Developmental patterns of doublecortin expression and white matter neuron density in the postnatal primate prefrontal cortex and schizophrenia.

Abstract

Postnatal neurogenesis occurs in the subventricular zone and dentate gyrus, and evidence suggests that new neurons may be present in additional regions of the mature primate brain, including the prefrontal cortex (PFC). Addition of new neurons to the PFC implies local generation of neurons or migration from areas such as the subventricular zone. We examined the putative contribution of new, migrating neurons to postnatal cortical development by determining the density of neurons in white matter subjacent to the cortex and measuring expression of doublecortin (DCX), a microtubule-associated protein involved in neuronal migration, in humans and rhesus macaques. We found a striking decline in DCX expression (human and macaque) and density of white matter neurons (humans) during infancy, consistent with the arrival of new neurons in the early postnatal cortex. Considering the expansion of the brain during this time, the decline in white matter neuron density does not necessarily indicate reduced total numbers of white matter neurons in early postnatal life. Furthermore, numerous cells in the white matter and deep grey matter were positive for the migration-associated glycoprotein polysialiated-neuronal cell adhesion molecule and GAD65/67, suggesting that immature migrating neurons in the adult may be GABAergic. We also examined DCX mRNA in the PFC of adult schizophrenia patients (n = 37) and matched controls (n = 37) and did not find any difference in DCX mRNA expression. However, we report a negative correlation between DCX mRNA expression and white matter neuron density in adult schizophrenia patients, in contrast to a positive correlation in human development where DCX mRNA and white matter neuron density are higher earlier in life. Accumulation of neurons in the white matter in schizophrenia would be congruent with a negative correlation between DCX mRNA and white matter neuron density and support the hypothesis of a migration deficit in schizophrenia.

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Developmental Patterns of Doublecortin Expression and
White Matter Neuron Density in the Postnatal Primate
Prefrontal Cortex and Schizophrenia
Samantha J. Fung1,2,3*, Dipesh Joshi1,2, Katherine M. Allen1,2,4, Sinthuja Sivagnanasundaram1,2,
Debora A. Rothmond1,2, Richard Saunders5, Pamela L. Noble6, Maree J. Webster7, Cynthia Shannon
Weickert1,2,4
1Schizophrenia Research Institute, Sydney, Australia, 2Neuroscience Research Australia, Sydney, Australia, 3School of Medical Sciences, University of New South Wales,
Sydney, Australia, 4School of Psychiatry, University of New South Wales, Sydney, Australia, 5Laboratory of Neuropsychology, National Institute of Mental Health (NIMH),
National Institutes of Health, Bethesda, Maryland, United States of America, 6National Institute of Mental Health (NIMH) IRP Non-Human Primate Core, Poolesville,
Maryland, United States of America, 7Stanley Medical Research Institute, Rockville, Maryland, United States of America
Abstract
Postnatal neurogenesis occurs in the subventricular zone and dentate gyrus, and evidence suggests that new neurons may
be present in additional regions of the mature primate brain, including the prefrontal cortex (PFC). Addition of new neurons
to the PFC implies local generation of neurons or migration from areas such as the subventricular zone. We examined the
putative contribution of new, migrating neurons to postnatal cortical development by determining the density of neurons
in white matter subjacent to the cortex and measuring expression of doublecortin (DCX), a microtubule-associated protein
involved in neuronal migration, in humans and rhesus macaques. We found a striking decline in DCX expression (human
and macaque) and density of white matter neurons (humans) during infancy, consistent with the arrival of new neurons in
the early postnatal cortex. Considering the expansion of the brain during this time, the decline in white matter neuron
density does not necessarily indicate reduced total numbers of white matter neurons in early postnatal life. Furthermore,
numerous cells in the white matter and deep grey matter were positive for the migration-associated glycoprotein
polysialiated-neuronal cell adhesion molecule and GAD65/67, suggesting that immature migrating neurons in the adult may
be GABAergic. We also examined DCX mRNA in the PFC of adult schizophrenia patients (n=37) and matched controls
(n=37) and did not find any difference in DCX mRNA expression. However, we report a negative correlation between DCX
mRNA expression and white matter neuron density in adult schizophrenia patients, in contrast to a positive correlation in
human development where DCX mRNA and white matter neuron density are higher earlier in life. Accumulation of neurons
in the white matter in schizophrenia would be congruent with a negative correlation between DCX mRNA and white matter
neuron density and support the hypothesis of a migration deficit in schizophrenia.
Citation: Fung SJ, Joshi D, Allen KM, Sivagnanasundaram S, Rothmond DA, et al. (2011) Developmental Patterns of Doublecortin Expression and White Matter
Neuron Density in the Postnatal Primate Prefrontal Cortex and Schizophrenia. PLoS ONE 6(9): e25194. doi:10.1371/journal.pone.0025194
Editor: Olivier Jacques Manzoni, Institut National de la Sante ´ et de la Recherche Me ´dicale, France
Received January 31, 2011; Accepted August 30, 2011; Published September 26, 2011
This is an open-access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for
any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.
Funding: This work was supported by the Schizophrenia Research Institute, utilizing funding from NSW Health and the Macquarie Group Foundation;
Neuroscience Research Australia; the University of New South Wales; and National Health and Medical Research Council of Australia (grant number 630452).
Tissues were received from the Australian Brain Donor Programs NSW Tissue Resource Centre, which is supported by the University of Sydney, National Health
and Medical Research Council of Australia, Schizophrenia Research Institute, National Institute of Alcohol Abuse and Alcoholism (grant number R24AA012725)
and NSW Department of Health. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: s.fung@neura.edu.au
Introduction
Interstitial white matter neurons (IWMNs) are a population of
neurons that reside among the fibres and glia of white matter,
particularly in primates [1,2,3]. These neurons are present in adult
animals and are thought to be remnants of the subplate, a
transient layer below the cortical plate in the developing brain that
provides guidance and a temporary target for thalamocortical
axons. Subplate neurons are also important in the development of
cortical columns and maturation of inhibitory circuitry [4,5,6,7]
and many of these subplate cells undergo apoptosis during normal
development. Some IWMNs persist in the adult [8,9] however
their function in the mature brain is not understood, although a
role in vasodilation and vasoconstriction has been suggested
[10,11]. It has also been suggested that some IWMNs may be
neurons of subventricular zone (SVZ) origin originally destined for
the cortex, however they remain in the white matter [12].
Interestingly, in schizophrenia there is an increased density of
IWMNs [13,14,15,16,17,18] which might indicate a deficit in the
cell death of subplate neurons and/or deficient migration of
neurons during development or at maturation.
