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Differential Hippocampal Expression of Glutamic Acid Decarboxylase 65 and 67 Messenger RNA in Bipolar Disorder and Schizophrenia
Stephan Heckers, MD;
David Stone, PhD;
John Walsh, MA;
John Shick;
Pamposh Koul;
Francine M. Benes, MD, PhD
Arch Gen Psychiatry. 2002;59:521-529.
ABSTRACT
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Background Expression of messenger RNA (mRNA) for the  -aminobutyric acid
(GABA)–synthesizing enzyme, glutamic acid decarboxylase (GAD), in the
prefrontal cortex and the number of GABAergic neurons in the hippocampus are
reduced in schizophrenia and bipolar disorder. We tested the hypothesis that
the expression of the 2 isoforms, one 65 kd (GAD65) and the other
67 kd (GAD67), is differentially affected in the hippocampus in
schizophrenia and bipolar disorder.
Methods Hippocampal sections from 15 subjects in 3 groups (control subjects
and subjects with schizophrenia and bipolar disorder) were studied using an
in situ hybridization protocol with sulfur 35–labeled complementary
riboprobes for GAD65 and GAD67 mRNA. Emulsion-dipped
slides were analyzed for the density of GAD mRNA–positive neurons in
4 sectors of the hippocampus and for the cellular expression level of both
GAD mRNAs.
Results The density of GAD65 and GAD67 mRNA–positive
neurons was decreased by 45% and 43%, respectively, in subjects with bipolar
disorder, but only 14% and 4%, respectively, in subjects with schizophrenia.
The decreased density of GAD65 mRNA–positive neurons in subjects
with bipolar disorder was significant in sectors CA2/3 and dentate gyrus,
and that of GAD67 mRNA–positive neurons was significant in
CA4, but not other hippocampal sectors. Cellular GAD65 mRNA expression
was significantly decreased in subjects with bipolar disorder, particularly
in CA4, but not in schizophrenic subjects. Cellular GAD67 mRNA
expression was normal in both groups.
Conclusion We have found a region-specific deficit of GAD65 and GAD67 mRNA expression in bipolar disorder.
INTRODUCTION
THE HUMAN hippocampus contains 2 nerve cell types, the glutamatergic
projection neuron and the -aminobutyric acid–transmitting (GABAergic)
interneuron.1 Various subtypes of the GABAergic
interneuron provide tonic and phasic inhibitory control over the projection
neurons.2 An intricate balance of excitation
and inhibition provides the cellular basis for the encoding and retrieval
of sensory information, relayed to hippocampal neurons via 2 pathways from
the entorhinal cortex.3-4
Previous studies of cell density, receptor expression, and messenger
RNA (mRNA) expression have demonstrated an abnormality of hippocampal interneurons
in schizophrenia and bipolar disorder.5 Earlier
studies had indicated that the expression of GABA A receptors on cells receiving
input from hippocampal interneurons is increased in schizophrenia.6-7 Subsequently, the density of interneurons
was found to be selectively decreased in hippocampal sector CA2 in bipolar
disorder and schizophrenia,8 whereas the density
of hippocampal pyramidal cells is normal in schizophrenia9-12
and bipolar disorder.12 Finally, preliminary
data point toward a decrease of 67-kd glutamic acid decarboxylase (GAD67) mRNA expression in the hippocampus in schizophrenia.13
The -aminobutyric acid (GABA)–synthesizing enzyme GAD is
the signature cellular protein that distinguishes the GABAergic interneurons
from non-GABAergic principal cells. Two isoforms of the enzyme, GAD65 and GAD67, are coded for by 2 different genes and vary in
cellular localization and function.14 The expression
of GAD67 mRNA is known to respond to environmental stimuli, making
it a valuable target for studies of gene regulation.14-15
Several previous studies have demonstrated a decreased expression of GAD67 mRNA in a subset of prefrontal cortex neurons in schizophrenia16-18 and bipolar disorder.18
Here we present, to our knowledge, the first comprehensive study of
GAD mRNA expression in the human hippocampus. The relative density of GAD
mRNA–positive neurons has been determined in all subsectors of the human
hippocampus, and the relative cellular expression within identified GAD mRNA–positive
neurons has been estimated. This approach has allowed us to test the hypothesis
that the expression of the 2 GAD mRNAs may be differentially affected in schizophrenia
and/or bipolar disorder.
SUBJECTS AND METHODS
SUBJECTS
Brain specimens were obtained from the Harvard Brain Tissue Resource
Center, Belmont, Mass, and included 15 control subjects, 15 subjects with
bipolar disorder (bipolar subjects), and 15 subjects with schizophrenia (schizophrenic
subjects) (Table 1). Each control
was matched with 1 schizophrenic and 1 bipolar subject based on age and postmortem
interval (PMI) to form 15 triplets. The mean (±SD) differences within
each triplet were less than 3.16 (±2.50) years for age and 3.23 (±2.34)
hours for PMI. The female-male ratios were 5:10 for the controls and 6:9 for
both patient groups.
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Description of Study Tripletsa
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During the microscopic analysis of the study material, 1 schizophrenic
subject (S3546, triplet 9) was found to have pathological changes in the hippocampal
CA1 sector consistent with a diagnosis of hippocampal sclerosis. A neuropathologist
had studied the contralateral hippocampus, and no signs of hippocampal abnormality
had been present. This subject was removed from the analysis.
