1 18
Brabt Research, t55 ti~J7~) I I,S 127 (~/Elsevier/North-Holland Bioiatedical Pres~
Uptake and retrograde axonal transport of horseradish peroxidase in botulinum-intoxicated mice
KRISTER KRISTENSSON and TOMAS OLSSON
Neuropathological Laboratory, Department oJ Pathology 11, LinkSping University (Sweden J (Accepted May 25th, 1978)
Axon terminals at neuromuscular junctions may incorporate macromolecules, which can subsequently be transported in a retrograde direction to the nerve cell bodies in the brain stem or spinal cord16, is. This phenomenon is now extensively used for studies of neuronal connectivity 19. Macromolecutar tracers, such as horseradish peroxidase (HRP), are taken up by endocytosis. The rate of H R P incorporation varies with the degree of synaptic activity, which may reflect recycling of vesicle membranes during transmitter release12,14. Studies on organotypic nervous tissue cultures and on frog muscle-nerve preparations in vitro have indicated that the synaptic activity also influences the amount of retrogradely transported material20, 2~. Since this may provide a feed-back mechanism by which the nerve cell body is informed of its synaptic function. it would be interesting to study if such influences on retrograde axonal transport also occur in other experimental models. The aim of the present study was to examine if an inhibited release of transmitter in vivo is also followed by an inhibition of axonaI uptake and retrograde transport of HRP. Inhibition of transmitter release was produced by botulinum toxin (BoTX) type A 4,26, which was injected under ether anaesthesia in a volume of 0.05 ml gelatin phosphate buffer solution, p H 6.6, into the region of the muscles of the vibrissae on the left side of male Swiss albino mice 50-70 days of age. In order to reduce diffusion of the toxin to the contralateral side a strip of surgical tape was applied in a midline incision on the previous day. BoTX was injected in 10-fold dilutions and the movements of the vibrissae recorded. The number of mice and the results are listed in Table I. In one set of experiments 5 mg H R P (Type II, Sigma. St. Louist in 0.05 ml physiological saline was injected into the region of the muscles of the vibrissae on both sides 4 (2) and 18 (4) h after injection of non diluted (50 pg) BoTX into the left side. The numbers in brackets refer to the number of mice. In addition 60 mg HRP in 0.6 ml physiological saline was injected into the tail-vein of one mouse injected with non-diluted BoTX 18 h previously and into one control mouse. After 9 h the mice were sacrificed and treated as described below. In another set of experiments, BoTX diluted l :10 was injected into the left side and 1 (4), 3 (4), 7 (2). 14 (2), 21 (2) and 28 (2) days later 60 mg H R P in 0.6 ml physiological saline was injected into the tail-vein. The LV. injections were made in order to obtain a more even and comparable diffusion of H R P into the muscles of the vibrissae on both sides and to avoid any trauma of i.m. injection 3,17. As
119 TABLE
I
BoTX dilution
l I:lO 1:102 1:10 z
No. o/'mice
10 45 3 3
Time,for onset of vibrissae paralysis
Time for
LeJt side
Right side
Death
Recovery
2 h 6 h 20 h .
18 20 h --.
25-27 h ---
5 6 days 5 6 days
.
.
* T h e r e s p e c t i v e d o s a g e s r e l a t i n g to t h e d i l u t i o n s a r e : 5 0 ; 5; 0 . 5 ; a n d 0.05 pg.
