Effects of a conditioning lesion on bullfrog sciatic nerve regeneration: Analysis of fast axonally transported proteins

Brain Research, 423 (1987) 1-12

1

Elsevier BRE 12917

Research Reports

Effects of a conditioning lesion on bullfrog sciatic nerve regeneration: analysis of fast axonally transported proteins G.W. Perry 1, Susan R. Krayanek I and David L. Wilson 1'2 1Department of Physiologyand Biophysics, School of Medicine and 2Department of Biology, Universityof Miami, Miami, FL (U.S.A.) (Accepted 3 March 1987)

Key words: Fast axonal transport; Nerve regeneration; Conditioning lesion; Protein; Nerve crush; 2D-gel; Frog

We have shown that bullfrog sciatic nerves respond to a conditioning lesion similarly to goldfish optic nerve and rat or mouse sciatic nerve; that is, following a crush the rate of regeneration is faster in nerves that have received a conditioning lesion compared to nerves that have not. Also, damaged nerve fibres show initial growth or sprouting earlier in a previously conditioned nerve compared to nerves that have not received a prior conditioning lesion. We have not detected changes in the transport of fast axonally transported proteins with the conditioning lesion paradigm, other than those changes seen in regenerating nerves after receiving a single lesion. However, more label was present in a few fast axonally transported proteins at the lesion site in conditioned nerves compared to nonconditioned nerves, and this difference is not apparently due to increased transport. It seems that changes in fast axonally transported proteins probably do not contribute directly to the mechanism underlying the conditioning lesion effect of higher out growth rates, although some of the fast transported proteins may be involved in functions, possibly at the growing tip of damaged fibres, which promote or result from the conditioning effect. INTRODUCTION Some nerve cells capable of regrowth after damage appear to possess the remarkable capacity for even greater growth when they are damaged for a second time. This 'conditioning lesion effect '7 has been known for some time t°, and has been demonstrated in mammalian peripheral nerves 10,15'19,22, in goldfish optic nerves 12,13,1s,2° and in retinal cells of amphibia 1. However, the mechanism underlying the enhanced regenerative effort by these nerves following two lesions is not known. One explanation for the effect is perhaps as simple as that the nerve is already in a 'state of growth' following the first lesion, and consequently there is minimal delay following a second lesion before the nerve is capable of regrowth. Even so, it seems unlikely that a reduced delay be-

fore initiating a growth state can account for the accelerated rate of outgrowth of fibres and increased metabolic activity seen in twice-damaged nerves. During the several days following a lesion, the nerve cell responds to the damage with a variety of changes 6'9, but not all nerve cells respond alike. Most notable among some nerve cells, for example retinal ganglion cells of lower vertebrates, is the large increase in overall protein synthesis and transport during regeneration following damage (see ref. 8), although such a response is not typical of all nerve cells of lower vertebrates, for instance the dorsal root ganglion ( D R G ) neurones of frogs 25,26. Following a second lesion many of the changes seen in the cell body of the neurone are further enhanced beyond that seen after the first lesion, for example, protein synthesis and transport are further increased 21. One pos-

Correspondence: G.W. Perry, Department of Physiology and Biophysics, University of Miami, School of Medicine, P.O. Box 016430, Miami, FL 33101, U.S.A. 0006-8993/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)

sible mechanism for the conditioning effect is that the increased rate of outgrowth reflects an increased delivery of transported materials to the growing axon. An especially important component of regenerating axons is the membrane needed for the formation and extension of the growing nerve fibre. The delivery of membrane components to the growing tips of the fibres occurs via fast axonal transport. The growth rate of fibres appears to be correlated with the rate of slow axonal transport 5.3s although it is possible that certain components of fast axonal transport may contribute to enhanced outgrowth 7. Following a single lesion specific rapidly transported proteins have been seen to be increased relative to other fast axonaUy transported proteins 2-4A1'25'27'29'31'32'35and conceivably these proteins could play a role in enhanced outgrowth following a conditioning lesion. The present study tests whether D R G neurones in the sciatic nerve of the bullfrog respond to a conditioning lesion comparable to that seen in some other nerve cells, and if so whether or not more dramatic changes might be elicited in the individual fast axonally transported proteins than seen hitherto (see ref. 25). A preliminary account of some of this work has appeared 2s. MATERIALS AND METHODS

