Normal cellular levels of synaptophysin mRNA expression in the prefrontal cortex of subjects with schizophrenia

Normal Cellular Levels of Synaptophysin mRNA 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 hybridization 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 schizophrenia reflect dysfunction of the prefrontal cortex (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.

© 2000 Society of Biological Psychiatry

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 synaptophysin 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 supported by reports of decreased gray matter volume (Andreasen 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 explanations 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 subjects reflects an abnormality in intrinsic axon terminals, then one might expect to see altered expression of synaptophysin messenger RNA (mRNA) in PFC neurons. For example, if the decreased dendritic spine density on PFC 0006-3223/00/$20.00 PII S0006-3223(00)00923-9

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Table 1. Characteristics of Matched Pairs of Subjects Control subjects Pair

Case Race

Schizophrenic subjects

Gender/ Brain Storage age PMI pH time

Cause of death

1

630

W

M/65

21.2 6.94

12

ASCVD

2

643

W

M/50

24.0 6.02

11

ASCVD

3

567

W

F/46

15.0 6.72

22

4

585

W

M/26

16.0 6.67

20

Mitral valve prolapse Trauma

5

685

W

M/56

14.5 6.57

5

6

557

W

M/47

15.9 6.77

24

Hypoplastic coronary artery disease ASCVD

7

546

W

F/37

23.5 6.74

26

ASCVD

8

551

W

M/61

16.4 6.63

25

Cardiac tamponade

9

659

W

M/46

22.3 6.77

8

10

592

B

M/41

22.1 6.72

18

47.5 11.5

19.1 6.66 3.8 0.24

17.1 7.6

Mean SD

Peritonitis

ASCVD

Case

Diagnosis

566 Schizophrenia, chronic undifferentiatedb 581 Schizophrenia, chronic paranoidc,d 597 Schizoaffective disorder 547 Schizoaffective disorder 317 Schizophrenia, chronic undifferentiated 537 Schizoaffective disordere 587 Schizophrenia, chronic undifferentiatedb 622 Schizophrenic, chronic undifferentiatede 625 Schizophrenia, chronic disorganizedf 533 Schizophrenia, chronic undifferentiated

Race

Gender/ Brain Storage age PMI pH timea

Cause of death

W

M/63

18.3 6.80

23

ASCVD

W

M/46

28.1 6.99

20

W

F/46

10.1 7.02

18

Accidental drug overdose Pneumonia

B

M/27

16.5 6.94

26

Heat stroke

W

M/48

8.3 6.07

75

Pneumonia

W

F/37

14.5 6.68

27

B

F/38

17.8 7.02

19

Suicide by hanging Myocardial hypertrophy

W

M/58

18.9 6.78

13

Right MCA infarction

B

M/49

23.5 7.05

13

ASCVD

W

M/40

29.1 6.82

18

Asphyxiation

45.2 10.4

18.5 6.82 6.9 0.29

25.2 18.1

PMI, postmortem interval in hours; W, white; M, male; ASCVD, atherosclerotic coronary vascular disease; F, female; B, black; MCA, middle cerebral artery. a Storage time (at ⫺80°C) is in months. b Alcohol abuse, in remission at time of death. c Alcohol dependence, current at time of death. d Other substance abuse, current at time of death. e Schizophrenic subjects off antipsychotic medications at time of death. f Alcohol abuse, current at time of death.

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 approved 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

Synaptophysin mRNA in Schizophrenia

study of GAD67 mRNA 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 at ⫺80°C. Brain pH, determined using the procedure of Harrison et al (1995), did not differ (t ⫽ 1.35, p ⫽ .21) between subject groups (Table 1). Blocks containing the superior frontal gyrus immediately anterior to the genu of the corpus callosum were then sectioned coronally at 20 ␮m and thaw-mounted onto gelatin-coated slides. Every tenth section was stained for Nissl substance and examined 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 at ⫺80°C until processed. Total tissue storage time (t ⫽ 1.12, p ⫽ .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 ␮m) 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 3⬘-labeled with 35S-dATP (NEN, Boston) by terminal deoxynucleotidyl transferase (Bethesda Research Laboratories, Gaithersburg, MD). Labeled oligonucleotides 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 temperature 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 ␮mol/L nonradioactive dATP in hybridization buffer (100 ␮L 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

