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Research Report

Injury to retinal ganglion cell axons increases polysialylatedneural cell adhesion molecule (PSA-NCAM) in the adult rodentsuperior colliculus

J.A. Murphya, P.E.B. Nickersona, D.B. Clarkea,b,⁎aNeuron Survival and Regeneration Laboratory, Department of Anatomy and Neurobiology, Faculty of Medicine, Dalhousie University,5850 College Street, Halifax, Nova Scotia, Canada B3H 1X5bDepartment of Surgery (Neurosurgery), Faculty of Medicine, Dalhousie University, 5850 College Street, Halifax, Nova Scotia,Canada B3H 1X5

A R T I C L E I N F O

⁎ Corresponding author. Fax: +1 902 494 1212E-mail address: d.clarke@dal.ca (D.B. ClarAbbreviations: CNS, central nervous syste

ganglion cell; SC, superior colliculus

0006-8993/$ – see front matter © 2007 Elsevidoi:10.1016/j.brainres.2007.05.069

A B S T R A C T

Article history:Accepted 21 May 2007Available online 14 June 2007

The adult mammalian central nervous system (CNS) exhibits a limited regenerativeresponse to injury. It is well established that polysialylated neural cell adhesion molecule(PSA-NCAM) contributes to nervous system plasticity. In the visual system, PSA-NCAMparticipates in retinal ganglion cell (RGC) axon growth during development and specificallyinfluences RGC innervation of its principle target tissue, the superior colliculus (SC). Thegoals of this study were to determine whether PSA-NCAM is expressed in the normal adultmouse SC and to evaluate PSA-NCAMexpression following RGC injury. In the normal rostral,but not caudal, SC we find that PSA-NCAM is present in the retinorecipient layers; however,PSA-NCAM and RGC axons do not co-localize. In the deeper collicular layers, PSA-NCAM isobserved as several distinct patches that occur at the same depth along the medial–lateralaxis throughout the colliculus. RGC axotomy denervates predominantly the contralateralcolliculus, where increased PSA-NCAM levels are seen at 7 and 10 days after the injury.Further evaluation of the retinorecipient layers of the partially denervated SC reveals thatsome intact CTB-traced RGC axons (less than 5%) labeled from the ipsilateral eye do co-localize with PSA-NCAM. This study is the first characterization of PSA-NCAM expression inthe normal and partially denervated adult SC and may indicate that PSA-NCAM is involvedin attempted visual system remodeling after injury.

© 2007 Elsevier B.V. All rights reserved.

Keywords:Polysialic acidRetinal ganglion cellSuperior colliculusInjury

1. Introduction

Injured adult mammalian retinal ganglion cell (RGC) axons donot regenerate intrinsically; however, experimental interven-tions may be taken to successfully regrow some injured axonsand induce sprouting of a few intact axons. For example, some

.ke).m; NCAM, neural cell adh

er B.V. All rights reserved

injured RGC axons will regrow through a peripheral nerve graftto their principal target structure, the superior colliculus (SC),and form functional synapses within this region (Richardson etal., 1980; Carter et al., 1998; Sauve et al., 2001). With respect tointact axon outgrowth, following near complete denervation byunilateral enucleation at birth, the remaining RGCaxons extend

esion molecule; ON, optic nerve; PSA, polysialic acid; RGC, retinal

.

Fig. 1 – PSA-NCAM is present in the retinorecipient layers ofthe rostral SC in adult mice. PSA-NCAM immuno-staining(red: A, C) was evaluated in coronal sections of the right SC 5days after RGC axonswere labeled by an intravitreal injectionof CTB (green: B, D). In the rostral SC (2.9 mm posterior tobregma), PSA-NCAM immuno-labeling (A) is found in thesuperficial retinorecipient layers (B) and is present as distinctpatches (A, arrows) along the medial–lateral axis in thedeeper layers. In the caudal SC (4.6 mm posterior to bregma),PSA-NCAM immuno-staining (C) is localized within the SZand as distinct patches (arrows) in the deeper layers;however, in contrast to the rostral SC, no PSA-NCAM ispresent in the retinorecipient layers (D). Control sections ofthe SCwhere the primary antibodywas omitted (E) and of theSC in NCAM−/− mice (F) are PSA-NCAM-negative. Scalebars=200 μm.

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into the deafferented area of the tectum (Lund and Lund, 1976).This phenomenon does not occur in the adult intrinsically;however, experimental manipulations such as exposure togrowth factors can enhance intact RGC axon outgrowth inresponse topartial denervationof the adult rodentSC (Tropeaetal., 2003). These studies indicate that the adult mammalian SCmay possess the capacity for plastic changes following injury iffostered in the appropriate environment. Despite concentratedinvestigation into facilitating the growth of RGC axons afterinjury, few studies have explored the endogenous molecularmechanisms in target structures that may influence thisregeneration, and none has specifically examined the role ofthe neural cell adhesion molecule (NCAM).

