401
J. Anat. (1979), 128, 2, pp. 401406 With 4 figures Printed in Great Brtain
The uptake of horseradish peroxidase by damaged autonomic nerves in vitro P. N. ANDERSON, J. MITCHELL AND D. MAYOR
Human Morphology, University of Southampton Medical and Biological Sciences Building, Bassett Crescent East, Southampton
(Accepted 14 April 1978) INTRODUCTION
The horseradish peroxidase (HRP) technique has been used extensively to study fibre pathways in the central nervous system, and more recently in the peripheral nervous system (Ellison & Clark, 1975; Elfvin & Dalsgaard, 1977). The technique involves the application of HRP to axons or their terminals, and its subsequent uptake and retrograde transport to the perikarya. LaVail & LaVail (1975) have shown that the uptake of HRP at nerve endings is by pinocytosis. However, Kristensson & Olsson (1976) reported a rapid, diffuse entry of HRP into damaged mouse sciatic nerve axons, but found little evidence for pinocytosis. The purpose of the present study was to examine the mechanism of the uptake of the HRP at the site of damage in ligated sympathetic nerves in vitro. Post-ganglionic sympathetic nerves were studied because the changes occurring around the site of ligation have been well documented (Kapeller & Mayor, 1969). An in vitro system was chosen since it allows greater scope for experimental investigation of both the uptake and the retrograde transport of the enzyme (Anderson, Mitchell & Mayor, 1978). MATERIALS AND METHODS
Male Hartley guinea-pigs weighing 280-350 g were anaesthetized with intraperitoneal Nembutal (40 mg/kg: Abbott). The hypogastric nerves were located in the mesentery of the colon and ligated with a fine (5/0) silk ligature approximately 2 5 cm distal to the inferior mesenteric ganglion (IMG). The inferior mesenteric artery was ligated distal to the ganglion. The ligated nerve/IMG preparation was removed from the animal and by means of the thread ligatures, which had been left long, was suspended in the incubation chamber. A double chamber similar in design to that used by Banks, Mayor, Mitchell & Tomlinson (1971), but with smaller compartments, was employed. 25 mg of Sigma type II HRP was dissolved in one drop of oxygenated tissue culture medium (Eagle's MEM; Gibco Bio-cult.). This concentrated HRP solution was applied around the ligation site on the hypogastric nerve. The top of the chamber was assembled, and tissue culture medium added to the ganglion side of the compartment. Ten minutes later 5 ml of tissue culture medium was added to the ligated nerve compartment. The chamber was placed in a water bath maintained at 37 °C for periods up to 24 hours. The tissue culture medium within each compartment of the chamber was gassed with 95 % oxygen/5 % carbon dioxide throughout the period of incubation. 26
ANA
I28
402
P. N. ANDERSON, J. MITCHELL AND D. MAYOR
Fig. 1. Electron micrograph of hypogastric nerve, between 05 and 1 mm proximal to the ligation, exposed to HRP for 24 hours. A myelinated axon is shown (starred arrow) containing very dense, non-vesicular peroxidase reaction product; non-myelinated axons containing nonvesicular HRP reaction product (arrow) are also shown. Bar = 1 gum.
The preparation was then removed and fixed for 5 hours at room temperature in a solution containing: 1% paraformaldehyde, 3 % glutaraldehyde, 7 % sucrose and phosphate buffer at pH 7-4. Following fixation the preparation was washed overnight in phosphate buffer/sucrose mixture at 4 'C. Half millimetre lengths of the hypogastric nerves were rinsed in distilled water and reacted for peroxidase activity, using the technique of Graham & Karnovsky (1966). The tissues were then osmicated, dehydrated in ethanol, passed through epoxy propane and embedded in Araldite. Unstained sections 2 ,um thick were examined, using phase contrast light microscopy. Ultrathin sections, both stained and unstained, were studied electron microscopically. RESULTS
The ultrastructural appearances proximal to the site of constriction of guinea-pig hypogastric nerves maintained in Eagle's MEM tissue culture medium for 24 hours in vitro were similar to those reported for ligated cat hypogastric and splenic nerves in vivo (Kapeller & Mayor, 1969). In the 0 5 mm segment of nerve immediately proximal to the site of ligation the most striking feature was the accumulation of axonal organelles. This was Fig. 2. Electron micrograph of hypogastric nerve less than 05 mm from the ligation, exposed to HRP for 24 hours. Several large swollen axons are shown (sa) which contain membranebound HRP reaction product. Some peroxidase-positive organelles resemble elongated cisternae of smooth endoplasmic reticulum (ec). The extracellular space contains HRP reaction product, much of which is adhering to collagen fibres (c) and some of which is seen within mesaxons (ma). Bar = 1 /tm. The inset shows a higher power micrograph of a swollen axon. A coated vesicle appears to be forming from the axonal membrane. Note that the peroxidase reaction product adheres to the membrane of the vesicle. Bar = 0 1 #um.
