95. 0 1973 by John Wiley & Sons, Inc. ..... of figures we thank Mr. Par-Anders Larsson. The technical assistance of Mr. John Reynolds is gratefully acknowledged.
JOURNAL OF NEUROBIOLOGY,
VOL.
4,
NO.
2,
PP.
95-103
SLOW ACCUMULATION OF CHOLINE ACETYLTRANSFERASE I N CRUSHED SCIATIC NERVES OF T H E RAT N. R. SAUNDERS, K. DZIEGIELEWSKA*, C . J. HAGGENDAL, and A. B. DAHLSTROM
Dept. of Physiology, University College, London England, and Dept. of Pharmacology and Institute of Neurobiology, University of Goteborg, Goteborg, Sweden.
SUMMARY
Choline Acetyltransferase (ChAc) activity has been estimated using a microradiochemical method in short segments of rat sciatic nerve at different times after crushing the nerve once or with a second crush 2 cm distal to the first. Above a single or proximal crush, the ChAc levels rose slowly in the 5 mm immediately above the crush to reach about 1 4 times control at 24 hr. Distal to a single crush and between two crushes no change was detected in ChAc-activity up to 18-24 hr. The results are discussed in relation to the rapid accumulation of acetylcholine (ACh) above a crush, and redistribution between two crushes which have previously been demonstrated in the same preparation. It is concluded that the rate of transport of ChAc is probably much slower than that of ACh, and that their mechanisms of transport are not the same. INTRODUCTION
The presence of choline acetyltransferase (ChAc) in peripheral nerve fibers is dependent upon continuity of the axons with their cell bodies, since nerve section results in disappearance of ChAc from the part of a nerve distal to a cut after several days (Hebb and Waites, 1956). Since the level of enzyme activity increases in the nerve immediately proximal to a cut or a crush, several authors (e.g. Hebb and Silver, 1961; Frizell, Hasselgren, and Sjostrand, 1970) have suggested that ChAc is transported from the perikaryon to the periphery. Earlier, we have demonstrated that acetylcholine (ACh) accumulates rapidly in crushed sciatic nerves of the rat (Haggendal, Saunders, and Dahlstrom, 1971). Three main possibilities for explaining this accumulation appear to be: (a) the crush has interfered with a proximo-distal transport of ACh in the axons, (b) the crush itself has induced an increased synthesis of ACh, or (c) a n accumulation of ChAc or of other material
* Present address:
Warsaw 22, 5 Prokuratorska, Poland.
95
01973 by John Wiley & Sons, Inc.
SAUNDERS ET A L involved in the synthesis of ACh in the axons may cause an increased synthesis of ACh. If the synthesis of ACh were accelerated locally by crushing, increased amounts of ACh would be expected to be present both above and below the crush. However, this was not the case; the increase was confined t o the part of nerve proximal to the crush. If the third alternative were true, then a rapid accumulation of ChAc, or another factor involved in synthesis, would be detectable above the crush, as observed for ACh. Previous estimations of changes in ChAc activity in cut or crushed nerves have given quantitatively variable results (see discussion). Thus, comparison of these results from other preparations with our ACh experiments in rat sciatic nerve may not be justified. The present study was undertaken in order to see if the accumulation of ACh was paralleled by a n accumulation of ChAc in rat sciatic nerves. It has been found that not only is the rate of accumulation of ChAc above a crush much slower than that of ACh, but also that above a second, more distal crush, no detectable rise in ChAc occurred in contrast to the rise which occurs in ACh (Haggendal et al., 1971). METHODS
Male Albino rats (Sprague-Dawley, 200-250 g) were used. Both sciatic nerves were crushed a t a high level 3-5 mm below for infrupiriformis, or, in another series, both at a high level and simultaneously a t a level 2 cm distally. The nerves were crushed by applying pressure on the nerve with a fine silk thread pulled against a glass rod for 5 sec. (Lubinska, 1959). The rats were killed by decapitation a t different time intervals after the operation (see Fig. 1 , 2 , and 3 ) , and the nerves were dissected out and cut into 5-mm lengths. The pieces of nerve were blotted on filter paper and cleaned from adherent connective tissues. No effort was made to strip off the epineurium. I n the double