controls, 3 mice not injected with BoTX served. After a circulation time of 24 h the mice were fixed by perfusion with 2.5 ~ glutaraldehyde in phosphate buffer, pH 7.4, with 0.01 ~ CaCl2 added. The brain stem at the level of the facial nuclei and the muscles of the vibrissae on both sides were dissected, postfixed for 4 h followed by a rinse overnight in phosphate buffer. Frozen sections, 40 #m thick, were cut and incubated with 3,3'diaminobenzidine and hydrogen peroxide for light microscopical demonstration of H R P 1°. A scoring procedure was used to compare H R P reaction product in neurons on the left and right side ~3. The muscle specimens were embedded in agar and chopped into sections 40 #m thick using a Sorvall TC-2 sectioner. To localize motor end-plate regions, one of two adjacent sections was reacted for acetylcholinesterase activity 9. From the parallel section areas rich in motor end-plates were selected and incubated with 3,3'-diaminobenzidine and glucose-glucose oxidase 29. The pieces were postfixed in 2 ~ osmium, dehydrated and embedded in Epon. Motor end-plates (50-60) were examined ultrastructurally from paralysed, non-paralysed and control muscles each. For demonstration of possible nerve sprouts, 40/~m thick frozen sections were incubated for non-specific cholinesterase with butyrylthiocholine as substrate v. The appearance of Nissl bodies in the facial neurons was studied in 3 mice perfused with 4 formalin 4 days after injection of BoTX diluted 1 : 10 by staining paraffin-embedded sections with cresyl violet-acetic acid. The movements of the vibrissae on the ipsilateral side ceased regularly 2 h after the injection of non-diluted BoTX. H R P injected 2 h after onset of paralysis appeared in synaptic vesicles, coated vesicles, larger vacuoles and cisternal structures in the axon terminals. H R P reaction product was seen in the nerve cell bodies of the ipsilateral facial nucleus to the same extent as in the contralateral one, where paralysis had not developed when the mice were sacrificed 13 h after injection of BoTX. A marked axonal uptake (Fig. 1) and accumulation of H R P in the nerve cell bodies (Fig. 2) occurred in mice injected i.m. or i.v. with H R P 16 h after onset of paralysis and sacrificed 9 h later, at a time when they were moribund. Injection of BoTX diluted 1 : 10 caused the movements of the ipsi[ateral vibrissae to cease after 6 h. They reappeared 5 6 days later. No paresis of the contralateral vibrissae was observed. H R P injected i.v. I or 3 days after BoTX injection diffused into the muscles of the vibrissae, was incorporated into the axon terminal at the neuromuscular junction and transported to the nerve cell bodies in the facial nuclei. No difference
120
Fig. I. Axon terminal outlined by dark reaction product of HRP, which also is incorporated in to several vesicles and cisternal structures; 27 h after non-diluted BoTX injection. ~,: 26,800. Fig. 2. Granular HRP reaction product in facial neurons 27 h after BoTX injection ; dark-field condensor. × 1200. Fig. 3. Projections containing synaptic vesicles (arrows) above an original axon terminal profile; 14 days after injection ofBoTX. 7," 1%000.
12l
Fig. 4. Motor end-plates of normal appearance 14 days after BoTX injection. Note the close proximity between the axon terminals and the blood vesselthrough which i.v. injected H R P has passed and diffused into the synaptic cleft. To the right the terminal segment ofa myelinated nerve fibre. ~ 12,200. between the paralysed and the non-paralysed side occurred, and the appearance was similar as in the control mice. After 7 and 14 days, when the movements of the vibrissae had reappeared, most neuromuscular junctions had an ordinary appearance (Fig. 4). In connection with some of them small projections filled with vesicles lie above and beside the axon terminals (Fig. 3). Only occasionally axon terminals rich in filaments and containing few synaptic vesicles occurred s. No significant sign of sprouting was seen light microscopically in cholinesterase preparations. No difference in occurrence of H R P reaction product was noted between the facial nuclei of the left and right side in mice sacrificed 7, 14, 21 and 28 days after BoTX injection. Thus, during BoTX intoxication, even with a lethal dose, incorporation of H R P into organdies of the axon terminal and retrograde transport to the nerve cell bodies occurred. These results were contrary to our expectations, since it has been proposed that BoTX prevents transmitter release by an impaired exocytosis 15 and, consequently, the compensatory endocytosis due to recycling of membranes ought to be inhibited 13. Our findings may be explained by (a) uptake and/or retrograde transport of H R P in this in vivo model is not mainly linked to exocytosis of synaptic vesicles but occurs as independent phenomena regulated by other mechanisms, (b) a few acetylcholine quanta may be released in spite of the paralysis, leading to miniature end-plate potentials11,')4; a concomitant H R P uptake may result in a quantitatively decreased transport of H R P not revealed by a histochemical method, (c) exocytosis occurs during BoTX intoxication, but the synaptic vesicles are unloaded with acetylcholinel; this hypothesis has, however, not gained any support in recent studieslS,21,22, at, (d) synaptic vesicle mem-
