopamine differentially regulates dynorphin, substance P, and enkaphalin expression in striatal neurons: in situ hybridization histochemical analysis. J Neurosci. 1991;111016- 1031
Neter  JKutner  MHNachtsheim  CJWasserman  W Applied Linear Statistical Models.  Chicago, Ill Irwin1996;
Harrison  PJHeath  PREastwood  SLBurnet  PWJMcDonald  BPearson  RCA The relative importance of premortem acidosis and postmortem interval for human brain gene expression studies: selective mRNA vulnerability and comparison with their encoded proteins. Neurosci Lett. 1995;200151- 154
Holm  S A simple sequentially rejective multiple test procedure. Scand J Stat. 1979;665- 70
Hendry  SHCSchwark  HDJones  EGYan  J Numbers and proportions of GABA-immunoreactive neurons in different areas of monkey cerebral cortex. J Neurosci. 1987;71503- 1519
Benes  FMMcSparren  JBird  EDSanGiovanni  JPVincent  SL Deficits in small interneurons in prefrontal and cingulate cortices of schizophrenic and schizoaffective patients. Arch Gen Psychiatry. 1991;48996- 1001
Selemon  LDRajkowska  GGoldman-Rakic  PS Abnormally high neuronal density in the schizophrenic cortex: a morphometric analysis of prefrontal area 9 and occipital area 17. Arch Gen Psychiatry. 1995;52805- 818
Woo  T-UMiller  JLLewis  DA Parvalbumin-containing cortical neurons in schizophrenia. Am J Psychiatry. 1997;1541013- 1015
Selemon  LDRajkowska  GGoldman-Rakic  PS Elevated neuronal density in prefrontal area 46 in brains from schizophrenic patients: application of a three-dimensional, stereologic counting method. J Comp Neurol. 1998;392402- 412
Thune  JJHofsten  DEUylings  HBMPakkenberg  B Total neuron numbers in the prefrontal cortex in schizophrenia [abstract]. Soc Neurosci Abstracts. 1998;24985
Glantz  LAAustin  MCLewis  DA Cellular levels of synaptophysin mRNA expression in the prefrontal cortex of subjects with schizophrenia [abstract]. Soc Neurosci Abstracts. 1998;24987
Woo  T-UWhitehead  REMelchitzky  DSLewis  DA A subclass of prefrontal γ-aminobutyric acid axon terminals are selectively altered in schizophrenia. Proc Natl Acad Sci U S A. 1998;955341- 5346
Lewis  DALund  JS Heterogeneity of chandelier neurons in monkey neocortex: corticotropin-releasing factor and parvalbumin immunoreactive populations. J Comp Neurol. 1990;293599- 615
Delfs  JMEllison  GDMercugliano  MChesselet  M-F Expression of glutamic acid decarboxylase mRNA in striatum and pallidum in an animal model of tardive dyskinesia. Exp Neurol. 1995;133175- 188
Chen  JFWeiss  B Irreversible blockade of D2 dopamine receptors by fluphenazine-N-mustard increases glutamic acid decarboxylase mRNA in rat striatum. Neurosci Lett. 1993;150215- 218
Jolkkonen  JJenner  PMarsden  CD GABAergic modulation of striatal peptide expression in rats and the alterations induced by dopamine antagonist treatment. Neurosci Lett. 1994;180273- 276
Delfs  JMAnegawa  NJChesselet  M-F Glutamate decarboxylase messenger RNA in rat pallidum: comparison of the effects of haloperidol, clozapine, and combined haloperidol-scopolamine treatments. Neuroscience. 1995;6667- 80
Camps  MCortés  RGueye  BProbst  APalacios  JM Dopamine receptors in human brain: autoradiographic distribution of D2 sites. Neuroscience. 1989;28275- 290
