Expression in the Prefrontal Cortex of Subjects
with Schizophrenia
Leisa A. Glantz, Mark C. Austin, and David A. Lewis
Background:
Previous studies have reported that the
38-kd synaptic vesicle-associated protein, synaptophysin,
is decreased in the prefrontal cortex of subjects with
schizophrenia.
Methods:
To determine whether the decreased protein
levels reflect diminished expression of the synaptophysin
gene by prefrontal cortex neurons, we used in situ
hybrid-ization histochemistry to determine the cellular levels of
synaptophysin messenger RNA in prefrontal cortex area 9
from 10 matched pairs of schizophrenic and normal
control subjects.
Results:
Neither the density of neurons with detectable
levels of synaptophysin messenger RNA nor the mean level
of synaptophysin messenger RNA expression per neuron
differed between schizophrenic and control subjects in any
cortical layer.
Conclusions:
These findings indicate that the expression
of synaptophysin messenger RNA is not altered in this
brain region in schizophrenia. Consequently, reduced
levels of synaptophysin protein in the prefrontal cortex of
subjects with schizophrenia are more likely to reflect
either posttranscriptional abnormalities of synaptophysin
in prefrontal cortex neurons or a diminished number of
axonal projections to the prefrontal cortex from other
brain regions.
Biol Psychiatry 2000;48:389 –397 ©
2000
Society of Biological Psychiatry
Key Words:
Prefrontal cortex, schizophrenia, synaptic
proteins, synaptophysin, thalamus
Introduction
A
variety of studies (for reviews, see Goldman-Rakic
and Selemon 1997; Weinberger et al 1994) have
demonstrated that certain cognitive symptoms of
schizo-phrenia reflect dysfunction of the prefrontal cortex (PFC).
Nonetheless, the absence of gross structural abnormalities
in this brain region suggests that the pathophysiology of
schizophrenia may involve subtle disturbances in PFC
connectivity (Lewis 1997; Selemon and Goldman-Rakic
1999). Consistent with this hypothesis, immunoreactivity
for synaptophysin, a 38-kd integral membrane protein of
small synaptic vesicles (Jahn et al 1985; Wiedenmann and
Franke 1985), has been reported to be decreased in the
rostral PFC (areas 9, 10, and 46) of schizophrenic subjects
(Glantz and Lewis 1997; Honer et al 1999; Karson et al
1999; Perrone-Bizzozero et al 1996). Because
synaptophy-sin appears to be present in virtually all presynaptic axon
terminals (Jahn et al 1985) and to serve as a reliable
marker of the number of cortical synapses (Hamos et al
1989; Masliah et al 1990), these findings may reflect a
decreased number of presynaptic terminals in the PFC of
subjects with schizophrenia. This interpretation is
sup-ported by reports of decreased gray matter volume
(An-dreasen et al 1994a; Goldstein et al 1999; Schlaepfer et al
1994; Shelton et al 1988; Sullivan et al 1998; Zipursky et
al 1992), increased cell packing density (Daviss and Lewis
1995; Selemon et al 1995, 1998), decreased dendritic
spine density (Garey et al 1998; Glantz and Lewis 2000),
and diminished levels of
N
-acetylaspartate (Bertolino et al
1996, 1998; a marker of neuronal/axonal integrity) in the
PFC of subjects with schizophrenia. Although other
ex-planations for each of these abnormalities are possible, all
would be expected to accompany a decrease in presynaptic
terminal number.
Understanding the pathophysiologic significance of a
decreased number of presynaptic terminals depends, in
part, on which populations of axons are affected. Axon
terminals can be divided into two general categories: those
that arise from neurons located within that region (intrinsic
terminals) and those that arise from neurons in other brain
regions (afferent terminals; White 1989). If the decreased
synaptophysin protein in the PFC of schizophrenic
sub-jects reflects an abnormality in intrinsic axon terminals,
then one might expect to see altered expression of
synap-tophysin messenger RNA (mRNA) in PFC neurons. For
example, if the decreased dendritic spine density on PFC
From the Departments of Neuroscience (LAG, DAL) and Psychiatry (MCA, DAL), University of Pittsburgh, Pittsburgh, Pennsylvania.
Address reprint requests to David A. Lewis, M.D., University of Pittsburgh, Western Psychiatric Institute & Clinic, 3811 O’Hara Street, W 1651 BST, Pittsburgh PA 15213.