Animals Bullfrogs (Rana catesbeiana) 15-21 cm in length were obtained from Lederberg, WI. Animals were housed together at room temperature (20-23 °C) in aquaria in a room with a light:dark cycle of 14:10 h, and fed live crickets. For surgical procedures the frogs were anesthetized in an aqueous solution of ether (5 % v/v). Frogs were killed by decapitation and pithing.

Nerve crush procedures Fig. 1 shows diagrammatically the various lesioning paradigms used in this study. Sciatic nerves of bullfrogs were crushed as described previously 25. Initially, the right sciatic nerve was crushed about 35 mm from the 9th D R G (conditioning lesion). In one group of frogs (group 1) a second crush (testing lesion) was administered 10 days later to the right sciatic nerve about 25 mm from the 9th D R G , and there-

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Fig. 1. Experimental paradigm, a: conditioning lesion paradigm: the right sciatic nerve was crushed at about 35 mm from the D R G and 10 days later crushed again (testing lesion) 25 mm from the DRG. b: testing lesion only: the left sciatic nerve was crushed 25 mm from the D R G at the same time as the right nerve received the testing (second) lesion. Four days after the testing lesion the sciatic nerves were dissected from the frogs and labelled in vitro with [35S]methionine as described in Materials and Methods. Following the 24-h transport period, the sciatic nerve was desheathed and segments of nerve taken at Site 1 (crush site) and Site 2 (intact nerve), c: 14 days of regeneration: the right sciatic nerve of another frog was crushed at the

same time as the conditioning lesion in (a). d: normal nerve: the left sciatic nerve of the frog from (c) was left intact. The sciatic nerves of the second frog from (c) and (d) were dissected 14 days after the right nerve crush, and labelled for transport as described above. Following the transport period, nerves were desheathed and nerve segments equivalent to Site 2 were taken. Fast axonally transported proteins in the nerve segments were analysed by 2D-gel electrophoresis. fore about 10 mm proximal to the first or conditioning crush. This is the conditioned nerve. We have determined that a 10-day interval between these two lesions results in optimal outgrowth rate of fibres compared to intervals of 5, 15 and 20 days (data not shown). At the same time as the second lesion was made on the right sciatic nerve, the left sciatic nerve of the same frog was crushed about 25 mm from the 9th D R G (testing nerve). No further crushes were administered to a second group (group 2) of frogs which received only the initial lesion (Fig. lc). Experiments were staggered such that a group 1 frog was processed on the same day as a group 2 frog.

Transport profiles to determine the extent of regeneration At 21 days following the testing (second) crush, one set of frogs were killed and their sciatic nerves and D R G removed. The D R G were selectively labelled with [35S]methionine (New England Nuclear, 200 BCi at >1000 Ci/mmol). The procedure was essentially that described previously25,34, except that the D R G were labelled continuously for 24 h with [35S]methionine. After the 24 h transport period, nerves were desheathed and cut into 2-mm segments. TCA-precipitable counts per minute (cpm) in each segment were then determined using liquid scintillation spectrophotometry25.

Electron microscopy At various times, following one or two sciatic nerve lesions, frogs were anaesthetized with 5% ether and the sciatic nerve was exposed by dissection. The lesion site, which was easily identifiable, was fixed in situ by dripping fixative (2% glutaraldehyde, 2% paraformaldehyde in 0.1 M cacodylate buffer, pH 7.4, and 10 mM calcium chloride) onto the site. After 20-30 min the lesion site was removed and immersed in the same fixation solution overnight. Following a rinse in 0.1 M cacodylate buffer, the tissue was postfixed for 1 h in 1% osmium tetroxide in 0.1 M cacodylate buffer. Dehydration in a graded series of ethyl alcohols was followed by infiltration with propylene oxide and Araldite, and Araldite embedding. After removing the lesion site the frog was killed by decapitation. Thick and thin sections of the lesion site were examined with a Philips 300 electron microscope.

