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ORIGINAL ARTICLES
Levels of mRNAs Encoding Synaptic Vesicle and
Synaptic Plasma Membrane Proteins in the Temporal
Cortex of Elderly Schizophrenic Patients
Boris P. Sokolov, Andrew A. Tcherepanov, Vahram Haroutunian,
and Kenneth L. Davis
Background: Electron microscopy and biochemical studies indicate that developmental abnormalities in synaptic
organization may be present in brains of schizophrenic
patients. This study determined whether these synaptic
abnormalities are reflected in differential or uniform
alterations in the expression of various synaptic protein
genes in the left superior temporal gyrus of schizophrenic
patients.
Methods: Levels of mRNAs encoding four synaptic vesicle
proteins (synaptotagmin I [p65], rab3a, synaptobrevin 1,
and synaptobrevin 2) and two synaptic plasma membrane
proteins (syntaxin 1A and SNAP-25) were measured postmortem in the left superior temporal gyrus from elderly
(58 –95 years) schizophrenic patients (n 5 14) and agematched control subjects (n 5 9).
Results: There were significant negative correlations
between age and levels of synaptotagmin I (p65), rab3a,
synaptobrevin 1, SNAP-25, and syntaxin 1A mRNAs in

schizophrenic patients (2.692 , r , 2.517, .003 , p ,
.030) but not in control subjects. Levels of all six synaptic
mRNAs studied were increased in the younger (58 –79
years) subgroup of schizophrenic patients compared to
control subjects and older (80 –95 years) subgroup of
schizophrenic patients.
Conclusions: That similar abnormalities were found for
mRNAs encoding different synaptic vesicle and synaptic
plasma membrane proteins suggests that they reflect
overall neurodevelopmental abnormalities in synaptic
connectivity in the temporal cortex of schizophrenic patients rather than changes in the number of synaptic
vesicles per synapse or abnormalities in a specific synaptic function. Biol Psychiatry 2000;48:184 –196 © 2000
Society of Biological Psychiatry
Key Words: Schizophrenia, temporal cortex, synaptic
proteins, gene expression, synaptic organization, mRNA

From Molecular Neurobiology Branch, National Institute on Drug Abuse, National
Institutes of Health, Baltimore, Maryland (BPS) and the Department of
Psychiatry, Mount Sinai School of Medicine, New York, New York (BPS,
AAT, VH, KLD).

Address reprint requests to Boris P. Sokolov, National Institute on Drug Abuse,
Molecular Neurobiology Branch, NIH, 5500 Nathan Shock Dr., Baltimore MD
21224.
Received October 13, 1999; revised January 28, 2000; revised February 16, 2000;
accepted March 6, 2000.

© 2000 Society of Biological Psychiatry

Introduction

S

ynaptic abnormalities may be involved in various
sensory processing deficits associated with schizophrenia (Feinberg 1982). Electron microscopy studies
have revealed ultrastructural changes in the synaptic organization of some brain regions of schizophrenic patients
(Miyakawa et al 1972; Ong and Garey 1993; Soustek
1989). Consistent with these data are biochemical findings
indicating that region, age, and gene-specific abnormalities in expression of some synaptic proteins may be
present in brains of schizophrenic patients. These studies
have shown a pattern of synaptic protein disregulation

with the levels of different synaptic proteins increased,
decreased, or unchanged in different brain regions
(Browning et al 1993; Eastwood et al 1995; Eastwood and
Harrison 1995; Glantz and Lewis 1997; Karson et al 1999;
Thompson et al 1998).
In one series of studies increased concentrations of
synaptophysin, as well as two other synaptic proteins
(SNAP-25 and syntaxin), were revealed in cingulate cortex from a group of chronically hospitalized elderly
schizophrenic patients (Gabriel et al 1997). A subsequent
mRNA study in the same cohort of elderly schizophrenic
patients provided further evidence that the expression of
synaptic protein genes may be increased in some cortical
regions of schizophrenic patients, but only in a subgroup
of patients younger than 75 years (Tcherepanov and
Sokolov 1997). Levels of mRNAs encoding three synaptic
vesicle-associated proteins (synaptophysin, synapsin 1A,
and synapsin 1B) were increased in the temporal cortex
(Brodmann’s areas 21 and 22) of 52–73-year-old schizophrenic patients, compared to age matched control subjects, and declined significantly with age in schizophrenic
patients, but not in control subjects. Coordinated ageassociated alterations in expression of mRNAs encoding
three major synaptic proteins (synaptophysin and synapsin

1A and synapsin 1B) were hypothesized to indicate overall
age-related alterations in synaptic function in the temporal
cortex of elderly schizophrenic patients (Tcherepanov and
Sokolov 1997). It remains unclear, however, whether these
alterations reflect overall abnormalities in synaptic densi0006-3223/00/$20.00
PII S0006-3223(00)00875-1

Synaptic Proteins in Schizophrenia

ty/activity or whether they are restricted to some specific
synaptic functions or structures. For example, synaptophysin and synapsin 1 are localized solely to synaptic vesicles;
thus, changes in their expression may reflect abnormalities
in the number of synaptic vesicles per synapse rather than
in the number of synapses.
One way to address these questions is to examine
expression of other synaptic protein genes in brains of
schizophrenic patients. Different synaptic proteins have
different functions and localization in nerve terminals
(synaptic vesicles or synaptic plasma membrane) and are
differentially expressed in different neurons (Jahn and

Su¨dhof 1994; Oyler et al 1989; Su¨dhof 1995; Ullrich and
Su¨dhof 1995). Therefore, differential alterations of different synaptic protein genes may reveal abnormalities in a
specific synaptic function/structure and in specific subclasses of synapses. Alternatively, similar abnormalities in
expression of many synaptic protein genes may indicate a
general failure of synaptic function or a generalized
reduction in synaptic specializations.
This study used measurements of mRNAs encoding
four synaptic vesicle proteins (synaptotagmin I [p65],
rab3a, synaptobrevin 1, synaptobrevin 2) and two synaptic
plasma membrane proteins (syntaxin 1A and SNAP-25) to
address the question of whether levels of these synaptic
protein mRNAs were differentially or uniformly altered in
elderly schizophrenic patients who had previously been
found to have abnormal levels of some other synaptic
protein mRNAs and synaptic proteins (Gabriel et al 1997;
Tcherepanov and Sokolov 1997). The superior temporal
gyrus was chosen for study because of multiple studies
showing structural abnormalities in this region in schizophrenia and suggesting a role for this region in the
mediation of thought disorders and auditory hallucinations
(Barta et al 1997; Hirayasu et al 1998; Levitan et al 1999;

Menon et al 1995; Nestor et al 1993; Pearlson 1997;
Pearlson et al 1996; Penfield and Perot 1963; Ross and
Pearlson 1996; Shenton et al 1992).

