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Asymmetry of protein transport in two branches of bifurcating axons
354
Brain Research, 139 (1978) 354-358 © Elsevier/North-Holland Biomedical Press
Asymmetry of protein transport in two branches of bifurcating axons
Y. KOM1YA and M. KUROKAWA Department of Biochemistry, Institute of Brain Research, Tokyo University Faculty of Medicine, Hongo, Tokyo 113 (Japan)
(Accepted August 8th, 1977)
Morphological and functional differences between the central and peripheral branches of the dorsal root ganglion (DRG) cell axon have received a continuous interest because of the repeated demonstration that D R G cells show profuse chromatolytic changes after section of their peripheral axons, but little if any reaction as seen by light microscopy after section of their central axons4, ~. Evidence has accumulated to indicate that the amount of rapidly migrating proteins within the central branch of the D R G cell axon is obviously less as compared with the peripheral one; flow rate of the fast transport proved identical between the central and peripheral branches2,13,18. However, no information has been available with respect to the slow transport that constitutes approximately 70 ~12 of the transport within the axon. We describe a marked asymmetry in the rate of slow transport of labelled proteins between the central and peripheral branches of the D R G cell axons. e-[U-14]leucine was injected into the D R G (LD of the adult rat and, at appropriate time intervals, distribution of radioactivity in the sciatic nerve (peripheral axon) and dorsal root (central axon) was determined. In the peripheral branch, a peak of radioactivity appeared 2 weeks after the isotope injection, and this shifted downwards as the injection-sacrifice interval increased (Fig. 1). Plot of the distance between the peak and ganglion as a function of the injection-sacrifice interval indicates a flow rate of 1.0 mm/ day (Fig. 2), which reasonably agrees with the value of 1.0-1.2 ram/day 12 estimated in a similar way to ours, and of 0.8 mm/day 6 estimated by means of radioautography in the sciatic nerve of the adult rat. In the central branch, however, the peak became apparent only after 4 weeks (Fig. 1), and this moved somatofugally at a definitely lower rate, i.e. 0.4 ram/day (Fig. 2). In the peripheral branch, the peak became broader and appeared to split into two subpeaks as the injection-sacrifice interval increased (Fig. 1), the observation being in common with that by Hoffman and Lasek 11. In the central branch, the broadening of the peak was less apparent, presumably because of its slower rate of migration. The amount of centrally directed transport is similar to or slightly larger, compared with the peripherally directed one, as evidenced by the central/peripheral ratio of |. 17 (Table I). In sharp contrast with the slow transport, rate of the fast transport was found
355
1°°I t
\ \ 2
\
O l Of< \
o
",,,
0
10 20 30 40 50 60 Distance from the ganglion (ram)
70
Fig. 1. Flow profiles of slow transport of labelled proteins in the central and peripheral axons of the rat dorsal root ganglion (DRG) cells. Adult albino rats (Wistar strain) weighing 250-300 g were used. Under ether anaesthesia, D R G (L~) was exposed by removing a portion of the spine, and 1.6 #Ci (in 0.4/~1 of saline) of L-[U-14C]leucine was carefully injected into the ganglion in two divided doses during a period of 10 min. After weeks indicated, animals were killed by decapitation, and the sciatic nerve, L~ ganglion, dorsal root together with a portion of the spinal cord (hemi-cord) were dissected out, placed on an ice-cold plastic plate, and cut into consecutive 3 mm segments. Each segment was washed 3 times each with 0.3 ml of ice-cold 5 ~ (w/v) trichloroacetic acid, 2 times each with 0.3 ml of absolute ethanol, then 3 times each with 0.3 ml of n-hexane, dried, dissolved in 0.2 ml of Soluene 350 (Packard Instrument, Downers Grove, Ill.) by heating at 50 °C for 2 h, and radioactivities were determined as described 1, with a counting efficiency of 85 %. Contralateral root and nerve used as control showed little radioactivity, less than 10 cpm per segment above the background level. Normalized value (vertical axis) denotes (radioactivity per 3 mm segment) × 100/(radioactivity of the ganglion), and this was plotted semilogarithmically as a function of the distance centrally and peripherally from the ganglion. 0 , dorsal root and the cord; ©, sciatic nerve. Injection-sacrifice interval was 2, 4 and 6 weeks in A, B and C, respectively. Vertical arrow indicates the site of entry of the dorsal root into the cord. A peak of radioactivity seen at the entry is most probably due to the retention of the rapidly migrating materials in axons and/or in axon collaterals terminating at this level of the cord, as suggested by Ochs TM. e q u a l b e t w e e n t h e c e n t r a l a n d p e r i p h e r a l b r a n c h e s (350 m m / d a y
