Neuroscience Vol. 112, No. 3, pp.

573^582, 2002
< 2002 IBRO. Published by Elsevier Science Ltd
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COMPLEXITY OF SENSORY ENVIRONMENT DRIVES THE EXPRESSION

OF CANDIDATE-PLASTICITY GENE, NERVE GROWTH FACTOR
INDUCED-A

R. PINAUD,a1 L. A. TREMERE,b M. R. PENNER,c F. F. HESS,a H. A. ROBERTSONc and

R. W. CURRIEa
a
Laboratory of Molecular Neurobiology, Department of Anatomy and Neurobiology, Dalhousie University,
Halifax, NS, Canada B3H 4H7
b
Laboratory of Molecular Neurobiology, Department of Physiology and Biophysics, Dalhousie University,
Halifax, NS, Canada B3H 4H7
c
Laboratory of Molecular Neurobiology, Department of Pharmacology, Dalhousie University, Halifax, NS, Canada B3H 4H7

Abstract3Exposure of animals to an enriched environment triggers widespread modi¢cations in brain circuitry and
function. While this paradigm leads to marked plasticity in animals chronically or acutely exposed to the enriched
environment, the molecular mechanisms that enable or regulate such modi¢cations require further characterization. To
this end, we have investigated the expression pro¢les of both mRNA and protein products of a candidate-plasticity gene,
nerve growth factor induced-A (NGFI-A), in the brains of rats exposed to increased environmental complexity. We found
that NGFI-A mRNA is markedly up-regulated throughout the brains of animals exposed to the enriched environment,
but not in the brains of either handled-only or undisturbed control groups. The most pronounced e¡ects were observed in
the somatosensory and visual cortices, in layers III and V, while more modest increases were observed in all other cortical
layers, with the exception of layer I. A striking NGFI-A mRNA up-regulation was also observed in the striatum and
hippocampal formation, notably in the CA1 sub¢eld, of animals exposed to the enriched environment paradigm.
Immunocytochemistry was also used to investigate the distribution of NGFI-A protein in response to the environ-
mental enrichment protocol. A marked increase in the number of NGFI-A positive nuclei was identi¢ed in the enriched
environment condition, as compared to undisturbed and handled-only controls, throughout the rat brain. While the
greatest number of NGFI-A immunolabeled neurons was found in cortical layers III and V, up-regulation of NGFI-A
protein was also detectable in layers II, IV and VI, in both the somatosensory and visual cortices. NGFI-A immuno-
positive neurons were also more numerous in the CA1 sub¢eld of the hippocampal formation of animals exposed to the
enriched environment, but remained at basal levels in both control groups.
Our results implicate NGFI-A as one of the possible early genetic signals that ultimately lead to plastic changes in the
CNS. < 2002 IBRO. Published by Elsevier Science Ltd. All rights reserved.

Key words: plasticity, environmental complexity, immediate early gene, NGFI-A, reorganization.

Lifelong functional modi¢cation of CNS sensory path-
ways can occur in response to development, use and
aging (Fox, 1992; Recanzone et al., 1992; Jones and
Pons, 1998; Florence et al., 1998). These changes have
been postulated to re£ect altered connectivity at the syn-
aptic, cellular and system levels (Buonomano and
Merzenich, 1998; Jones, 2000). Many paradigms that
induce CNS plasticity do so within a single sensory
1
Present address: Neurological Sciences Institute (NSI), Oregon
Health Sciences University (OHSU), 505 NW 185th Avenue, Bea-
verton, OR 97006-3499, USA.
*Corresponding author. Tel. : +1-902-494-3343; fax: +1-902-494-
1212.
E-mail address: wcurrie@is.dal.ca (R. W. Currie).
Abbreviations : ANOVA, analysis of variance ; CA1/CA2/CA3,
cornu ammonis, sub¢elds 1, 2 and 3; EE, enriched environment ;
HO, handled-only ; IEG, immediate early gene; LTP, long-term
potentiation ; NGFI-A, nerve growth factor induced gene-A;
NMDA, N-methyl-D-aspartate; PBS, phosphate-bu¡ered saline ;
SSC, saline sodium citrate; UD, undisturbed controls.
573
modality or sub-modality. However, acute or chronic
exposure to an enriched environment (EE) leads to wide-
spread morphological changes in primary sensory path-
ways, association areas and the hippocampal formation
(Rosenzweig et al., 1972; Volkmar and Greenough,
1972; Saito et al., 1994). An additional advantage of
this experimental approach is that plasticity can be
induced in the absence of injury or biologically abnormal
use.
The hypothesis that an EE induces CNS plasticity has
been well supported using both histological and electro-
physiological techniques. For example, animals housed
in an EE display a higher neuron to glia ratio, greater
somatic areas, enlarged presynaptic boutons and
enhanced neurotransmitter release (Rosenzweig et al.,
1972; Schrott, 1997). One ¢nding that may be particu-
larly important in understanding what processes partic-
ipate in functionally restructuring the CNS is that EE
exposure is associated with increased dendritic arboriza-
tions (Volkmar and Greenough, 1972; Globus et al.,
1973). One interpretation of this result is that increases