Methods and Materials
Patients
Postmortem brain specimens derived from elderly chronically
institutionalized schizophrenic patients (n 5 14) and normal
elderly control subjects (n 5 9) were obtained through the
Schizophrenia Brain Bank of the Department of Psychiatry at the
Mount Sinai School of Medicine, New York. Each case satisfied
DSM-III-R criteria for schizophrenia. Eight of the 14 schizophrenic patients had been assessed and diagnosed antemortem
within 18 months of death by a team of research psychiatrists; the
diagnoses for the remaining six cases were based on extensive
chart review by the same diagnosis and assessment team. The
antemortem assessment battery and assessment procedures have
been described in detail previously (Davidson et al 1995). The

BIOL PSYCHIATRY
2000;48:184 –196

185

general neuropathological characteristics of this cohort of subjects have been described in detail (Purohit et al 1998). On
neuropathological examination, evidence of an old infarct was
found in the temporal cortex of one of the control subjects (case
109) contralateral to the side used for the current study. Three
patients (cases 163, 106, and 170) had senile plaque counts in
temporal cortex ranging from 3.6 to 6.8 per mm2. One of these
patients (case 170) also had sparse neurofibrillary tangles in
temporal cortex. The densities of these neuropathological lesions
were normal for age and did not approach density criteria for
Alzheimer’s disease (Khachaturian 1985; Mirra et al 1991).
Exclusion of these three schizophrenic cases (163, 106, and 170)
from analysis produced essentially the same results as the
analysis of the total group of schizophrenic cases (data not
shown). Other cases were free of neuropathological lesions.
There were no histories of drug or alcohol abuse for any of the
cases. Demographic characteristics of the cases studied are
shown in Table 1. Five schizophrenic cases had very long

postmortem intervals (above 60 hours); however, exclusion of
these cases from analyses produced essentially the same results
as the analyses of the total schizophrenic group (see Results). In
addition, there were no significant correlations between postmortem interval (PMI) and synaptic protein mRNA levels (see
Results). All patients with schizophrenia had been treated with
antipsychotic drugs some time in their lives. Ten patients with
schizophrenia were free of neuroleptic drugs at least 4 weeks (up
to 5 years) prior to death. Four other patients were receiving
antipsychotic drugs at the time of death or until 72 hours prior to
death. The superior temporal gyrus was dissected from coronal
sections at the level of the anterior mammillary body at a level
roughly corresponding to coronal section 6 of the atlas published
in Damasio and Damasio (1989).

RNA Isolation
Total RNA was isolated from 200 mg of postmortem human brain
by the guanidinium isothiocyanate method (Chomczynsky and
Sacchi 1987). To remove DNA contamination, the RNA samples
were treated with 30 units of DNAse I (5 Prime-3 Prime, Boulder,
CO) in a 200 mL reaction mixture containing 5 mmol/L MgCl2, 30

mmol/L TrisHCl, pH 7.5; and 100 units of RNase inhibitor
(Clontech Laboratories, Palo Alto, CA) for 1 hour at 37°C, extracted
twice with phenol/chloroform/isoamyl alcohol, precipitated by the
addition of an equal amount of isopropanol and washed three times
with cold 70% ethanol. The yield of total RNA was in the range
between 48 and 180 mg per 200 mg of brain tissue.
Cultured fibroblasts from human kidney were isolated and
grown as described previously for skin fibroblasts (Sokolov et al
1995). Approximately 107 cells were used for RNA isolation.
RNA extraction and analysis were carried using the same
methods as for postmortem brain tissue.

Assay for mRNAs Encoding Synaptic Proteins
The limited amount of brain tissue and possible partial degradation
of mRNA in postmortem tissue favored the use of reverse transcription polymerase chain reaction (RT-PCR) with endogenously expressed internal standard for quantitative assays of synaptic

186

B.P. Sokolov et al

BIOL PSYCHIATRY
2000;48:184 –196

Table 1. Demographic Data for Autopsy Cases
Interval of time between
the last antipsychotic
medication and death
Group (case)

Weeks

Schizophrenic patients
163
.260
309
.260
271
.260
106
250

170
176
193
106
283
45
426
9
195
6
337
4
199
,0.4
254
,0.4

265
287
Meansa
Range
Control subjects
232
230
82
192
22
46
97
93
109
Meansa
Range

,0.4
,0.4

Neuroleptic

Other drugs

Gender

Age
(years)

PMI
(hours)

pH

Storage

Cause of
death

Thioridazine
NRb
NR
Chlorpromazine
Thioridazine
Haloperidol
Haloperidol
Thiothixene
Haloperidol
Thioridazine
Thioridazine
Haloperidol