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Fig. 3. An electron micrograph showing a non-myelinated axon within a hypogastric nerve, less than 0-5 mm proximal to the ligation, after exposure to peroxidase for 90 minutes. The axon is not grossly swollen, and within the axoplasm is a coated vesicle containing HRP reaction product (cv). The HRP reaction product is very dense in the extracellular space, and has penetrated the mesaxon (ma). Bar = 1 /,tm. Fig. 4. A section through a similar segment of hypogastric nerve to that in Fig. 3. Many axons, such as the arrowed non-myelinated axon, contain very electron-dense HRP reaction product. Bar = 1 ,um.
particularly marked in the non-myelinated axons, which were often swollen. Most numerous among the accumulated organelles were mitochondria, dense-cored vesicles, tubulovesicular profiles of smooth endoplasmic reticulum, and neurofilaments. More proximal still (0 5-1 0 mm from the ligation) the axons were less swollen, and in segments of nerve more than 2 mm proximal to the ligation the ultrastructural appearances could not be readily distinguished from those of normal nerves fixed in vivo. In control ligated nerves, to which HRP had not been applied, reaction product resulting from endogenous peroxidase activity was present in erythrocytes and some leucocytes. No reaction product was seen within axons or in the extracellular spaces. The perineurium in the millimetre of nerve immediately proximal to the ligation (the P1 segment) was often clearly damaged, and in experiments where peroxidase had been applied, HRP reaction product was seen to extend between the perineurial cells, indicating the failure of the normal diffusion barrier. The perineurial diffusion barrier was intact in regions more than 2 mm proximal to the ligation. HRP reaction product was also seen throughout the interstitial spaces, mainly
Uptake of horseradish peroxidase
405
adhering to the collagen fibres. Reaction product could be seen within connective tissue cells and Schwann cells, and in mesaxons and axons within the P1 segment. Between 0 5 and 1 mm proximal to the ligation many axons, both myelinated and non-myelinated, contained non-vesicular peroxidase reaction product (Fig. 1). HRP reaction product in a membrane-bound form was not commonly seen in this part of the nerve and there was little evidence of pinocytosis. The swollen axons in the 0 5 mm of nerve immediately proximal to the ligation contained varying amounts of diffuse HRP. The electron density of the reaction product was related to the size of the axons, being much less dense in the greatly swollen axons. The swollen axons also contained membrane-bound HRP reaction product (Fig. 2). Large vesicles and elongated cisternae containing the enzyme were particularly prominent. Many of the swollen axons also showed evidence of pinocytotic uptake of the peroxidase (Fig. 2), usually into coated vesicles. The reaction product within such vesicles was usually closely associated with the vesicle membrane, rather than diffused through the lumen. More than 2 mm from the ligation reaction product was occasionally seen within vesicles in axons, but was not seen in the interstitial spaces, indicating that HRP did not spread long distances through the interstitial spaces. To investigate the early stages of the uptake of HRP by axons, nerves were examined 90 minutes after the application of the enzyme. In these experiments there was much less swelling of the axons immediately proximal to the ligation than was apparent after 24 hours. However, HRP reaction product was present throughout the interstitial spaces of the P1 segment, in connective tissue cells, Schwann cells and mesaxons (Fig. 3). Many axons contained diffuse HRP reaction product (Fig. 4) which was usually more dense than that seen after 24 hours' exposure. Coated vesicles containing HRP reaction product were seen within many of the axons which did not contain the enzyme in a diffuse form (Fig. 3). DISCUSSION
