Molecular Brain Research 98 (2002) 93–101

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Research report

Plasticity-driven gene expression in the rat retina

Raphael Pinaud a , Liisa A. Tremere b , Marsha R. Penner c , Felipe F. Hess a,d , Steven Barnes b ,
Harold A. Robertson c , R. William Currie a , *
a
Department of Anatomy and Neurobiology, Laboratory of Molecular Neurobiology, Dalhousie University, Halifax, Nova Scotia B3 H 4 H7, Canada
b
Department of Physiology and Biophysics, Dalhousie University, Halifax, Nova Scotia B3 H 4 H7, Canada
c
Department of Pharmacology, Laboratory of Molecular Neurobiology, Dalhousie University, Halifax, Nova Scotia B3 H 4 H7, Canada
d
´
Laboratorio ´
de Neurobiologia II, Instituto de Biofısica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil

Accepted 26 November 2001

Abstract

Animals exposed to an enriched environment display features of neural plasticity such as an increased brain volume, enhanced number
of dendritic spines, as well as enlarged synapses. Here we report the first description of molecular plasticity in the mammalian retina, as
revealed by gene expression. A marked upregulation of both NGFI-A and Arc, two candidate-plasticity genes, was observed in adult rats
that had been exposed to an enriched environment for 3 weeks. This increase was paralleled by an increase in the expression of the late
genes GAP-43 and Synapsin I, which also indicated changes in retinal connectivity. Our results suggest that both NGFI-A and Arc may
regulate mechanisms of plasticity that had been invoked by heightened complexity of the visual environment.  2002 Elsevier Science
B.V. All rights reserved.

Theme: Neural basis of behavior

Topic: Neural plasticity

Keywords: Plasticity; Retina; NGFI-A; Arc; Transcription factor; Enriched environment

1. Introduction enhanced 18% visual acuity when compared to restrained

Altering the parameters of sensory experience can be
used to produce diverse morphological changes in the
central nervous system [12,20]. For example, exposing rats
to an enriched environment is an effective procedure for
inducing plasticity in the cerebral cortex, a phenomenon
that is paralleled by an increase in the number of neuronal
arborizations and dendritic spines, as well as an increase in
the size of neurons and their nuclei [10,20,24]. These
changes are postulated as both neuroprotective and as the
physical underpinnings of learning and memory [3,27,28].
Furthermore, Young and colleagues have demonstrated that
exposure to an enriched environment prevents seizures and
spontaneous apoptosis in adult rats [28]. More recently,
Prusky and co-workers have demonstrated that mice
exposed to an enriched environment from birth have an
*Corresponding author. Tel.: 11-902-494-3343; fax: 11-902-494-
1212.
E-mail address: wcurrie@is.dal.ca (R.W. Currie).
controls, suggesting that the performance of the visual
system is greatly influenced by the complexity of the
visual environment [19]. Even though a great deal of
evidence has buttressed the association between enriched
environmental complexity and morphological and func-
tional changes in the central nervous system, the molecular
mechanisms underlying such modifications remain to be
characterized.
Studying the expression of immediate early genes
(IEGs) has provided new insights into the molecular
mechanisms of plasticity. Furthermore, the proteins en-
coded by this class of genes can act as transcription
factors, regulating the expression of late genes and their
protein products, which are postulated to mediate more
stable changes in reconnectivity. Unlike late response
genes, IEGs are expressed rapidly and transiently, and do
not require de novo protein synthesis for their function [5].
One of the most interesting features of IEGs is their
responsiveness to neuronal depolarization, which has al-
lowed several research groups to utilize their expression as