Hydroxizine pameate
Notriptyline
Buspirone

F
F
F
F
F
M
M
F
M
F
F
M

84
65
82
86
95
84
80
79
69
69
76
58

32
5.8
111.0
6.9
60.0
6.2
91.3
20.0
4.5
14.0
8.5
30.0

6.2
5.9
6.2
5.8
6.7
6.5
6.4
7.1
6.4
6.2
6.1
6.9

50
30
33
62
49
45
32
12
37
25
43
35

CPF
CPF
CPF
ARI
MI
CPF
Sepsis, PN
CPF
MI
CPF
CPF
CPF

M
F
F9:M5

73
84
77.4 6 2.6
58 –95

72.0
111.0
41.0 6 10.7
4.5–111.1

6.5
6.4
6.4 6 0.1
5.8 –7.1

34
32
37.1 6 3.3
12– 62

CPF
CPF

F
F
F
F
M
M
M
M
M
F4:M5

74
96
86
79
69
88
55
70
77
77.1 6 4.0
55–96

3.0
3.2
4.7
3.0
6.0
4.8
10.0
8.0
4.3
5.2 6 0.8
3–10

6.0
6.7
6.5
5.5
5.8
5.9
5.7
6.0
6.3
6.0 6 0.13
5.5– 6.7

38
38
73
45
97
86
65
67
61
63.3 6 20.6
38 –97

CPF
CPF
Unknown
CPF
Sepsis
Unknown
Lymph
LGIB
MI

Haloperidol
Haloperidol

Benadryl

Iorazepam
Hydroxizine pameate,
desaril, dilantin
Iorazepam

Cases were partially described previously (Hernandez and Sokolov 1997a; Sokolov 1998). PMI, postmortem interval; NR, no record on neuroleptic treatment within the
last 5 years prior to death; CPF, cardiopulmonary failure; ARI, acute respiratory insufficiency; MI, myocardial infarction; PN, pneumonia; Lymph, lymphoma; LGIB, Lower
gastrointestinal bleeding.
a
Values are means 6 SEMs.

mRNAs. This highly specific and sensitive method incorporates
controls for possible variability in differential levels of RNA
degradation among samples, as well as for variations in efficiency of
the RT-PCR and pipetting errors (Ma et al 1994a, 1994b; Sokolov
1998; Stanta and Bonin 1998; Zamorano et al 1996).
Several studies reported on possible effects of PMI, brain pH,
or sample storage time on measurements of some mRNAs in
postmortem brain tissue (Barton et al 1993; Harrison et al 1995;
Johnston et al 1997). Accordingly, the following important
factors have been considered and accounted for in the design of
mRNA measurements in the current study. First, prolonged PMI,
changes in pH, or prolonged sample storage time may indeed
potentially lead to changes in the postmortem brain tissue,
possibly causing activation of ribonucleases that can non-specifically degrade RNA. Ribonucleases are normally compartmentalized within the cells, however, therefore degradation of
mRNA occurs mainly when the ribonucleases begin to diffuse
and access RNA molecules. The most critical step here is when
the tissue is being removed from the storage at 280°C, thawed

and homogenized for RNA extraction or prepared for in situ
hybridization. Thawing and homogenization disrupts cellular
membranes, enabling access of ribonucleases to RNA and
subsequently enabling RNA degradation. This process of degradation is very fast and mRNA may be degraded almost completely within minutes, even in freshly prepared tissue. Thus, the
level of mRNA degradation depends not only on the amount or
activity of ribonucleases, but by and large on the time of
ribonucleases’ action, which is the interval between the time of
disruption of cellular membranes and time of physical isolation
of RNA or inactivation of ribonucleases. Accordingly, our
protocol was developed to minimize RNA degradation in the
process of RNA isolation. The pulverized brain tissue stored at
280°C was immediately transferred into preheated solution of
guanidinium isothiocyanate and homogenized. Guanidinium isothiocyanate is a denaturing agent and strong ribonuclease inhibitor, which disrupts cells, releases RNA, and simultaneously
inactivates ribonucleases, thus preventing RNA degradation
(Chomczynsky and Sacchi 1987). After RNA isolation, all

Synaptic Proteins in Schizophrenia

subsequent manipulations with RNA were carried out in the
presence of RNase inhibitor from placenta (Clontech Laboratories). The second important consideration is that the relative
levels of synaptic mRNAs in the samples were estimated based
on amplification of short (below 600 bases) mRNA sequences.
Thus, even partially degraded mRNAs were accounted in the
measurements. Importantly, random hexamers were used in the
reverse transcriptase reaction instead of oligo-dT primers. Therefore, preservation of only those short sequences of mRNAs,
which are used for amplification, is necessary for detection,
whereas in the case of oligo-dT primers a larger sequence
including also a sequence between the amplified fragment and
polyA tail is necessary. Additionally, normalization with endogenously expressed b-actin mRNA was used to further account for
differential levels of RNA degradation among the samples (Ma et
al 1994a, 1994b; Sokolov 1998; Stanta and Bonin 1998; Zamorano et al 1996).
The conditions for RT-PCR were designed to allow simultaneous amplification of six synaptic mRNAs together with b-actin
mRNA in the same reaction with the same efficiency, permitting
the precise measurements of mRNA levels relative to one
another. b-Actin mRNA is a housekeeping mRNA that is widely
used as an internal standard in gene expression studies (Ma et al
1994a, 1994b; Sokolov 1998; Stanta and Bonin 1998; Zamorano
et al 1996). Previous studies have shown that its abundance does
not differ in postmortem brains of schizophrenic patients as
compared to control brains, is independent of the age of the
donors, and is not affected by postmortem delay (Sokolov 1998;
Tcherepanov and Sokolov 1997). In accordance with previous
studies (Sokolov 1998; Tcherepanov and Sokolov 1997), measurements of the raw b-actin mRNA levels in the current study
revealed no significant correlation with PMI (r 5 2.079, n 5 23,
p 5 .72) Importantly, no significant correlations with PMI were
found also for the raw (not normalized with b-actin) levels of any
of the synaptic protein mRNAs studied here (2.262 , r ,
2.055, .228 , p , .802).
Two mg of total RNA were used in 20 mL of reverse
transcription reaction to synthesize cDNA using a commercial kit
(SuperScript, GIBCO BRL, Grand Island, NY) and random
hexanucleotides as primers. Then 0.05 mg of the diluted cDNA
were amplified in 10 mL of PCR reaction using a mixture of
seven pairs of primers. Primers STGF (59-AATAGCCATAGTCGCAGTCC-39) and STGR (59-CCAATTCCGAGTATGGTACC-39) were designed to amplify a 473– base pair (bp)
fragment of synaptotagmin 1 mRNA using published sequence
of the gene (Perin et al 1991). Primers S25F (59-TGAGTCGCTGGAAAGCACC-39) and S25R (59-ATCAGCCTTCTCCATGATCC-39) were designed to amplify a 490 bp fragment of
SNAP-25 mRNA (Zhao et al 1994). Primers STX1F (59AGCTGGAAGAACTCATGTCC-39) and STX1R (59-GAACATGTCGTGTAGCTCAC-39) were designed to amplify a 428
bp fragment of syntaxin 1 mRNA (Zhang et al 1995). Primers
Syb1F (59-TGCTCCAGCTCAGCCACCT-39) and Syb1R (59AACTACCACGATGATGGCAC-39) were designed to amplify
a 331 bp fragment of synaptobrevin 1 mRNA (Archer et al 1990).
Primers Syb2F (59-CCTCACCAGTAACAGGAGAC-39) and
Syb2R (59-GAGGATGATGGCGACAATCA-39) were designed
to amplify a 249 bp fragment of synaptobrevin 2 mRNA (Archer

