Scuola Superiore di Studi Universitari e Perfezionamento SantAnna Cl a s se A cca demi ca di Sci enze Speri mental i SE T T ORE DI SCIE NZE

M E DICHE

CHONDROITINASE ABC - MEDIATED OCULAR DOMINANCE PLASTICITY

FR OM M OLECULAR BASES T O TH ER APEUTI C TAR G ETS

ALLIEVO:

TUTOR:

FIL IPP O QUAT T RO NE

A N T O N I O L A B B A T E

ANNO A CCAD E MI CO

2009/2010

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ABSTR ACT

At birth, sensory cortical circuits are immature and shape their connections, experience-dependently, only during specific critical periods in early infancy. Restoration of this neuronal plasticity in the adult central nervous system could fix disorders due to a lack of environmental stimuli during critical periods. One of these diseases is amblyopia, caused by a dominance of one eye stimulation in primary visual cortex during its critical period. This situation leads to reduction of visual acuity in the non-dominant eye and loss of stereopsis, the depth perception given by binocular vision. A molecular correlate of the closure of critical periods is the organization of the extracellular matrix of the nervous tissue in condensed layers, known as perineuronal nets, around cell bodies, dendrites and proximal segments of axons. Chondroitinase-ABC is a bacterial enzyme that degrades chondroitin sulfate glycosaminoglycans and hyaluronan, which are key constituents of the perineuronal nets. This digestion induces a reopening of the critical period and thus promotes recovery of ocular dominance and visual acuity in the animal model of amblyopia.

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CONTEN TS

ABSTRACT

ABBREVIATIONS

INTRODUCTION

NEURONAL PLASTICITY IN VISUAL CORTEX

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1.1 The concept of neuronal plasticity and its evolution 1.2 Critical periods: a limit to plasticity 9

1.3 Ocular dominance in visual cortex as model of neuronal plasticity Figure 1. Experiments on ocular dominance plasticity 1.4 Molecular correlates of plasticity in visual cortex 11 10

EXTRACELLULAR ENVIRONMENT AND VISUAL CORTICAL PLASTICITY

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2.1 The architecture of brain extracellular matrix

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2.2 The role of extracellular matrix in ocular dominance plasticity

Figure 2. The perineuronal nets in adult and immature brain and after chondroitinase ABC treatment 15

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CHONDROITINASE ABC: BIOCHEMICAL ASPECTS

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Figure 3: Molecular structure of chondroitinase-ABC and its active site

CHONDROITINASE ABC EFFECTS ON OCULAR DOMINANCE PLASTICITY

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4.1 Experimental protocols to assess chondroitinase skills

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Figure 4. Chondroitinase ABC reactivates ocular dominance plasticity Figure 5. ChABC does not modify functional properties of cortical neurons Figure 6. ChABC allows recovery of OD in adult RS rats Figure 7. ChABC normalizes spine density in adult RS rats 4.2 Putative mechanisms of action of chondroi tinase AB C Figure 8. Anatomical plasticity and tPA: a mechanism model 23 21 21 22

CHONDROITINASE ABC-EFFECTS IN OTHER REGIONS: AN OVERVIEW

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CONCLUSIONS

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REFERENCES

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ABBR EV I ATI ONS

Arg BDNF C-4-S C-6-S CaMKII ChABC CNS CREB Crtl1 CS DR DS ECM EGF ERK GABA GAG Glu

Arginine Brain derived neurotrophic factor Chondroitin-4-sulfate Chondroitin-6-sulfate Ca2+/calmodulin dependent protein kinases II Chondroitinase ABC I Central nervous system cAMP response element-binding Cartilage link protein 1 Chondroitin sulfate Dark rearing Dermatan sulfate Extracellular matrix Epidermal growth factor Extracellular-signal-regulated kinases -Aminobutyric acid Glycosaminoglycan Glutamic acid

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HA His HLT MD NCAM NMDA OD P-ase PG PKA PKC PNN RPTP RS tPA Tyr V1 VEP

Hyaluronan Histidine Hyaluronanlectican-tenascin Monocular deprivation Neural Cell Adhesion Molecule N-methyl-D-aspartic acid Ocular dominance Penicillinase Proteoglycan Protein kinase A Protein kinase C Perineuronal nets Receptor tyrosine phosphatase Reverse suture Tissue plasminogen activator Tyrosine Primary visual cortex Visual evocated potential

