Up-regulation of fast-axonally transported proteins in retinal ganglion cells of adult rats with optic–peroneal nerve grafts

Molecular Brain Research 53 Ž1998. 53–68

Research report

Up-regulation of fast-axonally transported proteins in retinal ganglion cells of adult rats with optic–peroneal nerve grafts Ben C. Wouters, Susan Bock-Samson, Kenneth Little, Jeanette J. Norden

)

Department of Cell Biology, Vanderbilt UniÕersity School of Medicine, Medical Center North C-2310, NashÕille, TN 37232, USA Accepted 12 August 1997

Abstract Metabolic labeling and quantitative 2D gel fluorography were used to assess changes in the synthesis and transport of five fast-axonally transported and developmentally regulated proteins ŽGAP-43, SNAP-25, and proteins of 18, 22, and 23r24 kDa. after grafting of a peroneal nerve segment onto a transected optic nerve in adult rats. After optic nerve transection alone, only GAP-43 was up-regulated significantly compared to normal adult controls. The other proteins showed little change or were down-regulated following axotomy. By 4 weeks following optic nerve transection and peroneal nerve grafting, however, GAP-43, proteins 22 and 23r24 kDa showed a sustained up-regulation in synthesis and transport compared to normal controls; SNAP-25 and protein 18 kDa showed levels of expression similar to or slightly greater than normal controls. Increased expression of GAP-43 in retinal ganglion cells was also examined with immunocytochemistry. While a transient up-regulation of GAP-43 in retinal ganglion cells was observed following optic nerve transection, a sustained increase in GAP-43 immunoreactivity was present only in animals with nerve grafts. Backfilling of retinal ganglion cells from the grafts with horseradish peroxidase combined with GAP-43 immunocytochemistry revealed that all retinal ganglion cells with axons growing into the grafts were positive for GAP-43, but not all retinal ganglion cells showing GAP-43 immunoreactivity were extending axons into the grafts. We conclude that the presence of a nerve graft sustains the up-regulation of a number of proteins including GAP-43, and that this up-regulation is correlated with an increased potential for nerve growth, but other as yet unknown factors or conditions appear to play a role in determining if this growth potential will be realized. q 1998 Elsevier Science B.V. Keywords: Growth-associated protein; GAP-43; SNAP-25; Development; Regeneration

1. Introduction Growth-associated proteins were first described as a class of fast-axonally transported proteins the synthesis and transport of which were up-regulated significantly during optic nerve regeneration in the toad w28x. Growthassociated protein-43 ŽGAP-43., the most prominent and acidic of these proteins, has been reported to increase its synthesis and transport by as much as 100-fold during regeneration of the optic nerve in both toad w28x and goldfish w2x. In rabbit, GAP-43 is expressed at very high levels during development of the optic nerve, but an augmented synthesis and transport are not re-induced following injury to the adult rabbit optic nerve which does not regenerate w29x. These data suggested that the expres-

)

Corresponding author. Fax: q1 Ž615. 343-4539.

0169-328Xr98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 0 1 6 9 - 3 2 8 X Ž 9 7 . 0 0 2 7 4 - X

sion of GAP-43 and possibly other growth-associated proteins might be a prerequisite for axonal growth to occur Žthe so-called ‘GAP’ hypothesis w28x. and that the inability of mammalian vertebrates to regenerate CNS axons might be related, at least in part, to the failure to up-regulate this class of proteins following nerve injury. During development of the rat optic nerve, GAP-43 and other growth-associated proteins are produced at very high levels by retinal ganglion cells ŽRGC. w10,31x. At about the time synapses are formed between developing optic axons and target structures such as the superior colliculus w19x, the synthesis and transport of GAP-43 and other growthassociated proteins begin a dramatic decline to just barely or un-detectable levels. While a sustained up-regulation of GAP-43 synthesis and transport does not occur following injury to the optic nerve in the adult rat, we found that crushing of the nerve near the orbit resulted in a transient increase in GAP-43 expression w10x. This finding appeared to be in contradiction to what had been found by Skene

and Willard w29x in the rabbit where no up-regulation of GAP-43 or other growth-associated proteins was seen following injury to the adult rabbit optic nerve near the chiasm. More recently, Doster et al. w8x have shown that there is a transient up-regulation of GAP-43 following axotomy of the optic nerve in adult rats, but only if the injury occurs within approximately 3 mm of the eye. Using both metabolic labeling of newly synthesized proteins and changes in GAP-43 immunoreactivity in RGCs, Doster et al. w8x reported that injury to the rat optic nerve near the eye results in a transient up-regulation of GAP-43 between approximately 6–25 days post-axotomy. Injury of the nerve at distances greater than 6 mm from the eye produced no detectable increase in GAP-43 immunoreactivity in the retina and only a slight increase in synthesis and transport as shown by metabolic labeling. These data demonstrated that an augmented GAP-43 expression can occur in the absence of regeneration and that GAP-43 expression may be influenced by a factor or factors present in the longer nerve lengths that inhibit or repress the expression of this class of proteins. The distance at which axotomy occurs also appears to influence the potential ability of RGC axons to grow into peripheral nerve grafts. It has been documented by a number of laboratories that adult rat RGCs can be induced to elongate axons into peripheral nerve grafts, but only if the graft is within a few millimeters of the eye or into the eye itself w1,5,32,34x. Thus, the factors which appear to influence the expression of GAP-43 in RGCs may also be important determining factors in the ability of axons to elongate or to regenerate. Here we report that grafting a piece of the sciatic nerve onto a transected optic nerve close to the eye results in a sustained up-regulation of GAP-43 synthesis and transport in RGC axons growing into the grafts, and a sustained GAP-43 immunoreactivity within some RGCs in the retina. Using double-labeling immunofluorescent methods, we have shown also that at least some of the RGCs showing an augmented GAP-43 immunoreactivity in the retina are extending axons into the grafts. Additionally, we present data on four other developmentally regulated polypeptides Žsynaptosomal-associated protein-25 ŽSNAP-25. and proteins of apparent molecular weights of 18, 22, and 23r24 kDa., which show consistent and reproducible quantitative changes in synthesis and transport during RGC growth into nerve grafts. A preliminary report of some of these data has been published as an abstract w36x.

2. Materials and methods A total of 65 adult male Ž225–330 g. and 15 neonatal Sprague–Dawley rats were used in this study.

2.1. Optic nerÕe grafts For all experiments involving nerve grafting, adult rats were anesthetized with xylazine Ž87 mgrkg. and ketamine Ž13 mgrkg.. At the appropriate time, animals were killed by an overdose of anesthesia Žxylazine, 174 mgrkg; ketamine, 26 mgrkg., unless otherwise specified. Sciatic nerve homografts were made onto a sectioned optic nerve similar to that described by Bray et al. w3x, and as shown in Fig. 1. The peroneal branch of the sciatic nerve was isolated and removed from the left hind limb and placed in saline at room temperature. The proximal end of the excised segment was suture-ligated to prevent any extraneous axon growth into the graft. The right optic nerve was then exposed by a superio-lateral approach, transecting the extraocular muscles. The nerve was completely transected within 1–3 mm from the eye, with great care taken not to damage the retinal artery. The distal end of the peroneal nerve graft was sutured to the optic nerve stump with 10-0 Proline suture. Hemostasis was obtained with a thermocautery pen. Chloromycetin Ž6.5 mgrkg. was injected i.m. and neomycin, polymyxin, and bactracin antibiotic ointment was placed on the cornea after surgery, and for 3 days thereafter. Animals survived for 3 days to 4 weeks following surgery. To determine if axons were growing into the nerve grafts, some animals were re-anesthetized with ketamine and xylazine, perfused transcardially with 10% formalin, and the optic nerve and graft processed and paraffin-embedded. Longitudinal sections were cut at 20 mm through the optic nerve, anastomotic site, and peroneal nerve graft, and stained with the Bodian silver stain for visualization of axons w4x. Once we were assured that the grafting procedure resulted in elongation of a substantial number of axons into the grafts, experimental animals were prepared for the studies described below.

