Brain Research, 114 (1976)293-303 © ElsevierScientificPublishing Company, Amsterdam - Printed in The Netherlands
293
SELECTIVE U P T A K E A N D 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 OF DOPAMINE-fl-HYDROXYLASE ANTIBODIES IN P E R I P H E R A L A D R E N ERGIC NEURONS
M. FILLENZ*, C. GAGNON, K. STOECKEL and H. THOENEN Department of Pharmacology, Biocenter of the University, Basel (Switzerland)
(Accepted February 10th, 1976)
SUMMARY In the present experiments the uptake and retrograde axonal transport of antibodies to dopamine fl-hydroxylase (DBH) in adrenergic neurons was studied. When partially purified labelled antibodies to DBH were injected unilaterally into the vicinity of the adrenergic nerve terminals in the iris, radioactive substances accumulated preferentially in the superior cervical ganglia of the injected side. By SDS (sodium dodecyl sulfate) gel electrophoresis and immunoprecipitation it could be shown that the accumulated radioactivity in the superior cervical ganglion represented antibodies to DBH. This retrograde accumulation was greatly reduced by colchicine, axotomy or destruction of the adrenergic nerve terminals by 6-hydroxydopamine. The rate of retrograde transport was the same as that of nerve growth factor ( N G F ) and tetanus toxin in sympathetic neurons. The retrograde transport of antibodies was confined to sympathetic neurons and could not be detected in either sensory or motor neurons.
INTRODUCTION In previous studies it has been shown that macromolecules are taken up with high selectivity by nerve terminals and are transported retrogradely to the corresponding cell bodies6,7,16,17. The selectivity of uptake can depend on properties which are inherent in all nerve terminals, as seems to be the case for the uptake and retrograde transport of tetanus toxin is, or the binding and uptake can be restricted to a selective population of neurons, as is the case for nerve growth factor (NGF)17, is. Thus, we can delineate varying degrees of selectivity of retrograde transport, i.e., a selectivity * Present address: University Laboratory of Physiology, Parks Road, Oxford OX1 3PT, Great Britain.
294 with respect to the macromolecules which are transportedX~, xr, and additionally a selectivity with respect to the species of neurons is. Recently, histochemical evidence has been presented for accumulation of antibodies to dopamine fl-hydroxylase (DBH) in adrenergic neurons after injection into the general circulation s. Since DBH, the enzyme which catalyzes the final step of noradrenaline synthesis, is exclusively located in adrenergic neurons and is predominantly located in noradrenaline storage vesicles 12the question arises as to whether this accumulation is confined to adrenergic neurons, whether the accumulation results from a direct accumulation by the adrenergic cell body or from an uptake by the nerve terminals followed by retrograde axonal transport, and whether the mechanism of retrograde transport is similar to that investigated previously for other macromoleculesT, is. The present experiments were designed to answer these questions. MATERIALS AND METHODS
Production of DBH-antibodies DBH was purified from the chromaffin granule lysate of beef adrenal medulla. Granules were isolated by the method of Smith and Winkler 14 and lysed with a low ionic strength (0.005 M) phosphate buffer. Each pellet of chromaffin granules (originating from 15 g of tissue) was resuspended in 2 ml of 0.005 M sodium phosphate buffer, pH 7.2 and centrigufed at 40,000 × g for 30 min. DBH from the lysate was purified by chromatography on DEAE cellulose and Concanavalin A - Sepharose 4B as will be described in detail elsewhere (Gagnon, in preparation). Purified DBH was mixed with the complete Freund's adjuvant in order to produce a water in oil emulsion. The mixture (0.8 ml) containing 200 #g of DBH was injected into the rear foot paws of 1.5 kg 'White Wiener' rabbits. Three weeks later the booster injection (50 #g of DBH in 0.15 M NaC1) was given subcutaneously. The antiserum was collected weekly starting 12 days after the booster injection. The antibodies against DBH were judged to be monospecific by double immunodiffusion and immunoelectrophoresis is against crude or purified DBH.
Partialpurification of DBH-antibodies DBH-antiserum was precipitated with an equal volume of saturated ammonium sulfate solution and stirred for 30 min at 0-4 °C. The precipitate was centrifuged at 10,000 × g for 10 min, the pellet dissolved in 0.05 M sodium phosphate buffer, pH 7.8 and extensively dialyzed against this buffer.
Labelling ofpartially purified DBH-antibodies and NGF The labelling was performed with Na125I and chloramine T according to Greenwood et al. 5 with minor modifications described in detail by Stoeckel et al? 6. Nal~5I was provided by EIR, Wiirenlingen, Switzerland, at a specific activity of 8-15 Ci/mg iodine. Each preparation was performed using either 200 #g of N G F or 200 #g protein of partially purfied antibodies (see above) in 25 #1 of 0.05 M phosphate buffer, pH 7.8 and 5 mCi of Nal25[.
295 The specific activity of the labelled N G F was 8 ffCi/fg; that of the labelled partially purified DBH-antibodies, 6 #Ci/#g of protein. The stock solution used for injection of [125I] N G F was about 350 #g/ml; that of the partially purified DBH-antibodies contained about 600 fig protein/ml.
Precipitation of labelled DBH-antibodies Serum DBH-antibodies were precipitated by an excess of crude DBH. To 40 #1 of iodinated partially purified serum was added 80 ffl of chromaffin granule lysate. The mixture was incubated for 1 h at 37 °C and 1 h at 0-4 °C, then centrifuged at 15,000 x g for 10 min. The supernatant was collected and 40 ffl of chromaffin granule lysate was added to it. After repetition of the incubation and centrifugation procedure the supernatant was collected and used as control serum.
Radioactivity The radioactivity of the samples was determined directly in a Packard 7-counter Mod. 3001 at a counting efficiency of 65~. The counts/rain values are not corrected for the counting efficiency.
SDS gel electrophoresis A pool of 5 ganglia were homogenized in 0.5 ml of 0.005 M Tris-HC1-Triton X-100 (0.1~) buffer, pH 7.4 and centrifuged at 15,000 × g for 20 rain. The supernatant was subjected to SDS (0.1~) polyacrylamide gel electrophoresis according to Laemmlii£ The slab gel electrophoresis was performed as described by Studier TM. As molecular weight markers were used: cytochrome C (13,000), chymotrypsinogen (25,000), ovalbumin (45,000) and serum albumin (67,000). After electrophoresis the slab gel was stained, destained and cut in 2 mm slices with an automatic gel slicer (Mickel Laboratory Engineering Co., Gomshall, England). Each slice was counted for 10 rain in the 7-counter.
