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Axonal uptake and retrograde transport of exogenous proteins in the hypoglossal nerve
BRAIN RESEARCH
399
A X O N A L U P T A K E A N D R E T R O G R A D E T R A N S P O R T OF E X O G E N O U S PROTEINS IN T H E H Y P O G L O S S A L NERVE
KRISTER KRISTENSSON, YNGVE OLSSON AND JOHAN SJOSTRAND Neuropathological Laboratory, Department of Pathology I, and Department of Histology, University of Gi~teborg, Gi~teborg (Sweden)
(Accepted March 26th, 1971)
INTRODUCTION Many neuronal constituents, which are synthesized in the cell body, are subsequently transported down the axon to reach the terminals25, 41. The mechanisms regulating this so-called 'anterograde axonal transport' are complex and various materials may be transported at different velocities presumably even within the same axon 24. It is possible that anterograde axonal transport is involved in the processes by which neurones exert so-called 'trophic influences' on their end-organs, i.e., nervous mechanisms not mediated by impulse activitylL The trophic relations between neurones and end-organs are presumably reciprocal, since the metabolism, growth and differentiation of nerve cells may be influenced by their peripheral field of innervation13, 31,39,40. The mechanisms by which various tissues exert such influences upon the neurones are largely unknown. Theoretically, signal substances may be taken up from the periphery by the axons and transported to the nerve cell bodies. However, our knowledge of a 'retrograde axonal transport' of materials is still very limited10,24,2L Recently, we have demonstrated that protein tracers such as fluorescent labelled albumin and horseradish peroxidase after injection into the gastrocnemius muscle of suckling mice can actually be taken up by axons and that they are subsequently transported to the perikaryon of the corresponding motor neurones in the spinal cord 21,23. The aim of the present study was to extend our observations on retrograde axonal transport to other species, age groups and kinds of nerve. The use of larger animals than the immature mouse made it possible to estimate the velocity of transport and to elucidate the effects of nerve crush and ischaemia, which inhibit anterograde axonal transport2S, 29. For this purpose the hypoglossal nerve was used since anterograde axonal transport has previously been extensively studied in this cranial nervea,35,a6. MATERIALSAND METHODS Animals Adult Sprague-Dawley rats (200-300 g), adult rabbits (2.0-2.5 kg), adult (35-40 g) and suckling (5-10 g) mice and adult guinea pigs (400-500 g) were used. Brain Research, 32 (1971) 399-406
400
K. KRISYENSSON et ~1[.
Protein tracers and histological techniques (1) One per cent (w/v) Evans blue (Merck AG, Darmstadt, Germany) in 5 % (w/v) bovine albumin (Fraction V, Nutritional Biochemical Co., Cleveland, Ohio, U.S.A.) abbreviated EBA. This complex shows a brilliant red fluorescence under ultraviolet irradiation, which permits its localization at the cellular level. The tissues were fixed in formalin overnight. Frozen sections, 10 ,urn thick, were cut and mounted in 50 % aqueous glycerine. The sections were viewed under a fluorescence microscope equipped with dark field condenser and light from an Osram HBO high pressure mercury lamp 37. (2) Horseradish peroxidase (P) in Ringer's solution (Type VI, Sigma Chemical Co., St. Louis, Mo., U.S.A.). To localize this tracer the animals were fixed by perfusion through the heart with a cacodylate-buffered mixture of glutaraldehyde and paraformaldehyde at pH 7.2 (ref. 17). The specimens were dissected out, washed in buffer and cut into sections 40 #m thick. The sections were incubated in a medium containing hydrogen peroxide and 5',5-diaminobenzidine tetrahydrochloride 11 and mounted in Entellan for light microscopy. To ascertain the specificity of the histochemical reaction, brain stem from normal rats and mice, and such injected with EBA into the tongue were fixed and incubated in the same manner.
