Neuropharmacology 62 (2012) 2388e2397

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Neuropharmacology
journal homepage: www.elsevier.com/locate/neuropharm

Enriched experience and recovery from amblyopia in adult rats: Impact

of motor, social and sensory components
Laura Baroncelli a,1, Joyce Bonaccorsi b,1, Marco Milanese c,1, Tiziana Bonifacino c, Francesco Giribaldi c,
Ilaria Manno b, Maria Cristina Cenni a, Nicoletta Berardi a, d, Giambattista Bonanno c, Lamberto Maffei a, b,
Alessandro Sale a, *
a

Institute of Neuroscience CNR, Pisa I-56100, Italy

Laboratory of Neurobiology, Scuola Normale Superiore, Pisa I-56100, Italy
Department of Experimental Medicine, Section of Pharmacology and Toxicology, University of Genova, Genova I-161100, Italy
d
Department of Psychology, Florence University, Florence I-50100, Italy
b
c

a r t i c l e i n f o

a b s t r a c t

Article history:
Received 17 November 2011
Received in revised form
23 January 2012
Accepted 12 February 2012

Amblyopia is one of the most common forms of visual impairment, arising from an early functional
imbalance between the two eyes. It is currently accepted that, due to a lack of neural plasticity,
amblyopia is an untreatable pathology in adults. Environmental enrichment (EE) emerged as a strategy
highly effective in restoring plasticity in adult animals, eliciting recovery from amblyopia through
a reduction of intracortical inhibition. It is unknown whether single EE components are able to promote
plasticity in the adult brain, crucial information for designing new protocols of environmental stimulation suitable for amblyopic human subjects. Here, we assessed the effects of enhanced physical exercise, increased social interaction, visual enrichment or perceptual learning on visual function recovery in
adult amblyopic rats. We report a complete rescue of both visual acuity and ocular dominance in
exercised rats, in animals exposed to visual enrichment and in animals engaged in perceptual learning.
These effects were accompanied by a reduced inhibition/excitation balance in the visual cortex. In
contrast, we did not detect any sign of recovery in socially enriched rats or in animals practicing a purely
associative visual task. These ndings could have a bearing in orienting clinical research in the eld of
amblyopia therapy.
2012 Elsevier Ltd. All rights reserved.

Keywords:
Amblyopia
Environmental stimulation
GABAergic inhibition
Perceptual learning
Plasticity

1. Introduction
Amblyopia is the most common impairment of visual function
affecting one eye in adults, with a prevalence of about 1e5% of the
total world population (Holmes and Clarke, 2002). This pathology is
caused by early abnormal visual experience with a functional
imbalance between the two eyes owing to anisometropia, strabismus or congenital cataract, resulting in a dramatic loss of visual
acuity in an apparently healthy eye and a broad range of other
perceptual abnormalities, including decits in contrast sensitivity
and in stereopsis (Lewis and Maurer, 2005; Levi, 2006). In animal
models, amblyopia can be articially caused by imposing a longterm reduction of inputs from one eye by lid suture (monocular

* Corresponding author. Institute of Neuroscience National Research Council (CNR)

via Moruzzi 1, Pisa I-56124, Italy. Tel.: 390 503 153196; fax: 390 503 153220.
E-mail address: sale@in.cnr.it (A. Sale).
1
These authors equally contributed to this work.
0028-3908/$ e see front matter 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.neuropharm.2012.02.010

deprivation, MD) (Smith, 1981; Harwerth et al., 1983; Prusky et al.,

2000a,b), or by inducing experimental anisometropia or strabismus
(Singer et al., 1980; Mitchell et al., 1984; Kiorpes et al., 1998). The
classic hallmarks of amblyopia in animal models are a permanent
loss of visual acuity (VA) in the affected eye and a pronounced
ocular dominance (OD) shift of visual cortical neurons in favor of
the normal eye (Singer et al., 1980; Timney, 1983; Mitchell et al.,
1984; Kiorpes et al., 1998; Maurer et al., 1999; Prusky et al., 2000a).
It is currently accepted that, due to a lack of sufcient residual
plasticity within the brain, amblyopia is untreatable in adulthood.
However, recent experimental results obtained both in animal
models and in clinical trials have challenged this traditional view,
unmasking a previously unsuspected potential for promoting
recovery after the end of the critical period for visual cortex plasticity
(Levi and Li, 2009; Bavelier et al., 2010; Baroncelli et al., 2011). A large
body of evidence converged in indicating the inhibitory tone as
a central hub for the restoration of plasticity in the adult visual cortex
showing that a decrease of GABAergic transmission levels is required
for the rescue of neural plasticity and recovery from amblyopic

