J. Phyeiol. (1978), 282, pp. 91-103 With 4 text-figure8 Printed in Great Britain
91
AXONAL TRANSPORT OF ACETYLCHOLINE, CHOLINE ACETYLTRANSFERASE AND CHOLINESTERASE IN REGENERATING PERIPHERAL NERVE
BY R. A. D. O'BRIEN* From the Department of Physiology, University College London, Cower Street, London WC1E 6BT
(Received 14 November 1977) SUMMARY
1. The axonal transport of acetylcholine (ACh), choline acetyltransferase (ChAc) and cholinesterase (ChE) was estimated in the peroneal nerves ofrabbits by measuring the accumulation of each against a nerve crush over a period of 20 hr. 2. Estimates were made of the amounts of these substances that were transported in nerves that had been regenerating for up to 111 days after being crushed or up to 13 days after being cut. 3. The initial response was the same whether the injury was a crush or a cut; the amount of ACh transported was increased, while ChAc and ChE transport was reduced. 4. The amounts of ACh, ChAc and ChE transported tended to return to normal levels when the nerves were allowed to reinnervate the denervated muscles. ChAc transport also showed an early recovery in the cut nerves. 5. The ACh content of the central nerve stump did not alter throughout regeneration but ChAc and ChE contents were reduced at the times when the transport of the enzymes was reduced. 6. These results are discussed in relation to the time course of nerve regeneration. INTRODUCTION
The mechanisms involved in the transport of materials along nerve axons have not yet been clearly determined, but the functions of axonal transport are becoming fairly well understood (for reviews see Heslop (1975) and Lubinska (1975)). In view of the importance of axonal transport in the maintenance of the nerve terminal it would be useful to understand its role in regenerating nerves, whose terminals presumably have different requirements from normal terminals since their function is to re-extend the axons to the target organ rather than to influence the activity of the target organ. Studies on the transport of radioactively labelled proteins in regenerating nerves often provide conflicting results. For example, Carlsson, Bolander & Sj6strand (1971) found a reduction in the speed at which labelled proteins were transported in the regenerating ventral roots of cats, while Grafstein & Murray (1969) found an increase in the speed of protein transport in the regenerating optic nerves of goldfish. Ochs (1976) found no change in the speed of protein transport in the injured *
Present address: Department of Anatomy & Embryology, University College London.
R. A. D. O'BRIEN 92 sciatic nerves of cats or in the amount of the label that was transported, while Frizell & Sj6strand (1974a, b) observed an increase in the amount of [3H]leucine labelled proteins that were transported but found no change in the speed at which
glycoproteins were transported. Since transmitters and related enzymes are concerned in the function of the normal nerve, it is of interest to know how their supply to the terminal is affected during regeneration. The axonal transport of acetylcholine (ACh) has not previously been investigated in regenerating nerves and it would be useful to compare any changes that occur in cholinergic nerves with the study by Karlstr6m & Dahlstrom (1973) on adrenergic nerves, in which a transient reduction in the amount of noradrenaline that was transported occurred between 7 and 17 days after crushing the sciatic nerves of rats; the reduction was permanent if the nerves were cut or tied, suggesting that the recovery of transport was dependent on the nerve's ability to reinnervate the target organ. Hebb & Silver (1966) showed that the capacity of goat sciatic nerves to produce and transport choline acetyltransferase (ChAc), the enzyme responsible for the synthesis of ACh, was reduced 3-4 weeks after crushing the nerves. Frizell & Sj6strand (1974a) found a reduction in the amounts of ChAc and acetylcholinesterase (AChE) transported in rabbit hypoglossal nerves 1 week after crushing, followed by a substantial recovery 3-5 weeks later. These experiments were repeated in the present study to see if there was a correlation between changes in the transport of the enzymes and the transmitter in the same preparation. An attempt is made to relate the observed changes with the time course of regeneration measured by Gutmann, Guttmann, Medawar & Young (1942) and Gutmann (1942). Part of this work has been communicated to the Physiological Society (O'Brien,
1975). METHODS
New Zealand White rabbits were used in these experiments. All the rabbits were adult males from the same stock, and weighed 2-5-3-0 kg. Thiopentone anaesthetic (2.5 g/100 ml.) was injected intravenously. The average dose was 2 ml./kg. Primary leeion. The purpose of the primary lesion was to induce a regenerative response in the injured nerve. The peroneal branch of the sciatic nerve was injured, under aseptic conditions, at the point where the sural blood vessels passed between the main branches of the sciatic nerve, just anterior to the lateral head of the gastrocnemius muscle. The nerve was either crushed, by pulling it against a glass rod with fine (4/0) silk thread, or it was cut with fine scissors. If the lesion was a cut, 1-2 cm of the distal stump was removed to prevent or delay reinnervation of the distal stump. In each animal the same operation was performed on both peroneal nerves. Te8t crushe8. Axonal transport was estimated by measuring the amount of ACh, ChAc or cholinesterase (ChE) that accumulated in the 5 mm of nerve immediately proximal to a crush made 20 hr before the nerve was removed for assaying. The sciatic nerve was exposed high up in the thigh, silk thread was passed around the peroneal branch, and two crushes were made, 2-4 mm apart, using the method described above. These double crushes were used to reduce the possibility that inaccurate slicing through a single crush might lead to contamination between the segments of nerve on either side of the crush. This was important because the part of the nerve immediately distal to the test lesion was also assayed; 'spill-over' from a large accumulation in the proximal segment of nerve would obviously affect the results obtained. The test crushes were 30-35 mm central to the primary lesion and were made in one peroneal nerve of each animal. Both the nerves were removed 20 hr after this operation.
AXONAL TRANSPORT IN REGENERATING NERVE
93
Removal and sampling of nerves. Both sciatic nerves were removed from the animal, starting with the side that had received the test crushes ('experimental nerve'). The peroneal branch was separated from the other branches of the sciatic by carefully cutting or peeling away the sciatic epineurium, and was laid without tension on the platform of a slicing frame. Slices were made through the primary lesion (if it was a crush) and through both of the test crushes, using a razor blade (Fig. 1). The nerve was then sliced into three 5 mm segments, immediately proximal to the test crushes (segment A), immediately distal to the test crushes (segment B) and immediately proximal to the primary lesion (segment E). The length of nerve between segments B and E was then divided into two equal segments (c and D), which were usually 10-15 mm in length. The contralateral nerve, which had not received the test crushes, was then mounted on the frame and sliced into segments whose lengths corresponded to those of the experimental nerve. The nerves were kept moist with saline (0.9 g/100 ml.) throughout this procedure.
Primary
Test crushes (one nerve)
Experimental
aA
E
B
lesion (both nerves) I
C
nerve
Central end
Contralateral nerve
Distal end A
B I
I
C I
l
IE
D
Fig. 1. Diagrammatical representation of the peroneal nerves in an experimental animal. The primary lesion (a crush or a cut) was made at one operation, and the test crushes were made after a specific time in the experimental nerve, 30-35 mm central to the primary lesion. After a further 20 hr both peroneal nerves were removed and sliced into the indicated segments. Segments A, B and E were each 5 mm in length; segments c and D were each 10-15 mm in length.
