Uptake and retrograde axonal transport of various exogenous macromolecules in normal and crushed hypoglossal nerves

Brain Research, 153 (1978) 477~493 :~ Elsevier/North-Holland Biomedical Press

477

UPTAKE AND RETROGRADE AXONAL TRANSPORT OF VARIOUS EXOGENOUS MACROMOLECULES IN NORMAL AND CRUSHED HYPOGLOSSAL NERVES

LESLIE MALMGREN, YNGVE OLSSON, TOMAS OLSSON and KRISTER KRISTENSSON

Neuropathological Laboratory, Institute of Pathology, Universityof Uppsala, Uppsalaand Neuropathological Laboratory, Department of Pathology H, University of LinkOping, LinkO'ping (Sweden) (Accepted January 12th, 1978)

SUMMARY

Macromolecular tracers were injected into the tongue or around a crush in mouse hypoglossal nerves. At various times thereafter, the tracers were histochemically localized on the basis of peroxidase activity. The distribution of reaction product was then examined using light microscopy in order to study the influence of molecular charge and size on uptake and retrograde axonal transport from the periphery or from the crushed axon. Of various proteins with peroxidase activity, horseradish peroxidase and cytochrome-c showed the greatest penetration into axons proximal to the crush. Following injection into the tongue, intra-axonal cytochrome-c was detectable in some of the peripheral branches but not any of the other proteins. Retrograde transport to the nerve cell bodies was demonstrated for horseradish peroxidase and cytochrome-c, both from the tongue and from the axonal crush but not for microperoxidase, myoglobin, hemoglobin, lactoperoxidase and catalase. The number of neuronal cell bodies having detectable reaction product was higher for peroxidase-injected than for cytochrome-c-injected animals. Ferritin and iron-dextran (Imferon) also accumulated in hypoglossal neurons, but this could be detected only after repeated injections into the tongue. Uptake and retrograde transport from the tongue or from the crush occurred both for anionic and for cationic horseradish peroxidase. This is interpreted as evidence against absolute specificity in the uptake and transport of macromolecules on the basis of electrical charge.

INTRODUCTION

Retrograde axonal transport of horseradish peroxidase (HRP) was originally

478 described in peripheral motor neurons by Kristensson and Olsson ~:~. Thi~ phenomenon is now routinely used as a tool to determine the location of cell bodies of the central and peripheral nervous systems (see review by Kristensson and Ol~son z~, La Vail 26 and KristenssonlS), and has also been used recently to map the topography of functional populations of axons in peripheral nerves by Malmgren et al.='~'~ The retrograde axonal transport of biologically active macromolecu tes to t he cell body can sometimes result in large alterations in the metabolism of the neuron 3'~,~. Retrograde transport may also be involved in the transmission of toxic and infectious agents to the central nervous system along axons 1~,38,2°,44 and in the mechanism of chromatolysis following axonal injury ~1 23 The mechanisms influencing the uptake of macromolecules into axons and subsequent transport to the neuronal cell body are of particular interest, since they determine the extent to which neurons have the capacity to sample their environment selectively. The uptake and retrograde transport of certain macromolecules such as nerve growth factor (NGF) and dopamine-/3-hydroxylase antibodies have been shown to be restricted to particular types of neurons ~,44. On the other hand, the uptake and transport of macromolecules such as HRP and tetanus toxin occurs in many, if not all, types of vertebrate neurons 26,44. Stoeckel et al. '~;~examined the possibility that the selective uptake and transport of N G F was determined by simple physicochemical characteristics such as molecular size and charge. Using various 12'51-labelled proteins that differed in these parameters, they found that, of the proteins examined, only N G F showed detectable transport in peripheral adrenergic neurons. Since there was no detectable transport of cytochrome-c, which is similar to N G F in size and charge, it was concluded that the mechanism for selective uptake of N G F did not involve a specificity based on simple physiochemical characteristics, it should be noted, however, that H R P was included among the proteins for which no transport could be detected, even though the transport of H R P has been demonstrated in neurons of this type using histochemical methods ~t. Thus, it is likely that some types of molecules such as N G F are very readily taken up into neurons by a mechanism that is extremely selective and not influenced by simple physicochemical characteristics, while other molecules such as H R P are also taken up and transported by some other much less efficient and poorly understood mechanism. Recently, Bunt et al. '2 have reported specificity in the uptake and retrograde axonal transport of HRP. Using histochemical methods, they detected the transport of cationic but not of anionic isoenzymes of HRP in CNS neurons. They suggested the possibility that this apparent specificity in uptake and transport may be based either on the electrical charge carried by the different isoenzymes or on differences in their carbohydrate moeity. In the present study we have examined the role of specificity in the uptake and retrograde axonal transport of proteins by motor neurons with particular reference to electrical charge and molecular size. Uptake and retrograde axonal transport of H R P to hypoglossal cell bodies may occur either after intramuscular injection or after uptake into injured axons at the site of a crush t9,'~1,2'~,23. Since it might be assumed that the mechanisms involved in uptake at neuromuscular junctions differ from those