122 b r a n e fuses with a x o n surface m e m b r a n e , but still acetylcholine is n o t or is only p a r t i a l l y released into the s y n a p t i c cleft; a l t e r n a t i v e l y new a n d less f a v o r a b l e sites at the ax0n t e r m i n a l for exocytosis m a y develop d u r i n g B o T X i n t o x i c a t i o n n . It has been suggested t h a t a x o n a l u p t a k e o f an extracellular p r o t e i n is prevented by B o T X a n d t h a t changes in the nerve cell b o d y ' s synthesis o f r i b o s o m a l R N A occur as a consequence o f an i n t e r r u p t e d ascent o f such a p r o t e i n in the axon 28. Since in o u r m o d e l a m a r k e d r e t r o g r a d e t r a n s p o r t o f H R P d u r i n g B o T X i n t o x i c a t i o n persisted, o u r findings d o n o t s u p p o r t this hypothesis. Recently, B o T X has been f o u n d t o be r e t r o , g r a d e l y t r a n s p o r t e d to the m o t o r n e u r o n s in the spinal c o r d following km. injection a~, a n d a nerve cell b o d y response m i g h t well be related to this. A s to fast axonal t r a n s p o r t in the other, a n t e r o g r a d e , direction, no effect o f B o T X has been recorded ~,'~a. W i t h a s u b ' l e t h a l dose o f B o T X we f o u n d no signs o f c h r o m a t o l y s i s in the facial n e u r o n s and, a l t h o u g h at the time o f f u n c t i o n a l recovery some u l t r a s t r u c t u r a l signs o f s p r o u t i n g were present, the m o r p h o l o g y o f the synapses was generally preserved: This s u p p o r t s the o p i n i o n t h a t f u n c t i o n m a y r e t u r n n o t only f r o m nerve s p r o u t s 2,s but also f r o m recovering original axon t e r m i n a l s ~7. This s t u d y was s u p p o r t e d by g r a n t s f r o m the Swedish M e d i c a l Research Council, Project B78-12X-04480-04A, a n d from Ollie and E l o f Ericssons F o r s k n i n g s f o n d .
I Boroff, D. A., Del Castitlo, J., Evoy. W. H. and Steinhardt. R. A.. Observations on the action of type A botulinum toxin on frog neuromuscular junctions, J. Physiol. (Lond.), 240 (i974) 227-253. 2 Bray, J. J. and Harris, A. J., Dissociation between nerve-muscle transmission and nerve trophic effects on rat diaphragm using type D botulinum toxin, J. Physiol. (Lond.), 253(1975) 53-77. 3 Broadwell, R. D. and Brightman, M. W., Entry of peroxidase into neurons of thecentrat and peripheral nervous systems from extracerebral and cerebral blood, J. comp. Neurol., 166 (1976) 257-284. 4 Brooks, V. B.. The action of botulinum toxin on motor-nerve filaments. J. Physiok (Lond.), 123 (1954) 510-515. 5 DahlstrSm, A., Personal communication. 6 Drachman, D. B., The role of acetylcholine as a neurotrc phic transmitter. Ann. N.. Y Acad. Sci.. 228 (1974) 160-175. 7 Duchen, L. W., Changes in motor innervation and cholinesterase localization induced by botulinum toxin in skeletal muscle of the mouse: differences between fast and slow muscles. J. Neurol. Neurosurg. Psychiat.. 33 (1970) 40-54. 8 Duchen. L. W., An electron microscopic study of the changes induced by botulinum toxin in the motor end-plates of slow and fast skeletal muscle fibres of the mouse, J. neurol. Sci., 14 (1971 ) 47-60. 9 Gomori, G., Microscopic Histochemistry. Principles and Practice, University of Chicago Press. Chicago, 1952, 210 pp. 10 Graham, R. C. and Karnovsky, M. J., The early stages of absorption of injected horseradish peroxidase in the proximal tubules of mouse kidney. Ultrastructural correlates by a new technique, J. Histochem. Cytochem., 14 (1966) 291-302. 11 Harris, A. J. and Miledi, R. ,The effect of type D botutinum toxin on frog neuromuscular juncti°ns" J. Physiol. (Lond.), 217 (1971) 497-515. 12 Heuser, J. E. and Reese, T. S., Evidence for recycling of synaptic vesicle membrane during transmitter release at the frog neuromuscular junction, J. Cell BioL, 57 (1973) 315-344. 13 Holtzman, E., The origin and fate of secretory packages, especially synaptic vesicles. Neuroscience, 2 (1977) 327-355. 14 Holtzman, E.,Freeman, A.R. andKashner, L.A..Stimulation-dependentalterationsin peroxidase uptake at lobster neuromuscular junctions, Science, t73 (1971) 733-736.