NCAM is a cell surface protein that exists as three mainisoforms (NCAM-120, -140 and -180) produced through alter-native splicing of a single gene product and distinguished bytheirmolecular weights (Cunninghamet al., 1987; Barbas et al.,1988). NCAM's function is believed to shift from a plasticitypromoting molecule during development to a mediator ofstructural stability within the adult (reviewed by Rutishauserand Landmesser, 1996; Bruses and Rutishauser, 2001; Durbecand Cremer, 2001). This modification in function is accom-plished primarily by the developmental regulation of PSA onNCAM. PSA reduces the adhesiveness between cells bydecreasing physical impedance or charge repulsion (Yang etal., 1992; Johnson et al., 2005), which is thought to render tissuepermissive to axon growth. During the embryonic period, PSA-NCAM is expressed throughout the entire nervous system(Rothbard et al., 1982; Chuong and Edelman, 1984); however, inthe adult, it is localized predominantly to regions character-ized by plasticity or neurogenesis (Miragall et al., 1988; Aaronand Chesselet, 1989; Bonfanti et al., 1992; Kiss et al., 1993).Furthermore, PSA-NCAM is upregulated in several neuralregions following injury (Daniloff et al., 1986; Bonfanti et al.,1996; Rodriguez et al., 1998; Dusart et al., 1999), and changes inPSA-NCAM expression may be functionally important sinceenzymatic removal of PSA delays neurite outgrowth fromhippocampal cultures (Muller et al., 1994) and affects prefer-ential motor reinnervation of regenerating femoral nerves(Franz et al., 2005).

In the visual system during development, NCAM has beenshown to play a critical role in RGC axon growth, guidance andfasciculation. PSA-NCAM regulates the guidance of developingintra-retinal chick RGC axons (Monnier et al., 2001), plays acritical role in RGC axon fasciculation (Yin et al., 1995) and tectalinnervation (Thanos et al., 1984; El Maarouf and Rutishauser,2003) and is expressed on mouse RGC axons as they growtoward their target structures (Bartsch et al., 1990; Chung et al.,2004). PSA-NCAM has also been localized on regeneratingmouse retinal neurites (Bates et al., 1999) and is functionallyimportant for chick retinal neurite outgrowth (Zhang et al.,1992). Together, these findings implicate PSA-NCAM as beingimportant in promoting and directing RGC axonal growth. Amore comprehensive knowledge of PSA-NCAM localizationfollowing injury is imperative to understand this molecule'spotential role following CNS insult and throughout recovery.

The goals of this study are to determine the distributionand localization of PSA-NCAM in the normal adult rodent SCand to determine whether its expression is altered in responseto RGC injury. Specifically, we have characterized the differ-

ences in localization of PSA-NCAM along the rostral–caudaland dorsal–ventral axes in the normal SC and have investi-gated PSA-NCAM levels and localization in the colliculus afteroptic nerve (ON) transection.

2. Results

2.1. PSA-NCAM distribution and localization in theuninjured adult mouse SC

We demonstrate the presence of PSA-NCAM within the SC ofadult (2–4months old)mice (n=6) relative to RGC axons (Fig. 1).Themorphology of our PSA-NCAM immuno-labeling is similarto that of published reports investigating other CNS regions(Aaron and Chesselet, 1989; Bonfanti et al., 1992; El Maarouf et

Fig. 2 – PSA-NCAM in the SC does not co-localize with RGC axons from the contralateral retina. PSA-NCAM immuno-staining(red) was evaluated in coronal sections of the rostral right SC 5 days after RGC axons were labeled by an intravitreal injection ofCTB (green). High power examination of the rostral SC using confocalmicroscopy reveals that PSA-NCAM (A) is not co-localizedwith CTB-traced RGC axons (B) despite the extensive amount of PSA-NCAM present in the same region as RGC axons. Panel Cdemonstrates merged image. M=midline, D=dorsal. Scale bar=50 μm.

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al., 2005) and the patterns of anterograde traced RGC axons incoronal sections of the SC are consistent with previous studies(Godement et al., 1984; Wu et al., 2000; Yakura et al., 2002). Inthe rostral SC, we demonstrate that PSA-NCAM is expressedubiquitously in the stratum zonale (SZ), the most superficiallayer of the SC which is located immediately dorsal to RGCaxons. In addition, PSA-NCAM is localized in a heterogeneouspattern throughout the retinorecipient stratum opticum (SO)and stratum griseum superficiale (SGS) layers which containCTB labeled RGC axons from the contralateral eye (Figs. 1A andB). PSA-NCAM is also present in the deeper layers of the SC,which we define as all layers ventral to the retinorecipientlayers, where it is expressed as several distinct patches oflabeling found along themedial–lateral axis at the same depthrelative to the surfaceof theSC. In contrast to the rostral SC, thecaudal SC has almost no PSA-NCAM in the regions innervatedby RGC axons (Figs. 1C and D). However, PSA-NCAM in the SZand deeper layers of the caudal SC is similar to the pattern ofimmuno-labeling found in these regions in the rostral SC (Figs.

Fig. 3 – PSA-NCAM is found adjacent to some RGC axons from tclearly, we investigated PSA-NCAM immuno-labeling (red) in theretina. Panels A–C demonstrate the presence of PSA-NCAM (A) in(C, merged image). (D–F) Higher power magnification with confocco-localized with CTB (E), patches of CTB labeling are found concmerged image). Scale bars=100 μm (A–C), 20 μm (D–F).