0169-328X / 02 / $ – see front matter  2002 Elsevier Science B.V. All rights reserved.
PII: S0169-328X( 01 )00328-X
94 R. Pinaud et al. / Molecular Brain Research 98 (2002) 93 – 101
Fig. 1. Schematic outline of experimental paradigm. EE animals were housed with toys and exploratory objects, and placed daily, for 60 min, into an
enriched environment setting. HO animals were manipulated by an experimenter for 5 min each day during the same experimental period, while UD
animals remained in home cages. These procedures were repeated daily for 3 weeks, between 15:00 and 16:00 h.
markers for relative rates of neuronal activation [11,13]. In
the present study, we have investigated the expression of
two IEGs; nerve growth factor induced gene-A (NGFI-A;
also known as zif268, egr-1, krox-24, ZENK) and the
activity-regulated cytoskeletal protein (Arc) in response to
3 weeks of exposure to an enriched environment. NGFI-A
is a zinc-finger transcription factor that has high affinity for
a specific DNA motif found in the promoter site of several
genes, including the gene encoding the vesicular phos-
phoprotein Synapsin I [23]. Arc is an IEG and growth
factor localized to neuronal dendrites. Furthermore, Arc
accumulation is correlated with higher rates of synaptic
activity in the dendrites of hippocampal cells [22]. In the
present study, we generated immunocytochemical profiles
for both candidate-plasticity genes in the retinas of adult
rats exposed daily, for 3 weeks, to 1 h of an enriched
environment. Data from enriched environment animals
(EE) was compared with undisturbed controls (UD), that
remained in their home cages throughout the 3 weeks of
the present experiment. To account for stress and expec-
tation-related non-specific gene expression, EE animals
were also compared to a second control group of animals
that were handled for 5 min during each day of the
experiment (Fig. 1). Our results constitute the first demon-
stration of genetic mechanisms associated with plasticity in
the mammalian retina.
2. Materials and methods
2.1. Animals
A total of 18 young adult male Sprague–Dawley rats
weighting between 200 and 225 g were used in the present
study. In order to minimize the confounding influences of
developmental plasticity, the present experiment was con-
ducted in young adult animals [4]. All procedures con-
formed with institutional regulations and the Guide to the
Care and Use of Experimental Animals from the Canadian
Council on Animal Care.
2.2. Experimental groups and enriched environment
setting
Animals were divided into three experimental groups:
enriched environment (EE; n56); handling only (HO;
n56) and undisturbed (UD; n56). For a period of 3
weeks, animals belonging to the EE group were exposed to
a ‘play area’ of 3 m 3 1 m 3 0.5 m, that contained a large
variety of toys, tubes, houses and food rewards. Animals
were placed in this maze every afternoon for 1 h, and
subsequently returned to their home cages, which also
contained toys. In the same period, animals in the HO
group were briefly lifted and held for a session lasting
approximately 5 min [18,25]. Undisturbed controls were
left in their home cages for the duration of the experiment,
until sacrificed (Fig. 1). Behavior of all three groups was
monitored daily, from outside the experimental rooms,
during the entire 60 min of the experiment.
2.3. Perfusion and histological procedures
Animals belonging to all groups were deeply anes-
thetized with an overdose of Somnotol (70 mg / kg, i.p.).
Once pain reflexes were suppressed, all animals were
transcardially perfused with 80 ml of 0.1 M phosphate
buffered saline (PBS, pH 7.4), followed by 250 ml of a
cold 4% paraformaldehyde solution (PFA) in 0.1 M PBS.
To avoid potential circadian effects on gene expression, all
animals were perfused between 15:00 and 16:00 h. The
anterior portion of the eye, including the lens, was
removed and this eye-cup preparation was post-fixed for an
additional 15 min, by immersion in 4% PFA solution.
Tissue was then rinsed in PBS and cryoprotected by
 R. Pinaud et al. / Molecular Brain Research 98 (2002) 93 – 101
equilibration in a 30% sucrose solution. Retinal tissue was
fast-frozen in a dry-ice / ethanol bath and cut vertically at
20 mm on a cryostat. Sections were thaw-mounted directly
onto Fisherbrand Superfrost Plus slides (Fisher Scientific,
USA), air-dried overnight, then stored at 270 8C.
2.4. Immunocytochemistry
We used anti-rabbit polyclonal antibodies, raised against
the carboxy-terminus of the rat Arc and NGFI-A proteins
(both from Santa Cruz Biotechnology, Santa Cruz, CA,
USA). GAP-43 and Synapsin I monoclonal antibodies
