Enriched experience and recovery from amblyopia in adult rats: Impact of motor, social and sensory components

Neuropharmacology 62 (2012) 2388e2397

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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 findings could have a bearing in orienting clinical research in the field of amblyopia therapy. Ó 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 deficits in contrast sensitivity and in stereopsis (Lewis and Maurer, 2005; Levi, 2006). In animal models, amblyopia can be artificially 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.: þ390 503 153196; fax: þ390 503 153220. E-mail address: [email protected] (A. Sale). 1 These authors equally contributed to this work. 0028-3908/$ e see front matter Ó 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 sufficient 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

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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 efficacy 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 efficacy, 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 first 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 specific 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 floors 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 fluorescent drum where specific 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 figures (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 modified version of the visual water box task (Prusky et al., 2000b; Sale et al., 2011). Briefly, 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 fixed 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 filled 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 fields. Electrical signals were amplified (10,000 fold), band-pass filtered (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 first 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). Briefly, animals are first 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 fitting the psychometric function in which the percentage of correct choices is plotted against spatial frequency (see Fig. 4).

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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 ¼ 0.442), motor enrichment (ME: paired t-test, p ¼ 0.926), visual stimulation (VE: Wilcoxon Signed Rank Test, p ¼ 0.938) and visual perceptual learning (PL: paired ttest, p ¼ 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 (Dunn’s Method) showed that visual acuity was not significantly 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 naïve 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 sacrificed 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 Teflon/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 stratified 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 stratified 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 final 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 filters 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 ¼ 36e39 min; basal outflow); one sample collected for 6-min (t ¼ 39e45 min; stimulus-evoked release); one sample collected for 3-min (t ¼ 45e48 min; basal outflow after stimulus-evoked release). A 90-sec period of stimulation was applied at t ¼ 39 min, after the first 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 filters 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 overflow was estimated by subtracting transmitter content of the two 3-min samples (basal outflow) 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 Dunn’s post hoc test

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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 naïve 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 ¼ 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 significance was p < 0.05.

Fig. 3. Perceptual learning in adult amblyopic rats. (A) Schematic diagram of the modified 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 significantly 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 first 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 first 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 ¼ 5), or left in standard cages for control (n ¼ 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 ¼ 0.926; Fig. 1A) or from that recorded in adult naïve animals (never deprived) (n ¼ 12, 0.92  0.02 c/deg; KruskaleWallis One-Way ANOVA on Ranks, post hoc Dunn’s Method; Fig. 1B). On the contrary, VA for the deprived eye in SC animals (0.62  0.05 c/deg) remained significantly lower than that for the fellow undeprived eye (1.04  0.03, paired t-test, t ¼ 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 ¼ 38.265 with 7 degrees of freedom, p < 0.001; post hoc Dunn’s Method, Q ¼ 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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rats (1.04  0.07) remained significantly lower than that recorded in adult naïve rats (One-Way ANOVA, F ¼ 7.435 with 7 degrees of freedom, p < 0.001; post hoc HolmeSidak method, t ¼ 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 ¼ 0.657). One-Way RM ANOVA with HolmeSidak method revealed that the visual acuity of the previously deprived eye measured after PL was significant increased with respect to that measured before visual training (p < 0.01) and remained unaltered 2 weeks after the end of PL (p ¼ 0.815). Visual acuity is obtained by extrapolation to 70% of correct choices on the sigmoidal function fitting 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, reflecting the predominance of crossed fibers 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 naïve controls (2.39  0.2; One-Way ANOVA, post hoc HolmeSidak method, p ¼ 0.315). No recovery was observed in SC rats (1.08  0.14; One-Way ANOVA, F ¼ 7.435 with 7 degrees of freedom, p < 0.001; post hoc HolmeSidak method, t ¼ 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 ¼ 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 ¼ 7.102 with 5 degrees of freedom, p < 0.001; Fig. 1A). A significant difference was detected when comparing the amblyopic eye VA of SS rats with that recorded in adult naïve animals (KruskaleWallis One-Way ANOVA on Ranks, H ¼ 38.265 with 7 degrees of freedom, p < 0.001; post hoc Dunn’s Method, Q ¼ 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 first step to characterize the role of visual stimulation, we investigated whether light stimulation of the amblyopic eye is necessary for the EE-induced recovery. Specifically, we measured visual functions in a group (n ¼ 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 significantly lower (0.61  0.02 c/deg) with respect to the other eye (1.00  0.07 c/deg; paired t-test, t ¼ 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 naïve animals (KruskaleWallis One-Way ANOVA on Ranks, H ¼ 38.265 with 7 degrees of freedom, p < 0.001; post hoc Dunn’s Method, Q ¼ 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 significantly lower than that recorded in naïve controls (One-Way ANOVA, F ¼ 7.435 with 7 degrees of freedom, p < 0.001; post hoc HolmeSidak method, t ¼ 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 ¼ 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 ¼ 0.938; Fig. 1A) and was completely comparable to VA recorded in adult naïve animals (KruskaleWallis One-Way ANOVA on Ranks, post hoc Dunn’s 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 naïve animals (One-Way ANOVA, post hoc HolmeSidak method, p ¼ 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