Doublecortin (DCX) is a microtubule associated protein
expressed in immature, migrating neurons [19,20]. DCX has
been identified as an important molecule in the proper lamination
of the cortex, with mutations in the Dcx gene causing lissencephaly
or double cortex syndrome in humans [21,22]. Such dramatic
phenotypes are not present in mouse models [23,24] likely due to
redundancy with doublecortin-like (DCL) and DCX-like kinase
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(DCLK) [25,26]; however, RNAi knockdown of DCX results in
migrational deficits in the rostral migratory stream or in
tangentially migrating interneurons destined for the cortex
[26,27,28,29,30]. DCX expression in new neurons is thought to
be transient [31] with expression beginning approximately 1 day
following the birth of a new cell and being maintained for 2–3
weeks after this time when cells begin to down-regulate DCX and
up-regulate markers of mature neurons (eg NeuN) [32], such that
DCX and NeuN may be co-expressed in the developing neuron
[33,34]. DCX therefore represents an endogenous marker of
immature, migrating neurons. In addition to DCX, the presence
of polysialic acid on neural cell adhesion molecule (PSA-NCAM)
reduces cell-cell interactions and plays an important role in cellular
plasticity as well as being required for the migration of neuroblasts
in the rostral migratory stream (reviewed by [35]), making PSA-
NCAM another marker of immature, migrating neurons.
Our previous studies have estimated that thousands of
immature neurons that express markers of migration, arranged
in clusters adjacent to the SVZ are present in the human infant
[36,37] and while the olfactory bulb may be among their targets,
their final destination is unknown. As cortical grey matter volume
increases from birth to about 5 years of age [38,39,40,41], we
hypothesise that the recruitment of new, migrating neurons may
contribute to postnatal cortical growth. In this study, we addressed
whether new neurons could contribute to increasing postnatal
cortical volume by examining whether IWMNs may represent a
population of migrating neurons in the postnatal primate brain,
and determining whether neuronal migration could be detected in
the overlying dorsolateral prefrontal cortex (DLPFC) during
postnatal development by analysing DCX expression [19,32].
We demonstrate that NeuN+ IWMN density and DCX expression
are at elevated levels in the early human and rhesus macaque
DLPFC prior to reaching adult levels, implying a robust
recruitment of new neurons to the primate cortex in early life.
In addition, several lines of evidence suggest that altered
neurogenesis may be linked to schizophrenia. Indeed, many
schizophrenia-associated genes such as neuregulin-1, ErbB4, and
reelin play a role in neuronal differentiation and migration (see
recent reviews by [42,43]). A reduction in cells positive for markers
of cell division or neuronal migration, Ki67 and PSA-NCAM, has
also been reported in the hippocampus of people with schizo-
phrenia [44,45]. Therefore, a second aim of this study was to test if
altered DCX mRNA, as a marker of neuronal migration, was
changed in the frontal cortex of people with schizophrenia and
relate DCX expression in the grey matter to IWMN density in the
white matter, that we have previously found to be increased in this
schizophrenia cohort [18].
Materials and Methods
Ethics statement
All non-human primate research procedures were carried out in
strict adherence to the laws and regulations of the U.S. Animal
Welfare Act, (USDA, 1990) and Public Health Service Policies,
(PHS, 2002) as well as non-governmental recommendations of the
National Research Council as published in the ILAR ‘‘Guide for
the Care and Use of Laboratory Animals’’. All research facilities
were approved by the International Association for the Assessment
and Accreditation of Laboratory Animal Care. The work was
carried out under an Animal Study Protocol approved by the
NIMH Animal Care and Use Committee. Therefore all research
practices were consistent with the recommendations of the
Weatherall Report (2006) on ‘‘The Use of Non-Human Primates
in Research’’.
Human post-mortem brain samples
Developing human post-mortem DLPFC tissue was obtained
from the University of Maryland Brain Tissue Bank for
Developmental Disorders (NICHHD contract # NO1-HD8-
3283). The human developmental cohort consisted of 68
individuals ranging in age from 6 weeks to 49 years (Summarised
in Table 1, details of subjects used for all analyses in Table S1).
These samples were a priori divided into seven developmental
periods: neonates, infants, toddlers, school age, teenagers, young
adults and adults, as described previously [46]. Neonates and
infants were full-term and all subjects were free of neurological and
gross behaviour changes at the time of death [36,47,48].
Moreover, toxicological analyses showed them to be free of drug
Table 1. Summary of developmental cohort demographics.
GroupAge (years)Gender PMI (hours) pHRIN#
Human
Neonate0.11–0.24 7M 4F22.4565.116.660.196.3761.6411
Infant0.25–0.91 8M 6F 16.9366.46.5860.206.9361.1814
Toddler1.58–4.865M 4F18.6765.296.7060.266.5161.219
School age5.39–12.985M 4F 15.1164.686.6360.27 6.6661.149
Teenage 15–17.826M 2F17.1364.166.7560.096.3461.01 8
Young adult20.14–25.38 6M 3F13.6768.266.6760.236.7360.679
Adult 35.99–49.225M 3F13.3864.606.6060.276.5360.768
Rhesus macaque
Neonate 0.04–0.161M 3F7.4160.274
Infant0.75–1.332M 2F6.2460.544
Juvenile 2–2.5 2M 3F 6.4461.035
Adolescent3.08–4.510M 3F6.5861.2813
Young Adult 6.33–7.586M 2F 7.2360.188
Adult8–12.087M 4F7.0260.63 11
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use [36,47,48]. Tissue from the DLPFC of patients with
schizophrenia/schizoaffective disorder (n=37) and matched
controls (n=37) was obtained from the New South Wales Tissue
Resource Centre (Table 2; Sydney, Australia, HREC 07261).
Groups within both cohorts were matched according to tissue pH,
PMI, RIN and, in the schizophrenia cohort, age [49].
Non-human primate brain samples
The non-human primate developmental cohort consisted of 2
week to 12 year old Macaca mulatta (rhesus macaques) (n=45) from
the NIMH, NIH (Table 1). Animals were euthanised, flushed with
saline and 1 cm coronal sections of brain were flash frozen and
stored at 280uC. For fluorescence immunohistochemistry for
NeuN and GAD65/67, 14 mm fresh frozen coronal sections from
the frontal cortex (containing the principal sulcus) of three
adolescent male animals (all 4.5 years old) from the rhesus
macaque developmental cohort were used. Fresh frozen coronal
sections containing the principal sulcus obtained from three adult
male animals (6.5, 7.6 and 9.6 years old) was used for additional
PSA-NCAM immunohistochemistry.
Rhesus macaque tissue was also obtained from one 12 day old, 1
month old, 3.7 year old, and 6 year old rhesus macaque for DCX
immunohistochemistry. Animals were euthanised, and flushed
with saline then 4% paraformaldehyde. Brains were then post-
fixed, cryoprotected, frozen and sectioned at a thickness of 40 mm
and stored in cryoprotectant solution [25% glycerol, 25% ethylene
glycol in phosphate buffered saline (PBS)] at 220uC.
Immunohistochemistry
DABimmunohistochemistry
DLPFC.