Animals Female Sprague-Dawley rats weighing 120-150 g were used in most of the experiments; in addition male Swiss mice weighing 25-30 g were used for the 6-hydroxydopamine (6-OHDA) experiments. The animals were kept at a constant temperature (23 °C) and given the usual lab chow diet and tap water ad libitum.
Injection schedulesand surgery General procedure. Nerve terminals of a symmetrical system were unilaterally exposed to high concentrations of labelled DBH antiserum. The accumulation of radioactivity in the corresponding cell bodies was then measured after various time intervals and the difference between injected and non-injected side taken as evidence for a retrograde axonal transport as shown in detail in previous studies with other macromoleculesT,l°,18. All surgical and injection procedures were performed under ether anesthesia. In order to study the retrograde axonal transport in adrenergic neurons, labelled DBH-antibodies were injected unilaterally into the anterior eye chamber
296 with a Hamilton glass syringe. The injection volume was 10 #1. The superior cervical ganglia of the injected and non-injected sides were removed between 1.5 and 48 h after injection. In order to study a possible retrograde axonal transport in sensory and m o t o r neurons the same procedure as previously described for N G F and tetanus toxin was used TM. Thirty #1 of labelled partially purified DBH-antibodies were injected unilaterally into the forepaw for the sensory neurons and into the musculus deltoideus for the motorneurons. Colchicine. Colchicine (Sandoz AG, Basel, Switzerland) in 0.9~ NaC1 was injected at a concentration of 40 mg/ml. Ten #1 of this solution were injected unilaterally into the anterior eye chamber 6 h before the injection of labelled antibodies. Controls were injected in an identical way with the same volume of 0.9~ NaC1. The superior cervical ganglia of the injected and non-injected side were removed 24 h after the injection of labelled antibodies. Transection. Under sterile conditions the right superior cervical ganglion of the animals was exposed. Care was taken not to damage the preganglionic sympathetic nerve trunk nor to touch the ganglion in order to prevent any changes in the blood supply of the ganglion. For these reasons only the external carotid nerve was completely transected whereas the internal carotid nerve was only squeezed with forceps at its exit from the ganglion. Twelve hours after surgery, 10 #1 of labelled partially purified DBH-antibody was injected into the anterior eye chamber of the transected side. The superior cervical ganglia of injected and non-injected side were removed 24 h later. 6 - O H D A . 6 - O H D A (Fluka, Buchs, SG, Switzerland) in 0.9~ NaC1 was used at a concentration of 30 mg/ml. Mice and rats were injected intravenously on 3 consecutive days with 100 mg/kg of 6 - O H D A • HC1. At various intervals after the last injection either labelled N G F or labelled DBH-antibodies were injected unilaterally into the anterior eye chamber.
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Fig. l. Time-course of accumulation of radioactivity in superior cervical ganglia after unilateral injection of 25/~Ci of 125I-labelled antibodies to DBH into the anterior eye chamber. One to 48 h after injection the animals were killed, the ganglia removed and their radiaoctivity determined. The values given represent the mean -J- S.E.M. of groups of 4-6 animals.
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Fig. 2. Comparison between the accumulation of radioactivity in superior cervical ganglia after unilateral injection of labelled DBH-antiserum, control-serum and DBH-antiserum after precipitation with DBH. The animals were injected unilaterally into the anterior eye chamber with 18 /~Ci of 125I-labelled DBH-antiserum, 16 #Ci of rabbit control-serum and 16 #Ci of DBH-antiserum after removal of the specific antibodies by precipitation with DBH. The superior cervical ganglia of the injected and non-injected side were dissected 24 h later and their radioactivity determined. The values given represent the means ± S.E.M. of groups of 4-5 animals. RESULTS
Evidencefor retrograde axonal transport After unilateral injection of l~SI-labelled antibodies to D B H into the anterior eye chamber there was no difference in the accumulation of radioactivity between the injected and non-injected side for the first 4 h (Fig. 1). The first difference appeared after 6 h, although it was not statistically significant (P > 0.05). However, after 8 h the accumulation on the injected side was 3 times that on the non-injected side (P < 0.005). It increased progressively up to 24 h when it reached a level about 10 times that on the non-injected side. The level on the non-injected side remained virtually unchanged throughout the experiment, which is in distinct contrast to previous studies with NGFT, is. The distance between the anterior eye chamber and the superior cervical ganglion is approximately 1.5 cm in the size of rats used in these experiments. Since the first detectable difference between injected and non-injected sides appears at 6-7 h, the rate of transport amounts to approximately 2-3 mm/h. This rate of retrograde transport is in agreement with that calculated for N G F and tetanus toxin in both mice 7 and ratsG,7,TM.
Naturevf the radioactive compound(s) accumulated in the ganglion Two groups of rats were injected with iodinated DBH-antiserum and with iodinated control serum, respectively. Fig. 2 shows that preferential accumulation of radioactivity on the injected side occurred only when DBH-antiserum was injected, indicating that DBH-antibodies are involved. However, since the control serum and
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slices (2mm) Fig. 3. Electrophoretic profiles of l~SI-labelled antibodies to DBH on SDS polyacrylamide gels. Five rats of 150 g body weight were injected unilaterally in the anterior eye chamber with 40 #Ci of 125I-labelled partially purified DBH-antibody. Superior cervical ganglia from both sides were removed 16 h later and homogenized separately in Tris-Triton solution. The homogenates were centrifuged at 15,000 × g for 20 rain and the supernatant subjected to SDS gel electrophoresis. The samples (40 F1) were mixed with 20 pl of 45~ glycerol, 15~ 2-mercaptoethanol, 7~ SDS, 0.003~ bromophenol blue and 0.2 M Tris-HCl, pH 6.8. The mixtures were heated for 5 rain at 95 °C and applied on a 0.1~ SDS-10~ polyacrylamide slab gel. The gel was stained in methanol-acetic acidwater (5:1:5, v/v/v) containing 0.25~ Coomassie brillant blue (R 250) and destained in the same solution. The gel strips were sliced in 2 mm fractions and counted in a 7-counter. (a) Partially purified DBH-antiserum; (b) superior cervical ganglia from injected side; and (c) superior cervical ganglia from non-injected side. Bovine serum albumin (67,000), ovalbumin (45,000), chymotrypsinogen (25,000) and cytochrome C (13,000) were used as molecular weight markers.