Diagnoses were made by means of retrospective review of medical records
and of an extensive questionnaire about social and medical history completed
by family members of the donor. Two psychiatrists (S.H. and F.M.B.) reviewed
all records and applied the criteria of Feighner et al19
for the diagnosis of schizophrenia and DSM-III-R
criteria20 for the diagnosis of schizoaffective
and bipolar disorders. During the course of the study, documentation for 1
bipolar subject (BP3256, triplet 3) was not sufficient to verify the diagnosis.
We therefore decided to exclude this subject from the study. The removal of
2 subjects resulted in 15 controls, 14 schizophrenic subjects, and 14 bipolar
subjects in the study.
TISSUE PREPARATION AND IN SITU HYBRIDIZATION
One hemisphere of each brain specimen was dissected fresh, and 3-mm-thick
blocks of a central portion of the hippocampus were removed. The hippocampal
blocks were fixed in 4% paraformaldehyde in ice-cold 0.1M phosphate buffer
(pH, 7.4) for 90 minutes, immersed in 30% sucrose in the same buffer overnight,
then frozen in medium (Tissue Tek OCT; Sakura Finetek, Torrance, Calif) on
dry ice and stored at -70°C. In 27 of the 45 cases, a piece of frozen
cerebellum was homogenized in 15 volumes of distilled and deionized filtered
water, and the acidity was measured using a pH meter (Corning Inc, Acton,
Mass). These measurements were used to assess whether brain pH may have affected
the integrity of GAD mRNA during the PMI.
The hippocampus of each subject was sectioned at 10 µm and mounted
on slides (Superfrost Plus; Fisher Scientific, Pittsburgh, Pa). Two sections
were used per subject, and sections were mounted (2 per slide) such that the
following 3 slides were used for each matched triplet: 1 slide containing
control and schizophrenic tissue; 1, control and bipolar tissue; and 1, schizophrenic
and bipolar tissue. This method of mounting sections was designed to control
for slide-to-slide variability associated with the in situ hybridization procedure.
The other hemisphere of each brain specimen was cut in serial sections
for a complete neuropathological examination. Results of gross and microscopic
examination did not reveal any evidence of Alzheimer disease, cerebrovascular
accident, or tumors.
The complementary RNA probes were transcribed in vitro from full-length
human complementary DNA (cDNA) clones inserted into bluescript vector (2.01-kilobase
[kb] human GAD65 and 2.7-kb human GAD67, provided by
Allan Tobin, PhD, University of California–Los Angeles). The cDNA probes
have been characterized previously and were found to hybridize with human
brain RNA.21 Probes were synthesized using
sulfur 35–labeled uridine triphosphate (NEN Life Sciences, Boston, Mass).
The probe specificity for GAD65 and GAD67 was demonstrated
by means of a control experiment in which sense probes failed to reveal any
specific hybridization signal. To ensure full penetration into tissue, the
2.01-kb GAD65 cRNA and 2.7-kb human GAD67 probes were
hydrolyzed with an equal volume of sodium carbonate buffer (pH, 10.2; 40mM
sodium bicarbonate and 60mM sodium carbonate) at 60°C for a resultant
average fragment size of 0.8 kb. The reaction was stopped by adding 0.08 volume
of 2M sodium acetate in 6.25% glacial acetic acid. Probes were then reconstituted
in a hybridization buffer consisting of 50% formamide, 0.1% yeast transfer
RNA, 10% dextran sulfate, 1x Dehardt solution, 0.5M EDTA, 0.02% sodium
dodecyl sulfate, 4x saline sodium citrate buffer (Sigma-Aldrich Corp,
St Louis, Mo), 10mM dithiothreitol, and 0.1% single-stranded DNA, at a final
probe concentration of 0.4 ng/µL of hybridization buffer.
Slides were fixed in 4% paraformaldehyde in 0.1M phosphate buffer (pH,
7.4) for 15 minutes, treated in proteinase K solution for 15 minutes, and
then incubated in 0.1M triethanolamine (pH, 8.0) for 5 minutes, followed by
0.1M triethanolamine and 0.25% acetic anhydride (pH, 8.0) for 10 minutes.
Slides were then dehydrated using a graded series of ethyl alcohol solutions
(50%-100%). Sections were hybridized for 3 hours at 55°C. After hybridization,
slides were incubated in 0.025 mg/mL RNase A in 0.5M sodium chloride and 0.05M
phosphate buffer for 1 hour at 37°C, and washed in 50% formamide, 0.5M
sodium chloride, 0.05M phosphate buffer at 63°C for 30 minutes, followed
by an overnight wash in 0.25 x silver sulfadiazine and chlorhexidine
at room temperature. Slides were then covered with NTB2 emulsion (Eastman
Kodak Co, Rochester, NY) and exposed for 21 days for cellular analysis. After
development, slides were counterstained with cresyl violet and dehydrated
through a graded series of ethanol and xylene. A coverslip was then applied.
QUANTIFICATION OF GAD mRNA EXPRESSION
All slides were coded to conceal subject identity throughout the study.