14 mm sections of the human middle frontal gyrus
were cut from frozen tissue blocks using a cryostat (Leica CM3050
S) and thaw-mounted onto gelatin-coated slides. Sections were
stored at 280uC and thawed at room temperature (RT) for
20 min prior to immunohistochemistry. Immunohistochemistry
for NeuN was performed on 14 mm fresh frozen sections, as
previously detailed [18]. Briefly, sections were fixed in 4%
paraformaldehyde in PBS (137 mM NaCl, 2.7 mM KCl, 8 mM
Na2HPO4, 2 mM KH2PO4, pH 7.4), 10 min at 4uC, washed in
PBS then endogenous peroxidases were quenched for 20 min at
RT with methanol+3% H2O2(3:1) solution. Sections were washed
and blocked with 10% normal goat serum in diluent [0.05%
bovine serum albumin (BSA), 0.3% triton X-100 in PBS] for 1 hr
at RT. Mouse anti-NeuN antibody (Millipore MAB377, 1:1000 in
diluent) was applied overnight at 4uC. Following washing, goat
for NeuNinhuman
anti-mouse
Laboratories, Cat # BA-9200; 1:500 in diluent) was applied for
1 hr at RT. Slides were washed, incubated at RT (1 hr) in avidin–
biotin–peroxidasecomplex(Vectastain
Laboratories) and treated with 3,39–diaminobenzidine (DAB;
Sigma; 12 mM final concentration in PBS with 0.003% H2O2)
for 5–7 min. Slides were Nissl stained (1.5 min exposure to 0.02%
thionin) and coverslipped. PSA-NCAM immunohistochemistry
was also performed in 14 mm sections of fresh frozen tissue from
adult rhesus macaques (6.5, 7.6 and 9.6 years of age) using the
DAB method (Table S2). Primary antibodies were omitted as a
negative control and did not show immunoreactivity.
DAB immunohistochemistry for DCX, GAD65/67 and
PSA-NCAM in rhesus macaque.
for DCX, GAD65/67 and PSA-NCAM was performed on 40 mm
rhesus macaque fixed-floating coronal sections at the level of the
head of the caudate with antibodies detailed in Table S2. Floating
sections were washed in PBS and endogenous peroxidases were
quenched with methanol+3% H2O2(3:1) solution, 20 mins at RT,
washed, then blocked with 10% goat or rabbit serum for 1 hr at
RT. Primary antibodies were applied in diluent at concentrations
specified in Table S2 for two nights at 4uC. Sections were washed
in PBS and secondary antibodies were applied in diluent, 1 hr at
RT, and avidin-biotin complex, DAB reaction and thionin
counterstaining were performed as for NeuN immunostaining.
Controls were performed where primary antibodies were omitted
and were negative for immunoreactivity.
Double-labelimmunohistochemistry
GAD65/67 in rhesus macaque.
tochemistrytodemonstrateco-localisation
GAD65/67 was performed on 14 mm thick fresh-frozen sections
from rhesus macaque. Tissue was thawed, fixed in 4%
paraformaldehyde for 10 min at 4uC, rinsed and blocked with
10% donkey serum in diluent for 1 hr at RT then primary
antibodies (1:1000 mouse anti-NeuN and 1:500 rabbit anti-
GAD65/67) were applied in diluent overnight at 4uC. Following
washing, secondary antibodies were applied, each at 1:1000
dilution (Alexa Fluor 488 donkey anti-mouse IgG, Invitrogen
A21202 and Alexa Fluor 594 conjugated donkey anti rabbit IgG,
Invitrogen A21207) for 1 hr at RT prior to washing in PBS, then
49,6-diamidino-2-phenylindole (DAPI, 1:1000) in PBS for 5 mins
and a further wash in PBS before slides were coverslipped.
Controls for binding of the secondary antibody were performed
where one primary antibody was omitted (ie only anti-NeuN or
only GAD65/67 were applied). Alexa Fluor 488 signal was absent,
IgG biotinylatedsecondaryantibody(Vector
ABCkit;Vector
DAB immunohistochemistry
forNeuNand
Fluorescence immunohis-
ofNeuN and
Table 2. Summary of demographics for control and schizophrenia groups.
control group (n=37)schizophrenia group (n=37)
Age (years)51.1 (18–78) 51.3 (27–75)
gender 7F, 30M13F, 24M
hemisphere23R, 14L17R, 20L
pH6.6660.296.6160.30
PMI (hrs)24.8610.9728.8614.07
RIN7.360.57 7.360.58
subclass-paranoid=16; undifferentiated=7; disorganised=5; residual=2;
schizoaffective, depressive type=4; schizoaffective, bipolar type=3
age of onset (years)-23.760.10
duration of illness (years)-27.662.3
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and some faint Alexa Fluor 594 signal was detected, but was below
intensity of the signal in the presence of the GAD65/67 antibody.
Quantification of NeuN+ IWMNs
Images of NeuN immunostaining were captured at 106
magnification and stitched together using the Virtual Slice facility
in the Stereo Investigator Software (MBF Biosciences). The grey
matter/white matter boundary was identified by a sharp change in
NeuN density, the presence of additional smaller astrocytic nuclei
and lighter NeuN neuropil staining (Figure S1) as detailed in [18].
IWMN density was quantified around the middle frontal gyrus of
cases from the human developmental cohort and data from
control cases from the schizophrenia cohort aged between 18 and
50 years [18] were included in this analysis to increase statistical
power within the older age groups (Table S1). Regions of white
matter sampled were selected on straight banks of white matter
avoiding the crown and the curve at the deep end of the sulcus,
where IWMN density can be inconsistent. Superficial IWMNs
were defined as those lying between 0 and 700 mm deep to the
grey/white matter border and 20 sampling boxes 4706470 mm
were placed within this region parallel to the grey/white matter
border at random distances between each other and from the
grey/white matter border within the 700 mm superficial compart-
ment. All NeuN+ IWMNs within boxes were counted, except
those touching the right and bottom sides of the counting box.
Slides were sampled and counted by two researchers. The final
NeuN+ density was calculated as the average of the 20 boxes.
Researchers were blind to developmental age group through the
experimental and quantification procedure.
Microarray analysis
Total RNA from human DLPFC tissue (grey matter) was
extracted from all subjects using the Trizol method (Invitrogen).
RNA quality was assessed using the 2100 Bioanalyser electropho-
resis system (Agilent Technologies). RNA was purified through a
Qiagen RNA mini Kit (Qiagen Inc, Valencia CA USA) from 45
individuals of the human developmental cohort (Table S1) was
prepared for microarray analysis according to Affymetrix protocol
(www.affymetrix.com) using HG-U133 version 2.0+ (GeneChips,
Affymetrix CA, USA) as described previously [50]. The Biocon-
ductor package was used to compute normalised expression values
from the Affymetrix.cel files and statistical analysis was performed
using R and Bioconductor software. Probe sets that met the
criteria of being 50% present in at least one of the age/gender
subgroups were retained in the analysis (33,210 probes sets
retained, 61% of total number). All data are MIAME compliant
and raw data has been deposited in the GEO database (NCBI)
with the accession number GSE13564.
Quantitative real time PCR analysis
cDNAwassynthesisedfromtotal RNAextractedfromthecortical
grey matter (human DLPFC) or frontal pole (rhesus macaque) using
the SuperScriptH First-Strand Synthesis kit and random hexamers
(Invitrogen) from 3 mg of total RNA per sample, repeated twice and
pooled. DCX transcript levels were measured by quantitative real
time-PCR (qPCR) using an ABI Prism 7900HT Fast Real time
PCR system with a 384-well format and TaqMan Gene Expression
Assays (Applied Biosystems) (Hs01035496_m1 for human and
Rh02829106_m1 for rhesus macaque). All measurements from
each subject were performed in triplicate and relative quantities
determined from a seven point standard curve. Transcript quantities
were normalised by the geometric mean of four housekeeping genes:
GUSB(Hs99999908_m1), PBGD
(Hs99999904_m1) and UBC (Hs00824723_m1) for the human
(Hs00609297_m1),PPIA
developmental series and UBC,ACTB (Hs99999903_m1), GAPDH
(Hs99999905_m1), TBP (Hs00427620_m1) for the schizophrenia
cohort [49] and TBP, SDHA (Hs01549169_m1) and ACTB for the
rhesus developmental series.