the D B H - a n t i s e r u m d i d n o t originate f r o m the same rabbit, there r e m a i n e d the possibility t h a t o t h e r substances c o u l d be involved. I n o r d e r to clarify this aspect we precipitated the D B H - a n t i b o d i e s in the a n t i s e r u m with crude D B H . As can be seen in Fig. 2 this p r o c e d u r e c o m p l e t e l y abolished the preferential a c c u m u l a t i o n o f r a d i o activity. The n a t u r e o f the r a d i o a c t i v e c o m p o u n d s a c c u m u l a t e d in the superior cervical ganglia 16 h after the unilateral injection o f p a r t i a l l y purified labelled D B H - a n t i b o d i e s into the a n t e r i o r eye c h a m b e r a n d salivary g l a n d was next investigated. F i v e ganglia were h o m o g e n i z e d in a T r i s - T r i t o n solution, centrifuged at 15,000 × g for
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Fig. 4. Effect of colchicine and transection of the post-ganglionic adrenergic fibers on the accumulation of radioactivity in superior cervical ganglia after unilateral injection of 125I-labelled DBH-antibodies into the anterior eye chamber. Both control and experimental animals were injected with 40/zCi of l~'SI-labelled DBH-antibodies. The ganglia were removed 24 h after the unilateral injection for determination of their radioactivity. One group of animals was injected into the anterior eye chamber with 400 #g of colchicine 6 h prior to the injection of lzsI-labelled DBH-antiserum. In another group of animals the postganglionic fibers of the superior cervical ganglia were transected unilaterally 24 h before th~ injection of [l~5I]DBH-antiserum into the anterior eye chamber of the same side. The values given represent the means ± S.E.M. of groups of 5-6 animals.
20 rain and the supernatant was subjected to SDS gel electrophoresis. As a control, labelled DBH-antiserum was also submitted to SDS gel electrophoresis. The profile of the antiserum revealed 3 major peaks of radioactivity with molecular weights of 67,000, 50,000 and 25,000 corresponding to serum albumin and the light and heavy chains ofimmunoglobulins (Fig. 3a). However, when the gel was stained with Coomassie brillant blue, more than 50 faint bands could be observed. Fig. 3b shows that in the superior cervical ganglion of the injected side the majority of the counts were found at the position of the light and heavy chains of immunoglobulins, i.e. at positions which correspond to molecular weights of 25,000 and 50,000. This indicates that the radioactivity transported retrogradely is selectively associated with the DBH-antibodies. On the non-injected side, the majority of the radioactivity also appeared in the two bands of immunoglobulins (Fig. 3c). However, the content (counts/rain) was 10 times smaller than on the injected side.
Lffect of colchicine and axotomy on retrograde transport In order to obtain further evidence that the preferential accumulation of antibodies to DBH results from retrograde axonal transport from the nerve terminals to the cell body, the effect of colchicine and axotomy were examined. Axotomy greatly reduced the preferential accumulation as can be seen in Fig. 4, whereas colchicine virtually abolished all accumulation of radioactivity. It is likely that the large dose of colchicine interfered with transport not only on the injected side but also on the noninjected side. On the other hand the smaller effect of axotomy probably results from
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Fig. 5. Accumulation of radioactivity in the spinal cord segment (C~-Cs) (motoneurons) and the dorsal root ganglia (Ca, C7) (sensory neurons) after unilateral injection of l~SI-labelled partially purified DBH-antibodies into either the musculus deltoideus (64 FCi) or the forepaw (60/tCi). In all the experiments the animals were killed 18 h after unilateral injection of the labelled DBH-antiserum. The spinal cord segments (C6-C8) or the dorsal root ganglia (C6, C7) of the injected and noninjected side were dissected and the radioactivity determined in a 7-counter. Values given represent means ~: S.E.M. for groups of 6-7 animals.
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Fig. 6. Effect of 6-hydroxydopamine (6-OHDA) on the accumulation of radioactivity in the superior cervical ganglia after unilateral injection of 40 pCi of 125I-labelledpartially purified DBH-antibodies into the anterior eye chamber. The animals were killed 9 or 16 h after the injection of the labelled antibodies. One group of animals was injected intravenously on 3 consecutive days with 100 mg/kg of 6-OHDA. The last dose was given 24 h before the injection of the labelled DBH-antibodies. The values given represent means ± S.E.M. of groups of 5-6 rats. i n c o m p l e t e a x o t o m y . Since special care was t a k e n to preserve the b l o o d supply to the ganglion, a c o m p l e t e a x o t o m y was p r o b a b l y n o t accomplished.
Specificity of uptake and retrogradetransportof DBH-antibody L a b e l l e d D B H - a n t i b o d i e s were injected into the vicinity o f nerve terminals o f sensory a n d m o t o r n e u r o n s in o r d e r to see whether 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 o f a n t i b o d i e s occurred. Fig. 5 shows that in c o n t r a s t to the preferential a c c u m u l a t i o n in the s u p e r i o r cervical ganglion o f the injected side there was no side difference in sensory o r m o t o r neurons. This lack o f preferential a c c u m u l a t i o n in the cell bodies o f sensory a n d m o t o r n e u r o n s is n o t the result o f experimental inadequacies since the
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Fig. 7. Effect of 6-hydroxydopamine on the accumulation of radioactivity in the superior cervical ganglia of mice and rats after unilateral injection of [I~SI]NGFinto the anterior eye chamber. Both mice and rats were injected intravenously on 3 consecutive days with 100 mg/kg of 6-OHDA. In mice the last dose of 6-OHDA was injected 1, 4 or 7 days prior to the unilateral intraocular injection of 4/~Ci of [I~SI]NGF.In rats the last dose of 6-OHDA was given 1 or 8 days prior to the injection of [125I]NGF.Both rats and mice were killed 16 h after the injection of [a25I]NGF. The ganglia of the injected and non-injected side were removed and their radioactivity was determined in a ycounter. The values given represent the means ± S.E.M. of groups of 7-8 animals. same injection procedure resulted in a preferential accumulation of tetanus toxin (motor and sensory neurons) and N G F (sensory neurons)17,18.