479 regulating uptake into injured axons 13,2z, the various macromolecular tracers were injected into the tongue as well as at the site of the hypoglossal nerve crush. METHODS AND MATERIALS Two-month-old male Swiss albino mice were used for all experiments. Tracers

In one of the laboratories (Uppsala) we concentrated on various proteins (Sigma, St. Louis) having peroxidase activity. They were used to examine the fate of macromolecules with differing physicochemical characteristics (Table 1). Electrophoretically purified isoenzymes of H R P (Sigma type IX Basic lsoenzyme and Sigma type VII Acidic lsoenzyme) were used to examine the effect of the electrical charge of the macromolecule on the specificity of the uptake and transport mechanisms. The cationic isoenzyme (type IX) is indicated by the manufacturer to represent the major basic isoenzyme of H R P and to migrate as a single band toward the anode during electrophoresis. The type VII anionic isoenzyme is indicated to migrate as a single band toward the cathode. We have confirmed the charges carried by these isoenzymes using isoelectric focusing. The pl of cationic H R P was found to be ~> pi 9.5, while that of anionic H R P was ~< pl 3.5. Consequently, there is no doubt that these isoenzymes are cationic and anionic respectively at physiological pH. In addition to the peroxidase proteins indicated above, two iron containing macromolecules, ferritin (type 1, Sigma) and iron-dextran (Imferon, Pharmacia) were also used as tracers (tool. wt. 572,000 and 73,000, respectively). These experiments were done in the Link6ping laboratory. Administration o f tracer proteins

Tracer proteins were administered under ether anesthesia either by injection into

TABLE

I

Physicochemical characteristics of compounds used for studies on specificity in uptake and retrograde transport of macromolecules in hypoglossal neurons Compound

Type

Source

MoL wt

pl

Microperoxidase Cytochrome-c Myoglobin Peroxidase Peroxidase Hemoglobin Lactoperoxidase Catalase lron-dextran

MP-II Ill, VI I IX VII 1

horse heart horse heart horse skeletal muscle horseradish horseradish beef blood milk beef liver synthetic

1900 12,000 18,000 40,000 40,000 68,000 82,000 240,000 73,000

Ferritin

I

horse spleen

572,000

5.4 10.5 7.0 >9.5 _<3.5 7.0 8.0, 9.2 5.7 anionic in pH range 3-12 4.5

C-40

480 the tongue or by application to the hypoglossal nerve immediately prior to crushing it. For the purpose of comparing the distribution of the various enzymatic tracers in the tongue and at the crush site, it was desirable to compare tissues ha~mg similar densities of reaction product (rp). The doses injected (0.5-18 rag) were varied to satisfy this requirement. The tongue injections were made into the dorsal midline of the tongue using a number 30 gauge needle and an injection volume of 0.06 ml (peroxidase tracers) or 0.1 ml (ferritin and iron--dextran). In experiments concerning the uptake and transport of the proteins from the crushed hypoglossal nerve, the Lracers were applied to the nerve and allowed to dissolve in the tissue fluid before crushing the nerve with jeweller's forceps.