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incubated overnight at 37°C in hybridization buffer (50 ␮L per slide) containing both 35S-dATP-labeled probes (1.5 ⫻ 106 dpm/section). Following four washes for 15 min each in 2⫻ saline-sodium citrate (SSC) (1⫻ 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 1⫻ SSC at 40°C, briefly rinsed in distilled water and 70% ethanol, and then allowed to dry. The sections were dipped in photographic 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 Quantitative assessments of synaptophysin mRNA expression were conducted using a previously described approach (Volk et al 2000). Using a Micro Computer Imaging Device (MCID; Imaging Research, Ontario, Can.) at a magnification of 1510⫻, a sampling frame (140 ⫻ 140 ␮m) was randomly placed along lines, tangential to the pial surface, that marked the midpoint of layers 2, superficial 3, deep 3, 4, 5, and 6. Layer 1 was not quantified because of the low density of labeled neurons in that layer, and layer 3 was subdivided because the superficial and deep pyramidal neurons in this layer have been reported to be differentially affected in other studies of schizophrenia (Rajkowska et al 1998; Glantz and Lewis 2000). Two frames in each layer were sampled in each of the five sections per subject. Neurons intersected by the upper or right borders of the sampling frame were included, whereas those intersected by the lower or left borders of the sampling frame were excluded. Because the overlying silver grains frequently made nucleoli difficult to detect, all Nissl-stained profiles with the characteristic features of neurons were sampled. Glial cells (identified by small size, intense Nissl staining, and high nuclear to cytoplasm ratio) were excluded. Within each sampling frame, the borders of each neuron with more than three grains over its soma were manually outlined, and the number of grains over the encircled cell and the cross-sectional somal area were determined. More than 8900 neurons were sampled in this study, ranging from 181 to 665 neurons per subject. Grain density per neuron was determined for each sampled cell by dividing the grain count by the somal area. To control for nonspecific background label, two sample frames were randomly placed over the white matter in each section and the grain density in each frame was determined. The mean grain density over the white matter was then subtracted from the neuronal measures for each section. Cells exhibiting grain densities greater than five times background levels were considered specifically labeled (Volk et al 2000) and included in the data analysis. All measures were conducted by one investigator (LAG).

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Figure 1. Brightfield photomicrograph from prefrontal cortex area 9 processed with 35Ssynaptophysin oligonucleotide probes and counterstained with cresyl violet. Silver grains are clustered over both pyramidal (open arrows) and nonpyramidal (solid arrows) cell bodies. In contrast, few grains are present over glial cells (arrowheads). Calibration bar, 50 ␮m.

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 correlated 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 covariance structure was reflected in a multivariate analysis of covariance (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 implemented in SAS PROC Mixed (Littell et al 1996) and F tests for the effect of diagnostic group were based on type III sum of squares.

Results On tissue sections hybridized with the 35S-labeled synaptophysin probes and counterstained with cresyl violet, silver grains were clustered over the cell bodies of both pyramidal and nonpyramidal neurons (Figure 1). In contrast, 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 control 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 densities well above most of the other subjects with schizophrenia in all of the layers. The somal size of synaptophysin mRNA-positive neurons 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 subjects, 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 schizophrenic 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 expression 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

Synaptophysin mRNA in Schizophrenia

Figure 2. Darkfield photomicrograph from prefrontal cortex area 9 illustrating the size and packing density of synaptophysin messenger RNA–positive neurons across cortical layers. Note the absence of grain clusters in the white matter (WM). Numerals indicate cortical layers. Calibration bar, 300 ␮m.

1998; Rodriguez et al 1998) or Northern blots (Karson et al 1999), of normal levels of synaptophysin mRNA in the PFC of schizophrenic subjects.

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Although the findings across studies are consistent in revealing normal levels of synaptophysin mRNA expression in schizophrenia, the possibility of a type 2 error needs to be considered. First, our sample size (n ⫽ 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 GAD67 mRNA 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 mRNAlabeled 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 synaptophysin-labeled neurons (Guillery and Herrup 1997). Third, the stringent threshold employed for specifically labeled cells (defined as those with grain densities at least 5⫻ background) may have excluded cells with decreased expression of synaptophysin mRNA in subjects with schizophrenia; however, the use of a threshold of 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 synaptophysin-labeled neurons in the subjects with schizophrenia actually represents a decrease in the number of neurons with detectable levels of synaptophysin expression. Nonetheless, other studies using tissue homogenate based techniques have also failed to find altered levels of synaptophysin mRNA expression in the PFC of schizophrenic subjects (Eastwood and Harrison 1998; Karson et al 1999; Rodriguez et al 1998). Finally, because this study examined the right hemisphere and our previous study of synaptophysin protein examined the left hemisphere, it is possible that the synaptophysin alterations in schizophrenia are lateralized; however, Perrone-Bizzozero et al(1996) also found decreased synaptophysin protein levels 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

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Figure 3. Scatter plots illustrating the (A) density of synaptophysin messenger RNA (mRNA)-labeled neurons, (B) grain density per synaptophysin mRNA-labeled neuron, and (C) cross-sectional somal size of synaptophysin mRNA-labeled neurons in control (E) and schizophrenic (䢇) subjects for each cortical layer measured. Hash marks indicate mean values. No significant differences between schizophrenic and control subjects were found for any measure in any layer. 3s, superficial layer 3; 3d, deep layer 3.