L. Baroncelli et al. / Neuropharmacology 62 (2012) 2388e2397

condition (Harauzov et al., 2010; Sale et al., 2010). In particular,

environmental enrichment (EE), a widely used paradigm whereby
the animals are given the opportunity for voluntary physical activity,
enhanced social interactions, and multi-sensory stimulation (van
Praag et al., 2000; Sale et al., 2009), turned out to be very effective
in restoring visual abilities in adult animals (Sale et al., 2007).
The possibility to reinstate plasticity in the adult visual cortex by
using a non-invasive procedure such as EE is appealing for its
potential clinical application. Recent results in humans, indeed, have
shown that experimental paradigms which could be considered akin
to EE, such as playing videogames or practicing visual perceptual
learning (PL), are able to promote recovery from amblyopia in
adulthood (Levi and Li, 2009; Li et al., 2011). However, an exhaustive
characterization of the relative contribution given by each of the EE
components to recovery from amblyopia, as well as the mechanisms
of action of EE-like protocols such as visual PL, is still lacking. This
information could be useful in order to elaborate new protocols of
environmental stimulation suitable for amblyopic human subjects.
Here, we assessed separately the efcacy of physical exercise,
increased levels of social interaction, enhanced visual stimulation,
or visual PL for their potential in promoting recovery from amblyopia in adult rats and investigated the molecular mechanisms
underlying their efcacy, focusing on the intracortical inhibition/
excitation balance.
2. Methods
2.1. Animal treatment and surgical procedures
A total of 101 Long-Evans hooded rats were used in this study, which has been
approved by the Italian Ministry of Public Health and was carried out in accordance
with the European Communities Council Directive of 24 November 1986 (86/609/
EEC). All efforts were made to minimize animal suffering, to reduce the number of
animals used, and to utilize alternatives to in vivo techniques, if available.
The animals were housed in a room with a temperature of 21  C and a 12/12 h
lightedark cycle. We rendered rats amblyopic by using the gold standard procedure
adopted for rodents, i.e. long-term monocular deprivation (MD) (Pizzorusso et al.,
2006; He et al., 2007; Maya Vetencourt et al., 2008; Silingardi et al., 2010;
Spolidoro et al., 2011). Rats were anesthetized with avertin (1 ml/hg) at P21, when
MD was performed through eyelid suturing. Animals were allowed to recover from
anesthesia and were returned to their cages. Eyelid closure was inspected daily until
complete cicatrisation. Rats showing occasional lid reopening were not included in
the experiments. Adult rats (P70) were subjected, under avertin anesthesia, to
reverse suture (RS) consisting in reopening of the long-term deprived eye and in the
closure of the other eye. Great care was taken to prevent opacities of the reopened
eye by topical application of Aureomicin cream (Wyeth Lederle, Italy) onto the
cornea during the rst 3 days of RS. After RS, rats were allowed to recover from
anesthesia and then returned, for 3 weeks, to their previous cage or transferred to
rearing conditions specic for the different components of EE. Subjects showing
spontaneous lid reopening or eye anomalies were excluded from the study.
2.2. Rearing environments
At P70 the animals were assigned to one of the following rearing conditions:
Classic EE: consisted of a large cage (82  50  100 cm) containing two or more
oors linked by stairs and several food hoppers, running wheels and differently
shaped objects (platforms, boxes, toys, tunnels, shelters and nesting material),
which were repositioned once a day and completely substituted with others once
a week. Every cage housed sixeeight adult rats. Standard conditions housing (SC):
consisted of a standard cage (40  25  20 cm), housing three adult rats. Classic EE
under dark conditions (DR-EE): consisted of the same environmental cage used for
Classic EE, placed in a completely dark room. Motor Enrichment (ME): consisted of
a SC cage equipped with a running wheel connected to an automatic device
recording the number of wheel turns. Social stimulation (SS): consisted of a slightly
bigger SC cage (60  40  20 cm), where six rats where housed together. Visual
enrichment (VE): consisted of a standard cage positioned at the center of a rotating
uorescent drum where specic visual patterns were drawn. The visual stimuli
presented on the rotating walls were: one horizontal sinusoidal gratings of 0.1 c/
deg; two series of black bars oriented along 8 different axes; one series of
geometrical gures (triangles, squares, rings, rhombi) of various sizes; one
vertically-oriented spatial lattice square ware of 0.4 c/deg. The drum rotated
clockwise at 0.1 Hz. When the drum lights were on, the mean luminance detected
inside the drum was around 60 cd/m2. The apparatus was connected to a device
controlled by a timer, that automatically switched the mechanical engine and the

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lights on or off, according to the following time-schedule: 6:00e12:00 OFF;

12:00e18:00 ON; 18:00e24:00 OFF; 24:00e6:00 ON. Visual perceptual learning (PL):
we used a modied version of the visual water box task (Prusky et al., 2000b; Sale
et al., 2011). Briey, the animals were trained to distinguish a low spatial
frequency grating (reference grating, 0.117 c/deg) from a higher spatial frequency
grating (test grating, 0.593 c/deg). When the animals achieved a level of >80% of
accuracy in at least two subsequent sessions (criterion level), the PL protocol was
started by gradually reducing the spatial frequency of the test grating in steps
consisting in the subtraction of one cycle on the screen. Thus, the test grating
became progressively more similar to the reference grating. For each animal, a daily
threshold was calculated as the lowest spatial frequency of the test grating that the
rat was able to distinguish (70% correct performance) from the reference grating.
The PL task ended when the animal performance reached a plateau (performance at
a given SF of the test grating oscillating around 70% of correct choices for three
consecutive days). A group of control animals was trained to distinguish the reference grating from a homogeneous gray (associative learning, AL rats). One further
control group (1st step PL rats) learned the PL task but was allowed to only distinguish the reference grating from a test grating whose SF was always maintained at
the starting value of 0.593 c/deg. Control rats were matched to PL animals in terms of
overall swim time and training days in the water maze.
In all conditions, food and water were provided ad libitum.
2.3. In vivo electrophysiology
Visual evoked potentials (VEPs) were recorded from the binocular portion of the
visual cortex (Oc1B). Rats were anesthetized with an intraperitoneal injection of 20%
urethane (Sigma, St.Louis, MO, USA; 0.7 ml/hg of body weight) and mounted in
a stereotaxic apparatus allowing full viewing of the visual stimulus. Additional doses
of urethane were used to keep the anesthesia level stable throughout the experiment. The closed eye was reopened using scissors and both eyes were restrained in
a xed position by means of adjustable metal rings surrounding the external portion
of the eye bulb. The pupil was always clearly observable between eyelid margins.
Body temperature was continuously monitored with a rectal probe and maintained
at 37.0  C with a thermostatic electric blanket during the experiment. An electrocardiogram was monitored and respiration was facilitated by means of an oxygen
mask. A portion of the skull (2  2 mm) overlying the OcB1 was carefully drilled and
the dura madre was removed. A resin-coated microelectrode (Harvard apparatus,
Edenbridge, UK) with tip impedance of 2 MU lled with NaCl (3 M) was inserted into
the cortex perpendicularly to the stereotaxic plane, 4.8e5.2 mm lateral to lambda
(intersection between sagittal- and lambdoid-sutures). Microelectrodes were
advanced 100 or 400 mm within the cortex. At those depths VEPs had their maximal
amplitude. Typical visual stimuli were horizontal sinusoidal gratings of different
spatial frequency generated by a VSG2/2 card (Cambridge Research System,
Cheshire, UK) and presented on the face of a monitor suitably linearized by gamma
correction. The display (mean luminance 25 cd/m2, area 24  26 cm) was placed
20 cm in front of the animal and centered on the previously determined receptive
elds. Electrical signals were amplied (10,000 fold), band-pass ltered
(0.1e100 Hz), digitized (12 bit resolution) and averaged (at least 50 events in blocks
of 10 events each) in synchrony with the stimulus contrast reversal. Transient VEPs
in response to abrupt contrast reversal (0.5 Hz) were evaluated in the time domain
by measuring the peak-to-baseline amplitude and peak latency of the major
component. We measured binocularity by calculating the contralateral to ipsilateral
VEP ratio (C/I ratio), i.e. the ratio of VEP amplitudes recorded by stimulating the eye
contralateral and ipsilateral, respectively, to the visual cortex where the recording is
performed. During recording through one eye, the other was covered by a black
adhesive tape. To prevent sampling bias for each animal, at least three well-spaced
penetrations were performed and at least ten series of responses from each eye were
alternatively recorded. Care was taken to equally sample VEPs across the two cortical
depths so that all layers contributed to the analysis. Visual acuity of each eye was
obtained by extrapolation to zero amplitude of the linear regression through the
data points in a curve where VEP amplitude is plotted against log spatial frequency.
2.4. Behavioral assessment of visual acuity
In a separate group of rats subjected to PL, we also measured visual acuity
through the behavioral method of the visual water maze task. Visual acuity was rst
measured at P60 through the non amblyopic (not deprived) eye; then, we measured
visual acuity through the amblyopic eye three times: immediately after RS, at the
end of the PL procedure and after a period of 15 further days.
Behavioral assessment of VA were performed as previously described (Prusky
et al., 2000b; Sale et al., 2007). Briey, animals are rst conditioned to distinguish
a low spatial frequency (0.117 c/deg) from homogeneous gray (training phase). After
animals have achieved near-perfect (80% or more) performance over 20e40 trials on
a pseudorandom schedule in this phase, testing of VA can begin. For the testing phase
small incremental changes in the spatial frequency of the stimulus are made between
successive trials until the ability of animals to distinguish a grating from gray falls to
chance. Visual acuity was taken as the spatial frequency corresponding to 70% of
correct choices on the sigmoidal function tting the psychometric function in which
the percentage of correct choices is plotted against spatial frequency (see Fig. 4).

