Colchicine-induced sprouting of the neuromuscular junction in the pigeon extensor digitorum longus muscle

Brain Research, 363 (1986) 156-160 Elsevier

156 BRE 21293

Colchicine-induced sprouting of the neuromuscular junction in the pigeon extensor digitorum longus muscle CHRISTIAN S FAHLMAN and DANNY A RILEY

Department o] Anatomy G-3, School of Medtcme, Umversuy of Pennsylvama, Phdadelphta, PA 19104 and Department of Anatomy, Medical College of Wlsconsm, Mdwaukee, WI ( U S A ) (Accepted September 3rd, 1985)

Key words colcbiclne - - sprouting - - neuromuscular junction - - pigeon

Colchlcme-mduced motor endplate sprouting in the extensor &g~torum longus muscle of the p~geonwas examined Ten days after the drug apphcatlon sprounng from the endplate arborizanons and nodes of Ranvler were observed No concomitant changes m endplate surface area or m the degree of terminal branching could be demonstrated Similarities between the sprouting patterns of the pigeon endplate and the mammahan endplate are discussed

Previous studies 2,3,5,11 have demonstrated differences in the sprouting patterns of motor axons which can be correlated with the structure of the terminal arborlzatlon of the neuromuscular junction (NMJ). Sprouts can arise from the nodes of Ranvier (internodal, IN) as well as from the terminal arbor (ultratermlnal, UT) Sprouting has also been reported within the confines of the NMJ ttself in response to presynaptlc block, leading to a more complex and branched termlnall0 In light of the mode of actton of colchicme, i.e , disruption of axoplasmic transport, and with regard to the toxic effects of colchicine, It was of Interest to observe whether colchlcine application would result in changes in the surface area of NMJs or in alterations of the terminal arbor branching pattern The main objective was to determine whether sprouting NMJs evmce retraction or expansion of the terminal arbor Since most sprouting studies have been confined to mammals and amphtbians, it was also of interest to undertake studies on a species from a different taxonomic order, wtth emphasis on determining ff sprouting patterns correlate with terminal morphology as seems to be the case for mammals ~1 To these ends the pigeon was chosen, as it offered sever-

al advantages for this work: its NMJs are very large, with arbors subdividing into n u m e r o u s branches conforming to the 'complex' and 'intermediate' types previously described in the rat 11 The surface area is easily measured and the n u m b e r of branches easily discerned because of their wide spread. The rat NMJ with its smaller size and thinner terminal arbor is less suitable for this type of study. Sciatic nerves of prepuberal pigeons (450-600 g) were exposed to a 5 mM colchicine solution for 30 min according to the previously described protocol tt. The contralateral side was sham-treated. A n o t h e r group served as unoperated controls Ten days later the birds were sacrificed and the extensor digitorum longus (EDL) muscles were sectioned and stained according to the silver-cholinesterase technique used prevtouslyll with the sole modification being incubation In the acetylthlochohne todide medium at 37 °C for 15 rain. Only mtact axon-arbor units were counted, with 200-400 samples from each E D L The number of branch points in the terminal arborization were counted as shown in Ftg 1. Surface areas were measured for a random sample of 100-150 NMJs per muscle. Examined under the light microscope at 1500× magnification, each NMJ was divided up into

Correspondence C S Fahlman, Department of Anatomy G-3, School of Medicine, Umverslty o! Pennsylvama, Philadelphia, PA 19104, U S A 0006-8993/86/$03 50 © 1986 Elsevier Soence Pubhshers B V (Biomedical Division)

157

Fig 1 Neuromuscular junction in the EDL muscle of a normal untreated pigeon. 'Branch pomts' (see text) are defined as the points of bifurcation of the axon's terminal arbor branches, as denoted by the arrows. Branch points were counted to quantify possible retraction of the arbor, or increased intratermlnal branching following colchlclne application or sham treatment in experimental animals as compared to normal animals. Calibration line, 50/~m