1A and C). We find no staining when the primary antibody isomitted (Fig. 1E) and sections from NCAM−/− mice (Fig. 1F) areimmuno-negative.

Since PSA-NCAM was localized in the same region as RGCaxons only in the rostral SC, we investigated whether PSA-NCAM and CTB labeling of RGC axons from the contralateraleye co-localize in this region (n=3). Within the superficiallayers of the SC, PSA-NCAM does not co-localize with CTB-traced RGC axons (Fig. 2). This finding is consistent with aprevious study that reported no PSA-NCAM on RGC axons inthe ON of adult mice (Bartsch et al., 1990). Next we wanted toevaluate PSA-NCAM localization in the rostral SC relative toRGC axons projecting from the ipsilateral retina (n=6). Thepurpose of this investigation was to allow us to distinguishindividual RGC axons more clearly since of all rodent RGCaxons that project to the SC, only 5–10% project from theipsilateral eye (Cusick and Lund, 1982; Simon and O'Leary,1992). Low magnification indicates that PSA-NCAM is presentin the rostral SC in the environment of RGC axons from the

he ipsilateral retina. To identify individual RGC axons moreSC and CTB-traced RGC axons (green) from the ipsilateral

the environment of RGC axons (B) at low powermagnificational microscopy reveals that, although PSA-NCAM (D) is notentrated around PSA-NCAM-positive cell bodies (arrows; F,

Fig. 5 – PSA-NCAM levels increase in the contralateral SCafter optic nerve transection. Partially denervated SC werecollected at various times (in days) after axotomy, andWestern blot analyses were performed for PSA-NCAM usingthe 5A5 anti-PSA antibody (A). No differences in proteinlevels were apparent after amido black stain. (B) PSA-NCAMlevels were quantified at various times after axotomy andincreased significantly at 7 (174±40% higher than C,P=0.014) and 10 (210±71% higher than C, P=0.005) days.Levels are expressed as a percent of PSA-NCAM in theuninjured right SC±standard error of the mean (control).C=control; 1, 2, 4, 7, 10 and 14 are the number of days afteraxotomy; and (−/−) represents NCAM−/− SC. 180 representsmolecular weight in kDa. AB=amido black. Different from C:* represents P≤0.05.

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ipsilateral retina (Figs. 3A–C). With higher magnification, wedetermined that although no CTB and PSA-NCAM co-localiza-tion was found (n=3), examples were observed where RGCaxons from the ipsilateral retina surround PSA-NCAM-positivecells in the rostral SC (Figs. 3D–F). Furthermore, most PSA-NCAM-positive cells in the dorsal, rostral SC surrounded NeuNpositive nuclei, which suggests that these cells are neurons(Fig. 4).

Together, these findings indicate that PSA-NCAM ispresent in the retinorecipient layers of the rostral but notcaudal SC. Although no co-localization of PSA-NCAM and RGCaxons was found in the SC, RGC axons or terminals are foundadjacent to PSA-NCAM-positive cells in the rostral SC wherepolysialic acid in the extracellular space may influence intactRGC axons.

2.2. Polysialylation of NCAM increases in the SC followingtransection of the contralateral optic nerve

In the normal uninjured SC, we detected PSA-NCAM levelswith Western blot analysis (Fig. 5A, lane 2). A faint non-specific line (∼160 kDa) that did not correspond with specificbands was present at all times and in the NCAM−/− tissue. Noother stainingwas detectedwith NCAM−/− tissue (Figs. 5A and6, lane 1). PSA-NCAM levels were also assessed using the 5A5antibody on control tissue treated with Endo-N and, asexpected, no PSA-NCAM was detected (data not shown). SCof mice with no surgery were used as controls for all analyses.Following transection of the left ON, PSA-NCAM levels in thepartially denervated right SC increased significantly(F6,14=3.292, P=0.031 for injury day effect; n=3/time afterinjury) at 7 (mean % increase relative to control±SEM=174±40%, P=0.014) and 10 (210±71%, P=0.005) days after the injury(Fig. 5B). This increase in PSA-NCAM was no longer significantat 14 days after injury (Fig. 5).

We then assessed NCAM levels in the normal uninjuredand injured SC, as well as the specific NCAM isoformlocalization of PSA in the uninjured SC (n=3/time) (Fig. 6).We detect the presence of all 3 isoforms of NCAM (NCAM-180,-140 and -120) in the adult mouse SC (Fig. 6A, lane 2). PSA isfound predominantly on NCAM-180, with little or no PSA onNCAM-140 and NCAM-120 in control uninjured SC (Fig. 6B).NCAM-180, -140 and -120 did not change significantly at anytime after injury (Fig. 6A). No differences in protein levels were

Fig. 4 – PSA-NCAM-positive cells in the superficial layers of thewere double immuno-labeled for PSA-NCAM (A, red) and the neimage and demonstrates that most PSA-NCAM-positive cell bod(arrows). Scale bars: 20 μm. All confocal images in this figure rep

apparent after amido black stain. Taken together, these dataindicate that PSA is localized predominantly on NCAM-180and increases following injury, whereas NCAM levels do notchange in response to ON axotomy.

normal rostral SC surround NeuN positive nuclei. Normal SCuronal marker NeuN (B, green). Panel C represents a mergedies in the rostral superficial SC contain NeuN positive nucleiresent single optical sections taken from z-stacks.

