Reactivation of Ocular Dominance Plasticity in the Adult Visual Cortex

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Science  08 Nov 2002:
Vol. 298, Issue 5596, pp. 1248-1251
DOI: 10.1126/science.1072699


In young animals, monocular deprivation leads to an ocular dominance shift, whereas in adults after the critical period there is no such shift. Chondroitin sulphate proteoglycans (CSPGs) are components of the extracellular matrix (ECM) inhibitory for axonal sprouting. We tested whether the developmental maturation of the ECM is inhibitory for experience-dependent plasticity in the visual cortex. The organization of CSPGs into perineuronal nets coincided with the end of the critical period and was delayed by dark rearing. After CSPG degradation with chondroitinase-ABC in adult rats, monocular deprivation caused an ocular dominance shift toward the nondeprived eye. The mature ECM is thus inhibitory for experience-dependent plasticity, and degradation of CSPGs reactivates cortical plasticity.

Cortical circuits are sensitive to experience during well-defined intervals of early postnatal development called critical periods (1). After the critical period, plasticity is reduced or absent. Monocular deprivation (MD) is a classic model of experience-dependent plasticity. MD during the critical period results in a shift of ocular dominance (OD) of cortical neurons in favor of the nondeprived eye (2, 3). No OD shift is seen after MD in adult animals. The factors responsible for the cessation of OD plasticity in adults are only partially known. There is some evidence that the developmental increase in intracortical inhibition reduces plasticity and contributes to the termination of the critical period (4–6). However, other factors present in the adult visual cortex could stabilize synaptic connections and limit experience-dependent plasticity. CSPGs are attractive candidates for this role. CSPGs are components of the ECM that inhibit axonal sprouting and growth (7–9). Their adult pattern of expression is reached during late development, when CSPGs condense around the soma and dendrites of a subset of neurons in the form of perineuronal nets (PNNs) (10, 11). For instance, CSPGs recognized by the CAT-301, -315, and -316 antibodies appear in the cat visual cortex around the end of the critical period and their expression is regulated by visual experience (12).

If CSPGs in PNNs are involved in limiting OD plasticity in adults, the formation of adult-like PNNs around visual cortical neurons should coincide with the end of the critical period. We examined PNN formation using wisteria floribunda agglutinin (WFA), which binds the CSPG glycosaminoglycan (GAG) chains, and an antibody to the NH2-terminal fragment of the CSPG neurocan, which is associated with PNNs (13). During the critical period [postnatal day 22 (P22)], very few neurons were surrounded by PNNs although CSPGs were diffusely distributed (Fig. 1, A and B). The number of PNNs strongly increased in all cortical layers at P35 and reached adult levels at P70, coincident with the end of the critical period. Perineuronal staining for neurocan also increased, a process that continued during adulthood (Fig. 1, A and C). PNNs were more numerous in layer 4 than in supragranular and infragranular layers either during or after the end of the critical period (14). As in other brain structures, most (77 ± 1%, n = 3 rats) of the cortical neurons possessing PNNs use γ-aminobutyric acid (GABA) as a neurotransmitter, as shown by double staining with antibodies to the GABA-containing marker parvalbumin (15).

Figure 1

Neurocan and WFA staining increases throughout the critical period and is decreased by DR. (A) Neurocan and (B) WFA staining in the primary visual cortex (Oc1b) of P22, P70, and P100 animals. (C and D) Density of cells positive for (C) Neurocan and (D) WFA in Oc1b at different developmental stages (P22 rats, n = 3; P35,n = 4; P70, n = 3; P100,n = 3). For both kinds of staining, there is a significant effect of age [one-way analysis of variance (ANOVA),P < 0.05] (E) Neurocan staining in the visual cortex of P70 dark-reared rats (DR, left), age-matched controls (Nor, center), and dark-reared rats reexposed to light for 7 days (D-L, right). (F) Quantitation of neurocan-positive cells in the same groups (DR, n = 4; Nor, n= 4; D-L,n = 2). The first three bars (Oc1b) refer to density values measured in Oc1b, whereas the last two (Nonvisual) refer to values measured in a nonvisual area. The density of positive cells in the Oc1b of dark-reared rats is statistically different from that in both the Nor and D-L groups (one way ANOVA, post-hoc Tukey test,P < 0.01). No difference is present between the Nor and DR groups in the nonvisual area (Student's ttest, P > 0.05). (G) The density of WFA-positive cells in dark-reared (n = 4) and control (n = 4) rats is statistically different in layers 2 to 3 and 5 to 6 (t test, P< 0.05 and P < 0.01, respectively). Scale bar, 50 μm.