123 15 Kao, K., Drachman, D. B. and Price, D. L., Botulinum toxin : mechanism of presynaptic Flockade, Science, 193 (1976) 1256 1258. 16 Kristensson, K., Transport of fluorescent protein tracer in peripheral nerves, Acta nearopath. (Berl.), 16 (1970) 293 300. 17 Kristensson, K., Retrograde axonal transport of horseradish peroxidase. Uptake at mouse neuromuscular junction following systemic injection, Acta net, ropath. (Berl.), 38 (1977) 143-147. 18 Kristensson, K., Retrograde transport of macromolecules in axons, Ann. Rev. Pharrnacol. Toxicol., 18 (1978) 97 I10. 19 LaVail, J. H. and LaVail, M. M., The retrograde intraaxonal transport of horseradish peroxidase in the chick visual system : a light and electron microscopic study, J. comp. Neurol., 157 (1974) 303 357. 20 Lichty, W. J., Uptake and retrograde transport of horseradish peroxidase in frog sartorius nerve in vitro, Brain Research, 56 (1973) 377 381. 21 Lundh, H., Cull-Candy, S. G., Leander, S. and Thesleff, S., Restoration of transmitter release in botulinum-poisoned skeletal muscle, Brain Research, 110 (1976) 194 198. 22 Lundh, H. and Thesleff, S., The mode of action of 4-aminopyridine and guanidine on transmitter release from motor nerve terminals, Europ. J. Pharmacol., 42 (1977) 411-412. 23 Olsson, T. P., Forsberg, 1. and Kristensson, K., Uptake and retrograde axonal transport of horseradish lzeroxidase in regenerating facial motor neurons of the mouse, J. Neuroo, tol. (1978) in press. 24 Spitzer, N., Miniature end-plate potentials at mammalian neuromuscular junctions poisoned by botulinum toxin, Nature New Biol., 237 (1972) 26 27. 25 Teichberg, S., Holtzman, E., Crain, S. M., and Peterson E.R., Circulation and turnover of synaptic vesicle membrane in cultured fetal mammalian spinal cord neurons, J. Cell Biol., 67 (1975) 215 230. 2.5 Thesleff, S., Supersensitivity of skeletal muscle produced by botulinum toxin, J. Physiol. (Lond.), 151 (1960) 598-607. 26a Thesleff, S., Dahlstr6m, A. and Heiwall, P. O., Axonal transport of acetylcholine and related enzymes in rat motor nerves after botulinum toxin treatment, in preparation. 27 Tonge, D. A., Chronic effects of botulinum toxin on neuromuscular transmission and senstivity to acetylcholine in slow and fast skeletal muscle of the mouse, J. Physiol. (Lond.), 241 (1974) 127-139. 28 Watson, W. E., Cellular responses to axotomy and related procedures, Brit. reed. Bull., 30 (1974) 112 115. 29 Westergaard, E., Go, G., Klatzo, I. and Spatz, M., Increased permeability of cerebral vessels to horseradish peroxidase induced by ischemia in mongolian gerbils, Acta neuropath. (Berl.), 35 (1976) 307-325. 30 Wiegand, H., Erdmann, G. and Wellh/Sner, H. H., t'~'~l-labelled botulinum A neurotoxin: pharmacokinetics in cats after intramuscular injection, Naunyn-Schmiedeberg's Arch. exp. Path. Pharmacol., 292 (1976) 161-165. 31 Wonnacott, S. and Marchbanks, R. M., Inhibition by botulinum toxin of depolarisation-evoked release of [i iC]acetylcholine from synaptosomes in vitro, Biochem. J., 156 (1976) 701-712.