Fig. 7 – PSA-NCAM and GFAP expression increases afteroptic nerve transection in the partially denervated SC. Lowmagnification microscopy of PSA-NCAM (A, red) and GFAP(B, green) in coronal sections through the partiallydenervated right SC at 7 days after a left ON transectionreveals increased expression of both PSA-NCAM and GFAPrelative to the left SC. Similar stainingwasobservedat 10daysafter RGC axotomy. Scale bar=200 μm.

Fig. 6 – PSA is localized predominantly on NCAM-180.Denervated SC were collected at various times (in days) afterON transection, and Western blot analyses were performedfor NCAM using an anti-NCAM antibody that bindsspecifically to NCAM and not PSA. (A) There is no significantchange in NCAM levels (-180, -140 and -120) at any time afterON transection. Comparison of NCAM bands in tissue treatedwith Endo-N to remove PSA (A) and tissue not treated withEndo-N (B) reveals that PSA is localized predominantly orexclusively on NCAM-180, whereas NCAM-140 andNCAM-120 are mostly or completely without polysialylationin control tissue. C=control; 1, 2, 4, 7, 10 and 14 are thenumber of days after ON transection. 180, 140 and 120represent molecular weights in kDa.

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2.3. PSA-NCAM is expressed on some intact RGC axonsfollowing partial denervation of the SC

Next, we wanted to investigate the anatomical source of theelevated PSA-NCAM in the SC following RGC axotomy at timesafter injury when PSA-NCAM levels were significantly greaterthan control levels. Co-immuno-labeling for GFAP and PSA-NCAM was performed to determine whether reactive astro-cyteswithin the SC express PSA-NCAM.At 7 and 10 days after aleft ON transection, PSA-NCAM expression appears greater inthe partially denervated right SC (Fig. 7A), which is consistentwith our quantification of PSA-NCAM after injury with West-ern blot analysis (Fig. 5). Consistent with our previous results(Krueger-Naug et al., 2002), we found an abundance of GFAPimmuno-labeling selectively in the partially denervated rightSC (Fig. 7B) (n=2/group). Despite the abundant upregulationof GFAP and PSA-NCAM throughout the colliculus (Figs. 8Aand B), analysis with confocal microscopy reveals distinct,non-overlapping patterns of GFAP and PSA-NCAM immuno-labeling within the retinorecipient and the deeper layers ofthe SC (Figs. 8C–F). GFAP and PSA-NCAM are, however,localized adjacent to each other throughout the SZ (Figs.8A and B).

To determine whether intact RGC axons projecting fromthe ipsilateral eye into the right SC express PSA-NCAM inresponse to near complete denervation, we performed anintravitreal injection of CTB into the right eye 5 days prior to aleft ON transection. We found no examples of co-localizationbetween PSA-NCAM and CTB-traced RGC axons from theipsilateral retina in control brains with no RGC axotomy (n=2)(Fig. 3). In the rostral right SC at 7 days after a left ONtransection, the majority of CTB labeling from the ipsilateralretina does not co-localize with PSA-NCAM; however, someexamples of fibers double-labeled for PSA-NCAM and CTB arepresent (less than 5%; n=2) (Fig. 9). This finding indicates that

at least some RGC axons from one eye upregulate PSA-NCAMin response to injury of RGC axons in the contralateral eye.

3. Discussion

In the present study, we have identified PSA-NCAM in thenormal adult SC and evaluated changes in its level andlocalization following partial denervation. In the normaluninjured rostral, but not caudal, SC we show that PSA-NCAM is present in the retinorecipient layers but does not co-localize with RGC axons and that some RGC axons areconcentrated around PSA-NCAM-positive cells. In the partiallydenervated SC, PSA-NCAM (but not NCAM) levels increasesignificantly at 7 and 10 days after RGC injury. PSA-NCAM inthese colliculi co-localizes with some intact RGC axons fromthe ipsilateral eye. This study is the first to characterize theexpression and localization of PSA-NCAM in the normal adultSC and after RGC axotomy, and provides evidence that PSA-NCAM may be important for remodeling of the retinalprojection system after injury.

3.1. PSA-NCAM localization

We observe distinct, non-overlapping patterns of PSA-NCAMand GFAP immuno-staining throughout the retinorecipientand deeper layers of the colliculus suggesting that they are not

Fig. 8 – PSA-NCAM in the retinorecipient and deeper layers of the SC does not co-localize with GFAP after optic nervetransection. GFAP (A and D; green) and PSA-NCAM (B and E; red) immuno-labeling were evaluated in the partially denervatedcontralateral SC at 10 days (A–F) after a left ON transection. Panel C demonstrates an example of a GFAP-positive glial cell withinthe retinorecipient layers that is not PSA-NCAM-positive. Additional analysis with confocal microscopy reveals distinct,non-overlapping patterns of GFAP and PSA-NCAM immuno-labeling within the retinorecipient and the deeper layers of thepartially denervated colliculus (D–F). Panel F demonstrates a merged image of panels D and E. The small amount of labelingoverlap observed in panel F is an artifact due to tissue thickness. Scale bars=50 μm (A, B and D–F), 10 μm (C).