NSC 5543 4-6-02

574 R. Pinaud et al.
in dendritic substrate enhance the potential for contact
between cells and could o¡er more precise control over
which input zones can be activated or selectively inhib-
ited (Rosenzweig et al., 1972; Volkmar and Greenough,
1972; Globus et al., 1973). The phenotypical changes
described here have been associated with the induction
of plasticity mechanisms in the CNS and have also been
postulated to constitute the physical underpinnings of
higher order neural processes such as learning and mem-
ory (Bailey and Kandel, 1993; Bailey et al., 1996).
Enhanced complexity of a sensory environment also
leads to marked biochemical changes within CNS neu-
rons. Relative to their counterparts in the control group,
animals exposed to an EE displayed increased levels of
neurotrophic factors, such as the brain-derived neurotro-
phic factor, a growth factor that has been strongly impli-
cated in mediating both long-term synaptic facilitation
and depression (Falkenberg et al., 1992; Ickes et al.,
2000; Brunig et al., 2001). In addition to these neuro-
physiological outcomes of EE exposure, behavioral and
psychophysical changes have also been documented that
support the hypothesis that EE alters CNS performance.
Complementary work from both these ¢elds has shown
that the phenotypic changes postulated to re£ect plastic-
ity are correlated with enhanced performance in learning
and memory tasks, as well as improved visual acuity
(Paylor et al., 1992; Prusky et al., 2000). Together,
these ¢ndings suggest that experience in the EE modi¢es
CNS circuitry and that these changes can be connected
with an improved performance of a sensory system as
well as storage and use of environmental information.
One of the central tenets in molecular plasticity is that
phenotypic changes result from activation of the genome
within each cell, such that the appropriate physical sub-
strate is produced in order to mediate altered perfor-
mance (Bailey et al., 1996; Bailey and Kandel, 1993). In
general, this process would involve up- or down-regula-
tion of relevant proteins, such as those in ion channels,
synaptic vesicle docking proteins or cytoskeletal proteins
(Staiger et al., 2000). Furthermore, the instruction to ini-
tiate phenotypic change results from the activation of
biochemical messengers and immediate early genes
(IEGs). The expression of this class of gene is quick and
transient after cell stimulation, not requiring new protein
synthesis (Hughes and Dragunow, 1995). In the ¢eld of
molecular neurobiology, IEGs have become popular can-
didates as the ¢rst genetic response to mediate plasticity
in neurons, but may also be important as transcription
factors that regulate the expression of late genes, the class
of genes connected with longer, more stable structural
modi¢cations (Hughes and Dragunow, 1995).
Certain IEGs such as c-fos and c-jun have become
useful tools for evaluating the recent history of neural
activity (Kaczmarek and Chaudhuri, 1997; Herdegen
and Leah, 1998). However, other IEGs such as the activ-
ity-regulated cytoskeletal gene (Steward et al., 1998;
Pinaud et al., 2001), and the nerve growth factor induced
gene-A (NGFI-A) (Milbrandt, 1987), are not reliable
activity markers. The latter IEG, NGFI-A (also known
as zif-268, Krox-24, Egr-1 and ZENK), has the intrigu-
ing property of responding only to novel stimuli and
NSC 5543 4-6-02
therefore has been postulated to be involved in the
early genetic steps that lead to the formation of cellular
memories (Mello and Clayton, 1994). This property of
the NGFI-A response pro¢le makes it an excellent can-
didate for a biological marker at the initiation of learn-
ing-related processes.
In order to explore the involvement of NGFI-A as a
genetic indicator of neural plasticity, mRNA and protein
levels of this IEG were characterized in an EE exposure
paradigm.
EXPERIMENTAL PROCEDURES
Animals and experimental groups
The methods describing this EE paradigm have been previ-
ously described in detail (Wallace et al., 1995; Pinaud et al.,
2001). The subjects in these experiments were all age-matched
adult male Sprague^Dawley rats that weighed 210 P 7 g (n = 27,
Charles River, Canada). Rats were exposed to a 12/12-h light^
dark cycle. Animals belonged to one of three experimental con-
ditions. (1) Animals belonging to the ‘enriched environment’
group (EE, n = 9) spent 1 h/day together in a 3 mU1.5
mU1 m basin that had been lined with standard bedding mate-