I NTR OD UCTI ON

In occasion of my approach to chondroitinase-ABC (ChABC), I was impressed by its property of freeing the brain from past experiences and restoring in adulthood the malleability typical of the child. This skill could be useful not only to treat spinal lesions, traumas and amblyopia but also could help to cope with aging, making for instance possible to learn easily lifelong. This essay will show the results of the application of this tool in primary visual cortex (V1) where chABC treatment was able to induce in the animal model a reorganization of ocular dominance digesting brain extracellular matrix (ECM). This discovery could lead to a treatment for amblyopia, a visual disorder due to bad development of V1 during early infancy. The choice of this topic was influenced by the courses on physiology of perception and cellular biology held respectively by Nicoletta Berardi at Scuola Normale, and by Gian Michele Ratto at Scuola SantAnna. My experience in his laboratory at the NEST center in Pisa was an opportunity to better understand the world of neurosciences and the experimental procedures used in this field. After elucidating the concept of neuronal plasticity and critical period, the peculiarities of ocular dominance plasticity and its possible molecular correlates will be analyzed. A whole section will focus on the description of the ECM in the brain and the role of some components, the chondroitin sulfate proteoglycans, in the closure of periods of plasticity. Afterwards chABC biochemical properties will be outlined. Eventually attention will be conferred to results of chABC application in V1 and, briefly, in other districts.

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NEUR ONAL PLASTI CI TY I N V I SUAL COR TEX

1.1

THE CON CEP T OF N EUR ONAL PLASTI CI TY A ND I TS EV OLUTI ON

Neuronal plasticity is a term with a wide meaning referring to the changes in neural organization which may account for various forms of behavioral modifiability, either short-lasting or enduring, including maturation, adaptation to a mutable environment, specific and unspecific kinds of learning, and compensatory adjustments in response to functional losses from aging or brain damage (Berlucchi and Buchte, 2008). Neuronal plasticity was studied at the end of 19th century by many scientists such as James, Tanzi, Lugaro and Cajal but its modern definition was given by Jerzy Konorski in 1948. A year after Donald Olding Hebb in his The Organization of Behavior (1949) proposed that it is the synchronic activity of pre and post synaptic element to give more stability to a synapse (Berlucchi and Buchte, 2008). This concept is often summarized as cells that fire together wire together. Two are the forms of neuronal plasticity: synaptic plasticity, based on activity-dependent changing in synaptic strength of an existing synapse, and anatomical plasticity (also known as structural plasticity or remodeling) based on formation of new synapses (Galtrey and Fawcett, 2007). Recent studies (Chklovskii et al., 2004) show that wiring changes and weight changes are not mutual exclusive but that structural plasticity could cause a huge expansion of memory storage capacity, compared with weight plasticity alone. The anatomical correlates of structural plasticity are not large-scale modifications of the dendritic arbor, but modification of a small part of it: the dendritic spines, mobile tiny protrusions that emanate from dendritic shafts and contact axonal synaptic boutons. According to this hypothesis many studies showed dendritic spines growth and retraction are experience-dependent (Holtmaat and Svoboda, 2009).

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1.2

CR I TI CAL PER I OD S : A LI MI T TO PLASTI CI T Y

Neuronal plasticity is not constant during life: not only decreases with aging (Berardi et al., 2003) but is often confined in determinate time windows: the so called critical periods. A critical period is a time in the early stages of an organism's life during which it displays a heightened sensitivity to certain environmental stimuli, and its development is conditioned by experiences made at this time. Critical periods were found in virtually every animal in the development of visual, auditory and somatosensory systems and also in learning complex skills such as language or song birds (Berardi et al., 2000). The role played by experience in the development of neuronal circuits during critical periods is probably based on a hebbian selection of appropriate synapses and elimination of inappropriate ones (Berardi et al., 2000; Hensch and Stryker, 2004).

1.3

OCULAR D OM I NANCE I N V I SUAL COR TEX AS M OD EL OF NEUR ONAL PLASTI CI TY

The first and most studied critical period is the development of ocular dominance (OD) in primary visual cortex, called also striate cortex (because of the presence of the stria of Gennari, a visible band of myelinated axon) or V1. It is the first district where information from both eyes arrives, allowing binocular vision. OD is the property of binocular neurons of V1 of being activated to different degrees by visual stimuli presented to one eye or the other. In human beings the critical period lasts about two years (Banks et al., 1975) and its end coincides with the gain of mature visual acuity. The concept itself of critical period was first proposed by David Hunter Hubel and Torsten Wiesel studying with electrophysiology V1 neurons shift of responsiveness when one eye was deprived of vision during the critical period (Hubel and Wiesel, 1970). These experiments based on monocular deprivation (MD) showed a reduction of neurons excited by the sutured eye and an increase of neurons responding to the

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non-deprived eye (Wiesel and Hubel, 1963; Hubel and Wiesel, 1970; Hubel et al., 1977; Fagiolini et al., 1994). The consequence of this shift of OD, possible only during the critical period, is reduction of visual acuity of the deprived eye and loss of binocular vision similarly to human amblyopia (see figure 1). Another experimental protocol to study V1 plasticity is dark rearing (DR) that delays onset of the critical period (Fagiolini et al., 1994) suggesting that the precritical period contributes to the activation of the critical period (Hooks and Chen, 2007). Recent studies, however, showed OD maturation, especially the formation of OD columns, in contrast with Hubel and Wiesel view, is not a simple activitydependent event but it is also strongly gene-determined (Leamey, 2009; Katz and Crowley, 2002; Hooks and Chen, 2007).