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L. Baroncelli et al. / Neuropharmacology 62 (2012) 2388e2397

Fig. 1. Impact of motor, social and visual stimulation on visual acuity restoration in adult amblyopic animals. (A) Electrophysiological assessment (by VEPs) revealed that the visual
acuity of the formerly deprived eye was not statistically different with respect to that of the fellow eye in rats exposed to classic enrichment under normal light conditions (EE:
paired t-test, p 0.442), motor enrichment (ME: paired t-test, p 0.926), visual stimulation (VE: Wilcoxon Signed Rank Test, p 0.938) and visual perceptual learning (PL: paired ttest, p 0.06). In contrast, no recovery of visual acuity was observed in standard condition (SC: paired t-test, p < 0.01), social stimulation (SS: paired t-test, p < 0.001) and classic
enrichment combined with dark-rearing animals (DR-EE: paired t-test, p < 0.01). (B) KruskaleWallis One-Way ANOVA on ranks revealed a statistical difference in the mean values
of visual acuity for the long-term deprived eye among the various groups (p < 0.001); a multiple comparison procedure (Dunns Method) showed that visual acuity was not
signicantly different from that recorded in adult animals in EE, ME, VE and PL rats, but not in SC, SS and DR-EE animals. The gray box denotes the visual acuity range in nave adult
animals. Representative examples of electrophysiological visual acuity assessment for an amblyopic and a normal eye are reported on the right in the (B) panel: visual acuity is
obtained by extrapolation to zero amplitude of the linear regression through the data points in a curve where VEP amplitude is plotted against log spatial frequency. * p < 0.05; error
bars, s.e.m.

2.5. Analysis of neurotransmitter release in V1 synaptosomes

Animals were sacriced and the cortical area corresponding to the primary
visual cortex was removed. Synaptosomes were prepared essentially as previously
described (Stigliani et al., 2006). The tissue was homogenized at 4  C, utilizing
a homogenizer Teon/glass (clearance 0.25 mm), in 10 volumes of sucrose 0.32 M,
buffered with TriseHCl at pH 7.4. The homogenized tissue was centrifuged (5 min,
1000  g a 4  C) in order to remove all nuclei and cellular fragments. Then, the
supernatant was gently stratied on a discontinuous Percoll gradient (2, 6, 10, 20% v/
v in triseHCl/sucrose) and again centrifuged (33,500  g per 5 min a 4  C). After
centrifugation, the stratied fraction of synaptosomes, leaning between 10% and 20%
Percoll, was collected, washed by centrifugation (20,200  g per 15 min a 4  C) and
then resuspended in a physiologic medium, containing: NaCl 140 mM; KCl 3 mM;
MgCl2 1.2 mM; CaCl2 1.2 mM; NaH2PO4 1.2 mM; HEPES 10 mM; glucose 10 mM;
pH 7.4.
Synaptosomes were incubated at 37  C for 15 min with the radioactive tracers
(3H)D-Asp (a widely used non-metabolizable tracer labeling endogenous glutamatergic pool of synaptic vescicles supporting GLU release) or (3H)GABA, at a nal
concentration of 0.05 mM. (3H)GABA labeling was performed in the presence of
50 mM of the GABA transaminase inhibitor amino-oxyacetic acid, to minimize GABA
catabolism. Aliquots of the synaptosomal suspensions were layered on microporous
lters at the bottom of a set of parallel superfusion chambers (Superfusion System,
Ugo Basile, Comerio, Varese, Italy) (Reiteri et al., 1984) maintained at 37  C. Superfusion was started at a rate of 0.5 ml/min with standard medium, supplemented
with 50 mM amino-oxyacetic acid in the case of (3H)GABA release. After 36 min of

superfusion, to equilibrate the system, samples were collected according to the

following scheme: one sample collected for 3-min (t 36e39 min; basal outow);
one sample collected for 6-min (t 39e45 min; stimulus-evoked release); one
sample collected for 3-min (t 45e48 min; basal outow after stimulus-evoked
release). A 90-sec period of stimulation was applied at t 39 min, after the rst
sample has been collected. Lower-intensity stimulation (i.e. 15 mM KCl, substituting
for equimolar concentration of NaCl) was applied in the case of (3H)D-Asp, since
augmentation of the release rate was eventually expected. Higher-intensity stimulation (i.e. 25 mM KCl) was applied in the case of (3H)GABA. Radioactivity was
determined in each sample collected and superfused lters by liquid scintillation
counting. Tritium released in each sample was calculated as percentage of the total
synaptosomal tritium content at the beginning of the respective sample collection
(fractional rate  100). The stimulus-evoked overow was estimated by subtracting
transmitter content of the two 3-min samples (basal outow) from release evoked in
the 6-min sample collected during and after the depolarization pulse (stimulusevoked release).
2.6. Statistics
All statistical analysis was done using SigmaStat Software. Differences between
visual acuity of the long-term deprived eye and that of the normal eye were evaluated with a paired t-test. Differences between two groups were assessed with
a two-tailed t-test. Differences between more than two groups were evaluated with
One-Way ANOVA followed by HolmeSidak test for data normally distributed and
with KruskaleWallis One-Way ANOVA (ANOVA on ranks) with Dunns post hoc test

