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Uptake and retrograde transport of HRP by axons of intact and damaged peripheral nerve trunks
Neuroseienee Letters, 6 (1977)135--141 @ Elsevier/North-Holland Scientific Publishers Ltd.
135
UPTAKE AND RETROGRADE TRANSPORT OF HRP BY AXONS OF INTACT AND DAMAGED PERIPHERAL NERVE TRUNKS B.J. OLDFIELD and ELSPETH M. MeLACHLAN
Department of Physiology, Monash University, Wellington Road, Clayton, Victoria (Austratia) (Received August 5th, 1977) (Accepted August 9th, 1977)
SUMMARY
A light microscope study has been made of the uptake and transport of horseradish peroxidase (HRP) by axons of intact and damaged peripheral nerve trunks. Topically applied HRP entered axons of guinea pig cervical vagi and hypoglossal nerves whether or not these were damaged by cmshL,g or local freezing at the site of application of HRP. If damaged, the corresponding cell bodies contained HRP, but little was detected in the cell bodies of undamaged axons. If the damage was distalto the siteof H R P application,there was no significant retrograde transport. The presence of H R P within axons appears to be insufficientfor itstransport; trafficis initiatedonly from axon terminals and regions which have been damaged.
It. is usually claimed that axons which travel through, but do not terminate in, a region in the central nervous system, into which horseradish peroxidase (HRP) has been injected, do not transport the enzyme to their cell bodies [17, 19]. However, macromolecules, including HRP, can enter both myelinated and unmyelinated axons after topical application along their length [9,11], but it is not clear whether HRP is retrogradely transported to cell bodies following entry along undamaged axon shafts. Direct uptake of HRP into damaged axons does result in retrograde labelling [ 1,4,5,14 ], but no attempt, has been made to quantitate the extent of such labelling. This question is relevant not only in experiments where axons are intentionally damaged, but also in estimating the extent to which damage produced by injection procedures [4,7,8,15] might lead to spurious identificati.~n of pathways. Experiments have been undertaken to examine the uptake and retrograde transport of HRP after its application to intact and damaged ~ripheral nerve trunks, and the labelling of cell bodies has been quantitated using light microscope techniques. Vagus and hypoglossal nerves were exposed in guinea pigs anaesthetized with
136
sodium thiopentone (35 m g ~ ) . A p p r o x ~ a t e l y 2 mm of cleared nerve trunk were loosely wrapped with a cuff of thin absorbent tissue paper soaked with 1 ~1 of a 20% solution of HRP (Sigma ~ Vl), and a piece of thin polyethylene film was sutured around the cuff t~ i~late it from the surrounding tissue. In 21 experiments (16 vagus and 5 hypoglossal), the nerve trunk was simply wrapped with the HRP soaked tissue cuff, sp~ial care being taken while manipulating the nerve to minimise mech~ical damage to axons. In 36 other experiments (23 vagus and 13 hypoglossal), the nerve trunk was intentionally damaged by crushing transversely with jewellers forceps over approx. 1 ram, and the HRP-soaked cuff positioned directly over the site of damage. Twenty-four hours postoperatively, the animals were sacrificed and frozen sections (35/~m) of nodose ganglion (NG), dorsal motor nucleus of vagus (DMN-X) and hypoglossal nucleus (HN) were treated and examined for the demonstration of HRP using conventional techniques [6]. Material from animals which had not received HRP, and contralateral ganglia and motor nuclei, did not show any peroxidase reaction product in the regions studied. Furthermore, all nerve trunks to which HRP had been applied showed peroxidase reaction product both in and around the nerves, so that failures to detect HRP granules were unlikely to be due to inadequate histochemical procedures. The percentage of cells containing HRP was estimated in the section from each experiment having the largest number of HRP-positive cells. The minimum total number of cells in a section sampled in this way was 370 (NG), 67 (HN) and 122 (DMN-X).