All sections undergoing in situ hybridization were used for analysis, which
was performed using a bright-field microscope (Leitz Laborlux; Leitz, Wetzlar,
Germany) interfaced with an image analysis system (Bioquant MEG IV; R &
M Biometrics, Nashville, Tenn). The microscopic analysis was conducted in
the following 3 stages. First, we outlined 4 sectors, ie, the dentate gyrus
(DG) and the cornu ammonis sectors CA4, CA2/3, and CA1, in each hippocampus.
Second, we counted all grain clusters within the sectors using an XYZ encoder
to establish the density of GAD mRNA–positive neurons in the hippocampus.
Finally, we counted grains in all or a subsample of the previously identified
clusters to assess the cellular expression of GAD mRNA in individual neurons.
All sections were inspected at low power using x4 and x10
objectives to outline the boundaries of the 4 hippocampal sectors. The DG
sector included the molecular and granule cell layers, but not the polymorph
cell layer. Sector CA4 was defined as the area encapsulated by the granule
cell layer, but included the polymorph cell layer of the DG. Because the granule-polymorph
cell layer border is very distinct, whereas the polymorph-hilus border is
rather ambiguous, this approach made possible the performance of the analysis
in a reproducible manner. The medial boundary of sector CA2/3 was defined
by a straight line that connected the 2 ends of the C-shaped granule cell
layer of the DG, because the anatomical CA2/3-CA4 border was difficult to
define in our material. The lateral boundary of the CA2/3 sector was defined
by a decrease in cell density and lighter counterstaining, indicating the
beginning of sector CA1. Pilot studies demonstrated a homogeneous density
of grain clusters in sector CA1, which allowed us to sample a part of the
large CA1 sector.
The position of each grain cluster was plotted using a x25 objective
within each area outlined for the various sectors. All clearly identifiable
clusters overlying a neuronal profile were counted. One section from each
triplet was analyzed twice for test-retest reliability of the cluster counting.
These 15 pairs demonstrated a high reliability of the grain cluster counting
in all 4 sectors (Spearman  s, 0.94, 0.95, 0.99, and 0.92 for DG, CA4,
CA2/3, and CA1, respectively).
Grain counting within individual clusters was performed using a x40
objective. All clusters were analyzed if a given sector contained up to 20
grain clusters. If a sector contained more than 20 clusters, the samples were
taken in a systematic fashion in the following regions: 20 clusters at the
polymorph-granule cell layer border for the CA4 sector and 10 clusters each
at the medial and lateral borders of the CA2/3 sector. For sector CA1, 20
clusters were sampled in a columnar fashion through the full width of the
pyramidal cell layer. First, we outlined each cluster using a cursor displayed
on the computer monitor. The cluster area was then assigned a threshold, and
the area covered by the grains within each cluster was determined as a pixel
count. Before each sector was evaluated, the light intensity was adjusted
to be consistent throughout the study. After each sector was evaluated, 2
areas within the sectors that were free of any grain clusters were sampled
to determine the local background level of grains. This local background level
was subtracted from the mean area covered by grains to give a corrected grain
count for each sector.
Overall, 37 373 clusters indicating GAD mRNA–positive neurons
were counted (2135 in DG, 9613 in CA4, 6510 in CA2/3, and 19 115 in CA1);
11 641 clusters (31% of all clusters) were sampled for grain counting
(1404 in DG, 3632 in CA4, 3242 in CA2/3, and 3363 in CA1).
STATISTICAL ANALYSIS
For each subject, the numerical density of clusters and grain counts
per cluster within each of the 4 hippocampal sectors were averaged across
the 2 sections studied. The mean cluster and grain densities were entered
into a repeated-measures analysis of variance (ANOVA), with diagnosis (2 levels,
control-bipolar or control-schizophrenia) and sector (repeated measure with
4 levels) as main effects and triplet as a blocking effect. When the omnibus
F test revealed a significant effect of diagnosis (P<.05),
we used a post hoc unpaired, 2-tailed t test. To
control for multiple comparisons (4 sectors per ANOVA), we adjusted all P values of the t test using a
Bonferroni correction (corrected P = P x 4). Corrected P values are presented
in the text and the figures. We calculated the effect sizes using the Cohen d for all significant results of the t tests.
We evaluated the effect of 8 confounding variables (hemisphere, age,
sex, medication type, chlorpromazine equivalents, brain pH, PMI, and storage
time) using simple linear regression, analysis of covariance (ANCOVA), or
unpaired t tests.
RESULTS
DISTRIBUTION OF GAD65 AND GAD67 mRNA-POSITIVE NEURONS
IN THE HUMAN HIPPOCAMPUS
Light microscopic analysis of the emulsion-dipped sections showed a
characteristic clustering of silver grains over Nissl-stained cell bodies
(Figure 1). Grain clusters were
traced within the DG, CA4, CA2/3, and CA1 hippocampal subdivisions (Figure 2). The GAD65 and GAD67 mRNA–positive neurons were found in all 4 subdivisions (Figure 2). The GAD65 mRNA–positive
neurons were more prevalent than GAD67 mRNA–positive neurons
in each of the 3 groups (Figure 3).
The highest density of GAD65 and GAD67 mRNA–positive
neurons was found in CA4, followed by CA2/3, CA1, and DG (Figure 3).