Effect of 6-OHDA on retrograde transport of DBH-antibody and NGF Two groups of rats were treated with 6 - O H D A prior to administration of labelled antiserum as described in the methods. One group was killed 9 h and the other 18 h later. A comparison with two groups of control rats is shown in Fig. 6: in the 6 - O H D A treated rats there is a considerable reduction of the accumulation of radioactivity in the ganglia on the injected side. In a similar experiment labelled N G F was injected after pretreatment with 6-OHDA. Here too there was a striking reduction in the accumulation of radioactivity. This observation is in conflict with the findings of Hendry et al. 7 of the effect of 6 - O H D A in mice. Since it is known that in mice the regeneration of adrenergic nerve terminals after 6 - O H D A is extremely rapid 9 we investigated the effect of 6-OHDA in mice and rats. As shown in Fig. 7. the accumulation of labelled N G F 24 h after the end of 6 - O H D A treatment is virtually abolished. In mice there is 75~ recovery by the end of 4 days and complete recovery by 7 days. However, in rats, retrograde accumulation is only 48~ of control value by 8 days. DISCUSSION
The preferential accumulation of radioactivity in the superior cervical ganglion after injection of labelled antiserum to DBH into the anterior eye chamber provides evidence of uptake and retrograde transport of some constituent of the antiserum. The abolition of this preferential accumulation of radioactivity after removal of DBHantibody by immunoprecipitation shows that it is the DBH-antibody which appears in the ganglion. Further evidence is provided by SDS gel electrophoresis which shows the radioactivity to be associated with two bands which correspond to molecular weights
302 of 25,000 and 50,000, respectively, suggesting that these represent light and heavy chains of immunoglobulins; there is no significant transport of other labelled molecules. The ganglion on the non-injected side shows the same selective accumulation of antibody, but in very much smaller amounts. The fact that the light chain (25,000) of the DBH-antibody is more heavily labelled than the heavy chain (50,000) might mean that the light chain either contains more tyrosine and histidine residues, or that these are more readily accessible for iodination than in the heavy chain. A comparison of DBH-antibody transport with N G F , for which a similar mechanism of uptake and transport has been demonstrated v shows important differences as well as similarities. The radioactivity on the non-i~ected side rises after injection of N G F but stays low after injection of labelled antiserum to D B H ; this means that the accumulation on the contralateral side results from an escape into the general circulation followed either by direct binding or uptake by nerve terminals and retrograde transport. The difference between the accumulation of radioactivity on the non-injected side after injection of labelled N G F and DBH-antibody is probably due to the fact that there is less escape into the circulation after local injection of DBH-antibodies, since this molecule is about 10 times larger than the fi-subunit of N G F in its monomeric form 1. The retrograde transport of DBH-antibody resembles that of N G F as well as tetanus toxin in that it is greatly reduced by colchicine and axotomy. The rate of transport calculated from the distance between the nerve terminals and the cell body together with the time lag of arrival is the same as for these other macromolecules, i.e., 2-3 mm/h7, TM. This indicates that there is probably a common transport mechanism. However, retrograde transport of DBH-antibodies is confined to sympathetic neurons and is not seen in either motor or sensory neurons. The experiments with 6-OHDA show that the uptake mechanism for both N G F and DBH antibodies is situated in the nerve terminals. The earlier results of Hendry et al. 7 can most plausibly be explained by the rapid regeneration of nerve terminals in mice after 6-OHDA treatmerit 9. The binding site for DBH antibody is likely to be DBH; this is the chief marker for noradrenergic vesicles. From these vesicles noradrenaline is released by exocytosis~, 15. It is not known, however, whether there is any incorporation of the vesicle membrane into the nerve terminal or whether DBH becomes accessible to the DBHantibody only during exocytosis. If this were the case the uptake of DBH-antibody into recently emptied vesicles could serve as a marker for their subsequent fate. The present experiments suggest that they return to the cell body either for reuse or for disposal, since lysosomes which are present in nerve cells are rarely seen in nerve terminals. There is independent evidence for a rapid retrograde transport of particle bound DBH2, 3. There are now available 3 tools for the study of retrograde transport in peripheral neurons: DBH-antibody which is specific for sympathetic neurons, N G F for sympathetic and sensory neurons and tetanus toxin for sympathetic, sensory and motor neurons.
303 ACKNOWLEDGEMENTS This work was supported by the Swiss N a t i o n a l F o u n d a t i o n for Scientific Research ( G r a n t Nr. 3.432.74) a n d the Medical Research Council of C a n a d a .