Experimental procedure EBA was injected into the tongue of rats (0.1 ml), rabbits (0.6 ml), adult (0.05 ml) and 7-day-old (0.01 ml) mice and guinea pigs (0.2 ml); and peroxidase was injected into rats (5-10 mg), adult (6 mg) and 7- and 12-day-old mice (1-2 mg). From these animals the hypoglossal nerves and the brain stems with the hypoglossal nuclei were examined with regard to the possible occurrence of tracers. The number of animals and some other important data are presented in Table I. To estimate the velocity of the retrograde transport of EBA two rats and one rabbit were killed 2, 6, 10, 14, 26 and 48 h after injection of this tracer. One additional rabbit was taken after 6 and one after 10 h. To find out whether the retrograde transport of the tracers could be blocked by interruption of the axons, the left hypoglossal nerve from 4 rabbits, two rats and four 12-day-old mice was crushed by pressing the nerve firmly between a silk thread and a needle before the injection of the tracers into the tongue. In two of the rabits and the two rats the nerve was crushed immediately before the injection of EBA. In one rabbit the injury was made 10 days and in another 35 days before the injection. The hypoglossal nerves of the suckling mice were crushed immediately before injection of peroxidase (2 mg) into the tongue. Two of the suckling mice were killed 16 h after injection of the tracer, the other animals after 24 h. To elucidate the effects of ischaemia on the uptake and transport of proteins, two rats were bled to death immediately before the injection of EBA into the tongue and two other rats 2 h after the injection. The rats were left at room temperature for 24 h, after which the hypoglossal nuclei were removed. Brain Research, 32 (1971) 399-406
401
AXONALTRANSPORTOF PROTEIN TABLE I SURVEY OF THE MAJOR EXPERIMENTAL GROUPS
Experimental procedure
Species
No. of animals Tracer
Survival time(h)
Injection of tracer in the tongue
Rats Rats Rabbits Mice (adult) Mice (suckling) Mice* Guinea pigs
12 4 8 7 3 2 6
EBA P EBA EBA EBA P EBA
2-48 24 2-48 26--48 26 24 26~8
Injection of tracer into the Rats tongue ÷ unilateral crush Rabbits Mice (suckling) of the hypoglossal nerve
2 4 4
EBA EBA P
24 24 16-24
Injection of tracer into the tongue + ischaemia
Rats
4
EBA
24
Injection of non-labelled Rats albumin into the tongue + i.v. injection of tracer
2
EBA
24
* One adult and one suckling mouse. To test the possibility of a vascular transfer of the tracers to the nerve cell bodies two rats were injected in the tongue with 0.1 ml of a non-labelled 5 ~o albumin solution followed by intravenous injection with EBA (1 ml/100 g body weight). This injection was repeated after 24 h. Two hours later the rats were killed. RESULTS The results obtained with EBA as a tracer were similar both in rats and in rabbits and can therefore be described together. Two and 6 h after the injection of EBA into the tongue no tracer was seen in the hypoglossal neurones, but after 10 h faint red fluorescent granules were present in the cytoplasm of some of the neurones in both hypoglossal nuclei. The fluorescence intensity appeared to have increased somewhat after 14 h. After 26 and 48 h there was a more marked red fluorescence in most of the hypoglossal neurones. The red fluorescence was present in numerous small cytoplasmic granules which were most frequent around the nucleus. Fluorescent granules also extended out into the dendrites. A faint red fluorescence was detected in the nucleus and particularly in the nucleolus. The red fluorescence was restricted to the nerve cell bodies of the hypoglossal nucleus; consequently there was no red fluorescence in the surrounding neuropil or in the neurones in other nuclei in the vicinity of the hypoglossal region. No tracer was detected in the blood vessels in the brain stem; nor was there any red fluorescence in the endoneurium of the hypoglossal nerve. In the guinea pigs and adult mice no red fluorescence could be seen in the hypoglossal neurones whereas all 3 suckling mice showed accumulated red fluorescent granules in these cells Neuronal accumulation of tracer was not restricted to EBA-injected animals Brain Research, 32 (1971) 399-406
402
I'., K R I S ' I ' I ! N S S O N t'! ~l/.
Fig. 1. Accumulation of fluorescent material in hypoglossal neurones 26 h after injection of IBA into the tongue of an adult rabbit. Fig. 2. Hypoglossal neurones containing numerous cytoplasmic granules of peroxidase. This tracer was injected into the tongue of a 12-day-old mouse. A l m o s t all hypoglossal neurones in the suckling 7-day-old m o u s e c o n t a i n e d n u m e r o u s b r o w n o r black c y t o p l a s m i c granules after injection o f p e r o x i d a s e into the tongue. In the a d u l t mouse only a few neurones c o n t a i n e d c o r r e s p o n d i n g granules. In two o f the rats a few neurones in the hypoglossal nucleus c o n t a i n e d the reaction p r o d u c t , whereas in two o t h e r rats no peroxidase could be detected in the nerve cells. N o r e a c t i o n p r o d u c t was seen in hypoglossal neurones o f the controls. In the experiments in which one hypoglossal nerve had been crushed i m m e d i a t e l y o r 10 days before the injection o f EBA no red fluorescence was observed in hypoglossal neurones on the ipsilateral side. The neurones in the c o n t r a l a t e r a l hypoglossal nucleus c o n t a i n e d the tracer in the same m a n n e r as described above. The same effect was seen in c o r r e s p o n d i n g experiments with peroxidase. In f o u r 12-day-old mice m a n y o f the hypoglossal neurones on the c o n t r a l a t e r a l side contained granules o f peroxidase in their c y t o p l a s m whereas no such deposits were f o u n d on the side ipsilateral to the nerve crush. In the r a b b i t subjected to a crush injury 35 days before the injection o f
Fig. 3. Crushing the left hypoglossal nerve has prevented the accumulation of peroxidase in the ipsilateral hypoglossal nucleus (L). On the contralateral side (R) a marked accumulation of the tracer is present. Brain Research, 32 (1971) 399406
AXONAL TRANSPORT OF PROTEIN
403
EBA, permitting time for regeneration, the ipsilateral hypoglossal neurones showed an intense red fluorescence, which appeared to be even more marked than that of the contralateral hypoglossal neurones. The neuronal accumulation of EBA could also be blocked by ischaemia: neither in the rats killed immediately before injection of EBA nor after 2 h delay could any red fluorescence be detected in the neurones of the hypoglossal nuclei. The control rats, injected intravenously with EBA, showed no red fluorescence in the hypoglossal neurones. There were no signs of increased vascular permeability with neuronal accumulation of the tracer in the brain stem when the intravenous injection of EBA was combined with injection of non-labelled albumin into the tongue. DISCUSSION