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 difference 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 synaptophysin 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 (Davidsson 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

Synaptophysin mRNA in Schizophrenia

Figure 4. Bar graph illustrating the mean (⫾ SD) grain density in control (open bars) and schizophrenic (solid bars) subjects across all of the layers for different cell size groupings. No significant differences in grain density were observed between schizophrenic and control subjects for any cell size.

mRNA that is too small to be detected by in situ hybridization 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 development, mRNA levels show a more modest increase, suggesting that the levels of synaptophysin protein are controlled 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

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synaptophysin mRNA expression in the PFC of subjects with schizophrenia could reflect posttranslational modifications of synaptophysin that impair the recognition of the protein by immunologic techniques. Third, our observation 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 synaptophysin protein with normal synaptophysin 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 Harrison 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 mediodorsal 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 expression 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.

References

Figure 5. Bar graph illustrating mean (⫾ SD) grain number per synaptophysin mRNA labeled neuron for control (open bars) and schizophrenic (solid bars) subjects. No significant differences between schizophrenic and control subjects were present in any layer. 3s, superficial layer 3; 3d, deep layer 3.

Akbarian S, Kim JJ, Potkin SG, Hagman JO, Tafazzoli A, Bunney WE Jr, et al (1995): Gene expression for glutamic acid decarboxylase is reduced without loss of neurons in prefrontal cortex of schizophrenics. Arch Gen Psychiatry 52:258 –266. Andreasen NC, Arndt S, Swayze V II, Cizaldo T, Flaum M, O’Leary D, et al (1994b): Thalamic abnormalities in schizo-

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phrenia visualized through magnetic resonance image averaging. Science 266:294 –298. Andreasen NC, Flashman L, Flaum M, Arndt S, Swayze V II, O’Leary DS, et al (1994a): Regional brain abnormalities in schizophrenia measured with magnetic resonance imaging. JAMA 272:1763–1769. Barbas H (1992): Architecture and cortical connections of the prefrontal cortex in the Rhesus monkey. Adv Neurol 57:91– 115. Bertolino A, Callicott JH, Nawroz S, Mattay VS, Duyn JH, Tecleschi G, et al (1998): Reproducibility of proton magnetic resonance spectroscopic imaging in patients with schizophrenia. Neuropsychopharmacology 18:1–9. Bertolino A, Nawroz S, Mattay VS, Barnett AS, Duyn JH, Moonen CTW, et al (1996): Regionally specific pattern of neurochemical pathology in schizophrenia as assessed by multislice proton magnetic resonance spectroscopic imaging. Am J Psychiatry 153:1554 –1563. Daly C, Ziff EB (1997): Post-transcriptional regulation of synaptic vesicle protein expression and the developmental control of synaptic vesicle formation. J Neurosci 17:2365–2375. Davidsson P, Gottfries J, Bogdanovic N, Ekman R, Karlsson I, Gottfries C-G, et al (1999): The synaptic-vesicle-specific proteins rab3a and synaptophysin are reduced in thalamus and related cortical brain regions in schizophrenic brains. Schizophr Res 40:23–29. Daviss SR, Lewis DA (1995): Local circuit neurons of the prefrontal cortex in schizophrenia: Selective increase in the density of calbindin-immunoreactive neurons. Psychiatry Res 59:81–96. Eastwood SL, Harrison PJ (1998): Hippocampal and cortical growth-associated protein-43 messenger RNA in schizophrenia. Neuroscience 86:437– 448. Gabriel SM, Haroutunian V, Powchik P, Honer WG, Davidson M, Davies P, et al (1997): Increased concentrations of presynaptic proteins in the cingulate cortex of subjects with schizophrenia. Arch Gen Psychiatry 54:559 –566. Garey LJ, Ong WY, Patel TS, Kanani M, Davis A, Mortimer AM, et al (1998): Reduced dendritic spine density on cerebral cortical pyramidal neurons in schizophrenia. J Neurol Neurosurg Psychiatry 65:446 – 453. Glantz LA, Lewis DA (1997): Reduction of synaptophysin immunoreactivity in the prefrontal cortex of subjects with schizophrenia: Regional and diagnostic specificity. Arch Gen Psychiatry 54:943–952. Glantz LA, Lewis DA (2000): Decreased dendritic spine density on prefrontal cortical pyramidal neurons in schizophrenia. Arch Gen Psychiatry 57:65–73. Goldman-Rakic PS (1987): Circuitry of primate prefrontal cortex and regulation of behavior by representational memory. In: Plum F, Mountcastle V, editors. Handbook of Physiology. Bethesda, MD: American Physiological Society, 373– 417. Goldman-Rakic PS, Selemon LD (1997): Functional and anatomical aspects of prefrontal pathology in schizophrenia. Schizophr Bull 23:437– 458. Goldstein JM, Goodman JM, Seidman LJ, Kennedy DN, Makris N, Lee H, et al (1999): Cortical abnormalities in schizophrenia identified by structural magnetic resonance imaging. Arch Gen Psychiatry 56:537–547.