Western Blot analysis
Western blot analysis for DCX was performed as previously
described [46]. Briefly, 40 mg of pulverised frozen tissue was
homogenised in 400 ml of homogenisation buffer [50 mM Tris
pH 7.5, 50% glycerol and 1:20 v/v of protease inhibitor cocktail
(Sigma, P8340) final concentration: 2 mM aminoethylbenzenesul-
fonylfluoride, 0.015 mMaprotinin,
0.030 mM, pepstatin A, 0.028 mM E-64, 0.08 mM bestatin].
Protein concentration per sample was determined using Bradford
(Sigma) and BCA (Thermo Scientific) protein assays. Analyses were
performed in duplicate with 12 mg of total protein for each human
sample and 30 mg total protein for each rhesus macaque sample,
heat denatured at 95uC in 1 volume of Lamelli buffer (BioRad) with
0.5% b-mercaptoethanol, analysed by SDS-PAGE on a 4–12% Bis-
Tris gel (BioRad) and transferred onto PVDF membrane (BioRad)
for 1 hr on ice. 10 ml of Dual colour Precision Plus Protein
Prestained Standard (BioRad) was loaded on each gel. Membranes
wereblocked(5%w/vnon-fatmilk,0.1%v/vTween-20inPBS)for
1 hr at RT with agitation, then incubated with goat polyclonal anti-
DCX (1:200, sc-8066, Santa Cruz Biotechnology) primary antibody
in 1% w/v non-fat milk, 0.1% v/v Tween-20 in PBS overnight at
4uC with agitation. Donkey anti-goat horse-radish peroxidase
(HRP)-conjugated secondary antibody (1:10 000, sc-2033, Santa
Cruz Biotechnology) secondary antibody in 1% w/v non-fat milk,
0.1% v/v Tween-20 in PBS, was applied for 1 hr at RT. Bands
were visualised using chemiluminescent HRP substrate (Immobi-
lonTMWestern, Millipore) and quantified by densitometry using
Image J [51]. As a loading control, membranes were probed with
mouse anti-actin (1:10,000; Chemicon; AB1501), followed by goat
anti-mouse HRP-conjugated secondary antibody (1:5,000).Average
intensity for each sample was normalised with the respective b-actin
average intensity and an internal control (protein from human
neonate and adult tissue or a pooled sample of all rhesus macaque
developmental cohort cases) from each gel.
0.038 mMleupeptin,
Statistical analysis
No group outliers of IWMN density were detected in any of the
human developmental groups by Grubbs test. Microarray data
examining gene expression across age were analysed in a linear
regression model including age. Samples with RINs less than 5.8
were excluded (n=11) from developmental qPCR data and
outliers within a developmental group (significant, p,0.05 by
Grubb’s method) or two standard deviations from the mean for the
schizophrenia cohort were removed for ANOVA/ANCOVA
(,5%). ANOVA was performed with Fisher-LSD post hoc
analyses to determine differences in IWMN density or DCX
mRNA/protein between developmental groups. Pearson’s corre-
lations were performed to examine the relationship between DCX
mRNA expression and IWMN density in the human. Statistical
tests were performed using Statistica software (version 7.1) and
data are reported as mean 6 standard deviation.
Results
IWMN density under the middle frontal gyrus declines
over postnatal age in the human
The density of NeuN+ IWMNs showed a significant develop-
mental change (ANOVA F=5.6, df=57, p=0.0001, Figure 1A)
being highest in the neonate group (85.9626.4 NeuN+ cells/mm2,
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representative image shown in Figure 1B) and being significantly
reduced by 36–46% in toddlers, teenagers, young adults and
adults (all p,0.005 compared to neonates and infants, with the
exception of toddlers and infants, being p=0.01 by Fisher LSD
post hoc analysis). Interestingly the NeuN+ cell density in school
age individuals (Figure 1C) is not significantly different from
infants, but shows a trend to be reduced in school aged individuals
compared to neonates (25.5% fewer cells, p=0.053) and a trend to
be elevated relative to adults (26.3% more cells, p=0.09,
representative image in Figure 1D) which may indicate that
IWMN density might increase slightly in the school age period (6–
13 years of age) prior to puberty.
Expression of DCX dramatically declines over early
human postnatal development
By microarray analysis, DCX (probe set 204850_s_at) showed a
dramatic reduction in mRNA levels in the human DLPFC early in
postnatal life with the largest decrease (p,0.001, r=0.925,
Figure 2A) in transcript levels from newborns to mature adults.
The relatively high expression in neonates compared to adults
represents the largest and most significant change in gene
expression found in the developing human brain with age out of
the 55,000 transcripts surveyed on the Affymetrix U133A chip
[50]. The extent and timing of this marked reduction in
developmental DCX expression was confirmed by qPCR analysis,
with an approximately 94% reduction between neonates and
adults for DCX mRNA (ANOVA F(6, 50)=15.5, p,0.00001,
Figure 2B). A significant difference was noted in DCX expression
levels between neonates/infants and the rest of the developmental
age groups (p,0.00001 for neonates, p,0.05 for infants)
demonstrating that the most dramatic change in expression takes
place within the first postnatal year of human life. Interestingly,
DCX mRNA levels are maintained throughout adult life at a level
above the limit of detection. Using Western blot analysis, we
observed a marked reduction in DCX protein levels across the
Figure 1. Interstitial white matter neuron (IWMN) density declines over postnatal development. (A) The mean density of NeuN
immunopositive IWMNs in the superficial white matter of the middle frontal gyrus was quantified in different developmental age groups: neonate (N,
n=7), infant (I, n=12), toddler (Tod, n=8), school age (SA, n=6), teenage (Teen, n=8), young adult (YA, n=8), adult (A, n=15). ***p,0.005
compared to neonates; +p,0.05, +++p,0.005 compared to infants. Error bars represent standard error. (B–D) Representative photos of NeuN+
IWMNs in (B, inset E) neonate, (C, inset F) school aged, and (D, inset G) adult individuals, line represents approximate grey matter/white matter
border. Scale bars=20 mm.
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developmental period examined with an approximate 94%
reduction between neonates and adults (ANOVA F(6, 51)=11.4,
p,0.000001, Figure 2C), with the most prominent band at the
predicted size of 40 kDa which was strongly expressed in younger
individuals (up to 13 years of age) and weakly expressed in older
age groups such as older teens, young adults and adults
(Figure 2D). Variability in band intensity was particularly evident
in the infant group (Figure 2D), which may be attributed to the
large change in expression over the first year of life, demonstrated
in Figure 2A.