Indications of the occurrence of bidirectional axonal transport have been obtained from studies on the accumulation of organelles and various chemical substances following sectioning or constriction of peripheral nerves. In such experiments a piling up of acetylcholinesterase25, norepinephrine 7, mitochondria2, la and labelled proteins 24 has been observed distal to the nerve injury. Such a phenomenon may well represent an interruption of a retrograde axonal transport, although objections have been raised that it may be the result of a local axonal reaction to the nerve injury 41. More conclusive evidence for the occurrence of a retrograde axonal transport came from the studies by Kerkut et al. is and Watson 8s. The former authors observed a transfer of glutamine at a velocity of about 15 mm per day from the gastrocnemius muscle to the spinal cord in frogs. Watson as also described the uptake of [3H]lysine into axons of the hypoglossal nerve after injection into the geniohyoid muscle. If the nerve were constricted, the radioactivity was found distal to the injury but not on the proximal side. Studies on nervous tissue in vitro have also shown that various organelles can move in both directions in axons 15,27,32. The results of the present study show, in accordance with our earlier observations on suckling miceel, 23, that exogenous proteins can be taken up by axons of peripheral nerves and then transported to the nerve cell bodies. Previously, peroxidase was detected ultrastructurally inside axons in the peripheral nerve of suckling mice 23, whereas in the present study no tracers could be seen intraaxonally owing to the limited resolution of the light microscopic technique. The theoretical possibility of a spread of the tracers to the nerve cell bodies via other compartments than the axons, i.e., blood or endoneurial spaces, could be ruled out since it could be prevented by acute crush injuries to the hypoglossal nerve. Furthermore, the vascular permeability to proteins in the brain stem did not change after injection of non-labelled protein into the tongue and there was no tracer in the surrounding neuropil. The neuronal uptake of peroxidase and EBA was much more marked in suckling than in adult mice. A similar age-dependent difference was also observed in the previous study 21 in which a marked uptake of EBA into the motor neurones of the spinal cord was found in suckling but not in adult mice after intramuscular injection of the tracer. In the present study, however, the axonal uptake and retrograde transBrain Research, 32 (1971) 399-406
404
i<. KRISTENSSON('l a].
port of proteins was not restricted to immature animals, since both adult rats and rabbits revealed a marked neuronal uptake of EBA. On the other hand no neuronal uptake of the tracer was found in adult guinea pigs, but precautions must be taken in comparing the degree of protein uptake between animals of different ages and species. For instance, differences in protein concentration at the site of injection (which is hard to control) may well influence the uptake of protein in axons. In studies on other cell populations the concentration of protein around the cells was a major factor determining the degree of protein uptake 6. In adult animals, contrary to immature ones, the perineurium constitutes a barrier for proteins to diffuse into the endoneurium and come in contact with the axons19,22, 30. However, it has recently been demonstrated that in some nerves the perineurium is open-ended peripherallyS, 34 and that peroxidase can reach the axonal ending at the neuromuscular junctions 4e. Though the detailed ways by which proteins enter the axons in the hypoglossal nerve are still unknown it is plausible that the portal of entry is in the vicinity of the motor end-plates. The length of the hypoglossal nerve from the tongue to the brain stem in the adult rabbit is approximately 50 ram. The appearance of the fluorescent tracer in the nerve cell bodies 10 h after injection shows that the rate of the retrograde transport is at least 120 mm per day in the rabbit. From experiments with double ligatures on dog peroneus nerve, Lubifiska and Niemierko '~6 estimated the velocity for a retrograde transport of acetylcholine esterase at 134 mm per day. Previous studies have shown a rapid transport of axonal proteins in the anterograde direction at a rate of approximately 300 mm per day in the hypoglossal nerve of the rabbit35, 36. Accordingly there seems to be a rapid two-way traffic of material in this nerve. The mechanism by which the retrograde axonal transport is maintained is not clear. There are strong indications that the anterograde fast transport is due to an oxygen-requiring process present locally in the nerve fibers 29. It is reasonable to assume that this also holds true for retrograde transport since it could be blocked by the arrest of blood circulation. The nerve cells of the central nervous system (CNS) were formerly considered unable to take up exogenous proteins 33. However, neuronal uptake of ferritin and peroxidase has recently been demonstrated in vitro and in neurones located outside the CNS 14,a'3, but in the CNS the study of neuronal uptake of proteins has been difficult since the so-called 'blood-brain barrier' prevents proteins from reaching the nerve cells after intravenous administration 4. The present results are thus of considerable interest since they show that m o t o r neurones in the CNS of normal animals also accumulate exogenous proteins via their axons located outside the CNS. The demonstrated capacity of motor neurones to take up and subsequently transport exogenous macromolecules in axons to the nerve cell body is of importance for the understanding of many neurobiological phenomena. For instance, it might provide a system by which an end-organ could exert 'trophic' influences on the nerve cell body. Furthermore this finding provides a strong argument for the hypothesis that certain neurovirulent viruses and toxins, which spread to the CNS along peripheral nerves, are actually transported intraaxonallyl,a,9, 20. Brain Research, 32 (1971) 399406
AXONAL TRANSPORT OF PROTEIN
405
SUMMARY
E x o g e n o u s p r o t e i n s ( a l b u m i n labelled with Evans blue (EBA) and h o r s e r a d i s h p e r o x i d a s e ) were injected into the t o n g u e o f v a r i o u s l a b o r a t o r y a n i m a l s (rats, rabbits, mice a n d g u i n e a pigs). A t v a r i o u s t i m e intervals t h e r e a f t e r the h y p o g l o s s a l nuclei were e x a m i n e d with r e g a r d to the cytological l o c a l i z a t i o n o f the tracers. In the h y p o g l o s s a l n e u r o n e s red fluorescent c y t o p l a s m i c granules were seen 10 h after injection o f E B A into a d u l t rats a n d r a b b i t s indicating the presence o f p r o t e i n tracer. P e r o x i d a s e also a c c u m u l a t e d in these n e u r o n e s o f suckling a n d a d u l t mice a n d rats. The n e u r o n a l u p t a k e o f the p r o t e i n tracers was b l o c k e d by crush injuries to the h y p o g l o s s a l nerve a n d by a r r e s t o f b l o o d circulation. O u r results show t h a t the h y p o g l o s s a l n e u r o n e s have the c a p a c i t y to a c c u m u l a t e exogenous p r o t e i n s after p e r i p h e r a l injection. This process m u s t be a consequence o f a fast r e t r o g r a d e a x o n a l t r a n s p o r t . A s shown in p r e v i o u s studies, a similar t r a n s p o r t also occurs in the p e r i p h e r a l m o t o r n e u r o n e s o f the spinal c o r d in suckling mice. This p h e n o m e n o n therefore exists in different species, ages a n d kinds o f nerve. ACKNOWLEDGEMENT
S u p p o r t e d by grants f r o m the Swedish M e d i c a l R e s e a r c h Council, Projects N o . B72-12X-3020-03A a n d B71-12X-82-07C.
