Differential expression of presynaptic genes in a rat model of postnatal hypoxia: relevance to schizophrenia.

Abstract

Obstetric complications play a role in the pathophysiology of schizophrenia. However, the biological consequences during neurodevelopment until adulthood are unknown. Microarrays have been used for expression profiling in four brain regions of a rat model of neonatal hypoxia as a common factor of obstetric complications. Animals were repeatedly exposed to chronic hypoxia from postnatal (PD) day 4 through day 8 and killed at the age of 150 days. Additional groups of rats were treated with clozapine from PD 120-150. Self-spotted chips containing 340 cDNAs related to the glutamate system ("glutamate chips") were used. The data show differential (up and down) regulations of numerous genes in frontal (FR), temporal (TE) and parietal cortex (PAR), and in caudate putamen (CPU), but evidently many more genes are upregulated in frontal and temporal cortex, whereas in parietal cortex the majority of genes are downregulated. Because of their primary presynaptic occurrence, five differentially expressed genes (CPX1, NPY, NRXN1, SNAP-25, and STX1A) have been selected for comparisons with clozapine-treated animals by qRT-PCR. Complexin 1 is upregulated in FR and TE cortex but unchanged in PAR by hypoxic treatment. Clozapine downregulates it in FR but upregulates it in PAR cortex. Similarly, syntaxin 1A was upregulated in FR, but downregulated in TE and unchanged in PAR cortex, whereas clozapine downregulated it in FR but upregulated it in PAR cortex. Hence, hypoxia alters gene expression regionally specific, which is in agreement with reports on differentially expressed presynaptic genes in schizophrenia. Chronic clozapine treatment may contribute to normalize synaptic connectivity.

Full text

Page 1

ORIGINAL PAPER
Differential expression of presynaptic genes in a rat model
of postnatal hypoxia: relevance to schizophrenia
J. U. Sommer•A. Schmitt•M. Heck•E. L. Schaeffer•M. Fendt•
M. Zink•K. Nieselt•S. Symons•G. Petroianu•A. Lex•M. Herrera-Marschitz•
R. Spanagel•P. Falkai•P. J. Gebicke-Haerter
Received: 17 April 2010/Accepted: 4 August 2010/Published online: 14 October 2010
? The Author(s) 2010. This article is published with open access at Springerlink.com
Abstract
pathophysiology of schizophrenia. However, the biological
consequences during neurodevelopment until adulthood are
unknown. Microarrays have been used for expression
profiling in four brain regions of a rat model of neonatal
hypoxia as a common factor of obstetric complications.
Animals were repeatedly exposed to chronic hypoxia from
postnatal (PD) day 4 through day 8 and killed at the age of
Obstetric complications play a role in the
150 days. Additional groups of rats were treated with
clozapine from PD 120–150. Self-spotted chips containing
340 cDNAs related to the glutamate system (‘‘glutamate
chips’’) were used. The data show differential (up and
down) regulations of numerous genes in frontal (FR),
temporal (TE) and parietal cortex (PAR), and in caudate
putamen (CPU), but evidently many more genes are
upregulated in frontal and temporal cortex, whereas in
parietal cortex the majority of genes are downregulated.
Because of their primary presynaptic occurrence, five dif-
ferentially expressed genes (CPX1, NPY, NRXN1, SNAP-
25, and STX1A) have been selected for comparisons with
clozapine-treated animals by qRT-PCR. Complexin 1 is
upregulated in FR and TE cortex but unchanged in PAR by
The authors J. U. Sommer and A. Schmitt contributed equally to this
article.
Electronic supplementary material
article (doi:10.1007/s00406-010-0159-1) contains supplementary
material, which is available to authorized users.
The online version of this
J. U. Sommer ? M. Heck ? M. Zink ? R. Spanagel ?
P. J. Gebicke-Haerter (&)
Department of Psychopharmacology,
Central Institute of Mental Health,
J5, 68159 Mannheim, Germany
e-mail: peter.gebicke@zi-mannheim.de
A. Schmitt ? P. Falkai
Department of Psychiatry, University of Go ¨ttingen,
von-Siebold-Str. 5, 37075 Go ¨ttingen, Germany
A. Schmitt ? E. L. Schaeffer
Laboratory of Neuroscience (LIM27), Institute of Psychiatry,
University of Sao Paulo, Rua Dr. Ovidio Pires de Campos 785,
05453-010 Sa ˜o Paulo, SP, Brazil
M. Fendt
Animal Physiology Zoological Institute, Faculty of Biology,
University of Tu ¨bingen, Auf der Morgenstelle 28,
72076 Tu ¨bingen, Germany
K. Nieselt ? S. Symons
Department of Information and Cognitive Sciences,
Center for Bioinformatics Tu ¨bingen, University of Tu ¨bingen,
Tu ¨bingen, Germany
G. Petroianu
Department of Cellular Biology and Pharmacology,
Florida International University, 11200 SW 8th Street,
GL 495E, Miami, FL 33199, USA
A. Lex
Animal Physiology, Biological Institute, University of Stuttgart,
Pfaffenwaldring 57, 70550 Stuttgart, Germany
M. Herrera-Marschitz
Programme of Molecular and Clinical Pharmacology,
ICBM, Medical Faculty, University of Chile,
Av. Independencia 1027, Santiago 7, Chile
Present Address:
J. U. Sommer
Department of Otorhinolaryngology, Head and Neck Surgery,
Klinikum Mannheim, University of Heidelberg,
Mannheim, Germany
Present Address:
M. Heck
Department of Dermatology, Klinikum Mannheim,
University of Heidelberg, Mannheim, Germany
123
Eur Arch Psychiatry Clin Neurosci (2010) 260 (Suppl 2):S81–S89
DOI 10.1007/s00406-010-0159-1