were used (both from Chemicon Inc., Canada). Slides were
removed from the 270 8C freezer and hydrated for 30 min
in PBS. Tissue was subsequently incubated in a blocking
buffer solution, containing 0.5% albumin and 0.3% Triton
X-100 in 0.1 M PBS, for 2 h, at room temperature (RT),
followed by a 30-min wash in PBS (three washes, 10 min
each). Sections were incubated in a solution containing the
Arc or NGFI-A primary antibodies, at a dilution of 1:1000
in blocking buffer, inside a humid chamber at 4 8C, where
they remained overnight. Synapsin I and GAP-43 anti-
bodies were used at a dilution of 1:800. The following day,
slides were washed for 30 min in PBS, in order to remove
excess, unbound antibody. Slides were then incubated in a
goat-anti-rabbit biotinylated antibody (IgG; Vector Lab-
oratories, Burlingame, CA, USA), at the dilution of 1:200
in blocking buffer, for 2 h at RT. Tissue was washed for
30 min in PBS, a step that was followed by a 2-h
incubation in avidin–biotin-complex, at RT (ABC; Vector
Laboratories), at the dilution recommended by the manu-
facturer. This step was followed by another 30-min wash
in PBS. We developed our sections by incubating them in a
filtered solution containing 0.03% diaminobenzidine
(DAB), 0.15% nickel-ammonium sulfate in 0.1 M PBS to
which 0.001% hydrogen peroxide (H 2 O 2 ) had been added.
Sections were then dehydrated in a standard series of
alcohols, exposed to xylenes and coverslipped with En-
tellan (BDH, Darmstadt, Germany). Cresyl-violet staining
was used in adjacent sections in order to assess cyto-
architectonic features. Controls for the immunocytoch-
emistry procedures were carried out by omitting the
primary antibodies, which resulted in no immunolabeling.
To avoid variations in concentrations and exposure to other
processing solutions, data presented in this paper were
photographed and quantified from tissue reacted as a single
batch.
2.5. Imaging
Images were captured by use of a Spot camera (Diag-
nostics Instruments Co., USA) coupled to an Axioplan
microscope, and digitized into a Power PC Macintosh
computer. Using the ‘levels’ command, grayscales of
photomicrographs were matched and plates were assem-
95
bled by using Adobe Photoshop software (Pantone Inc.,
USA).
2.6. Cell counts and optical density analysis
Data were collected from five animals in each ex-
perimental group. Two representative sections were select-
ed from each animal at approximately the same level of the
retina. NGFI-A positive nuclei were counted as profiles.
These counts were performed manually and conducted
twice by two independent observers. As NGFI-A-positive
nuclei were distributed evenly throughout the length of the
retina (data not shown), nuclei were counted from the GCL
and INL for the full extent of the section. The imaging of
Arc, GAP-43 and Synapsin I immunolabeled sections was
performed at a constant level of illumination and followed
a similar protocol to that of Mathern and colleagues [15].
Optical density was calculated using a constant threshold
level, with NIH Image software. Our calculations were
based on an area of 10,000 mm 2 (100 mm3100 mm) for
the labeling in the IPL and an area of 600 mm 2 (10
mm360 mm) in the OPL. Our results were quantified as
pixels / area.
2.7. Data analysis
Data were analyzed using one-way analysis of variance
(ANOVA). Significant treatment effects were followed by
post-hoc analysis, using Tukey tests (P,0.05).
3. Results
Animals exposed to the enriched environment displayed
a marked increase in the number of NGFI-A-expressing
neurons in the ganglion cell layer (GCL) (Fig. 2A). In
contrast, ‘handling-only’ animals (HO) and UD controls
displayed a few scattered NGFI-A immunoreactive cell
nuclei in the GCL (Fig. 2C and E). Likewise, large
numbers of NGFI-A-positive nuclei were observed in the
inner nuclear layer (INL) of the retina, in response to
environmental enrichment (Fig. 2A), while INL labeling
was at low levels in either control group (Fig. 2C and E).
In order to quantify the differences across all experimental
groups, cell counts were performed in both GCL and INL,
retinal layers where significant differences in NGFI-A
expression were observed (Fig. 3A and B). A statistical
comparison between NGFI-A-expressing cells in EE ani-
mals versus UD controls revealed a pronounced seven- and
13-fold increase in the GCL and INL, respectively. EE
animals also differ significantly in the number of NGFI-A-
expressing cells when compared to HO animals. In the
GCL, there was an eight-fold increase with a seven-fold
increase observed for the INL (Fig. 3A and B). No
96 R. Pinaud et al. / Molecular Brain Research 98 (2002) 93 – 101