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187

et al 1990). Primers Rab3F (59–TCGACTTCAAGGTCAAGACC-39) and Rab3R (59-TCTCGCAGATGACATCCACC-39)
were designed to amplify a 387 bp fragment of Rab3a mRNA
(Zahraoui et al 1989). Primers BA1 (59-ACGAAACTACCTTCAACTCC-39) and BAR (59-CTTCCTGTAACAATGCATCTC-39) were designed to amplify a 585 bp fragment of
b-actin mRNA using published sequence (Ponte et al 1984). All
RT-PCR products were verified by sequencing. The conditions
for PCR were: 30 sec at 94°C, 1 min at 54°C, and 1 min at 72°C
in a 9600 (Perkin Elmer) temperature cycler. Primers for PCR
were 59-end labeled with 32P. The kinetics of amplification were
assessed by withdrawing aliquots of the reaction mixture after
successive PCR cycles. The PCR products were separated on a
6% polyacrylamide gel containing 2 mol/L urea (Figure 1A). The
incorporated radioactivity in each DNA fragment was measured
using a phosphostimulable storage plate (Molecular Dynamics,
Sunnyvale, CA). As illustrated on Figure 1, amplification kinetics for synaptic protein mRNAs and b-actin mRNA were in the
exponential phase and were similar for different mRNAs up to 21
cycles of PCR reaction. In subsequent experiments, 20 cycles of
amplification were chosen for quantitative assays.

Statistical Analysis
The primary hypothesis (levels of synaptic protein mRNAs
decrease with age in schizophrenic patients) was examined using
Pearson product moment correlation analysis. One-tailed significance was used, as the hypothesis was directional and derived
from earlier findings (Tcherepanov and Sokolov 1997). Note,
however, that similar conclusions would have been reached even
if two-tailed tests had been used. The correlation between levels
of various synaptic protein mRNAs, as well as the effects of
PMI, brain pH, and sample storage time were examined using
Pearson’s correlation analysis (two-tailed). The effects of withdrawal from antipsychotic medication were examined using a
logarithmic model (synaptic protein mRNA vs. logarithm(10) of
continuous neuroleptic-free interval before death controlling for
age). The logarithmic model was used because of the wide range
of neuroleptic free interval (from 72 hours to 260 weeks) and in
view of logarithmic modes of correlations with neuroleptic-free
interval observed for a number of serotonin and glutamate
receptor mRNAs (Hernandez and Sokolov 1997a, 1997b;
Sokolov 1998). Differences among groups were examined using
two-way analysis of variance (ANOVA) with diagnosis and age
group (below or above 79.5 years) as the main factors. Statistical
analyses were performed using SPSS 7.5 and Statistica statistical
programs. A modification of the Newman–Keuls procedure
(Begun and Gabriel 1981; Ryan 1960) was used for post hoc
comparisons among means.

Results
Age-Related Differences between Schizophrenic
Patients and Control Subjects
Levels of mRNAs encoding three synaptic vesicle proteins—synaptotagmin 1, rab3a, and synaptobrevin 1 (measured as ratios to b-actin mRNA)—in the left superior

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B.P. Sokolov et al

Figure 1. Reverse transcription polymerase
chain reaction (RT-PCR) assay of synaptic
protein messenger RNA (mRNA) abundances
as a ratios to b-actin abundance. The kinetics
of synaptic protein mRNAs and b-actin
mRNA amplifications. Aliquots were withdrawn from PCR after consecutive cycles and
separated on 6% polyacrylamide gel. (A, B)
Autoradiography of the gel. (A) Total RNA
from temporal cortex. (B) Cultured kidney
fibroblasts. Cycle numbers are indicated. M,
molecular markers; C, control, amplification
of total RNA (26 cycles of PCR) without
reverse transcription; bp, base pair. (C) Plot of
incorporated radioactivity at the respective
cycles (measured using a phosphostimulable
storage plate [PhosphorImager, Molecular Dynamics, Sunnyvale, CA]).