REFERENCES 1 Angeletti, R. H., Bradshaw, R. A. and Wade, R. D., Subunit structure and amino acid composition of mouse submaxillary gland nerve growth factor, Biochemistry (Wash.), 10 (1971) 463-469. 2 Brimijoin, S., Retrograde axonal transport of dopamine fl-hydroxylase. In E. Usdin and S. Snyder (Eds.), Frontiers in Catecholamine Research, Pergamon Press, Oxford, 1973, pp. 201-203. 3 Brimijoin, S., Local changes in subcellular distribution of dopamine fl-hydroxylase after blockade of axonal transport, J. Neurochem., 22 (1974) 347-353. 4 Gewirtz, G. P. and Kopin, I. J., Release of dopamine fl-hydroxylase with norepinephrine during cat splenic nerve stimulation, Nature (Lond.), 227 (1970) 406-407. 5 Greenwood, F. C., Hunter, W. M. and Glover, J. S., The preparation of lalI-labelled human growth hormone of high specific radioactivity, Biochem. J., 89 (1963) 114-123. 6 Hendry, I. A., Stach, R. and Herrup, K., Characteristics of the retrograde axonal transport system for nerve growth factor in the sympathetic nervous system, Brain Research, 82 (1974) 117-128. 7 Hendry, I. A., Stoeckel, K., Thoenen, H. and Iversen, L. L., Retrograde axonal transport of nerve growth factor, Brain Research, 68 (1974) 103-121. 8 Jacobowitz, D. M., Ziegler, M. G. and Thomas, J. A., In vivo uptake of antibody to dopamine fl-hydroxylase into sympathetic elements, Brain Research, 91 (1975) 165-170. 9 Jonsson, G., Microfluorimetric and neurochemical studies on degenerating and regenerating adrenergic nerves. In K. Fuxe, L. Olson and Y. Zotterman (Eds.), Dynamics of Degeneration and Growth in Neurons, Pergamon Press, Oxford, 1974, pp. 61-75. 10 Iversen, L. L., Stoeckel, K. and Thoenen, H., Autoradiographic studies of the retrograde axonal transport of nerve growth factor in mouse sympathetic neurons, Brain Research, 88 (1975) 37-43. 11 Laemmli, U. K., Cleavage of structural proteins during the assembly of the head of bacteriophage T4, Nature (Lond.), 227 (1970) 680-685. 12 Molinoff, P. B. and Axelrod, J., Biochemistry of catecholamines, Ann. Rev. Biochem., 40 (1971) 465-500. 13 Ouchterlony, O., Handbook oflmmunodiffusion andbnmunoelectrophoresis, Ann. Arbor Sci. Publ., Ann. Arbor, Mich., 1970. 14 Smith, A. D. and Winkler, H., A simple method for the isolation of adrenal chromaffin granules on a large scale, Biochem. J., 103 0967) 480-482. 15 Smith, A. D., De Potter, W. P., Moerman, E. L and De Schaepdryver, A. F., Release of dopamine fl-hydroxylase and chromagranine A upon stimulation of the splenic nerve, Tissue and Cell, 2 (1970) 547-568. 16 Stoeckel, K., Paravicini, U. and Thoenen, H., Specificity of the retrograde axonal transport of nerve growth factor, Brain Research, 76 (1974) 413-421. 17 Stoeckel, K., Schwab, M. and Thoenen, H., Specificity of retrograde transport of nerve growth factor (NGF) in sensory neurons: A biochemical and morphological study, Brain Research, 89 (1975) 1-14. 18 Stoeckel, K., Schwab, M. and Thoenen, H., Comparison between the retrograde axonal transport of nerve growth factor and tetanus toxin in motor, sensory and adrenergic neurons, Brain Research, in press. 19 Studier, F. W., Analysis of Bacteriophage T 7 early RNAs and proteins on slab gels, J. tool. Biol., 79 (1973) 237-248.
293
SELECTIVE U P T A K E A N D 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 OF DOPAMINE-fl-HYDROXYLASE ANTIBODIES IN P E R I P H E R A L A D R E N ERGIC NEURONS
M. FILLENZ*, C. GAGNON, K. STOECKEL and H. THOENEN Department of Pharmacology, Biocenter of the University, Basel (Switzerland)
(Accepted February 10th, 1976)
SUMMARY In the present experiments the uptake and retrograde axonal transport of antibodies to dopamine fl-hydroxylase (DBH) in adrenergic neurons was studied. When partially purified labelled antibodies to DBH were injected unilaterally into the vicinity of the adrenergic nerve terminals in the iris, radioactive substances accumulated preferentially in the superior cervical ganglia of the injected side. By SDS (sodium dodecyl sulfate) gel electrophoresis and immunoprecipitation it could be shown that the accumulated radioactivity in the superior cervical ganglion represented antibodies to DBH. This retrograde accumulation was greatly reduced by colchicine, axotomy or destruction of the adrenergic nerve terminals by 6-hydroxydopamine. The rate of retrograde transport was the same as that of nerve growth factor ( N G F ) and tetanus toxin in sympathetic neurons. The retrograde transport of antibodies was confined to sympathetic neurons and could not be detected in either sensory or motor neurons.
INTRODUCTION In previous studies it has been shown that macromolecules are taken up with high selectivity by nerve terminals and are transported retrogradely to the corresponding cell bodies6,7,16,17. The selectivity of uptake can depend on properties which are inherent in all nerve terminals, as seems to be the case for the uptake and retrograde transport of tetanus toxin is, or the binding and uptake can be restricted to a selective population of neurons, as is the case for nerve growth factor (NGF)17, is. Thus, we can delineate varying degrees of selectivity of retrograde transport, i.e., a selectivity * Present address: University Laboratory of Physiology, Parks Road, Oxford OX1 3PT, Great Britain.
294 with respect to the macromolecules which are transportedX~, xr, and additionally a selectivity with respect to the species of neurons is. Recently, histochemical evidence has been presented for accumulation of antibodies to dopamine fl-hydroxylase (DBH) in adrenergic neurons after injection into the general circulation s. Since DBH, the enzyme which catalyzes the final step of noradrenaline synthesis, is exclusively located in adrenergic neurons and is predominantly located in noradrenaline storage vesicles 12the question arises as to whether this accumulation is confined to adrenergic neurons, whether the accumulation results from a direct accumulation by the adrenergic cell body or from an uptake by the nerve terminals followed by retrograde axonal transport, and whether the mechanism of retrograde transport is similar to that investigated previously for other macromoleculesT, is. The present experiments were designed to answer these questions. MATERIALS AND METHODS
Production of DBH-antibodies DBH was purified from the chromaffin granule lysate of beef adrenal medulla. Granules were isolated by the method of Smith and Winkler 14 and lysed with a low ionic strength (0.005 M) phosphate buffer. Each pellet of chromaffin granules (originating from 15 g of tissue) was resuspended in 2 ml of 0.005 M sodium phosphate buffer, pH 7.2 and centrigufed at 40,000 × g for 30 min. DBH from the lysate was purified by chromatography on DEAE cellulose and Concanavalin A - Sepharose 4B as will be described in detail elsewhere (Gagnon, in preparation). Purified DBH was mixed with the complete Freund's adjuvant in order to produce a water in oil emulsion. The mixture (0.8 ml) containing 200 #g of DBH was injected into the rear foot paws of 1.5 kg 'White Wiener' rabbits. Three weeks later the booster injection (50 #g of DBH in 0.15 M NaC1) was given subcutaneously. The antiserum was collected weekly starting 12 days after the booster injection. The antibodies against DBH were judged to be monospecific by double immunodiffusion and immunoelectrophoresis is against crude or purified DBH.
Partialpurification of DBH-antibodies DBH-antiserum was precipitated with an equal volume of saturated ammonium sulfate solution and stirred for 30 min at 0-4 °C. The precipitate was centrifuged at 10,000 × g for 10 min, the pellet dissolved in 0.05 M sodium phosphate buffer, pH 7.8 and extensively dialyzed against this buffer.