REFERENCES 1 BARINGER, J. R., AND GRIFFITH, J. F., Experimental herpes simplex encephalitis: early neuro-
pathologic changes, d. Neuropath. exp. Neurol., 29 (1970) 89-104. 2 BLUMCKE,S., NIEDORF, H. R., AND RODE, J., Axoplasmic alterations in the proximal and distal stumps of transsected nerves, Acta neuropath. (Bed.), 7 (1966) 44-61. 3 BODIAN, n., ANn HOWE, H. A., The rate of progression of poliomyelitisvirus in nerves, Bull. Johns Hopk. Hosp., 69 (1941) 79-85. 4 BRIGHTMAN,M. W., KLATZO,I., OLSSON,Y., AND REESE,T. S., The blood-brain barrier to proteins under normal and pathological conditions, J. neurol. Sci., I0 (1970) 215-239. 5 BURKEL, W. E., The histological fine structure of perineurium, Anat. Rec., 158 (1967) 177-190. 6 CHAPMAN-ANDRESEN,C., AND CHRISTENSEN, S., Pinocytic uptake of ferritin by the amoeba Chaos Chaos measured by atomic absorption of iron, C.R. Lab. Carlsberg, 38 (1970) 19-57. 7 DAHLSTR~M, A., Observations on the accumulation of noradrenaline in the proximal and distal parts of peripheral adrenergic nerves after compression, J. Anat. (Lond.), 99 (1965) 677-689. 8 FRIZELL,M., HASSELGREN,P. O., ANDSJOSTRAND,J., Axoplasmic transport of acetylcholinesterase and choline acetyltransferase in the vagus and hypoglossal nerve of the rabbit, Exp. Brain Res., 10 (1970) 526-531. 9 GOODPASTURE, E. W., The axis-cylinders of peripheral nerves as portals of entry to the central nervous system for the virus of herpes simplex in experimentally infected rabbits, Amer. J. Path., 1 (1925) 11-28. 10 GRAFSTEIN, B., Axonal transport: communication between soma and synapse, Advanc. Biochem. Psychopharmaeol., 1 (1969) 11-25. 11 GRAHAM, R. C., AND KARNOVSKY, M. J., The early stages of absorption of injected horseradish peroxidase in the proximal tubules of morse kidney: Ultrastructural correlates by a new technique, J. Histochem. Cytochem., 14 (1966) 291-299. 12 GUTH, L., 'Trophic' effects of vertebrate neurons, Neurosci. Res. Progr. Bull., 7 (1969) 1-73. 13 HAMBURGER,V., Trends in experimental neuroembryology. In H. WAELSCH(Ed.), Biochemistry of the Developing Nervous System, Academic Press, New York, 1955, pp. 52-71. Brain Research, 32 (1971) 399-406
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K. KRISTENSSON ~'! gl/.