Figure 1. Experiments on ocular dominance plasticity A) Ocular dominance distribution of single unit recordings from a large number of neurons in the primary visual cortex of normal adult cats. Cells in group 1 were activated only by the contralateral eye, cells in group 7 by the ipsilateral eye. Diagrams below these graphs indicate procedure, and bars indicate duration of deprivation (purple). Exp= time when experimental observations were made. Following closure of one eye from 1 week after birth until 2.5 months of age no cells could be activated by the deprived eye. Some cells could not be activated by either eye (NR, no response). A much longer period of monocular deprivation in an adult cat has little effect on ocular dominance (although overall cortical activity is diminished). In this case, the contralateral eye was closed from 12 to 38 months of age. (A after Hubel and Weisel, 1962; B after Wiesel and Hubel, 1963; C after Hubel and Wiesel, 1970.)

B) C)

(from D. Purves et al., Neuroscience, Sinauer associates, 2001)

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1.4

M OLECULAR COR R ELAT ES OF P LASTI CI TY I N V I SUAL COR TEX

Molecular mechanisms that regulate critical period in visual cortex are still unclear but many candidates have been proposed (Berardi et al., 2003). Some studies show that NMDA (N-methyl-D-aspartic acid) receptors, thought to be the neural implementation of hebbian hypothesis, switch their subunits 2B to 2A in coincidence with the closure of the critical period (Berardi et al., 2000) even if this changing is not essential for this process (Fagiolini et al., 2003). Moreover block of NMDA receptors avoids the effects of monocular deprivation (Bear et al., 1990). NMDA receptors allow Ca 2+ ions to enter in the neuron activating a complex network of kinases such as PKA, ERK, CaMKII and calcineurin phosphatase (Beaver et al., 2001; Di Cristo et al., 2001; Taha et al., 2002; Yang et al., 2005) that leads to activation of CREB transcription factor. Modification of this network causes absence of OD shift after MD (Berardi et al, 2004). Neurotrophins are secreted in response to electrical activity and enhance it. They are, therefore, able to strengthen the most active synapses (Berardi et al., 2003). In visual cortex BDNF (brain derived growth factor) overexpression accelerates both the development of visual acuity and the time course of OD plasticity. This is probably due to its action on intracortical inhibition. The intracortical inhibition, actually, must surpass a threshold before the critical period can start (Berardi et al., 2004). If intracortical inhibition is increased, either by early diazepam administration (Fagiolini and Hensch, 2000), that enhances the effect of the inhibitory neurotransmitter GABA, or by overexpression of BDNF (Hanover et al., 1999) the critical period starts and closes earlier. Another key element that regulates neuronal plasticity, tightly bound to the previous ones, is the extracellular matrix (ECM) and its principal components in CNS, the chondroitin-sulfate proteoglycans (CSPGs) (Berardi et al., 2003).

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EX TR ACELLULAR ENV I R ON M ENT AND V I SUAL COR TI CAL PLASTI CI TY

2.1

THE AR CHI TEC TUR E OF BR AI N EX TR ACELL ULAR M ATR I X

Histologically it is possible to identify in the brain three types of ECM: the first is a diffuse matrix that exists throughout the CNS, the second is defined by some cell surface-associated matrix molecules, the third is the dense organized matrix of perineuronal nets (PNNs), the most involved in ocular dominance plasticity (Sathyaseelan Deepa et al., 2006). PNNs are lattice like structure of neuronal and glyal origin that ensheathe neuronal bodies, proximal dendrites and axonal initial segments as far as the beginning of the myelin sheath. They are fenestrated at sites of synaptic contact (Fox and Caterson 2002, Karetko and Skangiel-Kramska, 2009). PNNs are present around specific types of neurons in the cortex (above all in motor and primary sensory areas), hippocampus, thalamus, brainstem and spinal cord. In the cortex they are associated with GABAergic interneurons, especially parvalbumin containing interneurons, and pyramidal cells (Murakami and Ohtsuka, 2003, Galtrey and Fawcett, 2007). PNNs were first described by Camillo Golgi and object of debate by first neuroscientists. After a long period of stagnation since 90s several studies have raised attention to PNNs (Celio et al., 1998). PNNs structure is still mysterious and it is known that their composition varies among different sets of neurons and between immature and mature brain. The most credited hypothesis on their shape is the HLT (hyaluronan lecticantenascin) matrix model (Yamaguchi, 2000) (see figure 2A). In this model PNNs are constituted of a ternary complex of hyaluronan, lecticans and tenascin. Hyaluronan (HA, also hyaluronic acid or hyaluronate) is a particular glycosaminoglycan (GAG). GAGs are usually large unbranched, negatively charged, variably sulfated polymers composed of 20200 repeating disaccharide units