Hall  HSedvall  GMagnusson  OKopp  JHalldin  CFarde  L Distribution of D1- and D2-dopamine receptors, and dopamine and its metabolites in the human brain. Neuropsychopharmacology. 1994;11245- 256
Guillery  RWHerrup  K Quantification without pontification: choosing a method for counting objects in sectioned tissues. J Comp Neurol. 1997;3862- 7
Shelton  RCKarson  CNDoran  ARPickar  DBigelow  LBWeinberger  DR Cerebral structural pathology in schizophrenia: evidence for a selective prefrontal cortical defect. Am J Psychiatry. 1988;145154- 163
Breier  ABuchanan  RWElkashef  AMunson  RCKirkpatrick  BGellad  F Brain morphology and schizophrenia: a magnetic resonance imaging study of limbic, prefrontal cortex, and caudate structures. Arch Gen Psychiatry. 1992;49921- 926
Zipursky  RBLim  KOSullivan  EVBrown  BWPfefferbaum  A Widespread cerebral gray matter volume deficits in schizophrenia. Arch Gen Psychiatry. 1992;49195- 205
Zipursky  RBLambe  EKKapur  SMikulis  DJ Cerebral gray matter volume deficits in first episode psychosis. Arch Gen Psychiatry. 1998;55540- 546
Andreasen  NCFlashman  LFlaum  MArndt  SSwayze  V  IIO'Leary  DSEhrhardt  JCYuh  WT Regional brain abnormalities in schizophrenia measured with magnetic resonance imaging. JAMA. 1994;2721763- 1769
Isaacson  JSSolis  JMNicoll  RA Local and diffuse synaptic action of GABA in the hippocampus. Neuron. 1993;10165- 175
Giros  BJaber  MJones  SRWightman  RMCaron  MG Hyperlocomotion and indifference to cocaine and amphetamine in mice lacking the dopamine transporter. Nature. 1996;379606- 612
Jones  SRGainetdinov  RRJaber  MGiro  BWightman  RM Profound neuronal plasticity in response to inactivation of the dopamine transporter. Proc Natl Acad Sci U S A. 1998;954029- 4034
Pakkenberg  B Pronounced reduction of total neuron number in mediodorsal thalamic nucleus and nucleus accumbens in schizophrenics. Arch Gen Psychiatry. 1990;471023- 1028
Manaye  KFLiang  C-LHicks  PBGerman  DYoung  KA Nerve cell numbers in thalamic anterior and mediodorsal nuclei are selectively reduced in schizophrenia [abstract]. Soc Neurosci Abstracts. 1998;241236
Jones  LMall  NByne  W Localization of schizophrenia-associated thalamic volume loss [abstract]. Soc Neurosci Abstracts. 1998;24985
Popken  GJBunney  WE  JrPotkin  SGJones  EG Neuron number and GABAergic and glutamatergic mRNA expression in subdivisions of the thalamic mediodorsal nucleus of schizophrenics [abstract]. Soc Neurosci Abstracts. 1998;24991
Glantz  LALewis  DA Decreased dendritic spine density on prefrontal cortical pyramidal neurons in schizophrenia. Arch Gen Psychiatry. 2000;5765- 73
Garey  LJOng  WYPatel  TSKanani  MDavis  AMortimer  AMBarnes  TREHirsch  SR Reduced dendritic spine density on cerebral cortical pyramidal neurons in schizophrenia. J Neurol Neurosurg Psychiatry. 1998;65446- 453
Cruz  DAMelchitzky  DSLewis  DA Parvalbumin-immunoreactive varicosities are selectively decreased in the thalamic recipient zone of the prefrontal cortex in schizophrenia [abstract]. Soc Neurosci Abstracts. 1999;25817
Hendry  SHCJones  EG Activity-dependent regulation of GABA expression in the visual cortex of adult monkeys. Neuron. 1988;1701- 712
Benson  DLHuntsman  MMJones  EG Activity-dependent changes in GAD and preprotachykinin mRNAs in visual cortex of adult monkeys. Cereb Cortex. 1994;440- 51