14 HOLTZMAN, E., AND PETERSON, E., Cytochemical studies of protein uptake, bsosomes and GERL in neurons, J. Histochem. Cvtochem., 16 (1968) 503. 15 HUGHES, A., The growth of embryonic neuritis. A study on cultures o!" chick neural tissue, .I. Anat. (Lond.), 87 (1953) 150 162. 16 KAPELLER, K., AND MAYOR, D., An electron microscopic study of the early changes distal to a constriction in sympathetic nerves, Proc. roy. Soc. B., 172 (1969) 53-63. 17 KARNOVSKV, M. J., A formaldehyde glutaraldehyde fixative of high osmolarity for use in electron microscopy, J. Cell Biol., 27 (1965) 137Z. 18 KERKUT, G. A., SHAPIRA, A., AND WALKER, R. J., The transport of 14C-labelled material from CNS muscle along a nerve trunk, Comp. Biochem. Physiol., 23 (1967) 729-748. 19 KLEMM, H. Das Perineurium als Diffusionsbarriere gegentiber Peroxydase bei epi- und endoneuraler Applikation, Z. Zellforsch., 108 (1970) 431-495. 20 KmSXENSSON, K., Morphological studies of the neural spread of herpes simplex virus to the central nervous system, Acta neuropath. (Berl.), 16 (1970) 54-63. 21 KRISTENSSON, K., Transport of fluorescent protein tracer in peripheral nerves, Acta neuropath. (Berl.), 16 (1970) 293-300. 22 KRISTENSSON, K., AND OLSSON, Y., The perineurium as a diffusion barrier to protein tracers, Acta neuropath. (Berl.), 17 (1971) 127-138. 23 KRmTENSSON, K., AND OLSSON, Y., Retrograde axonal transport of protein, Brain Research, 29 (1971) 363-365. 24 LASEK, R. J., Protein transport in neurons, Int. Rev. Neurobiol., 13 (1970) 289-324. 25 LUmRSKA, L., Axoplasmic streaming in regenerating and in normal nerve fibres. In M. SINGER AND J. P. SCHADk (Eds.), Mechanisms of Neural Regeneration, Progress in Brain Research, Vol. 13, Elsevier, Amsterdam, 1964, pp. 1-71. 26 LUmrqSKA, L., AND NIEMIERKO, S., Velocity and intensity of bidirectional migration of AChE in transected nerves, Brain Research, 27 (1971) 329-342. 27 NAKA~, J., Dissociated dorsal root ganglia in tissue culture, Amer. J. Anat.. 99 (t956) 81-129. 28 OCHS, S., AND RANISH, N., Characteristics of the fast transport system in mammalian nerve fibres, J. Neurobiol., 1 (1969) 247-261. 29 OCHS, S., AND RAMSH, N., Metabolic dependence of fast axoplasmic transport in nerve, Science, 167 (1970) 878 879. 30 OLSSON, Y., Studies on vascular permeability in peripheral nerves. I. Distribution of circulating fluorescent serum albumin in normal, crushed and sectioned rat sciatic nerve, Aeta neuropath. (Berl.), 7 (1966) 1-15. 31 PANNESE, E., Investigations on the ultrastructural changes of the spinal ganglion neurones in the course of axon regeneration and hypertrophy, Z. Zellforseh., 61 (1963) 561-586. 32 POMERAT, C. M., HENDELMAN, W. J., RMBORN, C. W., AND MASSEV, J. F., Dynamic activities of nervous tissue in vitro. In H. HYDkN (Ed.), The Neuron, Elsevier, Amsterdam, 1967, pp. 119-178. 33 ROSENBLUTH,J., AND WISSIG, S. L., The distribution of exogenous ferritin in toad spinal ganglia and the mechanism of its uptake of neurons, J. Cell Biol., 23 (1964) 307-325. 34 SAITO, A., AND ZACHS, S. J., Ultrastructure of Schwann and perineurial sbeeths at the mouse neuromuscular junction, Anat. Rec., 164 (1969) 379-390. 35 SJ0STRAND, J., Rapid exoplasmic transport of labelled protein in the vagus and hypoglossal nerves of the rabbit, Exp. Brain Res., 8 (1969) 105-112. 36 SJ0STRAND,J., Fast and slow components of axoplasmic transport in the hypoglossal and vagus nerves of the rabbit, Brain Research, 18 (1970) 461~-67. 37 STHNWALL, O., AND KLA'rZO, I., Double tracer methods in studies on bloodbrain barrier dysfunction and brain edema, Acta neurol, scand., 41 (1965) 591-595. 38 WATSON, W. E., Centripetal passage of labelled molecules along mammalian motor axons, J. Physiol. (Lond.), 196 (1968) 122-123P. 39 WATSON, W. E., Some metabolic responses of axotomized neurones to contact between their axons and denervated muscle, J. Physiol. (Lond.), 210 (1970) 321-343. 40 WHss, P. A., Peripheral effects on central development. In B. H. W~LLIER, P. A. WHSS AND V. HAMBURGER (Eds.), Analysis of Development, Saunders, Philadelphia, 1955, pp. 380-386. 41 WHSS, P. A., Neuronal dynamics and neuroplasmic ('axonal') flow, Syrup. Int. Soc. Cell Biol., 8 (1969) 3 34. 42 ZACKS, S. J., AND SAITO, A., Uptake of exogenous horseradish peroxidase by coated vesicles in mouse neuromuscular junctions, J. Histochem. Cytoehem., 17 (1969) 161-170.
Brain Research, 32 (1971) 399-406
399
A X O N A L U P T A K E A N D R E T R O G R A D E T R A N S P O R T OF E X O G E N O U S PROTEINS IN T H E H Y P O G L O S S A L NERVE
KRISTER KRISTENSSON, YNGVE OLSSON AND JOHAN SJOSTRAND Neuropathological Laboratory, Department of Pathology I, and Department of Histology, University of Gi~teborg, Gi~teborg (Sweden)
(Accepted March 26th, 1971)
INTRODUCTION Many neuronal constituents, which are synthesized in the cell body, are subsequently transported down the axon to reach the terminals25, 41. The mechanisms regulating this so-called 'anterograde axonal transport' are complex and various materials may be transported at different velocities presumably even within the same axon 24. It is possible that anterograde axonal transport is involved in the processes by which neurones exert so-called 'trophic influences' on their end-organs, i.e., nervous mechanisms not mediated by impulse activitylL The trophic relations between neurones and end-organs are presumably reciprocal, since the metabolism, growth and differentiation of nerve cells may be influenced by their peripheral field of innervation13, 31,39,40. The mechanisms by which various tissues exert such influences upon the neurones are largely unknown. Theoretically, signal substances may be taken up from the periphery by the axons and transported to the nerve cell bodies. However, our knowledge of a 'retrograde axonal transport' of materials is still very limited10,24,2L Recently, we have demonstrated that protein tracers such as fluorescent labelled albumin and horseradish peroxidase after injection into the gastrocnemius muscle of suckling mice can actually be taken up by axons and that they are subsequently transported to the perikaryon of the corresponding motor neurones in the spinal cord 21,23. The aim of the present study was to extend our observations on retrograde axonal transport to other species, age groups and kinds of nerve. The use of larger animals than the immature mouse made it possible to estimate the velocity of transport and to elucidate the effects of nerve crush and ischaemia, which inhibit anterograde axonal transport2S, 29. For this purpose the hypoglossal nerve was used since anterograde axonal transport has previously been extensively studied in this cranial nervea,35,a6. MATERIALSAND METHODS Animals Adult Sprague-Dawley rats (200-300 g), adult rabbits (2.0-2.5 kg), adult (35-40 g) and suckling (5-10 g) mice and adult guinea pigs (400-500 g) were used. Brain Research, 32 (1971) 399-406