rial and ¢lled with toys, tubes and food rewards. For the
remainder of their day, rats remained in their home cages.
(2) Animals in the ‘handled-only’ group (HO, n = 9) were lifted
and held repeatedly throughout 5 min then allowed to remain in
their home cages. This condition was designed to reveal any
changes in gene expression that may have been induced in EE
animals as a result of handling or as part of an expectation
response to the handler. (3) Undisturbed controls (UD, n = 9)
remained in their home cages throughout the experiment. The
duration of this experiment was 3 weeks. Variability in gene
expression related to circadian cycle was minimized by employ-
ing a consistent schedule with EE and HO protocols conducted
between 15.00 h and 16.00 h daily. During this time, behavior
was monitored for each experimental group in order to assess
levels of wakefulness and activity. Several rats in the HO and
UD groups were not included in the histological analyses
because of low activity levels. Rats included in the analyses
had approximately similar activity levels. Our experimental pro-
cedures conformed to institutional regulations and the Guide to
the Care and Use of Experimental Animals from the Canadian
Council on Animal Care.
Perfusion and histology
On the last experimental day (day 21), EE animals were
exposed to the EE setting, identical to previous experimental
days. However, at 16.00 h, when these animals should have
been returned to their home cages, they were anesthetized and
killed. HO animals were manipulated for 5 min starting at 15.00
h and returned to their home cages until 16.00 h, at which time
they were anesthetized. This 55-min period before perfusion of
the HO animals allowed time for any induced expression of
NGFI-A gene products to accumulate. UD controls remained
in their home cages until 16.00 h of day 21.
Immediately prior to death, all animals were deeply anesthe-
tized with sodium pentobarbital (70 mg/kg, i.p.), assessed for the
absence of re£exes, then perfused transcardially with a solution
of 0.1 M phosphate-bu¡ered saline (PBS). Brains were removed,
placed inside sterile Petri dishes and frozen in a 370‡C freezer.
Cryostat sections were taken in the coronal plane at 20 Wm.
Sections were directly mounted onto Fisherbrand Superfrost
slides and stored at 370‡C.
In situ hybridization
Mounted sections were thawed, dried at room temperature for
 NGFI-A expression in enriched environment
30 min then ¢xed in 4% paraformaldehyde solution for 5 min.
Subsequently, sections were washed in 150 mM sodium chloride/
15 mM sodium citrate (1USSC). A commercially synthesized
oligonucleotide probe, with the following sequence, was
designed to recognize NGFI-A transcripts (Genosys, Canada):
5P - CCGTTGCTCAGCAGCATCATCTCCTCCAGTTTGGG-
GTAGTTGTCC-3P. This sequence speci¢cally recognizes
nucleotides 355^399 of the rat NGFI-A mRNA (Milbrandt,
1987; GenBank accession number M18416). The cDNA oligo-
nucleotide probe was labeled at the 3P end with [33 P]dATP. The
NGFI-A probe was diluted in hybridization bu¡er (5U106
c.p.m./ml) that consisted of 50% deionized formamide, 50%
2Uhybridization bu¡er. Sections were incubated overnight, in
a hybridization oven, at 40‡C. The next day, sections were
washed in the following solutions: 4U15 min in 2USSC,
4U15 min in 1USSC, 4U15 min in 0.5USSC, 2U20 min in
0.25USSC. All washes were conducted at 55‡C, with only the
last wash occurring at room temperature. Finally, sections were
dipped in MilliQ water and air-dried overnight at room temper-
ature. Sections were then exposed to Kodak BioMax MR ¢lm
(Interscience, Markham, ON, Canada) for 2^5 days.
Immunocytochemistry
Sections were removed from the 370‡C freezer and hydrated
for 30 min in PBS at room temperature. Subsequently, tissue
was ¢xed in 4% paraformaldehyde for 10 min. Non-speci¢c
binding was minimized by incubating tissue in 0.5% albumin
and 0.3% Triton X-100 in 0.1 M PBS, for 2 h at room temper-
ature; this step was followed by three rinses in PBS (10 min per
wash). Sections were incubated in a solution containing the
NGFI-A primary antibody, at a dilution of 1:1000 in blocking
bu¡er, inside a humid chamber at 4‡C, where they remained
overnight. The primary antibody was a polyclonal antibody,
raised in rabbit against the carboxy-terminus of the rat NGFI-
A protein (Santa Cruz Biotechnology, Santa Cruz, CA, USA).
The following day, sections were washed for 30 min in PBS, in