Fixation The fixation procedure used with the peroxidase tracers was as follows. At various times (0.5-72 h) after the administration of the tracer, the mice were sacrified under ether anesthesia by perfusion fixation. The perfusion procedure involved a brief perfusion of saline followed by about 200 ml of 1.5~o glutaraldehyde (Polaron histochemical grade, A. Johnson, Malm5) in 0.1 M phosphate buffer (pH 7.4) at 37 C and 100 mg Hg. Following perfusion, the tissues were fixed by immersion in the same fixative for 4 h at room temperature before they were washed in 0.1 M phosphate buffer containing 5 ~o sucrose (4 °C) overnight. Mice injected with iron complexes were perfused with 4 ~ formaldehyde, after which the tissues were immediately sectioned. Sectioning procedure Frozen sections (20 #m thick) were cut from the brain stem, tongue and hypoglossal nerves. To avoid freezing artefacts the freezing process was carried out rapidly using a dry ice isopentane mixture. In some cases serial sections were successively distributed to separate groups in order to allow comparison of results obtained using different histochemical techniques. Histochemical procedures The various peroxidase tracers were localized histochemically using 3,3-diaminobenzidine tetrahydrochloride (DAB) and hydrogen peroxide as substrates. The buffer, pH and substrate concentrations of the incubation medium were varied as indicated in previous investigations concerning histochemical localization of each of the tracer proteins: microperoxidase TM, cytochrome-O 5, myoglobin 1, horseradish peroxidaseg,30,31, hemoglobin 8, lactoperoxidasO ° and catalase 37. For demonstration of iron-containing macromolecular tracers, sections were postfixed for 5 sec in absolute alcohoP 4 and stained according to Perl z6 for 5 min. The localization of the various protein tracers was determined by examination of the distribution of the DAB reaction product (rp) formed at sites of peroxidase activity. The contrast of the rp was selectively increased through the use of a Kodak Wratten No. 46 filter and bright-field microscopy 3°,31. Cell counts In order to examine the extent of detectable transport of the proteins to the cell

481 bodies, rp-labelled hypoglossal neurons were counted in coronal sections of the brain stem. Results were obtained from approximately 15 sections in each animal and expressed as the mean number of rp-labelled neurons per section. In cases of transport of proteins from the tongue, both hypoglossal nuclei were involved in contrast to experiments concerning transport from a hypoglossal nerve crush where transport was limited to the nucleus on the side of the crush. In order to facilitate the comparison of the results obtained in these two types of experiments, cell counts are expressed as the mean value for a single nucleus. RESULTS

Injection of protein in tracers into the tongue At 30 min after injection of cytochrome-c, myoglobin, anionic HRP, cationic HRP, hemoglobin, lactoperoxidase or catalase into the tongue, rp was present throughout the extracellular space of the tongue (Fig. 1). Rp was also present within the extracellular space of the endoneurium of most of the nerve branches in the tongue. No intra-axonal rp was detectable with the light microscope at this survival time. At 24 h the density of rp in the tongue was very much reduced, but the distribution of rp was in general not different from that at 30 rain apart from mice given cytochrome-c. Injection of this protein resulted in occasional intra-axonal rp in the nerve branches in the tongue which was not seen with any of the other tracers (Fig. 2). In order to examine the possibility that this was the result of mechanical injury associated with the injection into the tongue, experiments were carried out in which the tongue was compressed unilaterally with a hemostat. This resulted in an obvious unilateral increase in the number of axons containing diffuse rp at 24 h. Often the fibres containing rp were grouped together within the nerve branch. There was, however, no intra-axonal rp in the hypoglossal nerves either on the side of the injury or

Fig. 1. Nerve branch in the tongue at 24 h after injection of cationic HRP into the tongue. Note diffuse extracellular rp in endoneurium (arrows). x 891. Fig. 2. Nerve branch in the tongue at 24 h after injection of cytochrome-cinto the tongue. Note intraaxonal rp in some fibers (arrows). × 422.

482 in the contralateral side. At the tip o f the tongue, there was also a unilateral increase in the rp in certain muscle fibres, but this could not be clearly distinguished f r o m the endogenous peroxidase activity of muscle fibres at more proximal levels of the tongue. Counts o f rp-labelled neurons at 24 h did not indicate any clear change in the a m o u n t o f transport of cytochrome-c to either the ipsilateral or to the contralateral hypoglossal nucleus. After one injection of ferritin or iron-dextran a moderate degree of rp was seen t h r o u g h o u t the extracellular spaces and in granules of phagocytic cells 24 h later in the tongue. After 3 successive daily injections a more pronounced staining with similar distribution occurred while after 5-7 injections the staining was heavy. At various times after injection of protein tracers into the tongue, the hypoglossal nucleus was examined to determine if the neurons contained the foreign protein. After injection of myoglobin, hemoglobin or catalase there was no detectable rp in the cell bodies (Table II). Proteins for which transport was demonstrated included anionic H R P , cationic H R P and cytochrome-c (Table II and II!). All of the transported proteins showed the same granular localization of rp in the cell bodies (Figs. 3 and 4). The density of rp, particularly in the case of cytochrome-c, varied greatly between neurons. The n u m b e r of detectable rp-labelled neurons as well as the density o f labelling increased in the order of cytochrome-c:anionic H R P : c a t i o n i c H R P after injection of the same a m o u n t of protein.