each), a n d t h e
a m o u n t o f p e r i p h e r a l f l o w was 3-fold t h a t o f the c e n t r a l flow ( T a b l e I), in essential a g r e e m e n t w i t h t h e p r e v i o u s o b s e r v a t i o n s in o t h e r species o f animals2,1a, is. T h e r a t e o f s l o w a x o p l a s m i c t r a n s p o r t is k n o w n t o be g r e a t e r in D R G cells o f g r o w i n g a n i m a l s t h a n in a d u l t animals6,14. A l s o , t h e r a t e o f slow t r a n s p o r t was f o u n d to be a c c e l e r a t e d in r e g e n e r a t i n g a x o n s 8, i n d i c a t i n g a p o s s i b l e i n v o l v e m e n t o f this
356 ~80 g
60 ~40
Eo
20 m
2
4
6
;
1'0
Injection-sacrificeinterval (weeks) Fig. 2. Rates of the slow protein transport in the central and peripheral axons of the rat dorsal root ganglion cells. Flow profiles of labelled proteins in the dorsal root and the sciatic nerve were determined as in Fig. 1, and the distance between the peak and ganglion was plotted against the injection-sacrifice interval. O, dorsal root and the cord ; 63, sciatic nerve.
transport process in the growth and maintenance of axons. The present demonstration of a lower rate of the slow transport in the central branch may be of some interest in the light of observations that the regeneration rate of the dorsal root in the adult rat is 0.43 mm/day TM, in contrast with a faster rate (2.4-3.3 mm/day) reported for regeneration of the rat sciatic nerve 15. From small cells of the D R G emerge unmyelinated axons which are much finer in the central branch than in the peripheral one, and which may constitute 60-70 % of the total number of axons 9,t°,19,2°. This and our present observations raise the possibility that the rate of slow transport is lower in thinner axons than in thicker ones. Studying with the optic axons of flatfish that exhibit a marked right-left difference in TABLE I
Comparison o f slow and fast transports between the central and peripheral axons o f the rat dorsal root ganglion cells In the study of fast transport, 0.8/~Ci (0.2/d) of L-[U-~4C]leucine was injected into the L5 ganglion, and the flow rate was determined by plotting the distance between the radioactivity front and the ganglion against the injection-sacrifice interval, estimated at 10 time points between 1 and 6 hours. Ratio of radioactivity in the 3 mm peak segment in the central axon to that in the peripheral axon provided an estimate of the relative flow amount ; in this estimation, radioactivities were determined 6 weeks and 3 hours after the isotope injection for slow and fast transports, respectively. Values are means 4- S.E., with number of determinations in parentheses. Relative amount was also determined on the basis of radioactivities measured in three 3-mm segments from the peak region, which gave essentially identical results.
Slow transport Peripheral Central Fast transport Peripheral Central
Flow rate (mm/day)
Relative flow amount (central/peripheral)
1.0 0.4
1 1.17 ± 0.04 (12)
350 350
1 0.37 d_ 0.04 (12)
357 their length, M u r r a y 17 has obtained evidence suggesting that the rate o f slow (but not fast) transport is influenced by the length o f axons, being lower in shorter ones. This may also hold to explain the central-peripheral difference in the rate o f slow transport observed in our present experiment, although the matter is complicated by the lack o f any quantitative data concerning the length of axons of the D R G cells, particularly that o f centrally directed ones. With respect to the fast transport, the flow rate is considered to be independent of the diameter and length of axons is. In the case o f slow transport, however, the possibility remains that the rate is related to the diameter and/or length of axons, particularly in view of the observation that the great majority (95 ~ or more) of slowly moving labelled proteins is retained in the axons 7. The density of microtubules was reported to be significantly lower in the central branch than in the peripheral one in the rat 2a and frog el D R G cells, leading to a suggestion that the a m o u n t of fast transport may be related to the microtubule density in axonsa, 22. In slow transport, however, the a m o u n t o f centrally transported proteins is even larger, c o m p a r e d with peripherally directed ones (Table I), excluding the possibility o f direct correlation between the flow a m o u n t and the microtubule density as seen by electron microscopy. This work was supported by Research G r a n t 110702 f r o m the Ministry o f Education, Japan, and by a G r a n t f r o m the Mitsubishi Foundation.