Figures

Place holder to copy figure label and caption
Figure 1.

Nissl-stained coronal tissue section from prefrontal cortex area 9 showing the approximate placement of sampling frames (140 µm × 140 µm) in each cortical layer as well as in the subjacent white matter (WM) (scale bar, 200 µm).

Graphic Jump Location
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Figure 2.

Prefrontal cortex area 9 from a control subject (case 567) processed by in situ hybridization for glutamic acid decarboxylase67 messenger RNA and then counterstained with cresyl violet. Note the accumulation of silver grains over small- and medium-sized neurons (open arrows) but not over the larger pyramidal neurons (large arrows) or the smaller glial cells (small arrows) (scale bar, 50 µm).

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

Prefrontal cortex area 9 of a control subject (case 558) showing the laminar distribution of grain clusters, representing glutamic acid decarboxylase67 messenger RNA+ neurons. Note that the density of labeled neurons appears greatest in layers 2 and 4, intermediate in layers 3 and 5, and lowest in layers 1 and 6. WM indicates white matter (scale bar, 300 µm).

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

Layer 3 of prefrontal cortex area 9 showing glutamic acid decarboxylase67 messenger RNA expression in a matched pair of a control (top) and a subject with schizophrenia (bottom) (Table 1, pair 1). Note the apparent decrease in number of grain clusters in the subject with schizophrenia (scale bar, 150 µm).

Graphic Jump Location
Place holder to copy figure label and caption
Figure 5.

Bar graphs showing (top) the mean (SD) density of glutamic acid decarboxylase67 (GAD67) messenger RNA+ (mRNA+) neurons and (bottom) the mean grain density per GAD67 mRNA+ neuron in each prefrontal cortex area 9 layer of the schizophrenic and control groups. 3s indicates superficial layer 3.

Graphic Jump Location
Place holder to copy figure label and caption
Figure 6.

Comparison of glutamic acid decarboxylase67 (GAD67) messenger RNA+ (mRNA+) neuron density within each matched pair of subjects with schizophrenia and control subjects for superficial layer 3, layer 3-4 border, and layer 5. Eight of 10 pairs in superficial layer 3 and layer 5 and 9 of 10 pairs in layer 3-4 border show a decrease in GAD67 mRNA+ neuron density in the subject with schizophrenia. Closed circles indicate pairs in which the value for the control subject is greater than the value for the matched subject with schizophrenia; open triangles, pairs in which the value for the control subject is less than the value for the matched subject with schizophrenia.

Graphic Jump Location
Place holder to copy figure label and caption
Figure 7.

Bar graphs showing (top) the mean (SD) density of glutamic acid decarboxylase67 (GAD67) messenger RNA+ (mRNA+) neurons and (bottom) the mean grain density per GAD67 mRNA+ neuron in each layer of the prefrontal cortex in haloperidol-treated monkeys and control monkeys. No significant differences were observed for either measure in any layer. 3s indicates superficial layer 3.