Uptake o f HRP into undamaged and damaged axons Following the application of HRP around both intact and damaged cervical vagi and hypoglossal nerves (n = 42), dark brown reaction product w~s always present outside the perineurium (see Figs. 1 a,b,d) limited to a length of nerve corresponding approxima~ly to the size of the tissue paper wrap. Inside all nerve trunks diffuse light brown staining was detected within the endoneurial space, extending along the nerve beyond the limits of the darker stain outside the nerve. The barrier to diffusion of HRP presented by the perineurium [10] thus appeared by no means complete (cf. ref. 20). It appears that penetration can occur if the concentration of exogenous tracer is high (20% HRP cf. 10%~ ref. 13), and if theduration of exposure is prolonged (24 h cf. 2 h, ref. 10). In all nerve trunks, darker deposits of HRP reaction product, appeared as continuous columns at and near the si~ of application, (Figs. lb,e). It was confirmed that these were intro_s_xonal in semi-thin (1--5 ~m) transverse sections cut through the wrap site (Fig. ld). Linearly-arranged granules of HRP were observed more proximal to the site of application (Figs. lcj~), extending at least 1 cm central to the site of application in damaged axons, but only 3 mm proximal to the site of the wrap in undamaged axons. Granular deposits of HRP were also present in all axons distal to the site of application; the extent of the distal spread was not examined. This distribution of HRP is identical to that previously described in axons proximal to crush and cut lesions [5,14], but apparently has not been reported for undamaged nerve trunks.
137
a
,°
.
.~,
•
°
_
o
e
~
Fig. 1. Localisation of HRP in hypoglossal (a--d) and vagus (e--f) nerve trunks at and near the site of application. (a) Damaged region showing HRP outside the epineurium and the spread of HRP inside the nerve sheath attenuating distally (in direction of arrow}. (b) Undamaged wrapper region showing columnar HRP. (c) More p~-oximally, some evenly-fi|led axons and some containing granules of HRP reaction product. (d) Semi-thin section of undamaged wrapped region showing HRP inside axons. (e) Damaged region showing columnar HRP (~wrow). (f) More proximally, linearly-arranged granules (arrows). Densely stained RBC's pre~ent both outside and within the nerve sheath. Calibration bar in (a) represents 0.5 mm in (a), 80 ~m in (b), (c), (e) and (f), and 20 um in (d).
138
TABLE I Experiment
Site of cell bodies
HRP-positive cells/section -
4-
Total 4-4--i-
4-4-
(none)
(1-10%)
(10-40%)
(40-100%)
Wrap
NG DMN-X HN
14 1 4
2 1 0
0 0 1
0 0 0
16 2 5
Crush
NG DMN-X HN
6 0 1
9 2 2
5 2 3
3 2 7
23 6 13
Freeze
NG HN
3 1
3 0
2 2
1 1
9 4
Retrograde transport of HRP by undamaged and damaged axons In 86% of experiments (n = 21) in which HRP was applied around nerve trunks which were n o t intentionally damaged, no reaction product was detected in regions containing the appropriate cell bodies (see Table I). In the remaining cases, 2% and 4% of cells in NG. 4% of cells in DMN-X, and 14% of cells in HN gave a positive reaction for the presence of HRP. It seems likely t h a t these resulted from inadvertent damage to a few axons during placement of the wrap (see below). It appears t h a t insignificant amounts o f HRP are transported back and accumulated in granules in the cell bodies of undamaged axons, despite clear incorporation of the e n z y m e at sites of application. In contrast, in 81% of the experiments (n = 36) in which the nerve trunks were damaged b y transverse crushes at the site of application of HRP, cell bodies in appropriate regions contained typical granular deposits o f HRP. These results confirm the suggestion t h a t retrograde transport of HRP applied along the axon shaft only occurs if the axon membranes have been d i s r u p ~ d [4,7,14]. One remarkable aspect of the present data was the variability between experiments in the percentage of cells which were labelled {Table I). For example, in 17 experiments in which cells of NG were HRP-positive, a range of 1 to 86% staining was detected. The mean n u m b e r of ganglion cells containing detectable HRP transported from a crush site in all experiments on the vagus was 17 + 4.8% (S.E.M., n = 23). Similar variability was observed in the degree of labelling of preganglionic ceil bodies in DMN-X (mean 29 + 12.2%, S.E.M., n = 6) and of m o t o r neurons in HN {mean 47 + 9.1%, S.E.M., n = 13). In some experiments, particularly those on the vagus nerve, no HRP-positive cells were f o u n d after HRP application to a crushed nerve trunk. It seems possible t h a t crush damage migh ~_~:ve spared some of the smaller (and particu-
139
larly the unmyelinated) axons [3]. An alternative method of axonal damage, which does not interrupt the outer nerve sheaths, but produces extensive if not complete axonal damage is local freezing [ 18]. Local freezing prior to HRP application, using a steel hook cooled in liquid nitrogen (13 experiments, 9 vagus and 4 hypoglossal), was followed by labelling of cell bodies in NG and HN (69% of experiments), with variations in the percentage of HttP, positive cells comparable to those obtained after nerve crush (see Table I).