order to remove excess, unbound antibody. Sections were then
incubated in a goat anti-rabbit biotinylated antibody (Vector
Laboratories, Burlingame, CA, USA), at a dilution of 1:200
in blocking bu¡er, for 2 h at room temperature. Tissue was
subsequently rinsed for 30 min in PBS. Sections were incubated
in a PBS solution containing avidin^biotin complex (Vector
Laboratories), for 2 h at room temperature at the dilution rec-
ommended by the manufacturer. This step was followed by
another 30-min wash in PBS. Sections were developed by incu-
bation in a ¢ltered solution containing 0.03% diaminobenzidine
0.15% nickel^ammonium sulfate in 0.1 M PBS to which 0.001%
hydrogen peroxide had been added. After the chromogen reac-
tion, tissue was dehydrated in a standard series of alcohols,
exposed to xylenes and coverslipped with Entellan (BDH,
Darmstadt, Germany). Controls for the immunocytochemistry
procedure were carried out by omitting the primary antibody,
which resulted in no immunolabeling. To control for processing-
related variability, sections from all experimental groups were
reacted as a single immunocytochemical batch.
Imaging
To digitize images of the in situ hybridization autoradiograms,
¢lms were scanned at 300 d.p.i. with a Duoscan T1200 desktop
Table 1. E¡ects of EE on NGFI-A mRNA expression in the cerebral cortex
Group Level 1
EE 0.51 P 0.0001 a
HO 0.27 P 0.0005 b
UD 0.28 P 0.0004 b
Data are expressed as optical density (mean P S.E.M.), n = 6 per group. Using bregma as reference, approximate stereotaxic coordinates are:
level 1 (AP 0.70), level 2 (AP 33.80) and level 3 (AP 36.20), according to Paxinos and Watson (1998). a, b: groups with di¡erent letters
di¡er signi¢cantly (one-way ANOVA; P 6 0.01).
NSC 5543 4-6-02
575
scanner (Agfa). Digitized images of immunocytochemistry were
obtained with a digital Spot Camera (Diagnostics). Digitized
images were assembled into plates using Adobe Photoshop soft-
ware (Adobe, Pantone).
Quanti¢cation and statistical analysis
For quanti¢cation of NGFI-A mRNA expression, we used a
protocol previously described by us (Pinaud et al., 2001). In
brief, optical density analysis was performed using NIH Image
software. Data were obtained from six animals per group, in
which optical density was averaged across hemispheres. A
value between 0 (white) and 1 (black) was collected from cortical
regions at comparable stereotaxic levels presented in Fig. 1 and
hippocampi presented in Fig. 2. Background levels were col-
lected from the white matter and subtracted from the signal
obtained for cortical structures. One-way analysis of variance
(ANOVA) was used to analyze signi¢cant treatment e¡ects.
Tukey HSD test was used for post-hoc comparisons with the
criterion level set at P 6 0.01.
For immunocytochemistry quanti¢cation, a grid of 400 Wm by
400 Wm was placed over the supragranular, granular and infra-
granular layers of the somatosensory and visual cortices, at
stereotaxic levels corresponding to photomicrographs in Figs.
3 and 4. The total number of NGFI-A immunopositive neurons
in each cortical layer was counted as pro¢les and averaged for
three animals per group. The statistical signi¢cance for the dif-
ferences in these mean values was established using a two-sam-
ple for means t-test, that was two-tailed with an K value set to
P 6 0.01.
RESULTS
In situ hybridization
Serial coronal sections throughout the cerebral cortex
were used to assess the expression pro¢le of the IEG
NGFI-A in the rat brain. Sections from animals belong-
ing to all experimental groups were exposed to the same
histological conditions in order to eliminate variability in
labeling due to tissue processing. While UD controls
displayed basal NGFI-A mRNA expression, EE animals
underwent marked up-regulation in numerous cortical
regions. These e¡ects were more pronounced over the
somatosensory and visual cortices, as well as the hippo-
campal formation, motor cortex and striatum (Fig. 1).
HO animals displayed low levels of NGFI-A mRNA
labeling for the majority of these regions, although no
statistically signi¢cant di¡erences were detected between
this group and UD animals (Table 1).
A marked increase in NGFI-A expression was
detected in primary and secondary visual areas. The
strongest NGFI-A hybridization was identi¢ed in corti-
cal layers III and V, while a more modest, but still pro-
Level 2 Level 3
0.51 P 0.0003 a 0.46 P 0.0003 a
0.27 P 0.0001 b 0.31 P 0.0004 b
0.27 P 0.0001 b 0.30 P 0.0002 b
576 R. Pinaud et al.