TABLE I1 The number of hypoglossal neurons containing reaction product after injection 0[ various proteins into the tongue

Results are expressed as the mean number of cells containing reaction product per section for each animal. The number of labelled cells present in both hypoglossal nuclei is expressed as the mean value for a single nucleus in order to facilitate comparison with results obtained in other experiments where uptake and transport of proteins occurred unilaterally from a hypoglossal nerve crush. Tracer

Survival (h)

Dose (mg)

Cells~nucleus~section

Cyt-c |II

24 24 24 24 30 48

12 12 12 !8 18 18

0.22 1.70 0.18 0.34 0.20 3.35

0.05 1.15 0.08 0.25 0.13 2.10

0.00 0.00 0.00 0.08 0.05 0.29

Cyt-c VI

24 24 48 72

6 12 12 12

0.14 0.35 0.30 0,35

0.05 0.15 0.19 0.25

0.04 0.15 0.16

0.00 0.03 0.13

Catalase

24

18

0.00

0.00

0.00

0,00

Hemoglobin

24

18

0.00

0.00

0.00

Myoglobin

24 48

18 18

0.00 0.00

0.00 0.00

0.00 0.00

0.00 0.00

483 TABLE II[

Transport 0[" isoenzymes of horseradish peroxidase to the hypoglossal nucleus 24 h after injection into the tongue Results are expressed for each animal as the number of cells containing reaction product per section. The number of labelled cells in both hypoglossal nuclei is expressed as the mean value for a single nucleus in order to facilitate comparison with results obtained in other experiments where uptake and transport of proteins occurred unilaterally from a hypoglossal nerve crush. Sections were incubated either according to Malmgren and Olsson~°,31 (histochemical technique 1) or in some cases also according to Graham and Karnovsky 9 (histochemical technique 2).

Tracer

Dose (rag)

Histochemical technique

Cationic HRP 3.00 2 0.50 2 0.25 2 0.25 2 0.10

2 0.05 Anionic HRP 3.00 0.50

1 2 1 2

Cells/m~cleus/section

17.8 7.27 14.2 6.1 9.04 3.90 [.55 0.05 0.50 0.00 0.29 0.00 8.54 2.50 3.23 0.72

17.4 9.56 3.72 5.98 0.72

13.0 5.13 7.36 3.21 5.70

11.2

5.69 1.19

3.30 0.75

3.13 0.44

0.29 0.00 0.07 0.00 7.16 1.35 2.28 0.35

6.55 2.27 2.00 0.05

6.15 1.17 0.08

0.10 0.00

Fig. 3 and 4. Cell bodies containing granular accumulations ofrp (arrows) at 24 h after injection of 3 mg of either cationic or anionic HRP into the tongue. The ceils shown are representative of the most heavily labelled cells in experiments concerning the uptake and transport of cationic HRP (Fig. 3, × 1078) and anionic HRP (Fig. 4, x 1156). Although at the same dose the density of rp is much greater after transport of cationic HRP, the transport of anionic HRP is also clearly detectable. The rp in Fig. 3 appears to have a diffuse localization in the figure due to superposition of rp granules that were not in the focal plane, even though rp was actually limited to granular accumulations.