Time course of experiments. Uninjured (0 hr) nerves were used to estimate the levels of ACh, ChAc and ChE in normal nerves. Axonal transport in normal nerves was estimated by making the primary and test lesions simultaneously and removing the nerves 20 hr later. For nerves regenerating from a crush, both nerves of each animal were given the primary crush in one operation and were removed 6, 13, 51 or (except in the ACh experiments) 111 days later. 20 hr before the nerves were removed one of the nerves was given the test crushes. For nerves that were regenerating from a cut, both nerves of each animal were cut in one operation and removed 6 or 13 days later. 20 hr before the nerves were removed one of the nerves was given the test crushes. Measurement offunctional recovery. Reinnervation of the muscles originally innervated by the peroneal nerve was monitored by observing the return of the toe-spreading response, as described by Gutman et al. (1942). This reflex was tested every 1-3 days in the six rabbits that were used for the 51 day ACh experiments. The response was deemed positive if all the toes were spread apart when the animal was picked up by the scruff of the neck. Extraction and assay procedures ChAc and ChE were measured in the same nerve samples, using the same extraction procedure. ACh was extracted in an acid medium, the purpose of which was to denature the ChAc and ChE and thus minimize the synthesis and hydrolysis of ACh; a separate group of rabbits was therefore used for the ACh experiments. Extraction and assay of ACh. The extraction and assay of ACh was based on the method of
R. A. D. O'BRIEN
94
Goldberg & McCaman(1974). Quaternary ammonium ions (largely consisting of ACh and choline) were selectively extracted from homogenates of the nerve segments using tetraphenyl boron. The ACh was then assayed by hydrolysing it with AChE and converting the choline thus produced to [32P]phosphoryl choline, using choline kinase and [y_32P]ATP. Modifications had to be made to adapt the method for use with peripheral nerve, so the procedure will be described in some detail. After slicing, each nerve sample was dried on filter paper and transferred to a sealed conical glass homogenization tube containing a mixture of N-formic acid: acetone (15: 85 v/v) standing for longer segments. formic acid: acetone were used for 5 mm segments, 100 on ice. jl. 50 essential to minimize and immersion in the formic acid: acetone mixture were complete Speed the ACh synthesis known to occur within 1-2 minutes of cutting a nerve (Feldberg, 1943; Evans & Saunders, 1967) and the time taken between slicing and immersion was never more than 15 sec. Each sample was homogenized on ice using a ground glass pestle attached to a Gallenkamp stirrer. Complete homogenization took up to a minute due to the difficulty in homothe connective tissue, which was partially fixed by the formic acid: acetone mixture. g3nizing The tubes were left, sealed and on ice, for 30 min to allow extraction of the ACh. They were then centrifuged at 1900 x g for 10 min, and the supernatants were divided into two equal, measured aliquots, each of which was transferred to a clean plastic microcentrifuge tube (Hawksley), thus providing duplicate samples for each nerve segment. The samples were thenlyophilized, and the rest of the extraction (involving the tetraphenyl boron) was conducted in accordance with the method of Goldberg & McCaman (1974). The final extracts werelyophilized and stored at -20 'C until the assay was prepared; they were stored for up to 3 months without measurable loss of ACh. Samples from many experiments were assayed over a period of a few days because of the limited half-life of the isotope (14-2 days). The dried extracts were pre-incubated for 30 min at choline phosof a reaction mixture containing 0-5 mM-ATP, 5 38 'C with 10 phokinase (Sigma, crude extract) 0-6 units/ml., and 80 mM-sodium phosphate buffer, pH 8-0. The purpose of this step was to phosphorylate any choline present with unlabelled phosphate. The samples were then incubated for a further 30 min after the addition of 2-5 #I. of a reaction mixture containing 12 /tM-_[y_32P]ATP (Amersham or New England Nuclear, 20-30 Ci/m-mole on reference date), AChE (Sigma, type V) 40 units/ml., and 80 mM-sodium phosphate buffer, pH 8.0. After precipitation with 10 #sl. 300 mM-barium acetate the unreacted ATP was removed by eluting the samples through an anion exchange resin (Bio-Rad AG1X8, formate form) with 50 mM-NaOH. The eluates were collected in scintillation vials containing 10 ml. 2*9 mm aqueous 7-amino-1,3-naphthalene disulphonic acid (Eastman Kodak) and the resultant radiation was counted in the 14C channel of a Packard liquid scintillation spectrometer. External standards of 0 (blank), 10 and 100 p-mole ACh were taken through the entire extraction and assay procedure and were used to construct a standard curve. Internal standards of 100 p-mole ACh were added to some of the nerve samples before homogenization, but the loss of ACh from these standards was no greater than from the external standards. Extraction and of ChAc and C(hE. Each nerve segment was dried on filter paper, weighed on a torsion balance, and transferred to a homogenization tube standing on ice. The tube contained a 1% (w/v) solution of butanol in 0.9 % saline, 50 #1. for 5 mm segments, 100 for longer segments. The sampleswere then homogenized on ice, centrifuged at 1900 x g for 10 min, and aliquots of the supernatant were assayed in duplicate for ChAc and ChE activity, using butanol-saline blanks. ChAc activity was estimated from the conversion of [1-14C]acetyl coenzyme A to [1-14C]ACh, using the method of Glover & Green (1972). ChE activity was estimated from the hydrolysis of [1-14C]ACh to [1-14Cjacetate (Fonnum, 1969).
#sl.
mM-MgCl,,
jA.
6Cerenkov
assay
jul.
RESULTS
The ACh content of the nerves was expressed as p-mole/5 mm nerve; ChAc activity was expressed as p-mole units/5 mm nerve, where 1 p-mole unit represents the synthesis of 1 p-mole of ACh per min at 38 TC; ChE activity was expressed as n-mole units/5 mm nerve, where 1 n-mole unit represents the hydrolysis of 1 n-mole ACh
AXONAL TRANSPORT IN REGENERATING NERVE 95 per min at 38 'C. The nerve content was expressed per unit length rather than per unit weight because of local variations in the fluid content of the nerve after injury (Hebb & Silver, 1961; Lubin'ska & Niemierko, 1971). The levels of ACh, ChAc and ChE in the normal (0 hr) nerves are shown in Fig. 2. There was a small gradient of ACh content and ChAc activity, increasing proximo150
E 100 E La
0
a* 50
0 E 0 150
E
E100 La 0.
0 II
5 mm
C ChE E
4
S
L,
3
S
2
0
C
1 0
5 mm
Fig. 2. A, ACh; B, ChAc and C, ChE content of normal peroneal nerves (unbroken columns) and of nerves which had been crushed, 20 hr before removal, at the position indicated by the arrows (dashed columns). The segments are labelled as in Fig. 1. Vertical bars are ± s.E. of mean. Number of normal nerves used: ACh, 17; ChAe, 14; ChE, 14. Number of 20 hr nerves used: ACh, 11; ChAc, 6; ChE, 5.
R. A. D. O'BRIEN 96 distally, but there was no gradient of ChE activity. The mean levels along this length of nerve were: ACh, 76 p-mole/5 mm; ChAc, 62 p-mole units/5 mm; ChE, 1-79 nmole units/5 mm. To the author's knowledge this radio-enzymatic assay of ACh has not previously been applied to peripheral nerve, but the estimate of the ACh content of the normal nerves corresponds well with previous reports in which conventional bioassay procedures were used. The average mass per unit length of the nerves was 3-5 mg/5 mm, so the ACh content can be expressed as 21P7 n-mole/g or 3-2 ,ag/g. Evans & Saunders (1967) obtained a figure of 16-6 n-mole/g in rabbit sciatic nerves, and MacIntosh (1941) estimated a level of 4 ,ug/g in cat peroneal nerves. Superimposed on the 0 hr levels in Fig. 2 are the values obtained from nerves which had been crushed, at the position indicated by the arrow, 20 hr before removal from the animal. These values are from the contralateral nerves in the 20 hr experiments, so they had received the primary crush but not the test crushes. This shows that the amount of ACh, ChAc and ChE that accumulated against the lesion in 20 hr was entirely contained within the 5 mm of nerve proximal to the lesion (segment E in this case), as the levels in segment D were not raised above the 0 hr level. This is important because axonal transport was estimated from the 20 hr accumulations in the 5 mm nerve segment proximal to the test crushes in the experimental nerves (segment A).
