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Batrachotoxin induced axonal necrosis followed by regeneration
246
Brain Research, 279 (1983) 246-249 Elsevier
Batrachotoxin induced axonal necrosis followed by regeneration G. R. W. MOORE*, D. M. ROBERTSON and R. J. BOEGMAN l.** Departments" of Pathology and 1Pharmacology and 1Toxicology, Queen's University, and Kingston General Hospital, Kingston, Ont. K7L 3N6 (Canada) (Accepted July 19th, 1983) Key words: nerve regeneration - - axonal necrosis - - Wallerian degeneration - - batrachotoxin
Batrachotoxin (BTX), when applied to peripheral nerve in concentrations sufficient to block impulse transmission and axonal transport, causes axonal necrosis. Quantitation of this phenomenon reveals reduction of both myelinated and unmyelinated fibers 7 days post-BTX injection. Evidence for regeneration correlates with the previously reported recovery of postsynaptic events.
Batrachotoxin (BTX) is a steroidal neurotoxin which depolarizes electrogenic m e m b r a n e s by increasing sodium permeabilityl,2,10. A t relatively high concentrations (8 x 10-9 t o 9 x 10- 9 mol) it irreversibly blocks fast axonai transport in vitro ~4. Low concentrations (10-12 tool) reversibly block both anterograde and retrograde axonal transport in vivo3-L Prior exposure to tetrodotoxin (TI'X) which inhibits sodium influx into the cell, can prevent BTX-induced m e m b r a n e depolarization1,2, l0 and b l o c k a d e of axonal transport 14. This and more direct evidence 9 has lead to the conclusion that B T X acts by opening m e m b r a n e sodium channels. In a previous report we demonstrated necrosis of peripheral nerve axons after application of B T X in doses routinely used to block axonal transport and impulse conductionl2. The purpose of this communication is to further quantify this observation and d e m o n s t r a t e subsequent regeneration of the axon. Experiments were p e r f o r m e d on adult male Sprag u e - D a w l e y rats weighing from 322 to 460 g (average 370 g). U n d e r p e n t o b a r b i t a l anesthesia a single injection of 9 x 10-12 mol B T X in 1 #1 of 10% dextrose in saline was made into the peroneal nerve bilaterally in 6 animals. Two o t h e r animals were not in-
jected and served as controls. A total of 15 nerves were studied. Peroneal nerves were e x a m i n e d 7, 14 and 28 days post-BTX injection. A n i m a l s were anesthetized with methoxyflurane and perfused via the aortic root at a pressure of a p p r o x i m a t e l y 120 m m H g with fixative solution comprising 1% p a r a f o r m a l d e h y d e and 2% glutaraldehyde buffered to p H 7.2-7.4 with 0.1 M cacodylate buffer. A segment of peroneal nerve extending from a point at least 9 m m proximal to the injection site to the point of p e n e t r a t i o n o f the epimysium of the extensor digitorium brevis was removed. C o m p a r a b l e segments were taken from control animals. The nerves were post-fixed in osmium tetroxide, d e h y d r a t e d in graded alcohols and propylene oxide, and placed in Epon. The nerve segments were further cut into smaller segments for final e m b e d d i n g in E p o n blocks. Semi-thin (0.7 # m ) sections were cut from blocks with cross-sectional face, 8 m m from the distal end of the peroneal nerve segment. They were stained with Toluidine blue and examined under the light microscope. The total cross-sectional area of the nerve and the total n u m b e r of intact myelinated fibers present were measured from p h o t o m i c r o g r a p h s of known magnification.
* Present address: Department of Neuropathology, Albert Einstein College of Medicine, Bronx, NY 10461, U.S.A. ** To whom correspondence should be addressed at: Department of Pharmacology and Toxicology, Queen's University, Kingston, Ont. K7L 3N6, Canada. 0006-8993/83/$03.00 © 1983 Elsevier Science Publishers B.V.