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progressively finer discriminations of visual stimuli. A group of reverse-sutured amblyopic rats (PL rats, n ¼ 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 first 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 ¼ 22.791 with 9 degrees of freedom, p < 0.001; Fig. 3). After the PL training, VA and OD were first 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 ¼ 0.06) (Fig. 1A) and from that recorded in adult naïve animals (KruskaleWallis One-Way ANOVA on Ranks, post hoc Dunn’s 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 naïve animals (One-Way ANOVA, post hoc HolmeSidak method, p ¼ 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 ¼ 4) by using a standard behavioral method, the visual water box task. Behavioral data completely confirmed 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 ¼ 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 ¼ 26.117 with 2 degrees of freedom, p < 0.001; post hoc HolmeSidak method, t ¼ 6.378). The beneficial 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 ¼ 0.815; Fig. 4). To demonstrate that VA recovery was specifically elicited by visual PL, we performed two controls. In the first, 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 ¼ 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 significantly lower with respect to that of the fellow eye (0.91  0.06 c/deg; paired t-test, t ¼ 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 ¼ 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 ¼ 9.432 with 3 degrees of freedom, p < 0.01; Fig. 5).

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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 first 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 ¼ 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 quantified 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 ¼ 10: 7.26  0.40%), in the visual cortex of amblyopic rats reared under classical EE conditions (n ¼ 5: 4.24  0.19%), in those subjected to EE combined with dark exposure (n ¼ 6: 5.34  0.54%), in those experiencing high levels of voluntary physical activity (n ¼ 5: 3.47  0.26%), in those subjected to visual enrichment (n ¼ 6:

Fig. 6. Time-course of the release of a putative neurotransmitter from synaptosomes exposed to a stimulation pulse. Synaptosomes were stratified on microporous filter, and neurotransmitter release was monitored during superfusion. After 36 min to equilibrate the system, two 3-min samples (t ¼ 36e39 min and t ¼ 45e48 min) were collected before and after one 6-min sample (t ¼ 39e45 min). Synaptosomes were exposed to the stimulus (90 s; gray bar) at the end of the first sample collected (t ¼ 39 min). A cartoon of the synaptosome technique is also represented.

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5.18  0.41%) and in animals that practiced in PL training (n ¼ 8: 3.82  0.42%) (One-Way ANOVA, F ¼ 11.143 with 7 degrees of freedom, p < 0.001; post hoc HolmeSidak method, t ¼ 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 ¼ 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 ¼ 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 ¼ 11; 1.4  0.18%; EE rats, n ¼ 5: 1.68  0.25%; ME rats, n ¼ 5: 1.54  0.25%; SS rats, n ¼ 12: 1.42  0.5%; DR-EE rats, n ¼ 5: 2.03  0.10%; VE rats, n ¼ 7: 1.82  0.21%; PL rats, n ¼ 8: 2.17  0.35%) (One-Way ANOVA, p ¼ 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 significant 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 ¼ 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 ¼ 10.301 with 4 degrees of freedom, p < 0.001) and to normal adult values (t-test, t ¼ 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 significant difference among the group levels (p ¼ 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 significant difference among the group levels (p < 0.001); a multiple comparison procedure (HolmeSidak method) showed that levels of GABA were significantly 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 ¼ 0.06). * p < 0.05; error bars, s.e.m.

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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 naïve 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 ¼ 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 significantly lower than that of adult naïve rats (One-Way ANOVA, F ¼ 7.779 with 3 degrees of freedom, p < 0.01; HolmeSidak method, t ¼ 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 specifically examined the contribution given by each EE component in inducing plasticity in the adult brain. This analysis could be very

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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 first 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 firing 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 specific 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 reflected by positive effects on learning and memory abilities (Madroñal 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 specifically 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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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 first step of the discrimination procedure, but did not proceed with the task of incrementally finer 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 specificity 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 final 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 specific 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 specifically 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 modifications, 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 specific 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 significant 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 efficacy 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 beneficial 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).

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