Effect of injury on the ACh, ChAc and ChE content of the nerve The accumulation of ACh, ChAc or ChE against the primary lesion did not spread more than 25 mm central to the injury at any time. Segment A of the contralateral nerves was at least 30 mm central to the primary lesion, so the content of this segment of nerve shows the effect of the primary lesion on the content of the central stump, well clear of 'contamination' by accumulated material. The values obtained are shown by the spotted columns in Fig. 3. There was no significant change in the ACh content of the central nerve stump at any time, but the activities of ChAc and ChE were substantially reduced. ChAc activity was reduced to 40 % of the 0 hr level at 13 days after a crush injury, and to 50 % at 6 days in the cut nerves. ChE activity was reduced to 40 % at 51 days in the crushed nerves, and to 50 % at 13 days in the cut nerves. The loss of enzyme activity was therefore faster if the lesion was a cut, and, although the extent of the loss was similar for both enzymes, ChAc activity was reduced earlier than ChE activity. Axonal transport of ACh, ChAc and ChE The hatched columns in Fig. 3 show the levels in segment A of the experimental nerves. It seems likely that the content of the nerve in this region was affected by the primary lesion to the same extent as in the contralateral nerve, so the amount of ACh, ChAc and ChE that was transported in the 20 hr between making the test crushes and removing the nerves is given by the difference between the levels in segment A of the experimental nerve and segment A of the contralateral nerve (hatched and spotted columns, respectively, in Fig. 3). In the 20 hr nerves this difference is used as an estimate of the axonal transport in normal nerves, so the
AXONAL TRANSPORT IN REGENERATING NERVE Cut nerves
Crushed nerves Test
Exp. Con.
200
-
A
Test
Primary
4J
E" 1 Q e4:i -
O
97
Exp.
Primary
Itia--
Con. Ic-71
-I
I|
ACh
E
E %100
0
-
20 hr
6 days
13 days
51 days
6 days
13 days
6 days
13 days
51 days 111 days
6 days
13 days
6 days
13 days
51 days 111 dqys
6 days
13 days
150 -' B ChAc E E Lo
E
100 -
50 -
a 0-
C ChE
5
E E
20 hr
4
ILo
(;I
3
-
0 0a
2
-
I
1
c
E
_
0-
20 hr
Fig. 3. A, ACh; B, ChAc and C, ChE content of segment A in the experimental nerves (hatched columns) and contralateral nerves (spotted columns). Vertical bars are + s.E. of mean. The individual accumulations were calculated and are shown in Table 1. The 20 hr crushed nerves were used to estimate transport in normal nerves. The inset diagrams are as in Fig. 1. 4
P HY 282
R. A. D. O'BRIEN 98 results from the regenerating nerves are compared with the results from the 20 hr nerves to demonstrate changes in axonal transport. The accumulations were calculated for each individual experiment, and the results are shown in Table 1. In Fig. 4 the accumulations are normalized by expressing them as a percentage of the 20 hr accumulation. ACh transport was substantially increased during the two weeks after the primary injury, whether it was a crush or a cut. The apparent increase between 6 and 13 days TABLf 1. Axonal transport of ACh, ChAc and ChE in regenerating nerves Type of ACh ChAc ChE Time after primary accumulation accumulation accumulation primary lesion lesion (p-mole/5 mm) (p-mole units/5 mm) (n-mole units/5 mm) 20hr Crush 37± 11 (11) 77± 18 (6) 2.79±0-29 (5) 6 days Crush 89±16 (4) 27±16 (3) 0-69±0'12 (5) 13 days Crush 85 ± 15 (6) 1.7 ± 4-1 (4) 1*23 ± 0.26 (6) 51 days Crush 49 ± 14 (3) 71± 25 (6) 1-97 ±0 07 (3) 111 days Crush 40± 16 (3) 1D69±0.24 (4) 6 days Cut 85±15 (6) 32±3.1 (4) 1-09±0-14 (4) Cut 13 days 64 ± 10 (4) 123 ± 16 (4) 0*99 ± 0*09 (4)
The accumulation of ACh, ChAc or ChE against the test crushes (concentration in segment A of the experimental nerve - concentration in segment A of the contralateral nerve) was calculated for each animal. The 20 hr nerves give an estimate of transport in normal nerves. Values are mean + s.E. of mean, with the number of animals in brackets. These results are normalised in Fig. 4. A Crushed nerves - ACh B Cut nerves
--ChAc
300
ChE
200
-
100
20 hr
6 days
13 days
51 days 111 days Time after primary lesion
20 hr
6 days
13 days
Fig. 4. The accumulation of ACh, ChAc and ChE against the test crushes in the regenerating nerves are expressed as a percentage of the accumulation in the 20 hr nerves; A, up to 111 days after a crush primary lesion, and B, up to 13 days after a cut. Open circles: points not significantly different from the 20 hr accumulation. Filled circles: points significantly different from the 20 hr accumulation (P < 0-05, as determined with Student's two-tailed t test). Number of observations as in Table 1.
AXONAL TRANSPORT IN REGENERATING NERVE 99 in the cut nerves was not statistically significant. ChAc transport was reduced to an unobservable level 13 days after a crush injury, but recovered to a near-normal level by 51 days. In the cut nerves, however, ChAc transport was only transiently diminished at 6 days and showed a recovery by 13 days. ChE transport was at a minimum 6 days after a crush injury and showed a recovery at later times, although it did not return to the normal level even at 111 days. In the cut nerves ChE transport was reduced at 6 days and remained unchanged at 13 days. Inhibitors of non-specific esterases were not included in the ChE assay, but ChE accumulations can be considered to give reliable estimates of AChE transport since AChE constitutes about 90 % of the ChE activity in peripheral nerves and is responsible for virtually all of the transported ChE activity (Ranish & Ochs, 1972; Tucek, 1975).
Functional recovery The earliest sign of functional reinnervation was seen at 35 days in both feet of one rabbit and in one foot of another. Functional recovery in the other rabbits occurred on day 40 or 42, giving an average recovery time of 39 days. DISCUSSION
The method of measuring axonal transport from the accumulation of material against a crush has the advantage that its specificity is limited only by the specificity of the assay procedure, so it is useful for studying the transport of specific intra-axonal substances. This facility is not offered by the method of injecting radioactivelylabelled precursors into neuronal pools, the specificity of which is at best limited to the labelling of groups of related compounds. Another advantage of the crush method is that it gives a quantitative assessment of the amount ofmaterial that is transported. This is particularly appropriate in the present study since it shows changes in the amounts of ACh, ChAc and ChE that are delivered to the nerve terminals during the processes of nerve growth and reinnervation of the muscles. A disadvantage of the method is that, unlike studies with labelled precursors, the velocity of transport is difficult to calculate as the accumulation of a substance depends on the proportion of the substance in the axon that is available for transport, as well as on the transport velocity. Attempts have been made by others to estimate the mobile portions of ACh, ChAc and AChE, and hence to calculate their rates of transport, but these are not particularly relevant to this study because the mobile portions might change in magnitude during regeneration. The possibility that the lesion itself affects- axonal transport has been discussed in detail by Lubinska (1964, 1975). The fast axonal transport of radioactively labelled proteins continues unaffected even when the nerve is isolated from the cell body region (Ochs & Ranish, 1969), and the transport of [14C]choline-labelled phospholipids (mainly lecithin) is accelerated (Dziegielewska, Evans & Saunders, 1976). The accumulation of ACh, ChAc and AChE is linear for at least 18-20 hr after making the lesion (Frizell, Hasselgren & Sj6strand, 1970; Higgendal, Saunders & Dahlstrom, 1971; Lubiuska & Niemierko, 1971; Tucek, 1975). It is generally accepted that the accumulation of protein against a nerve lesion is 4-2
R. A. D. O'BRIEN due to the damming of axonal transport, since there is no appreciable protein synthesis in the axon (see Droz & Koenig, 1970), but when considering the accumulation of ACh it is important to distinguish between the damming of transported ACh and local synthesis of the transmitter in the region of the test crush. Dahlstr6m, Evans, Higgendal, Heiwall & Saunders (1974) considered the possibility that accumulated ChAc was responsible for the build-up of ACh against a nerve crush, but concluded that the relative rates of accumulation (ACh accumulation was the faster) were sufficiently different to discount this possibility. In support of this view it is significant that the ACh accumulation in the 13 days crushed nerves was more than twice the normal accumulation. while the accumulation of ChAc was abolished. Alternatively, local synthesis of ACh may be limited not by the level of ChAc but by the availability of substrate. For instance, choline could be taken up by the nerve at the site of the injury and converted into ACh. If this is so, one would expect to see a build-up of ACh distal to the test crush, and this was checked in the present experiments; only in the 13 day cut nerves was there a significant increase of ACh distal to the test crushes (measured by subtracting the level in segment B of the contralateral nerves from segment B of the experimental nerves). This distal accumulation amounted to 52 + 7-4 p-mole (mean + S.E. of mean, n = 4) compared with the proximal accumulation of 123 + 16 p-mole (n = 4). If the distal increase in ACh was due to local