Fig. 2. Expression of NGFI-A and Arc is markedly increased in EE animals, but not in HO and UD controls. NGFI-A immunoreactivity was increased in
EE animals (A), but not in HO or UD controls (C and E, respectively). Likewise, EE animals displayed an increase in immunoreactivity for the growth
factor Arc in both plexiform layers (B). In contrast, HO and UD animals did not display immunoreactivity (D and F, respectively). NGFI-A
immunopositive nuclei were distributed evenly throughout the INL and GCL of the retina of EE animals. Increased Arc labeling was restricted to the
dendrites of the IPL and OPL, and was evenly distributed across these layers. GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer;
IPL, inner plexiform layer; OPL, outer plexiform layer. Scale bar550 mm.

statistical significance was detected when NGFI-A-im-
munoreactivity in HO animals was compared with UD
controls for both GCL and INL.
It has been previously demonstrated that the protein
encoded by the immediate early gene arc is strongly
expressed in dendritic regions that have recently ex-
perienced relatively heightened rates of synaptic activity
[22]. Retinas taken from animals exposed to the enriched
environment displayed a remarkable increase in dendritic
labeling, most notably within the IPL and OPL (Fig. 2B),
when compared to controls, where basal levels of Arc
immunoreactivity are normally low for the inner and outer
plexiform layers (Fig. 2D and F).
We quantified the relative induction of Arc for all
experimental groups using optical density, according to a
protocol similar to the one described by Mathern and
co-workers [15]. Our study revealed an 844-fold and 122-
fold increase in Arc labeling for the IPL and OPL,
respectively, in animals that were exposed to the enriched
environment as compared to HO animals (Fig. 3C and D).
Similarly, when EE animals were compared with UD
controls, this difference represented approximately 1160-
fold for the IPL and 115-fold for the OPL (Fig. 3C and D).
In order to confirm that sites of NGFI-A and Arc
expression reliably reflected enhanced plasticity, we used
immunocytochemistry directed against two late genes that
have been implicated in central nervous system reorganiza-
tion: GAP-43 and Synapsin I. In the retinas of our EE
animals, GAP-43 expression was detected in the OPL and
IPL; the former layer displayed numerous stained beaded
dendrites (Fig. 4A). In contrast, both control groups had
basal levels of GAP-43 immunoreactivity in the IPL but
were devoid of immunostaining in the OPL (Fig. 4C and
E).
Synapsin I immunoreactivity was notably increased in
both the IPL and OPL of EE animals (Fig. 4B). In both
control groups, Synapsin I labeling was detectable at basal
levels in the IPL and was undetectable in the OPL (Fig. 4D
and F).
We also quantified the differences between our ex-
perimental groups for both GAP-43 and Synapsin I
expression. In the EE animals, GAP-43-immunoreactivity
 R. Pinaud et al. / Molecular Brain Research 98 (2002) 93 – 101 97

Fig. 3. Cell counts confirmed a massive upregulation of NGFI-A over either control group. Analysis of variance (ANOVA) was used to determine that
differences between EE and control groups were statistically significant, whereas differences between the two control groups (HO and UD) were not.
Comparisons were made for the GCL (A) and INL (B) for NGFI-A expression. Likewise, EE animals displayed a marked increase in Arc immunolabeling
in the IPL (C) and OPL (D) when compared to both control groups. Similarly, these differences were statistically significant (ANOVA, P,0.05) for EE
comparisons with controls, but not for comparisons between the control groups. Data are expressed as mean6S.D.