247 Thin sections (50-70 nM) of random areas of the cross-sections were stained with uranyl acetate and lead citrate. Random electron micrographs of known magnification were examined, their area measured, and total numbers of intact unmyelinated fibers within the sample counted. Based on the total cross-sectional area obtained from light micrographs, the total number of intact unmyelinated fibers in the entire cross-section of the nerve was computed. All measurements and counts in this experiment were performed with a Carl Zeiss MOP-3 digital analyzer. Statistical comparisons between fiber counts in control nerves and BTX-treated nerves at each time interval were performed for myelinated and unmyelinated fibers by means of t-ratios and the results expressed as a P-value. P-values < 0.05 were considered statistically significant. Light and electron microscopic findings 7 days post-BTX injection were essentially the same as reported previouslyl2; namely, extensive necrosis of BTX-treated nerve fibers. Seven days post-BTX injection there was a statistically significant reduction in both myelinated and unmyelinated fibers (Table I). Fourteen days post-BTX injection there was still a significant reduction in myelinated fibers. Although the raw data suggests an increase in fibers without myelin at 14 days, it was not statistically significant. This apparent increase in unmyelinated nerve fibers is due to the presence of numerous 'about to be myelinated' regenerating large diameter axons (Fig. 1). Early remyelination, as seen in nerve
fiber regeneration, was evident at 14 days post-BTX injection (Fig. 1). At 28 days post-BTX injection the myelinated fibers remained significantly reduced in number compared to controls. The data suggested an overall increase in their numbers and an overall reduction in 'unmyelinated' fibers compared to 14 days. This is probably due to increasing numbers of axons being myelinated in the regenerative process. The significant reduction of intact myelinated and unmyelinated fibers after BTX injection can account for the prolonged electrophysiologic changes noted in the muscle membrane after injecting BTX into the motor nerve 7. Thus the muscle membrane has been found to undergo partial depolarization between days 2 and 14, with almost complete recovery by day 22. Miniature end-plate potentials were absent for the first 7 days and their frequency was significantly reduced even beyond 22 daysT. Extrajunctional sensitivity to acetylcholine was demonstrable up to 22 days. Muscle atrophy was also evident. All these features are reminiscent of reinnervation of denervated muscle after a nerve crush injuryll. We suspect such findings are the results of denervation due to extensive axonal necrosis produced by BTX. The relatively intact basal lamina and connective tissue scaffold of the nerve during such an insult would essentially resemble a nerve crush injury rather than a complete transection of the nervel3,15. Wallerian degeneration of the nerve after BTX injection has been described previouslytL The prolonged elevation of lysosomal
TABLE I Nerve fiber counts at various time intervals after batrachotoxin injection Total fiber count for cross-sectional area
Control Myelinated: Unmyelinated: 7 days post-BTX Myelinated: Unmyelinated: 14 days post-BTX Myelinated: Unmyelinated: 28 days post-BTX Myelinated: Unmyelinated:
Mean total f b r e count + standard deviation
P-value
2055, 2158, 1077,2139 2732, 2055, 6196, 7317
2107+ 49 4575 + 2575
258, 279, 569, 1571 1119, 984, 534, 1598
667 + 618 1059 + 437
P < 0.005 P < 0.05
531, 479, 198, 1485 7096, 11150, 4354, 4289
673 + 561 6724 + 3226
P < 0.005 P < 0.25*
1436, 1510, 1757 2382, 3369, 5698
1568 + 168 3817 + 1702
P < 0.025 P > 0.35*
* Not statisticallysignificant.
248
Fig. 1. Electron micrograph of rat peroneal nerve 14 days after injection of batrachotoxin. Extensive endoneurial edema, fragments of myelin debris and many intracellular lipid droplets (asterisks) indicate nerve fiber necrosis. Single large diameter axons which will soon be myelinated are enveloped by Schwann cell processes (small arrows). Other nerve fibers show varying degrees of remyelination (large arrows). Magnification: ×7000.