Labelling ofpartially purified DBH-antibodies and NGF The labelling was performed with Na125I and chloramine T according to Greenwood et al. 5 with minor modifications described in detail by Stoeckel et al? 6. Nal~5I was provided by EIR, Wiirenlingen, Switzerland, at a specific activity of 8-15 Ci/mg iodine. Each preparation was performed using either 200 #g of N G F or 200 #g protein of partially purfied antibodies (see above) in 25 #1 of 0.05 M phosphate buffer, pH 7.8 and 5 mCi of Nal25[.
295 The specific activity of the labelled N G F was 8 ffCi/fg; that of the labelled partially purified DBH-antibodies, 6 #Ci/#g of protein. The stock solution used for injection of [125I] N G F was about 350 #g/ml; that of the partially purified DBH-antibodies contained about 600 fig protein/ml.
Precipitation of labelled DBH-antibodies Serum DBH-antibodies were precipitated by an excess of crude DBH. To 40 #1 of iodinated partially purified serum was added 80 ffl of chromaffin granule lysate. The mixture was incubated for 1 h at 37 °C and 1 h at 0-4 °C, then centrifuged at 15,000 x g for 10 min. The supernatant was collected and 40 ffl of chromaffin granule lysate was added to it. After repetition of the incubation and centrifugation procedure the supernatant was collected and used as control serum.
Radioactivity The radioactivity of the samples was determined directly in a Packard 7-counter Mod. 3001 at a counting efficiency of 65~. The counts/rain values are not corrected for the counting efficiency.
SDS gel electrophoresis A pool of 5 ganglia were homogenized in 0.5 ml of 0.005 M Tris-HC1-Triton X-100 (0.1~) buffer, pH 7.4 and centrifuged at 15,000 × g for 20 rain. The supernatant was subjected to SDS (0.1~) polyacrylamide gel electrophoresis according to Laemmlii£ The slab gel electrophoresis was performed as described by Studier TM. As molecular weight markers were used: cytochrome C (13,000), chymotrypsinogen (25,000), ovalbumin (45,000) and serum albumin (67,000). After electrophoresis the slab gel was stained, destained and cut in 2 mm slices with an automatic gel slicer (Mickel Laboratory Engineering Co., Gomshall, England). Each slice was counted for 10 rain in the 7-counter.
Animals Female Sprague-Dawley rats weighing 120-150 g were used in most of the experiments; in addition male Swiss mice weighing 25-30 g were used for the 6-hydroxydopamine (6-OHDA) experiments. The animals were kept at a constant temperature (23 °C) and given the usual lab chow diet and tap water ad libitum.
Injection schedulesand surgery General procedure. Nerve terminals of a symmetrical system were unilaterally exposed to high concentrations of labelled DBH antiserum. The accumulation of radioactivity in the corresponding cell bodies was then measured after various time intervals and the difference between injected and non-injected side taken as evidence for a retrograde axonal transport as shown in detail in previous studies with other macromoleculesT,l°,18. All surgical and injection procedures were performed under ether anesthesia. In order to study the retrograde axonal transport in adrenergic neurons, labelled DBH-antibodies were injected unilaterally into the anterior eye chamber
296 with a Hamilton glass syringe. The injection volume was 10 #1. The superior cervical ganglia of the injected and non-injected sides were removed between 1.5 and 48 h after injection. In order to study a possible retrograde axonal transport in sensory and m o t o r neurons the same procedure as previously described for N G F and tetanus toxin was used TM. Thirty #1 of labelled partially purified DBH-antibodies were injected unilaterally into the forepaw for the sensory neurons and into the musculus deltoideus for the motorneurons. Colchicine. Colchicine (Sandoz AG, Basel, Switzerland) in 0.9~ NaC1 was injected at a concentration of 40 mg/ml. Ten #1 of this solution were injected unilaterally into the anterior eye chamber 6 h before the injection of labelled antibodies. Controls were injected in an identical way with the same volume of 0.9~ NaC1. The superior cervical ganglia of the injected and non-injected side were removed 24 h after the injection of labelled antibodies. Transection. Under sterile conditions the right superior cervical ganglion of the animals was exposed. Care was taken not to damage the preganglionic sympathetic nerve trunk nor to touch the ganglion in order to prevent any changes in the blood supply of the ganglion. For these reasons only the external carotid nerve was completely transected whereas the internal carotid nerve was only squeezed with forceps at its exit from the ganglion. Twelve hours after surgery, 10 #1 of labelled partially purified DBH-antibody was injected into the anterior eye chamber of the transected side. The superior cervical ganglia of injected and non-injected side were removed 24 h later. 6 - O H D A . 6 - O H D A (Fluka, Buchs, SG, Switzerland) in 0.9~ NaC1 was used at a concentration of 30 mg/ml. Mice and rats were injected intravenously on 3 consecutive days with 100 mg/kg of 6 - O H D A • HC1. At various intervals after the last injection either labelled N G F or labelled DBH-antibodies were injected unilaterally into the anterior eye chamber.
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Fig. 2. Comparison between the accumulation of radioactivity in superior cervical ganglia after unilateral injection of labelled DBH-antiserum, control-serum and DBH-antiserum after precipitation with DBH. The animals were injected unilaterally into the anterior eye chamber with 18 /~Ci of 125I-labelled DBH-antiserum, 16 #Ci of rabbit control-serum and 16 #Ci of DBH-antiserum after removal of the specific antibodies by precipitation with DBH. The superior cervical ganglia of the injected and non-injected side were dissected 24 h later and their radioactivity determined. The values given represent the means ± S.E.M. of groups of 4-5 animals. RESULTS
Evidencefor retrograde axonal transport After unilateral injection of l~SI-labelled antibodies to D B H into the anterior eye chamber there was no difference in the accumulation of radioactivity between the injected and non-injected side for the first 4 h (Fig. 1). The first difference appeared after 6 h, although it was not statistically significant (P > 0.05). However, after 8 h the accumulation on the injected side was 3 times that on the non-injected side (P < 0.005). It increased progressively up to 24 h when it reached a level about 10 times that on the non-injected side. The level on the non-injected side remained virtually unchanged throughout the experiment, which is in distinct contrast to previous studies with NGFT, is. The distance between the anterior eye chamber and the superior cervical ganglion is approximately 1.5 cm in the size of rats used in these experiments. Since the first detectable difference between injected and non-injected sides appears at 6-7 h, the rate of transport amounts to approximately 2-3 mm/h. This rate of retrograde transport is in agreement with that calculated for N G F and tetanus toxin in both mice 7 and ratsG,7,TM.