Page 2

hypoxic treatment. Clozapine downregulates it in FR but
upregulates it in PAR cortex. Similarly, syntaxin 1A was
upregulated in FR, but downregulated in TE and unchan-
ged in PAR cortex, whereas clozapine downregulated it in
FR but upregulated it in PAR cortex. Hence, hypoxia alters
gene expression regionally specific, which is in agreement
with reports on differentially expressed presynaptic genes
in schizophrenia. Chronic clozapine treatment may con-
tribute to normalize synaptic connectivity.
Keywords
Clozapine ? PPI ? Animal model
Hypoxia ? Microarray ? Presynaptic genes ?
Introduction
In addition to genetic predisposition, the neurodevelop-
mental hypothesis of schizophrenia includes a number of
environmental factors like obstetric and birth complica-
tions with ensuing consequences on the pathophysiology of
the disease. Hence, the risk of schizophrenia increases with
the number and severity of hypoxia-associated obstetric
complications [5, 6, 22]. However, the pathogenetic
implications of obstetric complications are unclear. The
neurodevelopmental hypothesis of schizophrenia suggests
that the onset of abnormal brain development and neuro-
pathology occurs perinatally, whereas symptoms of the
disease appear in early adulthood [32].
Schizophrenia patients show deficits in attention and
processing of sensory information. A reliable test of sen-
sorimotor gating in vertebrates is prepulse inhibition (PPI)
of the acoustic startle reflex, which is disrupted in
schizophrenia patients and restored by antipsychotics,
mainly clozapine [2]. Several cortical brain regions and
striatum are involved in PPI-modulating circuitry [30]. We
previously reported on a PPI deficit in rats exposed to
neonatal hypoxia appearing only during adulthood. This
deficit was prevented and restored after chronic clozapine
treatment [12].
In our animal model of chronic neonatal hypoxia, rats
exhibited increased gene expression of the N-methyl-D-
aspartate (NMDA) receptor subunit NR1 in hippocampal
and cortical subregions [28], which suggests an involve-
ment of the NMDA receptor in the pathophysiology of
hypoxia-induced alterations. In schizophrenia, there is
indeed strong support for disturbances of the glutamatergic
and gamma-amino-butyric acid (GABA)ergic microcir-
cuitry, mainly affecting the expression of presynaptic
vesicle proteins including synaptosome-associated protein
25 (SNAP-25) and syntaxin, which form the soluble
N-fethylmaleimide-sensitive
receptor (SNARE) complex [9, 10, 15, 20, 29]. Complexin
(CPX) 1 and complexin 2 operate in synaptic vesicle
factorattachmentprotein
exocytosis by stabilizing the SNARE complex [4]. Neu-
ropeptide Y (NPY) and neurexins (NRXN) play a role in
cell adhesion and are hypothesized to play a role in the
pathophysiology of schizophrenia [14, 20, 23, 26].
There is only little information about the influence of
antipsychotic treatment on gene expression, as has been
shown for NPY and CPX in normal rats [8, 16]. Thus, the
present study investigates on the impact of neonatal
hypoxia and long-term treatment with the antipsychotic
clozapine on the expression of presynaptic genes in dif-
ferent cortical and subcortical brain regions in an animal
model. Furthermore, correlations between differentially
expressed genes and PPI deficits have been evaluated.
Materials and methods
Animals
A total of 12 Sprague–Dawley rats (Animal Facility,
Department of Animal Physiology, University of Tu ¨bin-
gen) were used for the experiments. The rats were housed
in groups of 3 animals under a 12-h light/dark cycle with
food and drinking water available ad libitum. The experi-
ments were carried out in accordance with the international
ethical guidelines for the care and use of laboratory animals
for experiments (declaration of Helsinki, NIH guidelines)
and were approved by the local animal care committee
(Regierungspra ¨sidium Tu ¨bingen, ZP 3/02).
Neonatal hypoxia
Chronic hypoxia (11% O2, 89% N2) was imposed from
postnatal day (PD) 4–8 on six animal pups and their
mothers by placing them in an air-tight plastic chamber for
a period of 6 h per day. The six other pups were subjected
to identical handling conditions and were placed in iden-
tical chambers but with regular oxygen concentrations
(21% O2= normoxia) [12, 28].
Clozapine treatment
Between PD 120 and PD 150, half of the rats were being
orally treated over a period of 4 weeks with 45 mg/kg/day
clozapine (Novartis Pharma AG, Basel, Switzerland) [12].
Test on prepulse inhibition and tissue preparation
The startle magnitude was measured as described in [12].
Since a deficit in prepulse inhibition had been shown to
surface on postnatal days 120 and 150, animals were killed
at postnatal day 150. Brains were removed and immersed
in -78?C cold isopentane and stored at -80?C until further
S82 Eur Arch Psychiatry Clin Neurosci (2010) 260 (Suppl 2):S81–S89
123