484

Fig. 5. Hypoglossal neurons showing granular rp 168 h after repeated injections of ferritin. :. 1200. Fig. 6. Hypoglossalneurons showing granular rp 192 h after repeated injections ofiron -dextran. 4000 After a single injection of ferritin of iron-dextran no rp could be detected in the hypoglossal neurons, while after 3 injections some neurons contained a clearly visible granular rp (Figs. 5 and 6). Mice injected 5-7 times showed many hypoglossal neurons containing rp. Administration of proteins at nerve crush Various protein tracers were applied to the hypoglossal nerve immediately before it was crushed with jeweller's forceps. At 30 min after the crush, all of the enzymatic proteins had diffused through the damaged perineurium and into the extracellular space of the endoneurium. However, at sites distant from the crush, none of the enzymes penetrated the perineurial diffusion barrier. The distribution of rp in the nerve proximal to the crush varied with the distance from the crush site. Zone 1, the immediate area of the crush, usually contained dense diffusely distributed rp. In nerves having only very little rp in this zone, fine granular rp was apparent. Zone 2 consisted of a relatively clear 0.1 mm region where only sparse finely granular rp was present with no detectable intra-axonal rp. This was bordered by another 0.1 mm zone, Zone 3, where large irregular rp accumulations were present. The most proximal region, Zone 4, was characterized by variable amounts of diffuse intra-axonal rp which extended to a distance of approximately 1.5 mm from the crush site. At 30 min all the enzymatic tracers were localized intra-axonalty in at least a few nerve fibres, but the number of fibres containing intra-axonal rp differed greatly for the various proteins (Figs. 7, 8 and 9). The distribution of rp differed markedly with the particular protein injected. Cationic HRP and cytochrome-c showed the greatest penetration into axons. With these proteins, densely labelled axons were found even in cases where rp at the crush site was barely detectable. In contrast anionic H R P and microperoxidase, the smaller anionic degradation product of cytochrome-c, showed a lesser affinity for the axoplasm of injured nerve fibres as did the other protein tracers examined. With such proteins only limited intra-axonal rp was present even in cases where rp at the crush site was very dense.

485

Figs. 7, 8 and 9. Hypoglossal nerve crush site at 30 rain after application of tracers to the nerve. Cytochrome-c is localized at the site of the crush (C) with intra-axonal accumulations of rp present more proximally (arrows) (Fig. 7, × 315). Fig. 8 shows intra-axonal accumulations of cytochrome-c at still more proximal levels (approx. 0.5-0.8 mm from crush) where the intra-axonal rp was more dense than that shown close to the crush in Fig. 7. x 1360. In experiments where microperoxidase was applied to the crush, relatively little intra-axonal rp was present, even though the density o f r p at the crush site (C) was heavy (Fig. 9, ~ 331).

486 TABLE IV Transport of iron-containing macromolecu&s to the hypoglossal nucleus after multiple ittjectiotts into the tongue

The dose given in this case is for the total amount of protein and iron administered. The number of cells in the hypoglossal nucleus containing detectable accumulations of iron are indicated as cells/ nucleus/section. Tracer

No. of injections

Survival (h)

Total dose (mg)

Fe:~ (mg)

Cells/nucleus/section

Ferritin

1 3 5 6

24 96 144 168

I0 30 50 60

2 6 10 12

0.00 4.55 13.8 14.2

0.00 9.45

Iron-dextran 1 3 7

24 96 192

14 42 245

2 6 35

0.00 1.20 13.40

0.00 0.20 9.70

0.15

The distribution of cytochrome-c and cationic H R P differed at 24 h from that seen at 30 min. By 24 h the intra-axonal rp had extended from 1.5 mm zone adjacent to the nerve crush to a distribution along the entire length of the hypoglossal nerve section (approx. 5 mm from the crush site). At this time the intra-axonal rp nearest the crush site remained very dark relative to that at more proximal levels which was generally faint. The distribution of cytochrome-c was examined also at 48 h and 72 h, At these longer survival times the intra-axonal rp became progressively more faint with a loss of the high density rp that had remained in the zone adjacent to the crush site at 24 h. Even though all the enzymatic tracers were localized intra-axonally in at least some fibres, evidence of transport to the hypoglossal nucleus was limited to the same proteins for which transport had been demonstrated after injection into the tongue, namely H R P VI, cationic HRP, anionic H R P and cytochrome-c (Table V), When equal or greater amounts of microperoxidase, myoglobin or hemoglobin were applied to the hypoglossal nerve immediately prior to crushing it, no rp was detectable in the hypoglossal nucleus at 24 h and 48 h survival times. As was also the case after injection into the tongue, the number of rp-labelled neurons as well as the density of labelling decreased in the order of cationic H R P :anionic H R P :cytochrome-c. In all cases and at all survival times the transported proteins were localized in the cell body as granular accumulations in contrast to the diffuse distribution that was seen in the axons of the crushed hypoglossal nerve. After application of ferritin and iron-dextran for 30 min and 24 h a faint bluish rp was seen in the crushed area. N o definite intra-axonal rp could be revealed. N o accumulation of the tracers in the nerve cell bodies was detected after a time interval of 24 or 48 h.