The uptake of HRP into damaged sympathetic axons probably occurs by more than one route. Its entry in a diffuse form near the site of injury has been observed by other workers (Kristensson & Olsson, 1976), and is certainly a rapid process by which large amounts of the enzyme can penetrate the axonal membrane in a short time. However, the significance of this mode of entry with regard to the subsequent retrograde transport of the enzyme towards the perikaryon remains uncertain. HRP reaction product present within axons several millimetres proximal to the ligation was enclosed within membrane-bound vesicles and elongated cisternae. It seems probable that such vesicular organelles are responsible for the transport of HRP towards the perikaryon in this, and in other systems (LaVail & LaVail, 1975). The possibility exists that the diffuse enzyme is incorporated into membranebound organelles within the axon, but in the present study no ultrastructural evidence for such a process was found. The reduced electron density of the diffuse HRP reaction product within the swollen axons may be a 'dilution' effect, resulting from the accumulation of axonal organelles. The abundance of membrane-bound HRP within the swollen axons, and the evidence of pinocytosis by these axons suggest an alternative route for the uptake of the enzyme by damaged axons. The pinocytotic uptake of HRP has been demonstrated in undamaged axons within a
406
P. N. ANDERSON, J. MITCHELL AND D. MAYOR
chick visual system (LaVail & LaVail, 1975) where it is also transported retrogradely toward the perikaryon. It seems likely that if pinocytotic uptake is responsible for the uptake of HRP prior to retrograde transport in the chick visual system, it could also be an effective method in damaged axons. It may be significant that in our system most presumptive pinocytotic invaginations were seen in swollen axons, which were located within 05 mm of the ligation. Such a short zone may be easily overlooked. It is possible that some of the pinocytotic activity of the swollen axons is initiated by the HRP, as it is known from other systems that positively charged molecules are good inducers of pinocytosis (Gingell, 1971), while the main isoenzyme of Sigma Type II peroxidase is a basic protein. SUMMARY The uptake of horseradish peroxidase by damaged autonomic nerves was studied in vitro. Large amounts of the enzyme penetrated the axons in a diffuse (nonvesicular) form. This ctcurred within 90 minutes of application of the enzyme. Twenty four hours after application of the enzyme diffuse peroxidase was still present in the axoplasm, but it was 'diluted' as a result of great axonal swelling. Pinocytotic uptake of the enzyme was observed both 90 minutes and 24 hours after application of the enzyme. The swollen axons close to the point of ligation showed most evidence of pinocytotic uptake, largely into coated vesicles, and much membrane-bound peroxidase reaction product was present. The uptake of peroxidase in a membrane-bound form may be of particular significance for the subsequent retrograde transport of the enzyme. REFERENCES ANDERSON, P. N., MITCHELL, J. & MAYOR, D. (1978). An in vitro method for studying the retrograde intra-axonal transport of horseradish peroxidase in sympathetic neurones. Brain Research 152,
151-156. BANKS, P., MAYOR, D., MITCHELL, M. & TOMLINSON, D. (1971). Studies on the translocation of noradrenaline-containing vesicles in post-ganglionic sympathetic neurones in vitro. Inhibition of movement by coichicine and vinblastine and evidence for the involvement of axonal microtubules. Journal of Physiology 216, 625-639. ELFVIN, L.-G., & DALSGAARD, C. J. (1977). Retrograde axonal transport of horseradish peroxidase in afferent fibres of the inferior mesenteric ganglion of the guinea pig. Identification of the cells of origin in dorsal root ganglia. Brain Research 126, 149-153. ELLISON, P. J. & CLARK, G. M. (1975). Retrograde axonal transport of horseradish peroxidase in peripheral autonomic nerves. Journal of Comparative Neurology 161, 103-114. GINGELL, D. (1971). Cell membrane surface potentials as a transducer. In Membranes and Ion Transport, vol. 3 (ed. E. E. Bittar), pp. 317-357. London, New York: Wiley-Interscience. GRAHAM, R. C. & KARNOVSKY, M. J. (1966). The early stages of absorption of injected horseradish peroxidase in the proximal tubules of the mouse kidney: ultrastructural correlates by a new technique. Journal of Histochemistry and Cytochemistry 14, 291-302. KAPELLER, K. & MAYOR, D. (1969). An electron microscope study of the early changes proximal to a constriction in sympathetic nerves. Proceedings of the Royal Society ofLondon B 172, 39-51. KRISTENSSON, K. & OLSSON, Y. (1976). Retrograde transport of horseradish peroxidase in transected axons. 3. Entry into injured axons and subsequent localisation in perikaryon. Brain Research 115, 201-213. LAVAIL, M. M. & LAVAIL, J. H. (1975). Retrograde intraaxonal transport of horseradish peroxidase in retinal ganglion cells of the chick. Brain' Research 85, 273-280.