Fig. 1. Optic–peroneal nerve grafting paradigm. The optic nerve was transected within 3 mm of the eye and a piece of the peroneal nerve grafted onto the cut end. The distal or free end of the graft was suture-ligated to prevent extraneous growth into the grafts. In animals with sham-grafts, the optic nerve was transected and a piece of the peroneal nerve was excised, but the graft was suture-ligated at both ends and placed extracranially under the scalp. The optic nerve stump in sham-grafted animals was sutured to the sclera. In all animals, care was taken during sectioning to ensure that the central retinal artery in the optic nerve stump was undamaged.

For the studies involving nerve grafting, controls consisted of normal and sham-grafted animals. Animals with sham grafts had optic nerve transections without the apposition of a nerve graft. These animals were anesthetized with ketamine and xylazine, and a segment of the peroneal nerve excised, and the optic nerve exposed and transected exactly as described for animals with nerve grafts. The peroneal nerve segment was then ligated at both ends using 4-O silk suture and placed extracranially under the scalp. The stump of the optic nerve was sutured to the sclera using 10-O Proline, and the incision was sutured closed. 2.2. Two-dimensional polyacrylamide gel electrophoresis and quantitatiÕe densitometry Two-dimensional polyacrylamide gel electrophoresis Ž2D-PAGE. was used to resolve newly synthesized membrane-associated fast-axonally transported proteins Žparticulate fractions. in the normal optic nerve, the optic nerve stump of animals with nerve transections Žshamgrafted., or the peroneal nerve segment in animals with nerve grafts. Animals were anesthetized with halothane and 2 ml of w 35 Sxmethionine Ž500 mCi. was injected into the right Žnormal, transected, or nerve-grafted. eye. After allowing 3.5–5 h for transport, animals were anesthetized with ketamine and xylazine and the optic nerve stump or a portion of the peroneal nerve graft was harvested and the animals killed by cardiac puncture and exsanguination. In some animals with nerve grafts, the graft was exposed and the part of the graft adjacent to the optic nerve stump was processed for electron microscopy as described below and the distal part of the graft was removed and processed for 2D-PAGE. The portion of the nerves to be used for 2D-PAGE was frozen on dry ice and stored at y708C. The first-dimension isoelectric focusing gel ŽIEF. was run as described previously w10x. The tissue was homogenized in 10 volumes of buffer Ž10 mM Tris ŽpH 7.4., 5 mM EDTA, 1 mM DTT, 1 mM each PMSF and o-phenanthroline. and centrifuged at 100 000 = g for 1 h at 48C. The resulting pellet was resuspended first in 1 volume of 0.5% SDS, 1 mM DTT then 2 volumes of 10% NP-40, 2 mM DTT followed by the addition of 0.078 g urear100 ml per sample with vortexing until dissolved. The sample was spun briefly Ž2 min at 10 000 = g ., and loaded onto the first-dimension tube gel containing 4% acrylamide: 0.21% bis-arcyl, 2% NP-40, 9 M urea, 6% ampholytes Ž1:1 pH 4–6 and 3.5–10 ŽPharmaciarLKB.., 0.12% TEMED and 0.014% ammonium persulfate, overlayed, and electrophoresed for 10 000 V-h using 20 mM NaOH and 10 mM phosphoric acid as electrolyte solutions. Aliquots of the first-dimension samples were taken to determine total radioactivity, radioactivity of trichloroacetic acid ŽTCA.precipitated proteins, and total protein in the samples as determined by Lowry w17x. The second dimension consisted of SDS-PAGE on 10% acrylamide gels Žwithout

urea.. These conditions were chosen specifically because they allow GAP-43 to be well resolved. Purified GAP-43 Ž1–2 mg. was added to each sample and visualized in gels stained with Coomassie brilliant blue w9x and used to identify unequivocally the position of GAP-43 in fluorographs. The gels were impregnated with fluor Ž0.4% PPO in APEX solution., rehydrated in H 2 O, dried, and exposed to Kodak XAR film preflashed to an optical density of 0.15 relative to unexposed film w15x. Fluorographs were exposed to 1.2 = 10 9 y 4.5 = 10 9 total disintegrations. Fluorographs exposed to similar densities were scanned with a Microscan 1000 densitometer ŽTechnology Resources, Nashville, TN.. The total optical density ŽO.D.. in the fluorographs and O.D. of individual protein spots were determined using the Microscan 1000 software after averaging and subtracting out background radioactivity. Only protein spots the O.D. values of which were within the linear range of the densitometer Žunsaturated at ; 0–2.5 O.D. range. were included in the quantitative analyses. Values obtained under the various conditions for each protein were compared statistically using a non-parametric Kruskal-Wallis test for small samples; post-hoc pairwise comparisons were made using the Newman-Keuls test ŽSAS versions, SAS Institute Inc., Cary, NC.. Particulate fractions of newly synthesized fast-transported proteins in 0-, 2- and 9-day-old rat pups and normal adults were also subjected to 2D-PAGE and fluorography. Normal adult animals were anesthetized with ketamine and xylazine and killed 3.5–5 h following w 35 Sxmethionine injections into the eye. Neonates were anesthetized with halothane and decapitated 2.5–3 h post-injection. The nerves from 3 to 5 neonatal animals were pooled for each sample. The first-dimension IEF gels were run as described above, and the second-dimension gels were 12% acrylamide Žwith 8 M urea.. These conditions were chosen to allow for the resolution of a variety of fast-axonally transported proteins and to allow comparison to be made with our previous developmental studies w10,31x. In normal and nerve-grafted animals, the area of the nerve or graft taken for analysis was sufficiently far away from the eye to ensure that diffusion of radioactivity, which might result in the local incorporation of label by glial cells, was not a factor in the labeling of proteins in the nerve. In sham-grafted animals, where proteins were analyzed from the optic nerve stump close to the eye, and in neonates, where there is a poorly developed blood–brain barrier with some systemic labeling of proteins w21x, labeling of glial proteins within the nerve is a potential problem. To ensure that the proteins analyzed in experimental animals represented only fast-axonally transported proteins and not locally labeled glial proteins, the profiles of proteins obtained in fluorographs prepared from the optic nerves and the peroneal nerve segments were compared to proteins identified from excised segments of optic and peroneal nerves, which had been incubated in Dulbecco’s