400
K. KRISYENSSON et ~1[.
Protein tracers and histological techniques (1) One per cent (w/v) Evans blue (Merck AG, Darmstadt, Germany) in 5 % (w/v) bovine albumin (Fraction V, Nutritional Biochemical Co., Cleveland, Ohio, U.S.A.) abbreviated EBA. This complex shows a brilliant red fluorescence under ultraviolet irradiation, which permits its localization at the cellular level. The tissues were fixed in formalin overnight. Frozen sections, 10 ,urn thick, were cut and mounted in 50 % aqueous glycerine. The sections were viewed under a fluorescence microscope equipped with dark field condenser and light from an Osram HBO high pressure mercury lamp 37. (2) Horseradish peroxidase (P) in Ringer's solution (Type VI, Sigma Chemical Co., St. Louis, Mo., U.S.A.). To localize this tracer the animals were fixed by perfusion through the heart with a cacodylate-buffered mixture of glutaraldehyde and paraformaldehyde at pH 7.2 (ref. 17). The specimens were dissected out, washed in buffer and cut into sections 40 #m thick. The sections were incubated in a medium containing hydrogen peroxide and 5',5-diaminobenzidine tetrahydrochloride 11 and mounted in Entellan for light microscopy. To ascertain the specificity of the histochemical reaction, brain stem from normal rats and mice, and such injected with EBA into the tongue were fixed and incubated in the same manner.
Experimental procedure EBA was injected into the tongue of rats (0.1 ml), rabbits (0.6 ml), adult (0.05 ml) and 7-day-old (0.01 ml) mice and guinea pigs (0.2 ml); and peroxidase was injected into rats (5-10 mg), adult (6 mg) and 7- and 12-day-old mice (1-2 mg). From these animals the hypoglossal nerves and the brain stems with the hypoglossal nuclei were examined with regard to the possible occurrence of tracers. The number of animals and some other important data are presented in Table I. To estimate the velocity of the retrograde transport of EBA two rats and one rabbit were killed 2, 6, 10, 14, 26 and 48 h after injection of this tracer. One additional rabbit was taken after 6 and one after 10 h. To find out whether the retrograde transport of the tracers could be blocked by interruption of the axons, the left hypoglossal nerve from 4 rabbits, two rats and four 12-day-old mice was crushed by pressing the nerve firmly between a silk thread and a needle before the injection of the tracers into the tongue. In two of the rabits and the two rats the nerve was crushed immediately before the injection of EBA. In one rabbit the injury was made 10 days and in another 35 days before the injection. The hypoglossal nerves of the suckling mice were crushed immediately before injection of peroxidase (2 mg) into the tongue. Two of the suckling mice were killed 16 h after injection of the tracer, the other animals after 24 h. To elucidate the effects of ischaemia on the uptake and transport of proteins, two rats were bled to death immediately before the injection of EBA into the tongue and two other rats 2 h after the injection. The rats were left at room temperature for 24 h, after which the hypoglossal nuclei were removed. Brain Research, 32 (1971) 399-406
401
AXONALTRANSPORTOF PROTEIN TABLE I SURVEY OF THE MAJOR EXPERIMENTAL GROUPS
Experimental procedure
Species
No. of animals Tracer
Survival time(h)
Injection of tracer in the tongue
Rats Rats Rabbits Mice (adult) Mice (suckling) Mice* Guinea pigs
12 4 8 7 3 2 6
EBA P EBA EBA EBA P EBA
2-48 24 2-48 26--48 26 24 26~8
Injection of tracer into the Rats tongue ÷ unilateral crush Rabbits Mice (suckling) of the hypoglossal nerve
2 4 4
EBA EBA P
24 24 16-24
Injection of tracer into the tongue + ischaemia
Rats
4
EBA
24
Injection of non-labelled Rats albumin into the tongue + i.v. injection of tracer
2
EBA
24
* One adult and one suckling mouse. To test the possibility of a vascular transfer of the tracers to the nerve cell bodies two rats were injected in the tongue with 0.1 ml of a non-labelled 5 ~o albumin solution followed by intravenous injection with EBA (1 ml/100 g body weight). This injection was repeated after 24 h. Two hours later the rats were killed. RESULTS The results obtained with EBA as a tracer were similar both in rats and in rabbits and can therefore be described together. Two and 6 h after the injection of EBA into the tongue no tracer was seen in the hypoglossal neurones, but after 10 h faint red fluorescent granules were present in the cytoplasm of some of the neurones in both hypoglossal nuclei. The fluorescence intensity appeared to have increased somewhat after 14 h. After 26 and 48 h there was a more marked red fluorescence in most of the hypoglossal neurones. The red fluorescence was present in numerous small cytoplasmic granules which were most frequent around the nucleus. Fluorescent granules also extended out into the dendrites. A faint red fluorescence was detected in the nucleus and particularly in the nucleolus. The red fluorescence was restricted to the nerve cell bodies of the hypoglossal nucleus; consequently there was no red fluorescence in the surrounding neuropil or in the neurones in other nuclei in the vicinity of the hypoglossal region. No tracer was detected in the blood vessels in the brain stem; nor was there any red fluorescence in the endoneurium of the hypoglossal nerve. In the guinea pigs and adult mice no red fluorescence could be seen in the hypoglossal neurones whereas all 3 suckling mice showed accumulated red fluorescent granules in these cells Neuronal accumulation of tracer was not restricted to EBA-injected animals Brain Research, 32 (1971) 399-406
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I'., K R I S ' I ' I ! N S S O N t'! ~l/.