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Figure 1. Light-microscopic view of emulsion-dipped
section through the granule (g) and polymorph (p) cell layer of the dentate
gyrus and the CA4 hippocampal sectors. Silver grains are clustered over 3
Nissl-stained cell bodies in each of the 3 regions. Bar indicates 20 µm.
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Figure 2. A, Tracing of a hippocampal section
of control subject C3374 (triplet 4) shows the dentate gyrus (DG) and cornu
ammonis CA4, CA2/3, and CA1 sectors of the hippocampus. The box overlying
DG indicates position from which Figure 1 was taken. Distribution of 65-kd
glutamic acid decarboxylase messenger RNA–positive neurons in the 4
hippocampal subdivisions of 3 matched subjects from triplet 4 is seen in the
remaining parts. B, Control subject C3374. C, Subject BP4237 with bipolar
disorder. D, Subject S3234 with schizophrenia.
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Figure 3. Bar graphs showing the mean (±SEM)
numerical density of 65-kd glutamic acid decarboxylase (GAD65)
(A) and GAD67 messenger RNA (mRNA)–positive neurons (B) in
control subjects (n = 15), subjects with schizophrenia (n = 14), and subjects
with bipolar disorder (n = 13 in hippocampal sector CA2/3; n = 14 in sectors
dentate gyrus [DG], CA4, and CA1).
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NUMERICAL DENSITY OF GAD65 AND GAD67 mRNA-POSITIVE
NEURONS
The numerical density of GAD65 mRNA–positive neurons
was decreased from 34% (CA1) to 55% (DG) in bipolar subjects and from 1% (CA1)
to 21% (CA4) in schizophrenic subjects (Figure
3), relative to controls. The decrease of hippocampal GAD65 mRNA–positive neurons was significant for bipolar subjects (main
effect of group, F1,14 = 17.4; P = .001)
but not for schizophrenic subjects (F1,14 = 1.57; P = .23) (Figure 3). The
changes in the bipolar subjects did not affect all hippocampal subdivisions
(region-by-diagnosis interaction, F1,3 = 6.7; P = .001). For example, the decreases were significant in CA2/3 (t = 3.25; P = .01; Cohen d = -1.23) and DG (t = 2.78; P = .04; Cohen d = -1.04),
but not in CA4 (t = 2.59; P
= .06) and CA1 (t = 2.24; P
= .14) (Figure 3). When each of
the bipolar subjects was compared with their matched controls in sector CA2/3,
the region with the most significant change, 11 of the 13 bipolar subjects
had a lower density of GAD65 mRNA–positive neurons (Figure 4).
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Figure 4. Numerical density of 65-kd glutamic
acid decarboxylase (GAD65) messenger RNA (mRNA)–positive
neurons in hippocampal sector CA2/3 in 13 subjects with bipolar disorder and
14 control subjects. The CA2/3 sector could not be analyzed in 1 bipolar subject.
Solid circles indicate the pairs in which the value for the control subject
is greater than that for the bipolar subject (11 of 13 pairs); open circles,
pairs in which the value for the bipolar subject is greater than that for
the control subject (2 of 13 pairs).
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The numerical density of GAD67 mRNA–positive neurons
was decreased from 33% (CA1) to 50% (CA4) in bipolar subjects relative to
controls (main effect of group, F1,14 = 7.73; P = .02), but did not show an overall decrease in
schizophrenic subjects (F1,14 = .16; P = .70)
(Figure 3). The changes in the bipolar subjects were regionally specific
(region-by-diagnosis interaction, F1,3 = 6.15; P = .002), with the decrease in sector CA4 being significant (t = 2.76; P = .04; Cohen d = -1.02), but not in CA2/3 (t = 2.47; P = .08), CA1 (t = 1.93; P = .26), or DG (t = 1.31; P = .80).
CELLULAR EXPRESSION OF GAD65 AND GAD67 mRNA
The area covered by grains within each cluster was a measure of the
cellular expression of GAD mRNA. The controls, but not the 2 patient groups,
showed the highest GAD65 mRNA expression in sector CA4 (Figure 5). Expression of GAD65
mRNA was decreased, relative to controls, from 21% (CA1) to 37% (CA4) in bipolar
subjects (main effect of group, F1,14 = 5.38; P = .04) and from 2% (CA2/3) to 24% (CA4) in
schizophrenic subjects (F1,14 = 1.65; P = .22)
(Figure 5). The decrease of cellular GAD65 mRNA expression
in bipolar subjects was regionally specific (region x diagnosis interaction,
F1,3 = 3.63; P = .02) and was most pronounced
in sector CA4 (24% decrease) (t = 2.10; P = .18; Cohen d = -0.78).
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Figure 5. Bar graphs showing the mean (±SEM)
area covered by grains over 65-kd glutamic acid decarboxylase (GAD65) (A) and 67-kd GAD (GAD67) (B) messenger RNA (mRNA)–positive
neurons in control subjects (n = 15), subjects with schizophrenia (n = 14),
and subjects with bipolar disorder (n = 13 in hippocampal sectors dentate
gyrus [DG] and CA2/3; n = 14 in sectors CA4 and CA1).
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Expression of GAD67 mRNA was not significantly decreased
in the bipolar (F1,14 = 1.36; P = .28)
or the schizophrenic (F1,14 = .005; P
= .95) group (Figure 5).