Analysis of fast axonally transported proteins Four days following the test lesion the frogs were killed by decapitation after which their sciatic nerves were removed and labelled with [35S]methionine as described above. The labelling patterns of fast axonally transported proteins were analysed to both qualitatively and quantitatively using 2D-gel electrophoresis as described previously25,26, with minor modifications as indicated below. Labelled, rapidly transported proteins were analysed at two sites along the desheathed sciatic nerves (Fig. 1). The first site (Site 1) included the crushed portion (testing crush; at 25 mm from DRG) of the

sciatic nerve, and 1-2 mm on either side of it (total = 4-mm segment). The second site (Site 2) was a nerve segment (4-mm) taken from nerve 10 mm closer to the DRG and included intact axons. Thus, the two nerve samples were different in that Site 1 includes growing fibre tips and represents protein reaching the crush site and growth cones; whereas Site 2 represents undamaged regions of axons containing proteins in transit to the growing fibre tips or incorporated into that nerve segment, e.g. axolemma 3°. The nerve segments were homogenized in 30 BI of a buffer containing 20 mM CHAPSO (Miles Laboratories), 50 mM dithiothreitol, 9.5 M urea 0.4% (v/v) 3-10 Bio-lyte (BioRad), 0.8% (v/v) 4-6 Bio-Lyte, and 0.8% (v/v) 5-7 Bio-Lyte in a small glass microhomogenizer which was washed with a further 30/A of the CHAPSO buffer. The homogenate was subjected to 2D-gel electrophoresis, after which the labelled, fast axonally transported proteins were located in the gel by fluorography. TCA-precipitable cpm were determined in an aliquot (5/A) of the homogenate prior to two-dimensional (2D) gel electrophoresis as described previously25. This determination of cpm allowsf0r an estimate to be made of the total labelled transported protein in the nerve sample, and also an estimate of the cpm loaded onto each gel, which subsequently determines the length of time of exposure of the gel to x-ray film (fluorograph). The cpm present in each protein spot on the gel was determined by liquid scintillation counting after cutting-out the spots from the gel as previously described 25. RESULTS The distribution of radiolabelled protein along the regenerating nerves following the one- or two-lesion paradigm are shown in Fig. 2. These profiles show that significant levels of label occur farther from the crush site in the conditioned nerve compared to the test nerves which receive only a single lesion (Fig. 2a, b). This suggests that fibres have regenerated farther in the 21 days following the test lesion in the conditioned nerve than in the test-lesion-only nerves, even though the nerves were last crushed at the same time and at similar points. In addition, the conditioned nerve shows significant levels of labelled protein at distances from the crush site that are comparable to

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Fig. 2. Transport profiles of labelled fast axonally transported proteins, a: testing lesion alone: left sciatic nerves were set up for labelling and transport (see Materials and Methods) 21 days after receiving only a testing lesion, b: conditioning lesion paradigm: right sciatic nerves previously crushed were crushed a second time 10 days later and left a further 21 days before being set up for labelling and transport, c: normal regeneration: both sciatic nerves of another frog were crushed at the same time as

the first nerve crush in (b), and were left undisturbed for a total of 31 days before labelling and transport. Following labelling and transport as described in Materials and Methods, nerves were cut into 2-ram sections and TCA-precipitable cpm was determined in each section by liquid scintillation counting. The arrows indicate the farthest distance along the nerve from the crush sites at which significant (P < 0.05) levels of radioactivity were measured. These were compared to a background level of radioactivity which was taken as the mean of all nerve segments distal to a nerve segment which showed significant radioactivity. Each data point represents a mean with n = 3 for the testing lesion alone (a), n = 3 for conditioning plus testing lesions (b), and n = 2 for normal regeneration (c). nerves (Fig. 2c) that were crushed at the time of the conditoning lesion, but not subsequently crushed a second time, and thus were allowed to regenerate for a longer period. This indicates that fibres in the conditioned nerve that were regenerating for only 21 days have grown as far as those in the nerve regenerating for 31 days. Taken together these data strongly suggest that the rate of outgrowth of fibres in the con-