Received January 7, 2000; revised April 26, 2000; accepted April 28, 2000.
layer 3 pyramidal cells in schizophrenia (Garey et al 1998;
Glantz and Lewis 2000) is due to a reduced number of
intrinsic excitatory synapses, then one might expect to see
altered synaptophysin expression in the sources of such
inputs—namely, pyramidal neurons in layers 2, 3, and 5
(Levitt et al 1993; Melchitzky et al 1998). To test this
hypothesis, we used in situ hybridization histochemistry to
examine the cellular levels of synaptophysin mRNA in
PFC area 9 from matched pairs of schizophrenic and
control subjects.
Methods and Materials
Characteristics of Subjects
Specimens from 20 human brains were obtained during autopsies conducted at the Allegheny County Coroner’s Office (Table 1) after informed consent for brain donation was obtained from the
next of kin. No neuropathologic abnormalities were detected except in two subjects. Subject 622 died from an acute infarction limited to the distribution of the inferior branch of the right middle cerebral artery; however, the cortical region of interest for the present study did not appear to be affected. In addition, thioflavin-S staining revealed a few senile plaques in one subject (685), but the density of plaques was insufficient to meet diagnostic criteria for Alzheimer’s disease (Mirra et al 1991), and there was no history of dementia in this subject. An independent committee of experienced clinicians made consensus DSM-III-R diagnoses for each subject using information obtained from clinical records and structured interviews conducted with one or more surviving relatives of the subject (Glantz and Lewis 1997). The University of Pittsburgh’s Institutional Review Board ap-proved all procedures for Biomedical Research.
Seven male and three female subjects with diagnoses of schizophrenia or schizoaffective disorder were examined in this study (Table 1). All of these subjects had been used in a previous Table 1. Characteristics of Matched Pairs of Subjects
Control subjects Schizophrenic subjects
Pair Case Race
of death Case Diagnosis Race Gender/
1 630 W M/65 21.2 6.94 12 ASCVD 566 Schizophrenia, chronic undifferentiatedb
W M/63 18.3 6.80 23 ASCVD
2 643 W M/50 24.0 6.02 11 ASCVD 581 Schizophrenia, chronic paranoidc,d
W M/46 28.1 6.99 20 Accidental drug overdose 3 567 W F/46 15.0 6.72 22 Mitral valve
prolapse
597 Schizoaffective disorder
W F/46 10.1 7.02 18 Pneumonia
4 585 W M/26 16.0 6.67 20 Trauma 547 Schizoaffective disorder
B M/27 16.5 6.94 26 Heat stroke
5 685 W M/56 14.5 6.57 5 Hypoplastic coronary
W M/48 8.3 6.07 75 Pneumonia
6 557 W M/47 15.9 6.77 24 ASCVD 537 Schizoaffective disordere
W F/37 14.5 6.68 27 Suicide by hanging 7 546 W F/37 23.5 6.74 26 ASCVD 587 Schizophrenia,
chronic undifferentiatedb
B F/38 17.8 7.02 19 Myocardial hypertrophy
9 659 W M/46 22.3 6.77 8 Peritonitis 625 Schizophrenia, chronic disorganizedf
B M/49 23.5 7.05 13 ASCVD
10 592 B M/41 22.1 6.72 18 ASCVD 533 Schizophrenia, chronic undifferentiated
W M/40 29.1 6.82 18 Asphyxiation
Mean 47.5 19.1 6.66 17.1 45.2 18.5 6.82 25.2
SD 11.5 3.8 0.24 7.6 10.4 6.9 0.29 18.1
PMI, postmortem interval in hours; W, white; M, male; ASCVD, atherosclerotic coronary vascular disease; F, female; B, black; MCA, middle cerebral artery.
aStorage time (at280°C) is in months. bAlcohol abuse, in remission at time of death. cAlcohol dependence, current at time of death. dOther substance abuse, current at time of death.
study of GAD67mRNA expression (Volk et al 2000). Four of these subjects also met diagnostic criteria for an alcohol-related disorder at some point during their life. Two subjects (537 and 622) had been off antipsychotic agents for 9.6 and 1.2 months, respectively, before death. Each schizophrenic subject was matched as closely as possible for gender, age, and postmortem interval (PMI) to one control subject who had no lifetime history of any neurologic or psychiatric disorder (Table 1). Subject groups did not significantly differ (t,1.77,p..11) in mean age or PMI (Table 1).