synthesis, a similar amount should have been synthesized in the proximal segment, so that the net amount of ACh transported can be corrected to 71 + 18 p-mole, which is about the same level as found in the 6 day cut nerves, and is still higher than the amount of ACh transported in the normal nerves. However, the possibility that the distal accumulation of ACh was at least partly due to retrograde axonal transport of the transmitter cannot be ruled out. The detailed work of Gutmann et al. (1942) included measurements of the rate of regeneration of rabbit peroneal nerves and their results are therefore directly applicable to the present experiments. The distance between the primary lesion and the most proximal of the denervated muscles (peroneus longus) was between 3 and 4 cm. From the data of Gutmann et al. (1942) the following time course of regeneration has been estimated for the crushed nerves: by 6 days the regenerating fibres should have crossed the site of the lesion and begun to reinnervate the distal stump; reinnervation of peroneus longus should have started about the 13th day, and functional reinnervation of all the denervated muscles should have been complete at about the 40th day. This corresponds well with the average of 39 days that was determined experimentally. Between 51 and 111 days regenerated fibres become more mature, but are still smaller and more numerous than normal (Aitken, Sharman & Young, 1947; Shawe, 1955). In the present experiments on cut nerves, reinnervation of the distal stump was prevented by the removal of a segment of nerve as well as by the natural retraction of the stumps. It was observed that the stumps were still completely separated 13 days after the lesion. The initial changes in axonal transport were the same whether the lesion was a crush or a cut; the transport of ACh was increased while that of the enzymes was reduced. This response did not depend on reinnervation of the distal nerve stump since steps were taken to prevent this in the cut nerves, so it is probably part of the 100
AXONAL TRANSPORT IN REGENERATING NERVE 101 general response of the nerve to injury. In the crushed nerves the tendency for transport to return to normal was first detected about 12 days after functional recovery, so it is not clear if it was in response to the 'morphological' reinnervation of the denervated muscles or to their functional reinnervation. The changes in enzyme transport correspond with the observations of Frizell & Sj6strand (1974a) and support the hypothesis that injury to a nerve causes a shift in the emphasis of the neuronal metabolism from the production and transport of transmitter-related materials to the production and transport of materials necessary to the reconstruction of the axon (see Watson, 1974). The reduction of noradrenaline transport observed by Karlstrdm & Dahlstr6m (1974) also supports this hypothesis, so it is surprising that the transport of ACh should be increased in an injured nerve. Most of the ACh in a normal axon is freely diffusible (Evans & Saunders, 1974), but there is also a small proportion of ACh which is contained in a particulate fraction carried at a fast rate of transport (see Saunders, 1975). The proportion of nondiffusible ACh increases in a regenerating nerve (Evans & Saunders, 1974), so the increased transport of ACh could correspond to an increase in the transport of particles with which the ACh is associated. This is also suggested by the observation that the amount of ACh in the nerve is unaffected by the initial injury; if some of the ACh in the nerve were to be transferred from a slow-moving, soluble pool to a rapidly-moving particulate pool the amount of ACh in the nerve could remain unchanged while the amount transported in a given time is increased. One can only speculate on the function of an increased transport of ACh in a regenerating nerve; for example, it may be essential for the regenerating nerve to release ACh and for the muscle, once reinnervated, to respond to the transmitter before functional contact can be established (see Gordon, Jones & Vrbova, 1976). According to Watson (1974) the formation of functional contact may provide a signal to the neurone causing the return of its metabolism to the provision of transmitterrelated enzymes rather than structural materials. The recovery of ChAc transport in the 13 day cut nerves is curious, particularly as ChE transport was still reduced; an explanation for this is not readily available. The presence of ChE transport in the 13 day crushed nerves (in which the transport of ChAc was reduced to an unobservable level) may reflect an association of AChE with transported membrane particles, as it is known that AChE is present in the axolemma of normal nerves (Lewis & Shute, 1966; Tennyson, Brzin & Duffy, 1968). This work was undertaken during the tenure of a Medical Research Council training award. I am very grateful to Dr Norman Saunders for his constant help and advice.
REFERENCES
AITKEN, J. T., SHARMAN, M. & YOUNG, J. Z. (1947). Maturation of regenerating nerve fibres with various peripheral connexions. J. Anat. 81, 1-22. CARLssoN, C. A., BOLANDER, P. & SJd8TMAND, J. (1971). Changes in axonal transport during regeneration of feline ventral roots. J. neural. Sci. 14, 75-93. DAHLSTROM, A. B., EvANs, C. A. N., HAGGENDAL, C. J., HEIwALL, P. 0. & SAUNDERS, N. R. (1974). Rapid transport of acetylcholine in rat sciatic nerve proximal and distal to a lesion. J. neural Transm. 35, 1-11.
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DROZ, B. & KOENIG, H. L. (1970). Localisation of protein metabolism in neurons. In Protein Metabolism of the Nervous System, ed. LAJTHA, A., pp. 93-106. New York: Plenum. DZIEGIELEWSKA, K., EvANs, C. A. N. & SAUNDERS, N. R. (1976). Effect of axotomy on rapid axonal transport of phospholipids in peripheral nerve of the rat. J. Physiol. 263, 200P. EvANs, C. A. N. & SAUNDERS, N. R. (1967). The distribution of acetylcholine in normal and regenerating nerves. J. Phy8iol. 192, 79-92. EvANs, C. A. N. & SAUNDERS, N. R. (1974). An outflow of acetylcholine from normal and regenerating ventral roots of the cat. J. Physiol. 240, 15-32. FELDBERG, W. (1943). Synthesis of acetylcholine in sympathetic and cholinergic nerve. J. Physiol. 101, 342-445. FoNNum, F. (1969). Radiochemical micro assays for the determination of choline acetyltransferase and acetylcholinesterase activities. Biochem. J. 115, 465-472. FRuzmLL, M. & SjOsTRAND, J. (1974a). Transport of proteins, glycoproteins and cholinergic enzymes in regenerating hypoglossal neurones. J. Neurochem. 22, 845-850. FR=zLL, M. & SJ6STRAND, J. (1974b). The axonal transport of [3H]fucose labelled glycoprotein in normal and regenerating peripheral nerves. Brain Res. 74, 109-123. FRIzELL, M., HASSELGREN, P. 0. & SJOSTRAND, J. (1970). Axoplasmic transport of acetylcholinesterase and choline acetyltransferase in the vagus and hypoglossal nerve of the rabbit. Expl Brain Res. 10, 526-531. GLOVER, V. & GREEN, D. P. L. (1972). A simple, quick microassay for choline acetyltransferase. J. Neurochem. 19, 2465-2466. GuTMANN, E. (1942). Factors affecting recovery of motor function after nerve lesions. J. Neurol. Psychiat., Lond. 5, 81-95. GuTMANN, E., GuMANN, L., MEDAWAR, P. B. & YOUNG, J. Z. (1942). The rate of regeneration of nerve. J. exp. Biol. 19, 14-43. GOLDBERG, A. M. & McCAMAN, R. E. (1974). An enzymatic method for the determination of picomole amounts of choline and acetylcholine. In Choline and Acetylcholine: Hand-book of Chemical Assay Methods, ed. HANIN, I., pp. 47-61. New York: Raven Press. GORDON, T., JONES, R. & VRBOVX, G. (1976). Changes in chemosensitivity of skeletal muscles as related to endplate formation. Prog. Neurobiol. 6, 103-136. GiF&sTIn;, B. & MURRAY, M. (1969). Transport of protein in goldfish optic nerve during regeneration. Expl Neurol. 25, 494-508. HAGGENDAL, C. J., SAUNDERS, N. R. & DAHLSTROM, A. B. (1971). Rapid accumulation of acetylcholine in nerve above a crush. J. Pharm. Pharmac. 23, 552-555. HEBB, C. 0. & SIVER, A. (1961). Gradient of choline acetylase activity. Nature, Lond. 189, 123-125. HEBB, C. & SILVER, A. (1966). Axoplasmic flow of protein. In Protides of the Biological Fluids, ed. PETERS, H., pp. 179-180. Amsterdam: Elsevier. HESLOP, J. P. (1975). Axonal flow and fast transport in nerves. Adv. comp. Physiol. Biochem. 6, 75-163. KARLSTROM, L. & DAHLSTROM, A. (1973). The effect of different types of axonal trauma on the synthesis and transport of amine storage granules in rat sciatic nerves. J. Neurobiol. 4, 191200. LEWIS, P. R. & SAuTE, C. C. D. (1966). The distribution of cholinesterase in cholinergic neurons demonstrated with the electron microscope. J. Cell Sci. 1, 381-390. LUBINSKA, L. (1964). Axoplasmic streaming in regenerating and normal nerve fibres. Prog. Brain Res. 13, 1-66. LUBIN'sKA, L. (1975). On axoplasmic flow. Int. Rev. Neurobiol. 17, 241-296. LuBIN'SKA, L. & NIEIE ERKO, S. (1971). Velocity and intensity of bidirectional migration of acetylcholinesterase in transacted nerves. Brain Res. 27, 329-342. MAcINTrosH, F. C. (1941). The distribution of acetylcholine inthe peripheralandthecentralnervous system. J. Physiol. 99, 436-442. O'BRIEN, R. A. D. (1975). Transport of cholinergic enzymes in regenerating peripheral nerve. J. Physiol. 252, 62-63P. OCHS, S. (1976). Fast axoplasmic transport in the fibres of chromatolysed neurones. J. Physiol. 255, 249-261. OCHS, S. & RANISH, N. (1969). Characteristics of the fast transport system in mammalian nerve fibres. .1. Neurobiol. 1, 247-261.