ditioned nerve (Fig. 2b) is greater than in those nerves lesioned only once (Figs. 2a, c). However, damaged fibres may begin growing or sprouting earlLer following a crush in the conditioned nerve compared to the test nerve. Growth cones were visible even after 1 day at the site of the test lesion in previously conditioned nerves (Fig. 3a), containing dense core vesicles, smooth endoplasmic reticulum, clear vesicles seen in tips regenerating a x o n s 13'14"24. At 2-2.5 days axon fascicles were prominent at the lesion site in previously conditioned nerves (Fig. 3b), but not in non-conditioned nerves, and numerous growth cones were seen, comparable to that seen after 6 days at a lesion site in a nerve receiving only a single lesion. A few bundles of regrowing axons and clusters of growth cones were present at the lesion site at 4 days after a single lesion, but were much more frequently seen at 6 days postlesion (Fig. 4). However, the faster initiation of regrowth is not enough to account for the greater distance of regrowth in conditioned nerves at 21 days (Fig. 2). After allowing for the different times for a large number of axons to initiate outgrowth (2.5 days after the second lesion, and 6 days after a single lesion), we have calculated a maximum rate of regrowth in the conditioned nerve of 1.20 ram/day after the second lesion as compared to a maximum rate of 0.93 ram/day in a nerve receiving only a single, test lesion. The difference, for n = 3, was highly significant. Analysis of rapidly transported protein by 2D-gel electrophoresis was performed at 4 days after the test lesions. Our assumption was that any changes in fast transported proteins that might support the earlier and/or increased rate of fibre outgrowth, seen in the conditioned nerves above, would be more readily detectable shortly after the test lesion. Indeed, were any changes in fast transported proteins to be responsible for the conditioning lesion effect, they would need to be displayed during this early stage. The analysis revealed no consistent qualitative differences in the patterns of fast transported proteins from analogous nerve sites between the various paradigms (Fig. 2), although comparison of the patterns from Site 1 with those of Site 2 did show some differences. Spot A30 was consistently seen in patterns from Site 2 but not Site 1. Conversely, spots A25 and C40 were consistently seen at Site 1 but not Site 2. Also, a protein spot lying above spot B14 and below

Fig. 3. Representative electron micrographs of crushed nerve segments from testing lesion site showing (a) growth cone (GC) at 1 day and (b) axon fascicles after 2-2.5 days following the testing lesion in previously conditioned nerves, dv, dense vesicles; ser, smooth endoplasmic reticulum; mt, microtubules; c, collagen fibres, a, x21,000; b, x 10,000.

Fig. 4. a: Electron micrograph showing axon fascicles, and b: growth cones normally seen at the lesion site in a crushed sciatic nerve at 6 days following a single lesion, x 19,000.

Table I shows that there were no significant differences in the overall a m o u n t of labelled protein rapid-

spot B4, which is not n u m b e r e d nor included in the map (Fig. 5f), was seen inconsistently in patterns

ly transported along the sciatic nerves (Site 2) u n d e r the various paradigms tested. However, quantitative

from u n d a m a g e d , n o r m a l nerve and from Site 2 of crushed nerves, but was never seen in transported protein patterns from Site 1 of damaged nerves. We

analysis of the majority of the spots seen in the fast transported protein patterns shown in Fig. 5, does

do not yet know whether to attach any significance to these latter observations.

show some differences in incorporation of label into

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8

TABLE I Total amount o f rapidly transported label present in a 4 m m segment o f Nerve (Site 2)

Cpm values are means +_S.D. Treatment

CPM

n

Normal transport Testing lesion (4 days after lesion) Conditioning + testing lesion (10 + 4 days after lesion) Regeneration for 14 days