1 Abe, T., Haga, T. and Kurokawa, M., Rapid transport of phosphatidylcholine occurring simul taneously with protein transport in the frog sciatic nerve, Biochem. J., 136 (1973) 731-740. 2 Anderson, L. E. and McClure, W. O., Differential transport of protein in axons: comparison between the sciatic nerve and dorsal columns of cats, Proc. nat. Acad. Sci. (Wash.), 70 (1973) 1521-1525. 3 Barker, J. L., Neale, J. H. and Gainer, H., Rapidly transported proteins in sensory, motor and sympathetic nerves of the isolated frog nervous system, Brain Research, 105 (1976) 497-515. 4 Carmel, P. W. and Stein, B. M., Cell changes in sensory ganglia following proximal and distal nerve section in the monkey, J. comp. Neurol., 135 (1969) 145-166. 5 Cragg, B. G., What is the signal for chromatolysis? Brain Research, 23 (1970) 1-21. 6 Droz, B., Fate of newly synthesized proteins in neurons. In C. P. Leblond and K. B. Warren (Eds.) The Use of Radioautography in Investigating Protein Synthesis, Academic Press, New York, 1965, pp. 159-174. 7 Droz, B., Koenig, H. L. and Di Giamberardino, L., Axonal migration of protein and glycoprotein to nerve endings. I. Radioautographic analysis of the renewal of protein in nerve endings of chicken ciliary ganglion after intracerebral injection of [ZH]lysine, Brain Research, 60 (1973) 93-127. 8 Grafstein, B. and Murray, M., Transport of protein in goldfish optic nerve during regeneration, Exp. Neurol., 25 (1969) 494-508. 9 Ha, H., Axonal bifurcation in the dorsal root ganglion of the cat : a light and electron microscopic study, J. comp. Neurol., 140 (1970) 227-240. 10 Hatai, S., Number and size of the spinal ganglion cells and dorsal root fibers in the white rat at different ages, J. comp. Neurol., 12 (1902) 107-124. 11 Hoffman, P. N. and Lasek, R. J., The slow component of axonal transport: identification of major structural polypeptides of the axon and their generality among mammalian neurons, J. Cell Biol., 66 (1975) 351-366. 12 Karlsson, J.-O. and Sjbstrand, J., Synthesis, migration and turnover of protein in retinal ganglion cells, J. Neurochem., 18 (1971) 749-767. 13 Lasek, R., Axoplasmic transport in cat dorsal root ganglion cells: as studied with [3Hl-L-leucine, Brain Research, 7 (1968) 360--377.
358 14 Lasek, R. J., Axonal transport of proteins ~n dorsal root ganglion cells of the growing cat: a comparison of growing and mature neurons, Brain Research, 20 (1970) 121-126. 15 Lubifiska, L., Axoplasmic streaming in regenerating and in normal nerve fibres : In M. Singer and J. P. Schad6 (Eds.), Mechanism of Neural Regeneration, Progress in Brain Research, Vol. 13, Elsevier, Amsterdam, 1964, pp. 1-66. 16 Moyer, E. K., Kimmel, D. L. and Winborne, L. W., Regeneration of sensory spinal nerve roots in young and in senile rats, J. comp. NeuroL, 98 (1953) 283-308. 17 Murray, M., Axonal transport in the asymmetric optic axons of flatfish, Exp. Neurol., 42 (1974) 636-646. 18 Ochs, S., Rate of fast axoplasmic transport in mammalian nerve fibres, J. Physiol. (Lond.), 227 (1972) 627-645. 19 Ranson, S. W., Non-myelinated nerve fibers in the spinal nerves, Amer. J. Anat., 12 (1911) 67-87. ~-0 Ranson, S. W. and Davenport, H. K., Sensory unmyelinated fibers in the spinal nerves, Amer. J. Anat., 48 (1931) 331-353. 21 Smith, R. S., Microtubule and neurofilament densities in amphibian spinal root nerve fibers: relationship to axoplasmic transport, Canad. J. physiol. Pharmacol., 51 (1973) 798-806. 22 Zenker, W. and H6gl, E., The prebifurcation section of the axon of the rat spinal ganglion cell, Cell Tiss. Res., 165 (1976) 345-363. 23 Zenker, W., Mayr, R. and Gruber, H., Neurotubules: different densities in peripheral motor and sensory nerve fibres, Experientia (Basel), 31 (1975) 318-320.