temporal gyrus of schizophrenic patients correlated negatively and significantly with the age of subjects at time of
death (2.692 , r , 2.517, .003 , p , .03; Figure 2).
Similarly, there were significant negative correlations
between age of schizophrenic patients and levels of
mRNAs encoding the synaptic plasma membrane proteins—syntaxin 1A (r 5 2.672, p 5 .004) and SNAP25
(2.639, p 5 .007). No correlation with age was revealed
for mRNA encoding another synaptic vesicle protein—
synaptobrevin 2 (r 5 .127, p 5 .333). In contrast to
schizophrenic patients, there were no significant correlations between age and levels of any of synaptic vesicle or
synaptic plasma membrane protein mRNAs in control
subjects (2.102 , r , .520, .099 , p , .397; Figure 2).
Comparisons of the slopes of the regression lines for these
synaptic proteins against age revealed significant slope
differences between schizophrenic patients and control
subjects for synaptotagmin [F(1,190) 5 6.87, p 5 .02],

and nearly significant differences for SNAP25 and syntaxin 1A [Fs(1,19) . 3.95, ps , .65], confirming the
impressions gleaned from Figure 2.
Examining of the raw (not normalized with b-actin)
levels of the synaptic protein mRNAs confirmed significant negative correlations with age in the schizophrenic
patients (2.549 , r , 2.499, .019 , p , .035), but not
in the control subjects (2.391 , r , 2.152, .149 , p ,
.348). The raw levels of b-actin mRNA did not correlate
significantly with age in both schizophrenic patients (r 5
2.395, p 5 .081) and control subjects (.332, p 5 .192).
Because of significant differences in correlation with
age in schizophrenic patients and control subjects (a
relationship that we had observed earlier in a similar
experiment [Tcherepanov and Sokolov 1997]), in further
comparisons of schizophrenic patients and control subjects
both diagnostic groups were subdivided according to age.
The median age for the schizophrenic group (79.5 years)

Synaptic Proteins in Schizophrenia

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2000;48:184 –196

189

Figure 2. Correlations between synaptic protein messenger RNA (mRNA) levels and age
at death of schizophrenic and control subjects.
Levels of synaptic protein mRNAs are represented as ratios to b-actin mRNA. ●, male
subjects; E, female subjects.

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B.P. Sokolov et al

Figure 3. Levels of synaptic protein messenger RNAs (mRNAs) in the left superior temporal gyrus (Brodmann’s area 22) from control
subjects and schizophrenic patients. Levels of synaptic protein mRNAs are represented as ratios to b-actin mRNA. Values are means 6
SEMs.

was used as the cutoff age. This cutoff age produces
relatively equal group sizes for ANOVA and is higher than
those we used previously (75 years) for other synaptic
proteins (Tcherepanov and Sokolov 1997). Note, however,
that similar conclusions would have been reached if the
age of 75 years had been used as the cutoff point.
Consistent with our previous findings for mRNAs encoding synaptophysin, synapsin 1A, and synapsin 1B, levels
of all six synaptic protein mRNAs studied here were
higher in the left superior temporal gyrus of “young”
(58 –79 years) schizophrenic patients compared to
“young” control subjects (55–79 years; Figure 3). In “old”
(80 –95 years) schizophrenic patients, levels of five synaptic mRNAs were slightly, but not statistically significantly, lower than in the “old” (86 –96 years) control

subjects, whereas the levels of synaptobrevin 2 mRNA
were increased. Two-way ANOVAs revealed significant
diagnosis by age– group (below or above 79.5 years)
interactions for synaptotagmin 1, rab3a, synaptobrevin 1,
SNAP-25, and syntaxin 1A [5.550 , F(1,19) , 11.304;
.003 , p , .029] but not for synaptobrevin 2 mRNA
[F(1,19) 5 0.014; p 5 .907]. Comparisons between
groups revealed that the levels of synaptotagmin 1, rab3a,
synaptobrevin 1, SNAP-25, and syntaxin 1A mRNAs were
significantly (ps , .05) higher in the left superior temporal
gyrus of young schizophrenic patients relative to the older
schizophrenic group. Furthermore, similar analyses revealed that the levels of synaptotagmin 1, synaptobrevin 1,
SNAP-25, and syntaxin 1A, but not rab3a, mRNAs were
significantly higher in the left superior temporal gyrus of

Synaptic Proteins in Schizophrenia

young schizophrenic patients than in the young normal
control subjects (ps , .05). Caution should be applied,
however, to the results of these latter statistical analyses
because the small sample sizes.

Confounding Factors
GENDER. The schizophrenic group had a higher female to male ratio (F9:M5) than the control group (F4:
M5), which might potentially have contributed to the
differences in correlations with age observed between
schizophrenic patients and control subjects; however,
separate analyses in gender subgroups revealed that correlations with age for synaptotagmin 1, rab3a, synaptobrevin 1, SNAP-25, and syntaxin 1A mRNAs were directionally similar in male (2.945 , r , 2.873, .008 , p ,
.027, n 5 5) and female schizophrenic patients (2.534 ,
r , 2.395, .069 , p , .171, n 5 9) and were of similar
magnitude as the correlations observed in the group as a
whole. In addition, no differences in synaptic protein
mRNA levels were found between males and females in
both the schizophrenic and control groups using analysis
of covariance (ANCOVA) with age as a covariate
[0.248 , F(1,10) , 1.012, .336 , p , .628 and 0.006 ,
F(1,5) , 1.288, .300 , p , .942, correspondingly).
POSTMORTEM INTERVAL. The PMI was greater in
the schizophrenic group (4.5–111 hours, mean 5 41.0,
SEM 5 10.7) than in the control group (3–10 hours,
mean 5 5.2, SEM 5 0.8; p , .001); however, no
significant correlations between PMI and synaptic protein
mRNAs measured as ratios to b-actin mRNA were found
(control subjects, 2.481 , r , .040, .190 , p , .919;
schizophrenic patients, 2.377 , r , 2.195, .109 , p ,
.504). Furthermore, no significant differences in levels of
synaptic protein mRNAs between schizophrenic patients
with long PMIs (60 –111 hours) and schizophrenic patients
with short PMIs (4.5–32 hours) were found using ANCOVA with age as a covariate [0.005 , F(1,13) , 3.74,
.079 , p , .994]. These data indicate that measurements
of synaptic protein mRNAs as ratios to b-actin mRNA
were not affected significantly by PMI. Nevertheless,
additional analyses of associations with age and diagnosis
were carried out excluding five schizophrenic cases with
the longest PMIs (above 32 hours). These analyses revealed essentially the same associations as the analyses of
the total group of schizophrenic patients. In this smaller
subset of schizophrenic cases (n 5 9), correlations with
age were significant for synaptotagmin 1, Rab3a, SNAP25, and syntaxin 1A (2.756 , r , 2.606, .009 , p ,
.042) but did not reach significance for synaptobrevin 1
mRNA (r 5 2.395, p 5 .146). Two-way ANOVAs
revealed significant diagnosis by age– group interactions