251624 + 89301

4

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4 4

particular proteins between the various paradigms. Table II shows the reduced quantitative data for labelling of 38 protein spots analysed from the 2D-gel patterns of the two nerve sites. The data analysis was as described previously 25 for the first 3 columns in Table II. That is, paired data for each spot were expressed as a ratio of experimental to control after normalization against a set of spots on the same gel, in this case all the spots analysed, and this ratio compared to unity for significant differences. Thus, the normalization values represent the amount of labelling relative to the total population of analysed spots. The first column in Table II shows a comparison of labelling for fast transported proteins present in normal, undamaged nerve and nerve regenerating for 14 days taken at Site 2. Although the nerve sampled in this case (Site 2) is somewhat different from that sampled previously (see ref. 25) which consisted of a 3-4mm nerve segment immediately proximal to a ligature placed immediately before a crushed region of nerve, these data confirm and extend those previous studies 25 and includes more protein spots in the analysis. As we saw previously, only a few of the analysed fast transported proteins change quantitatively during axon regrowth. Spots A1, A30, B8, C8 and C23 showed increased labelling, while spots A2, A45, B6, C l l and C50 showed decreased labelling in response to damage. The second column in Table II shows a comparison of labelling in the fast transported proteins at the crush site (Site 1) in test and conditioned nerves. There are a few differences. However, when labelling of the fast transported proteins from these paradigms are compared in a Site 2 segment of nerve (third column in Table II), all but one of the trans-

ported proteins show no significant differences between~the conditioned and test nerves. Only one spot, A~, shows a significant increase in labelling of about 60% in conditioned compared to test nerve. However, from the data shown in these columns, some ratios can be seen to be quite large but not significantly different from a ratio of 1.0. (With an 'n' of 4 the s.d. would have to be less than 54% for a ratio of 2.0 to be significant at a level o f p = 0.05.) Finally, the last column in Table II shows a comparison of labelling in the fast transported proteins present at Site 2 in the conditioned nerve compared to a nerve that has been in a regenerating state for as long as the conditioned nerve (14 days), that is, a nerve lesioned only once at the time the conditioning lesion was placed on the conditioned nerve. Because they were unpaired measurements, these data were analysed differently from that in the first 3 columns of Table II. In this case, the means of incorporation of label for each spot analysed in the transport patterns from Site 2 samples of the nerves were compared to one another for significant differences by an unpaired Student's t-test. As can be seen there are no significant differences in incorporation of label into any of the transported proteins. This final comparison not only shows that there are no further significant changes in labelling of fast transported proteins in a regenerating nerve as a result of a prior conditioning lesion, but also controls for any change that might have occurred in fast transported proteins in a nerve that has been in a 'regenerating state' for 14 days but not necessarily elaborating the same axons for that whole period. The data shown in the latter two columns of Table II therefore suggest that there is no increase in the amount of individual proteins transported in conditioned compared to normally regenerating nerves, and that those increases in a few proteins seen at the site of the crush (Site 1) represent some other differences, for example in the deposition or turnover of these proteins. DISCUSSION During the course of these studies we have confirmed and extended our earlier findings 25 showing that only a subset of fast axonally transported proteins change in abundance during regeneration of bullfrog sciatic nerves following a single lesion (Table

T A B L E II

Comparison of changes in labelling of fast axonally transported proteins in nerve samples from different experimental paradigms (see Fig. 1) Spot

14-Day regen./normal* Site 2

(C + T)/T* Site 1

(C + T)/T* Site 2

14-Day regen./(C + T)** Site 2

A1 A2 A4/10 A25 A30 A35 A45 A50 B1 B2 B3 B4 B6 B8 B14 B20 B35 B36 B37 C1 C2 C3 C4 C5/12 C6 C7 C8 C9 C10 Cll C23 C24/25 C30 C33 C35/36 C38 C40 C50