Brabt Research, t55 ti~J7~) I I,S 127 (~/Elsevier/North-Holland Bioiatedical Pres~
Uptake and retrograde axonal transport of horseradish peroxidase in botulinum-intoxicated mice
KRISTER KRISTENSSON and TOMAS OLSSON
Neuropathological Laboratory, Department oJ Pathology 11, LinkSping University (Sweden J (Accepted May 25th, 1978)
Axon terminals at neuromuscular junctions may incorporate macromolecules, which can subsequently be transported in a retrograde direction to the nerve cell bodies in the brain stem or spinal cord16, is. This phenomenon is now extensively used for studies of neuronal connectivity 19. Macromolecutar tracers, such as horseradish peroxidase (HRP), are taken up by endocytosis. The rate of H R P incorporation varies with the degree of synaptic activity, which may reflect recycling of vesicle membranes during transmitter release12,14. Studies on organotypic nervous tissue cultures and on frog muscle-nerve preparations in vitro have indicated that the synaptic activity also influences the amount of retrogradely transported material20, 2~. Since this may provide a feed-back mechanism by which the nerve cell body is informed of its synaptic function. it would be interesting to study if such influences on retrograde axonal transport also occur in other experimental models. The aim of the present study was to examine if an inhibited release of transmitter in vivo is also followed by an inhibition of axonaI uptake and retrograde transport of HRP. Inhibition of transmitter release was produced by botulinum toxin (BoTX) type A 4,26, which was injected under ether anaesthesia in a volume of 0.05 ml gelatin phosphate buffer solution, p H 6.6, into the region of the muscles of the vibrissae on the left side of male Swiss albino mice 50-70 days of age. In order to reduce diffusion of the toxin to the contralateral side a strip of surgical tape was applied in a midline incision on the previous day. BoTX was injected in 10-fold dilutions and the movements of the vibrissae recorded. The number of mice and the results are listed in Table I. In one set of experiments 5 mg H R P (Type II, Sigma. St. Louist in 0.05 ml physiological saline was injected into the region of the muscles of the vibrissae on both sides 4 (2) and 18 (4) h after injection of non diluted (50 pg) BoTX into the left side. The numbers in brackets refer to the number of mice. In addition 60 mg HRP in 0.6 ml physiological saline was injected into the tail-vein of one mouse injected with non-diluted BoTX 18 h previously and into one control mouse. After 9 h the mice were sacrificed and treated as described below. In another set of experiments, BoTX diluted l :10 was injected into the left side and 1 (4), 3 (4), 7 (2). 14 (2), 21 (2) and 28 (2) days later 60 mg H R P in 0.6 ml physiological saline was injected into the tail-vein. The LV. injections were made in order to obtain a more even and comparable diffusion of H R P into the muscles of the vibrissae on both sides and to avoid any trauma of i.m. injection 3,17. As
119 TABLE
I
BoTX dilution
l I:lO 1:102 1:10 z
No. o/'mice
10 45 3 3
Time,for onset of vibrissae paralysis
Time for
LeJt side
Right side
Death
Recovery
2 h 6 h 20 h .
18 20 h --.
25-27 h ---
5 6 days 5 6 days
.
.
* T h e r e s p e c t i v e d o s a g e s r e l a t i n g to t h e d i l u t i o n s a r e : 5 0 ; 5; 0 . 5 ; a n d 0.05 pg.
