~etwnscience Vol. 4, pp. 413 to 416 Pergamon Press Ltd 1979. Printed in Great Britain

FAST AXONAL TRANSPORT OF ACETYLCHOLINESTERASE AND PROTEIN IN COLD-BLOCKED FROG SCIATIC NERVES IN VITRO De~rtm~t

of Zoophysiology, University of Lund, Sweden

Ab&ract-Fast axonal transport

of isotopelabeled protein and acetylcbolinesterase has been studied in frog sciatic nerves in vitro. Labeled protein and acetylcholinesterase was allowed to accumulate in front of a cold-block and was then released as a pulse. The accumulation of acetylcholinesterase ceased after 4-5 h but continued for severai hours more if the spinal cord was included in the prep aration. Both the pulse of Iabeled protein and of acetylcholinesterase migrated at a rate of about 120 mm/day at 18°C. When the same nerve was assayed for both radioactivity and a~tylchotinester~, the pulses had a very similar appearance. The technique provides a convenient, direct method for the study of fast axonal transport of both isotope-labeled protein and endogenous enzymes. The transport rates so obtained are generally higher than those derived from studies on ligated nerves. The method also allows the simultaneous determination of transport in the sensory and motor fibers of the same nerve.

ACETYLCHOLINESTECRASE (AChE) was one of the first enzymes to be studied in connection with fast axonal transport (NIEMIERKO& LUBINSKA,1967). Part of the total AChE was found Lo accumulate in front of-and to a smaller degree distal t+-a ligature pinced on the mammalian sciatic nerve (LUBINSKA & NIEMIERKO, 1971). From the rapid rate of accumulation and the normal content of AChE in the nerve, the rate of transport was calculated to be about 260mm/ day. With a better knowledge of the size of the migrating fraction it has later been shown that the true rate is about 430mmlday in mammalian sciatic nerves (RANISH & OCHS, 1972). However, the ratio migrating/non-migrating enzyme is probably not constant between different types of nerves and between different species. This makes calculations of rates, based on estimates of the size of the migrating fraction, uncertain. We have therefore developed a method which avoids indirect calculations and instead measures the displacement of a pulse of enzyme activity or radioactivity towards the nerve terminals. The technique is based on the reversible inhibition of axonal transport by low temperature in the distal part of the nerve, while transport continues in the proximal part. When enough material has accumulated in front of the cold region, the temperature is raised and a pulse of activity migrates into the rewarmed part of the nerve. With this method we have studied the transport of [3H]ieucine labeled protein in frog sciatic nerves in vitro (ED~R~M & HANSON, 1973; HANSON& EIXTR~M, 1977). A similar technique was used to study the transport of AChE in rat and rabbit sciatic nerves in vivo (HANSON, 1978). In the present study we have compared the transport of a pulse of labeled protein in the sensory fibers of the

Abbreviation: AChE, a~tyicholin~terase. 413

frog sciatic nerve with the migration of AChE in the motor fibers of the same nerve. EXPERIMENTAL PROCEDURES Frogs, Rana remporari~, were used in this investigation. Before use they were acclimated to room temperature (EDSTR&I & HANSON, 1973). After decapitation, a prep aration consisting of the dorsal ganglia (nos 8 and 9), the dorsal and ventral roots, the nerve and the gastrocnemius muscle was dissected out. In some cases part of the spinal cord with the motor ceil bodies was included in the prep aration. Oxygenated, phosphate-buffers frog Ringer’s solution, pH 7.4 was used in the experiments. The preparations were placed in a special incubation apparatus with two compartments, separated by two pieces of l-mm thick glass on top of another. Two half circles were ground in each part of the glasses. When mounted on top of another, two holes with a diameter of about 2 mm were formed. The compartment on one side of the partition was kept at 18°C with a small heating element embedded in silicone rubber. The other compartment was kept at l-3°C with a cold-water jacket connected via a pump to a refrigerated bath. During incubation the prep arations were placed with the dorsal ganglia and roots in the warm, and the nerves and muscles in the cold compartment. The edges of the partition and the holes through which the nerves passed were sealed with silicone grease. L-[4,S3H]leucine (106 Ci/mmol, 1mCi/ml, The Radiochemical Centre, Amersham) was added to the ganglionic compartment and two small stirring motors (Milliperm, 6 V), one in each compartment, were started. To obtain a moderate running speed for the motors they were driven by pulses from a thyristor circuit (JOHNSON, 1976). Incubation with a temperature gradient was continued for various times and the temperature in the water jacket then quickly changed to 18°C. Simultaneously the solutions in the ganglionic and nerve compartments were replaced with fresh ones. To obtain a pulse, the migration of material into the nerve was allowed to continue for I h, after which the nerves were cut just distal to the barrier. After another