Effect o f lignocaine on retrograde HRP transport Retrograde transport of HRP has been thought to involve axonal micro° tubules [ 16] ;these structures are disrupted by lignocaine [ 2]. In 5 experiments, vagus nerves were wrapped with a tissue paper cuff soaked with 2% lignocaine for 45--60 min prior to crushing the ne~'ve. Subsequent application of HRP labelled cells in only one NG (10% of cells). The absence of retrograde labelling in this case may be attributed to the action of lignocaine on microtubules rather than its ability to block action potential firing, as damaged sensory axons which
Fig. 2. HRP granules in motor and sensory neurons after application to crushed nerve trunks. Cells in (a) hypoglossal nucleus, (b) dorsal m o t o r nucleus of vagus, (c), (d) and (e) nodose ganglion Calibration bar, 50 urn.
140
of local anaesthetic are also presumably silent. observations on colchicine-treated axons [16].
1 NH (27% ~ i n g
mtered the a x o n s , n o HRF was ,X (5 experiments), and in only of cel~ bodies)~m 6 experiments.
Differences in retrograde ~tRP-lab~lling by different types of axon. Table I shows that NG had consistently lower percentages of cells labelled than DMN-X or HN. In pz~icular, in 2 experiments, HN was 100% labelled. However, the appearar ~ of the granules of HRP-reaction product differed between cell bodies of motor ~md sensory axons. Granules within motor neurons o f HN and DMN-X were very prominent and were distributed throughout the soma and p r o x ~ d e n d r i t i ~ tree (Fig. 2a,b). In NG, by contrast, the monopolar cells contained higher densities of finer granules (Fig. 2c,d,e). Differences in degree of labelling and granule size were not related to neuron size. It seems possible that the lower percentages of labelled sensory neurons detected in the light microscope may be accounted for by difficulties in resolution of the consistently smaller particle sizes. These results imply that axons do not transport all HRP which enters them but only that which is sampled from axon terminals [12] or from sites of darnage. Thus cell bodies of 'en passant' axons damaged by an injection procedure might readily be labelled by HRP. However complete labelling of all neurons whose axons are intentionally disrupted is uncommon, so that the use of lesions t~ of cell bodies [ 1,4] might be expected to yield incomplete q REFERENCES
1 Adams, J.C. and Warr, W.B., Originso.f axons in the cats acoustic striae determined by injection of HRP into severed tracts, J. comp. Neurol., 170 (1976) 107--122. 2 Byers, M.R., Hendrickson, A.E., Fink, B.R., Kennedy, R,D. and Middaugh, M.E., Effects of lidocaine on axonal morphology, microtubuies and rapid transport in rabbit vagus nerve in vitro, J. Neu~obiol., 4 (1973) 125--143. 3 'Cajal, S.R., Degeneration and regeneration of the nervous system, Oxford University Press, London, 1928. 4 De Vito, J,L., Clausing, K.W. and Smith, O.A., Uptake and transport of horseradish peroxidame b y cut end of the vagus nerve, Brain Res., 82 (1974) 269--271. 5 Furstman, L., Saporta, S, and Kruger, L., Retrograde axonal transport o f horseradish peroxidssein s e n t r y nerves and g~glion cells of the rat, Brain Res:, 84(1975) 320--324. 6 Graham, R.C, Jr. and: Karnovsky, M.J., The early stages of absorption of injected horseradish peroxidase in the proximal tubules of mouse ki~iney: ultrastructural cytochemistry by a new technique, J. Histochem. Cytochem., 14 (1966) 291--302.