controls, 3 mice not injected with BoTX served. After a circulation time of 24 h the mice were fixed by perfusion with 2.5 ~ glutaraldehyde in phosphate buffer, pH 7.4, with 0.01 ~ CaCl2 added. The brain stem at the level of the facial nuclei and the muscles of the vibrissae on both sides were dissected, postfixed for 4 h followed by a rinse overnight in phosphate buffer. Frozen sections, 40 #m thick, were cut and incubated with 3,3'diaminobenzidine and hydrogen peroxide for light microscopical demonstration of H R P 1°. A scoring procedure was used to compare H R P reaction product in neurons on the left and right side ~3. The muscle specimens were embedded in agar and chopped into sections 40 #m thick using a Sorvall TC-2 sectioner. To localize motor end-plate regions, one of two adjacent sections was reacted for acetylcholinesterase activity 9. From the parallel section areas rich in motor end-plates were selected and incubated with 3,3'-diaminobenzidine and glucose-glucose oxidase 29. The pieces were postfixed in 2 ~ osmium, dehydrated and embedded in Epon. Motor end-plates (50-60) were examined ultrastructurally from paralysed, non-paralysed and control muscles each. For demonstration of possible nerve sprouts, 40/~m thick frozen sections were incubated for non-specific cholinesterase with butyrylthiocholine as substrate v. The appearance of Nissl bodies in the facial neurons was studied in 3 mice perfused with 4 formalin 4 days after injection of BoTX diluted 1 : 10 by staining paraffin-embedded sections with cresyl violet-acetic acid. The movements of the vibrissae on the ipsilateral side ceased regularly 2 h after the injection of non-diluted BoTX. H R P injected 2 h after onset of paralysis appeared in synaptic vesicles, coated vesicles, larger vacuoles and cisternal structures in the axon terminals. H R P reaction product was seen in the nerve cell bodies of the ipsilateral facial nucleus to the same extent as in the contralateral one, where paralysis had not developed when the mice were sacrificed 13 h after injection of BoTX. A marked axonal uptake (Fig. 1) and accumulation of H R P in the nerve cell bodies (Fig. 2) occurred in mice injected i.m. or i.v. with H R P 16 h after onset of paralysis and sacrificed 9 h later, at a time when they were moribund. Injection of BoTX diluted 1 : 10 caused the movements of the ipsi[ateral vibrissae to cease after 6 h. They reappeared 5 6 days later. No paresis of the contralateral vibrissae was observed. H R P injected i.v. I or 3 days after BoTX injection diffused into the muscles of the vibrissae, was incorporated into the axon terminal at the neuromuscular junction and transported to the nerve cell bodies in the facial nuclei. No difference
120
Fig. I. Axon terminal outlined by dark reaction product of HRP, which also is incorporated in to several vesicles and cisternal structures; 27 h after non-diluted BoTX injection. ~,: 26,800. Fig. 2. Granular HRP reaction product in facial neurons 27 h after BoTX injection ; dark-field condensor. × 1200. Fig. 3. Projections containing synaptic vesicles (arrows) above an original axon terminal profile; 14 days after injection ofBoTX. 7," 1%000.
12l
Fig. 4. Motor end-plates of normal appearance 14 days after BoTX injection. Note the close proximity between the axon terminals and the blood vesselthrough which i.v. injected H R P has passed and diffused into the synaptic cleft. To the right the terminal segment ofa myelinated nerve fibre. ~ 12,200. between the paralysed and the non-paralysed side occurred, and the appearance was similar as in the control mice. After 7 and 14 days, when the movements of the vibrissae had reappeared, most neuromuscular junctions had an ordinary appearance (Fig. 4). In connection with some of them small projections filled with vesicles lie above and beside the axon terminals (Fig. 3). Only occasionally axon terminals rich in filaments and containing few synaptic vesicles occurred s. No significant sign of sprouting was seen light microscopically in cholinesterase preparations. No difference in occurrence of H R P reaction product was noted between the facial nuclei of the left and right side in mice sacrificed 7, 14, 21 and 28 days after BoTX injection. Thus, during BoTX intoxication, even with a lethal dose, incorporation of H R P into organdies of the axon terminal and retrograde transport to the nerve cell bodies occurred. These results were contrary to our expectations, since it has been proposed that BoTX prevents transmitter release by an impaired exocytosis 15 and, consequently, the compensatory endocytosis due to recycling of membranes ought to be inhibited 13. Our findings may be explained by (a) uptake and/or retrograde transport of H R P in this in vivo model is not mainly linked to exocytosis of synaptic vesicles but occurs as independent phenomena regulated by other mechanisms, (b) a few acetylcholine quanta may be released in spite of the paralysis, leading to miniature end-plate potentials11,')4; a concomitant H R P uptake may result in a quantitatively decreased transport of H R P not revealed by a histochemical method, (c) exocytosis occurs during BoTX intoxication, but the synaptic vesicles are unloaded with acetylcholinel; this hypothesis has, however, not gained any support in recent studieslS,21,22, at, (d) synaptic vesicle mem-