Naturevf the radioactive compound(s) accumulated in the ganglion Two groups of rats were injected with iodinated DBH-antiserum and with iodinated control serum, respectively. Fig. 2 shows that preferential accumulation of radioactivity on the injected side occurred only when DBH-antiserum was injected, indicating that DBH-antibodies are involved. However, since the control serum and
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slices (2mm) Fig. 3. Electrophoretic profiles of l~SI-labelled antibodies to DBH on SDS polyacrylamide gels. Five rats of 150 g body weight were injected unilaterally in the anterior eye chamber with 40 #Ci of 125I-labelled partially purified DBH-antibody. Superior cervical ganglia from both sides were removed 16 h later and homogenized separately in Tris-Triton solution. The homogenates were centrifuged at 15,000 × g for 20 rain and the supernatant subjected to SDS gel electrophoresis. The samples (40 F1) were mixed with 20 pl of 45~ glycerol, 15~ 2-mercaptoethanol, 7~ SDS, 0.003~ bromophenol blue and 0.2 M Tris-HCl, pH 6.8. The mixtures were heated for 5 rain at 95 °C and applied on a 0.1~ SDS-10~ polyacrylamide slab gel. The gel was stained in methanol-acetic acidwater (5:1:5, v/v/v) containing 0.25~ Coomassie brillant blue (R 250) and destained in the same solution. The gel strips were sliced in 2 mm fractions and counted in a 7-counter. (a) Partially purified DBH-antiserum; (b) superior cervical ganglia from injected side; and (c) superior cervical ganglia from non-injected side. Bovine serum albumin (67,000), ovalbumin (45,000), chymotrypsinogen (25,000) and cytochrome C (13,000) were used as molecular weight markers.
the D B H - a n t i s e r u m d i d n o t originate f r o m the same rabbit, there r e m a i n e d the possibility t h a t o t h e r substances c o u l d be involved. I n o r d e r to clarify this aspect we precipitated the D B H - a n t i b o d i e s in the a n t i s e r u m with crude D B H . As can be seen in Fig. 2 this p r o c e d u r e c o m p l e t e l y abolished the preferential a c c u m u l a t i o n o f r a d i o activity. The n a t u r e o f the r a d i o a c t i v e c o m p o u n d s a c c u m u l a t e d in the superior cervical ganglia 16 h after the unilateral injection o f p a r t i a l l y purified labelled D B H - a n t i b o d i e s into the a n t e r i o r eye c h a m b e r a n d salivary g l a n d was next investigated. F i v e ganglia were h o m o g e n i z e d in a T r i s - T r i t o n solution, centrifuged at 15,000 × g for
299
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Fig. 4. Effect of colchicine and transection of the post-ganglionic adrenergic fibers on the accumulation of radioactivity in superior cervical ganglia after unilateral injection of 125I-labelled DBH-antibodies into the anterior eye chamber. Both control and experimental animals were injected with 40/zCi of l~'SI-labelled DBH-antibodies. The ganglia were removed 24 h after the unilateral injection for determination of their radioactivity. One group of animals was injected into the anterior eye chamber with 400 #g of colchicine 6 h prior to the injection of lzsI-labelled DBH-antiserum. In another group of animals the postganglionic fibers of the superior cervical ganglia were transected unilaterally 24 h before th~ injection of [l~5I]DBH-antiserum into the anterior eye chamber of the same side. The values given represent the means ± S.E.M. of groups of 5-6 animals.
20 rain and the supernatant was subjected to SDS gel electrophoresis. As a control, labelled DBH-antiserum was also submitted to SDS gel electrophoresis. The profile of the antiserum revealed 3 major peaks of radioactivity with molecular weights of 67,000, 50,000 and 25,000 corresponding to serum albumin and the light and heavy chains ofimmunoglobulins (Fig. 3a). However, when the gel was stained with Coomassie brillant blue, more than 50 faint bands could be observed. Fig. 3b shows that in the superior cervical ganglion of the injected side the majority of the counts were found at the position of the light and heavy chains of immunoglobulins, i.e. at positions which correspond to molecular weights of 25,000 and 50,000. This indicates that the radioactivity transported retrogradely is selectively associated with the DBH-antibodies. On the non-injected side, the majority of the radioactivity also appeared in the two bands of immunoglobulins (Fig. 3c). However, the content (counts/rain) was 10 times smaller than on the injected side.
Lffect of colchicine and axotomy on retrograde transport In order to obtain further evidence that the preferential accumulation of antibodies to DBH results from retrograde axonal transport from the nerve terminals to the cell body, the effect of colchicine and axotomy were examined. Axotomy greatly reduced the preferential accumulation as can be seen in Fig. 4, whereas colchicine virtually abolished all accumulation of radioactivity. It is likely that the large dose of colchicine interfered with transport not only on the injected side but also on the noninjected side. On the other hand the smaller effect of axotomy probably results from
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Fig. 5. Accumulation of radioactivity in the spinal cord segment (C~-Cs) (motoneurons) and the dorsal root ganglia (Ca, C7) (sensory neurons) after unilateral injection of l~SI-labelled partially purified DBH-antibodies into either the musculus deltoideus (64 FCi) or the forepaw (60/tCi). In all the experiments the animals were killed 18 h after unilateral injection of the labelled DBH-antiserum. The spinal cord segments (C6-C8) or the dorsal root ganglia (C6, C7) of the injected and noninjected side were dissected and the radioactivity determined in a 7-counter. Values given represent means ~: S.E.M. for groups of 6-7 animals.
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Fig. 6. Effect of 6-hydroxydopamine (6-OHDA) on the accumulation of radioactivity in the superior cervical ganglia after unilateral injection of 40 pCi of 125I-labelledpartially purified DBH-antibodies into the anterior eye chamber. The animals were killed 9 or 16 h after the injection of the labelled antibodies. One group of animals was injected intravenously on 3 consecutive days with 100 mg/kg of 6-OHDA. The last dose was given 24 h before the injection of the labelled DBH-antibodies. The values given represent means ± S.E.M. of groups of 5-6 rats. i n c o m p l e t e a x o t o m y . Since special care was t a k e n to preserve the b l o o d supply to the ganglion, a c o m p l e t e a x o t o m y was p r o b a b l y n o t accomplished.