Fig. 2. Axon with an mternodai sprout (arrows) extending down to ramify at the endplate 10 days after treating the sciatic nerve with 5 mM colchicine. This manner of sprouting is termed 'endplate elaboration'. Calibration line, 50/~m

p o i n t s are also p r e s e n t e d in T a b l e I. T h e r e w e r e n o differences m t h e n u m b e r of b r a n c h p o i n t s p e r N M J among either sprouting or non-sprouting terminals

triangles, t r a p e z o i d s and s q u a r e s , using t h e a r b o r ' s

f r o m the colchicinized, s h a m - t r e a t e d and u n o p e r a t e d

b r a n c h e s to d i l i n e a t e t h e s e p o r t i o n s . B y m e a s u r i n g

controls. N e i t h e r c o u l d any d i f f e r e n c e b e d i s c e r n e d

its d i m e n s i o n s , t h e a r e a of e a c h p o r t i o n c o u l d b e cal-

b e t w e e n s p r o u t i n g and n o n - s p r o u t i n g N M J s within

culated. S u m m i n g the areas of all t h e s e p o r t i o n s g a v e

the s a m e muscle. Surface a r e a m e a s u r e m e n t s

the total surface a r e a o f t h e N M J . A p p l i c a t i o n of colchicine r e s u l t e d in a significant

f e r e n c e s b e t w e e n muscles. T a b l e II shows t h e surface

(0.01 > P > 0.001) m c r e a s e in I N s p r o u t i n g (Fig. 2)

a r e a of t e r m i n a l s f r o m s p r o u t i n g and n o n - s p r o u t i n g

and significant (0.02 > P > 0.01) i n c r e a s e in U T

axons within the c o l c h i c i n e - t r e a t e d side. N o signifi-

s p r o u t i n g (Figs. 3 and 4). T h e v a l u e s for t h e s h a m - o p -

cant d i f f e r e n c e s c o u l d be d e m o n s t r a t e d b e t w e e n the

are

s h o w n in T a b l e I. M e a n areas s h o w no significant dif-

e r a t e d side w e r e n o t significantly d i f f e r e n t f r o m t h e

areas of N M J s f r o m n o n - s p r o u t i n g axons vs N M J s

u n o p e r a t e d c o n t r o l s ( T a b l e I). T h e d a t a for b r a n c h

f r o m s p r o u t i n g axons.

TABLE I Comparison o f sprouting rates, branch pomts and areas o f NMJs Colchtcme-treated (n = 4)

Axons bearing IN sprouts (%) NMJs bearing UT sprouts (%)

10.09 + 2.18" 9 38 + 2 74**

Sham-treated (n = 4)

Unoperated control (n = 4)

3.61 + 1.04"

4 68 + 2 29

1 73 + 0 49**

1 86_+ 0 72

1022+104 a

1007+101 a

9 5 4 + 1 10

Mean branch pomts/UT-sproutlng NMJ

10.1 + 0 328

10 O0 + 1 25b

9 25 + 0.92

Mean surface area of NMJ ~ m 2)

1716 + 278c

1639 + 313c

1556 +- 343

Mean branch points/non-UT sprouting NMJ

* 0 01 > P > 0 001 by Student's t-test ** 0 02 > P > 0 001 by Student's t-test a-c NO significant differences between colchlcine-treated, sham-treated, and unoperated sides, neither are the differences between sprouting and non-sprouting NMJs significant within each muscle.

