developmental processes including neurogenesis, neuronal migration, neurite outgrowth, and neurotransmitter signaling. Abnormal
neuronal morphology and cortical architecture are seen in human postmortem brain from patients with schizophrenia. However, the
point mutations (Q31L and L100P) and found a relative reduction in neuron number, decreased neurogenesis, and altered neuron
distribution compared to wild-type littermates. Frontal cortical neurons have shorter dendrites and decreased surface area and spine
Schizophrenia (SZ) is a common psychiatric disorder character-
ized by reduced hippocampal and cortical volume (Ross et al.,
2006), abnormal cytoarchitecture (Kovalenko et al., 2003), re-
duced neuronal density in superficial cortical layers (Akbarian et
dendritic arborization (Young et al., 1998) and dendritic spine
density (Garey et al., 1998). Many potential schizophrenia sus-
ceptibility genes have been identified (Wong and Van Tol,
2003; Ross etal.,2006),includingDisrupted-in-Schizophrenia1
(DISC1), first identified in a large Scottish family carrying a bal-
anced (1q42.1:11q14.3) translocation cosegregating with major
mental illnesses including SZ, bipolar disorder, and major de-
pression (Millar et al., 2000). The DISC1 locus shows genetic
linkage with SZ, and DISC1 variants show genetic association
with SZ (Nakata et al., 2009; Rastogi et al., 2009; Schumacher et
ing proteins that link DISC1 to important brain developmental
functions such as neurogenesis, neuron migration, neurite out-
margo et al., 2007; Brandon et al., 2009).
The cortical histology of transgenic mice expressing various
truncated mouse or human DISC1 fragments is similar to that
to express truncated DISC1 have morphological alterations in
medial prefrontal cortex and hippocampus (Kvajo et al., 2008).
Because the truncated DISC1 gene is unique to the original
translocation family and because common disease-associated
DISC1 variants are single-nucleotide polymorphisms (SNPs)
mice with Disc1 SNPs. Our group described previously two mu-
tant Disc1 mice, each with a different SNP: Q31L (127A/T) and
L100P (334T/C) (Clapcote et al., 2007). Both mutants have re-
duced brain volume, deficits in spatial working memory, and
decreased prepulse inhibition. In addition, the Q31L mutants
have abnormalities in social behavior and the forced swim test,
tical to human disease-associated variants, they may still provide
to the more drastic translocation mutations.
We undertook a comprehensive histological analysis of the
cerebral cortex of Disc1 Q31L and L100P mutant mice. Our mu-
tants have fewer neurons, decreased neuronal proliferation, and
altered cortical layer positioning compared to wild-type (WT)
littermates. Golgi staining showed shorter pyramidal neuron
dendrite length in frontal cortex and reduced spine density in
TheJournalofNeuroscience,March2,2011 • 31(9):3197–3206 • 3197
man studies of SZ and with transgenic
Disc1 mutant mouse models. Our results
provide evidence for the effects of DISC1
SNPs on neurodevelopment and cortical
ing DISC1 genetics in the general SZ pa-
tient population, and represent a starting
point for further investigations of molec-
ular disease mechanisms in SZ.
Mice. N-ethyl-N-nitrosurea-mutagenized Disc1
mutant mouse lines on a C57BL/6 background
(Q31L and L100P homozygous ?/?) were gen-
erated as described previously (Clapcote et al.,
2007), and additional mice were bred for histo-
logical analysis at the Toronto Centre for Phe-
nogenomics (TCP) (Toronto, Canada). WT
littermates from both Q31L and L100P groups
were combined and used as controls. All mouse
protocols were approved by the TCP Animal
Bromodeoxyuridine labeling. Timed pregnant
female mice were injected with bromodeoxyuri-
(E14) for proliferation experiments and at E12,
E15, or E18 for investigation of neuronal posi-
tioning. Embryonic brains were harvested 24 h
in 4% paraformaldehyde overnight, cryopro-
tected in 30% sucrose, and frozen at ?80°C be-
and frozen coronal sections of 5 and 10 ?m
thickness, respectively, were cut using a microtome cryostat system
ing solution (0.1 M PBS, 1% Triton X-100, 0.5% Tween 20, 2% skim
milk) or serum-free protein block (DakoCytomation) for 1 h at room
temperature to reduce nonspecific background and then incubated with
primary and secondary antibodies overnight at 4°C. The following pri-
mary antibodies were used: anti-NeuN (1:200; Millipore), anti-BrdU
Cux1 (1:200; Santa Cruz Biotechnology), and anti-Brn2 (1:200; Santa
Cruz Biotechnology). Fluorescent secondary antibodies conjugated to
Alexa 488 or Rhodamine Red-X (1:200; Invitrogen) or Cy3 (1:100; Jack-
son ImmunoResearch Laboratories) were used for detection of primary
Golgi–Cox staining. Golgi–Cox staining was performed as described
previously (Gibb and Kolb, 1998). In brief, adult mice (age 6–8 weeks)
were anesthetized with xylazene/ketamine (10 ml/kg) and intracardially
for 5 d. Sections of 200 ?m were sliced using a microtome (Leica
VT1000S) and placed on 2% gelatinized microscope slides. The slides
were stored in a humidified chamber for 3 d before further staining and
Analysis of immunohistochemistry: neuron number, distribution within
whole cortex were captured using a confocal microscope (Zeiss LSM510
converted to gray values and normalized to background staining. Sec-
tions chosen for analysis were anatomically matched along the rostral-
caudal axis for all samples. Regions of interest (ROIs) were positioned
over the cortex as sampling windows. A two-dimensional cell counting
approach of random sampling was used to provide accurate estimates of
counted using the ITCN plugin for ImageJ (http://rsb.info.nih.gov/ij/)
old, 1 pixel). Specific procedures for defining areas of analysis differed
slightly for each antibody, since each was chosen to address different
questions. These procedures are described in detail below.
NeuN antibody-labeled images were used to examine overall neuron
numbers in the cortex. Eight rectangular ROIs of fixed size (500 ?m
high ? 250 ?m wide), with the long axis perpendicular to the pial sur-
face, were outlined throughout the neocortex from medial to lateral (see
ber of neurons in the ROI was counted.
fluorescently labeled cells in the ventricular zone (VZ) and subventricu-
lar zone (SVZ) were counted in a fixed area ROI of 100 ? 120 ?m.
Similarly with P21 brains, BrdU-labeled cells in the frontal cortex were
counted in an ROI of fixed width (500 ?m) but of variable length, cor-
responding to the thickness of the cortex. Frontal cortical regions were
pendicular to the pia to assess the distribution of each wave of newly born
mally destined for the superficial cortical layers II and III would instead be
seen in deeper layers (IV-VI) in Disc1 mutant mice. In both Cux1- and
length spanning the thickness of the cortex were delineated. Each ROI was
subdivided into eight equal regions from the pia to the inner border of the
individual neurons, Golgi images at 40? magnification were captured
under brightfield illumination with a Nikon Eclipse E600 microscope.
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