141 7 Halperin, J.J. and LaVail, J.H., A study of the dynamics of retrograde transport and accumulation of HRF in injured, neurons, Brain Res., 100 (1975) 253--269. 8 Herkenham, M. and Nauta, W.J.H., Afferent connections of the habenular nuclei in the rat. A horseradish peroxidasestudy with a note on the fibre-of-passage problem, J. comp. Neurol., 173 (1977) 123--146. 9 Holtzman, E. and Peterson, E.R., Uptake of protein by mammalian neurons, J. Cell. Biol., 40 (1969) 863--869. t0 Klemm M., Das Perineurium als Diffusionbarriere gegenuber Peroxidase bei epi- und endoneuraler Applikation, Z. Ze]lforsch., 108 (1970) 431--445. 11 Krishnan, N. and Singer, M., Penetration of peroxidase into peripheral nerve fibres, Amer. J. Anat., 136 (1973) 1--14. 12 Kristensson, K. and Olsson, Y., Retrograde axonal transport of protein, Brain Res., 29 (1971) 363--365. 13 Kristensson, K. and Olsson, V., The perineurium as a diffusion barrier to protein tracers, Acta neuropath. (Berl~), 17 (1971) 127--138. 14 Kristensson, K. and Olsson, Y., Retrograde transport of horseradish peroxidase in transected axons. 3. Entry into injured axons and subsequent localization in perikaryon, Brain Res., 115 (1976) 201--213. 15 LaVail, J.H., Ftetrograde cell degeneration and retrograde transport techniques. In W.M. Cowan and M. Cuenod (Eds.), The U ~ of Axonal Transport for Studies of Neuronal Connectivity, Elsevier, Amsterdam, 1975, pp. 219--243. 16 LaVail, J.H., and LaVail, M.M., The retrograde intraaxonal transport of HRP in the chick visual system. A light and electron microscopic study, J. comp. Neurol., 157 (1974) 303--358. 17 LaVail, J.H., Winston, K.R. and Tish, A., A method based on retrograde intraaxonal transport of protein for identification of cell bodies of origin of axons terminating within the CNS, Brain Res., 58 (1973) 470--477. 18 Mira, J. and Pecot-Dechavassine, M., Effets d'une congelation localisee d'un neff peripherique sur la conduction d l'influx nerveux, au cours de la degenerescence et de la regeneration, Pflugers Arch., 330 (1971) 5--14. 19 Nauta, J~J.W., Pritz, M.E. and Lasek, R.J., ~ fferents to the rat caudoputamen studied with horseradish peroxidase. A~ evaluation of retrograde neuroanatomical r~.~arch method, Brain Res., 67 (1974) 219--234. 20 Olsson, Y. and Kristensson, K., The perineurium as a diffusion barrier to orot~.in tracers following trauma to nerves, Acta. neuropath. (Bed.), 23 (1973) 105--111.
135
UPTAKE AND RETROGRADE TRANSPORT OF HRP BY AXONS OF INTACT AND DAMAGED PERIPHERAL NERVE TRUNKS B.J. OLDFIELD and ELSPETH M. MeLACHLAN
Department of Physiology, Monash University, Wellington Road, Clayton, Victoria (Austratia) (Received August 5th, 1977) (Accepted August 9th, 1977)
SUMMARY
A light microscope study has been made of the uptake and transport of horseradish peroxidase (HRP) by axons of intact and damaged peripheral nerve trunks. Topically applied HRP entered axons of guinea pig cervical vagi and hypoglossal nerves whether or not these were damaged by cmshL,g or local freezing at the site of application of HRP. If damaged, the corresponding cell bodies contained HRP, but little was detected in the cell bodies of undamaged axons. If the damage was distalto the siteof H R P application,there was no significant retrograde transport. The presence of H R P within axons appears to be insufficientfor itstransport; trafficis initiatedonly from axon terminals and regions which have been damaged.