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usually bound to a proteic core to form proteoglycans (PGs). The principal GAGs in PGs are chondroitin sulfate (CS), dermatan sulfate (DS), heparan sulfate, keratan sulfate. CSs show different patterns of sulfation: chondroitin-4sulfate (C-4-S) and chondroitin-6-sulfate (C-6-S) are the two most common variants present on CSPG (Bandtlow and Zimmermann, 2000). HA, however, is not sulfated and it is present, protein free, on cell surfaces and in the ECM. Lecticans or hyalectans (hyaluronan plus lectin) are a group of CSPG characterized by the presence of a hyaluronan-binding domain and a C-type lectin domain in their core proteins (Yamaguchi, 2000). The interactions between hyaluronan and CSPGs are stabilized via link proteins (Galtrey and Fawcett, 2007). They are responsible, thanks to GAG chains, of the well-hydrated and strongly anionic microenvironment surrounding the neurons (Karetko and SkangielKramska, 2009). Four lecticans have been identified in CNS, namely aggrecan, versican, neurocan and brevican. Tenascin, present in the two forms R and C, is a large molecular weight dimeric or trimeric ECM glycoprotein expressed predominantly in CNS. It is the physiological ligand for the C-terminal globular domain of lecticans: tenascin-R shows higher affinity for brevican while tenascin-C binds strongly with neurocan. The presence of tenascin C in PNNs, however, is not essential for their stability (Yamaguchi, 2000; Galtrey and Fawcett, 2007). Other CSPGs not included but compatible with HLT model were described in CNS ECM: RPTPs and phosphacans, decorin and biglycan, NG2 and neuroglycan C (see figure 2B). RPTP (receptor-type protein-tyrosine phosphatase ) is a transmembrane protein that sometimes carries GAG chains (it is one of the so called part-time PGs). Phosphacan is a RPTP secreted splice variant that binds to tenascin and also to cell surface receptors such as neural cell adhesion molecule (NCAM) through its CS GAG chains. Decorin and biglycan are small leucine-rich proteoglycans. NG2 and neuroglycan C are other transmem-

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brane CSPGs able to bind to tenascin (Bandtlow and Zimmermann, 2000; Fox and Caterson, 2002; Galtrey and Fawcett, 2007). To these molecules a heparan sulfate should be added: agrin. Essential for the development and maintenance of the neuromuscular junction, agrin plays an important role also in anatomical plasticity (Dityatev et al., 2010).

2.2

THE R OLE OF EX TR ACE L LULAR M ATR I X I N OCULAR D OM I NANCE PLASTI CI TY

Nowadays it is common opinion that the ECM has not only a structural role but participates in many cell-functions. In CNS, ECM contributes to cell migration, axonal growth and neuronal plasticity both synaptic and anatomical (Bandtlow and Zimmermann, 2000, Dityatev and Schachner, 2003). In OD plasticity, a case of experience dependent structural plasticity, the role of CSPGs in the PNNs, and of the Tissue plasminogen activator (tPA) was proved (Berardi et al., 2003), but also other molecules of ECM, such as agrin, may play a role in this process (Dityatev et al., 2010). PNNs begin to condense during later development and are complete after the end of the critical period. Moreover it was demonstrated that DR, treatment that prevents the closure of the critical period, prevents also the formation of PNNs. It was therefore proposed a role of PNNs in the end of ocular dominance plasticity (Hockfield et al., 1990) (see figure 2C). The mechanism by which CSPGs inhibits plasticity in the adult visual cortex is still unknown. The CSPGs have an inhibitory action on axonal sprouting during development and in case of spinal lesion, so they are probably also non-permissive substrates for rearrangement of synaptic connections (Berardi et al., 2004). It is important to say, however, that the organization of CSPGs into perineuronal nets is the key event in the control of CNS plasticity by ECM: knockout animals for Crtl1 (cartilage link protein 1), a protein that binds lecticans to HA, despite having unchanged