defined media containing 250 mCi w 35 Sxmethionine at 378C in a humidified atmosphere of 5% CO 2r95% air for 3 h w30x, and processed as described above. 2.3. Electron microscopy In some of the sham- and nerve-grafted animals used for the metabolic labeling study, a portion of the peroneal nerve Žgraft or excised segment. was prepared for electron microscopy ŽEM. according to standard protocols used in our laboratory. Because the distal part of the graft in animals with optic–peroneal grafts was to be used for 2D-PAGE, the tissue processed for EM was immersion rather than perfusion fixed in 1P1G Ž1% paraformaldehyde, 1% glutaraldehyde. overnight. In sham-grafted animals, part of the unattached peroneal nerve segment was also fixed by immersion in 1P1G. The next day, tissues were washed, osmicated, dehydrated through a graded series of alcohols, and embedded in EPON 812. Thick Ž2 mm. and ultrathin Ž; 60 nm. sections were cut using a LKB Ultratome IV. Thick sections were stained with methylene bluerAzure II. Thin sections were stained with uranyl acetate and lead citrate, and examined in an Hitachi H-800 electron microscope. 2.4. Horseradish peroxidase labeling In a separate set of animals, horseradish peroxidase ŽHRP. was used to retrogradely label RGCs the axons of which were elongating into the grafts. Animals with nerve grafts were anesthetized with ketamine and xylazine, and the nerve graft was transected a few millimeters after it crossed over the supraorbital ridge, approximately 10–12 mm from the optic nervergraft anastomosis. The free end of the graft was placed within a plastic tube containing a pledget of HRP-soaked gelfoam Ž40% Type VI HRPr1% DMSO. and the tube sealed with silicon grease. Animals were killed 48 h later, and the retina removed, reacted with pyrocatechol and p-phenylenediamine dihydrochloride for visualization of the HRP reaction product, whole-mounted, and stained with cresyl violet as described in Stone w33x. 2.5. GAP-43 immunoreactiÕity in RGCs Animals were anesthetized with xylazine and ketamine, and perfused transcardially with saline followed by 70% methanol. Methanol was chosen as the fixative since cell body staining for GAP-43 is attenuated by aldehyde fixatives w6x. Each retina was dissected and flattened by placing it between two pieces of hardened filter paper and two wire mesh screens. The resulting sandwich was sewn together and processed through graded alcohols w33x. The retina was then removed from the filter paper, placed in xylene and subsequently embedded in paraffin. Sections were cut at 7 mm and collected onto chrom–alum-coated slides. The sections were processed for light-microscopic

immunocytochemistry for GAP-43 according to standard protocols used in our laboratory, using a polyclonal antibody raised in rabbit against purified rat GAP-43 at primary antibody dilutions of 1:250–1:500 w20x, or the monoclonal GAP-43 antibody 91E12 ŽBoehringer-Mannheim. at dilutions of 1:10–1:20. The sections were reacted with 3-3X-diaminobenzidine for visualization of GAP-43 antigentic sites and counterstained with cresyl violet. Control tissue was treated identically except that pre-immune serum was used in place of the primary GAP-43 antibody or the primary antibody was omitted. Animals with optic nerve transections Žsham-grafted. and optic–peroneal nerve grafts were yoked together as pairs, with surgery on the same day, and the tissues processed in parallel during every phase of the experiment. 2.6. Double-labeling of RGCs To determine if RGCs showing GAP-43 immunoreactivity within their cell bodies were extending axons into the nerve grafts, double-labeling immunofluorescence was done on paraffin-embedded sections of the retina. Two weeks following peroneal nerve grafting, RGCs were labeled by retrograde transport with HRP as described above. Two days later, animals were anesthetized with xylazine and ketamine, perfused with saline followed by 70% methanol, and their retinas embedded in paraffin. Six-micrometer sections were reacted with both GAP-43 antiserum Žrabbit. and peroxidase antiserum Žmouse.. Donkey anti-rabbit fluorescein-conjugated and donkey anti-mouse Texas red-conjugated antibodies ŽJackson Immunologicals. were used to visualize antigenic sites. Controls included incubating tissue under identical conditions except that the primary antibodies were omitted. The sections were examined using a Zeiss fluorescence microscope with a dual filter system.

3. Results 3.1. RGC axon elongation into peroneal nerÕe grafts We sought first to establish that RGC axons were growing into the peroneal nerve segments grafted onto the optic nerve as described above. Growth of RGC axons into the nerve grafts was confirmed using Bodian silver staining, labeling of RGCs via retrograde transport of HRP from the peroneal nerve segment, and EM. While the absolute number of axons extending into the grafts varied between animals, growth of axons into grafts was seen at 2 and 4 weeks in all animals. Fig. 2 shows Bodian-stained sections of an optic–peroneal nerve graft 4 weeks after grafting. Axons can be seen coursing from the optic nerve stump past the point of anastomosis and into the peroneal nerve segment. In other animals, retrograde transport of HRP was used to label RGCs in the retina which were

extending axons into the grafts. Fig. 3 shows HRP-labeled RGCs in an animal with a peroneal nerve graft at 4 weeks post-grafting. Rough estimates of the number of RGCs

labeled by this procedure indicated that between 1 and 10% of the normal number of RGCs in the rat retina w24x were elongating axons into the grafts.

Fig. 2. Bodian silver staining was used to visualize axons growing into the peroneal nerve grafts. ŽA. and ŽB. are two photomicrographs taken at different depths of focus in a 20 mm section from an animal 4 weeks following optic–peroneal nerve grafting. Numerous axons Ža few are indicated at arrows. can be seen crossing the anastomotic site Žsolid line. and entering the peroneal nerve graft. In both photomicrographs, the optic nerve segment ŽON. is to the left and the peroneal nerve graft ŽPNG. is to the right; the suture sites are also indicated ŽS.. Bar: 100 mm.

Additional evidence of axon growth into grafts was obtained by EM. Fig. 4A is a photomicrograph of a section through a peroneal nerve segment which was not sutured to the optic nerve but was ligated at each end and placed under the scalp and left in place for 2 weeks. Only degenerating myelinated axons are present. In contrast, numerous normal-appearing axons could be identified within the nerve grafts by 2 weeks following nerve-grafting. By 4 weeks, Schwann cells had remyelinated many of the RGC axons extending into the grafts ŽFig. 4B., as has been reported by others w11x. All of these data indicate that the procedures used to graft a segment of the peroneal nerve onto the optic nerve resulted in the successful growth of RGC axons into the grafts. 3.2. Synthesis and transport of radiolabeled proteins

Fig. 3. HRP was used to retrogradely label RGCs with axons growing into peroneal nerve grafts. Low-power photomicrograph of HRP labeled RGCs in an animal killed 4 weeks following optic–peroneal nerve grafting. Bar: 50 mm.

Metabolic labeling and 2D-PAGE were used to assess changes in the synthesis and transport of growth-associated proteins and other developmentally regulated fast-axonally transported group I proteins w35x under conditions of nerve grafting. In some animals, excised optic or peroneal nerves were incubated in w 35 Sxmethionine so that the profile of proteins synthesized locally in the nerves by glial cells could be determined and compared to the profiles seen from rapid axonal transport. Consistent with earlier reports, neither GAP-43 nor SNAP-25 were labeled from local incorporation of w 35 Sxmethionine directly into nerves w8x. Likewise, proteins resolving at 18, 22, and 23r24 kDa could not be identified in gels of proteins synthesized in

Fig. 4. Additional evidence of RGC axon growth into peroneal nerve grafts was obtained with EM. A: only degenerating myelinated axons can be seen in a section through an excised and ungrafted peroneal nerve segment 2 weeks after nerve resection. B: at 4 weeks following transection and optic–peroneal nerve grafting, normal appearing axons which have been remyelinated can be identified in the peroneal nerve segment. Bars: 1 mm.