249 enzymes within nerves t r e a t e d with B T X is also consistent with these observations 6. In the present experiments, we have evidence of axonal regeneration following application of B T X to the nerve at a p o i n t approximately 8 m m from the region of the m o t o r end-plate. R e g e n e r a t i n g m o t o r axons should be approaching the e n d - p l a t e by 18 days given a regeneration rate of 2 m m / d a y for rat sciatic m o t o r fibersS. Such a time frame would correlate with the recovery of the postsynaptic m e m b r a n e p a r a m e t e r s previously noted above 7. Recovery of fast axonal transport occurs much earlier than the muscle m e m b r a n e events. A n o r m a l transport rate of 400 m m / d a y was found 7 days after B T X injection 7. The reason for the dissociation between the recovery of postsynaptic events and axonal transport is unclear. O n e possible explanation might be the survival of some axons. A s shown in Table I, even though there had been extensive destruction of axons by BTX, a few were still intact at 7 days. It is possible that the concentration of B T X m a y vary at different points throughout the cross-sectional area of the nerve. Thus, in fibers e x p o s e d to a relatively high concentration of toxin n u m e r o u s sodium channels are o p e n e d , with massive influx of sodium and
water, resulting in extensive intracellular e d e m a and axonal disruption. In those fibers exposed to lower concentrations of B T X , relatively fewer sodium channels would o p e n resulting in a t e m p o r a r y blockade of axonal transport, but not sufficient intracellular e d e m a to lead to axonal death. The previously d e m o n s t r a t e d reduction in total radioactively labeled material present in axons showing a return to n o r m a l transport rate 7 would be consistent with axons present in reduced numbers. Thus, we have quantitated the destruction of myelinated and unmyelinated axons following an injection of B T X into p e r i p h e r a l nerve. Such axonal necrosis could explain many of the postsynaptic neuromuscular events seen after such injections. T h e r e is morphologic evidence for nerve fiber r e g e n e r a t i o n by 14 days after B T X injection.
1 Albuquerque, E. X., The mode of action of batrachotoxin, Fed. Proc., 31 (1972) 1133-1138. 2 Albuquerque, E. X., Daly, J. W. and Witkop, B., Batrachotoxin: chemistry and pharmacology, Science, 172 (1971) 995-1002. 3 Boegman, R. J. and Albuquerque, E. X., Batrachotoxin blocks fast axonal transport in vivo, Fed. Proc., 37 (1978) 525. 4 Boegman, R. J., Deshpande, S. S. and Albuquerque, E. X., Consequences of axonal transport blockade induced by batrachotoxin on mammalian neuromuscular junction. I. Early pre- and post-synaptic changes, Brain Research, 187 (1980) 183-196. 5 Boegman, R. J. and Riopelle, R. J., Batrachotoxin blocks slow and retrograde axonal transport in vivo, Neurosci. Len., 18 (1980) 143--147. 6 Boegman, R. J. and Scarth, B., Neurotoxin-induced hydrolase activity in peripheral nerve, Neurosci. Len., 24 (1981) 261-265. 7 Deshpande, S. S., Boegman, R. J. and Albuquerque, E. X., Consequences of axonal transport blockade by batrachotoxin on mammalian neuromuscular junction. II. Late pre- and post-synaptic changes, Brain Research, 225 (1981) 115-129. 8 Guth, L., Regeneration in the mammalian peripheral her-
vous system, Physiol. Rev., 36 (1956) 441-478. 9 Huang, L.-Y.M., Moran, N. and Ehrenstein, G., Batrachotoxin modifies the gating kinetics of sodium channels in internally perfused neuroblastoma cells, Proc. nat. Acad. Sci. U.S.A., 79 (1982) 2082-2085. 10 Khodorov, B. I. and Revenko, S. V., Further analysis of the mechanisms of action of batrachotoxin on the membrane of myelinated nerve, Neurosci., 4 (1979) 1315-1330. 11 McArdle, J. J. and Albuquerque, E. X., A study of the reinnervation of fast and slow mammalian muscles, J. gen. Physiol., 61 (1973) 1-23. 12 Moore, G. R. W., Boegman, R. J., Robertson, D. M. and Riopelle, R. J., Batrachotoxin induced axonal necrosis in peripheral nerves, Brain Research, 207 (1981) 481-485. 13 Nathaniel, E. J. H. and Pease, D. C., Regenerative changes in rat dorsal roots following Wallerian degeneration, J. ultrastruct. Res., 9 (1963) 533-549. 14 Ochs, S. and Worth, R., Batrachotoxin block of fast axoplasmic transport in mammalian nerve fibers, Science, 187 (1975) 1087-1089. 15 Schroder, J. M., Degeneration and regeneration of myelinated nerve fibres in experimental neuropathies. In P. J. Dyck, P. K. Thomas and E. H. Lambert (Eds.), Peripheral Neuropathy, Saunders, Philadelphia, 1975, pp. 337-362.
W e thank Dr. E. X. A l b u q u e r q u e for supplying the BTX, Dr. W. D. L y m a n and Dr. D. Bryant for statistical advice, Mr. N. Meyers, Mr. D. M o r e and Mrs. M. A r n o l d for technical assistance and Mrs. J. LeSarge for typing the manuscript. S u p p o r t e d by the Canadian Muscular D y s t r o p h y Association.