Figure 2. Doublecortin (DCX) is downregulated in the DLPFC of human brain during postnatal development. (A) mRNA expression
profile of DCX across chronological age by microarray (males=circles, females=triangles). (B) The developmental profile of DCX mRNA expression
was replicated by qPCR [DCX mRNA expression (mean 6 SEM) was normalised to the geometric mean of four housekeeper genes] (B), and (C) at the
protein level [expression (mean 6 SEM) normalised to b-actin]. (D) Representative western blot for DCX and b-actin in individuals from different
developmental age groups. ***p,0.001 compared to neonate; +p,0.05, ++p,0.005, +++p,0.001 compared to infants. Black, neonates (N); green,
infants (I); red, toddlers (Tod); dark blue, school age children (SA); light blue, teenagers (Teen); yellow, young adults (YA); pink, adults (A).
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Expression of DCX in the developing rhesus macaque
primate brain
In rhesus macaque, qPCR analysis revealed a similar reduction
in DCX expression over development to that found in humans,
although it was not as marked. An approximate reduction of 77%
in DCX mRNA expression was seen between neonate and adult
rhesus macaques (ANOVA F(5, 39)=5.74, p,0.0005, Figure 3A).
Western blot analysis revealed approximately an 86% reduction in
DCX protein expression between neonate and adult rhesus
macaques (ANOVA F(5,39)=5.6132, p,0.0005; Figure 3B) with
a band at the predicted size of ,40 kDa (Figure 3C). A significant
difference was noted in both DCX mRNA and protein expression
levels between neonates and the other developmental age groups
(p,0.001 for mRNA and p,0.005 for protein) and a marked
reduction in immunointensity on the Western blot can be seen in
some individuals 8 years and older.
Although DCX immunoreactivity was not reliably detected in
fresh frozen human tissue (including in a neonatal brain, using
antibodies in Table S3, data not shown), DAB immunohisto-
chemistry for DCX was successful on perfused, fixed rhesus
macaque brain sections. Dense DCX positive cells and fibre plexus
were observed across the different developmental ages. In the 12
day old rhesus macaque brain, DCX immunoreactivity was
present in the white matter and particularly robust around the
ventricle with clusters of DCX positive cells apparent in the VZ/
SVZ on both the dorsal and ventral sides (Figure 4A). Although it
is difficult to determine the morphology of individual cells in this
area due to the density of DCX+ cells, many cells elaborate
processes to the ventricle and/or into surrounding tissue
(Figure 4B). In the neonatal rhesus macaque brain, large masses
of DCX+ cells were also noted at the dorsal and ventral ends of the
ventricle and clusters of cells with long processes were also present
in the white matter around these masses (Figure 4C, Figure 5B)
and many DCX+ cells (,8 mm diameter, mostly with one or two
long processes) were present in layer II of the cortex in the
principal sulcus (Figure 4D), gyrus rectus (Figure 5D) and
particularly around the inferior arcuate sulcus and lateral orbital
sulcus. DCX positive cells were also observed in the corpus
callosum (not shown). In the 1 month-old animal, intense DCX
staining was also noted around the ventricle (Figure 4E),
particularly in immunopositive patches on the dorsal and ventral
sides (Figure 4F). Clusters and chains of DCX+ cells were present
in the white matter dorsal and ventral to the ventricle (Figure 4G,
Figure 5F) and a population of DCX+ cells was also present in
layer II of the cortex at 1 month of age (Figure 4H, Figure 5H).
In the adolescent rhesus macaque (3.7 years), small DCX+
clusters of cells could be detected around the ventral ventricle
(Figure 4J), however clusters of DCX+ cells dorsal or ventral to the
ventricle were absent, but some individual immunopositive cells/
processes could be seen in the white matter dorsal to the ventricle
and caudate (Figure 4K). DCX+ cells were still present in layer II
of the cortex, although less dense and with less elaborate processes
than earlier in life (Figure 4L, Figure 5K). Similarly, in the adult
rhesus macaque brain (6 years), there were relatively sparse DCX+
cell clusters around the ventral ventricle (Figure 4N) and few
DCX+ cells in the white matter around or adjacent to the ventricle
(Figure 4O, Figure 5M) and some DCX+ cells with 1–2 processes
were detected in layer II of the cortex (Figure 4P, Figure 5O).
IWMNs express markers of migration in the rhesus
macaque brain
Individual DCX+ cells with elongated cell bodies and processes
orientated parallel to the pial surface were present in the white
matter between sulci leading toward the principal sulcus
(Figure 6A) as well as to the gyrus rectus (Figure 5C) in the 12
day old animal, and some DCX+ cells could be detected in the
white matter between sulci, with somewhat less elaborate
processes in the 1 month old rhesus macaque brain (Figure 6B).
Distinct DCX+ cells were not observed in the white matter
between sulci in the 3.7 year old or 6 year old brains; however,
immunoreactivity for PSA-NCAM was observed on cells and
processes in the white matter in 3.7 year old and 6 year old
animals (Figure 6C), suggesting that these cells may be migrating
neurons in the white matter of the adolescent and adult rhesus
macaque. We confirmed this observation in an additional three
adult animals (6.5, 7.6 and 9.6 years old), where we observed
PSA-NCAM immunoreactivity in fresh-frozen tissue sections
(Figure 7A). Many PSA-NCAM immunopositive cells were
present in the white matter under the principal sulcus with
elongated cell bodies and a leading and/or trailing process
Figure 3. Doublecortin (DCX) is downregulated in the DLPFC of
rhesus macaque brain during postnatal development. (A) mRNA
expression profile of DCX (mean 6 SEM) across chronological age by
qPCR (DCX mRNA expression was normalised to the geometric mean of
three housekeeper genes) and (B) DCX protein expression (mean 6
SEM, normalised to b-actin). (C) Representative western blot for DCX
and b-actin in cases from different developmental age groups.
**p,0.005, ***p,0.001 compared to neonate. Black, neonates (N);
red, infants (I); dark blue, juveniles (J); light blue, adolescents (Ado);
yellow, young adults (YA); pink, adults (A). Pooled standard (S).
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parallel to the pial surface (Figure 7B, C; enlarged in Figure S2).
Many elongated PSA-NCAM positive cells were also directed
toward the crown of the gyrus (Figure 7F–H), some with unipolar
or bipolar processes oriented radially towards the crown, across
the thickness of the grey matter at the crown (arrowheads in
Figure 7H). Interestingly, we also observed some PSA-NCAM
positive cells with a process that was directed on an angle to the
pial surface, toward the grey matter (Figure 7J, K) or possibly
bifurcated (Figure 7L, M). PSA-NCAM+ cells were also in the
deeper layers of the cortex with a short process directed on an
angle, or perpendicular to the pial surface (arrows in Figure 7N–
Q). Immunoreactivity was diffuse in layer IV and several round,
multipolar and elongated, unipolar/bipolar PSA-NCAM positive
cells (,8–15 mm diameter) were present in the deeper layers of
the cortex (Figure 7D, E). In cortical layer II some smaller round
(,7–12 mm), PSA-NCAM cells whose morphology was difficult
to define due to diffuse immunoreactivity that may be associated
with processes were also present (Figure 7I).
