Environmental enrichmentprevents effects of dark-rearingin the rat visual cortexAlessandro Bartoletti1,4, Paolo Medini1,4, Nicoletta Berardi2,3 &Lamberto Maffei1,2

Environmental enrichment potentiates neural plasticity,enhancing acquisition and consolidation of memory traces1. In the sensory cortices, after cortical circuit maturation andsensory function acquisition are completed, neural plasticitydeclines and the critical period ‘closes’2. In the visual cortex,this process can be prevented by dark-rearing, and here weshow that environmental enrichment can promote physiologicalmaturation and consolidation of visual cortical connections indark-reared rats, leading to critical period closure.

During early postnatal development, visual cortical connections arehighly plastic. They consolidate progressively and become less modifiableby experience, in parallel with visual function maturation2. The absenceof visual experience from birth prevents this maturation. In particular,visual connections do not consolidate, remaining plastic well after thenormal critical period, and visual acuity does not develop2. Recently itbecame clear that environmental enrichment and exercise (EE, largecages with running wheels and toys) have strong effects on plasticity ofneural connections1. Exposure to EE increases the expression of severalfactors3, particularly insulin-like growth factor I (IGF-I)1,4, which in turnenhances the electrical activity of specific populations of CNS neurons1,4.An increase in electrical activity triggers a series of molecular eventsimportant for visual cortical plasticity, including an increase in neu-rotrophin expression, which is one result of exposure to EE1. Here weinvestigated whether dark-rearing effects, which stem from the lack ofvisually driven activity, could be counteracted by exposure to EE.

The present study used two experimental groups: dark-reared rats inconditions of EE (DR-EE rats) and dark-reared rats in standard housingconditions (DR-nonEE rats). If EE counteracts the effects of dark-rearingon visual cortical plasticity, one would expect to see a difference betweenthese two groups in susceptibility to monocular deprivation (MD) doneafter critical period closure (postnatal day 45, P45, in rats5).

We assessed the effectiveness of MD in shifting ocular dominance(OD) distribution in rats dark-reared from birth or in normal rats byextracellular recordings of single-unit spiking activity in the binocularvisual cortex (Oc1B) contralateral to the deprived eye. OD was quantita-tively attributed to each neuron (see Supplementary Methods online).

As expected, MD (1 week) from P50 was still effective in shifting OD ofvisual cortical neurons in favor of the ipsilateral non-deprived eye in DR-nonEE rats (n = 6; Fig. 1a,b). However, MD from P50 was ineffective in

DR-EE rats (n = 7, littermates of DR-nonEE rats, placed in EE at P18), asin rats with normal visual experience (Fig. 1a,b). The OD distribution ofeach animal was summarized with the contralateral bias index (CBI; Fig.1c; Supplementary Methods and Note). Mean CBI for DR-nonEE rats +MD was significantly lower than for DR-EE rats + MD and for normalnondeprived (n = 5, 0.6 ± 0.05) or MD (n = 5, 0.61 ± 0.06) adult rats; CBIsin these latter three groups did not differ.

Thus, EE promotes the consolidation of developing visual connec-tions, allowing a normal critical period closure in dark-reared rats.

Dark-rearing prevents the developmental organization into perineu-ronal nets (PNNs)6 of chondroitin sulphate proteoglycans (CSPGs),components of the extracellular matrix recently shown to be nonpermis-sive factors for visual cortical plasticity. Indeed, removal of CSPGsrestores OD plasticity6. We have now found that EE greatly reduces theeffects of dark-rearing on the development of PNNs (Fig. 2). In P50 DR-EE animals, which no longer show OD plasticity in response to MD, thedensity of neurons surrounded by PNNs was normal in layer 4 (Fig. 2

1Scuola Normale Superiore, Piazza dei Cavalieri, Pisa, Italy. 2Istituto di Neuroscienze del CNR, Via G. Moruzzi 1, Pisa, Italy. 3Dipartimento di Psicologia, Università diFirenze, Via San Niccolò 93, Florence, Italy. 4These authors contributed equally to this work. Correspondence should be addressed to N. B. (berardi@in.pi.cnr.it).