Preparation of Tissue
At the time of autopsy, the brain was removed and placed into cold phosphate buffer. Within 2 hours, coronal blocks (1.0 cm thick) were cut from the right hemisphere, frozen by immersion in 2-methylbutane on dry ice, and stored at280°C. Brain pH, determined using the procedure of Harrison et al (1995), did not differ (t 5 1.35, p5 .21) between subject groups (Table 1). Blocks containing the superior frontal gyrus immediately ante-rior to the genu of the corpus callosum were then sectioned coronally at 20mm and thaw-mounted onto gelatin-coated slides. Every tenth section was stained for Nissl substance and exam-ined to identify the location of area 9 using cytoarchitectonic criteria (Daviss and Lewis 1995; Rajkowska and Goldman-Rakic 1995). Slide-mounted tissue sections were stored at280°C until processed. Total tissue storage time (t51.12,p5.29) did not differ across subject groups (Table 1). For the range of sections determined to contain area 9, a random number table was used to select a starting section and then a total of five, equally spaced (200 mm) sections containing area 9 were selected from each subject. All 100 sections were processed for in situ hybridization simultaneously and in the same solutions.
In Situ Hybridization Procedure
Sections were processed for in situ hybridization histochemistry with a cocktail of two commercially synthesized (Oligos Etc., Wilsonville, OR) oligonucleotide probes complementary to bases 1–33 and 803– 842 of the synaptophysin cDNA sequence (Sudhof et al 1987). The probes were 39-labeled with35S-dATP (NEN, Boston) by terminal deoxynucleotidyl transferase (Be-thesda Research Laboratories, Gaithersburg, MD). Labeled oli-gonucleotides were separated from unincorporated nucleotides using Nuc-trap push columns (Stratagene, La Jolla, CA).
Tissue sections were immersed in 4% paraformaldehyde in 0.12 mol/L sodium phosphate-buffered saline at room tempera-ture for 5 min, washed, and then placed in a 0.1 mol/L triethanolamine/0.9% sodium chloride solution containing 0.25% acetic anhydride for 10 min. Next, sections were dehydrated and delipidated in a graded series of ethanol washes and chloroform and then incubated for 2 hours at 37°C with 50 mmol/L nonradioactive dATP in hybridization buffer (100mL per slide) consisting of 50% deionized formamide, 600 mmol/L sodium chloride, 80 mmol/L tris hydrochloric acid, 4 mmol/L EDTA, 0.1% tetrasodium pyrophosphate, 0.2% sodium dodecylsulfate, 0.2 mg/mL heparin, 10% dextran sulfate, and 100 mmol/L dithiothreitol. This buffer was removed and sections were then
incubated overnight at 37°C in hybridization buffer (50mL per slide) containing both 35S-dATP-labeled probes (1.5
3 106 dpm/section). Following four washes for 15 min each in 23
saline-sodium citrate (SSC) (13 SSC: 0.15 mol/L sodium chloride, 0.015 mol/L sodium citrate, pH 7.2)/50% deionized formamide at 40°C, slides were washed twice for 30 min in 13
SSC at 40°C, briefly rinsed in distilled water and 70% ethanol, and then allowed to dry. The sections were dipped in photo-graphic emulsion (NTB2, Kodak) diluted 1:1 with distilled water, and exposed for 2.5 weeks at 4°C. After the emulsion was developed, slides were lightly counterstained with cresyl violet and then coded so that the investigators were blind to subject number and diagnosis.
To assess the specificity of the hybridization signal, additional sections were pretreated either with RNase or excess unlabeled probe. No signal was detected in either case. In addition, other sections were treated using only one of the two synaptophysin probes. Identical patterns of labeling were observed with each probe used alone or in combination.
Analysis
Statistical Analyses
Within each layer of every section, the values of the three dependent variables (neuron density, grain density per neuron, and somal size) were averaged across the two sampling frames, with the value of each frame weighted by the number of observations in that frame. Thus, for each cortical layer of every subject, five observations were obtained for each of the three dependent variables. Because these values were possibly corre-lated and were also exchangeable within a given subject, the five observations were treated as repeated measures with a compound symmetric covariance structure (Neter et al 1996). This ance structure was reflected in a multivariate analysis of covari-ance (MANCOVA) that examined the effect of diagnostic group on each of the three dependent variables using age, gender, PMI, storage time, and brain pH as covariates. Analyses were imple-mented in SAS PROC Mixed (Littell et al 1996) andFtests for the effect of diagnostic group were based on type III sum of squares.
Results
On tissue sections hybridized with the
35S-labeled
synap-tophysin probes and counterstained with cresyl violet,
silver grains were clustered over the cell bodies of both
pyramidal and nonpyramidal neurons (Figure 1). In
con-trast, few grains were present over glial cells, and grain
clusters were virtually absent in the subjacent white matter
(Figure 2). Consistent with the neuronal localization of
synaptophysin, the distribution of grain clusters reflected
the relative size and packing density of neurons across the
layers of the cortex (Figure 2), although the density of grain
clusters was somewhat lower than expected in layer 2.