Brain Research, 139 (1978) 354-358 © Elsevier/North-Holland Biomedical Press
Asymmetry of protein transport in two branches of bifurcating axons
Y. KOM1YA and M. KUROKAWA Department of Biochemistry, Institute of Brain Research, Tokyo University Faculty of Medicine, Hongo, Tokyo 113 (Japan)
(Accepted August 8th, 1977)
Morphological and functional differences between the central and peripheral branches of the dorsal root ganglion (DRG) cell axon have received a continuous interest because of the repeated demonstration that D R G cells show profuse chromatolytic changes after section of their peripheral axons, but little if any reaction as seen by light microscopy after section of their central axons4, ~. Evidence has accumulated to indicate that the amount of rapidly migrating proteins within the central branch of the D R G cell axon is obviously less as compared with the peripheral one; flow rate of the fast transport proved identical between the central and peripheral branches2,13,18. However, no information has been available with respect to the slow transport that constitutes approximately 70 ~12 of the transport within the axon. We describe a marked asymmetry in the rate of slow transport of labelled proteins between the central and peripheral branches of the D R G cell axons. e-[U-14]leucine was injected into the D R G (LD of the adult rat and, at appropriate time intervals, distribution of radioactivity in the sciatic nerve (peripheral axon) and dorsal root (central axon) was determined. In the peripheral branch, a peak of radioactivity appeared 2 weeks after the isotope injection, and this shifted downwards as the injection-sacrifice interval increased (Fig. 1). Plot of the distance between the peak and ganglion as a function of the injection-sacrifice interval indicates a flow rate of 1.0 mm/ day (Fig. 2), which reasonably agrees with the value of 1.0-1.2 ram/day 12 estimated in a similar way to ours, and of 0.8 mm/day 6 estimated by means of radioautography in the sciatic nerve of the adult rat. In the central branch, however, the peak became apparent only after 4 weeks (Fig. 1), and this moved somatofugally at a definitely lower rate, i.e. 0.4 ram/day (Fig. 2). In the peripheral branch, the peak became broader and appeared to split into two subpeaks as the injection-sacrifice interval increased (Fig. 1), the observation being in common with that by Hoffman and Lasek 11. In the central branch, the broadening of the peak was less apparent, presumably because of its slower rate of migration. The amount of centrally directed transport is similar to or slightly larger, compared with the peripherally directed one, as evidenced by the central/peripheral ratio of |. 17 (Table I). In sharp contrast with the slow transport, rate of the fast transport was found
355
1°°I t
\ \ 2
\
O l Of< \
o
",,,
0
10 20 30 40 50 60 Distance from the ganglion (ram)
70
Fig. 1. Flow profiles of slow transport of labelled proteins in the central and peripheral axons of the rat dorsal root ganglion (DRG) cells. Adult albino rats (Wistar strain) weighing 250-300 g were used. Under ether anaesthesia, D R G (L~) was exposed by removing a portion of the spine, and 1.6 #Ci (in 0.4/~1 of saline) of L-[U-14C]leucine was carefully injected into the ganglion in two divided doses during a period of 10 min. After weeks indicated, animals were killed by decapitation, and the sciatic nerve, L~ ganglion, dorsal root together with a portion of the spinal cord (hemi-cord) were dissected out, placed on an ice-cold plastic plate, and cut into consecutive 3 mm segments. Each segment was washed 3 times each with 0.3 ml of ice-cold 5 ~ (w/v) trichloroacetic acid, 2 times each with 0.3 ml of absolute ethanol, then 3 times each with 0.3 ml of n-hexane, dried, dissolved in 0.2 ml of Soluene 350 (Packard Instrument, Downers Grove, Ill.) by heating at 50 °C for 2 h, and radioactivities were determined as described 1, with a counting efficiency of 85 %. Contralateral root and nerve used as control showed little radioactivity, less than 10 cpm per segment above the background level. Normalized value (vertical axis) denotes (radioactivity per 3 mm segment) × 100/(radioactivity of the ganglion), and this was plotted semilogarithmically as a function of the distance centrally and peripherally from the ganglion. 0 , dorsal root and the cord; ©, sciatic nerve. Injection-sacrifice interval was 2, 4 and 6 weeks in A, B and C, respectively. Vertical arrow indicates the site of entry of the dorsal root into the cord. A peak of radioactivity seen at the entry is most probably due to the retention of the rapidly migrating materials in axons and/or in axon collaterals terminating at this level of the cord, as suggested by Ochs TM. e q u a l b e t w e e n t h e c e n t r a l a n d p e r i p h e r a l b r a n c h e s (350 m m / d a y