487 TABLE V The number o f hypoglossal neurons coutabzing reaction product at times after application o f various proteins at a hypoglossal nerve crush

The results are expressed for each animal as the mean number of labelled neurons per section in the ipsilateral hypoglossal nucleus. Histochemical technique

Tracer

Survival (h)

Dose (rag)

Cells/nucleus~section

Cyt-c VI

24 24 48 72

3.0 1.0 3.0 3.0

4.00 1.36 12.2 2.70

HRP cationic

24

1.0

19.0

HRP anionic

24

1.0

12.6

7.83

5.50

Malmgren and Olsson3°,31

Microperoxidase 24

3.0

0.00 0.00

0.00 0.00

0.00 0.00

Feder5 Simoinescu et a142

Myoglobin

24 48

3.0 3.0

0.00 0.00

0.00 0.00

0.00 0.00

Anderson 1 Anderson ~

Hemoglobin

24 48

3.0 3.0

0.00 0.00

0.00 0.00

0.00 0.00

Ferritin

24 48

5.0 5.0

0.00 0.00

0.00 0.00

Perls 3~ Perls36

lron-dextran

24

17.5

0.00

0.00

Perls 36

3 . 0 8 1 . 0 8 0.20 7.75 1 . 5 7 1.08 0 . 9 1 18.1 1 4 . 5 1 3 . 5

0.00 0.00

Karnovsky and Karnovsky and Karnovsky and Karnovsky and

Rice 15 Rice a5 Rice 15 Rice 15

Malmgren and Olsson 3°,3I

Goldfischer et al. 8 Goldfischer et al. 8

DISCUSSION M a n y previous investigations have examined the capacity o f various cells to i n c o r p o r a t e m a c r o m o l e c u l e s f r o m the e n v i r o n m e n t in view o f the p r o b a b l e role o f this p h e n o m e n o n in the u p t a k e o f viruses, m a c r o m o l e c u l a r toxins, p r o t e i n - b o u n d drugs, peptide a n d p r o t e i n h o r m o n e s , nucleic acids a n d various o t h e r biologically active m a c r o m o l e c u l e s (see reviews by Ryser39, 4° a n d HabermannlZ). M o s t o f these investigations have been carried o u t in tissue culture, since this avoids the c o m p l i c a t i o n s inherent in in vivo studies. O u r w o r k has concerned the u p t a k e o f m a c r o m o l e c u l e s in vivo by the neuron, a m o r p h o l o g i c a l l y a n d physiologically complex cell type which is p r o t e c t e d in vivo by cellular diffusion barriers. M a c r o m o l e c u l a r u p t a k e has been studied at the p e r i p h e r y and at a lesion in a cellular process, a n d the intracellular m o v e m e n t o f the p r o t e i n tracers t o w a r d the p e r i k a r y o n has been followed using histochemical m e t h o d s . Despite the m a n y biological and technical difficulties with o u r system, several significant new findings have been obtained. F i r s t o f all, it was o f p a r t i c u l a r interest to determine the relative accessibilities of the different p r o t e i n s to the nerve fibres after injection into the tongue. Previous investigations have shown t h a t in large nerves proteins are excluded f r o m the e n d o n e u r i a l tissue space by diffusion barriers associated with the p e r i n e u r i u m a n d the

488 endoneurial blood vessels 17.'-'7,'.~4-46. However, when cytochrome-c or myoglobin are injected into the tongue of adult rats and mice, these proteins are subsequently localized throughout the extracellular space of the endoneurium '-'7,es. In the present study we have examined the diffusion of various macromolecules represen!ing a wide range of molecular size and charge. All of the macromolecules examined diffused at least to some extent into the endoneurial tissue space of the small intramuscular nerve branches in the tongue. However, these proteins diffuse proximally for only a limited distance, since rp was less frequent in the larger nerve branches in the tongue find was never present in the extracellular space of the main trunk of the hypoglossal nerve. regardless of survival time. Thus, it does not appear that charge or steric interactions exert strong differential effects on the diffusion of macromolecules through the extracellular matrix of the endoneurium. It can be assumed that these endoneurial diffusion pathways are also available to various pathogenetic and biologically active macromolecules, at least those falling within the size range examined (tool. wl.