Brain Research, 279 (1983) 246-249 Elsevier
Batrachotoxin induced axonal necrosis followed by regeneration G. R. W. MOORE*, D. M. ROBERTSON and R. J. BOEGMAN l.** Departments" of Pathology and 1Pharmacology and 1Toxicology, Queen's University, and Kingston General Hospital, Kingston, Ont. K7L 3N6 (Canada) (Accepted July 19th, 1983) Key words: nerve regeneration - - axonal necrosis - - Wallerian degeneration - - batrachotoxin
Batrachotoxin (BTX), when applied to peripheral nerve in concentrations sufficient to block impulse transmission and axonal transport, causes axonal necrosis. Quantitation of this phenomenon reveals reduction of both myelinated and unmyelinated fibers 7 days post-BTX injection. Evidence for regeneration correlates with the previously reported recovery of postsynaptic events.
Batrachotoxin (BTX) is a steroidal neurotoxin which depolarizes electrogenic m e m b r a n e s by increasing sodium permeabilityl,2,10. A t relatively high concentrations (8 x 10-9 t o 9 x 10- 9 mol) it irreversibly blocks fast axonai transport in vitro ~4. Low concentrations (10-12 tool) reversibly block both anterograde and retrograde axonal transport in vivo3-L Prior exposure to tetrodotoxin (TI'X) which inhibits sodium influx into the cell, can prevent BTX-induced m e m b r a n e depolarization1,2, l0 and b l o c k a d e of axonal transport 14. This and more direct evidence 9 has lead to the conclusion that B T X acts by opening m e m b r a n e sodium channels. In a previous report we demonstrated necrosis of peripheral nerve axons after application of B T X in doses routinely used to block axonal transport and impulse conductionl2. The purpose of this communication is to further quantify this observation and d e m o n s t r a t e subsequent regeneration of the axon. Experiments were p e r f o r m e d on adult male Sprag u e - D a w l e y rats weighing from 322 to 460 g (average 370 g). U n d e r p e n t o b a r b i t a l anesthesia a single injection of 9 x 10-12 mol B T X in 1 #1 of 10% dextrose in saline was made into the peroneal nerve bilaterally in 6 animals. Two o t h e r animals were not in-
jected and served as controls. A total of 15 nerves were studied. Peroneal nerves were e x a m i n e d 7, 14 and 28 days post-BTX injection. A n i m a l s were anesthetized with methoxyflurane and perfused via the aortic root at a pressure of a p p r o x i m a t e l y 120 m m H g with fixative solution comprising 1% p a r a f o r m a l d e h y d e and 2% glutaraldehyde buffered to p H 7.2-7.4 with 0.1 M cacodylate buffer. A segment of peroneal nerve extending from a point at least 9 m m proximal to the injection site to the point of p e n e t r a t i o n o f the epimysium of the extensor digitorium brevis was removed. C o m p a r a b l e segments were taken from control animals. The nerves were post-fixed in osmium tetroxide, d e h y d r a t e d in graded alcohols and propylene oxide, and placed in Epon. The nerve segments were further cut into smaller segments for final e m b e d d i n g in E p o n blocks. Semi-thin (0.7 # m ) sections were cut from blocks with cross-sectional face, 8 m m from the distal end of the peroneal nerve segment. They were stained with Toluidine blue and examined under the light microscope. The total cross-sectional area of the nerve and the total n u m b e r of intact myelinated fibers present were measured from p h o t o m i c r o g r a p h s of known magnification.
* Present address: Department of Neuropathology, Albert Einstein College of Medicine, Bronx, NY 10461, U.S.A. ** To whom correspondence should be addressed at: Department of Pharmacology and Toxicology, Queen's University, Kingston, Ont. K7L 3N6, Canada. 0006-8993/83/$03.00 © 1983 Elsevier Science Publishers B.V.