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191

for synaptotagmin 1, rab3a, synaptobrevin 1, SNAP-25,
and syntaxin 1A [6.491 , F(1,14) , 11.861, .004 , p ,
.023] but not for synaptobrevin 2 mRNA [F(1,14) 5
0.193, p 5 .667]. Levels of all six mRNAs were nominally
higher in young (58 –79 years) schizophrenic patients than
in young (55–79 years) control subjects (data not shown).
BRAIN PH AND SAMPLE STORAGE TIME. Brain pH
(a measure that is believed may reflect acidosis at agonal
state) in the subject groups studied here (Table 1) was
similar to that reported in other studies (for example,
mean 5 6.5 [Harrison et al 1995] and mean 5 5.9
[Johnston et al 1997]). Although brain pH in the control
subjects was lower than in schizophrenic patients (6.0 and
6.4, respectively, Table 1), these variations in pH are
unlikely to account for the age-associated differences in
synaptic protein mRNA levels observed between schizophrenic patients and control subjects. First, no significant
correlations between brain pH and synaptic protein mRNA
levels measured as ratios to b-actin mRNA were found in
control subjects (2.189 , r , .559, .118 , p , .681, n 5
9), schizophrenic patients (2.083 , r , .358, .209 , p ,
.778, n 5 14), and the combined subject groups (.098 ,
r , .340, .119 , p , .657, n 5 23). Second, elevated
levels of synaptic protein mRNAs in the younger (58 –79
years) subgroup of schizophrenic patients compared to the
older (80 –95 years) subgroup of schizophrenic patients
could not be associated with pH, because these two
schizophrenic subgroups are not different in brain pH
(6.4 6 0.2 and 6.3 6 0.1, respectively). Of note, analysis
of the overall quality of RNA, isolated in two different
laboratories from the same sample of control subjects and
schizophrenic patients and rated blindly based on agarose
gel electrophoresis, revealed no significant correlation
with pH (data not shown). In addition, similar lack of a
relationship between tissue pH and mRNA levels was
observed in previously published in situ hybridization and
RT-PCR studies of dopamine, serotonin, and glutamate
receptor transcripts in an overlapping cohort of subjects
(Hernandez and Sokolov 2000; Meador-Woodruff et al
1999; Sokolov 1998).
Similarly, no significant effect of sample storage time
on synaptic protein mRNA measurements was found using
correlational analysis (control subjects, .010 , r , .575,
.105 , p , .980, n 5 9; schizophrenic patients, 2.506 ,
r , .147, .065 , p , .616, n 5 14; combined groups,
2.234 , r , 2.156, .247 , p , .476, n 5 23).
NEUROLEPTIC TREATMENT. Effects of neuroleptic
treatment were examined by analyzing correlations between synaptic protein mRNAs and neuroleptic-free intervals prior to death. To remove effects of age, partial
correlation analysis controlling for age was used. There

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2000;48:184 –196

were nonsignificant trends toward negative correlations
between neuroleptic-free intervals and synaptotagmin 1
(r 5 2.461, p 5 .113), rab3a (r 5 2.383, p 5 .196),
SNAP-25 (r 5 2.490, p 5 .159), syntaxin 1A (r 5 2.414,
p 5 .159), and synaptobrevin 1 (r 5 2.227, p 5 .456), but
not synaptobrevin 2 (r 5 .242, p 5 .425).
Despite the fact that the analyses described above
showed that neuroleptic treatment did not exert a statistically significant influence on synaptic protein mRNAs, we
re-examined correlations between synaptic protein mRNAs and age using partial correlation analysis controlling
for neuroleptic-free intervals. When controlled for neuroleptic-free intervals, correlations with age remained significant for synaptotagmin 1 (r 5 2.584, p 5 .018),
SNAP-25 (r 5 2.506, p 5 .039), and syntaxin 1A (r 5
2.558, p 5 .024), but not for rab3a (r 5 2.362, p 5 .114)
and synaptobrevin 1 (r 5 2.400, p 5 .088). These data
indicate that the negative correlations between synaptic
protein mRNA levels and age of schizophrenic patients are
unlikely to have been due to treatment with neuroleptics.

Nonneuronal Expression of Synaptobrevin 2 mRNA
Unlike other synaptic protein mRNAs studied, levels of
synaptobrevin 2 mRNA did not correlate with age of
schizophrenic patients (Figure 2). In addition, levels of
five synaptic protein mRNAs were highly correlated with
each other, with r ranging from .761 to .943 (all ps ,
.001), whereas the levels of synaptobrevin 2 mRNA did
not correlate with the other synaptic protein mRNA levels
(.020 , r , .308, .153 , p , .928). These differences in
synaptobrevin 2 mRNA behavior might be in part related
to its nonneuronal expression. To test the hypothesis that
synaptobrevin 2 mRNA may be expressed in nonneuronal
cells, we examined the expression of all six synaptic
protein mRNAs in cultured fibroblasts isolated from human kidney. Fibroblasts were chosen as a readily available
extreme example of nonneuronal cells that are very unlikely to be contaminated by cells of neuronal origin.
Reverse transcription polymerase chain reaction amplification of total RNA isolated from fibroblasts produced
DNA bands with electrophoretic mobilities corresponding
to synaptobrevin 2 and b-actin mRNAs (Figure 1). Sequencing of these DNA bands confirmed that they derive
from synaptobrevin 2 and b-actin mRNAs, respectively.
No bands corresponding to other synaptic protein mRNAs
were amplified from fibroblasts (Figure 1). These data
indicate that synaptobrevin 2 mRNA, as well as housekeeping b-actin mRNA, are expressed in fibroblast at
significant levels, whereas no detectable expression of
other five synaptic protein mRNA occurs. Thus, differences in synaptobrevin 2 mRNA from other synaptic
protein mRNAs may be related to its abundant nonneuronal expression.