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co-localized after ON transection (see Fig. 8). In addition to thespatial discrepancy of PSA-NCAM and GFAP within the SC,their temporal elevated expressions are also distinct. PSA-NCAM increases transiently, exhibiting greater levels at 7 and10 days but not at 14 days after ON transection, whereas GFAPexpression is elevated as early as 4 days after RGC axotomy(our own unpublished observations inmice and Krueger-Nauget al., 2002 published results in rats) and remains high for amonth (Krueger-Naug et al., 2002). Clearly the changes in the

Fig. 9 – PSA-NCAM co-localizes with a few intact RGC axons or tethe vitreous of the right eye to label RGC axons, including the fewwas transected to partially denervate the contralateral SC. Mice wpartially denervated SC was examined using confocal microscopCTB-traced intact RGC axons (B, green); however, a few examplemagnification (A–C, inserts) identifies the PSA-NCAM-positive RGz-stack taken with confocal microscopy. Scale bars=5 μm (A–C),

injury-induced expression of GFAP and PSA-NCAM are inde-pendently regulated events. Our finding that PSA-NCAM doesnot appear to be localized on GFAP immuno-labeled astrocytesin the retinorecipient layers of the SC is unexpected given thatreactive astrocytes express PSA-NCAM in the hypothalamus(Alonso andPrivat, 1993; Theodosis et al., 1999;Monlezun et al.,2005), arcuate nucleus (Hoyk et al., 2001) and ON (Becker et al.,2001). However, consistent with our findings, other studieshave found significant differences in the temporal and spatial

rminals in the partially denervated SC. CTB was injected intothat project to the ipsilateral SC. Five days later, the left ONere sacrificed 7 days after ON transection and the right

y. Most PSA-NCAM (A, red) does not co-localize withs (arrows) of double-labeled axons were found (C). HigherC axons at four separate intervals of 0.22 μm through a1 μm (all inserts).

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expressions of PSA-NCAM and GFAP. For example, after injuryin the spinal cord, PSA-NCAM is upregulated transiently whileGFAP expression persists for much longer (Oumesmar et al.,1995; Bonfanti et al., 1996). Although injury-induced PSA-NCAM upregulation persists with GFAP expression for at least18 months after injury in the cerebellum, some GFAPexpressing cells do not label for PSA-NCAM (Dusart et al.,1999). Together, these findings demonstrate cell, tissue andtime specific differences in the expression of PSA-NCAM andGFAP. In the SZ of the colliculus, PSA-NCAM and GFAP (see Fig.8) are localized adjacent to each other. As previously noted inother similar studies (Varea et al., 2005), electron microscopywould be required to confirm PSA-NCAM's exact cellularlocalization.

3.2. Significance of PSA-NCAM in the normal SC

PSA-NCAM expression has been reported but not cha-racterized in the SC of 2- to 3-month-old adult rats (Aaronand Chesselet, 1989); however, a subsequent study investigat-ing all PSA-NCAM immuno-labeling throughout the brain didnot report its presence in the SC in adult rats 3–12 months ofage (Bonfanti et al., 1992). We find clear differences in PSA-NCAM immuno-staining between the rostral and caudalsuperficial layers of the SC (see Fig. 1). The primary functionsof the retinorecipient superficial layers of the SC are to form amap of the visual image by receiving input directly from theretina and indirectly from the visual cortex and to transmitthis signal to the deeper layers where motor commands arecommunicated to the brainstem and spinal cord (reviewed byMcLaughlin et al., 2003; May, 2005). In the rodent, RGCs in thetemporal retina innervate the rostral SC whereas RGCs in thenasal retina project axons to the caudal SC (Scalia and Arango,1979; Simon and O'Leary, 1992). Since temporal RGCs areinvolved in selective processing of the nasal visual field(Wagor et al., 1980; Espinoza and Thomas, 1983), the presenceof PSA-NCAM in the retinorecipient layers of the rostral SCmay function selectively in processing information from thenasal visual field. In addition, this distinction in PSA-NCAMlocalization may indicate that the cellular environmentaround axon terminals originating from RGCs in the temporalretina may be more permissive to axon growth and synapseformation than those originating from RGCs in the nasalretina; however, since RGC axon growth is absent after injury(Lund and Lund, 1976), post-synaptic PSA-NCAM alone isclearly not sufficient to initiate RGC axon growth.