247 Thin sections (50-70 nM) of random areas of the cross-sections were stained with uranyl acetate and lead citrate. Random electron micrographs of known magnification were examined, their area measured, and total numbers of intact unmyelinated fibers within the sample counted. Based on the total cross-sectional area obtained from light micrographs, the total number of intact unmyelinated fibers in the entire cross-section of the nerve was computed. All measurements and counts in this experiment were performed with a Carl Zeiss MOP-3 digital analyzer. Statistical comparisons between fiber counts in control nerves and BTX-treated nerves at each time interval were performed for myelinated and unmyelinated fibers by means of t-ratios and the results expressed as a P-value. P-values < 0.05 were considered statistically significant. Light and electron microscopic findings 7 days post-BTX injection were essentially the same as reported previouslyl2; namely, extensive necrosis of BTX-treated nerve fibers. Seven days post-BTX injection there was a statistically significant reduction in both myelinated and unmyelinated fibers (Table I). Fourteen days post-BTX injection there was still a significant reduction in myelinated fibers. Although the raw data suggests an increase in fibers without myelin at 14 days, it was not statistically significant. This apparent increase in unmyelinated nerve fibers is due to the presence of numerous 'about to be myelinated' regenerating large diameter axons (Fig. 1). Early remyelination, as seen in nerve
fiber regeneration, was evident at 14 days post-BTX injection (Fig. 1). At 28 days post-BTX injection the myelinated fibers remained significantly reduced in number compared to controls. The data suggested an overall increase in their numbers and an overall reduction in 'unmyelinated' fibers compared to 14 days. This is probably due to increasing numbers of axons being myelinated in the regenerative process. The significant reduction of intact myelinated and unmyelinated fibers after BTX injection can account for the prolonged electrophysiologic changes noted in the muscle membrane after injecting BTX into the motor nerve 7. Thus the muscle membrane has been found to undergo partial depolarization between days 2 and 14, with almost complete recovery by day 22. Miniature end-plate potentials were absent for the first 7 days and their frequency was significantly reduced even beyond 22 daysT. Extrajunctional sensitivity to acetylcholine was demonstrable up to 22 days. Muscle atrophy was also evident. All these features are reminiscent of reinnervation of denervated muscle after a nerve crush injuryll. We suspect such findings are the results of denervation due to extensive axonal necrosis produced by BTX. The relatively intact basal lamina and connective tissue scaffold of the nerve during such an insult would essentially resemble a nerve crush injury rather than a complete transection of the nervel3,15. Wallerian degeneration of the nerve after BTX injection has been described previouslytL The prolonged elevation of lysosomal
TABLE I Nerve fiber counts at various time intervals after batrachotoxin injection Total fiber count for cross-sectional area
Control Myelinated: Unmyelinated: 7 days post-BTX Myelinated: Unmyelinated: 14 days post-BTX Myelinated: Unmyelinated: 28 days post-BTX Myelinated: Unmyelinated:
Mean total f b r e count + standard deviation
P-value
2055, 2158, 1077,2139 2732, 2055, 6196, 7317
2107+ 49 4575 + 2575
258, 279, 569, 1571 1119, 984, 534, 1598
667 + 618 1059 + 437
P < 0.005 P < 0.05
531, 479, 198, 1485 7096, 11150, 4354, 4289
673 + 561 6724 + 3226
P < 0.005 P < 0.25*
1436, 1510, 1757 2382, 3369, 5698
1568 + 168 3817 + 1702
P < 0.025 P > 0.35*
* Not statisticallysignificant.
248
Fig. 1. Electron micrograph of rat peroneal nerve 14 days after injection of batrachotoxin. Extensive endoneurial edema, fragments of myelin debris and many intracellular lipid droplets (asterisks) indicate nerve fiber necrosis. Single large diameter axons which will soon be myelinated are enveloped by Schwann cell processes (small arrows). Other nerve fibers show varying degrees of remyelination (large arrows). Magnification: ×7000.