J. Anat. (1979), 128, 2, pp. 401406 With 4 figures Printed in Great Brtain
The uptake of horseradish peroxidase by damaged autonomic nerves in vitro P. N. ANDERSON, J. MITCHELL AND D. MAYOR
Human Morphology, University of Southampton Medical and Biological Sciences Building, Bassett Crescent East, Southampton
(Accepted 14 April 1978) INTRODUCTION
The horseradish peroxidase (HRP) technique has been used extensively to study fibre pathways in the central nervous system, and more recently in the peripheral nervous system (Ellison & Clark, 1975; Elfvin & Dalsgaard, 1977). The technique involves the application of HRP to axons or their terminals, and its subsequent uptake and retrograde transport to the perikarya. LaVail & LaVail (1975) have shown that the uptake of HRP at nerve endings is by pinocytosis. However, Kristensson & Olsson (1976) reported a rapid, diffuse entry of HRP into damaged mouse sciatic nerve axons, but found little evidence for pinocytosis. The purpose of the present study was to examine the mechanism of the uptake of the HRP at the site of damage in ligated sympathetic nerves in vitro. Post-ganglionic sympathetic nerves were studied because the changes occurring around the site of ligation have been well documented (Kapeller & Mayor, 1969). An in vitro system was chosen since it allows greater scope for experimental investigation of both the uptake and the retrograde transport of the enzyme (Anderson, Mitchell & Mayor, 1978). MATERIALS AND METHODS
Male Hartley guinea-pigs weighing 280-350 g were anaesthetized with intraperitoneal Nembutal (40 mg/kg: Abbott). The hypogastric nerves were located in the mesentery of the colon and ligated with a fine (5/0) silk ligature approximately 2 5 cm distal to the inferior mesenteric ganglion (IMG). The inferior mesenteric artery was ligated distal to the ganglion. The ligated nerve/IMG preparation was removed from the animal and by means of the thread ligatures, which had been left long, was suspended in the incubation chamber. A double chamber similar in design to that used by Banks, Mayor, Mitchell & Tomlinson (1971), but with smaller compartments, was employed. 25 mg of Sigma type II HRP was dissolved in one drop of oxygenated tissue culture medium (Eagle's MEM; Gibco Bio-cult.). This concentrated HRP solution was applied around the ligation site on the hypogastric nerve. The top of the chamber was assembled, and tissue culture medium added to the ganglion side of the compartment. Ten minutes later 5 ml of tissue culture medium was added to the ligated nerve compartment. The chamber was placed in a water bath maintained at 37 °C for periods up to 24 hours. The tissue culture medium within each compartment of the chamber was gassed with 95 % oxygen/5 % carbon dioxide throughout the period of incubation. 26
ANA
I28
402
P. N. ANDERSON, J. MITCHELL AND D. MAYOR
Fig. 1. Electron micrograph of hypogastric nerve, between 05 and 1 mm proximal to the ligation, exposed to HRP for 24 hours. A myelinated axon is shown (starred arrow) containing very dense, non-vesicular peroxidase reaction product; non-myelinated axons containing nonvesicular HRP reaction product (arrow) are also shown. Bar = 1 gum.
The preparation was then removed and fixed for 5 hours at room temperature in a solution containing: 1% paraformaldehyde, 3 % glutaraldehyde, 7 % sucrose and phosphate buffer at pH 7-4. Following fixation the preparation was washed overnight in phosphate buffer/sucrose mixture at 4 'C. Half millimetre lengths of the hypogastric nerves were rinsed in distilled water and reacted for peroxidase activity, using the technique of Graham & Karnovsky (1966). The tissues were then osmicated, dehydrated in ethanol, passed through epoxy propane and embedded in Araldite. Unstained sections 2 ,um thick were examined, using phase contrast light microscopy. Ultrathin sections, both stained and unstained, were studied electron microscopically. RESULTS
The ultrastructural appearances proximal to the site of constriction of guinea-pig hypogastric nerves maintained in Eagle's MEM tissue culture medium for 24 hours in vitro were similar to those reported for ligated cat hypogastric and splenic nerves in vivo (Kapeller & Mayor, 1969). In the 0 5 mm segment of nerve immediately proximal to the site of ligation the most striking feature was the accumulation of axonal organelles. This was Fig. 2. Electron micrograph of hypogastric nerve less than 05 mm from the ligation, exposed to HRP for 24 hours. Several large swollen axons are shown (sa) which contain membranebound HRP reaction product. Some peroxidase-positive organelles resemble elongated cisternae of smooth endoplasmic reticulum (ec). The extracellular space contains HRP reaction product, much of which is adhering to collagen fibres (c) and some of which is seen within mesaxons (ma). Bar = 1 /tm. The inset shows a higher power micrograph of a swollen axon. A coated vesicle appears to be forming from the axonal membrane. Note that the peroxidase reaction product adheres to the membrane of the vesicle. Bar = 0 1 #um.