Fig. 5. A number of developmentally regulated proteins can be resolved in fluorographs of w 35 Sxmethionine labeled fast-axonally transported optic nerve proteins from 0 ŽA. and 9 ŽB. day neonatal rats. Compared to fluorographs of proteins from the optic nerve of normal adult control animals ŽC,D., two proteins ŽGAP-43 and protein 23r24Ža. kDa. show a down-regulation in synthesis and transport during development; three proteins ŽSNAP-25 and proteins 18 and 22 kDa. show their greatest expression in adults. A second more basic spot resolving at 23r24 kDa Žprotein 23r24Žb.. increases in expression from 0 to 2 days, but undergoes little change from 2 to 9 days to adult. Protein 18 kDa also shows a marked p I shift during development from a more basic p I in the neonate Ž; 6.1–6.3. to a more acidic p I in the adult Ž; 5.5–5.7.. Different exposures of the same fluorograph are shown for adults ŽC,D. to indicate that GAP-43, a methionine-poor protein w14x is almost undetectable at exposures ŽC: 1.5 = 10 9 disintegrations. where SNAP-25, a methionine-rich protein, is robust. When fluorographs are over-exposed ŽD: 4.0 = 10 9 disintegrations., GAP-43 is detectable, but SNAP-25 is saturated, making it difficult to obtain accurate densitometric readings for this protein. Other proteins, particularly those between 50 and 70 kDa, appear to undergo developmental regulation. Data from gels of glial proteins labeled from systemic radioactivity indicate that a major component of some of these protein spots was from local labeling, and for this reason, these proteins were not analyzed further. Gel conditions Žsecond dimension. consisted of SDS-PAGE on 12% acrylamide gels Žwith urea..

either sciatic or optic nerves, indicating that they are not synthesized by PNS or CNS glia Ždata not shown.. Fluorographs of gels from developing Ž0, 9 day. and adult normal animals are shown in Fig. 5. Five proteins including GAP-43, SNAP-25 and proteins at 18, 22, and 23r24 kDa show a marked developmental regulation. Two protein spots resolving at a molecular weight of 23r24 kDa show changes developmentally. For reasons discussed below, we have referred to these as one protein, with the more acidic of the two spots designated protein 23r24Ža. kDa and the more basic spot as protein 23r24Žb. kDa. Two of the five developmentally regulated proteins ŽGAP-

43 and protein 23r24Ža. kDa. are expressed at their highest levels in optic nerve from 0 day animals ŽFig. 5A.. By 9 days ŽFig. 5B., GAP-43 expression is decreased slightly and protein 23r24Ža. kDa is decreased significantly. In adult optic nerve ŽFig. 5C,D., neither GAP-43 nor protein 23r24Ža. kDa can be resolved except in overexposed gels, where they are faint or barely detectable ŽFig. 5D.. Because GAP-43 and protein 23r24Ža. kDa show an increased synthesis and transport in the developing optic nerve compared to their expression in adults, they can be designated as growth-associated proteins w10,28x. Three proteins ŽSNAP-25, and proteins 18 and 22 kDa.

Fig. 7. Results of the quantitative densitometric analysis of 2D gel fluorographs for the different proteins under various conditions. For each protein, the percent volume of radioactivity represented by the O.D. of the protein spot on the fluorograph relative to the total O.D. measured in the fluorograph is shown for adult control animals, and animals with sham-grafts Žoptic nerve transection alone. or optic–peroneal nerve grafts at 2 and 4 weeks. In all graphs, values for normal adult controls are indicated by solid bars, for sham-grafts Žoptic nerve transection alone. by open bars, and for optic–peroneal nerve-grafted animals by striped bars; ) ) indicates that the protein was not detectable.

show a progressive increase in their expression developmentally with the highest levels seen in the adult animal. Protein 18 kDa also shows a consistent change in electrophoretic mobility during development, with a shift from

a more basic p I Ž; 6.1–6.3. in the neonate to a more acidic p I Ž; 5.5–5.7. in the adult. Protein 23r24Žb. kDa is expressed at a low level in 0 day animals, increases slightly in expression from 0 to 2 days Ždata not shown.,

Fig. 6. Fluorographs of optic nerve proteins in adult rats at 2 ŽC. and 4 ŽE. weeks following optic nerve transection alone, and at 2 ŽD. and 4 ŽF. weeks following optic nerve transection and peroneal nerve grafting. Of the five developmentally regulated proteins examined, only GAP-43 showed a significant increased expression in sham-grafted animals Žoptic nerve transection alone.. At 4 weeks following optic–peroneal nerve grafting, all of the proteins except SNAP-25 show a sustained increase in metabolic labeling indicating an increased synthesis and transport compared to normal adults or to axotomized control animals. By 4 weeks following nerve grafting, SNAP-25 was back to nearly normal levels. Fluorographs from two normal adults exposed to different exposures ŽA,B. show that GAP-43 and other proteins, which are methionine-poor or in low abundance, are detectable at exposures when SNAP-25 is saturated. Gel conditions Žsecond dimension. consisted of SDS-PAGE on 10% acrylamide gels Žwithout urea..

but undergoes little change in synthesis and transport from 0 to 2 days to adult. These data are similar both qualitatively and quantitatively with our earlier work identifying developmentally regulated proteins in rat visual system w10x. Representative fluorographs of gels from normal control animals, and animals with optic nerve transections Žshamgrafts. or optic–peroneal nerve grafts are shown in Fig. 6. In normal control animals ŽFig. 6A,B., GAP-43 and protein 23r24Ža. are barely or un-detectable even in over-exposed gels ŽFig. 6B.. In animals killed 2 weeks following optic nerve transections ŽFig. 6C., GAP-43 is transiently and significantly up-regulated, proteins 18 and 22 kDa show little change or slight up-regulation, and SNAP-25 and protein 23r24Žb. kDa are down-regulated compared to normal control animals. By 4 weeks following optic nerve transection alone ŽFig. 6E., only GAP-43 is still slightly up-regulated relative to values obtained for normal animals. SNAP-25 and proteins 22, 23r24Žb. and 18 kDa are undetectable or greatly decreased by 4 weeks following optic nerve transection alone Žsham-grafts. compared to normal controls. Protein 23r24Ža. is barely or un-detectable in normal adult animals, or in animals at 2 or 4 weeks following nerve transection. In animals with optic–peroneal nerve grafts killed at 2 weeks ŽFig. 6D., GAP-43 and proteins 22, and 23r24Ža. kDa showed an up-regulation, proteins 18 and 23r24Žb. kDa were unchanged, and SNAP-25 was down-regulated compared to normal control animals. Compared to the 2 week animals with optic nerve transection alone, in the 2 week animals with nerve grafts there was an increase in metabolic labeling of SNAP-25, proteins 23r24Ža. and 23r24Žb. kDa, and little difference in the expression of GAP-43 or proteins 18 and 22 kDa. By 4 weeks in the presence of a nerve graft ŽFig. 6F., GAP-43, proteins 18, 22, 23r24Ža. and 23r24Žb. kDa showed an up-regulation compared to normal control animals, and SNAP-25 showed levels comparable to normal controls. At 4 weeks, all of the proteins showed a significant increase in synthesis and transport compared to levels observed in animals at 4 weeks with optic nerve transections alone. In the presence of a nerve graft, protein 18 kDa also showed a p I shift from the acidic Ž; 5.5–5.7. back to more basic form Ž; 6.1–6.3. seen in developing optic nerve. Qualitatively, all of the fluorographs showed similar changes. For each group, fluorographs from three different animals were chosen for quantitation by densitometric analysis. The proteins were well-resolved in these fluorographs and there was a minimal labeling of glial proteins, particularly in nerve-grafted and normal animals. Results of the quantitative analyses are shown in Fig. 7 and the statistical analysis of these data is summarized in Fig. 8. In Fig. 7, the percent volume of radioactivity represented by the O.D. of the protein spot on the fluorograph relative to the total O.D. measured in the fluorograph is given for each protein in normal adult control animals, and in ani-