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levels of CSPGs and patterns of GAGs sulfation, retain juvenile levels of OD plasticity and their visual acuity remains sensitive to MD (Carulli et al., 2010). Moreover CS-chains are able to act as reservoir of many growth and inhibitory factors. They control localization of these factors promoting or inhibiting signaling and protect them from degradation (Crespo et al., 2007). tPA activity in the visual cortex is increased during MD in response to the unbalance between excitatory and inhibitory circuits. It was proposed that tPA cleaves adhesion molecules of inactive synapses. This initially causes spine motility and eventually spine retraction. This hypothesis is confirmed by the restoration of normal effects of MD in tPA-knockout mice by treatment with exogenous enzyme (Berardi et al., 2004) (see figure 8). Agrin is a heparan sulfate normally present in brain ECM. It is object of proteolytic cleavage by neurotrypsin, a serine protease secreted in an inactive form by the presynaptic terminal in case of action potential firing. The activation of neurotrypsin is possible only in presence of a NMDA receptors-dependent postsynaptic process. The cleavage of agrin unmasks a cryptic ECM-resident signal, a carboxy-terminal 22-kDa fragment (agrin 22), essential for the sprouting of filopodia, protrusions that can evolve in dendritic spines (Dityatev et al., 2010). One of the best ways of studying a biologic system is perturbing it. Therefore the effects of extracellular environment on neuronal plasticity were studied with an enzymatic tool able to deeply modify brain PNNs and, namely, GAG chains: chondroitinase ABC (see figure 2D).

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A.

B.

C.

D.

Figure 2. The structure of perineuronal nets (PNNs) in adult and immature brain and after chondroitinase treatment A) A schematic of the base pattern of mature PNNs based on a ternary complex of hyaluronan, tenascin-R and a member of the family of chondroitin sulfate proteoglycans (CSPGs) lecticans. B) Several subclasses of CSPGs (CS glycosaminoglycans are depicted as red lines) and their relations with other elements of the mature PNNs are showed. Members of the lectican subfamily neurocan (Nn) present with two cleavage isoforms neurocan-N, (Nn-N) and neurocan-C (Nn-C), versican (Vn), brevican (Bn), and aggrecan (An) associates with matrix hyaluronan (HA, pink) through globular hyaluronan-binding domains at their N-termini (yellow circles), while their C-terminal globular domains (white circles) binds to the matrix glycoprotein tenascin (T, triangles). Phosphacan (Pn), a secreted CSPG that carries also heparan sulfate (black lines in the figure), as well as to cell surface CSPGs such as neuroglycan C (NC) and NG2 takes part to PNNs binding to tenascin. Phosphacan can also bind to cell surface receptors such as neural cell adhesion molecule (NCAM). C) A model of the immature PNNs: a favorable environment for cell migration, axon growth and synaptogenesis is given by a loose matrix with a high amount of hyaluronan. D) Chondroitinase ABC is able to dramatically change extracellular environment degrading HA and CS glycosaminoglycans restoring the loose extracellular matrix present before the closure of the critical period. (modified from Yamaguchi, 2000; Fox and Caterson, 2002)

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CHOND R OI TI NASE ABC : BI OCHEM I CAL ASP ECTS

Chondroitin Sulfate ABC lyase I, usually called Chondroitinase ABC (ChABC sometimes also Chase), is a 997-amino-acid-residue endolytic enzyme from a soil bacterium, Proteus vulgaris, that uses it to digest cartilages in animal carcasses (Huang et al., 2003). ChABC shows chondroitin, C-4-S (in past called chondroitin sulfate A), DS (chondroitin sulfate B), chondroitin-6-sulfate (chondroitin sulfate C), and HA as substrates. It cleaves GAG chains to tetrasaccharides and disaccharides by -elimination of 1,4-hexosaminidic bond (Huang et al., 2003). Knowledge of its structure could be necessary to design modifications useful to have a more effective enzyme in future therapeutic applications (Huang et al., 2003). Nevertheless its mechanism of action is not still known also due to some mistakes in its published sequence (Huang et al., 2003; Prabhakar and Capila et al., 2005). Studies were not able to explain in a conclusive way, for example, how chABC is able to degrade both epimers of the uronic acid: the glucoronic acid of the CSs and the iduronic acid of DSs (Prabhakar and Capila et al., 2005; Prabhakar et al., 2005). A persuasive hypothesis could be the presence of two partially overlapping active sites catalyzing the respective reactions using the same substrate-binding site (Shaya et al., 2008). ChABC has three major domains. The N-terminal domain is the most mobile, involved in the bond with the glycosaminoglycan chains and contains a NA+ ion. The central one has a horseshoe shape and has catalytic properties. The Cterminal participates in giving to the molecule a C-shape and an extensive interacting surface (Huang et al., 2003) (see figure 3A). Phylogenetic and sitedirected mutagenesis studies elucidated, in part, the structure of the active site, showing the presence of a catalytic tetrad (see figure 3B and C) (Huang et al., 2003; Prabhakar and Capila et al., 2005; Prabhakar et al., 2005).