122 b r a n e fuses with a x o n surface m e m b r a n e , but still acetylcholine is n o t or is only p a r t i a l l y released into the s y n a p t i c cleft; a l t e r n a t i v e l y new a n d less f a v o r a b l e sites at the ax0n t e r m i n a l for exocytosis m a y develop d u r i n g B o T X i n t o x i c a t i o n n . It has been suggested t h a t a x o n a l u p t a k e o f an extracellular p r o t e i n is prevented by B o T X a n d t h a t changes in the nerve cell b o d y ' s synthesis o f r i b o s o m a l R N A occur as a consequence o f an i n t e r r u p t e d ascent o f such a p r o t e i n in the axon 28. Since in o u r m o d e l a m a r k e d r e t r o g r a d e t r a n s p o r t o f H R P d u r i n g B o T X i n t o x i c a t i o n persisted, o u r findings d o n o t s u p p o r t this hypothesis. Recently, B o T X has been f o u n d t o be r e t r o , g r a d e l y t r a n s p o r t e d to the m o t o r n e u r o n s in the spinal c o r d following km. injection a~, a n d a nerve cell b o d y response m i g h t well be related to this. A s to fast axonal t r a n s p o r t in the other, a n t e r o g r a d e , direction, no effect o f B o T X has been recorded ~,'~a. W i t h a s u b ' l e t h a l dose o f B o T X we f o u n d no signs o f c h r o m a t o l y s i s in the facial n e u r o n s and, a l t h o u g h at the time o f f u n c t i o n a l recovery some u l t r a s t r u c t u r a l signs o f s p r o u t i n g were present, the m o r p h o l o g y o f the synapses was generally preserved: This s u p p o r t s the o p i n i o n t h a t f u n c t i o n m a y r e t u r n n o t only f r o m nerve s p r o u t s 2,s but also f r o m recovering original axon t e r m i n a l s ~7. This s t u d y was s u p p o r t e d by g r a n t s f r o m the Swedish M e d i c a l Research Council, Project B78-12X-04480-04A, a n d from Ollie and E l o f Ericssons F o r s k n i n g s f o n d .
I Boroff, D. A., Del Castitlo, J., Evoy. W. H. and Steinhardt. R. A.. Observations on the action of type A botulinum toxin on frog neuromuscular junctions, J. Physiol. (Lond.), 240 (i974) 227-253. 2 Bray, J. J. and Harris, A. J., Dissociation between nerve-muscle transmission and nerve trophic effects on rat diaphragm using type D botulinum toxin, J. Physiol. (Lond.), 253(1975) 53-77. 3 Broadwell, R. D. and Brightman, M. W., Entry of peroxidase into neurons of thecentrat and peripheral nervous systems from extracerebral and cerebral blood, J. comp. Neurol., 166 (1976) 257-284. 4 Brooks, V. B.. The action of botulinum toxin on motor-nerve filaments. J. Physiok (Lond.), 123 (1954) 510-515. 5 DahlstrSm, A., Personal communication. 6 Drachman, D. B., The role of acetylcholine as a neurotrc phic transmitter. Ann. N.. Y Acad. Sci.. 228 (1974) 160-175. 7 Duchen, L. W., Changes in motor innervation and cholinesterase localization induced by botulinum toxin in skeletal muscle of the mouse: differences between fast and slow muscles. J. Neurol. Neurosurg. Psychiat.. 33 (1970) 40-54. 8 Duchen. L. W., An electron microscopic study of the changes induced by botulinum toxin in the motor end-plates of slow and fast skeletal muscle fibres of the mouse, J. neurol. Sci., 14 (1971 ) 47-60. 9 Gomori, G., Microscopic Histochemistry. Principles and Practice, University of Chicago Press. Chicago, 1952, 210 pp. 10 Graham, R. C. and Karnovsky, M. J., The early stages of absorption of injected horseradish peroxidase in the proximal tubules of mouse kidney. Ultrastructural correlates by a new technique, J. Histochem. Cytochem., 14 (1966) 291-302. 11 Harris, A. J. and Miledi, R. ,The effect of type D botutinum toxin on frog neuromuscular juncti°ns" J. Physiol. (Lond.), 217 (1971) 497-515. 12 Heuser, J. E. and Reese, T. S., Evidence for recycling of synaptic vesicle membrane during transmitter release at the frog neuromuscular junction, J. Cell BioL, 57 (1973) 315-344. 13 Holtzman, E., The origin and fate of secretory packages, especially synaptic vesicles. Neuroscience, 2 (1977) 327-355. 14 Holtzman, E.,Freeman, A.R. andKashner, L.A..Stimulation-dependentalterationsin peroxidase uptake at lobster neuromuscular junctions, Science, t73 (1971) 733-736.