Page 3

use. Slices of 120 um thickness were cut on a cryostat and
used to excise respective brain regions according to the
atlas of Paxinos and Watson [25] using a micropunching
tool. Resultant, cylindrical tissue blocks were collected on
dry ice and stored at -80?C until further processing.
Molecular investigations
RNA isolation and reverse transcription
One milliliter of Trizol was added to excised tissue, and
tissue was homogenized by 30 strokes of trituration. After
addition of 200 ul chloroform, the solution was transferred
to Phase-Lock tubes (Eppendorf) for phase separation.
Aqueous supernatants were purified and concentrated by
RNeasy mini columns (Qiagen). Quality control was per-
formed by OD measurements in a NanoDrop ND-1000
spectrophotometer (260/280 nm[1.8) and by electropho-
retic separation using an Agilent 2100 Bioanalyzer (R.I.N.
values[7.0).
Reverse transcription of purified total RNA was carried
out using oligo-dT primers with T7 promoter sequence and
Superscript II (Invitrogen).
In vitro transcription and labeling
Reverse-transcribed cDNAs were treated with RNAse and
with DNA polymerase to obtain double-stranded DNA.
Purified DNA was used for amplification reactions using
RNAMaxx High Yield Transcription Kit (Stratagene) with
T7 polymerase. Typically, this in vitro transcription
resulted in 40- to 50-fold amplification of mRNA/cDNA.
Subsequently, cRNA was reverse-transcribed. Labeled
DNA was purified and dried.
Preparation of DNA (‘‘glutamate’’) Chips
The chips represent a hypothesis driven selection of 340
cDNA sequences of genes related to glutamatergic and
GABAergic neurotransmission
proteins (Online Resource 1a–f), obtained from clones
purchasedfromDeutschen
Genomfoschung (RZPD, Berlin). The cDNA-inserts of
bacterial clones were PCR-amplified directly from aliquots
of bacterial cultures grown overnight (LB medium, con-
taining antibiotic selection). After electrophoretic quality
control, PCR mixtures were dried, resuspended in betaine-
containing spotting buffer [7], and spotted on amino silane
slides (Nexterion, PeqLab), using a MicroGrid II Arrayer.
Each DNA was double-spotted, and each array was spotted
three times on the same slide. The following control DNAs
were used: salmon sperm DNA, human COT-1 DNA, 4
Arabidopsis thaliana DNAs (for spike-ins), spotting buffer,
andsynaptic vesicle
Ressourcenzentrumfu ¨r
water, and a series of DNAs derived from artificial genes
(library of random external controls = LOREC) [19].
Altogether, a chip contained 4,608 spots, including
markers at the array corners to facilitate finding the correct
orientation after scanning.
After spotting, DNAs were UV-cross-linked with a
radiation of 250 mJ/cm2. Then, they were baked at 80?C
for 2 h and stored at RT.
Hybridization and scanning of slides
Spotted slides were sequentially washed in 1% SDS solution
in distilled water, immersed in 95?C distilled water, and
incubatedin55?Cprehybridizationsolutionfor45 min.Then,
they were washed in distilled water, in isopropanol (20 s,
each),anddriedbyastreamofnitrogenorair.Hybridizations
were done in SlideHyb Glass Array Hybridization buffer #1
(Ambion) overnight at 56?C. All subsequent washing steps at
increasing stringency were performed in the dark.
Scanning was carried out in a Packard ScanArray 5,000
scanner. Each slide was scanned twice at laser strengths of
90 and 100% to obtain a larger spectrum of fluorescence
intensities.
Data processing
Data quality was assessed by a number of procedures,
including visual inspection of intensities, MA-plots, and
correlation analysis of replicates. Within-array normaliza-
tion was performed according to locally weighted scatter-
plot smoothing (LOESS) method [34]. Only rank-invariant
probes were used for model generation during this proce-
dure [27]. For between-array normalization, scaling [34]
was used. After assessing normal distribution of replicate
spots using the Kolmogorov–Smirnov test, replicates were
pooled by their arithmetic means. To identify differentially
expressed genes, two-tailed t-tests and the RankProduct
(RP) [3] method were used. The false discovery rate (FDR)
method was employed to avoid accumulation of alpha-
error. Corrected P-values\0.05 were considered signifi-
cant. To limit the number of differentially expressed genes,
only those were used which were found by both t-test and
RP methods. Finally, to profile overall regulation, only
genes that were differentially regulated in at least two
regions were selected for further analyses.
Quantitative real-time PCR (qRT-PCR)
Quantitative RT-PCR was carried out in 384-well plates in
an ABI Prism 7900 HT Fast Real-Time PCR cycler
(Applied Biosystems, Foster City, Ca., USA). The primer
pairs used for the respective genes are listed in Table 1.
Beta actin was used as a reference (‘‘house-keeping’’) gene.
Eur Arch Psychiatry Clin Neurosci (2010) 260 (Suppl 2):S81–S89S83
123