158 TABLE II

Mean N M J areas m colchtcme-treated stde E D L s A comparison of NMJ surface areas w~thm the colchlcmetreated side. No slgmflcant differences were demonstrated between non-sprouting NMJs and the areas of NMJs bearing UT sprouts or NMJs whose axons bore IN sprouts

All NMJs (ltm 2)

Non-sproutmg NMJs (tim 2)

UT-sproutmg NMJs (ktm 2)

1N-sproutmg NMJs (l~m2)

1716 _+ 278

1712 ___278

1803 _+ 298

1729 _+ 312

Many UT sprouting NMJs bore multiple sprouts (17.35% + 7.94) and branched sprouts (19.59% + 4.93). To determine whether NMJs producmg multiple sprouts were supporting more or less synthesis and growth than terminals supporting branched sprouts, lengths of sprouts were measured. Total outgrowth for multt-sprouters was 57 0 + 28/zm vs 48 5 + 25.7 /~m for terminals with single branched sprouts The differences in frequency and quantity of sprouting between terminals bearing multiple or branched sprouts are not significant, indicating no preference between these two sprouting patterns. This suggests that elaborating multiple sprouts does not impose a greater metabohc burden on the arbor than supporting a stogie branched sprout IN sprouts often originated from nodes located a considerable distance from the NMJ, unlike the resuits prevtously seen tn the rat where the node closest to the NMJ was the 'preferred site' A large percentage of collateral IN sprouts were recorded (31.52% _+ 5 31 of total) Of the remaining IN sprouts 12.33% + 5 62 arborized over extra-junction acetylcholinesterase (ACHE) patches on the same muscle fiber mnervated by the parent axon (duplex formatton), and 56 15% + 7.40 extended down to the parent axon's terminal, arbortzmg over its AChE (endplate elaboration, illustrated m Fig 2) As was also noted prewously in the raO l, there were few (5 48% + 3.23) sprouting axons w~th multiple IN sprouts, and never more than 3 sprouts were seen emanating from a single locus Similar to the rat, branched IN sprouts were rare, comprising only 2.2% + 2.56 of sprouts Although exposure to colchicme significantly rinsed UT and IN sprouting, it did not reduce new branches withm the terminal arbor, nor cause retraction of exlstmg branches The lack of any slgmficant differences in branching patterns or surface area of

NMJs exposed to colchicme as compared to the sham/control series imply that sprouting can occur without any gross resorption of existing terminal arbor and subneural structure. This suggests that the distal axons possess the metabolic condiments for supporting sprouting. The initiation of sprouting may also be under local control, since colchicine would not only inhibit the delivery of precursor materials, but would also interrupt any signals for control of distal metabolic processes emanating from the soma. Secondly, these results imply that sprouts are primarily elongations of pre-existing arbor branches. In hght of the high percentage of sprouting terminals that bore multiple (2-5) sprouts, it would be expected that sprouting NMJs would show a higher mean number of branch points than non-sprouting NMJs if sprouts are tertiary branches synthesized de novo from the parent arbor. That there was no decrease in NMJ arbor size or surface area also indicates that at this dose and mode of admmistration colchlcine Is not neurotoxlc. Unlike presynaptlc impulse block, interruption of axoplasm~c transport apparently does not result in a gross change in termmal morphology and area. Impulse activity and axonally transported substances may regulate different aspects of muscle membrane physiology The maintenance of the subneural apparatus may be more dependent on impulses than chemical trophic factors. The hypothesis that the distal axon is capable of supporting sprout synthesis w~thout the participation of materials or s~gnals emanating from the level of the soma is supported by the observation of Rotshenker 13 that axotomized axons can be reduced to sprout by colch~clne

That the sprouting response obtained m the pigeon closely resembled that described for complex and intermediate type NMJs in the rat supports the hypothesis that endplate structure rather than muscle fiber type determines sprouting rates. The pauoty of multiple or branched IN sprouts seen m both the pigeon and rat ~mply hmttat~ons on sprouting that may be due to the availability of cytoskeletal constituents This constramt may explain the existence of 'preferred sites" of sprouting along the axonJ1 A higher percentage of collateral sprouts were found in this study than m the previous report This may be a reflection of the shorter exposure to colch~cine used and the greater thickness of the pigeon so-