Neither the density of neurons with detectable levels of
synaptophysin mRNA [Figure 3A;
F
(1,13)
,
2.16,
p
.
.17] nor the mean level of synaptophysin mRNA
expres-sion per neuron [Figure 3B;
F
(1,13)
,
2.84,
p
.
.12]
differed between schizophrenic and control subjects in any
layer. In addition, neither the schizophrenic subjects with
comorbid diagnoses of alcohol abuse or dependence or the
schizoaffective subjects differed from their matched
con-trol subjects on either measure. Interestingly, among the
subjects with schizophrenia, two of the schizoaffective
subjects (547 and 597) had the lowest values for mean
grain density in all layers except layer 6; however, the
third schizoaffective subject (537) had mean grain
densi-ties well above most of the other subjects with
schizophre-nia in all of the layers.
The somal size of synaptophysin mRNA-positive
neu-rons was decreased by 4.9% to 12% in superficial layer 3,
deep layer 3, layer 5, and layer 6 in the schizophrenic
subjects compared with the matched normal control
sub-jects, but none of these differences achieved statistical
significance [Figure 3C;
F
(1,13)
,
2.63,
p
.
.13]. In
addition, grain density did not differ between the
schizo-phrenic and control subjects when examined as a function
of somal size (Figure 4). Finally, mean grain number per
neuron did not differ between schizophrenic and control
subjects in any layer (Figure 5).
Discussion
The results of this study indicate that the cellular
expres-sion of synaptophysin mRNA, whether assessed by the
density of labeled neurons or by grain density or number
per labeled neuron, is not altered in PFC area 9 of
schizophrenic subjects. These observations are supported
by recent preliminary reports, using regional analyses of
film autoradiographic images (Eastwood and Harrison
1998; Rodriguez et al 1998) or Northern blots (Karson et
al 1999), of normal levels of synaptophysin mRNA in the
PFC of schizophrenic subjects.
Although the findings across studies are consistent in
revealing normal levels of synaptophysin mRNA
expres-sion in schizophrenia, the possibility of a type 2 error
needs to be considered. First, our sample size (
n
5
10
subjects per group) was relatively small; however, similar
numbers of subjects were examined in the studies that
found decreased levels of synaptophysin protein in the
PFC of schizophrenic subjects ( Glantz and Lewis 1997;
Honer et al 1999; Karson et al 1999; Perrone-Bizzozero et
al 1996). In addition, a recent investigation of the same
schizophrenic subjects examined in our study detected a
significant decrease in the expression of GAD
67mRNA in
layers 3, 4, and 5 of PFC area 9 (Volk et al 2000). Second,
the tendency for neuronal size to be smaller in layers 3, 5,
and 6 of the subjects with schizophrenia may have
contributed to less overcounting of synaptophysin
mRNA-labeled neurons in these subjects. Nonetheless, because
this difference was small relative to both the measured
somal size and section thickness, use of an Abercrombie
correction did not alter the relative densities of
synapto-physin-labeled neurons (Guillery and Herrup 1997). Third,
the stringent threshold employed for specifically labeled
cells (defined as those with grain densities at least 5
3
background) may have excluded cells with decreased
expression of synaptophysin mRNA in subjects with
schizophrenia; however, the use of a threshold of 3
3
background also failed to reveal differences between
schizophrenic and control subjects in any measure of
synaptophysin mRNA expression. Fourth, some (Selemon
et al 1995; 1998) but not all (Akbarian et al 1995) studies
suggest that neuronal density is increased in the dorsal
PFC of schizophrenic subjects. Thus, it is possible that our
failure to detect an increase in the density of
synaptophy-sin-labeled neurons in the subjects with schizophrenia
actually represents a decrease in the number of neurons
with detectable levels of synaptophysin expression.
None-theless, other studies using tissue homogenate based
tech-niques have also failed to find altered levels of
synapto-physin mRNA expression in the PFC of schizophrenic
subjects (Eastwood and Harrison 1998; Karson et al 1999;
Rodriguez et al 1998). Finally, because this study
exam-ined the right hemisphere and our previous study of
synaptophysin protein examined the left hemisphere, it is
possible that the synaptophysin alterations in
schizophre-nia are lateralized; however, Perrone-Bizzozero et
al(1996) also found decreased synaptophysin protein
lev-els in right area 9 of schizophrenic subjects, and two
studies failed to find decreased synaptophysin mRNA in
the left PFC of schizophrenic subjects (Eastwood and
Harrison 1998; Karson et al 1999).