1.67 + 0.42 a 0.35 + 0.10 ~ 0.70 + 0.34 1.81 + 0.42 a 0.67 4- 0.18 0.36 4- 0.21 b 0.78 4- 0.51 0.85 4- 0.38 2.12 4- 0.82 0.96 _.+0.60 1.24 + 0.61 0.62 _+ 0.19 a 1.92 + 0.51 a 1.02 + 0.38 1.07 4- 1.27 1.47 _+ 0.61 1.09 + 0.24 0.71 4- 0.45 0.99 + 0.13 2.51 + 1.11 1.18 4- 0.37 1.12 + 0.18 1.28 4- 0.55 1.42 4- 0.14 a 1.53 4- 1.33 0.77 + 0.37 0.46 + 0.06 d 1.96 + 0.38 a 1.22 + 0.22 0.74 + 0.36 1.28 4- 0.71 1.25 + 1.29 0.79 + 0.21 0.59 + 0.17 ~

1.76 + 0.53 a 1.10 + 0.31 0.62 + 0.35 1.43 + 0.33 a 1.14 + 0.14 0.68 4- 0.22 0.92 + 0.18 0.98 4- 0.61 1.09 4- 0.24 0.88 4- 0.13 0.90 + 0.29 0.74 4- 0.27 1.39 + 0.18 a 0.79 + 0.20 0.76 + 0.16 0.95 + 0.22 0.88 4- 0.26 1.42 + 0.50 1.23 + 0.35 0.71 + 0.29 1.13 + 0.37 1.64 4- 0.53 1.05 + 0.32 0.89 + 0.08 2.42 + 1.59 0.95 __. 0.29 1.01 + 0.75 0.72 + 0.37 1.27 4- 0.92 1.56 4- 0.50 0.88 4- 0.37 1.22 4- 0.13 1.86 + 0.39 0.97 + 0.31 0.94 + 0.49 1.53 + 0.48 0.99 4- 0.48

1.58 + 0.33 a 0.80 + 0.17 0.91 + 0.21 1.06 + 0.57 1.14 + 0.30 0.81 + 0.21 0.92 + 0.07 0.84 + 0.19 2.14 4- 1.88 0.90 + 0.16 0.83 + 0.34 0.86 _+ 0.18 1.34 4- 0.58 0.99 4- 0.23 1.00 + 0.35 0.60 + 0.01 0.82 _+ 0.35 1.09 + 0.55 1.10 + 0.24 0.69 + 0.24 1.10 4- 0.28 1.14 + 0.35 0.80 + 0.24 1.00 4- 0.56 1.24 + 0.41 1.05 + 0.68 0.70 + 0.41 1.08 4- 0.63 0.98 + 0.76 1.09 4- 0.41 0.95 + 0.29 0.69 + 0.23 1.82 + 0.32 1.25 + 0.88 0.95 + 0.51 1.32 + 0.53

n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s.

a-d Ratios significantly different from unity are indicated by a letter: a = P < 0.05; b = P < 0.02; c = P < 0.01; d = P < 0.001. All other ratios are not significantly different from unity. C p m ' s have been normalized relative to total cpm in all analyzed protein spots from each gel, before ratios were taken (see text for further explanation). * Paired analysis showing ratio of normalized c p m of experimental vs control condition + S.D. (n = 4 - 5 ); for a full description of the quantification procedure see ref. 25. C, conditioning lesion (10 days); T, testing lesion (4 days); regen., regeneration. ** Unpaired analysis showing comparison of m e a n s by Student's t-test (n = 4 - 5 ) ; n.s. = no significant difference.