It. is usually claimed that axons which travel through, but do not terminate in, a region in the central nervous system, into which horseradish peroxidase (HRP) has been injected, do not transport the enzyme to their cell bodies [17, 19]. However, macromolecules, including HRP, can enter both myelinated and unmyelinated axons after topical application along their length [9,11], but it is not clear whether HRP is retrogradely transported to cell bodies following entry along undamaged axon shafts. Direct uptake of HRP into damaged axons does result in retrograde labelling [ 1,4,5,14 ], but no attempt, has been made to quantitate the extent of such labelling. This question is relevant not only in experiments where axons are intentionally damaged, but also in estimating the extent to which damage produced by injection procedures [4,7,8,15] might lead to spurious identificati.~n of pathways. Experiments have been undertaken to examine the uptake and retrograde transport of HRP after its application to intact and damaged ~ripheral nerve trunks, and the labelling of cell bodies has been quantitated using light microscope techniques. Vagus and hypoglossal nerves were exposed in guinea pigs anaesthetized with
136
sodium thiopentone (35 m g ~ ) . A p p r o x ~ a t e l y 2 mm of cleared nerve trunk were loosely wrapped with a cuff of thin absorbent tissue paper soaked with 1 ~1 of a 20% solution of HRP (Sigma ~ Vl), and a piece of thin polyethylene film was sutured around the cuff t~ i~late it from the surrounding tissue. In 21 experiments (16 vagus and 5 hypoglossal), the nerve trunk was simply wrapped with the HRP soaked tissue cuff, sp~ial care being taken while manipulating the nerve to minimise mech~ical damage to axons. In 36 other experiments (23 vagus and 13 hypoglossal), the nerve trunk was intentionally damaged by crushing transversely with jewellers forceps over approx. 1 ram, and the HRP-soaked cuff positioned directly over the site of damage. Twenty-four hours postoperatively, the animals were sacrificed and frozen sections (35/~m) of nodose ganglion (NG), dorsal motor nucleus of vagus (DMN-X) and hypoglossal nucleus (HN) were treated and examined for the demonstration of HRP using conventional techniques [6]. Material from animals which had not received HRP, and contralateral ganglia and motor nuclei, did not show any peroxidase reaction product in the regions studied. Furthermore, all nerve trunks to which HRP had been applied showed peroxidase reaction product both in and around the nerves, so that failures to detect HRP granules were unlikely to be due to inadequate histochemical procedures. The percentage of cells containing HRP was estimated in the section from each experiment having the largest number of HRP-positive cells. The minimum total number of cells in a section sampled in this way was 370 (NG), 67 (HN) and 122 (DMN-X).
Uptake o f HRP into undamaged and damaged axons Following the application of HRP around both intact and damaged cervical vagi and hypoglossal nerves (n = 42), dark brown reaction product w~s always present outside the perineurium (see Figs. 1 a,b,d) limited to a length of nerve corresponding approxima~ly to the size of the tissue paper wrap. Inside all nerve trunks diffuse light brown staining was detected within the endoneurial space, extending along the nerve beyond the limits of the darker stain outside the nerve. The barrier to diffusion of HRP presented by the perineurium [10] thus appeared by no means complete (cf. ref. 20). It appears that penetration can occur if the concentration of exogenous tracer is high (20% HRP cf. 10%~ ref. 13), and if theduration of exposure is prolonged (24 h cf. 2 h, ref. 10). In all nerve trunks, darker deposits of HRP reaction product, appeared as continuous columns at and near the si~ of application, (Figs. lb,e). It was confirmed that these were intro_s_xonal in semi-thin (1--5 ~m) transverse sections cut through the wrap site (Fig. ld). Linearly-arranged granules of HRP were observed more proximal to the site of application (Figs. lcj~), extending at least 1 cm central to the site of application in damaged axons, but only 3 mm proximal to the site of the wrap in undamaged axons. Granular deposits of HRP were also present in all axons distal to the site of application; the extent of the distal spread was not examined. This distribution of HRP is identical to that previously described in axons proximal to crush and cut lesions [5,14], but apparently has not been reported for undamaged nerve trunks.
137
a
,°
.
.~,
•
°
_
o
e
~
Fig. 1. Localisation of HRP in hypoglossal (a--d) and vagus (e--f) nerve trunks at and near the site of application. (a) Damaged region showing HRP outside the epineurium and the spread of HRP inside the nerve sheath attenuating distally (in direction of arrow}. (b) Undamaged wrapper region showing columnar HRP. (c) More p~-oximally, some evenly-fi|led axons and some containing granules of HRP reaction product. (d) Semi-thin section of undamaged wrapped region showing HRP inside axons. (e) Damaged region showing columnar HRP (~wrow). (f) More proximally, linearly-arranged granules (arrows). Densely stained RBC's pre~ent both outside and within the nerve sheath. Calibration bar in (a) represents 0.5 mm in (a), 80 ~m in (b), (c), (e) and (f), and 20 um in (d).