123 15 Kao, K., Drachman, D. B. and Price, D. L., Botulinum toxin : mechanism of presynaptic Flockade, Science, 193 (1976) 1256 1258. 16 Kristensson, K., Transport of fluorescent protein tracer in peripheral nerves, Acta nearopath. (Berl.), 16 (1970) 293 300. 17 Kristensson, K., Retrograde axonal transport of horseradish peroxidase. Uptake at mouse neuromuscular junction following systemic injection, Acta net, ropath. (Berl.), 38 (1977) 143-147. 18 Kristensson, K., Retrograde transport of macromolecules in axons, Ann. Rev. Pharrnacol. Toxicol., 18 (1978) 97 I10. 19 LaVail, J. H. and LaVail, M. M., The retrograde intraaxonal transport of horseradish peroxidase in the chick visual system : a light and electron microscopic study, J. comp. Neurol., 157 (1974) 303 357. 20 Lichty, W. J., Uptake and retrograde transport of horseradish peroxidase in frog sartorius nerve in vitro, Brain Research, 56 (1973) 377 381. 21 Lundh, H., Cull-Candy, S. G., Leander, S. and Thesleff, S., Restoration of transmitter release in botulinum-poisoned skeletal muscle, Brain Research, 110 (1976) 194 198. 22 Lundh, H. and Thesleff, S., The mode of action of 4-aminopyridine and guanidine on transmitter release from motor nerve terminals, Europ. J. Pharmacol., 42 (1977) 411-412. 23 Olsson, T. P., Forsberg, 1. and Kristensson, K., Uptake and retrograde axonal transport of horseradish lzeroxidase in regenerating facial motor neurons of the mouse, J. Neuroo, tol. (1978) in press. 24 Spitzer, N., Miniature end-plate potentials at mammalian neuromuscular junctions poisoned by botulinum toxin, Nature New Biol., 237 (1972) 26 27. 25 Teichberg, S., Holtzman, E., Crain, S. M., and Peterson E.R., Circulation and turnover of synaptic vesicle membrane in cultured fetal mammalian spinal cord neurons, J. Cell Biol., 67 (1975) 215 230. 2.5 Thesleff, S., Supersensitivity of skeletal muscle produced by botulinum toxin, J. Physiol. (Lond.), 151 (1960) 598-607. 26a Thesleff, S., Dahlstr6m, A. and Heiwall, P. O., Axonal transport of acetylcholine and related enzymes in rat motor nerves after botulinum toxin treatment, in preparation. 27 Tonge, D. A., Chronic effects of botulinum toxin on neuromuscular transmission and senstivity to acetylcholine in slow and fast skeletal muscle of the mouse, J. Physiol. (Lond.), 241 (1974) 127-139. 28 Watson, W. E., Cellular responses to axotomy and related procedures, Brit. reed. Bull., 30 (1974) 112 115. 29 Westergaard, E., Go, G., Klatzo, I. and Spatz, M., Increased permeability of cerebral vessels to horseradish peroxidase induced by ischemia in mongolian gerbils, Acta neuropath. (Berl.), 35 (1976) 307-325. 30 Wiegand, H., Erdmann, G. and Wellh/Sner, H. H., t'~'~l-labelled botulinum A neurotoxin: pharmacokinetics in cats after intramuscular injection, Naunyn-Schmiedeberg's Arch. exp. Path. Pharmacol., 292 (1976) 161-165. 31 Wonnacott, S. and Marchbanks, R. M., Inhibition by botulinum toxin of depolarisation-evoked release of [i iC]acetylcholine from synaptosomes in vitro, Biochem. J., 156 (1976) 701-712.