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Figure 3. Molecular structure of chondroitinase-ABC and its active site. A) ChABC has a horseshoe structure and three domains: an N-terminal one (green), a central one (blue), a C-terminal one (yellow). The red sphere indicates a Na+ ion. B) The active site consists of a tetrad of residues: Arg560 (stabilizes the charged intermediate); His 501 (necessary for the abstraction of the C-5 proton from the uronic acid moiety); Tyr 508 and Glu653 (both position the substrate via hydrogen bonding). Enzymes mutant for one of these residues are unable to digest their substrates. C) Schematic of the different amino acids in the active site and their proximity to a C-4-S molecule. The cleavable bond is between the 1 (non-reducing side) and +1 (reducing side) sites The -elimination of 1,4-hexosaminidic bond needs (I) the abstraction of the C-5 proton from the uronic acid moiety (made by His 501), causing the formation of a double bond between C-4 and C5, (II) the stabilization of the carbanion intermediate (mediated by Arg 560) (III) the protonation of the anomeric oxygen that breaks the glycosidic bond. (modified from Huang et al., 2003; Prabhakar and Capila et al., 2005; Prabhakar et al., 2005)

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CHOND R OI TI NASE ABC EFF ECTS ON OCU LA R DOM I NANCE PLAS TI CI TY

Two studies of the same group (Pizzorusso et al., 2002 and Pizzorusso et al., 2006) showed that treatment of the visual cortex of rats with ChABC was able to reopen the OD critical period in adulthood and recover the effects of early MD. Furthermore the procedure not only did not interfere with functional properties of cortical neurons but also corrected them in case of early MD. There is no evidence of inflammation of the treated part. It was showed a correlation between these overall effects and increase of dendritic spine density.

4.1

EX PER I M ENTAL PR OTOCO L S TO ASS ESS CH ONDR OI TI NASE SKI LLS

To demonstrate chondroitinase ABC is able to reopen the OD critical period, three cohorts of adult rats (>100 postnatal days) were used: the first one had an intracortical injection of chABC in binocular primary visual cortex every three days and, at the moment of the first injection, was monocularly deprived suturing the eyelids of the eye contralateral to the treated cortex; the second one had the same treatment but was given penicillinase, a control enzyme with no endogenous substrate; the third had only MD. Shift in OD distribution was assessed after 7 or 15 days of MD by extracellular recordings of single-unit activity in the treated cortex. As expected, no shift was found in only monocularly deprived or penicillinase treated rats, but, according to the hypothesis, a MD shift toward the ipsilateral, non-deprived, eye was found in chABC treated rats with no significant differences between animals deprived for 7 or 15 days (see figure 4) . Immunofluorescence studies using OX42, an antibody that binds microglia and neutrophils, revealed an increase in the number of hypertrophic microglia only in the injection site but not in the recording area (Pizzorusso et al., 2002).

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Injection of chABC without MD did not induce a shift of OD toward the ipsilateral eye. Functional properties (visual acuity, the measure of the spatial resolution of sight, and receptive fields) of visual cortical neurons were studied with visual evoked potentials (VEPs) protocol, based on recording of neuronal activity after presentation of a visual stimulus (a flashing light). The results were not statically different from controls (Pizzorusso et al., 2002) (see figure 5). Chondroitinase was also able to induce a recovery from effects of MD (Pizzorusso et al., 2006). The protocol used was based on reverse suturing (RS, deprivation of the previously open eye and opening of the previously deprived eye) that during critical period is able to correct OD. In adult controls RS caused a limited spontaneous recovery of OD but no improvement in visual acuity. ChABC treatment allowed, on the contrary, complete recovery of ocular dominance and of visual acuity in adult RS rats (see figure 6). Visual acuity was assessed both by recording VEPs (used also for receptive field) and by a two-alternative forced-choice discrimination task (visual water box). In this task a grating is displayed randomly on one of two monitors at the end of a tank filled with water while in the other screen there is a gray-field. Animals are trained to swim toward the screens and choose the screen displaying the grating. In this way they reach a submerged platform hidden below the screen and escape from water (Pizzorusso et al., 2006). The spatial frequency of the gratings is increased until the animals do not choose randomly between the two monitors. This means that its maximal visual acuity was reached. Dendritic spine density was assessed by immunolabelling of frozen brain sections of rats treated with RS or RS and chABC. MD decreases spine density of visual cortex, especially the contralateral due to the small decussation in the visual system of the rodents. Treatment with chABC, not per se but after visual experience and RS, made the dendritic spine density similar to normal animals (Pizzorusso et al., 2006) (see figure 7).