Fig. 1. Accumulation of fluorescent material in hypoglossal neurones 26 h after injection of IBA into the tongue of an adult rabbit. Fig. 2. Hypoglossal neurones containing numerous cytoplasmic granules of peroxidase. This tracer was injected into the tongue of a 12-day-old mouse. A l m o s t all hypoglossal neurones in the suckling 7-day-old m o u s e c o n t a i n e d n u m e r o u s b r o w n o r black c y t o p l a s m i c granules after injection o f p e r o x i d a s e into the tongue. In the a d u l t mouse only a few neurones c o n t a i n e d c o r r e s p o n d i n g granules. In two o f the rats a few neurones in the hypoglossal nucleus c o n t a i n e d the reaction p r o d u c t , whereas in two o t h e r rats no peroxidase could be detected in the nerve cells. N o r e a c t i o n p r o d u c t was seen in hypoglossal neurones o f the controls. In the experiments in which one hypoglossal nerve had been crushed i m m e d i a t e l y o r 10 days before the injection o f EBA no red fluorescence was observed in hypoglossal neurones on the ipsilateral side. The neurones in the c o n t r a l a t e r a l hypoglossal nucleus c o n t a i n e d the tracer in the same m a n n e r as described above. The same effect was seen in c o r r e s p o n d i n g experiments with peroxidase. In f o u r 12-day-old mice m a n y o f the hypoglossal neurones on the c o n t r a l a t e r a l side contained granules o f peroxidase in their c y t o p l a s m whereas no such deposits were f o u n d on the side ipsilateral to the nerve crush. In the r a b b i t subjected to a crush injury 35 days before the injection o f
Fig. 3. Crushing the left hypoglossal nerve has prevented the accumulation of peroxidase in the ipsilateral hypoglossal nucleus (L). On the contralateral side (R) a marked accumulation of the tracer is present. Brain Research, 32 (1971) 399406
AXONAL TRANSPORT OF PROTEIN
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EBA, permitting time for regeneration, the ipsilateral hypoglossal neurones showed an intense red fluorescence, which appeared to be even more marked than that of the contralateral hypoglossal neurones. The neuronal accumulation of EBA could also be blocked by ischaemia: neither in the rats killed immediately before injection of EBA nor after 2 h delay could any red fluorescence be detected in the neurones of the hypoglossal nuclei. The control rats, injected intravenously with EBA, showed no red fluorescence in the hypoglossal neurones. There were no signs of increased vascular permeability with neuronal accumulation of the tracer in the brain stem when the intravenous injection of EBA was combined with injection of non-labelled albumin into the tongue. DISCUSSION
Indications of the occurrence of bidirectional axonal transport have been obtained from studies on the accumulation of organelles and various chemical substances following sectioning or constriction of peripheral nerves. In such experiments a piling up of acetylcholinesterase25, norepinephrine 7, mitochondria2, la and labelled proteins 24 has been observed distal to the nerve injury. Such a phenomenon may well represent an interruption of a retrograde axonal transport, although objections have been raised that it may be the result of a local axonal reaction to the nerve injury 41. More conclusive evidence for the occurrence of a retrograde axonal transport came from the studies by Kerkut et al. is and Watson 8s. The former authors observed a transfer of glutamine at a velocity of about 15 mm per day from the gastrocnemius muscle to the spinal cord in frogs. Watson as also described the uptake of [3H]lysine into axons of the hypoglossal nerve after injection into the geniohyoid muscle. If the nerve were constricted, the radioactivity was found distal to the injury but not on the proximal side. Studies on nervous tissue in vitro have also shown that various organelles can move in both directions in axons 15,27,32. The results of the present study show, in accordance with our earlier observations on suckling miceel, 23, that exogenous proteins can be taken up by axons of peripheral nerves and then transported to the nerve cell bodies. Previously, peroxidase was detected ultrastructurally inside axons in the peripheral nerve of suckling mice 23, whereas in the present study no tracers could be seen intraaxonally owing to the limited resolution of the light microscopic technique. The theoretical possibility of a spread of the tracers to the nerve cell bodies via other compartments than the axons, i.e., blood or endoneurial spaces, could be ruled out since it could be prevented by acute crush injuries to the hypoglossal nerve. Furthermore, the vascular permeability to proteins in the brain stem did not change after injection of non-labelled protein into the tongue and there was no tracer in the surrounding neuropil. The neuronal uptake of peroxidase and EBA was much more marked in suckling than in adult mice. A similar age-dependent difference was also observed in the previous study 21 in which a marked uptake of EBA into the motor neurones of the spinal cord was found in suckling but not in adult mice after intramuscular injection of the tracer. In the present study, however, the axonal uptake and retrograde transBrain Research, 32 (1971) 399-406
404
i<. KRISTENSSON('l a].