Fig. 1. Autoradiograms showing NGFI-A mRNA hybridization at similar stereotaxic levels in examples of brains from all
experimental groups. Sections are presented from rostral to caudal, left to right. NGFI-A up-regulation is more pronounced
over sensory cortices, striatum and hippocampi of EE animals. Scale bar = 500 Wm.

nounced increase was detectable in all other layers, rep-
resenting monocular and binocular vision (Fig. 1). Both
UD and HO controls displayed basal NGFI-A mRNA
signal in the visual cortex. These basal levels were higher
in layers III and V, while the other cortical layers failed
to exhibit increased signal (Fig. 1). Signi¢cant di¡erences
were detected between EE animals and both control
groups, however, no statistically signi¢cant di¡erences
were detected across either control group (Table 1).
As observed for the visual cortex, a striking NGFI-A
mRNA up-regulation was found in the somatosensory
cortex of EE animals. While this up-regulation was evi-
dent for all cortical layers, similar to the visual cortex,
layers III and V of primary and secondary somatosen-
sory areas underwent more advanced rates of NGFI-A
hybridization. Both HO and UD controls displayed
basal NGFI-A levels. However, higher NGFI-A hybrid-
ization was detected in layers III and V, as compared to
the expression in other cortical layers, for both of these
control groups (Fig. 1).
While NGFI-A basal levels were detected in all sub-
¢elds of the hippocampal formation of UD animals, a
marked up-regulation of NGFI-A expression was identi-
¢ed in the CA2 and CA3 regions of EE animals, with a
higher density of NGFI-A hybridization in the CA1
region (Figs. 1 and 2). HO animals displayed low hybrid-
ization levels in the hippocampal formation (Fig. 2). Fur-
thermore, statistical analysis revealed signi¢cant
di¡erences in NGFI-A mRNA hybridization levels in
the hippocampus of all experimental groups (Table 2).
NGFI-A expression was found at low levels in the den-
tate gyrus of UD animals. A modest increase in NGFI-A
expression was observed for this region in EE animals,
while HO controls also displayed low hybridization levels
(Fig. 2).
EE animals also displayed enhanced NGFI-A hybrid-
ization signal in the striatum, when compared to HO and
UD controls, where NGFI-A expression was detected at
basal levels (Fig. 1).
In all other cortical regions, NGFI-A expression also
Table 2. E¡ects of EE on NGFI-A mRNA expression in the
hippocampus
Group
EE 0.24 P 0.0001 a
HO 0.10 P 0.0001 b
UD 0.06 P 0.0004 c
Data are expressed as optical density (mean P S.E.M.), n = 6 per
group. a, b, c: groups with di¡erent letters di¡er signi¢cantly
(one-way ANOVA ; P 6 0.01).

NSC 5543 4-6-02

 NGFI-A expression in enriched environment 577

Fig. 2. NGFI-A mRNA hybridization in the hippocampus and dentate gyrus of EE, HO and UD animals. NGFI-A is
expressed in all sub¢elds of the hippocampus of animals from all experimental groups. However, EE animals displayed a
marked increase in NGFI-A mRNA hybridization signal in CA2 and CA3 sub¢elds, while the highest increase was observed
for CA1. In the dentate gyrus, NGFI-A mRNA was found in basal levels in HO and UD controls, while a modest increase
in signal was detected for EE animals. Scale bar = 200 Wm.

followed the same layering pattern. While the strongest
labeling was detected in the EE group for cortical layers
III and V, all other layers displayed more modest NGFI-
A mRNA hybridization. Furthermore, animals from
either control group displayed basal levels of NGFI-A
expression, with relatively stronger labeling in layers III
and V (Fig. 1).
Immunocytochemistry
Immunocytochemistry directed against the NGFI-A
protein was used in tissue from all experimental groups.
To minimize histological variability, all sections pre-
sented here were reacted in the same immunocytochem-
ical batch.
Immunolabeled neurons were identi¢ed in the primary
visual cortex of EE, HO and UD animals (Fig. 3). In UD
animals, NGFI-A immunopositive neurons were found
at high basal levels in cortical layers II and III (supra-
granular), V and VI (infragranular). Lower levels were
found in the thalamo-recipient layer IV (granular), while
no immunopositive neurons were found in layer I. A
similar immunoreactive pro¢le was observed in primary
visual cortex of HO animals. Infragranular and supra-
granular layers, with the exception of layer I, displayed
high NGFI-A basal levels, while these levels were mark-
edly reduced in layer IV. In contrast, a marked NGFI-A
up-regulation was found in all cortical layers of animals
exposed to an EE, with the exception of cortical layer I.
An increased number of NGFI-A positive nuclei was
found in layers II, III, V and VI, as compared to UD
and HO animals. Furthermore, enhanced numbers of

Fig. 3. NGFI-A protein levels are increased in all cortical layers of the primary visual cortex in EE animals. Compared to
UD and HO animals, EE animals displayed an enhanced number of immunopositive nuclei in cortical layers II, III, V and
VI, while a more modest increase was found in layer IV (A). Both HO (B) and UD (C) controls displayed basal levels of
NGFI-A immunoreactivity in all cortical layers, layer IV being the cortical layer with fewest NGFI-A positive nuclei. No
NGFI-A immunolabeled nuclei were detected in cortical layer I for all experimental groups. Scale bar = 200 Wm.

NSC 5543 4-6-02

578 R. Pinaud et al.

Fig. 4. The number of NGFI-A immunopositive nuclei is increased throughout the layers of somatosensory cortex of EE
animals. In the somatosensory cortex, EE animals displayed a striking increase in the number of NGFI-A positive cells in
layers II, III, V and VI, with a less pronounced enhancement in the number of immunolabeled cells in layer IV (A). Basal
NGFI-A levels were found in HO (B) and UD controls (C) in all cortical layers, with the exception of layer I. A modest
increase in the number of immunoreactive cells was found in layer III of HO animals, as compared to UD controls. Scale
bar = 200 Wm.