Fig. 8. Summary of the statistical analysis of the relative density of protein spots ŽO.D. values. in fluorographs after optic nerve transection alone or optic–peroneal nerve grafting. Overall significance levels are indicated by P values listed below each protein; single asterisks Ž ) . indicated on the first line indicate groups that were significantly different from the normal control animals. Double asterisks Ž ) ) . indicate statistically significant differences obtained for indicated post-hoc comparisons between groups. Not shown: values for GAP-43, SNAP-25, and proteins 18 and 22 kDa were significantly lower at 4 weeks compared to 2 weeks following optic nerve transection. For all post-hoc comparisons, P - 0.05.

mals at 2 and 4 weeks following optic nerve transection alone or with optic–peroneal nerve grafting. These analyses confirm that of the five developmentally regulated proteins, only GAP-43 shows a significant up-regulation in synthesis and transport after axotomy alone compared to normal control animals. Protein 23r24Ža. kDa is barely or un-detectable in normal adult animals, and is undetectable after optic nerve transection at either 2 or 4 weeks; SNAP25 and protein 23r24Žb. are down-regulated significantly in animals with sham-grafts at both 2 and 4 weeks; protein 18 kDa shows little change in expression at 2 weeks, but is significantly down-regulated 4 weeks following optic nerve transection. Protein 22 is slightly up-regulated at 2 weeks, but significantly down-regulated by 4 weeks in shamgrafted animals. Two weeks after optic–peroneal nerve grafting, GAP-43 and protein 23r24Ža. kDa are both up-regulated significantly compared to normal control animals; SNAP-25 is still down-regulated significantly compared to normal controls. All five proteins, however, show a significant and sustained increase in synthesis and trans-

Fig. 9. GAP-43 immunocytochemistry was used as an independent measure of the continued upregulation of GAP-43 synthesis in RGCs in the presence of a peroneal nerve graft. A: 10 days following optic nerve transection alone, HRP immunoreactive RGCs can be identified in the retina. B: 4 weeks after optic nerve transection alone, no RGCs positive for GAP-43 were seen in the retina. In animals with transection and peroneal nerve grafts, numerous GAP-43 immunoreactive RGCs were present in the retinas at 10 days ŽC. and 4 weeks ŽD. following grafting. Bar: 50 mm. Fig. 10. GAP-43 immunocytochemistry Žgreen fluorescence. was combined with retrograde transport of HRP from peroneal nerve grafts Žred fluorescence. to determine if RGCs growing axons into the grafts were the same cells showing a sustained expression of GAP-43. A: GAP-43 positive neurons could be identified throughout the retinas in animals killed at 2 weeks following optic–peroneal nerve grafting. B: many of the GAP-43 positive neurons also transported HRP from the peroneal nerve graft indicating that these cells had grown axons into the grafts. The punctate immunofluorescence in ŽA. represents GAP-43 immunoreactivity which is present in the inner plexiform layer in normal control animals and in animals after optic–peroneal nerve grafting. Bar: 50 mm.

port by 4 weeks following optic nerve transection and optic–peroneal nerve grafting compared to axotomized Žungrafted. controls. Also, by 4 weeks, all of the proteins except SNAP-25 and protein 18 kDa are significantly up-regulated in expression in animals with optic–peroneal nerve grafts when compared to normal controls. SNAP-25, which was significantly down-regulated after optic nerve transection alone, was expressed at nearly normal levels 4 weeks after transection in the presence of a peroneal nerve graft. Finally, all proteins except GAP-43 and protein 18 kDa showed a significant increase in expression at 4 weeks when the 2 and 4 week groups with optic–peroneal nerve grafts were compared. Protein 18 kDa was up-regulated at 4 weeks in animals with optic–peroneal nerve grafts compared both to normal controls and to animals at 2 weeks with optic–peroneal nerve grafts, but the variability between animals in the expression of this protein prevented this difference from reaching statistical significance. Like all of the other proteins, however, protein 18 kDa was up-regulated significantly at 4 weeks following grafting when compared to the 4 week sham-grafted animals. 3.3. Expression of GAP-43 in RGCs under conditions of nerÕe grafting After axotomy of the optic nerve close to the eye, GAP-43 immunoreactivity is expressed transiently within the retina between approximately 6–25 days post-lesion w8x. We examined whether this response would be sustained under conditions of nerve grafting. To address this question, we reacted tissue which had been fixed using methanol to maximize the probability of seeing GAP-43 in cell bodies w6x. In the present study, no GAP-43 positive RGCs were seen in the retinas of any of the normal adult animals. In animals with optic nerve transections alone, however, a few GAP-43 immunoreactive RGCs were seen within 3 days after optic nerve transection. Fig. 9A shows a GAP-43 immunoreactive RGC 10 days following optic nerve transection. The number of GAP-43 positive RGCs increased until approximately 14 days, and declined thereafter. No GAP-43 immunoreactive RGCs were seen at 4 weeks in any sham-grafted Žoptic nerve transected. animals ŽFig. 9B.. GAP-43 immunoreactive RGCs were first seen at 3 days in animals with nerve grafts as well. In contrast to animals with nerve transections alone, however, GAP-43 immunoreactive RGCs could be identified over the entire time period examined Ž3 days to 4 weeks.. Fig. 9 shows GAP-43 immunoreactive RGCs in animals with a peroneal nerve graft at 10 days ŽFig. 9C. and 4 weeks ŽFig. 9D. following surgery. 3.4. Double-labeling of RGCs for HRP and GAP-43 Although we had shown by 2D-PAGE and gel fluorography an augmented synthesis and transport of GAP-43 within RGC axons extending through the nerve grafts, and