CONFOUNDING VARIABLES
The 8 confounding variables were evaluated with respect to the numerical
density of GAD mRNA–positive neurons and expression of GAD mRNA. There
were no significant correlations (P>.05 for all sectors)
of the 2 dependent variables with PMI, storage time, or chlorpromazine equivalents.
Furthermore, patients treated with atypical antipsychotic medication did not
differ from those treated with typical antipsychotic medication.
The overall effects of hemisphere and sex were evaluated by comparing
all female (n = 16) and male (n = 27) subjects and all left (n = 28) and right
(n = 15) hemisphere specimens. We found no effect of hemisphere. Male subjects
had a higher density of GAD65 mRNA–positive neurons in CA1
(t = 2.51; P = .02) and
a higher expression of GAD mRNA expression in CA4 neurons (t = 2.05; P = .047), but all other parameters
showed no effect of sex. The 3 diagnostic groups had similar male-female and
left-right hemisphere ratios, and all of our findings from the ANOVA using
diagnosis and sector as main effects were confirmed by means of ANCOVAs that
included sex and hemisphere as single or dual covariates.
Age was negatively associated with the density of GAD65 mRNA–positive
neurons (P<.05) and the cellular expression levels
of GAD65 mRNA in all sectors (P<.02)
and GAD67 mRNA in sector CA4 (P<.02).
Because the 3 subjects of each triplet were matched by age, we controlled
for the effect of age by including triplet as a blocking effect in our ANOVA.
Brain tissue suitable for pH measurements was available in 27 of the
43 subjects studied (Table 1).
Mean (±SD) brain pH did not differ among the 3 groups (F2,24
= .06; P = .94) that included 10 controls (6.47 ±
0.3), 9 bipolar subjects (6.51 ± 0.26), and 8 schizophrenic subjects
(6.50 ± 0.37). The pH was positively associated with the mean density
of GAD65 and GAD67 mRNA–positive neurons in DG
and CA4 (P<.04), but not in CA2/3 and CA1 (P>.05). Brain pH also correlated with the expression of
GAD65 and GAD67 mRNA in all 4 hippocampal subdivisions
(P<.01).
Since pH could explain a significant variance component for the 2 variables
of interest, ANCOVAs were performed in the subset of 27 subjects using pH
and age as covariates. The decreased density of GAD mRNA–positive neurons
in bipolar disorder was confirmed for GAD65 mRNA–positive
neurons in CA2/3 (P = .002), CA4 (P = .006), and DG (P<.001), and for GAD67 mRNA–positive neurons in CA4 (P =
.02) and CA2/3 (P = .03). Furthermore, the trend
of decreased expression of GAD65 mRNA in CA4 in bipolar subjects
also became significant (P = .01). All other results
from the main analysis remained unchanged.
COMMENT
The findings reported herein indicate that bipolar disorder, but not
schizophrenia, is associated with a significant decrease of GAD mRNA–positive
neurons and of GAD65 mRNA expression in the hippocampus. The loss
of GAD mRNA–positive neurons in bipolar disorder was most pronounced
in hippocampal subdivision CA2/3, whereas the decrease of GAD65
mRNA expression occurred preferentially in subdivision CA4. These findings
provide further evidence of an abnormality of GABAergic neurons in bipolar
disorder. The pronounced reduction of GAD65 mRNA expression in
bipolar disorder could help to explain the clinical observation that GABA-mimetic
anticonvulsants show efficacy as mood-stabilizing agents.22
Similar but nonsignificant changes in the numerical density of GAD65 mRNA–positive neurons in schizophrenia were found, and previous
studies demonstrated abnormalities of hippocampal interneurons8
and of GAD67 mRNA expression in schizophrenia.13, 16-18
On the basis of studies in rat,23 approximately
95% of GABAergic neurons express both GAD genes. However, the relative expression
of GAD65 and GAD67 mRNA and the translation into protein
differ at the regional and cellular levels.24
This study is, to our knowledge, the first description of the distribution
of GAD65 and GAD67 mRNA–positive neurons in the
human hippocampus. The highest density of GAD mRNA–positive neurons
was found in the polymorph cell layer (included in the CA4 sector in this
study), a pattern similar in the human and nonhuman primate hippocampus.24
The mechanisms responsible for the differential expression of the 2
GAD mRNA isoforms are not fully understood, but most likely involve separate
mechanisms, since the regulatory regions for their respective genes show less
than 25% homology.14 Previous studies in rat
striatum demonstrated a preferential modulation of GAD65 and GAD67 mRNA expression via D1 and D2 dopamine receptors,
respectively.25-26 Previous studies
also suggested that GAD67 is more concentrated in neurons that
fire tonically, whereas GAD65 is more concentrated in neurons with
a low basal firing rate, whose activation is under strong synaptic control.14, 27 The most prominent decrease in GAD65 mRNA expression per cell was found in subdivision CA4, whereas that
for GAD67 was not different. The GABAergic neurons in this region
are known to provide local inhibitory control in the sector DG.2
Thus, decreased expression of GAD65 mRNA in this subset of neurons
could indicate a decreased control over information arriving via the perforant
pathway, resulting in increased activity conducted along the trisynaptic pathway.