Graphic Jump Location

Tables

Table Graphic Jump LocationDemographic Characteristics of Subjects*

References

Simpson  MDCSlater  PDeakin  JFWRoyston  MCSkan  WJ Reduced GABA uptake sites in the temporal lobe in schizophrenia. Neurosci Lett. 1989;107211- 215
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Pesold  CImpagnatiello  FCaruncho  HJCosta  EGuidotti  A Changes in GABAA receptor subunit expression in schizophrenic prefrontal cortex [abstract]. Soc Neurosci Abstracts. 1998;241274
Huntsman  MMTran  BVPotkin  SGBunney  WEJones  EG Altered ratios of alternatively spliced long and short γ 2 subunit mRNAs of the γ-amino butyrate type A receptor in prefrontal cortex of schizophrenics. Proc Natl Acad Sci U S A. 1998;9515066- 15071
Akbarian  SKim  JJPotkin  SGHagman  JOTafazzoli  ABunney  WE  JrJones  EG Gene expression for glutamic acid decarboxylase is reduced without loss of neurons in prefrontal cortex of schizophrenics. Arch Gen Psychiatry. 1995;52258- 266
Lewis  DA Neural circuitry of the prefrontal cortex in schizophrenia. Arch Gen Psychiatry. 1995;52269- 273
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Glantz  LALewis  DA Reduction of synaptophysin immunoreactivity in the prefrontal cortex of subjects with schizophrenia: regional and diagnostic specificity. Arch Gen Psychiatry. 1997;54943- 952
Mirra  SSHeyman  AMcKeel  DSumi  SMCrain  BJBrownlee  LMVogel  FSHughes  JPvan Bell  G The consortium to establish a registry for Alzheimer's disease (CERAD), part II: standardization of the neuropathological assessment of Alzheimer's disease. Neurology. 1991;41479- 486
Daviss  SRLewis  DA Local circuit neurons of the prefrontal cortex in schizophrenia: selective increase in the density of calbindin-immunoreactive neurons. Psychiatry Res. 1995;5981- 96
Bu  DFErlander  MGHitz  BCTillikaratne  NKaufman  DLWagner-McPherson  CBEvans  GATobin  AJ Two human glutamate decarboxylases, 65-kDa GAD and 67-kDa GAD, are each encoded by a single gene. Proc Natl Acad Sci U S A. 1992;892115- 2119
Gao  BMoore  RY The sexually dimorphic nucleus of the hypothalamus contains GABA neurons in rat and man. Brain Res. 1996;742163- 171
Austin  MCBradley  CCMann  JJBlakely  RD Expression of serotonin transporter messenger RNA in the human brain. J Neurochem. 1994;622362- 2367
Austin  MCO'Donnell  SM Regional distribution and cellular expression of tryptophan hydroxylase messenger RNA in postmortem human brainstem and pineal gland. J Neurochem. 1999;722065- 2073
Akil  MPierri  JNWhitehead  REEdgar  CLMohila  CLewis  DA Lamina-specific alteration in the dopamine innervation of the prefrontal cortex in schizophrenic subjects. Am J Psychiatry. 1999;1561580- 1589
Pierri  JNChaudry  ASWoo  T-ULewis  DA Alterations in chandelier neuron axon terminals in the prefrontal cortex of schizophrenic subjects. Am J Psychiatry. 1999;1561709- 1719
Janicak  PGDavis  JMPreskorn  SHAyd  FJ Principles and Practice of Psychopharmacotherapy.  Baltimore, Md Williams & Wilkins1993;
Gerfen  CRMcGinty  JFYoung  WS Dopamine differentially regulates dynorphin, substance P, and enkaphalin expression in striatal neurons: in situ hybridization histochemical analysis. J Neurosci. 1991;111016- 1031
Neter  JKutner  MHNachtsheim  CJWasserman  W Applied Linear Statistical Models.  Chicago, Ill Irwin1996;
Harrison  PJHeath  PREastwood  SLBurnet  PWJMcDonald  BPearson  RCA The relative importance of premortem acidosis and postmortem interval for human brain gene expression studies: selective mRNA vulnerability and comparison with their encoded proteins. Neurosci Lett. 1995;200151- 154
Holm  S A simple sequentially rejective multiple test procedure. Scand J Stat. 1979;665- 70
Hendry  SHCSchwark  HDJones  EGYan  J Numbers and proportions of GABA-immunoreactive neurons in different areas of monkey cerebral cortex. J Neurosci. 1987;71503- 1519
Benes  FMMcSparren  JBird  EDSanGiovanni  JPVincent  SL Deficits in small interneurons in prefrontal and cingulate cortices of schizophrenic and schizoaffective patients. Arch Gen Psychiatry. 1991;48996- 1001
Selemon  LDRajkowska  GGoldman-Rakic  PS Abnormally high neuronal density in the schizophrenic cortex: a morphometric analysis of prefrontal area 9 and occipital area 17. Arch Gen Psychiatry. 1995;52805- 818
Woo  T-UMiller  JLLewis  DA Parvalbumin-containing cortical neurons in schizophrenia. Am J Psychiatry. 1997;1541013- 1015
Selemon  LDRajkowska  GGoldman-Rakic  PS Elevated neuronal density in prefrontal area 46 in brains from schizophrenic patients: application of a three-dimensional, stereologic counting method. J Comp Neurol. 1998;392402- 412
Thune  JJHofsten  DEUylings  HBMPakkenberg  B Total neuron numbers in the prefrontal cortex in schizophrenia [abstract]. Soc Neurosci Abstracts. 1998;24985