Rearing animals in complete darkness from birth prolongs the critical period for OD plasticity. We tested whether dark rearing (DR) up to P70 would inhibit the developmental maturation of PNNs (12). Condensation of neurocan into PNNs was almost completely prevented by DR (Fig. 1, E and F), although diffuse neurocan immunoreactivity was unaffected (14). The effect of DR appeared specific for visual cortical areas, because DR did not affect neurocan labeling in a nonvisual area (the retrosplenial cortex) located in the same sections used for the counts in the visual cortex. DR also caused a reduction in the number of PNNs in the deep and superficial layers, but not in layer 4 (Fig. 1G). Reintroducing dark-reared animals into a normal light/dark cycle rapidly terminates OD plasticity. One week of normal visual experience after DR completely restored neurocan immunoreactivity in PNNs to control values (Fig. 1, E and F).

These results, together with the known inhibitory properties of CSPGs toward axonal sprouting, prompted us to test whether the presence of CSPGs in the adult visual cortex inhibits experience-dependent plasticity. Much of the inhibitory activity of CSPGs toward axon growth can be prevented by degradation or inhibition of GAG chain synthesis (16, 17). Degradation of CSPG GAG chains in vivo with chondroitinase-ABC (chABC) reduces the inhibitory properties of the damaged central nervous system (CNS) sufficiently to promote axon regeneration (18, 19). We intracortically injected the adult visual cortex of rats with chABC every 3 days (14). Seven days after the first chABC injection, immunohistochemistry using 2B6, an antibody that selectively labels digested CSPGs, showed a complete removal of GAG chains over the primary visual cortex (area Oc1b, Fig. 2A). Neurocan staining in PNNs, staining of diffusely distributed neurocan (Fig. 2A), and binding of WFA (15) were also not present in the chABC-treated area. Control injections of penicillinase, an enzyme with no endogenous substrate, did not affect CSPG integrity (14). Treatment with chABC had no effect on neuronal or astrocytic survival (14, 20). We also controlled inflammatory response to the treatments using OX42, an antibody that reveals microglia and neutrophils. Penicillinase and chABC injections increased the number of hypertrophic microglia around the injection sites, but the appearance and number of OX42-positive cells within the recording zone in Oc1b [which is at least 1 mm away from the injection sites (14)] were not different from normal (Fig. 2, B and C).

Figure 2

Effects of chABC injection into the visual cortex. (A) 2B6 (top) and neurocan (bottom) staining after chABC treatment show CSPG degradation in the visual cortex treated with chABC. (B) OX42 staining of microglia in Oc1b of normal rats or rats injected with penicillinase (P-ase) or chABC. Inflammatory reaction to chABC or penicillinase injections was minimal within area Oc1b. (C) Density of OX42-positive cells in Oc1b of the three groups (n = 3 animals for each group) is not significantly different (one-way ANOVA, P > 0.05).

OD plasticity was assessed in adult (>P100) rats well after the end of the critical period. One eye was closed at the time of the first injection with chABC or penicillinase. The effectiveness of MD in shifting OD distribution was assessed after 7 or 15 days of MD by extracellular recordings of single-unit activity in the treated cortex (contralateral to the deprived eye). Cortical neuronal activity in untreated rats of the same age, either monocularly deprived or nondeprived, was also recorded. OD was attributed to each neuron according to the Hubel and Wiesel classification system or by computation of an OD score (14, 21,22). Seven days of MD were totally ineffective in shifting OD distributions in adult rats who were either untreated or treated with penicillinase (Fig. 3, A through D). By contrast, a pronounced shift of OD toward the ipsilateral, nondeprived eye was induced in rats treated with chABC (Fig. 3, E to G). Fifteen days of MD did not further increase the OD shift induced by 7 days of MD in the chABC-treated rats (Fig. 3, E to G). The OD distribution of the animals in each different group was summarized with the contralateral bias index (CBI) (6, 23), on which 0 represents complete ipsilateral dominance and 1 represents complete contralateral dominance. The CBIs of all rats treated with chABC were reduced with respect to normal rats or control monocularly deprived rats (Fig. 3H). To investigate whether CSPGs are involved in very early events activated by MD, we studied the effects of 2 days of MD. During the critical period (beginning at P22 to P23), 2 days of MD induced a nonsaturating shift of OD (CBI, 0.38 ± 0.03; four animals, 96 cells). MD of the same duration did not affect OD in adult rats treated with chABC (CBI, 0.6 ± 0.04; four animals, 82 cells).