crush experiments, the distance between the two crushes varied between 18-22 rnm. The nerve between the crushes was always cut into four pieces of between 4.55.5 mm. Values were corrected to be equivalent to 5 mm. Nerves which were crushed immediately before killing the rats, were used as controls (zero hr). Each length of nerve was homogenized mechanically (Gallenkamp, variable speed geared stirrer) in 100 of 1 % buthanol in 0.9% saline, using conical-shaped all glass homogenizers. The homogenate was centrifuged at 4°C for 5 min, because it was found t h a t the supernatant gave more reproducible results; the pellet contained < 5 % of the ChAc activity found in the supernatant. The ChAc was assayed radiochemically using the method of Glover and Green (1972), with some very small modifications. ‘4C-acetyl CoA was used to produce I4C-ACh which was then selectively removed from the substrate with the reagent K2HgIa dissolved in octanone. The values obtained for samples of sciatic nerve were similar to those of Fonnum (1969). Chemicals. Acetyl-l-14C-CoA,59 mCi/mmole (Radiochemical Centre, Amersham) choline iodide and eserine sulphate (British Drug Houses Ltd) ; potassium chloride; phosphate buffer prepared from equimolar K2HP04and KH,POa, pH 7.0; disodium EDTA (B.D.H. “Analar” grade). The extraction reagent, 100 mM K2Hg14,was prepared by the method of Glover and Green (1972). Procedure. Ten pl of reaction mixture (185 pM ‘4C-Ac CoA; 10 mM choline iodide; 200 mM KCI; 20 mM phosphate buffer, pH 7.0; 0.2 m M EDTA; 0.2 m M eserine sulphate) were placed in small capped plastic tubes (Hawksley Ltd) on ice. Ten pl of each of the supernatants of the centrifuged homogenates were added to each tube. The
ACCUMULATION OF CHOLINE ACETYLTRANSFERASE 97
/ /
pmol of ACh formed
min. 5 mm.. o f nerve
0 5
3
0
6
600
4
300
__
12
18
24
Hours after crush
Fig. 1. Accumulation of choline acetyltransferase (ChAc) activity in the 5 mm above a crush in rat sciatic nerve at, different times after crushing. Mean values f S.E.M. are shown. Numbers indicate the number of observations. 0 = double crushed nerves; 0 = single crushed nerves. The line drawn joins the mean values. At zero h r a weighed mean has been calculated taking into account the different n values for the two control series. tubes were mixed on a Vortex mixer and placed in a water bath a t 37OC. After 5 min the reaction was stopped by addition of 10 p1 of 1 N formic acid. One hundred pl of K2Hg14in octanone were then added to each tube: the tubes were mixed in the Vortex mixer and spun for a few minutes in a centrifuge to separate the layers. Forty pl of the octanone layer were withdrawn, using a Hamilton microsyringe, and were added to 10 ml of scintillation mixture (4.5 L toluene, 0.5 L methanol, 20 g PPO) in a vial. 14Cactivity was counted in a Packard Tricarb with an efficiency of 80%. Duplicate analyses were usually performed and agreed within about 5 % . RESULTS
Single crushed nerves. The results are shown in Figs. 1and 2. In control crushed nerves the ChAc activity was significantly lower in the more distal parts of the nerve; this was probably a t least partly dependent upon the branching which occurs along the nerve (Fig. 2, c.f. Hebb, 1963). The main finding was that by 24 hr after crushing there was an increase of about 60% in ChAc-activity in the 5 mm immediately proximal to the crush (Figs. 1and 2d). At 6 hr and 12 hr the ChAc activity in this region above the crush was not significantly greater than control; nevertheless, an upward trend is apparent in Fig. 1. In the parts of the nerve distal to the crush no significant change in enzyme activity was observed (Fig. 2). Double crushed nerves. The results are shown in Fig. 3. Above the proximal crush a small but significant (P < .001) increase of about
SAUNDERS ET A L
98
p m o l o f A C h forrned/min
/5mm
600
600
300
300
60(Ih' 300
r;
5mm pieces of nerve
!m d
2 L HR
t
Fig. 2. Distribution of choline acetyltransferase (ChAc) activity in rat sciatic nerves a t different times after a single crush. Means f S.E.M. are shown. Arrows indicate the position of the crush. n = 7-10 except at 6 hr when n = 4. The diagram shows the branching pattern of the length of nerve used and the position of the crush. The times indicate the interval between crushing the nerve and its removal from the rat.