In the deeper SC layers, we visualize PSA-NCAM as severaldistinct patches of immuno-staining at the same depth acrossthe medial to lateral axis throughout the entire colliculus (seeFigs. 1A and C). Numerous studies have previously reportedlocalization of other neurochemical substances in discontin-uous patches of labeling within the deeper collicular layers,which appear similar to the PSA-NCAM immuno-labeling weobserve. These substances include acetyl cholinesterase (Siou,1958; Ramon-Moliner, 1972), choline acetyltransferase (Illing,1990), enkephalin (Graybiel et al., 1984), parvalbumin (Illing,1990), somatostatin (Spangler and Morley, 1987), calretinin(Illing, 1996), substance-P (Behan et al., 1993), cytochrome oxi-dase (Sandell, 1984; Wallace, 1986) and hexokinase (Sandell,1984). This patterning is likely due to the structural organiza-

tion of the deeper layers of the SC where specific afferents andefferents are known to form distinct clusters of projections(Illing and Graybiel, 1985; Wallace, 1986; Illing, 1990; Illing,1992; Jeon and Mize, 1993; Illing, 1996; Mana and Chevalier,2001). In contrast to the superficial SC layers, which functionexclusively in processing vision, the deeper layers receivesensory inputs for somatosensory, auditory and visualinformation; however, with respect to vision it is wellestablished that the primary role is saccadic eye movements(reviewed by Isa and Saito, 2001; Krauzlis et al., 2004).Localization of PSA-NCAM on specific afferents or efferentsby anatomical tracing and subsequent lesioning studies, aswell as analysis of saccadic eye movements after removal ofcollicular PSA, may reveal a role for PSA-NCAM in the deeperlayers of the adult colliculus.

3.3. Significance of altered PSA-NCAM expression intarget tissue of injured RGCs

Changes in PSA-NCAM levels are generally associated withstructural plasticity. In the optic tectum of Xenopus, PSA-NCAM expression is higher during the distinct critical periodswhen ipsilateral isthmotectal axons possess the ability to shifttheir connections (Williams et al., 1996). Furthermore, axonalbranching of motor axons in the developing chick limb isreduced by enzymatic removal of PSA, and thus PSA ondeveloping motoneurons is necessary for appropriate targetinnervation (Landmesser et al., 1990; Tang et al., 1992; Tang etal., 1994; Rafuse and Landmesser, 2000). Our data demonstrat-ing a transient increase in PSA-NCAM within the SC after ONtransection (see Figs. 5 and 7) suggest that the RGC targetenvironment may be permissive to axon growth; however,this phenomenon does not occur in the adult (Lund and Lund,1976; Gan and Harvey, 1986; Tan and Harvey, 1997). Sinceapplication of growth factors to the colliculus following partialdenervation induces some sprouting within 7 days (Tropea etal., 2003), the fact that PSA-NCAM levels increased onlytransiently likely does not explain the absence of adult RGCaxon outgrowth. The absence of RGC axon outgrowth in theadult SC may be explained, at least in part, by the lack of PSA-NCAM on GFAP immuno-reactive astrocytes throughout theretinorecipient layers of the SC. Consistent with this explana-tion, transfection of glial scar astrocytes with the polysialyl-transferase PST, which produces increased PSA-NCAMexpression, induces regrowth of some corticospinal fibersafter lesion (El Maarouf et al., 2006).

Another potential explanation as to why adult RGC axonssubsequently fail to sprout into the denervated area is the factthat most (more than 95%) intact RGC axons or terminals lackPSA-NCAM. Consistent with this, neurite outgrowth from anembryonic retina transplanted over the SC at birth occursfollowing removal of the contralateral eye at 1 month of age(Radel et al., 1991). One would predict that these neuritesexpress polysialic acid since PSA-NCAM is present on mouseRGC axons during development (Bartsch et al., 1990; Chung etal., 2004), on regenerating retinal neurites in vitro (Bates et al.,1999) and is functionally important for chick RGC growth andtarget innervation (Yin et al., 1995; El Maarouf and Rutishau-ser, 2003). Furthermore, PSA-NCAM has been localized on CNSaxons that are regenerating through peripheral nerve grafts in

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other regions of the brain (Zhang et al., 1995). It remains to bedetermined whether over-expression of PSA-NCAM specifi-cally on RGC axons and terminals would induce plasticchanges and recovery following injury.

Although, at least in our model, the presence of PSA-NCAM on post-synaptic cells is not sufficient to induceoutgrowth from intact RGCs in response to injury, wespeculate that PSA-NCAM in the adult colliculus maycontribute to the ability of RGC axons, which have success-fully regenerated through a peripheral nerve graph (Vidal-Sanz et al., 1987) to functionally reinnervate the SC (Carter etal., 1989; Keirstead et al., 1989; Thanos, 1992; Sasaki et al.,1993; Sauve et al., 1995; Sasaki et al., 1996). Although thereappears to be no preference for regenerating RGC axons ofadult hamsters to grow into the rostral versus the caudal SC,they do tend to grow into their appropriate topologicaldistribution (Sauve et al., 2001). In the peripheral nervoussystem, PSA-NCAM has been found to influence interactionsbetween motoneuron axons and guidance molecules (Tang etal., 1992). Therefore, the selective presence of PSA-NCAM inthe rostral SC, or lack of PSA in the caudal SC, may interactwith axon guidance molecules to influence the topologicaldistribution of reinnervating RGC axons. In addition, sincePSA-NCAM plays a key role in synaptogensis in other CNSregions (Seki and Rutishauser, 1998; Dityatev et al., 2004), thepresence of PSA-NCAMwithin the retinorecipient layers of therostral colliculus may facilitate synapse formation followingRGC axon regeneration.