L.A. Glantz et al

Guillery RW, Herrup K (1997): Quantification without pontification: Choosing a method for counting objects in sectioned tissues. J Comp Neurol 386:2–7. Hamos JE, DeGennaro LJ, Drachman DA (1989): Synaptic loss in Alzheimer’s disease and other dementias. Neurology 39: 355–361. Harrison PJ, Heath PR, Eastwood SL, Burnet PWJ, McDonald B, Pearson RCA (1995): 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 200:151– 154. Honer WG, Falkai P, Chen C, Arango V, Mann JJ, Dwork AJ (1999): Synaptic and plasticity-associated proteins in anterior frontal cortex in severe mental illness. Neuroscience 91: 1247–1255. Jahn R, Schiebler W, Ouimet C, Greengard P (1985): A 38,000-dalton membrane protein (p38) present in synaptic vesicles. Proc Natl Acad Sci U S A 82:4137– 4141. Karson CN, Mrak RE, Schluterman KO, Sturner WQ, Sheng JG, Griffin WST (1999): Alterations in synaptic proteins and their encoding mRNAs in prefrontal cortex in schizophenia: A possible neurochemical basis for “hypofrontality”. Mol Psychiatry 4:39 – 45. Levitt JB, Lewis DA, Yoshioka T, Lund JS (1993): Topography of pyramidal neuron intrinsic connections in macaque monkey prefrontal cortex (areas 9 & 46). J Comp Neurol 338: 360 –376. Lewis DA (1997): Development of the prefrontal cortex during adolescence: Insights into vulnerable neural circuits in schizophrenia. Neuropsychopharmacology 16:385–398. Littell RC, Milliken GA, Stroup WW, Wolfinger RD (1996): SAS System for Mixed Models. Cary, NC: SAS Institute. Manaye KF, Liang C-L, Hicks PB, German D, Young KA (1998): Nerve cell numbers in thalamic anterior and mediodorsal nuclei are selectively reduced in schizophrenia. Soc Neurosci Abstr 24:1236. Masliah E, Terry RD, Alford M, DeTeresa R (1990): Quantitative immunohistochemistry of synaptophysin in human neocortex: An alternative method to estimate density of presynaptic terminals in paraffin sections. J Histochem Cytochem 38:837– 844. Melchitzky DS, Sesack SR, Pucak ML, Lewis DA (1998): Synaptic targets of pyramidal neurons providing intrinsic horizontal connections in monkey prefrontal cortex. J Comp Neurol 390:211–224. Melloni RH Jr, Hemmendinger LM, Hamos JE, DeGennero LJ (1993): Synapsin I gene expression in the adult brain with comparative analysis of mRNA and protein in the hippocampus. J Comp Neurol 327:507–520. Mirra SS, Heyman A, McKeel D, Sumi SM, Crain BJ, Brownlee LM, et al (1991): The consortium to establish a registry for Alzheimer’s disease (CERAD). Part II. Standardization of the neuropathological assessment of Alzheimer’s disease. Neurology 41:479 – 486. Mitchell IJ, Cross AJ, Sambrook MA, Crossman AR (1985): Sites of the neurotoxic action of 1-methyl-4-phenyl-1,2,3,6tetrahydropyridine in the macaque monkey include the ventral tegmental area and the locus coeruleus. Neurosci Lett 61:195–200.