50% above the equivalent control segment was observed after 18 hr. I n the part separated by the two crushes no change or redistribution in enzyme activity could be observed at 18 h r after operation. I n particular there was no significant increase (P > .lo) in ChAc activity above the distal crush. DISCUSSION
The amount of ChAc activity in normal rat sciatic nerve has previously been found to be 7-10 pmole of ACh formed per hr per g of tissue (Fonnum, 1969). The figure obtained in crushed control nerves in the present study was about 500 pmole of ACh synthesized per min per 5 mm nerve. Since 5 mm of rat sciatic nerve weigh about 4 mg, our value corresponds to about 7 pmole of ACh per hr per g of tissue which agrees well with earlier estimations. Similar values have been found for sciatic nerves of other species, using different methods (e.g. Hebb, 1962). In the present study we found that ChAc-activity in the 5 mm of nerve proximal t o a crush (part A, Figs. 1and 2) appeared to increase steadily up to a t least 24 hr when it reached 607; higher than control. No change in enzyme activity could be detected in the 5-mm part further proximal to this piece (part A' in Fig. 2) within the time period studied. This
A C C U M U L A T I O N OF CHOLINE A C E T Y L T R A N S F E R A S E i 9 9 pmol of ACh formed/min/5mm.
'1 T
300
A' A t B C D E f F
G
300
f
t
Fig. 3. Distribution of choline acetyltransferase (ChAc) activity in rat sciatic nerves which were crushed twice simultaneously (double crushed nerves). Arrows indicate the positions of the crushes. Means i S.E.M. are shown. n = 4-5 at zero hr and 7-8 a t 18 hr.
indicates that any accumulation of ChAc-activity that occurs in rat crushed sciatic nerves, is confined to within 5 mm above the crush in the time period studied. This agrees with the results obtained by Frizell et al. (1970). Previous studies of alterations in ChAc-activity proximal to a nerve lesion have shown rather variable increases which are summarized in Table 1. Some of this variation is, of course, due to the different time intervals and lengths of nerve used. Differences in species and type of nerve may also be important. The only results which suggest a greater rate of accumulation than we have found, are those of Frizell et al. (1970). Here comparison is difficult, because the control levels of ChAc-activity in their experiments were two orders of magnitude less than ours. Presumably much of this difference was due to the different homogenization and estimation techniques which were used. The slow accumulation of ChAc in rat crushed sciatic nerves contrasts with the rapid accumulation of ACh in the same nerve (Haggendal et al., 1971)). The ACh level can be about four times control a t 24 h r in the 5 mm above a crush (Dahlstrom, Evans, Haggendal and Saunders, in preparation) whereas ChAc-activity increases to only about 1; times control at this time. However, any discussion of axonal transport rates of different substances should consider the possibility that not necessarily all of a substance is transported at the same rate, or even that a proportion of it may be stationary. Information about the availability of a substance
SAUNDERS ET A L
100
TABLE 1 Accumulation of choline acetyltransferase (ChAc) activity in nerve proximal to a lesion. A survey of earlier reports.
Species
Nerve
Operation
Time after operation
Amount of nerve Increase studied in ChAc
+ 30%
sheep
cut cervical sympathetic
2 days
sheep
cut cervical sympathetic
4 days
goat
sciatic
cut
7 days
per g of acetone dried tissue per g of acetone dried tissue 25-30 mm
rabbit
sciatic
7 days
30-60 mm
+
rabbit rabbit
vagus hypoglossal
cut or crush crush crush
20 h r 20 hr
5 mm 5 mm
+ 245% + 130%
rabbit
phrenic
ligation
3 days
15 mm
+ 50%
rat
sciatic
crush
24 h r
5 mm
+ 60%
+ 55%
Authors (reference) Hebb and Waites (1956) Hebb and Waites (1956)
+loo%
Hebb and Silver (1961) 100% Hebb (1962) Frizell, Hasselgren and Sjostrand (1970) Ekstrom and Emmelin (1971) present paper
for transport may be obtained by measuring the decrease in amount of the substance distal to a nerve lesion, unless the transport is dependent upon continuity with the perikarya. I n the case of ACh, it seems that only about 20% of the total amount of ACh in a nerve is rapidly transported at a rate of several mm per hour (Dahlstrom, Evans, Haggendal, and Saunders, in preparation). I n the present experiments there was no significant decrease in ChAc activity distal t o the crush even by 24 hr. Earlier investigations have shown that ChAc activity decreases markedly, distal to a cut several days after operation (Hebb and Silver, 1961; Ekstrom and Emmelin, 1971). This loss in ChAc activity is likely to have been due to degeneration of the axons. The lack of any reduction in enzyme activity 24 hr after operation in our experiments contrasts with the findings of Frizell et al. (1970), but possibly this could be due to the different types of nerve fibers investigated. Frizell et al., themselves, found that the estimated rate of transport for ChAc in the autonomic fibres of the rabbit vagus was about three times that for the motor axons of the rabbit hypoglossal nerve. I n the rat sciatic nerve we have calculated a rate of about 3 mm/24 hr (provided that all of the enzyme is transported a t the same rate), compared with the 6 mm/24 h r calculated for the rabbit hypoglossal nerve.