Rhesus macaque white matter neurons express
GABAergic markers
In the rhesus macaque brain, cells in the white matter between
sulci were positive for GAD65/67, the rate limiting enzyme
required for the synthesis of GABA, being present in the cell body
and processes, suggesting that these white matter cells may be
GABAergic neurons (Figure 6D), and we demonstrate co-
localisation of GAD65/67 immunoreactivity in NeuN+ IWMNs
in the principal sulcus of adolescent (4.5 years) rhesus macaques
(n=3) (Figure 8). GAD65/67 immunoreactivity was present in the
majority of NeuN+ cells, however some GAD65/67+ cells
displayed intense GAD65/67 immunoreactivity and less intense
or no NeuN immunoreactivity (Figure 8, arrow head and asterisk).
Expression of DCX mRNA in schizophrenia
qPCR was used to determine expression of DCX mRNA in
total RNA from the DLPFC of patients with schizophrenia/
Figure 4. Doublecortin (DCX) is highly expressed in infant and expression continues into adulthood in rhesus macaque. In a 40 mm
coronal section of a 12 day old rhesus macaque brain (A–D) DCX immunoreactivity is abundant around the lateral ventricle, particularly at the dorsal
and ventral ends and in layer II of the cortex, particularly around the inferior arcuate sulcus (arrow) and orbital cortex. DCX cells are also present
around the lateral ventricle in a 1 month old (E–H), 3.7 year old (I–L), and 6 year old (M–P) rhesus macaque brain. Higher power images show DCX
positive cells and fibre plexus in the subventricular zone (B, F, J, N), DCX immunoreactivity in clusters in the white matter dorsal to the lateral
ventricle in young brains (C, G), and DCX immunoreactivity in several cells or processes in the white matter adjacent to the dorsal ventricle in 3.7 and
6 year old brains (arrows K, O) and DCX immunoreactivity in layer II cells in the principal sulcus (with pial surface at top of the image in D, L and top-
left of image in H, P). Scale bar=1 mm (A, E, I, M), 20 mm (B–D, F–H, J–L, N–P).
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schizoaffective disorder (n=37) and controls (n=37). Analysis of
covariance was then performed with DCX mRNA and diagnosis,
co-varying for age, pH, PMI and RIN. DCX mRNA did not
display differences in expression in adult schizophrenia patients
compared with adult controls, with mean schizophrenia expression
being 97.4% of controls (ANCOVA F (1, 65)=0.31, p=0.56;
Figure 9A). No significant differences in expression by gender
(t=1.1, df=69, p=0.28) or hemisphere (t=0.79, df=69,
p=0.43) were noted. Performing Pearson’s correlation to clinical
variables (age of onset, duration of illness, daily chlorpromazine
mean, last recorded chlorpromazine dose, and lifetime chlor-
promazine exposure), we did not detect any significant correlation
between expression of DCX with disease demographics or
neuroleptic exposure (r=20.22 to 0.07, all p.0.21).
Relationship between DCX mRNA and IWMN density is
altered in schizophrenia
We have previously reported that superficial IWMN density is
elevated in people with schizophrenia in this cohort [18].
Therefore, to examine the relationship between DCX mRNA
expression in the grey matter and superficial IWMN density,
qPCR data for DCX in the grey matter was correlated with
superficial IWMN density [18]. In adult controls from the
schizophrenia cohort aged 18–78 years, there was no correlation
of DCX mRNA with the density of superficial white matter
neurons (r=20.04, p=0.80, Figure 9B); however , DCX mRNA
expression was negatively correlated with superficial IWMN
density in patients with schizophrenia (r=20.39, p=0.016,
Figure 9C) [19]. In the human developmental cohort, in stark
contrast, there was a positive relationship between grey matter
DCXmRNAandsuperficial
p=7.061024, Figure 9D).
IWMN density(r=0.51,
Discussion
Our data suggest that the arrival of new neurons may play a
significant role in the protracted postnatal development of the
prefrontal cortex and, consequently, the behavioural and cognitive
development associated with this region [52]. There is growing
Figure 5. Doublecortin (DCX) expression in the developing rhesus macaque rectus gyrus. DCX immunoreactivity in a 40 mm coronal
section from a 12 day old (A–D), 1 month old (E–H), 3.7 year old (I–K), and 6 year old (L–O) rhesus macaque brain. DCX immunoreactivity is
abundant around the lateral ventricle, and in clusters and chains of cells ventral to the ventricle in the 12 day old (B) and 1 month old (F) animal. DCX
cells (arrows) are also present ventral to the ventricle in a 3.7 year old (J) and a 6 year old animal (M). DCX positive cells are also present in the gyrus
rectus of young animals (arrows C, G) and processes in the adult white matter (arrow N) and layer II cells in the gyrus rectus (D, H, K, O) (pial surface
on left). Scale bar=1 mm (A, E, I, L), 20 mm (B–D, F–H, J, K, M–O).
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evidence to support the presence of immature neurons in the
postnatal and adult primate neocortex; however, the origin of
these cells is currently unclear. Our finding that DCX, a marker of
immature neurons, is highly expressed in the first few years of life
and continues to be expressed at lower levels in adulthood
supports the hypothesis that the arrival of new, immature neurons
into the DLPFC and gyrus rectus in postnatal life contributes to
overall growth and maturation or attainment of abilities and/or
learning. The observation of DCX+ cells surrounding the ventricle
persisting into adulthood, the developmental down-regulation of
IWMN density, and the presence of many PSA-NCAM+ cells in
the white matter and cortex of adult non-human primate brains
suggests that migration of immature neurons from the SVZ to the
cortex, or to the white matter subjacent to the cortex could last for
several years after birth in primate, and even into adulthood.
New neurons could contribute to postnatal growth of
the cortex
These findings demonstrate high expression of the neuronal
migration marker DCX and high IWMN density soon after birth.
DCX expression then dramatically declines in the first two years of
life, and while cell death may account for some of the decrease in
IWMN density, it is unlikely to explain the whole decrease, as very
few nuclei are TUNEL positive early in human postnatal
development [37]. Our observations replicate the expression of
DCX in the human occipital cortex [20] and follow a similar
developmental trajectory to that described for PSA-NCAM in the
prefrontal cortex [53], supporting a developmental down-regula-
tion of migrating neurons in postnatal life in the human cortex.
This decrease in IWMN density is unlikely to be explained solely
by a dilution effect caused by the expanding cortical volume and
surface area during early development [41,54]. The human brain
increases in volume from birth to approximately 5 years of age
[38,39,40,41] and the brain weight nearly doubles from the
neonate to the infant stage [55,56]. Taking into account the
expansion of the cortex and subcortical white matter over the first
few years of life, the lack of significant change in IWMN density
that we report between neonates and infants may even support an
increase in the number of putatively migrating neurons at these
early developmental time points. Indeed, cortical growth,
particularly in the grey matter which increases across childhood
[57,58], may, in part, represent the arrival of new neurons in early
postnatal development, as an increase in neuronal density has
been reported in the human cerebral cortex between 15 months
and 6 years of age [54].