Synaptophysin mRNA in Schizophrenia

Neter J, Kutner MH, Nachtsheim CJ, Wasserman W (1996): Applied linear statistical models, 4th ed. Chicago: Irwin. Pakkenberg B (1990): Pronounced reduction of total neuron number in mediodorsal thalamic nucleus and nucleus accumbens in schizophrenics. Arch Gen Psychiatry 47:1023–1028. Perrone-Bizzozero NI, Sower AC, Bird ED, Benowitz LI, Ivins KJ, Neve RL (1996): Levels of the growth-associated protein GAP-43 are selectively increased in association cortices in schizophrenia. Proc Natl Acad Sci U S A 93:14182–14187. Pierri JN, Edgar CL, Lewis DA (1999): Somal size of pyramidal neurons in deep layer 3 of prefrontal cortex of subjects with schizophrenia. Schizophr Res 36:84. Popken GJ, Bunney WE Jr, Potkin SG, Jones EG (1998): Neuron number and GABAergic and glutamatergic mRNA expression in subdivisions of the thalamic mediodorsal nucleus of schizophrenics. Soc Neurosci Abstr 24:991. Rajkowska G, Goldman-Rakic PS (1995): Cytoarchitectonic definition of prefrontal areas in the normal human cortex: I. Remapping of areas 9 and 46 using quantitative criteria. Cereb Cortex 5:307–322. Rajkowska G, Selemon LD, Goldman-Rakic PS (1998): Neuronal and glial somal size in the prefrontal cortex: A postmortem morphometric study of schizophrenia and Huntington disease. Arch Gen Psychiatry 55:215–224. Rodriguez RM, Weickert CS, Herman MM, Hyde TM, Webster MJ, Weinberger DR, et al (1998): Synaptophysin mRNA in normal postnatal prefrontal cortex development and in schizophrenia versus normal control. Soc Neurosci Abstr 24:987. Schlaepfer TE, Harris GJ, Tien AY, Peng LW, Lee S, Federman EB, et al (1994): Decreased regional cortical gray matter volume in schizophrenia. Am J Psychiatry 151:842– 848. Selemon LD, Goldman-Rakic PS (1999): The reduced neuropil hypothesis: A circuit based model of schizophrenia. Biol Psychiatry 45:17–25.

BIOL PSYCHIATRY 2000;48:389 –397

397

Selemon LD, Rajkowska G, Goldman-Rakic PS (1995): Abnormally high neuronal density in the schizophrenic cortex: A morphometric analysis of prefrontal area 9 and occipital area 17. Arch Gen Psychiatry 52:805– 818. Selemon LD, Rajkowska G, Goldman-Rakic PS (1998): Elevated neuronal density in prefrontal area 46 in brains from schizophrenic patients: Application of a three-dimensional, stereologic counting method. J Comp Neurol 392:402– 412. Shelton RC, Karson CN, Doran AR, Pickar D, Bigelow LB, Weinberger DR (1988): Cerebral structural pathology in schizophrenia: Evidence for a selective prefrontal cortical defect. Am J Psychiatry 145:154 –163. Sudhof TC, Lottspeich F, Greengard P, Mehl E, Jahn R (1987): The cDNA and derived amino acid sequences for rat and human synaptophysin. Nucleic Acids Res 15:9607. Sullivan EV, Lim KO, Mathalon D, Marsh L, Beal DM, Harris D, et al (1998): A profile of cortical gray matter volume deficits characteristic of schizophrenia. Cereb Cortex 8:117–124. Volk DW, Austin MC, Pierri JN, Sampson AR, Lewis DA (2000): Decreased GAD67 mRNA expression in a subset of prefrontal cortical GABA neurons in schizophrenia. Arch Gen Psychiatry 57:237–245. Weinberger DR, Aloia MS, Goldberg TE, Berman KF (1994): The frontal lobes and schizophrenia. J Neuropsychiatry Clin Neurosci 6:419 – 427. White EL (1989): Cortical Circuits. Synaptic Organization of the Cerebral Cortex. Structure, Function and Theory. Boston: Birkha¨user. Wiedenmann B, Franke WW (1985): Identification and localization of synaptophysin, an integral membrane glycoprotein of Mr 38,000 characteristic of presynaptic vesicles. Cell 41: 1017–1028. Zipursky RB, Lim KO, Sullivan EV, Brown BW, Pfefferbaum A (1992): Widespread cerebral gray matter volume deficits in schizophrenia. Arch Gen Psychiatry 49:195–205.