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RANISH, N. & OCHS, S. (1972). Fast axoplasmic transport of acetylcholinesterase in mammalian nerves. J. Neurochem. 19, 2641-2649. SAUNDERS, N. R. (1975). Axonal transport of acetylcholine. In Cholinergic Mechanism8, ed. WASER, P., pp. 177-185. New York: Raven Press. SHAwE, G. D. H. (1955). On the number of branches formed by regenerating nerve-fibres. Br. J. Surg. 42, 474-488. TENNYSON, V. M., BIziN, M. & DuFiy, P. (1968). Electron microscopic cytochemistry and microgasometric analysis of cholinesterase in the nervous system. Prog. Brain Res. 29, 41-61. Tu6EK, S. (1975). Transport of choline acetyltransferase and acetylcholinesterase in the central stump and isolated segments of a peripheral nerve. Brain ReB. 86, 259-270. WATSON, W. E. (1974). Cellular response to axotomy and to related procedures. Br. med. Bull. 30, 112-115.
91
AXONAL TRANSPORT OF ACETYLCHOLINE, CHOLINE ACETYLTRANSFERASE AND CHOLINESTERASE IN REGENERATING PERIPHERAL NERVE
BY R. A. D. O'BRIEN* From the Department of Physiology, University College London, Cower Street, London WC1E 6BT
(Received 14 November 1977) SUMMARY
1. The axonal transport of acetylcholine (ACh), choline acetyltransferase (ChAc) and cholinesterase (ChE) was estimated in the peroneal nerves ofrabbits by measuring the accumulation of each against a nerve crush over a period of 20 hr. 2. Estimates were made of the amounts of these substances that were transported in nerves that had been regenerating for up to 111 days after being crushed or up to 13 days after being cut. 3. The initial response was the same whether the injury was a crush or a cut; the amount of ACh transported was increased, while ChAc and ChE transport was reduced. 4. The amounts of ACh, ChAc and ChE transported tended to return to normal levels when the nerves were allowed to reinnervate the denervated muscles. ChAc transport also showed an early recovery in the cut nerves. 5. The ACh content of the central nerve stump did not alter throughout regeneration but ChAc and ChE contents were reduced at the times when the transport of the enzymes was reduced. 6. These results are discussed in relation to the time course of nerve regeneration. INTRODUCTION
The mechanisms involved in the transport of materials along nerve axons have not yet been clearly determined, but the functions of axonal transport are becoming fairly well understood (for reviews see Heslop (1975) and Lubinska (1975)). In view of the importance of axonal transport in the maintenance of the nerve terminal it would be useful to understand its role in regenerating nerves, whose terminals presumably have different requirements from normal terminals since their function is to re-extend the axons to the target organ rather than to influence the activity of the target organ. Studies on the transport of radioactively labelled proteins in regenerating nerves often provide conflicting results. For example, Carlsson, Bolander & Sj6strand (1971) found a reduction in the speed at which labelled proteins were transported in the regenerating ventral roots of cats, while Grafstein & Murray (1969) found an increase in the speed of protein transport in the regenerating optic nerves of goldfish. Ochs (1976) found no change in the speed of protein transport in the injured *
Present address: Department of Anatomy & Embryology, University College London.
R. A. D. O'BRIEN 92 sciatic nerves of cats or in the amount of the label that was transported, while Frizell & Sj6strand (1974a, b) observed an increase in the amount of [3H]leucine labelled proteins that were transported but found no change in the speed at which
glycoproteins were transported. Since transmitters and related enzymes are concerned in the function of the normal nerve, it is of interest to know how their supply to the terminal is affected during regeneration. The axonal transport of acetylcholine (ACh) has not previously been investigated in regenerating nerves and it would be useful to compare any changes that occur in cholinergic nerves with the study by Karlstr6m & Dahlstrom (1973) on adrenergic nerves, in which a transient reduction in the amount of noradrenaline that was transported occurred between 7 and 17 days after crushing the sciatic nerves of rats; the reduction was permanent if the nerves were cut or tied, suggesting that the recovery of transport was dependent on the nerve's ability to reinnervate the target organ. Hebb & Silver (1966) showed that the capacity of goat sciatic nerves to produce and transport choline acetyltransferase (ChAc), the enzyme responsible for the synthesis of ACh, was reduced 3-4 weeks after crushing the nerves. Frizell & Sj6strand (1974a) found a reduction in the amounts of ChAc and acetylcholinesterase (AChE) transported in rabbit hypoglossal nerves 1 week after crushing, followed by a substantial recovery 3-5 weeks later. These experiments were repeated in the present study to see if there was a correlation between changes in the transport of the enzymes and the transmitter in the same preparation. An attempt is made to relate the observed changes with the time course of regeneration measured by Gutmann, Guttmann, Medawar & Young (1942) and Gutmann (1942). Part of this work has been communicated to the Physiological Society (O'Brien,
1975). METHODS
New Zealand White rabbits were used in these experiments. All the rabbits were adult males from the same stock, and weighed 2-5-3-0 kg. Thiopentone anaesthetic (2.5 g/100 ml.) was injected intravenously. The average dose was 2 ml./kg. Primary leeion. The purpose of the primary lesion was to induce a regenerative response in the injured nerve. The peroneal branch of the sciatic nerve was injured, under aseptic conditions, at the point where the sural blood vessels passed between the main branches of the sciatic nerve, just anterior to the lateral head of the gastrocnemius muscle. The nerve was either crushed, by pulling it against a glass rod with fine (4/0) silk thread, or it was cut with fine scissors. If the lesion was a cut, 1-2 cm of the distal stump was removed to prevent or delay reinnervation of the distal stump. In each animal the same operation was performed on both peroneal nerves. Te8t crushe8. Axonal transport was estimated by measuring the amount of ACh, ChAc or cholinesterase (ChE) that accumulated in the 5 mm of nerve immediately proximal to a crush made 20 hr before the nerve was removed for assaying. The sciatic nerve was exposed high up in the thigh, silk thread was passed around the peroneal branch, and two crushes were made, 2-4 mm apart, using the method described above. These double crushes were used to reduce the possibility that inaccurate slicing through a single crush might lead to contamination between the segments of nerve on either side of the crush. This was important because the part of the nerve immediately distal to the test lesion was also assayed; 'spill-over' from a large accumulation in the proximal segment of nerve would obviously affect the results obtained. The test crushes were 30-35 mm central to the primary lesion and were made in one peroneal nerve of each animal. Both the nerves were removed 20 hr after this operation.