1900-240,000). Previous investigations concerning the effects of" molecular size and charge have indicated that cationic macromolecules are taken up into cells in tissue culture more readily than neutral or anionic macromolecules and also that the rate of uptake increases with molecular size as,ag. However, at the axon terminal, the rate of macromolecular uptake is determined by the rate of membrane turnovei that takes place as a result of synaptic transmission ta. Consequently, the mechanism for uptake of macromolecules at the axon terminal may differ from that of other cell types. The effect of molecular charge on the uptake and transport of macromolecules from CNS axon terminals was recently examined by Bunt et al. ~. They found histochemical evidence for the uptake and retrograde axonal transport of two different isoenzymes of HRP, but there was not detectable transport of an anionic isoenzyme of HRP. It was suggested that these differences were related either to charge differences between the isoenzymes or to differences in the carbohydrate moeity of H R P isoenzymes. Our findings differ from those obtained by this group. We have observed uptake and retrograde axonal transport not only of cationic H R P but also of anionic HRP. However, it is important to note that our results have been obtained for peripheral motor neurons while Bunt et al. ~ have examined the specificity of uptake and transport in CNS neurons where such mechanisms could conceivably differ. It is also possible that some transport of anionic H R P took place in Bunt's study 2 that was not detected. They used 4 ~'~iparaformaldehyde as a fixative which greatly reduces the enzymatic activity of H R P 16,a°,al, as compared to fixation with similar concentrations of glutaraldehyde. In addition, they used the standard Graham and Karnovsky incubation medium 9, which has been shown in the present study to result in a lower number of detectable rp-labelled cell bodies with either anionic or cationic H R P than when sections are incubated, according to Malmgren and Olssona°,aL Although these procedures may have reduced the sensitivity for the detection of transport of anionic HRP, this would not account for their finding that one of the cationic isoenzymes having similar peroxidase activity to anionic H R P was detectably transported, unless there were differences in the uptake and/or transport of these isoenzymes. However,

489 since we were able to detect the uptake and transport of both the cationic and the anionic isoenzymes, the mechanism that distinguishes between these two molecules must operate with a rather limited specificity. This would appear similar to the level of specificity reported for the uptake of charged macromolecules by other cell types. In such cases the relative rates of uptake of anionic proteins such as albumin and ferritin are only one to several orders of magnitude lower than very cationic proteins such as lysine-rich histone and arginine-rich histone 39. Although large differences in the relative amount of uptake are seen, this mechanism is not absolutely selective. This type of specificity would differ markedly from the much more efficient and specific uptake of macromolecules such as nerve growth factor. In such cases the uptake mechanism selects on the basis of characteristics specific to a particular molecule, rather than on the basis of general physicochemical properties such as size or charge, and the uptake may be limited to only certain types of neurons 4a,44. Our results show that the number of detectable rp-labelled neuronal cell bodies is considerably greater after injection of cationic HRP than after injection of the same dose of anionic HRP. The number of cells labelled per mg tracer injected is much lower for cytochrome-c and ferritin than with either of the HRP isoenzymes, both after injection into the tongue and after application of the tracer to the nerve crush. These results may be of interest to those concerned with the possible use of these tracers in neuroanatomical studies. However, the data should not be interpreted as an indication of the exact amount of each protein transported to the cell body for several reasons. The sensitivities for the detection of the peroxidase tracers are not equivalent due to differences in enzymatic activity (anionic HRP -- 95 purpurogallin units/mg*, cationic HRP = 330 purpurogallin units/mg*, cytochrome-c -- 0.00003-0.08 the activity of HRPa2,45), and the localization of ferritin and Imferon are carried out using entirely different histochemical procedures. Furthermore, in histochemical studies the differential loss of sensitivity by various tracers as a result of fixation must also be considered. More precise data may be possible using quantitative techniques for the assay of uptake and transport of various radiolabelled macromolecules, although in past studies it has not been possible to obtain sufficient sensitivity for the detection of the retrograde axonal transport of proteins such as horseradish peroxidase, cytochrome-c and ferritin using such techniques 4a. Histochemical procedures, on the other hand, permit the distribution of these tracers to be examined with higher resolution than is possible with other techniques and allow these tracers to be detected at extremely low concentrations. We have also demonstrated the uptake and retrograde axonal transport of cytochrome-c both after injection into the tongue and after application at a nerve crush. Bunt et al. 2 have discussed the possibility that specificity in uptake and transport of HRP isoenzymes was related to differences in their carbohydrate moeities. Cytochrome-c lacks a carbohydrate moeity a, and this might be interpreted as evidence that there is not an absolute requirement for a carbohydrate group in the uptake and

* Activities supplied by Sigma.