138
TABLE I Experiment
Site of cell bodies
HRP-positive cells/section -
4-
Total 4-4--i-
4-4-
(none)
(1-10%)
(10-40%)
(40-100%)
Wrap
NG DMN-X HN
14 1 4
2 1 0
0 0 1
0 0 0
16 2 5
Crush
NG DMN-X HN
6 0 1
9 2 2
5 2 3
3 2 7
23 6 13
Freeze
NG HN
3 1
3 0
2 2
1 1
9 4
Retrograde transport of HRP by undamaged and damaged axons In 86% of experiments (n = 21) in which HRP was applied around nerve trunks which were n o t intentionally damaged, no reaction product was detected in regions containing the appropriate cell bodies (see Table I). In the remaining cases, 2% and 4% of cells in NG. 4% of cells in DMN-X, and 14% of cells in HN gave a positive reaction for the presence of HRP. It seems likely t h a t these resulted from inadvertent damage to a few axons during placement of the wrap (see below). It appears t h a t insignificant amounts o f HRP are transported back and accumulated in granules in the cell bodies of undamaged axons, despite clear incorporation of the e n z y m e at sites of application. In contrast, in 81% of the experiments (n = 36) in which the nerve trunks were damaged b y transverse crushes at the site of application of HRP, cell bodies in appropriate regions contained typical granular deposits o f HRP. These results confirm the suggestion t h a t retrograde transport of HRP applied along the axon shaft only occurs if the axon membranes have been d i s r u p ~ d [4,7,14]. One remarkable aspect of the present data was the variability between experiments in the percentage of cells which were labelled {Table I). For example, in 17 experiments in which cells of NG were HRP-positive, a range of 1 to 86% staining was detected. The mean n u m b e r of ganglion cells containing detectable HRP transported from a crush site in all experiments on the vagus was 17 + 4.8% (S.E.M., n = 23). Similar variability was observed in the degree of labelling of preganglionic ceil bodies in DMN-X (mean 29 + 12.2%, S.E.M., n = 6) and of m o t o r neurons in HN {mean 47 + 9.1%, S.E.M., n = 13). In some experiments, particularly those on the vagus nerve, no HRP-positive cells were f o u n d after HRP application to a crushed nerve trunk. It seems possible t h a t crush damage migh ~_~:ve spared some of the smaller (and particu-
139
larly the unmyelinated) axons [3]. An alternative method of axonal damage, which does not interrupt the outer nerve sheaths, but produces extensive if not complete axonal damage is local freezing [ 18]. Local freezing prior to HRP application, using a steel hook cooled in liquid nitrogen (13 experiments, 9 vagus and 4 hypoglossal), was followed by labelling of cell bodies in NG and HN (69% of experiments), with variations in the percentage of HttP, positive cells comparable to those obtained after nerve crush (see Table I).
Effect o f lignocaine on retrograde HRP transport Retrograde transport of HRP has been thought to involve axonal micro° tubules [ 16] ;these structures are disrupted by lignocaine [ 2]. In 5 experiments, vagus nerves were wrapped with a tissue paper cuff soaked with 2% lignocaine for 45--60 min prior to crushing the ne~'ve. Subsequent application of HRP labelled cells in only one NG (10% of cells). The absence of retrograde labelling in this case may be attributed to the action of lignocaine on microtubules rather than its ability to block action potential firing, as damaged sensory axons which
Fig. 2. HRP granules in motor and sensory neurons after application to crushed nerve trunks. Cells in (a) hypoglossal nucleus, (b) dorsal m o t o r nucleus of vagus, (c), (d) and (e) nodose ganglion Calibration bar, 50 urn.
140
of local anaesthetic are also presumably silent. observations on colchicine-treated axons [16].
1 NH (27% ~ i n g
mtered the a x o n s , n o HRF was ,X (5 experiments), and in only of cel~ bodies)~m 6 experiments.