had confirmed the continued presence of GAP-43 immunoreactivity in RGCs under the same conditions, it did not necessarily mean that these were overlapping populations of cells. For example, GAP-43 could potentially be accumulating abnormally within some cell bodies and proximal axons because of some alteration in the cell’s transport mechanism. To determine if the specific RGCs extending axons into the grafts would also show increased GAP-43 immunoreactivity, we used double-labeling methods to demonstrate the degree of overlap between these two cell populations. Consistent with our previous findings discussed above, a large population of GAP-43 immunoreactive neurons could be identified 2 weeks following peroneal nerve grafting. Some of these GAP-43 immunoreactive RGCs were also positive for HRP after backfilling from the grafts. Although all neurons might not have transported HRP, the consistent finding that all retinas contained significantly more GAP-43 immunoreactive RGCs than could be backfilled with HRP, suggests that not all neurons showing an increased cell body immunoreactivity for GAP-43 were extending axons into the grafts. Examples of RGCs showing both GAP-43 immunoreactivity Žgreen fluorescence. and retrograde transport of HRP Žred fluorescence. are shown in Fig. 10. In retinas examined from six animals with nerve grafts, no HRP immunofluorescent cell was identified which was not also positive for GAP-43. 4. Discussion The major new finding of the present study is that RGC axon growth into a peroneal nerve grafted onto a transected optic nerve is correlated with a sustained up-regulation of a number of developmentally regulated proteins, including GAP-43. This sustained up-regulation is reflected in an increase in the specific metabolic labeling of these proteins under conditions of nerve grafting indicating an increase in their synthesis andror transport. A sustained expression of GAP-43 immunoreactivity was also seen within RGCs of the retina up to 4 weeks following nerve grafting. All of the RGCs extending axons into the grafts as indicated by retrograde labeling with HRP also showed sustained GAP-43 immunoreactivity. These data add additional support to the hypothesis that the increased synthesis and transport of GAP-43 is correlated with the propensity for axon growth, and the continued expression of GAP-43 in cell bodies may reflect these cells’ enhanced or sustained potential for axon growth, as has been suggested by others w8x. These data also indicate that a number of other proteins are likely to play a role in initiating or maintaining axon growth in development or regeneration. 4.1. GAP-43 The results presented here confirm and extend findings reported previously by others and by our own laboratory.

Here we confirm the finding by Doster et al. w8x that GAP-43 is transiently and significantly up-regulated following optic nerve injury close to the eye as evidenced both by metabolic labeling and by the presence of GAP-43 immunoreactivity in RGCs. This also confirms our previous data showing that in the adult rat, intraorbital optic nerve crush produced a transient up-regulation of GAP-43 w10x. Our observations of a transient up-regulation appeared to be in contradiction to the finding in adult rabbits where optic nerve injury did not result in an up-regulation of GAP-43 w29x. We agree with Doster et al. w8x that the difference in the expression of GAP-43 is likely to be due to a difference in the placement of the lesions, with lesions close to the eye inducing an up-regulation and lesions at greater distances not inducing an up-regulation of GAP-43, and possibly other growth-associated proteins. We have also extended these findings by showing that grafting of a peroneal nerve segment onto the optic nerve results in a sustained up-regulation in both the synthesis and transport of GAP-43 and a sustained increase in GAP-43 immunoreactivity in RGCs. This confirms our preliminary data w23,36x and that of others w7,18,27x. Thus, during development of the rat optic nerve when RGC axons are actively growing, and in adult animals under conditions of nervegrafting where axons are elongating into grafts, GAP-43 shows a sustained up-regulation in synthesis and transport. One interesting and potentially important difference in the expression of GAP-43 in development w8,20x and during RGC axon elongation through nerve grafts in adult animals, is the appearance under the latter condition of GAP-43 immunoreactivity within cell bodies. Doster et al. w8x and Schaden et al. w27x have also reported a transient GAP-43 immunoreactivity in RGCs after axotomy. In the present study, we show that GAP-43 immunoreactivity in RGCs is sustained in the presence of a peroneal nerve graft, and that cells expressing GAP-43 in their cell bodies were growing axons into the grafts. In an earlier study, Schaden et al. w27x had shown that GAP-43 immunoreactive RGCs also showed fluorogold labeling from grafts. Taken together, these data would suggest that RGCs which are regrowing axons after injury show a greatly increased synthesis of GAP-43 over the normally low levels of constitutive expression of this protein in the visual system w10,22,31x. This conclusion is further supported by the finding that GAP-43 can be visualized in cell bodies of mature neurons in systems in which there is a high level of constitutive synthesis and transport in the adult w6,16x. Under conditions which increase the synthesis and transport of GAP-43 in the visual pathway, such as after axotomy or nerve grafting, GAP-43 immunoreactive RGCs can be visualized by 2–3 Žpresent study and w27x. to 6 days w8x. 4.2. Protein 23 r 24 kDa Another protein which is developmentally regulated and also shows changes in synthesis and transport under condi-

tions of nerve grafting is a protein of apparent molecular weight of 23r24 kDa and p I of ; 5.8–6.0. A protein of similar molecular weight was first described as a growthassociated protein in the regenerating optic nerve of the toad ŽGAP-24. w28x. A protein with similar characteristics resolved well on 1D gels at ; 23 kDa in developing rabbit optic nerve ŽGAP-23. w29x, and was developmentally regulated similar to GAP-43 with high levels in early development and very low or undetectable levels in adults. Skene and Willard w29x, however, saw no increase in the expression of any 23r24 kDa protein resolved in 1D gels after axotomy and regeneration of the hypoglossal or vagus nerves. They concluded that while GAP-23 was a growthassociated protein in the developing rabbit optic nerve, an up-regulation of its synthesis and transport was not re-induced during PNS regeneration. This finding weakened the argument that growth-associated proteins Žas originally defined. were a general class of molecules both necessary and sufficient for axon growth. Redshaw and Bisby w25x later reported, however, that a 23 kDa protein Ždesignated protein S2b. showed an increased synthesis and transport during regeneration of the hypoglossal nerve in rat. In 2D gels the S2b protein appeared to resolve into two spots, which they designated S2bŽa. and S2bŽb.. Redshaw and Bisby w25x concluded that these two protein spots were likely to be homologous to GAP-23r24 identified in developing rabbit and regenerating toad optic nerve. In fluorographs of developing optic nerve, we have identified two protein spots resolving at 23r24 kDa which undergo developmental regulation. The more acidic of the two spots Žprotein 23r24Ža. kDa. shows its highest expression in development and is barely or un-detectable in nerves from normal adult control animals ŽFig. 5C,D and Fig. 6A,B.. The second protein spot, at a similar molecular weight but slightly more basic Žprotein 23r24Žb. kDa. increases its expression developmentally but undergoes little change in expression from 2 days to adult. From these developmental data, we would conclude that protein 23r24Ža. kDa is most likely to be homologous to rabbit GAP-23 and toad GAP-24 and that protein 23r24Žb. is a distinct developmentally regulated protein. Both protein 23r24Ža. kDa and protein 23r24Žb. kDa, however, increase significantly their synthesis and transport under conditions of nerve grafting Žpresent study. and during PNS regeneration w25x, but not after axotomy alone. 4.3. SNAP-25 SNAP-25, a major protein of synaptosomal fractions w12x, has recently been reported to be a t-snare associated with molecular complexes involved in synaptic vesicle docking and release w13x. Previously designated Superprotein for its high abundance in the adult brain w8,12x, SNAP-25 is a fast-axonally transported protein which is developmentally regulated in the mammalian optic nerve