Although different PFC regions (areas 9 and 10, right
hemisphere [Perrone-Bizzozero et al 1996]; areas 9 and
46, left hemisphere [Glantz and Lewis 1997]; area 10, left
hemisphere [Karson et al 1999]; and anterolateral inferior
or middle frontal gyrus, hemisphere unspecified [Honer et
al 1999]) were examined, four of four studies found
decreased levels of synaptophysin protein in the rostral
PFC of schizophrenic subjects. In addition, another study
of left PFC areas 45 and 46 found a 19% decrease in
synaptophysin levels in schizophrenia, although this
dif-ference did not achieve statistical significance (Davidsson
et al 1999). The only study that did not find a decrease in
synaptophysin protein examined a caudal PFC region
(area 8; Gabriel et al 1997). Thus, decreased
synaptophy-sin protein in the rostral PFC appears to be common
bilaterally in schizophrenia, but it does not appear to be
attributable to diminished synaptophysin gene expression
by PFC neurons (Eastwood and Harrison 1998; Karson et
al 1999; Rodriguez et al 1998; the study under discussion);
however, these observations do not necessarily rule out an
abnormality in PFC neurons as a cause of decreased
synaptophysin protein levels in schizophrenia for several
reasons. First, it is possible that the 15 to 40% decrease in
synaptophysin protein reported in previous studies
(Dav-idsson et al 1999; Glantz and Lewis 1997; Honer et al
1999; Karson et al 1999; Perrone-Bizzozero et al 1996)
may be associated with a reduction in synaptophysin
Figure 3. Scatter plots illustrating the (A)
density of synaptophysin messenger RNA (mRNA)-labeled neurons, (B) grain density per synaptophysin mRNA-labeled neuron, and
mRNA that is too small to be detected by in situ
hybrid-ization techniques. Indeed, synaptophysin mRNA and
protein levels may not be tightly correlated. For example,
although the amount of synaptophysin protein in cultured
hippocampal cells increases substantially during
develop-ment, mRNA levels show a more modest increase,
sug-gesting that the levels of synaptophysin protein are
con-trolled posttranscriptionally (Daly and Ziff 1997). In
addition, the mRNA and protein levels of synapsin,
another synaptic-vesicle associated protein, do not always
correspond in the rat hippocampus (Melloni et al 1993).
Second, abnormal protein levels but normal levels of
synaptophysin mRNA expression in the PFC of subjects
with schizophrenia could reflect posttranslational
modifi-cations of synaptophysin that impair the recognition of the
protein by immunologic techniques. Third, our
observa-tion of a trend toward a 5% to 12% decrease in somal size
in layers 3, 5, and 6 of the schizophrenic subjects may be
consistent with an intrinsic PFC abnormality. This slight
reduction in somal size is in agreement with recent reports
of decreased layer 3 pyramidal neuron size in the PFC of
subjects with schizophrenia (Pierri et al 1999; Rajkowska
et al 1998).
Another possible explanation of decreased
synaptophy-sin protein with normal synaptophysynaptophy-sin mRNA levels in the
PFC of subjects with schizophrenia may be that one or
more afferent projections to the PFC are abnormal in this
disease. For example, schizophrenic subjects have been
reported to have decreased levels of synaptophysin mRNA
in anterior cingulate (area 24) and superior temporal (area
22) cortices ( Davidsson et al 1999; Eastwood and
Harri-son 1998; Mitchell et al 1985), cortical areas known to
project to PFC area 9 (Barbas 1992; Goldman-Rakic
1987). In addition, subcortical sites, such as the
mediodor-sal thalamic nucleus, may also contribute fewer afferents
to the PFC in schizophrenia. For example, this nucleus has
been reported to be smaller in size and to have fewer
neurons in subjects with schizophrenia (Andreasen et al
1994b; Manaye et al 1998; Pakkenberg 1990; Popken et al
1998). The decreased density of dendritic spines on
pyramidal neurons in deep layer 3 of the PFC, the thalamic
recipient zone, may also reflect a decreased number of
thalamic afferents to the PFC (Glantz and Lewis 2000).
In summary, although the source(s) of a decreased
synaptophysin protein in the rostral PFC of subjects with
schizophrenia remains to be determined, diminished
ex-pression of synaptophysin mRNA by PFC neurons does
not appear to be a contributing factor.
This work was supported by USPHS Grants Nos. MH00519 and MH45156 and the Scottish Rite Schizophrenia Research Program, N.M.J. We thank Dr. Allan Sampson and Ms. Sungyoung Auh for statistical consultations, Mrs. Mary Brady for photographic assistance, and Sandra O’Donnell and David Volk for assistance with the methodology of this study.
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