port of proteins was not restricted to immature animals, since both adult rats and rabbits revealed a marked neuronal uptake of EBA. On the other hand no neuronal uptake of the tracer was found in adult guinea pigs, but precautions must be taken in comparing the degree of protein uptake between animals of different ages and species. For instance, differences in protein concentration at the site of injection (which is hard to control) may well influence the uptake of protein in axons. In studies on other cell populations the concentration of protein around the cells was a major factor determining the degree of protein uptake 6. In adult animals, contrary to immature ones, the perineurium constitutes a barrier for proteins to diffuse into the endoneurium and come in contact with the axons19,22, 30. However, it has recently been demonstrated that in some nerves the perineurium is open-ended peripherallyS, 34 and that peroxidase can reach the axonal ending at the neuromuscular junctions 4e. Though the detailed ways by which proteins enter the axons in the hypoglossal nerve are still unknown it is plausible that the portal of entry is in the vicinity of the motor end-plates. The length of the hypoglossal nerve from the tongue to the brain stem in the adult rabbit is approximately 50 ram. The appearance of the fluorescent tracer in the nerve cell bodies 10 h after injection shows that the rate of the retrograde transport is at least 120 mm per day in the rabbit. From experiments with double ligatures on dog peroneus nerve, Lubifiska and Niemierko '~6 estimated the velocity for a retrograde transport of acetylcholine esterase at 134 mm per day. Previous studies have shown a rapid transport of axonal proteins in the anterograde direction at a rate of approximately 300 mm per day in the hypoglossal nerve of the rabbit35, 36. Accordingly there seems to be a rapid two-way traffic of material in this nerve. The mechanism by which the retrograde axonal transport is maintained is not clear. There are strong indications that the anterograde fast transport is due to an oxygen-requiring process present locally in the nerve fibers 29. It is reasonable to assume that this also holds true for retrograde transport since it could be blocked by the arrest of blood circulation. The nerve cells of the central nervous system (CNS) were formerly considered unable to take up exogenous proteins 33. However, neuronal uptake of ferritin and peroxidase has recently been demonstrated in vitro and in neurones located outside the CNS 14,a'3, but in the CNS the study of neuronal uptake of proteins has been difficult since the so-called 'blood-brain barrier' prevents proteins from reaching the nerve cells after intravenous administration 4. The present results are thus of considerable interest since they show that m o t o r neurones in the CNS of normal animals also accumulate exogenous proteins via their axons located outside the CNS. The demonstrated capacity of motor neurones to take up and subsequently transport exogenous macromolecules in axons to the nerve cell body is of importance for the understanding of many neurobiological phenomena. For instance, it might provide a system by which an end-organ could exert 'trophic' influences on the nerve cell body. Furthermore this finding provides a strong argument for the hypothesis that certain neurovirulent viruses and toxins, which spread to the CNS along peripheral nerves, are actually transported intraaxonallyl,a,9, 20. Brain Research, 32 (1971) 399406
AXONAL TRANSPORT OF PROTEIN
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SUMMARY
E x o g e n o u s p r o t e i n s ( a l b u m i n labelled with Evans blue (EBA) and h o r s e r a d i s h p e r o x i d a s e ) were injected into the t o n g u e o f v a r i o u s l a b o r a t o r y a n i m a l s (rats, rabbits, mice a n d g u i n e a pigs). A t v a r i o u s t i m e intervals t h e r e a f t e r the h y p o g l o s s a l nuclei were e x a m i n e d with r e g a r d to the cytological l o c a l i z a t i o n o f the tracers. In the h y p o g l o s s a l n e u r o n e s red fluorescent c y t o p l a s m i c granules were seen 10 h after injection o f E B A into a d u l t rats a n d r a b b i t s indicating the presence o f p r o t e i n tracer. P e r o x i d a s e also a c c u m u l a t e d in these n e u r o n e s o f suckling a n d a d u l t mice a n d rats. The n e u r o n a l u p t a k e o f the p r o t e i n tracers was b l o c k e d by crush injuries to the h y p o g l o s s a l nerve a n d by a r r e s t o f b l o o d circulation. O u r results show t h a t the h y p o g l o s s a l n e u r o n e s have the c a p a c i t y to a c c u m u l a t e exogenous p r o t e i n s after p e r i p h e r a l injection. This process m u s t be a consequence o f a fast r e t r o g r a d e a x o n a l t r a n s p o r t . A s shown in p r e v i o u s studies, a similar t r a n s p o r t also occurs in the p e r i p h e r a l m o t o r n e u r o n e s o f the spinal c o r d in suckling mice. This p h e n o m e n o n therefore exists in different species, ages a n d kinds o f nerve. ACKNOWLEDGEMENT
S u p p o r t e d by grants f r o m the Swedish M e d i c a l R e s e a r c h Council, Projects N o . B72-12X-3020-03A a n d B71-12X-82-07C.
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