in a manner inversely to GAP-43 w10,22,29x. The present data show that 2 weeks following transection of the optic nerve near the eye, there is a decline in SNAP-25 expression of 3–4-fold. At 4 weeks following transection, SNAP25 is significantly down-regulated Ž; 85%.. In animals with peroneal nerve grafts, however, SNAP-25 levels are decreased by only ; 40% at 2 weeks and ; 13% at 4 weeks. These data suggest that the presence of the nerve graft influences the degree of down-regulation of SNAP-25 synthesis and transport which occurs as the result of axotomy. SNAP-25 also appears to increase its synthesis and transport as a function of the time since grafting, with levels in the 4 week nerve-grafted animals comparable to the levels seen consistently in normal adult controls. Changes in SNAP-25 synthesis and transport also become important when discussing the degree of up-regulation of GAP-43 and other GAPs in development, regeneration, or axon elongation through grafts. To allow for comparison between different gels, which may have been exposed for different times or show variable local labeling of glial proteins, GAP-43rSNAP-25 ratios are often used to normalize data, assuming that SNAP-25 levels are not affected by the various experimental conditions. There are a number of problems, however, in comparing the expression of proteins such as GAP-43 to that of SNAP-25. In our experience, SNAP-25 is so methionine-rich w12x that it saturates quickly in autoradiograms, making changes in the expression of the protein difficult to quantitate at exposures where methionine-poor proteins like GAP-43 w14x are detectable Žrefer to Fig. 5C,D and Fig. 6A,B.. Moreover, SNAP-25 shows variable changes in labeling after axotomy w8,25,26,28,29x. The present data, obtained from both optic nerve-transected and nerve-grafted animals, indicates that SNAP-25 does undergo a significant decrease in synthesis and transport after axotomy, at least under the conditions used in the present study. For this reason, we have not compared the expression of GAP-43 to SNAP-25 since this would give an overestimation of the up-regulation of GAP-43. Regardless, however, of whether the percent volume GAP-43rtotal radioactivity or GAP43rSNAP-25 ratios are used, or how these numbers are normalized or corrected for increased rates of incorporation w28x, GAP-43 shows a clear up-regulation during development, regeneration, and under conditions of nerve grafting. The absolute magnitude of this up-regulation, however, is dependent on how the data are compared. Our data, which represent a conservative estimate under conditions of nerve grafting, indicates that GAP-43 is up-regulated approximately 10-fold compared to normal controls. 4.4. Protein 22 kDa A developmentally regulated protein resolving at 22 kDa and p I of ; 6.4 also showed quantitative changes in synthesis and transport in adult animals under conditions of nerve grafting. At 2 weeks following optic nerve tran-

section alone or in the presence of a nerve graft, protein 22 kDa is slightly up-regulated compared to normal controls. At 4 weeks, however, protein 22 kDa shows a sustained up-regulation only in the presence of a nerve graft. Protein 22 kDa is similar in its molecular weight Ž; 20–22 kDa., p I Ž; 6.4., and appearance in 2D gels to protein S2a reported by Redshaw and Bisby w25x in regenerating rat hypoglossal nerve. While not defined as a growth-associated protein because it does not show its highest levels in development, it is up-regulated in adults under conditions where neurons are growing axons either during regeneration w25x, or under conditions of nerve grafting Žpresent results.. 4.5. Protein 18 kDa The protein 18 kDa identified in the present study is likely to be the 18 kDa protein identified in Skene and Willard w29x and protein T identified by Redshaw and Bisby w25x. Like protein 22 kDa, protein 18 kDa is not significantly up-regulated at 2 weeks in animals with optic nerve transection alone or after peroneal nerve grafting. It does show a significant and sustained up-regulation, however, at 4 weeks in animals with nerve grafts compared to sham-grafted animals. During optic nerve development in both rabbit w29x and rat Žpresent results; w10x., protein 18 kDa appears to undergo a change or post-translational modification which affects the charge of the protein. Thus, the protein undergoes a shift in p I from ; 6.2 in neonates to ; 5.5 in adults. The present data show that under conditions of nerve grafting in adult rats, the protein undergoes a significant shift in p I towards the basic end, resulting in a p I similar to that seen in the immature optic nerve. 4.6. Conclusions Our data show that under conditions of optic–peroneal nerve grafting, RGCs show a sustained increase in the synthesis and transport of a number of fast-axonally-transported proteins previously shown to be developmentally regulated in rat visual system. In general, the overall changes in protein expression observed between 2 and 4 weeks post-grafting appear to recapitulate changes observed during neonatal development. For example, for all proteins except proteins 23r24Ža. and 23r24Žb. kDa, the direction of change from 2 to 4 weeks after grafting is the same as observed from 0 to 9 days in development. Protein 18 kDa also shows a shift in p I to the more basic neonatal form. Finally, SNAP-25 levels increase under nerve grafting conditions at a time when GAP-43 levels are declining, just as occurs in development. The latter finding is also of interest because it suggests that there may be a programmed regulation of the expression of these RGC proteins independent of target innervation. While these observations are correlative only, they do suggest that regenera-

tion under nerve grafting conditions does induce changes in synthesis and transport, at least of some proteins, in a manner similar to that observed during development. Moreover, the data also suggest that even under the grafting conditions, GAP-43 and SNAP-25 expression may be regulated in a coordinate fashion. When the pattern of expression of individual proteins is examined, however, interesting differences in response to axotomy or grafting emerge. For example, 2 weeks following optic nerve transection alone, proteins respond to injury variably, showing increased ŽGAP-43, protein 22 kDa., decreased ŽSNAP-25, protein 23r24Žb. kDa., or similar Žproteins 18 and 23r24Ža. kDa. levels of metabolic labeling compared to normal controls. Even proteins showing a change in the same direction are responding to the injury differently. Thus, by 2 weeks post-axotomy, protein 23r24Žb. kDa expression is shut-off or at least is below detectable levels, whereas SNAP-25 continues to be synthesized and transported Žalbeit at lower levels. even in injured optic nerves. Finally, all proteins except GAP-43 show a continued increase in metabolic labeling between 2 and 4 weeks post-grafting. GAP-43 expression is decreased somewhat between 2 and 4 weeks after grafting, but is still quite high in expression at 4 weeks compared to normal adult levels. GAP-43 also shows a sustained upregulation in cell bodies even at 4 weeks post-grafting. These and other differences in expression suggest that different regulatory signals may be orchestrating the overall observed changes in protein expression. Under grafting conditions, RGCs are likely to be responding to substances released by Schwann cells; similar substances may be synthesized and released by CNS glia during development. The future identification of these regulatory molecules should provide insight into how different gene products are regulated and to whether or to what degree regeneration recapitulates development.

w16x

Acknowledgements

w17x

We would like to thank Dr. George Reed for help with the statistical analysis, and Dr. Gary Olsen and Ginger Winfrey for use of their fluorescence microscope. This work was supported by NIH Grant EY01117 and Vanderbilt University School of Medicine Biomedical Research Support Grants 523706 and 523862.