490 transport mechanism of motor neurons. However, a possible positive or negative effect of carbohydrate groups on uptake or transport is not ruled out. Unlike other proteins injected into the tongue, cytochrome-c was occasionally localized diffusely within some of the axons in the nerve branches in the tongue of injected animals. This diffuse localization of protein tracers is typically associated with injury to the membrane~l,9'3,m~,3z, 41. Thus, it is conceivable that the transport observed after injection of cytochrome-c into the tongue was restricted to a small number of injured fibres resulting from the mechanical damage produced by the injection procedure but, on the other hand, similar fibres were not seen after injection of HRP. The results from the tongue compression experiments support the view that such fibres might be injured but, in addition, cytochrome-c appears to have a particular affinity for axoplasm. Ferritin, also, could be taken up by axons and transported to the hypoglossal neurons. However, in order to detect the accumulation of this tracer in neuronal cell bodies, it was necessary to use repeated injections into the tongue, giving a high local concentration for relatively long periods of time. lron-dextran could also be localized in the cell bodies of the hypoglossal nucleus when administered in this way. This agrees well with studies carried out with organotypic cultures of nervous tissue in which very high concentrations of iron-dextran were needed to induce a histochemically detectable accumulation in mouse spinal cord and ganglia neurons. When the explants were exposed to lower concentrations, accumulations of tracer occurred only in macrophages, glial cells and fibroblasts "4. Since iron--dextran is used for intramuscular injections in clinical practice, the possibility therefore exists that this heavy metal may reach lower motor neurons also in humans. The possible long-term effects on motor neurons of such an accumulation is presently being experimentally tested. The tracers that could be detected in the cell bodies after application to the nerve crush site were the same ones that could be detected after single injections into the tongue, namely anionic HRP, cationic H R P and cytochrome-c. As in the tongue injections, no transport of lmferon and ferritin could be detected after single applications to the crush. We did not attempt multiple applications of these iron-containing macromolecules in the case of the nerve crush, since it has been shown that the uptake of macromolecules at a nerve crush occurs almost entirely during the first 30 min "3. The number of detectable labelled cell bodies increased in the order of cytochrome-c: anionic H R P : cationic HRP, but it should be emphasized once again that this is not a precise indication of the exact amount of protein that accumulated in the cell bodies due to technical considerations. The various tracers showed striking differences with regard to the extent to which they penetrated into injured axons at the hypoglossal nerve crush. Cytochromec (pl 10.5) and cationic H R P (pl 9.5) showed much greater affinity for the axoplasm than other proteins, even though some very limited intra-axonal rp could be detected in at least a few fibres in experiments where the other peroxidase tracers were used. Even in cases where the amount of extracellular rp at the crush was very faint, with these proteins dark rp could be found in some of the axons. This selective accumulation of protein in injured axons was not seen with microperoxidase (mol. wt. 1900; pl

491 5.4), a smaller anionic degradation product of cytochrome-c. Similar differences were seen between anionic a n d cationic H R P with, again, much greater b i n d i n g of the cationic tracer to the axoplasm. These differences in the d i s t r i b u t i o n of cationic and a n i o n i c protein tracers at the nerve crush may be related to differences in their b i n d i n g to the various c o m p o n e n t s of the axoplasm. It has been shown, for example, that charge effects result in the b i n d i n g of cytochrome-c to neurofilament protein 7. Similar charge interactions between macromolecules that enter injured axons a n d various axoplasmic c o m p o n e n t s may c o n t r i b u t e to a selective d i s t r i b u t i o n of macromolecules to different ' c o m p a r t m e n t s ' on the basis of charge. ACKNOWLEDGEMENTS This study was supported by G r a n t 1 F32 NS 05348-1, U n i t e d States N I N C D S , N I H and by the Swedish Medical Research Council Project No. B78-12X-3020-09A and B78-12X-04480-04A. Technical assistance was provided by ~,ke Pettersson a n d Benita Andersson.

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