ACCUMULATION OF CHOLINE ACETYLTRANSFERASE 101 The absence of redistribution of ChAc activity between two crushes contrasts with the redistribution of ACh (Haggendal et al., 1971) and noradrenaline (NA) (Dahlstrom, 1967) demonstrated to occur in double crushed nerves. This may indicate that the transport of ChAc is dependent upon continuity with the perikarya, whereas the transport mechanisms of ACh and NA apparently are independent of such a continuity. The behavior of ChAc in rat sciatic nerves, crushed either once or twice, is thus very different from the behaviour of both ACh (Haggendal et al., 1971) and AChE (Lubinska, Niemierko, Oderfelt and Szwarc, 1966) of sciatic nerves. ACh, AChE, and NA all increase rapidly above a crush, and in double crushed nerves a clear redistribution occurs, with an increased amount in the distal parts of the separated segment. I n contrast, ChAc accumulates slowly above a crush, and no sign of redistribution between two crushes could be detected. ACh may, at least partly, be located in vesicles in axons, AChE may be partly confined to the smooth endoplasmic reticulum (c.f. Kasa, 1968), and NA in adrenergic nerves is stored in amine granules. The fast transport for the probably particlebound fractions of these substances may be due to specific transport mechanisms, which are independent of the perikarya. Mitosis inhibiting substances such as colchicine and vinblastine, seem to interfere with the transport of NA (Dahlstrom, 1968; 1971), AChE (Kreutzberg, 1969) and also ACh (Heiwall, Dahlstrom, Haggendal and Saunders, in preparation), and thus it may be that microtubules are involved in the transport of these three substances, since this organelle is destroyed by such mitosis inhibitors. The most likely explanation for the difference in rate of accumulation central to, and decrease distal to, a crush for ACh and ChAc would seem to be that all the ChAc is transported at a slow rate (approximately 3 mm/24 hr), and that this transport is interrupted by axotomy so that there will be no decrease in ChAc until the axons degenerate. This slow rate of transport of ChAc would thus be very similar to the slow flow of axoplasm (Weiss and Hiscoe, 1948) and of some proteins (e.g. Droz and Leblond, 1962; Ochs and Johnson, 1969). Fonnum (1970) has shown that ChAc is probably a soluble enzyme which would, therefore, be expected to move with the bulk of the axoplasm. The possibility cannot yet be entirely discounted that a very small (less than e.g. 5%) proportion of the ChAc is rather rapidly transported, and the rest is either stationary or transported very slowly. The results of the double crushed nerve experiments could in fact support either possibility (i.e., slow transport of all of the ChAc or rapid transport of a very small proportion of ChAc and slow transport of the rest) since no increase in ChAc activity could be detected above the crush at 18 hr, in contrast to the rise which occurred in ACh as early as 6 hr (Haggendal et al., 1971). It is possible that accumulation of some substance other than ChAc could account for the rapid accumulation of ACh above a crush, e.g.
102
SAUNDERS ET A L
acetyl CoA (cf. Glover and Potter, 1972). However, mitochondria, which probably represent the main source of acetyl CoA in brain as well as in other tissues (for review see Tutek, 1970), are likely to be transported intra-axonally at rates of about 6-14 mm/24 hr (Banks, Mangnall and Mayor, 1969; Karlsson and Sjostrand, 1971) which would be too slow to produce the large and rapid increase of ACh observed in crushed nerves. The rapid accumulation of ACh is also unlikely to be due to a n accumulation of ChAc, partly because their rates of accumulation are so different, but mainly because axotomy appears to interrupt the transport of ChAc but not of ACh, at least in the rat sciatic nerve. REFERENCES