II c o l u m n 1 ) . W e h a v e n o w a n a l y s e d m o r e t h a n t w i c e

mall

as many proteins as before, and have shown similar

comigration studies one of these additional proteins,

undamaged

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From

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spot(s) A45, appears to be one of the subunits of the

t o c h a n g e 25 w A 1 , B 6 a n d C 2 3 . I n a d d i t i o n , w e h a v e

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s e e n c h a n g e s in l a b e l l i n g f o r t h e n e w l y a n a l y s e d s p o t s

published observation)

A2, A30, A45, B8, Cll

ling of A45 (see Table II) following nerve damage

and C50, with the remaining

spots showing no significant differences between nor-

(B. Tedeschi and D.L. Wilson, unand thus the decreased label-

is n o t s u r p r i s i n g . O f p a r t i c u l a r s i g n i f i c a n c e is o u r f a i l -

10 ure to detect dramatic changes in any fast transported protein in acidic regions of the gel. In other systems, an acidic protein (GAP-43) has been shown to increase dramatically in regenerating nerves 3'27' 29,31,32 although no such increase in an acidic protein has been seen during frog optic nerve regeneration (see ref. 35; Perng and Perry, unpublished observation). Also, separation of the fast transported proteins from regenerating frog sciatic nerve on more acidic gels (pI range 2.5-6.0) has not revealed a protein that changes so dramatically (data not shown). However, a possible candidate for the counterpart to GAP-43 in the frog fast transported protein pattern is spot B8, which does show a nearly two-fold increase in labelling during sciatic nerve regeneration (Table II). It is possible that such a dramatic increase in this protein, as seen in nerves from some species, is not prerequisite for successful regeneration of frog nerves, although it could still play an important role in regeneration. Such an increase, however, may still be necessary for regeneration of nerves from other animals. We have also shown that further dramatic changes in the abundance of the fast transported proteins are not elicited in nerves crushed for a second time, following a prior conditioning lesion (Table II, columns 3, 4). The data in Table II alone do not allow us to conclude anything about absolute, as opposed to relative, levels of change. However, when coupled with the data in Table I, showing no overall change in absolute levels of labelling of fast transported proteins, we conclude that the normalization procedure done to reduce scatter has not masked any dramatic absolute changes. Furthermore, the sum of cpm's in the protein spots used to normalize the data (all spots in this case) also does not show significant differences among the experimental paradigms. The DRG neurones of the frog sciatic nerve do respond to the conditioning lesion similarly to other nerves, in that the rate of axonal outgrowth after damage is greater if the nerve has received a prior conditioning lesion (Fig. 2), and in that the initiation of outgrowth or sprouting of fibres occurs earlier (see also refs. 13 and 16) in a conditioned nerve (Fig. 3). Fast axonally transported proteins were analysed at two sites along the nerve (see Fig. 1). The first site (Site 1) consisted of the nerve on either side of the test lesion and therefore contained growing nerve fi-

bre tips; whilst the second site (Site 2) was taken from nerve between the D R G and testing lesion site, and therefore contains intact bundles of nerve fibres. Thus, fast transported proteins in transit along the axon (Site 2), and those delivered to the growing fibre tips (Site 1) were analysed. The only consistent qualitative differences seen in the transported protein patterns between Site 1 and Site 2 in these experiments concern spots A25, A30, and C40. The presence of A25 at the site of damage (Site 1) has been described previously36. A30 and C40 were not readily apparent on gels from earlier studies using sodium dodecyl sulphate (SDS) as a solubilizing agent. However, with CHAPSO in the homogenization buffer rather than SDS, these two polypeptides are consistently present on the gels. Intriguingly, A30 is present in intact nerve segments (Site 2) whereas A25 and C40 are not present, and A30 is absent when A25 and C40 are present in damaged nerve segments (Site 1). A possible relationship between A30, and A25 and C40 is presently being investigated. When fast transported proteins are quantitatively analysed at the crush site (Site 1) shortly after damage, there appears to be greater labelling of a few fast transported proteins present in nerves that have received a conditioning lesion compared to non-conditioned nerves (Table ii, column 2). This suggests that these few proteins may be preferentially retained at the growing tips compared to other transported proteins. It is possible that their turnover rate is altered or that they are selectively incorporated or secreted to compartments within the growing axon tip and its milieu, rather than being retrogradely transported. The fast transported polypeptides that appear to be selectively retained (A1, A25, and B8) are not among those seen to be selectively released from the sciatic nerve during axon regrowth 37. Since there is an increased rate of formation of new axon after a condition lesion, there will be a need for additional membrane components. However, because we see no selective increase in fast orthograde transport, these membrane components could be supplied by reduced retrograde transport from the growing axon tips. Thus, the 'retained' polypeptides may be part of the membrane system of the growing axon, and enhanced build-up of these proteins could as easily be the result of the conditioning lesion as contributors to it.