3.4. Conclusions

The transient increase in PSA-NCAM levels and the fewexamples of PSA-NCAM co-localized with intact RGC axonsin the partially denervated colliculus suggest a muted attemptat remodeling in response to injury. The observation thatmostintact RGC axons do not express PSA-NCAM after injury mayhelp to explain the limited RGC axon plasticity within the SCand indicates that targeted upregulation of PSA-NCAMselectively on RGC axons or terminals may enhance visualsystem remodeling and promote functional recovery follow-ing injury.

4. Experimental procedures

4.1. Animals

Adult female C57BL/6 mice (18–20 g; Charles River Canada, St.Constant, PQ, Canada) were used for all experimental proce-dures and were cared for by the Dalhousie UniversityCommittee on Laboratory Animals in accordance with theCanadian Council on Animal Care. Adult NCAM−/− micegenerated from the C57BL/6 strain (8–14 weeks; Case WesternReserve University, Cleveland, Ohio, USA) were originallycreated as previously reported (Cremer et al., 1994) and wereused as a negative control throughout the experimentalprocedures. All surgical procedures were performed undergeneral anesthesia using a mixture of ketamine (135 mg/kg),rompun (7.2 mg/kg) and acepromazine (1.17 mg/kg) in 0.9%saline, administered by intraperitoneal (i.p.) injection.

4.2. Anterograde tracing of RGC axons

Cholera toxin-β (CTB) is commonly used for tracing projec-tions in the visual system (Ling et al., 1997; Wu et al., 1999,2000; Tropea et al., 2003) and is known to label axons, andtheir terminals and varicosities (Ling et al., 1998; Vercelli etal., 2000). To label RGC axons with CTB, the sclera of the eyewas exposed as previously described (Mansour-Robaey et al.,1994). Briefly, a 30-gauge needle was inserted through thesclera and retina into the vitreous chamber of the eye by aposterior approach. A unilateral injection of CTB conjugatedwith Alexa Fluor-488 (2 μl at 2 μg/μl in sterile 0.1 M PBS;Molecular Probes, Eugene, OR) was slowly administered intothe vitreous of one eye through the opening created by theneedle, using a 2 μl Hamilton syringe fitted with a drawn-outglass micropipette. Care was taken to ensure that no injurywas made to other structures of the eye, specifically the lensand the anterior portions of the eye, which both promoteregeneration when punctured (Mansour-Robaey et al., 1994;Leon et al., 2000; Pernet and Di Polo, 2006). After injection, theopening through the sclera was sealed with Histo-acryl tissueadhesive. Subsequent experimental procedures were per-formed 5 days after CTB injection. High levels (5 mg) of CTB-HRP tracers have been shown to transfer transsynaptically inthe visual system at only 6 days after injection (Trojanowskiand Schmidt, 1984). However, interneuronal transfer of CTB isunlikely in our study since we used only a small amount ofthe tracer (4 μg) and we did not find CTB-labeled cell bodies inthe SC.

4.3. Optic nerve transection

The left ON was transected with microscissors at approxi-mately 0.5 mm from the globe of the eye as describedpreviously (Mansour-Robaey et al., 1994). The right eyes servedas an internal control and animals with no surgery were usedas naive controls. Animals whose eyes showed evidence ofischemia or infection were excluded from the analysis.Animals were sacrificed at 1, 2, 4, 7, 10 and 14 days after ONtransection.

4.4. SDS–PAGE Western blot analysis

Following the experimental period, mice were anesthetizedwith an i.p. injection of a lethal dose of general anesthesiaand were perfused transcardially with 20 ml of chilled 0.1 Mphosphate buffer (PB). In one set of experiments, right andleft SC were dissected and separated using one mouse foreach time. This procedure was repeated in two other expe-riments using pooled tissue from two mice, instead of onemouse, for each time (n=3/injury time). All tissues werestored at −80 °C. Tissue was homogenized in extraction buffer(50 mM HEPES, 150 mM NaCl, 1 mM EDTA, 2 mM PMSF,100 μg/ml leupeptin, 0.2 TIU/ml aprotinin, 1% NP-40), placedon ice for 1 h and then centrifuged at 14,000 rpm for 1 h at4 °C. Supernatant was extracted and protein analysis wasperformed using bicinchoninic acid method (BCA proteinassay reagent; Pierce, Rockford, IL). To visualize NCAM, tissue(0.5 μg) was incubated for 2 h at room temperature (Rt) inendoneuraminidase (Endo-N; 2 μl of 6.7 U/μl in 50% PBS/