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Figure 4. Chondroitinase ABC reactivates ocular dominance plasticity The first histogram shows the distribution of ocular dominance (OD) dominance classes in a non treated adult rat (NOR). Monocular deprivation (MD, black histogram) or MD with injection of penicillinase (Pase, blue histogram) had no effects on OD in adult animals while chondroitinase ABC (chABC) treatment induced a shift toward the non deprived ipsilateral eye with no significant differences between the cohort treated for a week (red histogram) or for two weeks (green histogram). (modified from Pizzorusso et al., 2002)

(modified from Pizzorusso et al. 2002)

Figure 5. ChABC does not modify functional properties of cortical neurons A) B) C) D) Histogram distribution of the classes of ocular dominance in rat treated with chABC but without MD is the same of controls: chABC does not induce a shift in OD without MD. Receptive fields (RF) were not altered by treatment with penicillinase or chABC in monocularly deprived or non deprived rats. (data presented by boxplots, square symbol denotes mean value). Cell responsiveness was assessed as the ratio of peak response to baseline. There is no statistical difference between the groups (data presented by boxplots, square symbol denotes mean value). One week chABC treatment does not affect visual acuity. A representative example of visual acuity measurement by means of 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) is not different from normal values.

(modified from Pizzorusso et al., 2002)

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Figure 6. ChABC allows recovery of OD in adult RS rats A) Ocular dominance distribution of RS chABC-treated rats (red histogram) is within the normal values (Nor, blue line). The photo on the right shows the abolishment of WFA staining (Wisteria Floribunda Agglutinin, a marker for perineuronal nets) after chABC treatment in visual cortex. Treatment with penicillinase (P-ase) of RS rats does not induce any shift towards the first deprived eye while its distribution of OD class is similar to the one characteristic of monocularly deprived rats (MD, yellow line). On the right PNNs labeled with WFA appear intact after treatment with P-ase. Visual acuity of the two eyes (in black the deprived one) in controls (nor and P-ase) and chABC treated rats was assessed with VEPs technique. Treatment with chABC promoted recovery of visual acuity of the formerly deprived eye. Asterisks indicate a statistically different group. Also behavioral test (visual water box) gave similar results, showing a functional recovery of visual acuity of the amblyopic eye

B)

C)

D)

(modified from Pizzorusso et al., 2006)

Figure 7. ChABC normalizes spine density in adult RS rats In control rats there is no difference in spine density between the two cortex (Nor, one blue column). RS causes a decrease of spine density in the contralateral cortex to the formerly treated eye (RS, black columns). P-ase injection does not avoid this process (RS P-ase, green columns). ChABC-treated rats showed, however, a spine density not different from normal rats (RS chABC, red columns). The chABC treatment per se does not promote increase in spine density (chABC). (modified from Pizzorusso et al., 2006)

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4.2

PUTATI V E M ECHANI SM S OF ACTI ON OF CH ONDR O I TI NASE ABC

There are different hypotheses on the way digestion of ECM with chABC induces neuronal plasticity. It is likely that more factors play a role in such a complex process (Pizzorusso et al., 2006; Berardi et al., 2004; Crespo et al., 2007). Ocular dominance plasticity is thought be a case of anatomical plasticity based on rearrangements of dendritic spines. This is confirmed by the stability of spines underlined by the study of in vivo two-photon imaging in V1 (Grutzendler et al., 2002). It was shown that dynamic modifications of dendritic spines require extracellular proteolysis (Mataga et al., 2004; Oray et al., 2004). So ChABC, changing extracellular environment, could restore spine plasticity in adult. This action is probably direct to either the two principal neuronal species coated by PNNs: pyramidal neurons and GABAergic parvalbumin containing inhibitory interneurons. ChABC treatment could restore in this way an intracortical inhibition similar to the one present during critical period (Pizzorusso et al. 2006). In the loose matrix generated by chABC treatment also tPA is probably able again to depress non-active synapses (Berardi et al., 2004) (see figure 8). Other possible mechanisms that can take part in chABC effects are correlated to the release of growth bound to CSPGs chains or effects of some of their digestion products. Likely also the digestion of HA and its digestion products promote neuronal plasticity but not as much as CSPGs (Crespo et al., 2007). It was also proposed that CSPGs inhibit directly neurons via a receptor and this could be supported by the evidence of CSPG-mediated increases of intracellular Ca2+ with inhibitory effects on growth cone. In vitro experiments showed CSPGs activate protein kinase C (PKC) in cerebellar granule neurons in vitro. Active PKC has an inhibitory effect on neurite growth mediated by the activation of the small GTPase Rho. Recently, it has been discovered that suppression of the kinase activity of the epidermal growth factor (EGF) diminishes the

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inhibitory effects of CSPGs that are able to phosphorylate the EGF-receptor in a calcium-dependent manner (Crespo et al., 2007).