Differences in retrograde ~tRP-lab~lling by different types of axon. Table I shows that NG had consistently lower percentages of cells labelled than DMN-X or HN. In pz~icular, in 2 experiments, HN was 100% labelled. However, the appearar ~ of the granules of HRP-reaction product differed between cell bodies of motor ~md sensory axons. Granules within motor neurons o f HN and DMN-X were very prominent and were distributed throughout the soma and p r o x ~ d e n d r i t i ~ tree (Fig. 2a,b). In NG, by contrast, the monopolar cells contained higher densities of finer granules (Fig. 2c,d,e). Differences in degree of labelling and granule size were not related to neuron size. It seems possible that the lower percentages of labelled sensory neurons detected in the light microscope may be accounted for by difficulties in resolution of the consistently smaller particle sizes. These results imply that axons do not transport all HRP which enters them but only that which is sampled from axon terminals [12] or from sites of darnage. Thus cell bodies of 'en passant' axons damaged by an injection procedure might readily be labelled by HRP. However complete labelling of all neurons whose axons are intentionally disrupted is uncommon, so that the use of lesions t~ of cell bodies [ 1,4] might be expected to yield incomplete q REFERENCES
1 Adams, J.C. and Warr, W.B., Originso.f axons in the cats acoustic striae determined by injection of HRP into severed tracts, J. comp. Neurol., 170 (1976) 107--122. 2 Byers, M.R., Hendrickson, A.E., Fink, B.R., Kennedy, R,D. and Middaugh, M.E., Effects of lidocaine on axonal morphology, microtubuies and rapid transport in rabbit vagus nerve in vitro, J. Neu~obiol., 4 (1973) 125--143. 3 'Cajal, S.R., Degeneration and regeneration of the nervous system, Oxford University Press, London, 1928. 4 De Vito, J,L., Clausing, K.W. and Smith, O.A., Uptake and transport of horseradish peroxidame b y cut end of the vagus nerve, Brain Res., 82 (1974) 269--271. 5 Furstman, L., Saporta, S, and Kruger, L., Retrograde axonal transport o f horseradish peroxidssein s e n t r y nerves and g~glion cells of the rat, Brain Res:, 84(1975) 320--324. 6 Graham, R.C, Jr. and: Karnovsky, M.J., The early stages of absorption of injected horseradish peroxidase in the proximal tubules of mouse ki~iney: ultrastructural cytochemistry by a new technique, J. Histochem. Cytochem., 14 (1966) 291--302.
141 7 Halperin, J.J. and LaVail, J.H., A study of the dynamics of retrograde transport and accumulation of HRF in injured, neurons, Brain Res., 100 (1975) 253--269. 8 Herkenham, M. and Nauta, W.J.H., Afferent connections of the habenular nuclei in the rat. A horseradish peroxidasestudy with a note on the fibre-of-passage problem, J. comp. Neurol., 173 (1977) 123--146. 9 Holtzman, E. and Peterson, E.R., Uptake of protein by mammalian neurons, J. Cell. Biol., 40 (1969) 863--869. t0 Klemm M., Das Perineurium als Diffusionbarriere gegenuber Peroxidase bei epi- und endoneuraler Applikation, Z. Ze]lforsch., 108 (1970) 431--445. 11 Krishnan, N. and Singer, M., Penetration of peroxidase into peripheral nerve fibres, Amer. J. Anat., 136 (1973) 1--14. 12 Kristensson, K. and Olsson, Y., Retrograde axonal transport of protein, Brain Res., 29 (1971) 363--365. 13 Kristensson, K. and Olsson, V., The perineurium as a diffusion barrier to protein tracers, Acta neuropath. (Berl~), 17 (1971) 127--138. 14 Kristensson, K. and Olsson, Y., Retrograde transport of horseradish peroxidase in transected axons. 3. Entry into injured axons and subsequent localization in perikaryon, Brain Res., 115 (1976) 201--213. 15 LaVail, J.H., Ftetrograde cell degeneration and retrograde transport techniques. In W.M. Cowan and M. Cuenod (Eds.), The U ~ of Axonal Transport for Studies of Neuronal Connectivity, Elsevier, Amsterdam, 1975, pp. 219--243. 16 LaVail, J.H., and LaVail, M.M., The retrograde intraaxonal transport of HRP in the chick visual system. A light and electron microscopic study, J. comp. Neurol., 157 (1974) 303--358. 17 LaVail, J.H., Winston, K.R. and Tish, A., A method based on retrograde intraaxonal transport of protein for identification of cell bodies of origin of axons terminating within the CNS, Brain Res., 58 (1973) 470--477. 18 Mira, J. and Pecot-Dechavassine, M., Effets d'une congelation localisee d'un neff peripherique sur la conduction d l'influx nerveux, au cours de la degenerescence et de la regeneration, Pflugers Arch., 330 (1971) 5--14. 19 Nauta, J~J.W., Pritz, M.E. and Lasek, R.J., ~ fferents to the rat caudoputamen studied with horseradish peroxidase. A~ evaluation of retrograde neuroanatomical r~.~arch method, Brain Res., 67 (1974) 219--234. 20 Olsson, Y. and Kristensson, K., The perineurium as a diffusion barrier to orot~.in tracers following trauma to nerves, Acta. neuropath. (Bed.), 23 (1973) 105--111.