increased 155-fold and 156-fold in the OPL compared with
HO and UD controls (Fig. 5B). In the IPL, a more modest,
but significant increase in GAP-43 expression was detected
in EE animals; 0.26- and 0.45-fold compared to HO and
UD controls, respectively (Fig. 5A).
In the EE animals, Synapsin I expression in the OPL
was significantly enhanced 486-fold over HO animals and
474-fold over UD controls, respectively (Fig. 5D). Similar-
ly, Synapsin I labeling was significantly enhanced 0.33-
and 0.47-fold in the IPL of animals exposed to the EE
when compared to HO and UD controls, respectively (Fig.
5C). Although all differences were statistically significant
for the comparisons between EE and either of our control
groups, no statistical significance was detected between
HO and UD animals.
Our results indicate that NGFI-A and Arc may partici-
98 R. Pinaud et al. / Molecular Brain Research 98 (2002) 93 – 101

Fig. 4. Differential expression patterns of GAP-43 and Synapsin I in EE animals and HO and UD control groups. Following exposure to the enriched
environment, GAP-43 labeling of beaded dendrites could be detected in the OPL (A), but was undetectable in HO and UD controls (C and E). In addition,
a marked increase was detected for Synapsin I labeling in the OPL of EE animals (B). In control animals, the OPL was unlabeled for GAP-43 (C and E).
Similarly, Synapsin I immunolabeling was not detected in the OPL of animals belonging to either control group (D and F). Strong bands of GAP-43
immunoreactive puncta were visible in the IPL of all experimental groups, with significantly higher intensity in EE animals (A, C and E). Synapsin I
immunolabeling in the IPL was also prominent in all animal groups, with higher density in EE animals (B) when compared to HO and UD controls (D and
F, respectively). OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer. Scale bar550 mm.

pate in signaling pathways that contribute to long-term 4. Discussion

modifications in cellular morphology, as revealed by GAP-
43 and Synapsin I late gene products, in response to Our results provide the first evidence that the genes
heightened complexity of visual experience. NGFI-A and Arc are regulated in the adult mammalian
 R. Pinaud et al. / Molecular Brain Research 98 (2002) 93 – 101 99

Fig. 5. Exposure to the enriched environment also increased the levels of plasticity markers GAP-43 and Synapsin I. Based on optical density measures,
the effect of exposure to an enriched environment was quantified for both plasticity markers. GAP-43 immunoreactivity is found at high levels in EE
animals when compared to HO and UD controls in both the IPL (A) and OPL (B). Likewise, EE animals displayed a marked increase in Synapsin I
immunolabeling in the IPL (C) and OPL (D) when compared to both control groups. Analysis of variance (ANOVA) revealed that the effects were
statistically significant when comparing EE and either control group. The differences between control groups failed to meet the criteria for statistical
significance (P,0.05). Data are expressed as mean6S.D.