Figure 8. Anatomical plasticity and tPA: a mechanism model A) In vivo studies show that in V1 during the critical period changes in spines (indicated by arrows) happen very often while in the adult they are very scarce. This changing is probably mediated by molecules of the ECM like tPA. During the critical period, the imbalance in electrical activity caused by MD activates tPA release. Active synapses probably express adhesion molecules insensitive to tPA, while the ones present in inactive synapses are cleavable by it. tPA cleavage initially induces spine motility but eventually causes spine retraction. This generates a shift of OD in favor of the non-deprived eye (represented by the white eye on the schematic scale). tPA knock-out mice have not a shift in OD even if monocularly deprived. In the adult, the ECM is strongly non-permissive for morphological rearrangements, and tPA is no longer activated following MD. As a result, no changes in spine dynamics or in OD are present. ChABC changes the perineuronal environment promoting spine mobility and modifies the intracortical inhibitory circuits responsible of the detection of imbalance in electric activity. This could be a mechanism to explain how chABC induces shift in OD.

B)

C) D) E)

(modified from Berardi et al., 2004)

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CHOND R OI TI NASE ABC - EFFE CTS I N OT HER R EG I ONS : AN OV ER VI EW

PNNs, as previously stated, are widely diffuse in the CNS and in the last few years several studies have explored the effects on neuronal plasticity of the digestion of these structures in many different districts. The aim of these researches is to study the role of ECM in plasticity in the hope to find a way of promoting recovery from spinal injuries or traumas. Following CNS injury (e.g. a spinal cord lesion) a glial scar develops, containing ECM molecules including CSPGs that act as an inhibitory substrate for axonal growth. Treatment with chondroitinase ABC in rat spinal cord promotes restoration of plasticity in both injured and intact axons of ascending sensory projections and descending corticospinal tract (Bradbury et al., 2002; Barritt et al., 2006; Karetko and Skangiel-Kramska, 2009). CSPGs digestion promoted the regeneration of damaged dorsal root fibers and sprouting of intact dorsal ones restoring sensory function (Cafferty et al., 2007, 2008; Karetko and SkangielKramska, 2009). These results make the use of chABC in the treatment of spinal cord lesions the most promising application of this enzyme. Also digestion of the PNNs present in the amygdale after fear conditioning experiments gave interesting results. Fear conditioning in adult rats leads to the formation of memories that cannot be erased: spontaneous recovery of conditioned fear responses after extinction training shows that extinction involves new learning and not an erasure of previous fear memories. In young rats, however, extinction of conditioned fear induces memory erasure. This is probably due to the active protection against erasure given by PNNs that are present in the adult amygdale. ChABC treatment digesting PNNS was able to make fear memory erasable. These observations could lead to new strategies in treating post-traumatic stress disorders (Gogolla et al., 2009; Pizzorusso, 2009).

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CONCLUSI ONS

Studies on ChABC effects on OD plasticity are a fascinating example on how basic research could lead to findings of great clinical potential. They outline a new paradigm in treating disorders of brain development based on the search of factors promoting restoration of the plasticity typical of the immature brain. They show, besides, a pivotal role of brain ECM in many essential neuronal functions and so it is probable that alteration of PNNs could be involved in the pathogenesis of some neurological and neuropsychiatric disorders. Even if these results are very promising many questions have to be solved before thinking of therapeutic application of ChABC. Histological studies have to go on: the composition and the architecture of brain ECM are still mysterious and our staining methods reveal probably only a part of the PNNs present in the brain (Ajmo et al., 2008). Besides, we know still too little about the role of ECM molecules in both synaptic and anatomical plasticity and we do not understand the functional meaning of the large variety of GAGs pattern of sulfation and of PGs splicing variants. Use of other enzymes with different substrates and of knockout organisms will help to comprehend such a complex issue. An even more intriguing topic to solve is elucidating the role and possibilities of the endogenous regulation of ECM represented by some enzymes as neurotrypsin and metalloproteinases (Dityatev et al., 2010). Use of these enzymes could overwhelm immune response against chABC or its cleavage products and lead to more specific actions on ECM than the wide destruction oper-

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ated by ChABC. Such a deep modification of neuronal environment could lead to detrimental side effects such facilitation of tumor diffusion (Fox and Caterson, 2002). Neuronal plasticity is a complex process that involves many different kinds of molecules: it is likely that chABC treatment alone will not be able to give a complete recovery from development disorders or CNS injuries, but should be supported by supply of neurotrophins and other plasticity promoting factors. Among these factors the effect on V1 of agrin 22 injection should be studied with attention, due to its involvement in experience dependent plasticity. Experiments combining different factors could assess the effectiveness of a multiple approach to neuronal plasticity. Studies should also be conducted about dosage, frequency, and timing of the treatment to understand better its action and discover its best potentialities. A caveat for the application of ChABC in the treatment of amblyopia is represented by the deep differences between the almost monocular rodent visual system and the widely binocular human one. An important test for this therapeutic target will be the use of primates as animal models in experiments on OD plasticity.

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