New cortical neurons may originate from the adult SVZ
In the early human brain, the majority of interneuron genesis
takes place in the ganglionic eminence, however at 20 gestational
weeks (gw) many calretinin positive interneurons are also present
in the cortical VZ/SVZ [59] and by 25 gw the majority of
interneurons in the cortical plate (65%) are Mash1 positive [60],
suggesting they may have originated in the dorsal VZ/SVZ
[59,60,61,62]. We have previously shown neuronal clusters
positive for ErbB4, PSA-NCAM and TuJ1 that appear to be
migrating away from the ventral SVZ in the developing, postnatal
human brain which may be the source of origin of new neurons in
the cortex [36,37]. Our finding here of numerous DCX positive
cell clusters in the ventral SVZ of the monkey, even in the adult, is
indicative of an immature population of cells derived from the
ventral VZ/SVZ and is consistent with this notion.
It has been suggested that cells that arise from the ventral SVZ
may take a route like that of the rostral migratory stream to
locations such as the amygdala and prefrontal, parietal, piriform,
Figure 6. Non-human primate interstitial white matter neurons (IWMNs) express markers of migrating neurons and interneurons.
Some individual IWMNs were immunopositive for DCX in 12 day old (arrows A) and 1 month old (arrows B) brains. PSA-NCAM immunoreactivty was
apparent in some white matter neurons and their processes in the adult (40 mm sections) (arrows C). Some IWMNs were also positive for GAD65/67
immunoreactivity in the adult (arrows D) and there was also diffuse GAD65/67 immunoreactivity in the white matter of adults. Scale bars=20 mm.
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entorhinal and temporal cortices in rodents, rabbits, and non-
human primates [63,64,65,66,67,68,69]. This observation has
recently been extended to the adult human, where Wang and
colleagues showed continued proliferation of neuroblasts in the
anterior ventral SVZ and immature cells expressing DCX and
PSA-NCAM in the RMS [33]. Further to this, Inta and colleagues
used time-lapse imaging of transgenic 5-HT3-EGFP mice to
demonstrate the postnatal migration (in juveniles) of neurons from
the SVZ to the cortex and subcortical regions, demonstrating
DCX expression in these cells and an ultimate GABAergic
phenotype [70]. The abundance of DCX positive cells around the
ventricle, and the presence of DCX and PSA-NCAM positive
neurons in the white matter of the principal sulcus in infant rhesus
macaques that we report here provide further support of postnatal
migration of neurons from the SVZ to the cortex.
Although we did not find any DCX immunopositive cells in
the white matter of adult primates, the expression of PSA-NCAM
by rhesus macaque adult IWMNs and their bipolar, tangential
morphology suggests that these immature cells may be migrating
through the white matter. It is possible that DCX expression is
below the level of detection in these cells, or that there may be
another DCX-like molecule being expressed due to possible
molecular redundancy as has been suggested with DCL and
DCLK [25,26]. We suggest that neurons migrating from the
SVZ, or generated in the white matter may contribute to
immature PSA-NCAM expressing neurons in the deeper layers of
the cortex as we observed several PSA-NCAM+ cells that
appeared to be ‘‘turning’’ from the white matter with a process
directed toward the grey matter and many unipolar and bipolar
PSA-NCAM positive cells in the deeper cortical layers oriented
toward the crown of the gyrus in the adult rhesus macaque
cortex.
Immature neurons in the adult cortex
Our observations indicate that expression of DCX mRNA is
persistent at detectable levels in the grey matter throughout adult
life. In the adult, neurons that are positive for DCX protein and/
or PSA-NCAM have been reported in the cortex, particularly
layer II, of several species including rodents, cats, non-human
primates, and humans [31,63,67,71,72,73,74,75,76]. It is thought
that these cells are immature neurons due to their co-localisation
with neuronal markers such as TUC-4 (a neuronal lineage marker)
[72], TuJ1 [76] and expression of NeuN in some cells
[31,72,76,77]. Interestingly, the density of these immature neurons
Figure 7. PSA-NCAM is expressed in multiple white matter neurons and immature cortical neurons in the adult rhesus macaque.
Representative photos of DAB immunohistochemistry for PSA-NCAM in 14 mm coronal sections show diffuse immunoreactivity in layer IV of the
cortex in the frontal pole of the adult (6.5, 7.6 and 9.6 years) rhesus macaque brain (A). Higher power images show multiple PSA-NCAM+ cells are also
present in the white matter under the principal sulcus (B, C, J–M), and the numerous immunopositive cells are detected in the deeper cortical layers
(B, D, E, N–Q) as well as layer II of the principal sulcus (I). Additionally, many PSA-NCAM+ cells could be seen in the white matter near the crown of
the gyrus, often with elongated cell bodies (arrows in G) and some with one or two processes (arrowheads in H) along the long axis of the cell body
(arrow in H). Some PSA-NCAM+ processes in the white matter were orientated on an angle to the pial surface (J, arrows in K), or may have bifurcated
(L, arrows in M), and some PSA-NCAM+ cells with elongated cell bodies and a short process directed toward the pial surface were present in the deep
cortical layers (arrows in O, Q). Scale bars: 1 mm (A), 40 mm (B, F, J, L, N, P), 20 mm (C–E, G–I, K, M, O, Q). gm=grey matter, wm=white matter.
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is reduced in ageing [31,78], which may indicate that these cells
become depleted over time [79]. There is, however, controversy as
to whether these DCX+ cells may become GABAergic or
glutamatergic as some studies report expression of GABA,
GAD67, calretinin and parvalbumin with DCX [31,76], while
others report no co-localisation with GABAergic markers, and the
expression of Tbr-1 (a transcription factor of dorsal SVZ neurons)
in these immature DCX+ neurons [72,74,77]. Varea and
colleagues show immature DCX+/PSA-NCAM+ neurons in layer
II express Tbr-1, although BrdU labelling in adult cats indicates
that the majority of PSA-NCAM+ cells may not be born in adult
life [77], and, in the rodent, the birth of layer II cells peaks around
E15.5 [72]. However, new neurons in the adult are expected to be
less than 0.03% of total neurons (as reviewed by [80]), and thus
may be difficult to detect with the BrdU regimens used (2–4
injections of BrdU over a two day period). A further population of
larger PSA-NCAM positive cells are also found in deeper cortical
layers (as we find here), some of which expressed GAD67 and/or
calbindin and calretinin, suggesting that these deeper cortical
immature neurons may be GABAergic [77]. This would be
consistent with the inhibitory nature of the IWMNs we report here
in the rhesus macaque brain (GAD65/67+) and our previous
observation of somatostatin and neuropeptide Y expression in
human IWMNs [18].
Figure 8. NeuN positive IWMNs express GABAergic markers. Double-label immunofluorescence in adolescent (4.5 year old) rhesus macaque
frontal pole coronal 14 mm sections shows co-localisation of NeuN (green) with GAD65/67 (red) in white matter neurons (DAPI staining for nuclei in
blue). Some neurons show faint immunoreactivity for GAD65/67 (arrow) and some neurons express relatively more GAD65/67 and less NeuN
(arrowhead) or no NeuN (asterisk). Scale bars=25 mm.