Page 4

Results
Microarray experiments
After statistical evaluations of the 768 genes spotted on the
glass slides by Students t tests, many of them turned out to
be significantly regulated.
Table 2 provides an overview of differentially expressed
genes in frontal (FR), parietal (PAR), and temporal (TE)
cortex as well as in caudate putamen (CPU). At first sight,
there are apparently large regional differences in terms of
up- or downregulation, i.e. many more genes are upregu-
lated in FR [44 vs. 4 (ratio11)] and in TE [51 vs. 22 (ratio
2.32)] compared with PAR [10 vs. 33 (ratio 0.3)]. A more
balanced situation seems to occur in CPU.
Clozapine treatment and qRT-PCR results
To obtain a clearer picture, the focus was laid upon genes
primarilybeingexpressedpresynaptically.Thosewerefurther
investigated by qRT-PCR in the four brain regions in norm-
oxic and hypoxic animals treated with clozapine (Table 3).
Evidently, CPX1 is upregulated in frontal and temporal
cortex as well as in caudate putamen, but not in parietal
cortex in hypoxic animals (Table 2). Treatment with clo-
zapine resulted in an upregulation also in parietal cortex.
NPY was upregulated by hypoxia in frontal but downreg-
ulated in temporal cortex. Neurexin1 was upregulated in
both frontal cortex and CPU in hypoxic animals. SNAP25
was also upregulated in frontal cortex but downregulated in
CPU. Additionally, it was upregulated in temporal cortex.
Another counterregulation was observed with syntaxin1A
in frontal and temporal cortex (Table 2). Interestingly, in
parietal cortex, where hypoxia had no effect, STX1A was
upregulated by clozapine (Table 3).
Discussion
Complexin1
Upregulation of CPX1 upon postnatal hypoxia in frontal,
temporal cortex, and CPU has not been reported yet
(Fig. 1). In the light that clozapine additionally upregu-
lated the gene in parietal cortex, it may be assumed that
hypoxia-induced upregulation may be a compensatory
mechanism to ensure proper vesicular transmitter release.
Associations of complexins with schizophrenia have been
repeatedly reported [9, 10, 13]. A recent report comes to
the conclusion that complexins facilitate neurotransmitter
release [33]. Specifically, it has been suggested that
CPX1downregulationduring
GABAergic neurons facilitates glutamate release and loss
of overstimulated neurons—which subsequently results in
reduced glutamatergic projections [24]. The upregulation
of CPX1 in parietal cortex by clozapine is supported by
the findings of Eastwood [8]. Our results showing the
very strong correlation of CPX1 with PPI in hypoxia-
treated animals (Table 4) support the view that this gene/
protein is a key player in the development of Schizo-
phrenia-like symptoms.
perinatalhypoxia in
Neurexin1
Neurexins and neuroligins are emerging as central orga-
nizing molecules for excitatory glutamatergic and inhibi-
tory GABAergic synapses. They function as cell adhesion
molecules, bridging the synaptic cleft. Neurexin 1beta and
neuroligin normally form trans-synaptic complexes [1].
Finally, there is a recent report showing that disruption of
the neurexin 1 gene is associated with schizophrenia [26].
Preconditioningischemiacandownregulatetheincreased
neurexin–neuroligin1–PSD-95
ischemia injury and exerts a neuroprotective effect. These
resultsindicatethattheneurexin-neuroligin1isanimportant
signaling module in hypoxic injury and a novel possible
target in therapeutics of brain ischemia [21].
interactioninducedby
SNAP25
Recent genetic studies implicate alterations in SNAP-25
gene structure, expression, and/or function in contributing
directly to neuropsychiatric and neurological disorders.
Protein-by-protein analyses indicated a significant reduc-
tion in SNAP-25 immunoreactivity in the schizophrenia
non-suicide group [11, 15].
Table 1 Primers used in
qRT-PCR
Sequences reading from 50-end
Gene Forward primerReverse primer
CPLX1 GCAGGGCATACGAGATAAGT TGGTGGGGTCAGTGATGGCAGTA
NPYCGCTCTGCGACACTACAT CTCAGGGCTGGATCTCTTG
NRXN1AGGGCGTCAGCTCACAATCTTCAATCTGCCGAGCCTGGGTATGGT
SNAP25 AGTGGCGTTTGCTGAATGACTGCTGGTATGACTTAATCTTGACA
STX1ATGTCCCGAAAGTTTGTG GCGTTCTCGGTAGTCT
S84Eur Arch Psychiatry Clin Neurosci (2010) 260 (Suppl 2):S81–S89
123