L. Baroncelli et al. / Neuropharmacology 62 (2012) 2388e2397

2391

Fig. 2. Impact of motor, social and visual stimulation on ocular dominance restoration in adult amblyopic animals. The graph shows the contralateral to ipsilateral eye (C/I) VEP ratio
mean values for all different groups. The gray box denotes the C/I VEP ratio range in nave adult animals. One-Way ANOVA revealed a statistical difference in the mean values among
the various groups (p < 0.001); a multiple comparison procedure (HolmeSidak method) showed that ocular dominance recovered to normal adult values in EE, ME, VE and PL
animals (p 0.829, 0.315, 0.105, 0.863, respectively), but not in SC, SS and DR-EE rats (p < 0.05). Typical VEPs recorded in response to the stimulation of either the contralateral or
the ipsilateral eye in amblyopic and normal animals are reported in the top inset. Calibration bars: 25 mV, 100 ms. * p < 0.05; error bars, s.e.m.
for data not normally distributed. The progressive reduction in the minimum
discriminable SF difference between the reference and the test grating across the
days of PL procedure was evaluated with a One-Way RM ANOVA. Behavioral visual
acuity measured through the amblyopic eye of PL rats immediately after RS, at the
end of the PL procedure and after a period of 15 further days was compared with
a One-Way RM ANOVA. Level of signicance was p < 0.05.

Fig. 3. Perceptual learning in adult amblyopic rats. (A) Schematic diagram of the
modied version of the visual water box used for perceptual learning (PL) in amblyopic
rats. (B) Improvement of discrimination threshold in adult amblyopic rats performing
the PL task. The threshold, calculated as the minimum spatial frequency difference
between the reference and the test gratings discriminated (MDSFD), decreased
signicantly with the training days (One-Way RM ANOVA, p < 0.001). The MDSFD
obtained in the sixth day of the PL task was statistically different from that obtained in
the rst day (HolmeSidak method, p < 0.01). Examples of the discriminative difference
between the reference and the test grating across the training days are also represented. Error bars, s.e.m.

3. Results
3.1. Physical activity induces amblyopia recovery in adult rats
We rst investigated whether enhanced levels of physical
exercise are able to promote recovery from amblyopia. A group of
rats rendered amblyopic by monocular deprivation (MD) carried
out at the peak of the critical period (postnatal day 21, P21) were
subjected to reverse suture (RS) in adulthood (>P60) and then
either transferred, for three weeks, in standard cages endowed
with a running wheel connected to an automatic wheel turn
recording device (n 5), or left in standard cages for control (n 6).
At the end of the differential rearing period, we measured VA of
both eyes using electrophysiological recordings of visual evoked
potentials (VEPs) from the binocular portion of the primary visual
cortex (V1). Visual acuity of animals subjected to motor enrichment
(ME rats) was completely restored, while that of animals left in
standard cage (SC rats) did not recover (Fig. 1A). Visual acuity
through the amblyopic eye for the ME group (1.02  0.08 cycles per
degree, c/deg) was not statistically different either from that of the
not deprived eye (1.03  0.10 c/deg; paired t-test, p 0.926; Fig. 1A)
or from that recorded in adult nave animals (never deprived)
(n 12, 0.92  0.02 c/deg; KruskaleWallis One-Way ANOVA on
Ranks, post hoc Dunns Method; Fig. 1B). On the contrary, VA for the
deprived eye in SC animals (0.62  0.05 c/deg) remained signicantly lower than that for the fellow undeprived eye (1.04  0.03,
paired t-test, t 6.421 with 5 degrees of freedom, p < 0.01;
Fig. 1A) and that in normal adult animals (KruskaleWallis One-Way
ANOVA on Ranks, H 38.265 with 7 degrees of freedom, p < 0.001;
post hoc Dunns Method, Q 3.571; Fig. 1B).
In the same animals, we also evaluated the ocular dominance
(OD) by calculating the contralateral to ipsilateral (C/I) VEP ratio. C/I

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L. Baroncelli et al. / Neuropharmacology 62 (2012) 2388e2397

rats (1.04  0.07) remained signicantly lower than that recorded

in adult nave rats (One-Way ANOVA, F 7.435 with 7 degrees of
freedom, p < 0.001; post hoc HolmeSidak method, t 3.597;
Fig. 2).
3.3. Recovery from amblyopia in classic EE conditions is lightdependent

Fig. 4. Behavioral measure of visual acuity recovery in rats subjected to visual

perceptual learning. Visual acuity of both the long-term deprived and the open eye was
measured using the visual water box task. At the end of the PL procedure, visual acuity
of the previously deprived eye was not different from that of the other eye (paired ttest, p 0.657). One-Way RM ANOVA with HolmeSidak method revealed that the
visual acuity of the previously deprived eye measured after PL was signicant
increased with respect to that measured before visual training (p < 0.01) and remained
unaltered 2 weeks after the end of PL (p 0.815). Visual acuity is obtained by
extrapolation to 70% of correct choices on the sigmoidal function tting the psychometric function in which the percentage of correct choices is plotted against spatial
frequency. * p < 0.05; error bars, s.e.m.

VEP ratio is an accepted measure of OD properties of cortical

neurons (Porciatti et al., 1999). In normal adult rats C/I VEP ratio is
between 2.0 and 3.0, reecting the predominance of crossed bers
in retinal projections, but it decreases to around 1.0 in amblyopic
subjects, owing to the pronounced OD shift in favor of the open eye
caused by long-term MD (Sale et al., 2007). Exposure to ME induced
a full recovery of visual functions also at level of OD (Fig. 2): the C/I
VEP ratio of ME rats (1.98  0.21) was not statistically different from
that recorded in nave controls (2.39  0.2; One-Way ANOVA, post
hoc HolmeSidak method, p 0.315). No recovery was observed in
SC rats (1.08  0.14; One-Way ANOVA, F 7.435 with 7 degrees of
freedom, p < 0.001; post hoc HolmeSidak method, t 3.744,
p < 0.01).
3.2. Potentiation of social interactions is not effective in inducing
recovery from amblyopia in adult animals
In order to unravel the effects elicited by increased social
interactions on amblyopia recovery, we transferred for three weeks
a group (n 6) of reverse-sutured adult amblyopic rats to a cage
housing six animals together (see Methods for further details).
No recovery of visual functions was detected in rats exposed to
social stimulation (SS rats). VA of the long-term deprived eye
(0.67  0.02 c/deg) remained statistically lower with respect to that
of the fellow eye (0.95  0.04 c/deg; paired t-test, t 7.102 with 5
degrees of freedom, p < 0.001; Fig. 1A). A signicant difference was
detected when comparing the amblyopic eye VA of SS rats with that
recorded in adult nave animals (KruskaleWallis One-Way ANOVA
on Ranks, H 38.265 with 7 degrees of freedom, p < 0.001; post
hoc Dunns Method, Q 2.993; Fig. 1B). Also the C/I VEP ratio of SS