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Glycerol; a gift from Dr. Urs Rutishauser), an enzyme thatspecifically degrades linear homopolymers of sialic acid withalpha-2, 8-ketosidic linkages and requires a minimum chainlength of at least 7–9 residues (Silver and Rutishauser, 1984;Vimr et al., 1984). To visualize PSA-NCAM, tissue (0.5 μg) wasnot incubated in Endo-N. Next, tissue samples were incubatedin sample buffer (250mMTris–HCl [pH 6.8], 4% sodiumdodecylsulfate [SDS], 10% glycerol, 2% β-mercaptoethanol and 0.003%bromophenol blue), boiled for 5 min at 100 °C, separated ondenaturing 6% SDS–polyacrylamide gels at 75 V and trans-ferred onto a polyvinylidene fluoride membrane at 120 V for1 h (Millipore, Mississauga, ON, Canada). Membranes were air-dried, wet with methanol and rinsed in Tris-buffered saline(TBS) for 5 min. Membranes were then blocked in 5% dry,nonfat milk in TBS with 0.1% Tween 20 (TBS/T) for 1 h, washed3 times for 5 min each in TBS/T (all washes followed thisprotocol) and incubated overnight in monoclonal rat anti-NCAM (1:200, MAB310, Chemicon International; Temecula,CA) or monoclonal mouse anti-PSA (1:20; 5A5, DevelopmentalStudies Iowa City, IA) (Dodd et al., 1988), each in TBS/Twith 5%bovine serum albumin. Blots were then washed as describedabove, incubated for 1 h at Rt in peroxidase-conjugated goatanti-rat IgG (1:1000, Chemicon), or goat anti-mouse IgM(1:2000; Chemicon), and washed again. To visualize primaryantibodies, membranes were reacted with enhanced chemilu-minescence system (ECL, Amersham Biosciences; Little-Chal-font, Buckingshire, UK) according to the manufacturer'sprotocol and exposed to ECL Hyperfilm (for anti-NCAM pri-mary), or reacted with ECL-Plus (for anti-PSA primary; Amer-sham Biosciences) according to the manufacturer's protocoland scanned for fluorescence with STORM-840 (AmershamBiosciences). Images were edited for brightness and contrastonly using Adobe Photoshop 7.0. Protein levels were quanti-fied by scanning blots and applying densitometry (ScionImage, Frederick, Maryland). The membranes were stainedwith amido black to ensure that equal amounts of proteinwere loaded into the lanes on each gel.

4.5. Immunohistochemistry

Mice were perfused transcardially with 20 ml of chilled PBfollowed by 20 ml of chilled 4% paraformaldehyde (PFA) in PB.Brains were dissected, post-fixed overnight at 4 °C and cryo-protected in 30% sucrose. Coronal brain sections (30 μm)through the SCwere cut on a freezingmicrotome and stored inMillonig's solution (16.88 mg/ml NaH2PO4H2O, 3.86 mg/mlNaOH, 0.006%Na azide in distilledwater) at 4 °C. Sectionswerewashed 3 times for 5 min each (all washes followed thisprocedure) and placed in blocking buffer (10% serum in 0.1 MPBS/3% Triton-X 100) for 30 min at Rt on a shaker. Sectionswere then incubated in primary antibody for monoclonalmouse anti-PSA (1:5, 5A5) alone or with monoclonal mouseanti-neuronal nuclear protein (NeuN, 1:500; MAB377, Chemi-con) or polyclonal rabbit anti-glial fibrillary acidic protein(GFAP, 1:500; Dako Cytomation; Mississauga, ON, Canada). Allprimary antibody incubations were stored overnight at 4 °C ona shaker. Sections were then washed and incubated in theirappropriate secondary: cy2-conjugated goat ant-rabbit IgG(Jackson Immuno-Research Labs; West Grove PA); or rhoda-mine-conjugated goat anti-mouse IgM (AP128R, Chemicon).

All secondaries were diluted to 1:200 in PB and were incubatedfor 1 h at Rt on a shaker. Sections were washed, floated ontogelatinized slides, air-dried overnight and coverslipped withVectashield (Vectashield Laboratories; Burlington, CA) orCitifluor (Marivac Limited; Montreal, Quebec, Canada). Slideswere coverslipped using 1-ounce micro-cover glasses at #0thickness.

4.6. Microscopy

Sections were visualized using an inverted Zeiss microscope(Axiovert 200) or with a Zeiss LSM 510 confocalmicroscope. Forconfocal microscopy, pinhole diameter was maintained at 2.0airy units for all wavelengths. Laser outputs were set at 5%(488 nm), 80% (543 nm) and 50% (633 nm). Emission filters,including band-pass filters, were used to control for channelbleed through as follows: LP N633 nm (Cy5), band-pass 505–530 nm (Cy2) and band-pass 560–615 nm (Cy3). Co-localizationin multi-labeled sections was confirmed by confocal micro-scopy with orthogonal analysis or with a focal plane thicknessset at b0.25 μm.Where appropriate, sections were adjusted forbrightness and contrast.

4.7. Statistical analysis

Statistics are expressed as mean percentages±standard errorof the mean (SEM), relative to control. The Levene test wasused to determine the homogeneity of variances. Statisticalsignificance was assessed with a one-way analysis of variance(ANOVA). For parametric or non-parametric statistics with asignificant main effect, a Fischer's LSD or Games–Howell posthoc test was used respectively to assess differences betweengroupswith SPSS 9.0 software (SPSS, IL, USA). Differences wereconsidered statistically significant if P≤0.05.

Acknowledgments

This research has been supported by funding from the NaturalSciences and Engineering Research Council of Canada(NSERC), the Capital Health Research Fund and the Depart-ment of Surgery at Dalhousie University. The authors wish tothank Dr. Urs Rutishauser for kindly providing Endo-N, Ms.Tanya Myers and Ms. Margaret Luke for their technical assis-tance and Dr. Victor Rafuse for his assistance in data analysis.

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