AXONAL TRANSPORT IN REGENERATING NERVE
93
Removal and sampling of nerves. Both sciatic nerves were removed from the animal, starting with the side that had received the test crushes ('experimental nerve'). The peroneal branch was separated from the other branches of the sciatic by carefully cutting or peeling away the sciatic epineurium, and was laid without tension on the platform of a slicing frame. Slices were made through the primary lesion (if it was a crush) and through both of the test crushes, using a razor blade (Fig. 1). The nerve was then sliced into three 5 mm segments, immediately proximal to the test crushes (segment A), immediately distal to the test crushes (segment B) and immediately proximal to the primary lesion (segment E). The length of nerve between segments B and E was then divided into two equal segments (c and D), which were usually 10-15 mm in length. The contralateral nerve, which had not received the test crushes, was then mounted on the frame and sliced into segments whose lengths corresponded to those of the experimental nerve. The nerves were kept moist with saline (0.9 g/100 ml.) throughout this procedure.
Primary
Test crushes (one nerve)
Experimental
aA
E
B
lesion (both nerves) I
C
nerve
Central end
Contralateral nerve
Distal end A
B I
I
C I
l
IE
D
Fig. 1. Diagrammatical representation of the peroneal nerves in an experimental animal. The primary lesion (a crush or a cut) was made at one operation, and the test crushes were made after a specific time in the experimental nerve, 30-35 mm central to the primary lesion. After a further 20 hr both peroneal nerves were removed and sliced into the indicated segments. Segments A, B and E were each 5 mm in length; segments c and D were each 10-15 mm in length.
Time course of experiments. Uninjured (0 hr) nerves were used to estimate the levels of ACh, ChAc and ChE in normal nerves. Axonal transport in normal nerves was estimated by making the primary and test lesions simultaneously and removing the nerves 20 hr later. For nerves regenerating from a crush, both nerves of each animal were given the primary crush in one operation and were removed 6, 13, 51 or (except in the ACh experiments) 111 days later. 20 hr before the nerves were removed one of the nerves was given the test crushes. For nerves that were regenerating from a cut, both nerves of each animal were cut in one operation and removed 6 or 13 days later. 20 hr before the nerves were removed one of the nerves was given the test crushes. Measurement offunctional recovery. Reinnervation of the muscles originally innervated by the peroneal nerve was monitored by observing the return of the toe-spreading response, as described by Gutman et al. (1942). This reflex was tested every 1-3 days in the six rabbits that were used for the 51 day ACh experiments. The response was deemed positive if all the toes were spread apart when the animal was picked up by the scruff of the neck. Extraction and assay procedures ChAc and ChE were measured in the same nerve samples, using the same extraction procedure. ACh was extracted in an acid medium, the purpose of which was to denature the ChAc and ChE and thus minimize the synthesis and hydrolysis of ACh; a separate group of rabbits was therefore used for the ACh experiments. Extraction and assay of ACh. The extraction and assay of ACh was based on the method of
R. A. D. O'BRIEN
94
Goldberg & McCaman(1974). Quaternary ammonium ions (largely consisting of ACh and choline) were selectively extracted from homogenates of the nerve segments using tetraphenyl boron. The ACh was then assayed by hydrolysing it with AChE and converting the choline thus produced to [32P]phosphoryl choline, using choline kinase and [y_32P]ATP. Modifications had to be made to adapt the method for use with peripheral nerve, so the procedure will be described in some detail. After slicing, each nerve sample was dried on filter paper and transferred to a sealed conical glass homogenization tube containing a mixture of N-formic acid: acetone (15: 85 v/v) standing for longer segments. formic acid: acetone were used for 5 mm segments, 100 on ice. jl. 50 essential to minimize and immersion in the formic acid: acetone mixture were complete Speed the ACh synthesis known to occur within 1-2 minutes of cutting a nerve (Feldberg, 1943; Evans & Saunders, 1967) and the time taken between slicing and immersion was never more than 15 sec. Each sample was homogenized on ice using a ground glass pestle attached to a Gallenkamp stirrer. Complete homogenization took up to a minute due to the difficulty in homothe connective tissue, which was partially fixed by the formic acid: acetone mixture. g3nizing The tubes were left, sealed and on ice, for 30 min to allow extraction of the ACh. They were then centrifuged at 1900 x g for 10 min, and the supernatants were divided into two equal, measured aliquots, each of which was transferred to a clean plastic microcentrifuge tube (Hawksley), thus providing duplicate samples for each nerve segment. The samples were thenlyophilized, and the rest of the extraction (involving the tetraphenyl boron) was conducted in accordance with the method of Goldberg & McCaman (1974). The final extracts werelyophilized and stored at -20 'C until the assay was prepared; they were stored for up to 3 months without measurable loss of ACh. Samples from many experiments were assayed over a period of a few days because of the limited half-life of the isotope (14-2 days). The dried extracts were pre-incubated for 30 min at choline phosof a reaction mixture containing 0-5 mM-ATP, 5 38 'C with 10 phokinase (Sigma, crude extract) 0-6 units/ml., and 80 mM-sodium phosphate buffer, pH 8-0. The purpose of this step was to phosphorylate any choline present with unlabelled phosphate. The samples were then incubated for a further 30 min after the addition of 2-5 #I. of a reaction mixture containing 12 /tM-_[y_32P]ATP (Amersham or New England Nuclear, 20-30 Ci/m-mole on reference date), AChE (Sigma, type V) 40 units/ml., and 80 mM-sodium phosphate buffer, pH 8.0. After precipitation with 10 #sl. 300 mM-barium acetate the unreacted ATP was removed by eluting the samples through an anion exchange resin (Bio-Rad AG1X8, formate form) with 50 mM-NaOH. The eluates were collected in scintillation vials containing 10 ml. 2*9 mm aqueous 7-amino-1,3-naphthalene disulphonic acid (Eastman Kodak) and the resultant radiation was counted in the 14C channel of a Packard liquid scintillation spectrometer. External standards of 0 (blank), 10 and 100 p-mole ACh were taken through the entire extraction and assay procedure and were used to construct a standard curve. Internal standards of 100 p-mole ACh were added to some of the nerve samples before homogenization, but the loss of ACh from these standards was no greater than from the external standards. Extraction and of ChAc and C(hE. Each nerve segment was dried on filter paper, weighed on a torsion balance, and transferred to a homogenization tube standing on ice. The tube contained a 1% (w/v) solution of butanol in 0.9 % saline, 50 #1. for 5 mm segments, 100 for longer segments. The sampleswere then homogenized on ice, centrifuged at 1900 x g for 10 min, and aliquots of the supernatant were assayed in duplicate for ChAc and ChE activity, using butanol-saline blanks. ChAc activity was estimated from the conversion of [1-14C]acetyl coenzyme A to [1-14C]ACh, using the method of Glover & Green (1972). ChE activity was estimated from the hydrolysis of [1-14C]ACh to [1-14Cjacetate (Fonnum, 1969).
#sl.
mM-MgCl,,
jA.
6Cerenkov
assay
jul.