403
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Fig. 3. An electron micrograph showing a non-myelinated axon within a hypogastric nerve, less than 0-5 mm proximal to the ligation, after exposure to peroxidase for 90 minutes. The axon is not grossly swollen, and within the axoplasm is a coated vesicle containing HRP reaction product (cv). The HRP reaction product is very dense in the extracellular space, and has penetrated the mesaxon (ma). Bar = 1 /,tm. Fig. 4. A section through a similar segment of hypogastric nerve to that in Fig. 3. Many axons, such as the arrowed non-myelinated axon, contain very electron-dense HRP reaction product. Bar = 1 ,um.
particularly marked in the non-myelinated axons, which were often swollen. Most numerous among the accumulated organelles were mitochondria, dense-cored vesicles, tubulovesicular profiles of smooth endoplasmic reticulum, and neurofilaments. More proximal still (0 5-1 0 mm from the ligation) the axons were less swollen, and in segments of nerve more than 2 mm proximal to the ligation the ultrastructural appearances could not be readily distinguished from those of normal nerves fixed in vivo. In control ligated nerves, to which HRP had not been applied, reaction product resulting from endogenous peroxidase activity was present in erythrocytes and some leucocytes. No reaction product was seen within axons or in the extracellular spaces. The perineurium in the millimetre of nerve immediately proximal to the ligation (the P1 segment) was often clearly damaged, and in experiments where peroxidase had been applied, HRP reaction product was seen to extend between the perineurial cells, indicating the failure of the normal diffusion barrier. The perineurial diffusion barrier was intact in regions more than 2 mm proximal to the ligation. HRP reaction product was also seen throughout the interstitial spaces, mainly
Uptake of horseradish peroxidase
405
adhering to the collagen fibres. Reaction product could be seen within connective tissue cells and Schwann cells, and in mesaxons and axons within the P1 segment. Between 0 5 and 1 mm proximal to the ligation many axons, both myelinated and non-myelinated, contained non-vesicular peroxidase reaction product (Fig. 1). HRP reaction product in a membrane-bound form was not commonly seen in this part of the nerve and there was little evidence of pinocytosis. The swollen axons in the 0 5 mm of nerve immediately proximal to the ligation contained varying amounts of diffuse HRP. The electron density of the reaction product was related to the size of the axons, being much less dense in the greatly swollen axons. The swollen axons also contained membrane-bound HRP reaction product (Fig. 2). Large vesicles and elongated cisternae containing the enzyme were particularly prominent. Many of the swollen axons also showed evidence of pinocytotic uptake of the peroxidase (Fig. 2), usually into coated vesicles. The reaction product within such vesicles was usually closely associated with the vesicle membrane, rather than diffused through the lumen. More than 2 mm from the ligation reaction product was occasionally seen within vesicles in axons, but was not seen in the interstitial spaces, indicating that HRP did not spread long distances through the interstitial spaces. To investigate the early stages of the uptake of HRP by axons, nerves were examined 90 minutes after the application of the enzyme. In these experiments there was much less swelling of the axons immediately proximal to the ligation than was apparent after 24 hours. However, HRP reaction product was present throughout the interstitial spaces of the P1 segment, in connective tissue cells, Schwann cells and mesaxons (Fig. 3). Many axons contained diffuse HRP reaction product (Fig. 4) which was usually more dense than that seen after 24 hours' exposure. Coated vesicles containing HRP reaction product were seen within many of the axons which did not contain the enzyme in a diffuse form (Fig. 3). DISCUSSION