immunopositive nuclei were found in layer IV (Fig. 3).
Signi¢cant di¡erences were detected in the number of
NGFI-A positive nuclei for the EE group; however,
HO and UD animals did not di¡er signi¢cantly in the
primary visual cortex (Table 3).
In the somatosensory cortex of UD controls, basal
levels of NGFI-A protein were identi¢ed in supra- and
infragranular layers, with the exception of layer I, while
layer IV displayed minimal numbers of NGFI-A positive
nuclei (Fig. 4). The immunoreactive pro¢le of HO ani-
mals di¡ered very little from UD controls. Basal NGFI-
A immunoreactivity was identi¢ed in layers II, III, V and
VI, while a few immunoreactive nuclei were detected in
layer IV (Fig. 4). Similar to the results obtained for the
visual cortex, a striking up-regulation of the levels of
NGFI-A protein was detected in the somatosensory cor-
tex of EE animals. Increased numbers of immunoreactive
nuclei were identi¢ed in layers II, III, V and VI.
Although a clear increase in the number of NGFI-A
positive nuclei was detected in layer IV, this enhance-
ment was more modest as compared to the increases
experienced by all other cortical layers in EE animals
(Fig. 4). As described for HO and UD animals, no
immunolabeled nuclei were identi¢ed in cortical layer I
of EE animals. Signi¢cant di¡erences in the numbers of
NGFI-A positive nuclei were detected in EE animals, as
compared to HO and UD controls. No signi¢cant di¡er-
ences were detected in comparisons across both control
groups in the primary somatosensory cortex (Table 4).
Exposure to an EE was also e¡ective in markedly
increasing the number of NGFI-A positive cells in the
hippocampal formation and dentate gyrus. EE animals
displayed a striking increase in the numbers of immuno-
positive nuclei in the CA1 sub¢eld, while a more modest,
but still high, NGFI-A up-regulation was detected in the
CA2 and CA3 sub¢elds (Fig. 5). UD animals displayed
basal NGFI-A levels in all hippocampal sub¢elds, with
no apparent asymmetry for each of these regions. Com-
pared to UD controls, HO animals displayed a very
Table 3. Distribution of NGFI-A immunopositive nuclei at
di¡erent cortical depths within the primary visual cortex
Group Supragranular Granular Infragranular
EE 207.0 P 1.5 a 98.0 P 1.7 a 157.7 P 2.0 a
HO 86.7 P 2.0 b 32.7 P 2.9 b 78.0 P 2.0 b
UD 75.7 P 1.8 b 29.7 P 2.6 b 79.3 P 2.7 b
Data are expressed as number of NGFI-A immunopositive nuclei
(mean P S.E.M.), n = 3 per group. a, b: groups with di¡erent let-
ters di¡er signi¢cantly. Two-tailed, paired t-test, two-sample for
means P 6 0.01.

NSC 5543 4-6-02

 NGFI-A expression in enriched environment 579

Fig. 5. Photomontages showing NGFI-A immunoreactive neuronal nuclei in the hippocampus and dentate gyrus of all exper-
imental groups. A marked increase in the number of NGFI-A positive nuclei was identi¢ed in the CA1, CA2 and, to a lesser
extent, in the CA3 sector of the hippocampus of EE animals (A). HO animals also displayed a modest increase in the num-
ber of NGFI-A immunolabeled cells in all sub¢elds of the hippocampal formation (B), as compared to UD controls, where
basal NGFI-A expression was detected (C). Increased NGFI-A protein levels were also detected in the dentate gyrus of EE
animals (A). Similarly to the data collected from the hippocampus, the dentate gyrus of HO animals displayed a small
increase in the number of NGFI-A positive cells (B), while UD controls displayed basal NGFI-A levels in this region. Scale
bar = 500 Wm.

modest increase in the number of NGFI-A positive
nuclei in CA1 sub¢eld, while no obvious di¡erence was
detected for the CA2 and CA3 regions (Fig. 5). A
marked increase in the number of NGFI-A immunola-
beled nuclei was also found in the dentate gyrus of EE
animals. While basal levels were identi¢ed in the dentate
gyrus of UD controls, HO animals displayed a modest,
but detectable increase in the number of NGFI-A pos-
itive nuclei.
DISCUSSION
Up-regulation of the candidate-plasticity gene NGFI-
A, in the EE setting was initially demonstrated by Wallace
et al. (1995) at the level of mRNA. In the present work,
this original ¢nding was con¢rmed and the line of inves-
tigation extended to include cortical layering for NGFI-A
mRNA and protein following EE exposure.
A high a⁄nity consensus sequence for NGFI-A has
been identi¢ed in the promoter region of a number of
genes that are expressed in the CNS. Therefore, this IEG
could potentially regulate the expression of several genes
in response to sensory stimulation. Two well-character-
ized examples of genes that are regulated by NGFI-A are
synapsins I and II (Thiel et al., 1994; Petersohn et al.,
1995). The synapsin family of phosphoproteins play
important roles in synaptic function as docking proteins