One possible explanation for the decrease of GAD mRNA in bipolar disorder
is an overt loss of GABAergic interneurons, rather than a decrease of expression
in an otherwise intact neuron. Hippocampal cell loss in psychosis appears
to occur preferentially in the population of interneurons, since studies of
total cell number or pyramidal cell density have not found differences in
schizophrenia9-12
or bipolar disorder.12 We found a selective
decrease of GAD65 mRNA–positive neurons in sector CA2 in
bipolar disorder, but a similar decrease in schizophrenia was not observed.
The more pronounced decrease of GAD65 mRNA–positive neurons
compared with GAD67 mRNA–positive neurons in bipolar disorder
suggests a preferential decrease in GAD65 expression. Furthermore,
the pronounced decrease of GAD65 mRNA expression per cell in sector
CA4 is consistent with the notion that decreased GAD mRNA expression is, in
part, due to abnormal regulation in existing GABAergic interneurons in bipolar
disorder.
We found no correlation of GAD mRNA expression and the dosage of antipsychotic
medication taken by all of the schizophrenic subjects and some of the bipolar
subjects before death. Therefore, the observed changes in GAD mRNA expression
in bipolar disorder are unlikely to be due to the treatment with antipsychotic
drugs. Previous studies, however, have demonstrated that treatment with haloperidol
can increase GABA-immunoreactive axon terminals in rat medial prefrontal cortex28 and that the number of GAD65-immunoreactive
terminals correlates positively with the dose of typical antipsychotic drugs
in schizophrenic subjects.29 Since the average
chlorpromazine equivalent dose in schizophrenic subjects was double that of
bipolar subjects, these drugs may have stimulated the hippocampal GABA system
more in the schizophrenic subjects. Schizophrenic subjects showed a marked
up-regulation of the GABA A receptor in the hippocampus,6
particularly in the sectors CA4, CA3, and CA2, which is consistent with the
possibility that an inherent defect exists in this system in schizophrenia,
possibly one that is partially compensated by antipsychotic drugs.
Our finding that GAD67 mRNA expression is not significantly
decreased in schizophrenia differs from a preliminary report of decreased
hippocampal GAD67 mRNA expression in schizophrenia.13
Our study also suggests that a decrease of GAD67 mRNA expression,
previously documented in subsets of neurons in the prefrontal cortex,16-18 is not ubiquitously
found in schizophrenia. Although details of the experimental protocols could
explain some of the differences between the studies, the decrease of GAD67 mRNA expression in schizophrenia likely affects isocortex more than
limbic allocortex, including the hippocampal formation. This finding is consistent
with the differential modulation of GAD expression, which varies across brain
regions and most likely involves multiple mechanisms.
The lack of marked changes of hippocampal GAD mRNA expression in schizophrenia
is of interest for the interpretation of recent neuroimaging studies that
have demonstrated increased hippocampal activity during rest,30-34
during the experience of auditory hallucinations,35
and during word retrieval36-37
in schizophrenia. Our study makes it less likely that the increased baseline
activity and the lack of normal modulation of hippocampal activity seen in
schizophrenia36 are due to a decreased inhibitory
tone, as assessed by the expression of GAD mRNA. The GABAergic activity might
still be decreased, even with a normal expression of GAD mRNA, if the translation
into GAD protein is abnormal. However, the number of GAD65 immunoreactive
puncta on hippocampal neurons was found to be normal in schizophrenia, although
schizophrenic subjects free of neuroleptic drugs showed a significant reduction.29
A recent study of GAD65 immunoreactive terminals in the cingulate
and prefrontal cortices found decreases in bipolar disorder but not in schizophrenia.38 With our finding of decreased GAD65 mRNA
expression in bipolar disorder, this finding seems to indicate that abnormalities
of hippocampal GAD expression are more prominent in bipolar disorder than
in schizophrenia. Studies of correlates of this GABAergic abnormality at the
level of neural circuitry and cognition would be of interest. Such studies
could provide insights into the link between GABAergic abnormalities of the
hippocampus and the clinical features of bipolar disorder, potentially leading
to new targets for pharmacological intervention.
AUTHOR INFORMATION
Submitted for publication February 23, 2001; final revision returned
August 16, 2001; accepted September 11, 2001.
This study was supported by grants MH00423, MH42261, MH31152, MH/NS31862
(Dr Benes), and MH01763-02 (Dr Heckers) from the National Institute of Mental
Health, Rockville, Md, and a grant from the National Alliance for Research
on Schizophrenia and Depression, Great Neck, NY (Dr Heckers).
We thank Christine Konradi, PhD, for characterization of the cDNA clones.
Corresponding author and reprints: Francine M. Benes, MD, PhD, McLean
Hospital, 115 Mill St, Belmont, MA 02429 (e-mail: benesf{at}mclean.harvard.edu).
From the Laboratory of Structural Neuroscience, McLean Hospital, Belmont,
Mass (Drs Heckers, Stone, and Benes; Messrs Walsh and Shick; and Ms Koul),
and Department of Psychiatry (Drs Heckers and Benes) and Program of Neuroscience
(Dr Benes), Harvard Medical School, Boston, Mass.