Among the various sensory modalities, visual stimulation can be

supposed to be critically involved in amblyopia recovery under EE
conditions. Therefore, as a rst step to characterize the role of visual
stimulation, we investigated whether light stimulation of the
amblyopic eye is necessary for the EE-induced recovery. Specically, we measured visual functions in a group (n 5) of adult
amblyopic rats reverse-sutured and reared for three weeks in
a classic EE setting placed in a completely dark room (DR-EE rats).
In DR-EE rats, VA of the deprived eye remained signicantly
lower (0.61  0.02 c/deg) with respect to the other eye
(1.00  0.07 c/deg; paired t-test, t 5.006 with 4 degrees of
freedom, p < 0.01; Fig. 1A). Moreover, VA of DR-EE rats was
statistically different from that recorded in adult nave animals
(KruskaleWallis One-Way ANOVA on Ranks, H 38.265 with 7
degrees of freedom, p < 0.001; post hoc Dunns Method, Q 3.619;
Fig. 1B). No recovery of OD was detected in the visual cortex
contralateral to the formerly deprived eye: the C/I VEP ratio
(0.89  0.12), indeed, was signicantly lower than that recorded in
nave controls (One-Way ANOVA, F 7.435 with 7 degrees of
freedom, p < 0.001; post hoc HolmeSidak method, t 3.992;
Fig. 2). Thus, light stimulation is necessary for the effects of visual
function recovery elicited by classic EE in adult amblyopic rats.
3.4. Amblyopia recovery in adult rats exposed to enhanced visual
stimulation
Since exposure to EE is likely to enhance visually-driven stimulation owing to the complexity of the available structured visual
patterns, we next assessed visual function recovery in a group of
animals (n 7) subjected to enriched visual stimulation (visual
enrichment, VE). Immediately after RS, VE rats were transferred to
a SC cage placed inside a mechanical backlit-rotating drum where
various visual stimuli of different spatial frequencies and orientations were drawn on the walls (see Methods for details).
After three weeks of treatment, we found that VA of the previously deprived eye (0.94  0.05 c/deg) did not statistically differ
from that of the other eye (0.95  0.02 c/deg; Wilcoxon Signed Rank
Test; p 0.938; Fig. 1A) and was completely comparable to VA
recorded in adult nave animals (KruskaleWallis One-Way ANOVA
on Ranks, post hoc Dunns Method; Fig. 1B). We also detected
a marked OD recovery in VE rats (Fig. 2), with their C/I VEP ratio
(1.80  0.22) being not statistically different (even if slightly lower)
from that recorded in adult nave animals (One-Way ANOVA, post
hoc HolmeSidak method, p 0.105).
3.5. Visual perceptual learning induces a full recovery of visual
functions in adult amblyopic rats
An increasing number of clinical studies have reported that
visual training eliciting perceptual learning (PL) processes may be
a very useful approach for the treatment of amblyopia in humans,
providing a substantial improvement in a variety of visual tasks
(see Levi and Li, 2009 for a recent review). The promising results
obtained using our protocol of visual enrichment in the rotating
drum prompted us to investigate whether recovery of visual
functions might be achievable by a stimulation paradigm in which
adult amblyopic rats undertake visual PL by performing

L. Baroncelli et al. / Neuropharmacology 62 (2012) 2388e2397

progressively ner discriminations of visual stimuli. A group of

reverse-sutured amblyopic rats (PL rats, n 7) practiced in
a forced-choice visual task, discriminating between two vertical
gratings differing only for their spatial frequency (SF). The SF of
the test grating was made progressively more similar to that of the
reference grating (0.117 c/deg), starting from a SF of the test
grating of 0.593 c/deg. Practice in this task caused a progressive
improvement of visual discrimination abilities, as evidenced by
the reduction in the minimum discriminable SF difference
(MDSFD) between the reference and the test grating across the
days: while, on the rst day, the mean MDSFD was
0.203  0.022 c/deg, this value reached 0.005  0.009 c/deg at the
end of the test (One-Way RM ANOVA, F 22.791 with 9 degrees of
freedom, p < 0.001; Fig. 3).
After the PL training, VA and OD were rst evaluated electrophysiologically by recording VEPs. The VA of the long-term MD eye
(0.88  0.04 c/deg) did not statistically differ from that of the
normal eye (1.01  0.06 paired t-test, p 0.06) (Fig. 1A) and from
that recorded in adult nave animals (KruskaleWallis One-Way
ANOVA on Ranks, post hoc Dunns Method; Fig. 1B).
A very good recovery of OD was also found: the C/I VEP ratio of
PL animals (2.45  0.26) was not statistically different from that
recorded in adult nave animals (One-Way ANOVA, post hoc
HolmeSidak method, p 0.863; Fig. 2).
Given that visual PL is currently considered one of the most
promising procedures to treat amblyopia in adult human subjects,
we repeated VA assessments in a separate group of long-term
monocularly deprived and reverse-sutured animals subjected to
visual PL training (n 4) by using a standard behavioral method,
the visual water box task. Behavioral data completely conrmed
the electrophysiological outcome: a full VA recovery was evident in
the amblyopic eye of rats subjected to PL (VA of the not deprived
eye: 0.89  0.04 c/deg, VA of the previously deprived eye:
0.91  0.05 c/deg; paired t-test, p 0.657; Fig. 4). Moreover, VA of
the previously deprived eye after PL was statistically higher than
that measured before the beginning of the training (0.62  0.02 c/
deg; One-Way RM ANOVA, F 26.117 with 2 degrees of freedom,
p < 0.001; post hoc HolmeSidak method, t 6.378). The benecial
effect elicited by PL was long-lasting: indeed, VA recovery in the
formerly deprived eye persisted unaltered 2 weeks after the end of
the PL procedure (VA of amblyopic eye 2 weeks after PL:
0.90  0.03 c/deg; One-Way RM ANOVA, post hoc HolmeSidak
method, p 0.815; Fig. 4).
To demonstrate that VA recovery was specically elicited by
visual PL, we performed two controls. In the rst, to rule out the
possibility that the recovery effects in PL rats were simply due to
the practice of associative learning in the water box instead than to
practice in discriminating visual stimuli, we performed VA analysis
in a group of control animals that were trained to distinguish the
reference grating from a homogeneous gray, matching this control
group (associative learning, AL rats, n 4) to PL rats in terms of
overall swim time and training days in the water maze. We found
that VA of the long-term deprived eye (0.62  0.01 c/deg) remained
signicantly lower with respect to that of the fellow eye
(0.91  0.06 c/deg; paired t-test, t 4.630 with 3 degrees of
freedom, p < 0.05; Fig. 5). In the second, to assess that simply
practicing visual stimuli discrimination, but not undergoing visual
PL, was not enough to promote recovery, we repeated the behavioral assessment of VA in a group of rats that learned the grating
discrimination task but was allowed to practice only with the easy
initial discrimination (test grating 0.593 c/deg, see also Sale et al.,
2011). These animals (1st step PL rats, n 4) showed no VA
recovery (VA of the non deprived eye: 0.90  0.03 c/deg; VA of the
previously deprived eye: 0.60  0.01 c/deg, paired t-test, t 9.432
with 3 degrees of freedom, p < 0.01; Fig. 5).