Published online 15 February 2004; doi:10.1038/nn1201

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Figure 1 Environmental enrichment promotes consolidation of visual corticalconnections in dark-reared rats. (a) Ocular dominance distributions (ODD) innormal adult rats (n = 5, 115 cells), in adult rats subjected to 1 week MD (n = 5, 117 cells) and in DR-EE + MD and DR-nonEE + MD rats (n = 7, 159cells and n = 6, 138 cells, respectively). Only in DR-nonEE + MD rats was therea significant (P < 0.001) ODD shift toward the open eye (open circle). (b) Cumulative fractions for OD scores. For each cell, an OD score wascomputed (Supplementary Methods and Note). Number of animals as in a, cells114, 115, 122 and 105 for normal, adult + MD, DR-EE + MD and DR-nonEE +MD, respectively. Only the curve for DR-nonEE animals significantly differedfrom that in normals (Kolmogorov-Smirnov test, P < 0.05). (c) Summary of MDeffects in all DR animals. CBI scores in DR-nonEE + MD rats differ from thosein DR-EE + MD rats, which do not differ from those in normal adults (shadowedrectangle) (one-way ANOVA, P < 0.001, post-hoc Tukey’s test). Error barsrepresent s.e.m.©

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and Supplementary Note) and nearly normal in layers 2/3 and 5/6. Onthe contrary, in DR-nonEE animals, still susceptible to MD effects, den-sity of PNNs-surrounded neurons was much lower than normal6.

There is growing evidence that maturation of inhibition in the visualcortex is a determinant of critical period2. We quantified the expressionof GAD65 (glutamic acid decarboxylase, the biosynthetic enzyme for theinhibitory transmitter GABA) in the presynaptic boutons of GABAgergicinterneurons around the soma of target neurons (see ref. 7 andSupplementary Note online) in P50 normal (n = 4), DR-nonEE (n = 4)and DR-EE (n = 5) animals. We found that, as known for other markersof GABAergic function8, dark-rearing decreased GAD65 expression;however, GAD65 expression was normal in DR-EE rats.

The developmental decline of plasticity in the visual cortex is corre-lated with the maturation of visual acuity. Visual acuity in rats is low at eye-opening and reaches adult value within 1 month of age5. Dark-rearing prevents visual acuity development5. We assessed EE effects onvisual acuity maturation in rats dark-reared from birth (Fig. 3).

Electrophysiological recordings were made from Oc1B at P60, andvisual acuity was estimated by visual evoked potentials (VEPs)5,6. VEPsare a sensitive measure of visual cortical development (SupplementaryMethods). In P60 DR-nonEE rats (n = 8), visual acuity was low (0.58 ±0.04 cycles per degree, cpd) as expected; on the contrary, in P60 DR-EErats (n = 11), visual acuity was normal (1.03 ± 0.02 cpd; Fig. 3).

We conclude that EE promotes visual acuity development in dark-reared rats. It should be noted that these EE effects observed in dark-reared rats may not apply in the same measure to other species withmuch higher visual acuities, as monkeys and humans, in which lack ofvisual experience has more pronounced effects on visual performance9.However, we have shown in a mammalian species that it is possible tomodulate the outcome of visual deprivation by varying the environmen-tal conditions. This suggests that factors not under the control of visualexperience may contribute to visual cortical development.

EE promotes the expression of several factors that could control visualcortical development and plasticity.A particularly good candidate is IGF-I; IGF-I receptors are present in the occipital cortex10 and IGF-I couldtherefore influence the expression of molecules relevant for visual corti-cal plasticity such as NGF and BDNF. Indeed, EE increases NGF andBDNF expression in the visual cortex11, and BDNF overexpression andNGF supply prevent dark-rearing effects2,12.

A causal relation between the protective effects of exposure to EE andthe cascade from increased IGF-I to increased BDNF expression has beenshown in several models of neurodegeneration4. Whether this relationholds also for EE effects in dark-reared rats remains to be seen.