The uptake of HRP into damaged sympathetic axons probably occurs by more than one route. Its entry in a diffuse form near the site of injury has been observed by other workers (Kristensson & Olsson, 1976), and is certainly a rapid process by which large amounts of the enzyme can penetrate the axonal membrane in a short time. However, the significance of this mode of entry with regard to the subsequent retrograde transport of the enzyme towards the perikaryon remains uncertain. HRP reaction product present within axons several millimetres proximal to the ligation was enclosed within membrane-bound vesicles and elongated cisternae. It seems probable that such vesicular organelles are responsible for the transport of HRP towards the perikaryon in this, and in other systems (LaVail & LaVail, 1975). The possibility exists that the diffuse enzyme is incorporated into membranebound organelles within the axon, but in the present study no ultrastructural evidence for such a process was found. The reduced electron density of the diffuse HRP reaction product within the swollen axons may be a 'dilution' effect, resulting from the accumulation of axonal organelles. The abundance of membrane-bound HRP within the swollen axons, and the evidence of pinocytosis by these axons suggest an alternative route for the uptake of the enzyme by damaged axons. The pinocytotic uptake of HRP has been demonstrated in undamaged axons within a
406
P. N. ANDERSON, J. MITCHELL AND D. MAYOR
chick visual system (LaVail & LaVail, 1975) where it is also transported retrogradely toward the perikaryon. It seems likely that if pinocytotic uptake is responsible for the uptake of HRP prior to retrograde transport in the chick visual system, it could also be an effective method in damaged axons. It may be significant that in our system most presumptive pinocytotic invaginations were seen in swollen axons, which were located within 05 mm of the ligation. Such a short zone may be easily overlooked. It is possible that some of the pinocytotic activity of the swollen axons is initiated by the HRP, as it is known from other systems that positively charged molecules are good inducers of pinocytosis (Gingell, 1971), while the main isoenzyme of Sigma Type II peroxidase is a basic protein. SUMMARY The uptake of horseradish peroxidase by damaged autonomic nerves was studied in vitro. Large amounts of the enzyme penetrated the axons in a diffuse (nonvesicular) form. This ctcurred within 90 minutes of application of the enzyme. Twenty four hours after application of the enzyme diffuse peroxidase was still present in the axoplasm, but it was 'diluted' as a result of great axonal swelling. Pinocytotic uptake of the enzyme was observed both 90 minutes and 24 hours after application of the enzyme. The swollen axons close to the point of ligation showed most evidence of pinocytotic uptake, largely into coated vesicles, and much membrane-bound peroxidase reaction product was present. The uptake of peroxidase in a membrane-bound form may be of particular significance for the subsequent retrograde transport of the enzyme. REFERENCES ANDERSON, P. N., MITCHELL, J. & MAYOR, D. (1978). An in vitro method for studying the retrograde intra-axonal transport of horseradish peroxidase in sympathetic neurones. Brain Research 152,
151-156. BANKS, P., MAYOR, D., MITCHELL, M. & TOMLINSON, D. (1971). Studies on the translocation of noradrenaline-containing vesicles in post-ganglionic sympathetic neurones in vitro. Inhibition of movement by coichicine and vinblastine and evidence for the involvement of axonal microtubules. Journal of Physiology 216, 625-639. ELFVIN, L.-G., & DALSGAARD, C. J. (1977). Retrograde axonal transport of horseradish peroxidase in afferent fibres of the inferior mesenteric ganglion of the guinea pig. Identification of the cells of origin in dorsal root ganglia. Brain Research 126, 149-153. ELLISON, P. J. & CLARK, G. M. (1975). Retrograde axonal transport of horseradish peroxidase in peripheral autonomic nerves. Journal of Comparative Neurology 161, 103-114. GINGELL, D. (1971). Cell membrane surface potentials as a transducer. In Membranes and Ion Transport, vol. 3 (ed. E. E. Bittar), pp. 317-357. London, New York: Wiley-Interscience. GRAHAM, R. C. & KARNOVSKY, M. J. (1966). The early stages of absorption of injected horseradish peroxidase in the proximal tubules of the mouse kidney: ultrastructural correlates by a new technique. Journal of Histochemistry and Cytochemistry 14, 291-302. KAPELLER, K. & MAYOR, D. (1969). An electron microscope study of the early changes proximal to a constriction in sympathetic nerves. Proceedings of the Royal Society ofLondon B 172, 39-51. KRISTENSSON, K. & OLSSON, Y. (1976). Retrograde transport of horseradish peroxidase in transected axons. 3. Entry into injured axons and subsequent localisation in perikaryon. Brain Research 115, 201-213. LAVAIL, M. M. & LAVAIL, J. H. (1975). Retrograde intraaxonal transport of horseradish peroxidase in retinal ganglion cells of the chick. Brain' Research 85, 273-280.