2393

Fig. 5. Visual acuity recovery in PL rats is dependent on visual training. The histogram
shows behavioral visual acuity of both eyes measured in animals subjected to PL, in
associative learning rats (AL) and in animals trained only in the rst step of PL training
(1st step PL). Visual acuity of the previously deprived eye was different from that of the
other eye in AL and 1st step PL animals (p < 0.001), but not in the PL group (p 0.750)
(Two-Way RM ANOVA, HolmeSidak method). * p < 0.05; error bars, s.e.m.

3.6. Recovery from amblyopia is associated with reduced inhibition/

excitation balance in the primary visual cortex
Since converging evidence points to the maturation of cortical
inhibitory circuits as the crucial brake limiting plasticity in the adult
brain (Morishita and Hensch, 2008) and EE has been shown to
decrease the inhibitory tone in the visual cortex of adult animals
(Sale et al., 2007; Baroncelli et al., 2010a), we investigated whether
recovery of VA and OD elicited by the selective EE procedures tested
in this study, was accompanied by a change in the intracortical
inhibition/excitation balance. To this purpose, we quantied via
synaptosome analysis (Fig. 6) the release of (3H)GABA and (3H)DAspartate ((3H)D-Asp) in Oc1B of animals reared under the same
conditions of selective environmental stimulation described so far.
We found that the depolarization-evoked (3H)GABA release was
markedly reduced, compared to rats reared in SC (n 10:
7.26  0.40%), in the visual cortex of amblyopic rats reared under
classical EE conditions (n 5: 4.24  0.19%), in those subjected to
EE combined with dark exposure (n 6: 5.34  0.54%), in those
experiencing high levels of voluntary physical activity (n 5:
3.47  0.26%), in those subjected to visual enrichment (n 6:

Fig. 6. Time-course of the release of a putative neurotransmitter from synaptosomes

exposed to a stimulation pulse. Synaptosomes were stratied on microporous lter,
and neurotransmitter release was monitored during superfusion. After 36 min to
equilibrate the system, two 3-min samples (t 36e39 min and t 45e48 min) were
collected before and after one 6-min sample (t 39e45 min). Synaptosomes were
exposed to the stimulus (90 s; gray bar) at the end of the rst sample collected
(t 39 min). A cartoon of the synaptosome technique is also represented.

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L. Baroncelli et al. / Neuropharmacology 62 (2012) 2388e2397

5.18  0.41%) and in animals that practiced in PL training (n 8:

3.82  0.42%) (One-Way ANOVA, F 11.143 with 7 degrees of
freedom, p < 0.001; post hoc HolmeSidak method, t 3.522, 2.632,
4.751, 2.833 and 5.053, respectively; Fig. 7B). On the contrary, the
release of (3H)GABA detected in the visual cortex of rats that
experienced social stimulation (SS rats, n 9: 8.25  0.65%) was not
statistically different from that measured in SC animals, even if
slightly increased (One-Way ANOVA, post hoc HolmeSidak method
p 0.06; Fig. 7B). We did not detect any difference in the
depolarization-evoked release of (3H)D-Asp, mimicking glutamate
release, among the different groups of animals (SC rats, n 11;
1.4  0.18%; EE rats, n 5: 1.68  0.25%; ME rats, n 5:
1.54  0.25%; SS rats, n 12: 1.42  0.5%; DR-EE rats, n 5:
2.03  0.10%; VE rats, n 7: 1.82  0.21%; PL rats, n 8:
2.17  0.35%) (One-Way ANOVA, p 0.232; Fig. 7A). Thus, visual
function recovery from amblyopia was tightly related to a reduced
inhibitory tone in the visual cortex.
In one case, however, this association was not detected. Indeed,
even if synaptosome analysis revealed a signicant decrease of

GABAergic transmission in the visual cortex of DR-EE rats, these

animals did not recover from amblyopia. Since this was the only
condition in which vision was totally prevented (due to exposure to
full darkness), we hypothesized that a reduced level of GABAergic
inhibition could allow amblyopia recovery only when the animals
have the possibility to actively use their amblyopic eye. To further
analyze this issue, we measured VA and OD in a group of adult longterm deprived rats which were exposed to classic EE in normal light
conditions, but which were prevented from vision through their
amblyopic eye since this was maintained closed during the three
weeks of EE exposure (i.e., we did not perform RS in this group:
noRS-EE rats, n 5). We recorded VEPs in V1 contralateral to the
deprived eye, which was reopened only at the time of the electrophysiological recordings. No recovery was detected in noRS-EE
rats: indeed, VA of the long-term deprived eye (0.71  0.03 c/
deg) remained statistically lower compared to that of the fellow eye
(1.02  0.03 c/deg; paired t-test, t 10.301 with 4 degrees of
freedom, p < 0.001) and to normal adult values (t-test, t 5.924
with 15 degrees of freedom, p < 0.001; Fig. 8A). Furthermore, in

Fig. 7. Excitation-inhibition balance regulates plasticity in adult amblyopic rats. (A) Depolarization-evoked release of (3H)D-Asp from synaptosomes. 15 mM KCl evoked (3H)D-Asp
release from visual cortex synaptosomes of the various groups. One-Way ANOVA did not show a signicant difference among the group levels (p 0.232). (B) Depolarization-evoked
release of (3H)GABA from synaptosomes. 25 mM KCl evoked GABA release from visual cortex synaptosomes. One-Way ANOVA showed a signicant difference among the group
levels (p < 0.001); a multiple comparison procedure (HolmeSidak method) showed that levels of GABA were signicantly lower with respect to SC animals in EE, ME, DR-EE, VE and
PL animals (p < 0.05), while no statistical difference was present between SC animals and SS rats (p 0.06). * p < 0.05; error bars, s.e.m.