Page 5

Table 2 Differentially expressed genes in some regions of normoxic and hypoxic rat brains
CPU
FR
PAR
TE
Genelist t-test fdr correction
Genelist t-test fdr correction
Genelist t-test fdr correction
Genelist t-test fdr correction
The factor ‘‘hypoxia’’ regulated the
following genes in comparison with‘‘normoxia’’
The factor ‘‘hypoxia’’ regulated the following
genes in comparison with ‘‘normoxia’’
The factor ‘‘hypoxia’’ regulated the following
genes in comparison with ‘‘normoxia’’
The factor ‘‘hypoxia’’ regulated the following
genes in comparison with ‘‘normoxia’’
UP
DOWN
UP
DOWN
UP
DOWN
UP
DOWN
ATF2
ATF2
CAPN2
ADCY3
ATF4
AURKB
CAPON
ADCY6
ATP2A2
CALM2
CASK
ATP2A2
CPX1
CAP2
EAAT3
BDNF
CPX2
CAPN8
GABBR1
BNPI
GAP43
CPX1
GAPDH
CAPN2
GAPDH
DEAD BOX
GNA12
CAPN6
GATA4
DLGAP2
LOC294679
CASQ2
GRM2
DUSP1
LOC362704
CPX1
ITPR1
FYN
MAPK3
DAT1
MARK1
GAPDH
PIPS
EAAT3
MPPS7
GNAO
PLD2
ERK7
MYD116
MAPK14
PLEKHC1
GABRG3
NM_017261
MAPK3
POU3F4
GAD67
NOS2
MRPS7
PPP1R14A
GADD45A
NRXN1
NELL2
PPP2R2C
GAP43
PKN1
NPY
PPP5C
GATA4
PLD1
NOS2-1149
PREFOLDIN5
GNAO
PLEKHC1
NRXN1
PRKCZ
GPSM1
PPM1B
PACSIN2
PTPRA
GRM8
PPP1CC
PIM3
PTPRR
ID3
PPKCC
PLAT
RAB14
ITPR3
PTP4A1
PPP1R14B
RAB3A
LOC294679
PTPRA
PPP1R14C
RAB3B
LOC362704
PTPRN
PTP4A2
RN.25045
MGC105762
PTPRN2
PTPRD
RN.28284
NPY
RAB10
PTPRN
SHANK1
NR2F2
RN.28284
PTPRN2
SLC2A1
NRXN3
SACM1L
RAB3A
SLC6A12
NUP54
SLC1A3
SLC1A3
SLK
PIPS
Eur Arch Psychiatry Clin Neurosci (2010) 260 (Suppl 2):S81–S89 S85
123