REFERENCES
| |
1. Somogyi P, Tamas G, Lujan R, Buhl EH. Salient features of synaptic organisation in the cerebral cortex. Brain Res Brain Res Rev. 1998;26:113-135.
FULL TEXT
| PUBMED
2. Freund TF, Buzsaki G. Interneurons of the hippocampus. Hippocampus. 1996;6:347-470.
FULL TEXT
|
ISI
| PUBMED
3. Witter MP, Wouterlood FG, Naber PA, Van Haeften T. Anatomical organization of the parahippocampal-hippocampal network. In: Scharfman HE, Witter MP, Schwarcz R, eds. The Parahippocampal Region: Implications for Neurological and Psychiatric
Diseases. Vol 911. New York: Annals of the New York Academy of Sciences;
2000:1-24.
4. Sybirska E, Davachi L, Goldman-Rakic PS. Prominence of direct entorhinal-CA1 pathway activation in sensorimotor
and cognitive tasks revealed by 2-DG functional mapping in nonhuman primate. J Neurosci. 2000;20:5827-5834.
FREE FULL TEXT
5. Benes FM, Berretta S. GABAergic interneurons: implications for understanding schizophrenia
and bipolar disorder. Neuropsychopharmacology. 2001;25:1-27.
FULL TEXT
|
ISI
| PUBMED
6. Benes FM, Khan Y, Vincent SL, Wickramasinghe R. Differences in the subregional and cellular distribution of GABAA receptor
binding in the hippocampal formation of schizophrenic brain. Synapse. 1996;22:338-349.
FULL TEXT
|
ISI
| PUBMED
7. Benes FM, Wickramasinghe R, Vincent SL, Khan Y, Todtenkopf M. Uncoupling of GABA(A) and benzodiazepine receptor binding activity
in the hippocampal formation of schizophrenic brain. Brain Res. 1997;755:121-129.
FULL TEXT
|
ISI
| PUBMED
8. Benes FM, Kwok EW, Vincent SL, Todtenkopf MS. A reduction of nonpyramidal cells in sector CA2 of schizophrenics and
manic depressives. Biol Psychiatry. 1998;44:88-97.
FULL TEXT
|
ISI
| PUBMED
9. Benes FM, Sorensen I, Bird ED. Morphometric analyses of the hippocampal formation in schizophrenic
brain. Schizophr Bull. 1991;17:597-608.
10. Heckers S, Heinsen H, Geiger B, Beckmann H. Hippocampal neuron number in schizophrenia: a stereological study. Arch Gen Psychiatry. 1991;48:1002-1008.
FREE FULL TEXT
11. Arnold SE, Franz BR, Gur RC, Gur RE, Shapiro RM, Moberg PJ, Trojanowski JQ. Smaller neuron size in schizophrenia in hippocampal subfields that
mediate cortical-hippocampal interactions. Am J Psychiatry. 1995;152:738-748.
FREE FULL TEXT
12. Benes FM, Kwok EW, Vincent SL, Todtenkopf MS. A reduction of nonpyramidal cells in sector CA2 of schizophrenics and
manic depressives. Biol Psychiatry. 1998;44:88-97.
13. Costa E, Pesold C, Auta J, Caruncho H, Davis JM, Davidkova G, Dwivedi Y, Grayson DR, Rodriguez M, Uzunov DP, Guidotti A. Reelin and GAD67 downregulation and psychosis vulnerability [abstract]. Biol Psychiatry. 2000;47(suppl 8S):68S.
14. Soghomonian JJ, Martin DL. Two isoforms of glutamate decarboxylase: why? Trends Pharmacol Sci. 1998;19:500-505.
FULL TEXT
| PUBMED
15. Benson DL, Isackson PJ, Hendry SHC, Jones EG. Activity-dependent changes in GAD and preprotachykinin mRNAs in visual
cortex of adult monkeys. Cereb Cortex. 1994;4:40-51.
FREE FULL TEXT
16. Akbarian S, Kim JJ, Potkin SG, Hagman JO, Tafazzoli A, Bunney WE Jr, Jones EG. Gene expression for glutamic acid decarboxylase is reduced without
loss of neurons in prefrontal cortex of schizophrenics. Arch Gen Psychiatry. 1995;52:258-278.
FREE FULL TEXT
17. Volk DW, Austin MC, Pierri JN, Sampson AR, Lewis DA. Decreased glutamic acid decarboxylase67 messenger RNA expression
in a subset of prefrontal cortical -aminobutyric acid neurons in subjects
with schizophrenia. Arch Gen Psychiatry. 2000;57:237-245.
FREE FULL TEXT
18. Guidotti A, Auta J, Davis JM, Gerevini VD, Dwivedi Y, Grayson DR, Impagnatiello F, Pandey G, Pesold C, Sharma R, Uzunov D, Costa E. Decrease in reelin and glutamic acid decarboxylase67 (GAD67) expression in schizophrenia and bipolar disorder. Arch Gen Psychiatry. 2000;57:1061-1069.
FREE FULL TEXT
19. Feighner JP, Robins E, Guze SB. Diagnostic criteria for use in psychiatric research. Arch Gen Psychiatry. 1972;26:57-63.
FREE FULL TEXT
20. Spitzer RL, Williams JBW, Gibbon M, First MB. Structured Clinical Interview for DSM-III-R. Washington, DC: American Psychiatric Press; 1991.