BANKS,P., MANGNALL, D., and MAYOR, D. (1969). The redistribution of cytochrome oxidase, noradrenaline and adenosine triphosphate in adrenergic nerves constricted a t two points. J . Physiol. (Lond.) 200: 745-762. DAHLSTROM, A. (1967). The transport of noradrenaline between two simultaneously performed ligations of the sciatic nerves of rat and cat. Actu physiol. scand. 69: 158-166. DAHLSTROM, A. 11968). Effect of colchicine on the transport of amine storage granules in sympathetic nerves of rat. Europ. J. Pharmacol. 5 : 111-113. DAHLSTROM, A. (1971). Effects of vinblastine and colchicine on monoamine containing neurons of the rat with special regard to the axoplasmic transport of amine granules. Actu neuroputh. (Berl.) Suppl. V: 226-237. DAHLSTROM, A. B., EVANS,C. A. N., HAGGENDAL, C . J. and SAUNDERS, N. R. (1971). The hehaviour of acetylcholine in rat sciatic nerve above and below a crush. (In preparation). DAHLSTROM, A. and HAGGENDAL, J. (1966). Studies on the transport and life-span of amine storage granules in a peripheral adrenergic neuron system. Actu physiol. scand. 6 7 : 278-288. DROZ,B. and LEBLOND, C. P. (1963). Axonal migration of proteins in the central. nervous system and peripheral nerves as shown by autoradiography. J . comp. Neurol. 121: 325-346. EKSTROM,J. and EMMELIN,N. (1971). Movement of choline acetyltransferase in axons disconnected from their cell bodies. J . Physiol. 216 : 247-256. FONNUM, F. (1969). Radiochemical micro-assays for the determination of choline acetyltransferase and acetylcholinesterase activities. Biochem. J . 115 : 465-472. FONNUM, F. (1970). Surface charge of choline acetyltransferase from different species. J . Neurochem. 17 : 1905-1100. FRIZELL,M., HASSELGREN, P.-0. and SJOSTRAND, J. (1970). Axoplasmic transport of acetylcholine esterase and choline acetyltransferase in the vagus and hypoglossal nerve of the rabbit. E x p . Bruin Res. 10: 526-531. V. A. S. and GREEN,D. P. (1972). A simple quick micro-assay for choline GLOVER, acetyltransferase. J . Neurochem. 19: 2465-2466. GLOVER, V. A. S. and POTTER,L. T. (1971). Purification and properties of choline acetyltransferase from ox brain striate nuclei. J . Neurochem. 18 : 571-580. HAGGENDAL, J., SAUNDERS, N. R. and DAHLSTROM, A. (1971). Rapid accumulation of acetylcholine in nerve above a crush. J . Pharm. Pharmucol. 23 : 552-555. HEBB,C . 0. (1962). Acetylcholine content of the rabbit plantaris muscle after denervation. J . Physiol. 163 : 294-306. HEBB,C. 0. 11963). Formation, storage and liberation of acetylcholine. In: Handbuch der exp. Pharmacol. (ed. G. B. Koelle) Springer-Verlag, Berlin. 15 : 55-88.
ACCUMULATION OF CHOLINE A C E T Y L T R A N S F E R A S E 103 HEBB,C. 0. and SILVER,A. (1961). Gradient of choline acetylase activity. Nature (Lond.) 189 : 123-125. HEBB,C. 0. and WAITES,G. M. H. (1956). Choline acetylase in antero- and retrograde degeneration of a cholinergic nerve. J.Physiol. 132 :667-671. A., HAGGENDAL, J. and SAUNDERS, N. R. (1973). Effect Heiwall, P.-O., DAHLSTROM, of vinblastine and colchine on the rapid accumulation of acetylcholine in rat sciatic nerve. (In preparation). KARLSSON, J.-0. and SJOSTRAND, J. (1971). Synthesis, migration and turnover of protein in retinal ganglion cells. J . Neurochem. 18 : 749-767. KASA, P. (1968). Acetylcholine esterase transport in the central and peripheral nervous tissue: The role of tubules in the enzyme transport. Nature (Lond.) 218 : 1265-1267. KREUTZBERG, G. (1969). Neuronal dynamics and axonal flow. IV. Blockage of intraaxonal enzyme transport by colchicine. Proc. Nut. Acad. Sci. (Wash.) 62 : 722-728. LUBINSKA, L. (1959). Region of transition between preserved and regenerating parts of myelinated nerve fibres. J . comp. Neurol. 113 : 315-335. B. and SZWARC, L. (1964). Behaviour of LUBINSKA, L., NIEMIERKO,S., ODERFELD, acetylcholinesterase in isolated nerve segments. J . Neurochem. 11 : 132-138. OCHS,S. and JOHNSON, J. (1969). Fast and slow phases of axoplasmic flow in ventral root fibres. J . Neurochem. 16 : 845-853. T U ~ E KS., (1970). Subcellular localization of enzymes generating acetyl-CoA and their possible relation to the biosynthesis of acetylcholine. In: (Eds.) E. Heilbronn and A. Winter, Drugs and Cholinergic Mechanisms i n the C N S Almqvist & Wicksell, pp. 117-131. WEISS,P. A. and HISCOE,H. (1948). Experiments on the mechanism of nerve growth. J. exp. Zool. 107: 315-395. This study was supported by grants from the Wellcome Trust, from the Swedish Medical Research Council (grants Nos. B 72-14X-166-08-A and B 72-14X-2207-06-B) and from the Medical Faculty, University of Goteborg, Sweden. The technical assistance of Mr. John Reynolds is gratefully acknowledged. For preparation of figures we thank Mr. Par-Anders Larsson.