each), a n d t h e
a m o u n t o f p e r i p h e r a l f l o w was 3-fold t h a t o f the c e n t r a l flow ( T a b l e I), in essential a g r e e m e n t w i t h t h e p r e v i o u s o b s e r v a t i o n s in o t h e r species o f animals2,1a, is. T h e r a t e o f s l o w a x o p l a s m i c t r a n s p o r t is k n o w n t o be g r e a t e r in D R G cells o f g r o w i n g a n i m a l s t h a n in a d u l t animals6,14. A l s o , t h e r a t e o f slow t r a n s p o r t was f o u n d to be a c c e l e r a t e d in r e g e n e r a t i n g a x o n s 8, i n d i c a t i n g a p o s s i b l e i n v o l v e m e n t o f this
356 ~80 g
60 ~40
Eo
20 m
2
4
6
;
1'0
Injection-sacrificeinterval (weeks) Fig. 2. Rates of the slow protein transport in the central and peripheral axons of the rat dorsal root ganglion cells. Flow profiles of labelled proteins in the dorsal root and the sciatic nerve were determined as in Fig. 1, and the distance between the peak and ganglion was plotted against the injection-sacrifice interval. O, dorsal root and the cord ; 63, sciatic nerve.
transport process in the growth and maintenance of axons. The present demonstration of a lower rate of the slow transport in the central branch may be of some interest in the light of observations that the regeneration rate of the dorsal root in the adult rat is 0.43 mm/day TM, in contrast with a faster rate (2.4-3.3 mm/day) reported for regeneration of the rat sciatic nerve 15. From small cells of the D R G emerge unmyelinated axons which are much finer in the central branch than in the peripheral one, and which may constitute 60-70 % of the total number of axons 9,t°,19,2°. This and our present observations raise the possibility that the rate of slow transport is lower in thinner axons than in thicker ones. Studying with the optic axons of flatfish that exhibit a marked right-left difference in TABLE I
Comparison o f slow and fast transports between the central and peripheral axons o f the rat dorsal root ganglion cells In the study of fast transport, 0.8/~Ci (0.2/d) of L-[U-~4C]leucine was injected into the L5 ganglion, and the flow rate was determined by plotting the distance between the radioactivity front and the ganglion against the injection-sacrifice interval, estimated at 10 time points between 1 and 6 hours. Ratio of radioactivity in the 3 mm peak segment in the central axon to that in the peripheral axon provided an estimate of the relative flow amount ; in this estimation, radioactivities were determined 6 weeks and 3 hours after the isotope injection for slow and fast transports, respectively. Values are means 4- S.E., with number of determinations in parentheses. Relative amount was also determined on the basis of radioactivities measured in three 3-mm segments from the peak region, which gave essentially identical results.
Slow transport Peripheral Central Fast transport Peripheral Central
Flow rate (mm/day)
Relative flow amount (central/peripheral)
1.0 0.4
1 1.17 ± 0.04 (12)
350 350
1 0.37 d_ 0.04 (12)
357 their length, M u r r a y 17 has obtained evidence suggesting that the rate o f slow (but not fast) transport is influenced by the length o f axons, being lower in shorter ones. This may also hold to explain the central-peripheral difference in the rate o f slow transport observed in our present experiment, although the matter is complicated by the lack o f any quantitative data concerning the length of axons of the D R G cells, particularly that o f centrally directed ones. With respect to the fast transport, the flow rate is considered to be independent of the diameter and length of axons is. In the case o f slow transport, however, the possibility remains that the rate is related to the diameter and/or length of axons, particularly in view of the observation that the great majority (95 ~ or more) of slowly moving labelled proteins is retained in the axons 7. The density of microtubules was reported to be significantly lower in the central branch than in the peripheral one in the rat 2a and frog el D R G cells, leading to a suggestion that the a m o u n t of fast transport may be related to the microtubule density in axonsa, 22. In slow transport, however, the a m o u n t o f centrally transported proteins is even larger, c o m p a r e d with peripherally directed ones (Table I), excluding the possibility o f direct correlation between the flow a m o u n t and the microtubule density as seen by electron microscopy. This work was supported by Research G r a n t 110702 f r o m the Ministry o f Education, Japan, and by a G r a n t f r o m the Mitsubishi Foundation.