Discussion
This study demonstrates that levels of mRNAs encoding
three synaptic vesicle proteins (synaptotagmin 1, rab3a,
and synaptobrevin 1) and two synaptic plasma membrane
proteins (SNAP-25 and syntaxin 1A) decline significantly
with age in the left superior temporal gyrus (BA22) of
elderly schizophrenic patients. By contrast, no significant
decreases with age were found in age-matched elderly
control subjects. The levels of synaptic protein mRNAs in
schizophrenic patients younger than 79.5 years were
significantly higher than in control subjects and schizophrenic patients older than 79.5 years.
These abnormalities in synaptic protein mRNAs are
unlikely to be an effect of antipsychotic medication. First,
no significant correlations were found between synaptic
protein mRNAs and neuroleptic-free intervals before
death. Second, negative correlations with age remained
significant or nearly significant when possible effects of
neuroleptic were removed using partial correlation analysis controlling for neuroleptic-free intervals. Similarly, no
effects of gender, brain pH, or sample storage time were
found. It is also unlikely that the synaptic protein mRNA
abnormalities noted above were associated with long
postmortem intervals in some of schizophrenic cases.
Correlational analysis showed no significant effect of PMI
on synaptic protein mRNA levels measured as ratios to
b-actin mRNA. In addition, similar conclusions were
reached even when schizophrenic cases with long PMIs
(above 32 hours) were excluded from the analyses. Consistent with findings here, previous human and rat studies
revealed no, or at most a modest effect of PMI on
measurements of many different mRNAs up to about 100
hours postmortem (Barton et al 1993; Harrison et al 1995;
Johnston et al 1997; Schramm et al 1999; Sokolov 1998),
although some evidence suggest that certain individual
mRNA species may undergo postmortem degradation
(Barton et al 1993; Harrison et al 1995). The lack of
significant effects of PMI (as well as brain pH or sample
storage time) on the measurements of synaptic protein
mRNAs here is likely due to the specifics of the assay
protocol employed (see Methods and Materials for details), which allow us 1) to minimize degradation of RNA
in the process of its isolation; 2) to minimize effects of
partial degradation on mRNA measurements; and 3) to
effectively standardize for the differential levels of RNA
degradation among samples (Ma et al 1994a, 1994b;
Sokolov 1998; Stanta and Bonin 1998; Zamorano et al
1996). It is important, therefore, to note that the lack of
effects of PMI, brain pH, or storage time on mRNA
measurements here does not imply that these factors have
no effect on mRNA preservation and measurements of
mRNA by using other methods.

Synaptic Proteins in Schizophrenia

The results for mRNAs encoding three synaptic vesicle
associated proteins (synaptotagmin 1, rab3a, and synaptobrevin 1) are consistent with previous analysis of three
other mRNAs encoding synaptic vesicle proteins (synaptophysin, synapsin 1A, and synapsin 1B; Tcherepanov and
Sokolov 1997). Similar age-related correlations in schizophrenia were revealed for mRNAs encoding synaptic
plasma membrane proteins—SNAP-25 and syntaxin 1A.
The synaptic proteins examined here and previously have
different functions and are involved in different steps of
synaptic vesicle cycle. Furthermore, they have different
subcellular localization and are differentially expressed in
different neurons (Jahn and Su¨dhof 1994; Oyler et al 1989;
Su¨dhof 1995; Ullrich and Su¨dhof 1995). Synaptotagmin 1
is localized to synaptic vesicles where it functions in the
calcium-triggered release of neurotransmitters, possibly
serving as a Ca21 sensor for exocytosis (Shao et al 1997).
Rab3a, a small GTP-binding protein, is concentrated on
the surface of synaptic vesicles, unlike integral membrane
proteins, such as synaptophysin, synaptobrevin, or synaptotagmin. Rab3a is not permanently associated with the
synaptic vesicle membrane and dissociates from synaptic
vesicles after exocytosis (Stahl et al 1996). Rab3a is
thought to guide membrane fusion between a transport
vesicle and the target membrane, and to determine the
specificity of docking (Geppert et al 1997). Synaptobrevin
is a synaptic vesicle protein that binds tightly to the
complex of plasma membrane proteins syntaxin and
SNAP-25 (Hayashi et al 1994; Pevsner et al 1994). The
synaptobrevin–syntaxin–SNAP-25 complex bridges synaptic vesicle and plasma membranes and may function in
the docking and/or fusion of synaptic vesicles (McMahon
and Su¨dhof 1995). That similar abnormalities were found
for multiple mRNAs encoding these different synaptic
proteins with different functions and localizations suggests
an overall abnormality in synaptic organization in the left
superior temporal gyrus of schizophrenic patients rather
than abnormalities in specific synaptic functions. The
finding that levels of synaptic vesicle and synaptic plasma
membrane protein mRNAs correlate highly with each
other and reveal similar changes in brains of schizophrenic
patients studied here is more consistent with a suggestion
of an abnormal synaptic density or functional activity than
with alterations in the number of synaptic vesicles per
synapse. Future direct studies of synaptic density and
synaptic vesicle per synapse ratios in brains from the same
group of schizophrenic patients and control subjects would
help to test this hypothesis.
Normal cortical development is accompanied by substantial (30 – 40%) decline in synaptic density during
adolescence (synaptic pruning; Huttenlocher 1979) with
further, though less dramatic (;20 –25%), decrease between ages 16 and 60 years (Masliah et al 1993). No