11 It appears then that changes in fast axonally transported proteins do not form the basis for the conditioning lesion effect, although our data do not rule out a possible role for the fast component. Also, con"ditioning effects might result from proteins transported in low abundance which are altered or modified, and would not be detected on our gels. Such a conclusion was reached by McQuarrie and Grafstein 21 after they demonstrated an early but transiently greater increase in total fast transported protein in conditioned compared to non-conditioned goldfish optic nerves. The labelling of fast transported proteins in the conditioned goldfish optic nerves was seen to return to those levels normally seen after a single lesion, whilst the increased rate of nerve elongation was maintained. On the other hand, following a conditioning lesion, slow axonal transport was also seen to increase and remained elevated 21. This suggests that slow axonal transport is more likely to contribute to the conditioning lesion effectl7; however, changes in the fast component could play a role in the production of growth cones and earlier sprouting 21. Such a notion is in accord with evidence that the maximum rate of axon regeneration is correlated with the rate of slow axonal transport 5,38. While we do not see further changes in the proteins in fast transport after a conditioning lesion, one should not infer that fast axonally transported proteins play no role in nerve regeneration or growth cone production. On the contrary, it is generally accepted that fast transport supplies the bulk of material necessary for membrane addition in growing nerves and membrane maintenance in mature, intact nerves. We have presented evidence that the fast transported proteins are unlikely to cause the increased rate of outgrowth seen after a conditioning and testing lesion. The shorter time to initiation of outgrowth after such lesions could be caused by the changes in fast transport that are seen after a single lesion (but see ref. 21). Our data do not allow us to conclude anything about the role of fast transport in reducing the latent period before initiation of outgrowth after a conditioning lesion.

REFERENCES 1 Agranoff, B.W., Field, P. and Gaze, R.M., Neurite out-

The results presented here for individual fast transported proteins are, however, very different from those seen in regenerating goldfish optic nerve, where not only do all the fast transported proteins increase their incorporation of label following a single lesion, and some more dramatically than others 2'29, but the majority also show a marked response to the conditioning lesion paradigm (J.R. Sparrow et al., in preparation). The difference between the goldfish optic nerve and the bullfrog sciatic nerve might not be unexpected since all fast transported proteins increase considerably in regenerating goldfish optic nerves following one lesion 29, unlike the frog sciatic nerve where only a subset of the fast transported proteins change in response to injury 25. Thus the two systems would appear to be very different in their metabolic response to both a single lesion and a conditioning lesion, although both systems regenerate well, particularly if the nerve has been previously conditioned. The conditioning lesion paradigm is an interesting one because it suggests that nerve cells capable of regrowth do not respond maximally after a single lesion. With subsequent lesions they show enhanced regrowth, although there is evidence to suggest that a maximal response is attained after repeated lesions 33. Elucidation of the mechanism behind the conditioning lesion effect could have important clinical applications; however at present, the increased rate of outgrowth of conditioned nerves seems unlikely to be due to changes in fast axonally transported proteins, although perhaps the selective retention of some of these proteins with special functions at the growth cone plays some role in the conditioning lesion effect. ACKNOWLEDGEMENTS We are grateful to Philip Garcia for excellent technical help, particularly with running the gels. This research was supported by NIH Grants EY06449 to G.W.P. and NS18263 to D.L.W.; S.R.K. was supported by a NIH postdoctoral traineeship (NS07044). growth from explanted Xenopus retina: an effect of prior optic nerve section, Brain Research, 113 (1976) 225-234. 2 Benowitz, L.I., Shashoua, V.E. and Yoon, M., Specific

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