RESULTS
The ACh content of the nerves was expressed as p-mole/5 mm nerve; ChAc activity was expressed as p-mole units/5 mm nerve, where 1 p-mole unit represents the synthesis of 1 p-mole of ACh per min at 38 TC; ChE activity was expressed as n-mole units/5 mm nerve, where 1 n-mole unit represents the hydrolysis of 1 n-mole ACh
AXONAL TRANSPORT IN REGENERATING NERVE 95 per min at 38 'C. The nerve content was expressed per unit length rather than per unit weight because of local variations in the fluid content of the nerve after injury (Hebb & Silver, 1961; Lubin'ska & Niemierko, 1971). The levels of ACh, ChAc and ChE in the normal (0 hr) nerves are shown in Fig. 2. There was a small gradient of ACh content and ChAc activity, increasing proximo150
E 100 E La
0
a* 50
0 E 0 150
E
E100 La 0.
0 II
5 mm
C ChE E
4
S
L,
3
S
2
0
C
1 0
5 mm
Fig. 2. A, ACh; B, ChAc and C, ChE content of normal peroneal nerves (unbroken columns) and of nerves which had been crushed, 20 hr before removal, at the position indicated by the arrows (dashed columns). The segments are labelled as in Fig. 1. Vertical bars are ± s.E. of mean. Number of normal nerves used: ACh, 17; ChAe, 14; ChE, 14. Number of 20 hr nerves used: ACh, 11; ChAc, 6; ChE, 5.
R. A. D. O'BRIEN 96 distally, but there was no gradient of ChE activity. The mean levels along this length of nerve were: ACh, 76 p-mole/5 mm; ChAc, 62 p-mole units/5 mm; ChE, 1-79 nmole units/5 mm. To the author's knowledge this radio-enzymatic assay of ACh has not previously been applied to peripheral nerve, but the estimate of the ACh content of the normal nerves corresponds well with previous reports in which conventional bioassay procedures were used. The average mass per unit length of the nerves was 3-5 mg/5 mm, so the ACh content can be expressed as 21P7 n-mole/g or 3-2 ,ag/g. Evans & Saunders (1967) obtained a figure of 16-6 n-mole/g in rabbit sciatic nerves, and MacIntosh (1941) estimated a level of 4 ,ug/g in cat peroneal nerves. Superimposed on the 0 hr levels in Fig. 2 are the values obtained from nerves which had been crushed, at the position indicated by the arrow, 20 hr before removal from the animal. These values are from the contralateral nerves in the 20 hr experiments, so they had received the primary crush but not the test crushes. This shows that the amount of ACh, ChAc and ChE that accumulated against the lesion in 20 hr was entirely contained within the 5 mm of nerve proximal to the lesion (segment E in this case), as the levels in segment D were not raised above the 0 hr level. This is important because axonal transport was estimated from the 20 hr accumulations in the 5 mm nerve segment proximal to the test crushes in the experimental nerves (segment A).
Effect of injury on the ACh, ChAc and ChE content of the nerve The accumulation of ACh, ChAc or ChE against the primary lesion did not spread more than 25 mm central to the injury at any time. Segment A of the contralateral nerves was at least 30 mm central to the primary lesion, so the content of this segment of nerve shows the effect of the primary lesion on the content of the central stump, well clear of 'contamination' by accumulated material. The values obtained are shown by the spotted columns in Fig. 3. There was no significant change in the ACh content of the central nerve stump at any time, but the activities of ChAc and ChE were substantially reduced. ChAc activity was reduced to 40 % of the 0 hr level at 13 days after a crush injury, and to 50 % at 6 days in the cut nerves. ChE activity was reduced to 40 % at 51 days in the crushed nerves, and to 50 % at 13 days in the cut nerves. The loss of enzyme activity was therefore faster if the lesion was a cut, and, although the extent of the loss was similar for both enzymes, ChAc activity was reduced earlier than ChE activity. Axonal transport of ACh, ChAc and ChE The hatched columns in Fig. 3 show the levels in segment A of the experimental nerves. It seems likely that the content of the nerve in this region was affected by the primary lesion to the same extent as in the contralateral nerve, so the amount of ACh, ChAc and ChE that was transported in the 20 hr between making the test crushes and removing the nerves is given by the difference between the levels in segment A of the experimental nerve and segment A of the contralateral nerve (hatched and spotted columns, respectively, in Fig. 3). In the 20 hr nerves this difference is used as an estimate of the axonal transport in normal nerves, so the
AXONAL TRANSPORT IN REGENERATING NERVE Cut nerves
Crushed nerves Test
Exp. Con.
200
-
A
Test
Primary
4J
E" 1 Q e4:i -
O
97
Exp.
Primary
Itia--
Con. Ic-71
-I
I|
ACh
E
E %100
0
-
20 hr
6 days
13 days
51 days
6 days
13 days
6 days
13 days
51 days 111 days
6 days
13 days
6 days
13 days
51 days 111 dqys
6 days
13 days
150 -' B ChAc E E Lo
E
100 -
50 -
a 0-
C ChE
5
E E
20 hr
4
ILo
(;I
3
-
0 0a
2
-
I
1
c
E
_
0-
20 hr
Fig. 3. A, ACh; B, ChAc and C, ChE content of segment A in the experimental nerves (hatched columns) and contralateral nerves (spotted columns). Vertical bars are + s.E. of mean. The individual accumulations were calculated and are shown in Table 1. The 20 hr crushed nerves were used to estimate transport in normal nerves. The inset diagrams are as in Fig. 1. 4
P HY 282
R. A. D. O'BRIEN 98 results from the regenerating nerves are compared with the results from the 20 hr nerves to demonstrate changes in axonal transport. The accumulations were calculated for each individual experiment, and the results are shown in Table 1. In Fig. 4 the accumulations are normalized by expressing them as a percentage of the 20 hr accumulation. ACh transport was substantially increased during the two weeks after the primary injury, whether it was a crush or a cut. The apparent increase between 6 and 13 days TABLf 1. Axonal transport of ACh, ChAc and ChE in regenerating nerves Type of ACh ChAc ChE Time after primary accumulation accumulation accumulation primary lesion lesion (p-mole/5 mm) (p-mole units/5 mm) (n-mole units/5 mm) 20hr Crush 37± 11 (11) 77± 18 (6) 2.79±0-29 (5) 6 days Crush 89±16 (4) 27±16 (3) 0-69±0'12 (5) 13 days Crush 85 ± 15 (6) 1.7 ± 4-1 (4) 1*23 ± 0.26 (6) 51 days Crush 49 ± 14 (3) 71± 25 (6) 1-97 ±0 07 (3) 111 days Crush 40± 16 (3) 1D69±0.24 (4) 6 days Cut 85±15 (6) 32±3.1 (4) 1-09±0-14 (4) Cut 13 days 64 ± 10 (4) 123 ± 16 (4) 0*99 ± 0*09 (4)
The accumulation of ACh, ChAc or ChE against the test crushes (concentration in segment A of the experimental nerve - concentration in segment A of the contralateral nerve) was calculated for each animal. The 20 hr nerves give an estimate of transport in normal nerves. Values are mean + s.E. of mean, with the number of animals in brackets. These results are normalised in Fig. 4. A Crushed nerves - ACh B Cut nerves
--ChAc
300
ChE
200
-
100
20 hr
6 days
13 days
51 days 111 days Time after primary lesion
20 hr
6 days
13 days
Fig. 4. The accumulation of ACh, ChAc and ChE against the test crushes in the regenerating nerves are expressed as a percentage of the accumulation in the 20 hr nerves; A, up to 111 days after a crush primary lesion, and B, up to 13 days after a cut. Open circles: points not significantly different from the 20 hr accumulation. Filled circles: points significantly different from the 20 hr accumulation (P < 0-05, as determined with Student's two-tailed t test). Number of observations as in Table 1.