L. Baroncelli et al. / Neuropharmacology 62 (2012) 2388e2397

Fig. 8. Amblyopia recovery in enriched animals depends on the use of the impaired
eye. (A) Electrophysiological assessment revealed that the visual acuity of the formerly
deprived eye remained lower with respect to that of the other eye in rats exposed to
three weeks of EE without reopening of their long-term deprived eye (noRS-EE rats)
(paired t-test, p < 0.001). Moreover, in noRS-EE rats the visual acuity of the long-term
deprived eye was lower than that recorded in nave adult animals (t-test, p < 0.001).
The gray box denotes the visual acuity range in adult normal animals. For comparison,
data of visual acuity in long-term deprived rats exposed to either standard condition or
EE are also reported. (B) Contralateral to ipsilateral eye (C/I) VEP ratio mean values in
SC, EE and noRS-EE animals. One-Way ANOVA showed a statistical difference in the
mean values among the three groups (p < 0.05); a multiple comparison procedure
(HolmeSidak method) showed that ocular dominance was recovered to normal adult
values in EE animals (p 0.847), but not in SC and noRS-EE rats (p < 0.05). The gray
box denotes the C/I VEP ratio range in adult normal animals. * p < 0.05; error
bars, s.e.m.

noRS-EE animals the C/I VEP ratio (1.17  0.08) was signicantly
lower than that of adult nave rats (One-Way ANOVA, F 7.779
with 3 degrees of freedom, p < 0.01; HolmeSidak method,
t 2.929; Fig. 8B).
These results indicate that different environmental stimulation
procedures are able to reopen visual cortex plasticity through
a reduction of GABAergic inhibition levels and that this reduction is
effective in inducing visual function recovery in adult amblyopic
rats only if the animals have the opportunity to use their long-term
deprived eye.
4. Discussion
4.1. Effects elicited by motor, social and visual stimulation on
amblyopia recovery
It is widely held that the positive effects elicited by EE are due to
the combination of the various stimulating factors (motor, social,
sensory) included in this protocol. However, very few studies have
specically examined the contribution given by each EE component
in inducing plasticity in the adult brain. This analysis could be very

2395

helpful not only in a basic research perspective, but also for the
elaboration of novel protocols of environmental stimulations suitable to be applied to human patients. Here, we demonstrated that
procedures aimed at the potentiation of single components typically present in EE are able to reproduce the effect of recovery of
visual functions from amblyopia previously reported in classicallyenriched animals (Sale et al., 2007).
We rst focused our analysis on the characterization of the
effects elicited by a protocol aimed at stimulating voluntary physical activity, placing the animals in cages where they had free access
to a running wheel. Exercised rats fully recovered visual functions
both at level of VA and OD. The strong activation of the primary
motor cortex induced in ME animals by physical activity may
eventually result in the activation of cross-modal plasticity in V1.
Accordingly, it has been shown that the visually evoked ring rate
of V1 neurons is strongly enhanced, in awake mice, when the
animals transit from standing still to running (Niell and Stryker,
2010). In addition, the positive effects elicited by exercise on V1
plasticity might depend on the up-regulation of peripheral or
central growth factors and their relative intracellular cascades,
which may drive structural and functional changes in V1 circuitry.
For example, it is well known that IGF-1 levels increase both at
peripheral level (Schwarz et al., 1996) and in the brain (Carro et al.,
2000) in exercised rats.
In contrast, social enrichment per se was not able to induce
restoration of normal VA and OD in adult amblyopic rats. It has to
be underlined that, to analyze this component, we employed
a protocol in which two variables were changed compared to the
SC, i.e. rat number and cage size. Even if this rendered hard to
estimate the specic effects deriving from the social variable alone,
we point out that we used the protocol originally adopted in
literature for reproducing social enrichment (Rosenzweig et al.,
1978). Moreover, as the social stimulation group did not differ
from the controls, we consider unlikely an effect deriving from the
larger size cage. In agreement with our results, a recent study
pointed out a weak contribution of social stimulation on brain
plasticity, demonstrating that while the number of newly generated hippocampal neurons is increased in animals subjected to
enhanced social interactions, this augment is not reected by
positive effects on learning and memory abilities (Madroal et al.,
2010).
The animals exposed to the protocol of visual enrichment
showed a marked recovery from amblyopia. The apparatus that we
used for visual stimulation was specically designed to maximize
stimulation of V1 cortical neurons, which are particularly sensitive
to gratings of different spatial frequencies and to the orientation of
the stimuli (Maffei et al., 1977). This visual enrichment protocol was
not a passive stimulation paradigm, since the animals could choose
when and how much to watch the visual stimuli. We also
demonstrated that adult amblyopic animals placed under classic EE
conditions, but completely deprived from visual stimulation, failed
to recover normal visual functions. Quinlan and colleagues (He
et al., 2007) recently demonstrated that the loss of VA resulting
from chronic MD is reversible in animals reared in darkness in
adulthood. In this previous report, however, light deprivation
preceded the reopening of the deprived eye, which coincided with
the animals being returned to normal light conditions. Conversely,
in our experimental paradigm the animals were reverse-sutured
and placed in dark-rearing (and in EE) at the same moment.
In agreement with evidence on human subjects (for recent
reviews, see Levi and Li, 2009; Astle et al., 2011), a marked recovery
of visual functions was evident in amblyopic rats subjected to visual
PL. Electrophysiological and behavioral data concordantly documented a full recovery of VA in PL rats. The recovery outlasted the
end of the treatment, as is the case for EE (Sale et al., 2007),

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L. Baroncelli et al. / Neuropharmacology 62 (2012) 2388e2397

persisting for at least 15 days: this effect is remarkable, if one

considers that, in the timescale of human life, it is as the functional
improvement lasted for 20 months or more. We previously showed
that visual PL is accompanied by long-term potentiation (LTP) of
intracortical synaptic responses in rat V1 (Sale et al., 2011), in
agreement with Cooke and Bear (2010). Thus, practice with the
long-term deprived eye might induce recovery of visual functions
via potentiation of synaptic transmission in the visual connections
subserving the long-term deprived eye. Accordingly, no recovery
from amblyopia was evident in two control groups in which the
treatment did not induce LTP in V1, i.e. in rats who only learned the
association stimulus-escape platform and in animals that were
trained only until the rst step of the discrimination procedure, but
did not proceed with the task of incrementally ner discrimination
leading to improvement in performance (Sale et al., 2011).
It has been pointed out that one caveat to the therapeutic value
of PL procedures in the treatment of amblyopia is the narrow
specicity of the achievable improvements, which are frequently
limited to the selected trained stimulus, condition or task (Levi and
Li, 2009). Our results show that even if PL rats practiced in
discriminating visual gratings in the 0.1e0.6 c/deg range, they
displayed a discrimination improvement in a range of higher spatial
frequencies, with nal VA values of 0.9e1.0 c/deg.