21. Bu DF, Erlander MG, Hitz BC, Tillakaratne NJ, Kaufman DL, Wagner-McPherson CB, Evans GA, Tobin 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;89:2115-2119.
FREE FULL TEXT
22. Bowden CL. New concepts in mood stabilization: evidence for the effectiveness
of valproate and lamotrigine. Neuropsychopharmacology. 1998;19:194-199.
FULL TEXT
|
ISI
| PUBMED
23. Stone DJ, Walsh J, Benes FM. Localization of cells preferentially expressing GAD67 with negligible
GAD65 transcripts in the rat hippocampus: a double in situ hybridization study. Mol Brain Res. 1999;71:201-209.
PUBMED
24. Jongen-Rêlo AL, Pitkänen A, Amaral DG. Distribution of GABAergic cells and fibers in the hippocampal formation
of the macaque monkey: an immunohistochemical and in situ hybridization study. J Comp Neurol. 1999;408:237-271.
FULL TEXT
|
ISI
| PUBMED
25. Laprade N, Soghomonian JJ. Differential regulation of mRNA levels encoding for the two isoforms
of glutamate decarboxylase (GAD65 and GAD67) by dopamine receptors in the
rat striatum. Mol Brain Res. 1995;34:65-74.
PUBMED
26. Laprade N, Soghomonian JJ. Glutamate decarboxylase (GAD65) gene expression is increased by dopamine
receptor agonists in a subpopulation of rat striatal neurons. Mol Brain Res. 1997;48:333-345.
PUBMED
27. Feldblum S, Erlander MG, Tobin AJ. Different distributions of GAD65 and GAD67 mRNAs suggest that the two
glutamate decarboxylases play distinctive functional roles. J Neurosci Res. 1993;34:689-706.
FULL TEXT
|
ISI
| PUBMED
28. Vincent SL, Adamec E, Sorensen I, Benes FM. The effects of chronic haloperidol administration on GABA-immunoreactive
axon terminals in rat medial prefrontal cortex. Synapse. 1994;17:26-35.
FULL TEXT
|
ISI
| PUBMED
29. Todtenkopf MS, Benes FM. Distribution of glutamate decarboxylase65 immunoreactive puncta on
pyramidal and nonpyramidal neurons in hippocampus of schizophrenic brain. Synapse. 1998;29:323-332.
FULL TEXT
|
ISI
| PUBMED
30. Liddle PF, Friston KJ, Frith CD, Jones T, Hirsch SR, Frackowiak RSJ. Patterns of cerebral blood flow in schizophrenia. Br J Psychiatry. 1992;160:179-186.
FREE FULL TEXT
31. Friston KJ, Liddle PF, Frith CD, Hirsch SR, Frackowiak RS. The left medial temporal region and schizophrenia: a PET study. Brain. 1992;115:367-382.
FREE FULL TEXT
32. Kawasaki Y, Suzuki M, Maeda Y, Urata K, Yamaguchi N, Matsuda H, Hisada K, Suzuki M, Takashima T. Regional cerebral blood flow in patients with schizophrenia: a preliminary
report. Eur Arch Psychiatry Clin Neurosci. 1992;241:195-200.
FULL TEXT
|
ISI
| PUBMED
33. Gur RE, Mozley PD, Resnick SM, Mozley LH, Shtasel DL, Gallacher F, Arnold SE, Karp JS, Alavi A, Reivich M, Gur RC. Resting cerebral glucose metabolism in first-episode and previously
treated patients with schizophrenia relates to clinical features. Arch Gen Psychiatry. 1995;52:657-667.
FREE FULL TEXT
34. Kawasaki Y, Maeda Y, Sakai N, Higashima M, Yamaguchi N, Koshino Y, Hisada K, Suzuki M, Matsuda H. Regional cerebral blood flow in patients with schizophrenia: relevance
to symptom structures. Psychiatry Res. 1996;67:49-58.
FULL TEXT
|
ISI
| PUBMED
35. Silbersweig DA, Stern E, Frith C, Cahill C, Holmes A, Grootoonk S, Seaward J, McKenna P, Chua SE, Schnorr L, Jones T, Frackowiak RSJ. A functional neuroanatomy of hallucinations in schizophrenia. Nature. 1995;378:176-179.
FULL TEXT
| PUBMED
36. Heckers S, Rauch SL, Goff D, Savage CR, Schacter DL, Fischman AJ, Alpert NM. Impaired recruitment of the hippocampus during conscious recollection
in schizophrenia. Nat Neurosci. 1998;1:318-323.
FULL TEXT
|
ISI
| PUBMED
37. Heckers S, Goff D, Schacter DL, Savage CR, Fischman AJ, Alpert NM, Rauch SL. Functional imaging of memory retrieval in deficit vs nondeficit schizophrenia. Arch Gen Psychiatry. 1999;56:1117-1123.
FREE FULL TEXT
38. Benes FM, Todtenkopf MS, Logiotatos P, Williams M. Glutamate decarboxylase65-immunoreactive terminals in cingulate and
prefrontal cortices of schizophrenic and bipolar brain. J Chem Neuroanat. 2000;20:259-269.
FULL TEXT
|
ISI
| PUBMED
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