249 enzymes within nerves t r e a t e d with B T X is also consistent with these observations 6. In the present experiments, we have evidence of axonal regeneration following application of B T X to the nerve at a p o i n t approximately 8 m m from the region of the m o t o r end-plate. R e g e n e r a t i n g m o t o r axons should be approaching the e n d - p l a t e by 18 days given a regeneration rate of 2 m m / d a y for rat sciatic m o t o r fibersS. Such a time frame would correlate with the recovery of the postsynaptic m e m b r a n e p a r a m e t e r s previously noted above 7. Recovery of fast axonal transport occurs much earlier than the muscle m e m b r a n e events. A n o r m a l transport rate of 400 m m / d a y was found 7 days after B T X injection 7. The reason for the dissociation between the recovery of postsynaptic events and axonal transport is unclear. O n e possible explanation might be the survival of some axons. A s shown in Table I, even though there had been extensive destruction of axons by BTX, a few were still intact at 7 days. It is possible that the concentration of B T X m a y vary at different points throughout the cross-sectional area of the nerve. Thus, in fibers e x p o s e d to a relatively high concentration of toxin n u m e r o u s sodium channels are o p e n e d , with massive influx of sodium and
water, resulting in extensive intracellular e d e m a and axonal disruption. In those fibers exposed to lower concentrations of B T X , relatively fewer sodium channels would o p e n resulting in a t e m p o r a r y blockade of axonal transport, but not sufficient intracellular e d e m a to lead to axonal death. The previously d e m o n s t r a t e d reduction in total radioactively labeled material present in axons showing a return to n o r m a l transport rate 7 would be consistent with axons present in reduced numbers. Thus, we have quantitated the destruction of myelinated and unmyelinated axons following an injection of B T X into p e r i p h e r a l nerve. Such axonal necrosis could explain many of the postsynaptic neuromuscular events seen after such injections. T h e r e is morphologic evidence for nerve fiber r e g e n e r a t i o n by 14 days after B T X injection.
1 Albuquerque, E. X., The mode of action of batrachotoxin, Fed. Proc., 31 (1972) 1133-1138. 2 Albuquerque, E. X., Daly, J. W. and Witkop, B., Batrachotoxin: chemistry and pharmacology, Science, 172 (1971) 995-1002. 3 Boegman, R. J. and Albuquerque, E. X., Batrachotoxin blocks fast axonal transport in vivo, Fed. Proc., 37 (1978) 525. 4 Boegman, R. J., Deshpande, S. S. and Albuquerque, E. X., Consequences of axonal transport blockade induced by batrachotoxin on mammalian neuromuscular junction. I. Early pre- and post-synaptic changes, Brain Research, 187 (1980) 183-196. 5 Boegman, R. J. and Riopelle, R. J., Batrachotoxin blocks slow and retrograde axonal transport in vivo, Neurosci. Len., 18 (1980) 143--147. 6 Boegman, R. J. and Scarth, B., Neurotoxin-induced hydrolase activity in peripheral nerve, Neurosci. Len., 24 (1981) 261-265. 7 Deshpande, S. S., Boegman, R. J. and Albuquerque, E. X., Consequences of axonal transport blockade by batrachotoxin on mammalian neuromuscular junction. II. Late pre- and post-synaptic changes, Brain Research, 225 (1981) 115-129. 8 Guth, L., Regeneration in the mammalian peripheral her-
vous system, Physiol. Rev., 36 (1956) 441-478. 9 Huang, L.-Y.M., Moran, N. and Ehrenstein, G., Batrachotoxin modifies the gating kinetics of sodium channels in internally perfused neuroblastoma cells, Proc. nat. Acad. Sci. U.S.A., 79 (1982) 2082-2085. 10 Khodorov, B. I. and Revenko, S. V., Further analysis of the mechanisms of action of batrachotoxin on the membrane of myelinated nerve, Neurosci., 4 (1979) 1315-1330. 11 McArdle, J. J. and Albuquerque, E. X., A study of the reinnervation of fast and slow mammalian muscles, J. gen. Physiol., 61 (1973) 1-23. 12 Moore, G. R. W., Boegman, R. J., Robertson, D. M. and Riopelle, R. J., Batrachotoxin induced axonal necrosis in peripheral nerves, Brain Research, 207 (1981) 481-485. 13 Nathaniel, E. J. H. and Pease, D. C., Regenerative changes in rat dorsal roots following Wallerian degeneration, J. ultrastruct. Res., 9 (1963) 533-549. 14 Ochs, S. and Worth, R., Batrachotoxin block of fast axoplasmic transport in mammalian nerve fibers, Science, 187 (1975) 1087-1089. 15 Schroder, J. M., Degeneration and regeneration of myelinated nerve fibres in experimental neuropathies. In P. J. Dyck, P. K. Thomas and E. H. Lambert (Eds.), Peripheral Neuropathy, Saunders, Philadelphia, 1975, pp. 337-362.
W e thank Dr. E. X. A l b u q u e r q u e for supplying the BTX, Dr. W. D. L y m a n and Dr. D. Bryant for statistical advice, Mr. N. Meyers, Mr. D. M o r e and Mrs. M. A r n o l d for technical assistance and Mrs. J. LeSarge for typing the manuscript. S u p p o r t e d by the Canadian Muscular D y s t r o p h y Association.