Note: Supplementary information is available on the Nature Neuroscience website.

ACKNOWLEDGMENTSThis work was supported by MIUR COFIN, Fondazione Telethon, FIRB and FISR.

COMPETING INTERESTS STATEMENTThe authors declare that they have no competing financial interests.

Received 4 November 2003; accepted 6 January 2004Published online at http://www.nature.com/natureneuroscience/

1. Cotman, C.W. & Berchtold, N.C. Trends Neurosci. 25, 295–301 (2002).2. Berardi, N., Pizzorusso, T., Ratto, G.M. & Maffei, L. Trends Neurosci. 26, 369–378

(2003).3. Molteni, R., Ying, Z. & Gomez-Pinilla, F. Eur. J. Neurosci. 16, 1107–1116 (2002).4. Carro, E., Trejo, J.L., Busiguina, S. & Torres-Aleman, I. J. Neurosci. 21, 5678–5684

(2001).5. Fagiolini, M., Pizzorusso, T., Berardi, N., Domenici, L. & Maffei L. Vision Res. 34,

709–720 (1994).6. Pizzorusso, T. et al. Science 298, 1248–1251 (2002).7. Huang, Z.J. et al. Cell 98, 739–755 (1999).8. Benevento, L.A., Bakkum, B.W. & Cohen, R.S. Brain Res. 689, 172–182 (1995).9. Maurer, D., Lewis, T.L., Brent, H.P. & Levin, A.V. Science 286, 108–110 (1999).10. Frolich, L. et al. J. Neural Transm. 105, 423–438 (1998).11. Pham, T.M., Winblad, B., Granholm, A.C. & Mohammed, A.H.. Pharmacol. Biochem.

Behav. 73, 167–175 (2002).12. Gianfranceschi, L. et al., Proc. Natl. Acad. Sci. USA 100, 12486–12491 (2003).

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Figure 2 Environmental enrichment prevents dark-rearing effects on CSPGdevelopmental organization into perineuronal nets (PNNs) in the visual cortex.(a–c) Examples of staining for WFA (wisteria floribunda agglutinin, which labelsPNNs, green) and NeuN (neuronal marker, red) in Oc1B of a normal, a DR-nonEE and a DR-EE rat at P50. The decrease caused by dark-rearing in the numberof PNNs was counteracted in EE-DR animals. Calibration bar: 100 µm. (d) Quantification of the density of PNNs surrounded neurons in layers 2/3, 4and 5/6 of normal (n = 4), DR-non EE (n = 4) and DR-EE (n = 5) animals. Thereduction in PNN density caused by dark-rearing and the increase in PNNdensity in DR-EE with respect to DR-nonEE animals is significant in all layers(one-way-ANOVA on ranks, Dunn’s post-hoc test, P < 0.001). Error barsrepresent s.e.m. See Supplementary Note online.

Figure 3 Environmental enrichment promotes development of visual acuity indark-reared rats. (a) Examples of VEPs recorded from Oc1B in response tovisual stimulation with gratings of spatial frequencies 0.2 and 0.6 cpd, in P60DR-nonEE and DR-EE rats. Gratings are temporally modulated at 4 Hz (period250 ms); the principal component of the VEP response is on a temporalfrequency twice the stimulus temporal frequency (second-harmonic). VEPresponse to a blank field reported to show noise level. (b) Example of visualacuity estimate in a DR-EE and a DR-nonEE rat. Experimental points arenormalized VEP second-harmonic amplitudes; thick lines are linear fits to thedata. Estimated visual acuities are indicated by arrows. The gratings in figurehave spatial frequencies in the ratio 1:2:4. (c) Summary of visual acuities in allDR animals; shadowed rectangle is the range of visual acuity in normal adults(1.05 ± 0.1 cpd, not different from DR-EE rats, one-way ANOVA, P < 0.001,post-hoc Tukey’s test). Error bars represent s.e.m.

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