1 Abe, T., Haga, T. and Kurokawa, M., Rapid transport of phosphatidylcholine occurring simul taneously with protein transport in the frog sciatic nerve, Biochem. J., 136 (1973) 731-740. 2 Anderson, L. E. and McClure, W. O., Differential transport of protein in axons: comparison between the sciatic nerve and dorsal columns of cats, Proc. nat. Acad. Sci. (Wash.), 70 (1973) 1521-1525. 3 Barker, J. L., Neale, J. H. and Gainer, H., Rapidly transported proteins in sensory, motor and sympathetic nerves of the isolated frog nervous system, Brain Research, 105 (1976) 497-515. 4 Carmel, P. W. and Stein, B. M., Cell changes in sensory ganglia following proximal and distal nerve section in the monkey, J. comp. Neurol., 135 (1969) 145-166. 5 Cragg, B. G., What is the signal for chromatolysis? Brain Research, 23 (1970) 1-21. 6 Droz, B., Fate of newly synthesized proteins in neurons. In C. P. Leblond and K. B. Warren (Eds.) The Use of Radioautography in Investigating Protein Synthesis, Academic Press, New York, 1965, pp. 159-174. 7 Droz, B., Koenig, H. L. and Di Giamberardino, L., Axonal migration of protein and glycoprotein to nerve endings. I. Radioautographic analysis of the renewal of protein in nerve endings of chicken ciliary ganglion after intracerebral injection of [ZH]lysine, Brain Research, 60 (1973) 93-127. 8 Grafstein, B. and Murray, M., Transport of protein in goldfish optic nerve during regeneration, Exp. Neurol., 25 (1969) 494-508. 9 Ha, H., Axonal bifurcation in the dorsal root ganglion of the cat : a light and electron microscopic study, J. comp. Neurol., 140 (1970) 227-240. 10 Hatai, S., Number and size of the spinal ganglion cells and dorsal root fibers in the white rat at different ages, J. comp. Neurol., 12 (1902) 107-124. 11 Hoffman, P. N. and Lasek, R. J., The slow component of axonal transport: identification of major structural polypeptides of the axon and their generality among mammalian neurons, J. Cell Biol., 66 (1975) 351-366. 12 Karlsson, J.-O. and Sjbstrand, J., Synthesis, migration and turnover of protein in retinal ganglion cells, J. Neurochem., 18 (1971) 749-767. 13 Lasek, R., Axoplasmic transport in cat dorsal root ganglion cells: as studied with [3Hl-L-leucine, Brain Research, 7 (1968) 360--377.
358 14 Lasek, R. J., Axonal transport of proteins ~n dorsal root ganglion cells of the growing cat: a comparison of growing and mature neurons, Brain Research, 20 (1970) 121-126. 15 Lubifiska, L., Axoplasmic streaming in regenerating and in normal nerve fibres : In M. Singer and J. P. Schad6 (Eds.), Mechanism of Neural Regeneration, Progress in Brain Research, Vol. 13, Elsevier, Amsterdam, 1964, pp. 1-66. 16 Moyer, E. K., Kimmel, D. L. and Winborne, L. W., Regeneration of sensory spinal nerve roots in young and in senile rats, J. comp. NeuroL, 98 (1953) 283-308. 17 Murray, M., Axonal transport in the asymmetric optic axons of flatfish, Exp. Neurol., 42 (1974) 636-646. 18 Ochs, S., Rate of fast axoplasmic transport in mammalian nerve fibres, J. Physiol. (Lond.), 227 (1972) 627-645. 19 Ranson, S. W., Non-myelinated nerve fibers in the spinal nerves, Amer. J. Anat., 12 (1911) 67-87. ~-0 Ranson, S. W. and Davenport, H. K., Sensory unmyelinated fibers in the spinal nerves, Amer. J. Anat., 48 (1931) 331-353. 21 Smith, R. S., Microtubule and neurofilament densities in amphibian spinal root nerve fibers: relationship to axoplasmic transport, Canad. J. physiol. Pharmacol., 51 (1973) 798-806. 22 Zenker, W. and H6gl, E., The prebifurcation section of the axon of the rat spinal ganglion cell, Cell Tiss. Res., 165 (1976) 345-363. 23 Zenker, W., Mayr, R. and Gruber, H., Neurotubules: different densities in peripheral motor and sensory nerve fibres, Experientia (Basel), 31 (1975) 318-320.