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Figure 9. Doublecortin (DCX) is unaltered in schizophrenia cortex but related to white matter neuron (IWMN) density. DCX mRNA
expression [normalised to the geometric mean of four housekeeping genes, shown as a % of control mean for control and schizophrenia groups
(bar=mean)] was not altered in the brains of people with schizophrenia (n=37) compared to control subjects (n=37, p=0.5, co-varying for age, pH,
PMI, and RIN) (A). (B) DCX mRNA expression did not correlate with the density of superficial IWMNs in controls, and was negatively correlated with
superficial IWMNs in patients with schizophrenia (C). (D) In development there was a positive correlation between DCX mRNA expression (normalised
to the geometric mean of four housekeeping genes, shown as a % of the mean expression of the neonate group) and superficial IWMN density.
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The high expression of grey matter DCX in the first few years of
life is consistent with DCX involvement in cortical growth and it
follows that differentiation of newly arrived cells may result in
delayed up-regulation of the interneuron markers that are late
developing. Indeed, we have recently reported within the same
cohort that the most dramatic changes, either up-regulation or
reduction, in mRNA expression of multiple biochemical markers
of interneurons occur within the first five postnatal years in the
human DLPFC [44]. Protracted increases in interneuron markers
parvalbumin, cholecystokinin, calbindin and vasoactive intestinal
peptide in the DLPFC over the first years of life were shown [46],
that are reciprocal to the reduction in DCX, implying that some
immature migrating neurons may down-regulate DCX and up-
regulate markers of differentiated interneurons in the human
frontal cortex. This is consistent with other reports, such as up-
regulation of parvalbumin in cartridges and neurons, and
calbindin positive neurons that display protracted development
in the primate [81,82], and in human frontal [83] and entorhinal
cortex [84].
DCX in schizophrenia
Putative candidate schizophrenia genes like neuregulin/ErbB4
and reelin have important roles in migration of new neurons, and
some studies have implied a reduction in neurogenesis in the
schizophrenic brain [44,45]. While we have previously reported an
increase in the density of IWMNs in schizophrenia using this
cohort [18], we do not find a change in DCX mRNA in the
DLPFC in schizophrenia here. However, even though DCX is
expressed in the cortex of the adult it may be an imperfect marker
for neuronal migration in the adult due to its relatively low
expression level and variable intensity in layer II neurons. This
may be supported by the wide spread in DCX mRNA expression
in the normal adults, and variable immunointensity within a given
neuron, such that DCX mRNA could be regulated by factors like
cell activity which may differ between individuals. Additionally,
the age of individuals in the control and schizophrenia population
(spanning from 18–80 years) may make subtle alterations in DCX
mRNA expression difficult to detect.
Interestingly, while in the normal adult brain there is no
correlation between DCX grey matter mRNA and IWMN
density, in people with schizophrenia there is a negative
correlation, such that individuals with less DCX mRNA in the
grey matter tend to have more subjacent IWMNs. This change
parallels our previous finding of reduced interneuron marker
mRNA (somatostatin) being correlated with increased IWMN
density in schizophrenia [18], and is consistent with a failure of
migration of some newly born neurons to the cortex in
development. We hypothesise that NeuN expression may overlap
with that of some immature markers, such as DCX (as this also
occurs in layer II) [77,78], as immature neurons down-regulate
immature neuron markers and up-regulate NeuN while they begin
to differentiate [18,85]. Therefore, NeuN expressing neurons in
the white matter would correlate with the increase in DCX in the
grey matter at a time of high neuronal migration. In the adult
controls, there appears to be an uncoupling of DCX mRNA
expression and IWMN density that could be due to low levels of
neuronal migration and the variable presence of DCX in many
layer II neurons. If developmental neuronal migration is deficient
in the brains of people with schizophrenia, this could lead to an
accumulation of IWMNs under the cortex, and higher numbers of
IWMNs in individuals with schizophrenia that may constrain the
immature neurons reaching the cortex and drive the negative
correlation between IWMN density and DCX mRNA expression
in the disease state.
Although adult cortical neurogenesis is controversial, our results
support the hypothesis that neuronal migration to the cortex may
be robust in the early postnatal primate brain. We also
demonstrate that molecules associated with immature neurons,
neuronal migration and/or plasticity can still be found in the adult
primate brain in grey matter and in the white matter, and we
suggest that migration of immature inhibitory neurons continues
to occur into the adult primate frontal cortex albeit at much lower
levels. Our results support seminal findings of Gould and others
[63,64,65] that raise the question as to whether or not the olfactory
bulb is the sole destination of newly born neurons of the primate
SVZ. While we did not detect a change in the expression of DCX
in the brains of people with schizophrenia, more sensitive or direct
methods may be required to detect altered migration of neurons in
the disease (which would likely affect a small population of the
total number of neurons in the cortex) and further lines of
evidence, such as altered IWMN density and positioning, and the
involvement of several schizophrenia susceptibility genes in
neuronal migration indicate that altered neuronal migration may
be implicated in schizophrenia pathology. Understanding postna-
tal neurogenesis and persistent migration of immature neurons in
the juvenile and adult brain suggests that cortical neurogenesis
may represent an important therapeutic target for intervention in
schizophrenia.
Supporting Information
Figure S1
density. Representative photomicrograph showing grey matter
and white matter boundary (line) used for quantification of
superficial IWMNs. The representative image shows DAB
immunohistochemistry for NeuN in a normal adult human brain.
Some examples of IWMNs are indicated with arrows.
(TIF)
Interstitial white matter neuron (IWMN)
Figure S2
neurons (IWMNs) express PSA-NCAM. PSA-NCAM immu-
noreactivity was apparent in multiple white matter neurons in
adult rhesus macaques at several ages. PSA-NCAM + INWMs in
(A) 6.5 year old, (B) 7.6 year old and (C) 9.6 year old animals.
Some examples of PSA-NCAM+ IWMNs are indicated with
arrows. Scale bars=50 mm.
(TIF)
Non-human primate interstitial white matter
Table S1
used
RIN=RNA integrity number, M=male, F=female, IWMN=in-
terstitial white matter neuron.
(XLSX)
Summary of human developmental cohort
experiments.
PMI=post-mortem
for
interval,
Table S2
try. DAB=3,39–diaminobenzidine, GAD=glutamic acid decar-
boxylase, PSA-NCAM=polysialyated neuronal cell adhesion
molecule.
(XLSX)
Antibodies used in DAB immunohistochemis-
Table S3
tissue pilot.
(XLSX)
Antibodies used for DCX in fresh-frozen
Acknowledgments
We would like to thank Shan-Yuan Tsai, Duncan Sinclair, and David Yu
for technical support. We acknowledge the assistance of Dr. H. Ronald
Zielke and Robert Vigorito of the University of Maryland Brain and Tissue
Bank for Developmental Disorders. Tissues were received from the New
South Wales Tissue Resource Centre at the University of Sydney.
Postnatal Neuronal Migration in the Primate Brain
PLoS ONE | www.plosone.org13 September 2011 | Volume 6 | Issue 9 | e25194

Page 14

Author Contributions
Conceived and designed the experiments: SJF SS MJW CSW. Performed
the experiments: SJF DJ KMA SS DAR RS PLN. Analyzed the data: SJF
DJ KMA SS DAR. Contributed reagents/materials/analysis tools: RS
PLN MJW CSW. Wrote the paper: SJF DJ KMA SS MJW CSW.
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