Specificity of uptake and retrogradetransportof DBH-antibody L a b e l l e d D B H - a n t i b o d i e s were injected into the vicinity o f nerve terminals o f sensory a n d m o t o r n e u r o n s in o r d e r to see whether 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 o f a n t i b o d i e s occurred. Fig. 5 shows that in c o n t r a s t to the preferential a c c u m u l a t i o n in the s u p e r i o r cervical ganglion o f the injected side there was no side difference in sensory o r m o t o r neurons. This lack o f preferential a c c u m u l a t i o n in the cell bodies o f sensory a n d m o t o r n e u r o n s is n o t the result o f experimental inadequacies since the
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Fig. 7. Effect of 6-hydroxydopamine on the accumulation of radioactivity in the superior cervical ganglia of mice and rats after unilateral injection of [I~SI]NGFinto the anterior eye chamber. Both mice and rats were injected intravenously on 3 consecutive days with 100 mg/kg of 6-OHDA. In mice the last dose of 6-OHDA was injected 1, 4 or 7 days prior to the unilateral intraocular injection of 4/~Ci of [I~SI]NGF.In rats the last dose of 6-OHDA was given 1 or 8 days prior to the injection of [125I]NGF.Both rats and mice were killed 16 h after the injection of [a25I]NGF. The ganglia of the injected and non-injected side were removed and their radioactivity was determined in a ycounter. The values given represent the means ± S.E.M. of groups of 7-8 animals. same injection procedure resulted in a preferential accumulation of tetanus toxin (motor and sensory neurons) and N G F (sensory neurons)17,18.
Effect of 6-OHDA on retrograde transport of DBH-antibody and NGF Two groups of rats were treated with 6 - O H D A prior to administration of labelled antiserum as described in the methods. One group was killed 9 h and the other 18 h later. A comparison with two groups of control rats is shown in Fig. 6: in the 6 - O H D A treated rats there is a considerable reduction of the accumulation of radioactivity in the ganglia on the injected side. In a similar experiment labelled N G F was injected after pretreatment with 6-OHDA. Here too there was a striking reduction in the accumulation of radioactivity. This observation is in conflict with the findings of Hendry et al. 7 of the effect of 6 - O H D A in mice. Since it is known that in mice the regeneration of adrenergic nerve terminals after 6 - O H D A is extremely rapid 9 we investigated the effect of 6-OHDA in mice and rats. As shown in Fig. 7. the accumulation of labelled N G F 24 h after the end of 6 - O H D A treatment is virtually abolished. In mice there is 75~ recovery by the end of 4 days and complete recovery by 7 days. However, in rats, retrograde accumulation is only 48~ of control value by 8 days. DISCUSSION
The preferential accumulation of radioactivity in the superior cervical ganglion after injection of labelled antiserum to DBH into the anterior eye chamber provides evidence of uptake and retrograde transport of some constituent of the antiserum. The abolition of this preferential accumulation of radioactivity after removal of DBHantibody by immunoprecipitation shows that it is the DBH-antibody which appears in the ganglion. Further evidence is provided by SDS gel electrophoresis which shows the radioactivity to be associated with two bands which correspond to molecular weights
302 of 25,000 and 50,000, respectively, suggesting that these represent light and heavy chains of immunoglobulins; there is no significant transport of other labelled molecules. The ganglion on the non-injected side shows the same selective accumulation of antibody, but in very much smaller amounts. The fact that the light chain (25,000) of the DBH-antibody is more heavily labelled than the heavy chain (50,000) might mean that the light chain either contains more tyrosine and histidine residues, or that these are more readily accessible for iodination than in the heavy chain. A comparison of DBH-antibody transport with N G F , for which a similar mechanism of uptake and transport has been demonstrated v shows important differences as well as similarities. The radioactivity on the non-i~ected side rises after injection of N G F but stays low after injection of labelled antiserum to D B H ; this means that the accumulation on the contralateral side results from an escape into the general circulation followed either by direct binding or uptake by nerve terminals and retrograde transport. The difference between the accumulation of radioactivity on the non-injected side after injection of labelled N G F and DBH-antibody is probably due to the fact that there is less escape into the circulation after local injection of DBH-antibodies, since this molecule is about 10 times larger than the fi-subunit of N G F in its monomeric form 1. The retrograde transport of DBH-antibody resembles that of N G F as well as tetanus toxin in that it is greatly reduced by colchicine and axotomy. The rate of transport calculated from the distance between the nerve terminals and the cell body together with the time lag of arrival is the same as for these other macromolecules, i.e., 2-3 mm/h7, TM. This indicates that there is probably a common transport mechanism. However, retrograde transport of DBH-antibodies is confined to sympathetic neurons and is not seen in either motor or sensory neurons. The experiments with 6-OHDA show that the uptake mechanism for both N G F and DBH antibodies is situated in the nerve terminals. The earlier results of Hendry et al. 7 can most plausibly be explained by the rapid regeneration of nerve terminals in mice after 6-OHDA treatmerit 9. The binding site for DBH antibody is likely to be DBH; this is the chief marker for noradrenergic vesicles. From these vesicles noradrenaline is released by exocytosis~, 15. It is not known, however, whether there is any incorporation of the vesicle membrane into the nerve terminal or whether DBH becomes accessible to the DBHantibody only during exocytosis. If this were the case the uptake of DBH-antibody into recently emptied vesicles could serve as a marker for their subsequent fate. The present experiments suggest that they return to the cell body either for reuse or for disposal, since lysosomes which are present in nerve cells are rarely seen in nerve terminals. There is independent evidence for a rapid retrograde transport of particle bound DBH2, 3. There are now available 3 tools for the study of retrograde transport in peripheral neurons: DBH-antibody which is specific for sympathetic neurons, N G F for sympathetic and sensory neurons and tetanus toxin for sympathetic, sensory and motor neurons.
303 ACKNOWLEDGEMENTS This work was supported by the Swiss N a t i o n a l F o u n d a t i o n for Scientific Research ( G r a n t Nr. 3.432.74) a n d the Medical Research Council of C a n a d a .
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