AXONAL TRANSPORT IN REGENERATING NERVE 99 in the cut nerves was not statistically significant. ChAc transport was reduced to an unobservable level 13 days after a crush injury, but recovered to a near-normal level by 51 days. In the cut nerves, however, ChAc transport was only transiently diminished at 6 days and showed a recovery by 13 days. ChE transport was at a minimum 6 days after a crush injury and showed a recovery at later times, although it did not return to the normal level even at 111 days. In the cut nerves ChE transport was reduced at 6 days and remained unchanged at 13 days. Inhibitors of non-specific esterases were not included in the ChE assay, but ChE accumulations can be considered to give reliable estimates of AChE transport since AChE constitutes about 90 % of the ChE activity in peripheral nerves and is responsible for virtually all of the transported ChE activity (Ranish & Ochs, 1972; Tucek, 1975).
Functional recovery The earliest sign of functional reinnervation was seen at 35 days in both feet of one rabbit and in one foot of another. Functional recovery in the other rabbits occurred on day 40 or 42, giving an average recovery time of 39 days. DISCUSSION
The method of measuring axonal transport from the accumulation of material against a crush has the advantage that its specificity is limited only by the specificity of the assay procedure, so it is useful for studying the transport of specific intra-axonal substances. This facility is not offered by the method of injecting radioactivelylabelled precursors into neuronal pools, the specificity of which is at best limited to the labelling of groups of related compounds. Another advantage of the crush method is that it gives a quantitative assessment of the amount ofmaterial that is transported. This is particularly appropriate in the present study since it shows changes in the amounts of ACh, ChAc and ChE that are delivered to the nerve terminals during the processes of nerve growth and reinnervation of the muscles. A disadvantage of the method is that, unlike studies with labelled precursors, the velocity of transport is difficult to calculate as the accumulation of a substance depends on the proportion of the substance in the axon that is available for transport, as well as on the transport velocity. Attempts have been made by others to estimate the mobile portions of ACh, ChAc and AChE, and hence to calculate their rates of transport, but these are not particularly relevant to this study because the mobile portions might change in magnitude during regeneration. The possibility that the lesion itself affects- axonal transport has been discussed in detail by Lubinska (1964, 1975). The fast axonal transport of radioactively labelled proteins continues unaffected even when the nerve is isolated from the cell body region (Ochs & Ranish, 1969), and the transport of [14C]choline-labelled phospholipids (mainly lecithin) is accelerated (Dziegielewska, Evans & Saunders, 1976). The accumulation of ACh, ChAc and AChE is linear for at least 18-20 hr after making the lesion (Frizell, Hasselgren & Sj6strand, 1970; Higgendal, Saunders & Dahlstrom, 1971; Lubiuska & Niemierko, 1971; Tucek, 1975). It is generally accepted that the accumulation of protein against a nerve lesion is 4-2
R. A. D. O'BRIEN due to the damming of axonal transport, since there is no appreciable protein synthesis in the axon (see Droz & Koenig, 1970), but when considering the accumulation of ACh it is important to distinguish between the damming of transported ACh and local synthesis of the transmitter in the region of the test crush. Dahlstr6m, Evans, Higgendal, Heiwall & Saunders (1974) considered the possibility that accumulated ChAc was responsible for the build-up of ACh against a nerve crush, but concluded that the relative rates of accumulation (ACh accumulation was the faster) were sufficiently different to discount this possibility. In support of this view it is significant that the ACh accumulation in the 13 days crushed nerves was more than twice the normal accumulation. while the accumulation of ChAc was abolished. Alternatively, local synthesis of ACh may be limited not by the level of ChAc but by the availability of substrate. For instance, choline could be taken up by the nerve at the site of the injury and converted into ACh. If this is so, one would expect to see a build-up of ACh distal to the test crush, and this was checked in the present experiments; only in the 13 day cut nerves was there a significant increase of ACh distal to the test crushes (measured by subtracting the level in segment B of the contralateral nerves from segment B of the experimental nerves). This distal accumulation amounted to 52 + 7-4 p-mole (mean + S.E. of mean, n = 4) compared with the proximal accumulation of 123 + 16 p-mole (n = 4). If the distal increase in ACh was due to local synthesis, a similar amount should have been synthesized in the proximal segment, so that the net amount of ACh transported can be corrected to 71 + 18 p-mole, which is about the same level as found in the 6 day cut nerves, and is still higher than the amount of ACh transported in the normal nerves. However, the possibility that the distal accumulation of ACh was at least partly due to retrograde axonal transport of the transmitter cannot be ruled out. The detailed work of Gutmann et al. (1942) included measurements of the rate of regeneration of rabbit peroneal nerves and their results are therefore directly applicable to the present experiments. The distance between the primary lesion and the most proximal of the denervated muscles (peroneus longus) was between 3 and 4 cm. From the data of Gutmann et al. (1942) the following time course of regeneration has been estimated for the crushed nerves: by 6 days the regenerating fibres should have crossed the site of the lesion and begun to reinnervate the distal stump; reinnervation of peroneus longus should have started about the 13th day, and functional reinnervation of all the denervated muscles should have been complete at about the 40th day. This corresponds well with the average of 39 days that was determined experimentally. Between 51 and 111 days regenerated fibres become more mature, but are still smaller and more numerous than normal (Aitken, Sharman & Young, 1947; Shawe, 1955). In the present experiments on cut nerves, reinnervation of the distal stump was prevented by the removal of a segment of nerve as well as by the natural retraction of the stumps. It was observed that the stumps were still completely separated 13 days after the lesion. The initial changes in axonal transport were the same whether the lesion was a crush or a cut; the transport of ACh was increased while that of the enzymes was reduced. This response did not depend on reinnervation of the distal nerve stump since steps were taken to prevent this in the cut nerves, so it is probably part of the 100
AXONAL TRANSPORT IN REGENERATING NERVE 101 general response of the nerve to injury. In the crushed nerves the tendency for transport to return to normal was first detected about 12 days after functional recovery, so it is not clear if it was in response to the 'morphological' reinnervation of the denervated muscles or to their functional reinnervation. The changes in enzyme transport correspond with the observations of Frizell & Sj6strand (1974a) and support the hypothesis that injury to a nerve causes a shift in the emphasis of the neuronal metabolism from the production and transport of transmitter-related materials to the production and transport of materials necessary to the reconstruction of the axon (see Watson, 1974). The reduction of noradrenaline transport observed by Karlstrdm & Dahlstr6m (1974) also supports this hypothesis, so it is surprising that the transport of ACh should be increased in an injured nerve. Most of the ACh in a normal axon is freely diffusible (Evans & Saunders, 1974), but there is also a small proportion of ACh which is contained in a particulate fraction carried at a fast rate of transport (see Saunders, 1975). The proportion of nondiffusible ACh increases in a regenerating nerve (Evans & Saunders, 1974), so the increased transport of ACh could correspond to an increase in the transport of particles with which the ACh is associated. This is also suggested by the observation that the amount of ACh in the nerve is unaffected by the initial injury; if some of the ACh in the nerve were to be transferred from a slow-moving, soluble pool to a rapidly-moving particulate pool the amount of ACh in the nerve could remain unchanged while the amount transported in a given time is increased. One can only speculate on the function of an increased transport of ACh in a regenerating nerve; for example, it may be essential for the regenerating nerve to release ACh and for the muscle, once reinnervated, to respond to the transmitter before functional contact can be established (see Gordon, Jones & Vrbova, 1976). According to Watson (1974) the formation of functional contact may provide a signal to the neurone causing the return of its metabolism to the provision of transmitterrelated enzymes rather than structural materials. The recovery of ChAc transport in the 13 day cut nerves is curious, particularly as ChE transport was still reduced; an explanation for this is not readily available. The presence of ChE transport in the 13 day crushed nerves (in which the transport of ChAc was reduced to an unobservable level) may reflect an association of AChE with transported membrane particles, as it is known that AChE is present in the axolemma of normal nerves (Lewis & Shute, 1966; Tennyson, Brzin & Duffy, 1968). This work was undertaken during the tenure of a Medical Research Council training award. I am very grateful to Dr Norman Saunders for his constant help and advice.
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