References w1x M. Benfrey, A.J. Aguayo, Extensive elongation of axons from rat brain into peripheral nerve grafts, Nature 296 Ž1982. 150–152. w2x L.I. Benowitz, E.R. Lewis, Increased transport of 44 000- to 49 000dalton acidic proteins during regeneration of the goldfish optic nerve: a two-dimensional gel analysis, J. Neurosci. 3 Ž1983. 2153– 2163. w3x G.M. Bray, M. Vidal-Sanz, A.J. Aguayo, Regeneration of axons

w4x w5x

w6x

w7x

w8x

w9x

w10x

w11x

w12x

w13x

w14x

w15x

w18x

w19x w20x

w21x

w22x

w23x

from the central nervous system of adult rats, Prog. Brain Res. 71 Ž1987. 373–379. G. Clark, M.P. Clark, A Primer in Neurological Staining Procedures. CC Thomas, Publ., Springfield, IL, 1971. S. David, A.J. Aguayo, Axonal elongation into peripheral nervous system bridges after central nervous system injury in adult rats, Science 214 Ž1981. 931–933. S.M. De la Monte, H.J. Federoff, S.-C. Ng, E. Grabczyk, M.C. Fishman, GAP-43 gene expression during development: persistence in a distinct set of neurons in the mature cental nervous system, Dev. Brain Res. 46 Ž1989. 161–168. S.K. Doster, A.M. Lozano, M. Willard, A.J. Aguayo, Axonal transport of GAP-43 in injured and regenerating retinal ganglion cell axons of adult rats, Soc. Neurosci. Abstr. 14 Ž1988. 802. S.K. Doster, A.M. Lozano, A.J. Aguayo, M.B. Willard, Expression of the growth-associated protein GAP-43 in adult rat retinal ganglion cells following axon injury, Neuron 6 Ž1991. 635–647. G. Fairbanks, T.L. Steck, D.F.H. Wallach, Electrophoretic analysis of the major polypeptides of the human erythrocyte membrane, Biochemistry 10 Ž1971. 2606–2617. J.A. Freeman, S. Bock, M. Deaton, B. McGuire, J.J. Norden, G.J. Snipes, Axonal and glial proteins associated with development and response to injury in the rat and goldfish optic nerve, Exp. Brain Res. 13 Ž1986. 34–47. S. Hall, M. Berry, Electron microscopic study of the interaction of axons and glia at the site of anastomosis between the optic nerve and cellular or acellular sciatic nerve grafts, J. Neurocytol. 18 Ž1989. 171–184. D.T. Hess, T.M. Slater, M.C. Wilson, J.H.P. Skene, The 25 kDa synaptosomal-associated protein SNAP-25 is the major methioninerich polypeptide in rapid axonal transport and a major substrate for palmitoylation in the adult CNS, J. Neurosci. 12 Ž1992. 4634–4641. H.P.M. Horikawa, H. Saisu, T. Ishizuka, Y. Sekine, A. Tsugita, S. Odani, T. Abe, A complex of rab3A, SNAP-25, VAMPrsynaptobrevin-2 and syntaxins in brain presynaptic terminals, FEBS 330 Ž1993. 236–240. L.R. Karns, S.-C. Ng, J.A. Freeman, M.C. Fishman, Cloning of complementary DNA for GAP-43, a neuronal growth-related protein, Science 236 Ž1987. 597–599. R.A. Laskey, A.D. Mills, Quantitative film detection of 3 H and 14 C in polyacrylamide gels by fluorography, Eur. J. Biochem. 56 Ž1975. 335–341. L.H. Lin, S. Bock, K. Carpenter, M. Rose, J.J. Norden, Synthesis and transport of GAP-43 in entorhinal cortex neurons and perforant pathway during lesion-induced sprouting and reactive synaptogenesis, Mol. Brain Res. 14 Ž1992. 147–153. O.H. Lowry, N.J. Rosebrough, A.L. Farr, R.J. Randall, Protein measurement with the Folin phenol reagent, J. Biol. Chem. 193 Ž1951. 265–275. A.M. Lozano, S.K. Doster, A.J. Aguayo, M.B. Willard, Immunoreactivity to GAP-43 in axotomized and regenerating retinal ganglion cells of adult rats, Soc. Neurosci. Abstr. 13 Ž1987. 1389. R.D. Lund, J.S. Lund, Development of synaptic patterns in the superior colliculus of the rat, Brain Res. 42 Ž1972. 1–20. C.B. McGuire, G.J. Snipes, J.J. Norden, Light-microscopic immunolocalization of the growth- and plasticity-associated protein GAP-43, Dev. Brain Res. 41 Ž1988. 277–291. K.L. Moya, L.I. Benowitz, S. Jhaveri, G.E. Schneider, Enhanced visualization of axonally transported proteins in the immature CNS by suppression of systemic labeling, Dev. Brain Res. 31 Ž1987. 183–191. K.L. Moya, L.I. Benowitz, S. Jhaveri, G.E. Schneider, Changes in rapidly transported proteins in developing hamster retinofugal axons, J. Neurosci. 8 Ž1988. 4445–4454. J.J. Norden, B. Wouters, J. Knapp, S. Bock, J.A. Freeman, The role of GAP-43 in axon growth and synaptic plasticity in the visual system: evolutionary implications. in: J.R. Cronly-Dillon ŽEd.., Vi-

w24x w25x

w26x

w27x

w28x

w29x

w30x

sion and Visual Dysfunction, Vol. 11, Macmillan Press, London, 1991, pp. 219–231. V.H. Perry, Evidence for an amacrine cell system in the ganglion cell layer of the rat retina, Neuroscience 6 Ž1981. 931–944. J.D. Redshaw, M.A. Bisby, Proteins of fast axonal transport in the regenerating hypoglossal nerve of the rat, Can. J. Physiol. Pharmacol. 62 Ž1984. 1387–1393. T.A. Reh, J.D. Redshaw, M.A. Bisby, Axons of the pyramidal tract do not increase their transport of growth-associated proteins after axotomy, Mol. Brain Res. 2 Ž1987. 1–6. H. Schaden, C.A.O. Stuermer, M. Bahr, GAP-43 immunoreactivity and axon regeneration in retinal ganglion cells of the rat, J. Neurobiol. 25 Ž1994. 1570–1578. J.H.P. Skene, M.B. Willard, Changes in axonally transported proteins during axon regeneration in toad retinal ganglion cells, J. Cell Biol. 89 Ž1981. 86–95. J.H.P. Skene, M.B. Willard, Axonally transported proteins associated with axon growth in rabbit central and peripheral nervous systems, J. Cell Biol. 89 Ž1981. 96–103. G.J. Snipes, Studies on the Roles of the Nerve Injury-Induced 37

w31x

w32x

w33x w34x

w35x

w36x

kilodalton Protein and the Growth-Associated Protein, GAP-43 in Nerve Regeneration. Dissertation, Vanderbilt University, Dept. of Cell Biology, 1986. G.J. Snipes, B. Costello, C.B. McGuire, B.N. Mayes, S.S. Bock, J.J. Norden, J.A. Freeman, Regulation of specific neuronal and nonneuronal proteins during development and following injury in the rat central nervous system, Prog. Brain Res. 71 Ž1987. 155–175. K.-F. So, A.J. Aguayo, Lengthy regrowth of cut axons from ganglion cells after peripheral nerve transplantation into the retina of adult rats, Brain Res. 328 Ž1985. 349–354. J. Stone, The Whole Mount Handbook. Maitland Publ., 1981. M. Vidal-Sanz, G.M. Bray, M.P. Villegas-Perez, S. Thanos, A.J. Aguayo, Axonal regeneration and synapse formation in the superior colliculus by retinal ganglion cells in the adult rat, J. Neurosci. 7 Ž1987. 2894–2909. M. Willard, W.M. Cowan, R. Vagelos, The polypeptide composition of the intra-axonally transported proteins: evidence for four transport velocities, Proc. Natl. Acad. Sci. USA 71 Ž1974. 2183–2187. B.C. Wouters, J.J. Norden, GAP-43 upregulation in regenerating retinal ganglion cells in the rat, Soc. Neurosci. Abstr. 16 Ž1990. 338.