NSC 5543 4-6-02

580 R. Pinaud et al.
Table 4. Distribution of NGFI-A immunopositive nuclei at
di¡erent cortical depths within the primary somatosensory cortex
Group Supragranular Granular Infragranular
EE 146.7 P 0.9 a 58.0 P 3.2 a 131.0 P 4.2 a
HO 51.3 P 1.8 b 45.0 P 1.2 b 73.7 P 2.4 b
UD 53.7 P 1.7 b 33.3 P 1.9 b 62.3 P 3.8 b
Data are expressed as number of NGFI-A immunopositive nuclei
(mean P S.E.M.), n = 3 per group. a, b: groups with di¡erent let-
ters di¡er signi¢cantly. Two-tailed, paired t-test, two-sample for
means P 6 0.01.
that mediate the release of neurotransmitter-containing
synaptic vesicles (Greengard et al., 1993). For these rea-
sons, it may not be surprising that increased levels of
NGFI-A mRNA were observed in all regions of neocor-
tex of EE animals, since animals belonging to this group
would have experienced heightened activation in all sen-
sory systems, as a result of the increased complexity of
stimuli available in the EE setting.
NGFI-A expression has often been used as a marker
for neuronal activation in sensory pathways (for reviews
see Kaczmarek and Chaudhuri, 1997; Herdegen and
Leah, 1998). However, neural activity does not always
predict the induction of NGFI-A (Mello and Clayton,
1994). In the present work, HO animals were exposed,
on a daily basis, to sensory stimulation that accompanied
experimental manipulation, yet expressed considerably
less NGFI-A mRNA and protein, as compared to EE
animals.
It has been established that exposure to an EE induces
CNS plasticity (Rosenzweig et al., 1972; Volkmar and
Greenough, 1972; Wallace et al., 1995). As a result of
the present work, we postulate that the elevated levels of
NGFI-A expression, characteristic of EE animals in this
paradigm, re£ect an early component of the genetic
response that may mediate neural plasticity.
In the brains of animals exposed to the EE, the spatial
distributions of both NGFI-A mRNA and protein
expression were nearly identical. This ¢nding suggests,
at least in this experimental paradigm, that NGFI-A
expression is una¡ected by post-transcriptional modi¢ca-
tions, as demonstrated in other paradigms and species
such as the songbird song system (Whitney et al., 2000).
In the present study we have shown that enhanced
environmental complexity increases the number of
NGFI-A expressing neurons in both the hippocampus
and dentate gyrus. One intriguing possibility is that neu-
rogenesis, driven by exposure to an EE, may account for
the increase in the overall number of NGFI-A positive
cells. For example, Kempermann et al. (1997) demon-
strated an enlargement of the hippocampal granule cell
layer in mice exposed to an EE. In addition, these
authors found a 15% increase in the number of granule
cells in the dentate gyrus of EE animals. In our present
study, we observed an increase of 200^300% in the num-
ber of NGFI-A expressing cells in cortical structures.
While neurogenesis after exposure to the EE may
account for some increase in the number of NGFI-A
expressing cells, it is unlikely to account for the two-
to three-fold increase observed here.
NSC 5543 4-6-02
The altered neurochemistry that corresponds to di¡er-
ent behavioral states, such as alertness, exploration or
novelty detection, may also be important considerations
in understanding how candidate-plasticity gene expres-
sion may be evoked (Wallace et al., 1995; Mello and
Clayton, 1994; Staiger et al., 2000). To some extent,
these behavioral states have been correlated with ele-
vated cortical levels of neuromodulators such as acetyl-
choline and noradrenaline (Inglis et al., 1994; Inglis and
Fibiger, 1995; Foote et al., 1983). In the visual and audi-
tory systems, it has already been demonstrated that nor-
adrenaline plays a critical role in NGFI-A induction
(Pinaud et al., 2000; Ribeiro and Mello, 2000). Together
these ¢ndings may be useful in implicating neuromodu-
lators as potential mediators for the expression of candi-
date plasticity genes in the CNS.
Exercise is an obvious variable that may be mediating
or causing the changes in the CNS produced following
exposure to the EE. Increased motor activity and its
potential physiological e¡ects on the organism, including
changes in blood perfusion rate and blood pressure,
changes in circulating hormone levels, particularly as
this a¡ects the mobilization of glucose from storage,
may all in£uence the performance of neural tissue and
augment on-going physiological processes such as those
that underlie learning or memory (Delp et al., 2001;
Dishman et al., 2000; Ide and Secher, 2000). The cate-
cholamine noradrenaline, for example, has an established
role in NGFI-A expression (Cirelli et al., 1996; Pinaud et
al., 2000). In the present study, to control for locomotor
activity experienced by EE animals and sleep-related
gene expression, two activities that a¡ect IEG expression
in the cerebral cortex and hippocampus, rats in the HO
and UD groups with low activity levels were excluded
from histological analysis. Rats included in the analyses
had approximately similar activity levels. While more
detailed investigations of exercise-induced changes on
NGFI-A expression are clearly warranted, in our study
normal basal activity does not seem to be associated with
increased expression of NGFI-A.
In the present study, NGFI-A expression was exam-
ined at the end of 3 weeks of exposure to EE for 1 h/day.
Whether NGFI-A expression results from daily exposure
to EE or remains up-regulated throughout the 3-week
exposure to EE has not been examined here. However,
two considerations suggest a non-sustained expression of
NGFI-A in response to the EE setting. First, NGFI-A is
an IEG and its mRNA has a short half-life of about 30
min (Chaudhary et al., 1996). This fast degradation time
would be a major contributor for a down-regulation of
NGFI-A after cell stimulation and would not support
the idea that NGFI-A expression is sustained after EE
exposure. Second, NGFI-A expression is down-regulated
4^6 h after stimulus onset, regardless of whether the
stimulus is continuously presented (Mello and Clayton,
1994; Mello and Ribeiro, 1998). This suggests that
sustained NGFI-A expression for periods longer than
6 h is unlikely. The same rationale would apply to the
current study where the interval between EE exposures
was 24 h. Therefore, we believe that NGFI-A expression
reported in the present work occurs following the expo-
 NGFI-A expression in enriched environment 581