159

Fig. 4. Example of collateral sprouting An ultratermlnal sprout (small arrow) has crossed over to a neighboring muscle fiber to arbonze over a small patch of extra-junctional acetylchohnesterase (large arrow) Cahbrat~on hne, 50/~m

Fig. 3. Example of an ultratermlnal sprout (arrow) growing out from the terminal arbor of an NMJ 10 days after treatment wtth 5 mM colchtcme Cahbratlon hne, 501tm

atic nerve. In the previous studyll, most sprouts tended to grow along the muscle fibers innervated by their parent axons, possibly because a greater proportion of axons had been affected and the sprouting signal from the parent axon's muscle fiber may have masked that of neighboring muscle fibers. In the thicker pigeon nerve, fewer axons may have b e e n exposed to colchicine, mcreasing the likehhood of collaterals sprouting from unaffected neighbours responding to sprouting signals from muscle fibers of

1 Barker, D and Ip, M C , Sprouting and degeneration of mammahan motor axons m normal and de-afferented skeletal muscle, Proc R Soc. London Ser B, 163 (1966) 538-554 2 Brown, M C , Holland, R L and Hopkins, N G , Motor nerve sproutmg. Annu Rev. Neuroscl, 4 (1981) 17-42 3 Brown, M.C and Ironton, R , Motor neurons sprouting induced by prolonged tetrodotoxin block of nerve action potentials, Nature (London), 265 (1977) 459-461 4 Diamond, J Cooper, E , Turner, C and Maclntyre, L , Trophic regulation of nerve sprouting, Scwnce, 193 (1976) ' 371-377 ,

affected axons. It may also reflect a species difference in the susceptibility to the effects of colchicine, as such differences have been observed (J. D i a m o n d , personal communication and refs. 2, 6, 11-13). Possibly, these differences may reflect differences in permeabllity of the drug, or may have a physiological basis These problems could be solved by observing the depth of penetration of labelled colchiclne in nerves of different species (as well as in nerves of different calibers within a species), and correlating tt with the magnitude of the sprouting response ehclted. This study was supported by N A S A Cooperative A g r e e m e n t NCC-2-266. Dr. Walter F r e e m a n of U C Berkeley is thanked for providing the pigeons Dr. Matthew Lavail of U C S F is thanked for assistance with the photography. Lonnie Schilling ts thanked for typing parts of the manuscript.

5 Duchen, L.W, Changes in motor mnervatlon and chohnesterase localization reduced by botuhnum toxin in skeletal muscle of the mouse: difference between fast and slow muscles,J Neurol Neurosurg Psychiatry, 33 (1970)40-54 6 Edds, M V , Collateral nerve regeneration. Q Rev Btol, 28 (1953) 260-276 7 Guth, L , Smith, S, Donati, E J and Albuquerque, E X., Induction of intramuscular collateral nerve sprouting by neurally applied colchicme, Exp Neurol, 67 (1980) 513-523 8 Harris, J B , The relatlonsh~p between endplate size and transmitter release in normal and dystrophic muscles of the

160 mouse, J Phystol (London), 296 (1979) 245-265 9 Holland, R L and Brown, M C., Postsynaptic transmission block can cause terminal sprouting of a motor nerve, Sctence, 207 (1980) 649-651 10 Pestronk, A and Drachman, D , Motor nerve sprouting and acetylcholine receptors, Sctence, 199 (1978) 1223 - 1225 11 Riley, D A and Fahlman, C S , ColcMcme-mduced differential sprouting of the endplates on fast and slow muscle fl-

bers an rat extensor d~g~torum longus, soleus and ttbtalls antenor muscles, Brain Research, 329 (1985) 83-95 12 Rotshenker, S , Sprouting of intact motor neurons reduced by neuronal leston an the absence of denervated muscle fibers and degenerating axons, Bram Research, 155 (1978) 354-356 13 Rotshenker, S , Transneuronal and peripheral mechamsms for the mductton of motor neuron sprouting, J. Neurosct, 2 (1982) 1359-1368