retina by non-invasive, and biologically meaningful, stimu-
lation of the sensory pathway.
The gene products that result from the expression of
NGFI-A have been used as molecular markers for neuronal
activation in the visual system, as well as other sensory
systems [11,13]. Even though the expression of NGFI-A is
highly sensitive to neuronal depolarization, caution is
warranted as electrophysiological activity and NGFI-A
expression are not paralleled [6,16]. Therefore, NGFI-A
expression is not strictly a marker for neural activity. We
believe that NGFI-A expression may more accurately
reflect signaling linked to heightened molecular plasticity
for networks under restructural pressure. This hypothesis is
consistent with previous demonstrations that NGFI-A
expression is dependent on the activation of the NMDA
type of glutamatergic receptors, which have been repeated-
100 R. Pinaud et al. / Molecular Brain Research 98 (2002) 93 – 101
ly implicated in the induction of long-term changes in
synaptic efficacy [6]. In addition, experimental protocols
that are linked to plasticity in the visual system, such as
dark adaptation followed by light stimulation are extremely
effective in inducing NGFI-A in the visual cortex
[11,13,14].
Furthermore, the discovery of polyribosomal complexes
located near activated post-synaptic regions in the de-
ndrites of neuronal cells [21], and an increase in the
aggregation of polyribosomes within synaptoneurosomes
treated with metabotropic glutamatergic agonists suggest
that translational events happen locally at the dendritic
level and that these changes may be activity-dependent
[26]. One implication of the present research is that
candidate-plasticity genes such as NGFI-A and Arc, iden-
tify both the induction of neural plasticity and suggest
locations where the system is experiencing restructural
pressure.
Exposure of animals to an enriched environment is
strongly associated with upregulation of candidate-plastici-
ty genes in the cerebral cortex [25]. In the present study,
while EE animals had toys in their cages and the HO and
UD animals did not, the enriched environment is visually
complex in comparison with typical laboratory home
cages. We postulate that it is the number and diversity of
stimuli in the visual environment that has similarly in-
voked mechanisms of plasticity within the retina. We
interpret the present findings (i.e. NGFI-A and Arc expres-
sion) as indicators of plasticity, rather than an isolated
effect of altering any single psychophysical aspect within
the enriched visual environment. Some possible factors,
that may drive the expression of these two gene products
would include illumination, contrast, spatial frequency and
the widened area of the accessible environment, that may
increase the range of depth perception [9].
In our study, levels of illumination could not account for
differences across experimental groups as this parameter
was held constant for all conditions. However, the in-
creased number of objects in the visual field may have
increased the occurrence of edges or other high contrast
visual features, which would increase the frequency of
spatial processing. Fischer and colleagues have demon-
strated an upregulation of the chick homolog of NGFI-A as
a result of increasing the range of depths, over which the
organism could focus on an object [9]. This finding may be
especially relevant to the present work as the placement of
rats in the enriched condition provided them with a much
larger visual environment, over which they would navigate
and interact with objects in their visual world. The
individual influence of these parameters on retinal process-
ing in the retina is currently unknown for both NGFI-A
and Arc expression.
Although the focus of the present study was to char-
acterize the involvement of IEGs during enriched en-
vironment-induced restructural pressure in the retina, we
extended the present work to include the late genes
synapsin I and GAP-43. Changes in the levels of these
proteins has been strongly correlated with functional
reorganization of sensory pathways in response to deaf-
ferentation [1,2,7]. Synapsin I is regulated by the IEG
NGFI-A [23]. NGFI-A and GAP-43 are related in that the
expression of both genes results from stimulation by NGF
in PC-12 cells [8,17]. Therefore, due to the increase in
NGFI-A expression, we anticipated an upregulation in
protein levels for both Synapsin I and GAP-43 in the
enriched environment paradigm. In the present study, the
greatest difference in the expression of either protein was
identified in the OPL. According to our present under-
standing of retinal anatomy, our EE paradigm appeared to
have the most impact on the circuitry of photoreceptors,
bipolar cell neurites and / or horizontal cell processes.
The robust increase in the expression of both IEGs
provide the basis for in depth studies on molecular
plasticity in the earliest stages of central sensory infor-
mation processing. To our knowledge, the present paper
provides the first evidence of retinal plasticity in an adult
mammal, induced by heightened complexity of the sensory
information processing, as revealed by molecular markers.
Future research will be directed at describing the contribu-
tions of candidate-plasticity genes to functional restructur-
ing of the retina, which would be expected to accompany
heightened complexity of the visual environment.
Acknowledgements
We would like to thank Kay Murphy and Brenda Ross
for technical assistance and Dr. William Baldridge for
useful discussions. This work was supported by the Heart
and Stroke Foundation of New Brunswick (grant to
R.W.C), Canadian Stroke Network (grants to R.W.C and
H.A.R.), Canadian Institutes of Health Research (grants to
H.A.R. and S.B.) and an Izaak Walton Killam Memorial
Scholarship (to R.P.). Raphael Pinaud would like to
dedicate this paper to the friend, mentor and NGFI-A
disciple, Professor Claudio V. Mello.
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