sure to the EE setting and is not sustained over the 24-h
day.
The role of NGFI-A in mediating neural plasticity is
further indicated by its dependence on N-methyl-D-aspar-
tate (NMDA) receptor activity (Cole et al., 1989). Both
neural plasticity, in the form of long-term potentiation
(LTP), and NGFI-A expression are coupled to activity of
this type of glutamatergic receptor. Antagonists to this
receptor block both NGFI-A induction and LTP forma-
tion (Wisden et al., 1990). Furthermore, NGFI-A is
highly induced in response to the trains of stimulation
that invoke LTP formation (Wisden et al., 1990). The
intimate relationship between NMDA receptor activity,
LTP formation and NGFI-A expression further strength-
ens the argument that these processes all re£ect some
aspect of neural plasticity (Wisden et al., 1990).
Higher levels of NGFI-A expression observed for the
EE animals may re£ect enhanced rates of sensory pro-
cessing due to both increased levels of arousal and an
increased diversity and complexity of the sensory envi-
ronment. Based on our ¢ndings, it would appear that EE
animals might have experienced biologically meaningful
visual events that did not occur for animals that
remained in their home cages. The increased up-regula-
tion of NGFI-A in the visual cortex of EE animals could
be driven by the higher number of visual events, such as
increased number of borders, frequency of stimuli,
increased depth of the environment, variety of colors,
and other psychophysical aspects of the EE setting, to
which these animals were exposed. Speci¢cally, animals
exposed to the EE might have used their visual system to
collect the visual information required for constructing a
new spatial map of the accessible territory.
While a similar argument could be made for the diver-
sity of tactile experience in both EE and HO animals,
only EE animals displayed a pronounced NGFI-A up-
regulation. One hypothesis that has been put forward by
Wallace et al. (1995) is that NGFI-A expression is driven
by exploratory behavior, which would be characteristic
of rats in EE environments, but not in HO and UD
conditions.
NGFI-A expression was evident in the hippocampus
of EE animals, as compared to HO and UD controls.
One interpretation for this ¢nding is that exposure to an
EE results in new spatial map construction that, with
repeated exposure of the animals to the EE condition,
became consolidated as spatial memory. The hippocam-
pus has been strongly implicated as a site of storage for
spatial maps, however, behavioral experiments would be
required in order to explore this hypothesis.
Finally, the relative density of NGFI-A mRNA,
detected by in situ hybridization, and NGFI-A immuno-
positive nuclei varied for di¡erent cortical layers. The
consistent ¢nding was that the greatest amounts of label-
ing were detected in cortical layers III and V. There is
some experimental evidence that layers II/III and V may
normally express a higher degree of neural plasticity than
is found for the other cortical layers, and that synaptic
potentiation is more reliably produced at this cortical
depth (Castro-Alamancos et al., 1995; Glazewski and
Fox, 1996; Petersen and Sakmann, 2001). In both visual
and somatosensory cortices, these layers also display
higher densities of GABAergic interneurons (Jones,
2000). Inhibitory interneurons have been postulated to
mediate some forms of neural plasticity, particularly
within CNS reorganization of sensory pathways in
adult mammals (Jones, 2000; Tremere et al., 2001a,b).
The present data further support a role for NGFI-A in
mediating EE-induced CNS plasticity in the adult mam-
mal. There is a strong spatial correlation between the
expression pro¢le for this IEG and cortical regions,
more speci¢cally cortical layers, that have characteristi-
cally undergone experimentally induced changes in
responsiveness postulated to re£ect neural plasticity.
Acknowledgements#Work supported by the Canadian Institutes
of Health Research, the Canadian Stroke Network, the Heart
and Stroke Foundation of New Brunswick and a Killam Schol-
arship to R.P. The authors would like to thank Ms. Kay Mur-
phy and Ms. Brenda Ross for excellent technical support.
Raphael Pinaud dedicates this paper to the memory of his men-
tor and friend, Professor Gustavo de Oliveira Castro.

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(Accepted 22 February 2002)

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