414

M. HANSON b. protein

a. AChE

normal nerve

1020

20 10 mm from barrier

mm from barrier

30

c. AChE A2.5h

1

20 10 mm from barrier

30 mm from barrier

Ftc. 1. (a) Normal distribution of acetylcholinesterase in frog sciatic nerve. Collaterals were cut at various levels and the decreased activity distally may be due both to an actual gradient and fewer fibers. The values plotted refer to the middle of each nerve segment. (bf Protein transport in two nerves from the same frog. [3H]leucine (2O~Ci/ml Ringer’s ~lution) was added to the ganghonic compartment. The pulses migrated for 4 or 5 h respectively. The plotted values refer to the middle of each nerve segment. (c) Distribution of AChE activity in four different nerves at various times after rewarming. The piotted values refer to the middle of each nerve segment. (d) Migration of 3H-Iabeled protein and AChE in the same nerve for 4.5 h. The plotted values refer to the middle of each nerve segment. few hours the nerves were cut into 2-4mm pieces and assayed for radioactivity or AChE. For radioactivity each nerve piece was treated with 5% trichloracetic acid at 80°C for 1h, washed in distilled water and solubilimd in Soluene-100 (Packard Co.). The proteinincorporated radioa~ivity was determined in a 0.55% Permablend (Packard Co.) solution in toiuene with a Packard 3375 liquid s~ntiliat~on spectrometer. To determine AChE activity each nerve piece was homogenized in 4001.11tris HCI buffer, pH 8.0, containing 0.2% (v/v) Triton X-109 and assayed according to ELLMAN, COURTNEY,ANDREW& FEATHERSTONE(1961). Non-specific esterases were determined with butyrylthiocholine instead of acetylthiocholine as substrate, When both radioactivity and AChE were measure&in the same nerve piece loOn of the homogenate was used for each assay. RESULTS

The temperature gradient in the cold-blocked nerves is very steep-from 2 to 3°C to 18°C in a

2-mm length of nerve. The background radioactivity in the nerve remains very low since no local protein synthesis can take place during the cold period and all solutions are changed before rewarming. The transport apparently starts within a few minutes when the temperature is raised. The rate of ovation of was about incorporated ra~oacti~ty protein 120mmfday at 18°C (Fig. lb) which is in agreement with earlier observations (EDSTMJM & HANSON,1973). In untreated nerves the AChE activity was less in the distal than in the proximal part of the nerves (Fig. la). Collaterals were cut during the dissection and the number of fibers thus less in the distal part of the nerve. However, also when the branches are

included and the total number of fibers is constant, a gradient exists (LUBINSKA, NIFMIERKO, ODERWLD& SZWARC,1963). A few hours of cold-block changes the distribution. In front of the barrier AChE a~umuiates. The increase continues for 4-5 h. If the

Axonal transport in frog nerves

415

ment of AChE takes place distal to the barrier. When the temperature is raised, both the pulse of AChE and the enzyme distal to the barrier (Fig. la) starts migrating towards the nerve endings. Since all fibers except those to the gastrocnemius muscle are cut, most of the migrating material will accumulate at their ends. The subsequent fate of the AChE which arrives first is not clear; it might be gradually lost to the medium or be transported in a reverse direction after some delay (BISBY, 1976). The rate of AChE transport was very similar to that of the bulk of 3H-labeled protein. A closely similar rate for protein and AChE has also been found in cat sciatic nerves by RANISH& OCHS (1972). With the present direct technique of measuring rates of transport, the values are generally higher than those obtained from experiments using ligatures or other methods (EDSTR~~M & MATTSON, 1972; PARTJ_OW, Ross, MOTWANI & MCDOUGAL, 1972). BRIMIJOIN (1975) compared the rates obtained from ligature and DISCUSSION cold-block experiments and found a large difference even if the size of the mobile fraction was accounted The fastest mi~ating fraction of a~tylcho~in~terase activity seems to move to the distal part of the for. Probably the ligature ex~riment only measures an average rate of transport. It is also possible that nerve and to the terminals but a considerable amount of activity is deposited behind the pulsed (Fig. 1~). a permanent block (ligature or crush affects the behaviour of the AChE in the axoplasm far from the The decrease in size of a pulse of ‘H-labeled protein site of injury. NIEMIERKO& LUBINSKA(1967) found is much less (Fig. lb). The ventral roots had a limited that repeated cutting of the ends of isolated nerve pool of AChE available for transport. The cessation segments made more AChE available for transport. of accumulation at the cold-block after 4-5 h indicates that the mobile fraction in the ventral roots was Together with experiments demonstrating a reversal exhausted at that time. With a rate of transport of of transport direction at ligatures of crushes (BISBY, 1976), these results indicate the unreliability of esti120mm/day, 4 h corresponds approximately to the mates of the amount of AChE or other enzymes availmigration time for AChE From the cut ends of the able for transport. With the cold-block technique ventral roots to the cold-block. When the spinal cord was present, the amount of available AChE was these difficulties are avoided and rates for different substances can be directly compared. The method is larger, either depending on a supply of newly synthesized enzyme or transport from a pool of AChE in also directly applicable to mammalian nerves by changing the temperature (M. HANSON,unpublished the cell bodies. The increased AChE activity at the distal end of observations). the nerve (Fig. lc) at 2.5, 3 and 3.5 h might indicate Acknowledgements-This work was supported by Statens that some activity had migrated at a very fast rate Naturvetenskapliga Forskningsrid, grant no. 2535-15 to (a 200 mm/day). However, another explanation seems A. EDSTRBW, Magnus Ber@;vallsStiftelse and Kungl. Fysiomore likely. During the cold-block phase no move- grafiska Slllskapet, Lund.