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further decrease with normal aging (but rather a nonsignificant trend toward an increase in synaptic density) was
found between ages 60 and 98 years (Masliah et al 1993).
This finding is consistent with no decrease, (but rather a
nonsignificant trend toward an increase) in levels of
multiple synaptic protein mRNAs with aging in our cohort
of 55–96-year-old normal control subjects (here and previously [Tcherepanov and Sokolov 1997]), which provides
an additional support to the view that levels of synaptic
protein mRNAs may be valuable markers of synaptic
density (Eastwood et al 1995). As such, increased levels of
multiple synaptic protein mRNAs found in the temporal
cortex of relatively young schizophrenic patients are
consistent with the hypothesis that irregularities in programmed synaptic elimination may be involved in schizophrenia (Feinberg 1982). In contrast to elderly control
subjects, the levels of synaptic protein mRNAs in elderly
schizophrenic patients declined significantly with increasing age, suggesting that synaptic pruning may possibly be
delayed in schizophrenia. Interestingly, computer simulation analysis of a speech perception neural network indicates that eliminating up to 65% of working memory
connections improves perceptual ability (Hoffman and
McGlashan 1997). Thus, a delay in synaptic elimination in
Brodmann’s area 22 (an area involved in auditory perception and interpretation of auditory stimuli and implicated
in thought disorders and auditory hallucinations (Barta et
al 1997; Hirayasu et al 1998; Levitan et al 1999; Menon et
al 1995; Nestor et al 1993; Pearlson 1997; Pearlson et al
1996; Penfield and Perot 1963; Ross and Pearlson 1986;
Shenton et al 1992) may be among the mechanisms
involved in impaired speech perception, auditory hallucinations, and thought disorder in schizophrenia.
Alternatively, the decreases in synaptic protein mRNAs
with aging in schizophrenic patients may reflect brain
abnormalities not related to synaptic pruning or other
developmental events. It is possible that the decreases in
synaptic protein mRNA may indicate a decrease in functional activity of the synapses or survival effect. Interestingly, schizophrenic subjects studied here had higher
dementia rates than the control cases (Davidson et al 1995;
Purohit et al 1998). No distinctive histopathology was
found in this group of chronic elderly schizophrenic
patients (Purohit et al 1998). This may suggest that
cognitive impairment in this group of patients may be
associated with synaptic alterations reported here. The
increased levels of synaptic protein mRNAs in the
younger schizophrenic patients might possibly reflect an
increased synaptic activity or sprouting during the early
phases of a dementing process, whereas the subsequent
declines in levels of these mRNAs might possibly represent a reduction in synaptic function or neuronal connections. It should be noted, however, that no significant

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associations between synaptic protein mRNA levels and
clinical dementia rating was found (data not shown).
A previous immunochemical study of an overlapping
cohort of elderly (52–95 years) chronically institutionalized schizophrenic and control cases revealed significant
increase in the levels of synaptophysin, syntaxin and
SNAP-25 in the cingulate cortex of schizophrenic patients
with similar trends in Brodmann’s areas 20 and 8 but not
in Brodmann’s area 7 (Gabriel et al 1997). Analysis of
synaptic mRNAs in the temporal cortex (here and previously [Tcherepanov and Sokolov 1997]) provides additional evidence that the expression of synaptic proteins
may be increased in some areas of the brains of elderly
schizophrenic patients and suggests that this increased
expression is present mostly in schizophrenic patients
between the ages of 52 and 75 years. Studies of other brain
regions in different groups of schizophrenic patients similar to our cohort of younger schizophrenic patients (mean
ages 54.5 6 15.0 and 57.0 6 5.0 years) revealed decreased
synaptophysin expression in CA4, CA3, subiculum, parahippocampal gyrus (Eastwood and Harrison 1995; Easwood et al 1995) and prefrontal cortex (Brodmann’s areas
9 and 46) but not in the primarily visual cortex (Brodmann’s area 17; Glantz and Lewis 1997). Another study
reported a decrease of synapsin 1 but not synaptophysin
immunoreactivity in hippocampi of schizophrenic patients
with mean age of 61.3 6 11.2 years (Browning et al 1993).
Significant decrease in synaptophysin and SNAP-25 but
not their encoding mRNAs was reported in Brodmann’s
area 10 of schizophrenic patients (Karson et al 1999).
Increased levels of SNAP-25 in Brodmann’s area 9,
decreased in Brodmann’s areas 10 and 20, and normal
levels in Brodmann’s area 17 were reported in schizophrenia (Thompson et al 1998). These data indicate that
synaptic abnormalities in schizophrenia may be region
specific (increased synaptic density or function in some
brain regions, decreased in others and not changed in still
other regions) and may derive from differences in patterns
of synaptic density development in the normal and diseased states.
Recent morphometric studies have revealed abnormally
high neuronal density in the cortexes of schizophrenic
patients with no changes in glial density (Rajkowska et al
1998; Selemon et al 1995). These findings may suggest
that increased expression of synaptic protein genes reported here and previously (Gabriel et al 1997;
Tcherepanov and Sokolov 1997) may reflect an increased
neuronal or neuropil density in schizophrenia.
In conclusion, this study describes age specific abnormalities in the abundance of mRNAs encoding six synaptic proteins in the temporal cortex of postmortem brains
obtained from patients with schizophrenia. The data revealed that in schizophrenic patients, age-related changes

occur in the abundance of mRNAs that encode both for
synaptic vesicle and synaptic plasma membrane associated
proteins. These findings are consistent with the hypothesis
that overall changes in synaptic function, rather than
changes in a specific synaptic function or in the number of
synaptic vesicles per synapse, may be present in the
temporal cortex of schizophrenic patients. These results
replicate and extend our previous findings on age-related
abnormalities in abundance of various multiple synaptic
protein mRNAs in the temporal cortex of elderly schizophrenic patients and provide additional support for the
hypothesis that developmental synaptic abnormalities may
contribute to the pathophysiology of schizophrenia. Future
studies using independent methods are necessary to confirm the findings in this group of elderly chronic schizophrenic patients.

This work was supported in part by MHCRC (NIH R2NH56083).
The authors gratefully acknowledge Drs. D. Perl and D. Purohit for
their neuropathological characterization of the cases studied, and Drs. M.
Davidson and P. Powchik for antemortem assessment of schizophrenic
cases. We thank Dr. J. Schmeidler for his helpful comments on the
statistical analysis.
A preliminary report on the work described here was presented at the
Society of Biological Psychiatry Meeting in San Diego, CA, May 1997.

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