Figure 3

CSPG degradation restores OD plasticity in the adult visual cortex. (A to D) MD has no effect in adult rats. OD distributions of (A) adult rats left nondeprived (Nor, six rats, 149 cells) or MD for 7 days either (B) untreated (MD, five rats, 163 cells) or (D) treated with penicillinase (MD + P-ase, five rats, 185 cells) are not statistically different (χ2 test, P > 0.05). (C) Statistical comparison of the cumulative distributions of the OD score confirms that MD has no effect in adult rats either untreated or treated with penicillinase [Kolmogorov-Smirnov (K-S) test, P> 0.05; Nor, 114 cells; MD, 115 cells; MD + P-ase, 127 cells]. (E and F) chABC treatment of the visual cortex of adult MD rats for (E) 7 days (MD + chABC 7d, five rats, 120 cells) or (F) 15 days (MD + chABC 15d, three rats, 73 cells) restores an OD shift. OD distributions of animals treated with chABC for 7 or 15 days significantly differ from those of the MD + P-ase, Nor, and MD groups (χ2test, P < 0.05). (G) Cumulative distribution of the OD score of MD, MD + P-ase, and MD + chABC treatment groups (7 days, 74 cells; 15 days, 49 cells). MD + chABC groups are statistically different from the MD and the MD + P-ase groups (P < 0.05, K-S test). No difference is present between the MD + chABC groups treated for 7 or 15 days (P > 0.05, K-S test). (H) The CBIs (23) of normal rats, rats monocularly deprived for 7 days during the critical period (Crit per), adult monocularly deprived rats (Ad), and adult monocularly deprived rats treated with penicillinase or chABC for 7 or 15 days. Solid circles represent the average CBI ± SEM; open circles represent the CBI of single rats. The CBIs of MD + chABC 7d and 15d rats are statistically different from those of MD + P-ase, Nor, and MD rats (one-way ANOVA, P < 0.001; post-hoc Tukey test, P < 0.05), whereas these latter groups do not differ among each other.

Treatment with chABC but without MD did not cause a shift of OD toward the ipsilateral eye (Fig. 4A). Functional properties of visual cortical neurons (Fig. 4, B and C) in chABC-treated rats (either nondeprived or monocularly deprived) were not different from those of normal adult rats or control rats. Visual acuity was also not affected (Fig. 4D). This indicates that chABC treatment restored OD plasticity without interfering with many important functional properties of cortical neurons.

Figure 4

chABC per se does not alter OD and other functional properties of visual cortical neurons. (A) OD distribution of adult rats treated with chABC for 7 days without MD (chABC, four rats, 68 cells). This distribution does not differ from that of normal rats (χ2 test, P > 0.05). (B) Receptive field (RF) size was not altered by penicillinase or chABC in either monocularly deprived or nondeprived rats (Nor, 79 cells; chABC, 54 cells; MD + chABC, 7 and 15 days, 112 cells; MD + P-ase, 109 cells). Data are represented as box charts: Horizontal lines denote the 25th, 50th, and 75th percentiles; error bars denote the 5th and 95th percentiles; square symbol denotes mean value. None of the treatments significantly altered RF size (one-way ANOVA on ranks, P = 0.186). (C) Cell responsiveness for each unit of the different treatment groups (Nor, 93 cells; chABC, 47 cells; MD + chABC, 116 cells; MD + P-ase, 109 cells) was expressed as peak response compared with baseline activity. No statistical difference is present between all groups (one-way ANOVA on ranks, P = 0.218). (D) Normal visual acuity after 7 days of chABC treatment of adult nondeprived rats. A representative example of visual acuity measurement in a chABC-treated rat by means of visually evoked potentials (VEPs) is shown. Estimated visual acuity is indicated on the abscissa by an arrowhead. Visual acuity of chABC-treated animals (0.95 ± 0.05 cycle/deg, n = 3 animals, inset) is within normal values, either estimated with VEPs or behaviorally [about 1 cycle/deg (3)].

Our results show that degradation of CSPG GAG chains restores OD plasticity in the adult visual cortex, demonstrating that the mature ECM is a crucial factor in regulating experience-dependent plasticity. The condensation of CSPGs into PNNs correlates with the end of the critical period for OD plasticity and is prolonged by DR. This suggests that CSPG assembly in PNNs is important for the restriction of OD plasticity to the critical period.

How can CSPG removal restore OD plasticity to the adult visual cortex? CSPG inhibitory action on axonal sprouting suggests that degradation of PNNs could remove nonpermissive substrates for experience-dependent generation or rearrangement of synaptic connections, two processes that are thought to underlie cortical plasticity (24–26). Another possible mechanism is suggested by colocalization of PNNs with parvalbumin-positive GABA-containing interneurons. Maturation of GABA-containing interneurons has been proposed to terminate the critical period for OD plasticity. Indeed, a reduction of inhibitory transmission in the adult cortex reactivates OD plasticity (27). Treatment with chABC did not alter several functional properties of visual cortical neurons that are known to be affected by pharmacological treatments with GABA antagonists (28), indicating that general disinhibition of cortical neurons is not the mechanism by which chABC reactivates OD plasticity. Further experiments are needed to understand whether CSPGs modulate plasticity by curbing the experience-dependent formation of new synapses on and by inhibitory neurons.

Because PNNs are found throughout the CNS, targeting the PNN-forming CSPGs could be a promising strategy to promote plastic mechanisms underlying recovery from amblyopia in the visual system (29), as well as from many forms of damage in many parts of the CNS. Recent experiments in which rat spinal cord injuries were treated with chABC showed behavioral improvements that may have been partly due to increased plasticity (19).

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