(Kasamatsu, 1982; Maya Vetencourt et al., 2008; Morishita et al.,

2010; for a review, see Baroncelli et al., 2011). Another key actor
might be the brain-derived neurotrophic factor (BDNF) and its
specic TrkB receptor signaling, which are increased in EE rodents
(e.g. Ickes et al., 2000; Baroncelli et al., 2010b) and have been
shown to be specically required for recovery of cortical responses
following monocular deprivation (Kaneko et al., 2008). Environmental changes can also alter the brain chromatin status (Fischer
et al., 2007), and epigenetic modications, such as the acetylation
of histones, have been recently implicated in the regulation of
plasticity in the adult visual cortex (Putignano et al., 2007;
Silingardi et al., 2010). Moving to the extracellular level, it has been
shown that infusion in the mature cortex of amblyopic rats of an
enzyme (chondroitinase ABC) that degrades chondroitin sulfate
proteoglycans (CSPGs), an essential component of the brain extracellular matrix, produces a marked reinstatement of both visual
acuity and binocularity (Pizzorusso et al., 2006). Since we previously showed that EE reduces the density of CSPG perineuronal
nets in the visual cortex of adult amblyopic rats exposed to classic
EE conditions (Sale et al., 2007), it is possible the a similar process
could also take part in the functional and structural remodeling of
visual cortex circuitries promoted by the specic environmental
components analyzed in the present work.

4.2. Amblyopia recovery and intracortical inhibition

5. Conclusions

The excitatory-inhibitory balance is well known to be crucially

involved in the regulation of plasticity during development and in
adulthood (Morishita and Hensch, 2008; Sale et al., 2010; Baroncelli
et al., 2011). The results reported in the present study are in full
agreement with this concept. Recovery of VA and OD detected in
adult amblyopic animals subjected to voluntary physical activity,
visual enrichment and PL, indeed, was associated with a marked
reduction of GABAergic intracortical inhibition, as revealed by
reduced GABA release in synaptosome analysis. No decrease of
intracortical inhibition was present for protocols which did not
induce recovery from amblyopia, with only one exception:
a signicant reduction in the GABAergic inhibitory tone was
detected in enriched rats housed under dark-rearing conditions
which did not recover from amblyopia. We favor the interpretation
that while EE per se decreases GABAergic inhibition in V1 (Sale
et al., 2007), thus increasing cortical plasticity and giving the
potential for vision rescue, an effective process of visual function
recovery is strictly dependent on active use of the amblyopic eye. In
agreement with this explanation, we showed that no recovery was
observed in amblyopic rats exposed to classic EE but in which the
amblyopic eye was maintained occluded.
Interestingly, the balance between excitation and inhibition has
been suggested to be impaired during development in amblyopic
human subjects and cortical over-inhibition could underlie the
degradation of spatial vision abilities (Polat, 1999; Levi et al., 2002;
Wong et al., 2005). Repetitive transcranial magnetic stimulation,
which increases cortical excitability, transiently improves contrast
sensitivity in adult amblyopes, likely acting on the excitation/
inhibition balance (Thompson et al., 2008).
Even if our results point toward a reduction of brain inhibition
levels as a crucial mediator for the effects elicited by sensory and
motor enrichment, it is likely that other mechanisms could also
underlie visual function recovery in amblyopic rats exposed to
enriched conditions. Potential candidates are brainstem neuromodulatory systems involved in the regulation of the arousal state
of the brain (Gu, 2002), such as the noradrenergic, serotonergic and
cholinergic systems, which are particularly sensitive to environmental stimuli (Baroncelli et al., 2010b) and whose permissive
action on neural plasticity has been repeatedly documented

Environmental enrichment is a complex paradigm, since an

increased stimulation is provided at multiple sensory, motor, and
social levels. Although most humans do experience a high degree of
environmental complexity, levels of physical, social and sensory
stimulation vary greatly among individuals and in different periods
of life. Our results obtained using motor enrichment and protocols
of visual stimulation show that these components are crucially
involved in amblyopia recovery elicited by EE and that they may act
through a reduction of the intracortical inhibition/excitation
balance in V1. In addition, our results indicate that the efcacy of PL
in promoting recovery from amblyopia in human subjects (Levi and
Li, 2009) might be related to the effects that PL exerts on the
intracortical inhibition/excitation balance, promoting the longterm strengthening of practiced intracortical visual connections
(Sale et al., 2011).
It should be noted that the effects reported in the present work
in a rodent model of amblyopia may not apply in the same measure
to other species with much higher visual acuities, as monkeys and
humans, in which an impairment of early visual experience has
more pronounced effects on visual abilities. However, our results
encourage efforts in the application of paradigms based on
enriched experience, such as physical exercise, videogames (Li
et al., 2011) or virtual reality, for the therapy of amblyopia in
adulthood and offers an insight into the underlying mechanism of
action.
Finally, it should be pointed out that our results can also have
implications for other nervous system disorders different from
amblyopia. Exposure to EE, indeed, has remarkably benecial
effects in rodent models of various nervous system injuries and
diseases (Will et al., 2004; Nithianantharajah and Hannan, 2006;
Baroncelli et al., 2010b). In some cases, the positive impact of EE on
brain disorders can still derive form its ability to reduce GABAergic
inhibition. This is likely to be the case, for instance, for a group of
developmental disorders characterized by excessive inhibition
levels and severe brain disabilities, such as the Down syndrome
(Fernandez and Garner, 2007). Accordingly, we recently showed
that EE reduces the cortical inhibitory tone and induces a marked
functional recovery in the murine Down syndrome model Ts65Dn
(Begenisic et al., 2011).

L. Baroncelli et al. / Neuropharmacology 62 (2012) 2388e2397

Conict of interest
The authors declare that they have no conicts of interest.
Acknowledgments
Work supported by the grant Progetto di Ateneo 2008 to AS
and a Scuola Normale Superiore grant to LM and LB.
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