spinal cord is included in the preparation the accumulated AChE activity increases for several hours more. After rewarming the accumulated AChE activity migrates distally at approximately the same rate as the radioactivity incorporated into protein (Fig. lc). Calculations of rates based on the position of the front of activity after a certain time gives slightly higher values than measurements of the displacement of the pulse at various times. The remaining activity just distal to the barrier is higher in nerves assayed for AChE (Fig. lc and d) than in nerves assayed for radioactivity (Fig. 1b). Part of this activity might represent a slower moving fraction and the rest retrogradely migrating AChE. When both AChE and [3H]leucine labeled protein are assayed in the same nerve, very similar pulses are seen. The slight difference in rate lies probably within the errors of measurement (Fig. Id).

REFERENCES BISBY M.

A. (1976) Orthograde and retrograde axonal transport of labeled protein in motoneurons. Expf Neurd. 50, 628-640. BRIMLJOIN S. (1975) Stop-flow: A new technique for measuring axonal transport and its application to the transport of dopamine B-hydroxylase. J. Neurobioi. 6, 379-394.

EDSTROM A. & HANSONM. (1973) Temperature effects on fast axonal transport of proteins in vitro in frog sciatic nerves. Brain Res. 58, 345-354. A. & MATIXWN H. (1972) Fast axonal transport

EDSWIM

in vitro in the sciatic system of the frog. J. Neurochem.

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205-221. ELLMAN G. L., COURTNEY K. D., ANDREX V., JR 8:

FEATHERSTONE R. M. (1961) A new and rapid calorimetric determination of acetylcholinesterase activity. Biochem. Pharmac. 7, 88-95. HANSONM. (1978) A new method to study fast axonal transport in Y~DO.Brain Res. 153, 121-126. HANSON M. & EDSTR~ A. (1977) Fast axonal transport: Effect of antimitotic drugs and inhibitors of energy metabolism on the rate and amount of transported protein in frog sciatic nerves. J. Neurobiol. 8, 97-108. JOHNSONP. D. (1976) Model train speed controller. Practicat E~ec~o~ics 12, 988.

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M. HANSON

LUBINSKAL. & NIEMERKOS. (1971) Velocity and intensity of bidjre~tioRa1 migration of a~tylcho~n~terase in transected nerves. Brain Res. 27. 329-342. LUBINSKAL., NIEMIERKOS., ODERFELDB. & SZWARCL. (1963) The distribution of acetylcholinesterase in peripheral nerves. J. Neurochem. 10, 25-41. NIEMIERKO S. & LUBINSKAL. (1967) Two fractions of axonal acetylcholinesterase exhibiting different behavior in severed nerves. J. Neurochem. 14, 761-767. PARTLOWL. M., Ross C. D., MOTWANIR. & MCDOUGALD. B. JR (1972) Transport of axonal enzymes in surviving segments of frog sciatic nerve. J. gen. F~ys~o~.60, 388-405. RANISHN. & OCHS S. (1972) Fast axoplasmic transport of acetylcholinesterase in mammalian nerve fibers. J. New-o&em. 19, 2641-2649. (Accepted 9 October 1978)