Glantz  LAAustin  MCLewis  DA Cellular levels of synaptophysin mRNA expression in the prefrontal cortex of subjects with schizophrenia [abstract]. Soc Neurosci Abstracts. 1998;24987
Woo  T-UWhitehead  REMelchitzky  DSLewis  DA A subclass of prefrontal γ-aminobutyric acid axon terminals are selectively altered in schizophrenia. Proc Natl Acad Sci U S A. 1998;955341- 5346
Lewis  DALund  JS Heterogeneity of chandelier neurons in monkey neocortex: corticotropin-releasing factor and parvalbumin immunoreactive populations. J Comp Neurol. 1990;293599- 615
Delfs  JMEllison  GDMercugliano  MChesselet  M-F Expression of glutamic acid decarboxylase mRNA in striatum and pallidum in an animal model of tardive dyskinesia. Exp Neurol. 1995;133175- 188
Chen  JFWeiss  B Irreversible blockade of D2 dopamine receptors by fluphenazine-N-mustard increases glutamic acid decarboxylase mRNA in rat striatum. Neurosci Lett. 1993;150215- 218
Jolkkonen  JJenner  PMarsden  CD GABAergic modulation of striatal peptide expression in rats and the alterations induced by dopamine antagonist treatment. Neurosci Lett. 1994;180273- 276
Delfs  JMAnegawa  NJChesselet  M-F Glutamate decarboxylase messenger RNA in rat pallidum: comparison of the effects of haloperidol, clozapine, and combined haloperidol-scopolamine treatments. Neuroscience. 1995;6667- 80
Camps  MCortés  RGueye  BProbst  APalacios  JM Dopamine receptors in human brain: autoradiographic distribution of D2 sites. Neuroscience. 1989;28275- 290
Hall  HSedvall  GMagnusson  OKopp  JHalldin  CFarde  L Distribution of D1- and D2-dopamine receptors, and dopamine and its metabolites in the human brain. Neuropsychopharmacology. 1994;11245- 256
Guillery  RWHerrup  K Quantification without pontification: choosing a method for counting objects in sectioned tissues. J Comp Neurol. 1997;3862- 7
Shelton  RCKarson  CNDoran  ARPickar  DBigelow  LBWeinberger  DR Cerebral structural pathology in schizophrenia: evidence for a selective prefrontal cortical defect. Am J Psychiatry. 1988;145154- 163
Breier  ABuchanan  RWElkashef  AMunson  RCKirkpatrick  BGellad  F Brain morphology and schizophrenia: a magnetic resonance imaging study of limbic, prefrontal cortex, and caudate structures. Arch Gen Psychiatry. 1992;49921- 926
Zipursky  RBLim  KOSullivan  EVBrown  BWPfefferbaum  A Widespread cerebral gray matter volume deficits in schizophrenia. Arch Gen Psychiatry. 1992;49195- 205
Zipursky  RBLambe  EKKapur  SMikulis  DJ Cerebral gray matter volume deficits in first episode psychosis. Arch Gen Psychiatry. 1998;55540- 546
Andreasen  NCFlashman  LFlaum  MArndt  SSwayze  V  IIO'Leary  DSEhrhardt  JCYuh  WT Regional brain abnormalities in schizophrenia measured with magnetic resonance imaging. JAMA. 1994;2721763- 1769
Isaacson  JSSolis  JMNicoll  RA Local and diffuse synaptic action of GABA in the hippocampus. Neuron. 1993;10165- 175
Giros  BJaber  MJones  SRWightman  RMCaron  MG Hyperlocomotion and indifference to cocaine and amphetamine in mice lacking the dopamine transporter. Nature. 1996;379606- 612
Jones  SRGainetdinov  RRJaber  MGiro  BWightman  RM Profound neuronal plasticity in response to inactivation of the dopamine transporter. Proc Natl Acad Sci U S A. 1998;954029- 4034
Pakkenberg  B Pronounced reduction of total neuron number in mediodorsal thalamic nucleus and nucleus accumbens in schizophrenics. Arch Gen Psychiatry. 1990;471023- 1028
Manaye  KFLiang  C-LHicks  PBGerman  DYoung  KA Nerve cell numbers in thalamic anterior and mediodorsal nuclei are selectively reduced in schizophrenia [abstract]. Soc Neurosci Abstracts. 1998;241236
Jones  LMall  NByne  W Localization of schizophrenia-associated thalamic volume loss [abstract]. Soc Neurosci Abstracts. 1998;24985
Popken  GJBunney  WE  JrPotkin  SGJones  EG Neuron number and GABAergic and glutamatergic mRNA expression in subdivisions of the thalamic mediodorsal nucleus of schizophrenics [abstract]. Soc Neurosci Abstracts. 1998;24991
Glantz  LALewis  DA Decreased dendritic spine density on prefrontal cortical pyramidal neurons in schizophrenia. Arch Gen Psychiatry. 2000;5765- 73
Garey  LJOng  WYPatel  TSKanani  MDavis  AMortimer  AMBarnes  TREHirsch  SR Reduced dendritic spine density on cerebral cortical pyramidal neurons in schizophrenia. J Neurol Neurosurg Psychiatry. 1998;65446- 453
Cruz  DAMelchitzky  DSLewis  DA Parvalbumin-immunoreactive varicosities are selectively decreased in the thalamic recipient zone of the prefrontal cortex in schizophrenia [abstract]. Soc Neurosci Abstracts. 1999;25817
Hendry  SHCJones  EG Activity-dependent regulation of GABA expression in the visual cortex of adult monkeys. Neuron. 1988;1701- 712
Benson  DLHuntsman  MMJones  EG Activity-dependent changes in GAD and preprotachykinin mRNAs in visual cortex of adult monkeys. Cereb Cortex. 1994;440- 51

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