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The use of wheat germ agglutinin-horseradish peroxidase conjugates for studies of anterograde axonal transport
Journal of Neuroscience Method~'. 7 (1983) 117-128 Elsevier Biomedical Press
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The use of wheat germ agglutinin-horseradish peroxidase conjugates for studies of anterograde axonal transport Patrick W. Mantyh I and Marc Peschanski (P. blLM. and M.P.) Department of A naton~v, Universi O' of Cahfornia, San Francisco, San Francis~'o. ('A 94143. (P. IJLM.) Biomedical Research Division, Ames Research Center, NASA, Moffett Field. CA 94035 (IZ S.A.), and (M. P,) Unite de Recherches de Neurophysiologie Pharma~'ologique, I N S E R M U161, 2 rue d'Alesia. Paris, (France) (Received June 14th, 1982) (Accepted June 16th, 1982)
Keywords: wheat germ a g g l u t i n i n - H R P - - a n t e r o g r a d e transport--transneuronal transport--spinothalamic tract--dorsal column nuclei Injection of wheat germ agglutinin conjugated to horseradish peroxidase ( W G A - H R P ) into the hemisected spinal cord of the rat, cat and monkey consistently resulted in the intense anterograde labeling of ascending spinal projections such as the spinothalamic tract and spinocerebellar tracts and theiT terminal fields. Injections of W G A - H R P in the dorsal column nuclei resulted in the anterograde labeling of the medial lemniscus and its terminal fields in the thalamus. Injection of similar amounts of horseradish peroxidase alone (HRP) in hemisected animals or the dorsal column nuclei resulted in little anterograde labeling. The rate of the anterograde transport of W G A - H R P in cut axons appears to be greater than 200 r a m / d a y . Small amounts of transneuronal labeling appeared to occur after iniection of W G A - H R P in both cut axons and undamaged cell bodies. These results suggest that the amount of anterograde labeling observed after injection of W G A - H R P into both cut axons and undamaged cell bodies is significantly greater than the anterograde labeling observed after injections of H R P alone. Therefore, in the central nervous system W G A - H R P appears to be a far more effective anterograde tracer than H R P alone.
Introduction Since the introduction of wheat germ agglutinin (Schwab et al., 1978) (WGA) and wheat germ agglutinin conjugated to horseradish peroxidase (Gonatas et al., 1979) (WGA-HRP) as a neuroanatomical tracer, several authors have successfully applied these techniques to studies of retrograde axonal transport (Ruda and Coulter, 1980;
i To whom correspondence should be addressed at: M R C Neurochemical Pharmacology Unit, Medical Research Council Centre, Medical School, Hills Road, Cambridge CB2 2AH, U.K. 0165-0270/83/0000-0000/$03.00 ~ 1983 Elsevier Biomedical Press
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Staines et al., 1980; Steindler and Deniau, 1980; Streit, 1980; Bull et al.. 1981, Saper, 1981). Recently Gonatas et al. (1979) have reported that the retrograde transport of the W G A - H R P conjugate is up to 40 × as avid as H R P alone. In some of these published reports using W G A - H R P or W G A alone for retrograde tracing, the authors have incidentally commented that W G A - H R P a n d / o r W G A is also transported in an anterograde direction (Ruda and Coulter, 1980; Staines et al., 1980: Steindler and Deniau, 1980; Streit, 1980; Bull et al., 1981). Mesulam and Mufson (1980) have previously demonstrated that H R P is transported in an anterograde direction in the rat retino-tectal pathway. Craig and Burton (1981) have confirmed this finding by showing that anterograde transport of H R P can also take place in the cat spinal cord. However, in both studies the amounts of H R P used to accomplish the necessary labeling seem quite large if one were to apply this H R P anterograde technique to the various regions of the central nervous system (CNS). Recently, it has been reported that [~25I]WGA (Margolis et al., 1981) and H R P conjugates of cholera toxin and W G A (Trojanowski et al., 1981) appear to be transported in an anterograde direction in the chick (Margolis et al., 1981) and rat (Trojanowski et al., 1981) retino-tectal pathway respectively. These results suggest the possibility that W G A - H R P might be superior to H R P alone for neuroanatomical studies which utilize anterograde axonal transport. We have therefore investigated the anterograde transport of W G A - H R P in the central nervous system of the rat, cat and monkey. The present series of experiments were designed to test whether W G A - H R P could be a more effective tracer for anterograde connections in the CNS than H R P alone,whether this anterograde transport of W G A - H R P could occur from cut axons a n d / o r intact cell bodies and whether transneuronal transport of the tracer occurs.
Materials and Methods
Forty-one rats, 5 cats and 5 monkeys were used in these experiments.
W G A - H R P injections in the spinal cord Twenty-six rats, 5 cats and 5 monkeys received W G A - H R P injections into their spinal cords after hemisection. The rats were anesthetized with chloral hydrate (400 m g / k g ) while the cats and monkeys were first tranquilized with ketamine and then anesthetized with pentobarbital (40 mg/kg). The spinal column was exposed at either C7-C8 or L2-L3. A laminectomy was then performed, the dura reflected and the cord hemisected. A solution of 10% W G A - H R P (Sigma) in saline was injected into the spinal cord 1 m m rostral to the hemisection. In the rat 0.02-1.0 /zl was injected while in the cat and monkey 1-5 ~1 was injected. By hemisecting the cord and then injecting, we attempted to ensure that all cut fibers in the cord at the level of the hemisection would be exposed to the W G A - H R P . One to five clays after the injection of W G A - H R P into the cord, the animals were anesthetized and perfused transcardially with a 0.1 M phosphate-buffered (pH 7.4) saline solution followed by a 0.1 M phosphate-buffered solution (pH 7.4) containing
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2% glutaraldehyde and 1% paraformaldehyde fixative. After perfusion with the fixative a solution of 0.1 M phosphate-buffered solution (pH 7.4) containing 5% sucrose was perfused through the vasculature to remove any excess fixative (Mesulam, 1978). The brain and spinal cord were then removed and carried through a series of graded sucrose solutions to cryoprotect the brain. Serial 50 #m frozen sections were cut, collected, and every other section was reacted for the presence of W G A - H R P using tetramethyl benzidine (TMB) as the chromagen (Mesulam, 1978). Every other reacted section was counterstained with Neutral red. All sections were examined under both light- and dark-field optics and all areas containing W G A - H R P in fibers or terminals were plotted onto a drawing of the section that had been made with the aid of an overhead projector.
Control experiments Fifteen rats were used for various control experiments as described below. In six rats, 0.1-1.0 ~1 of 10% solution of H R P alone (Sigma type VI) was injected into the cervical cord (3 rats) or the lumbar cord (3 rats) after hemisection. The objective was to compare the anterograde transport of H R P to that of WGA-HRP. In these experiments all aspects of the surgery, amounts injected, recovery period and histochemical processing were identical to the W G A - H R P injected material outlined above. In 3 rats the cisterna magna was opened, the dura retracted and 0.2/LI of 10% W G A - H R P was placed on the dorsal column nuclei (DCN). The objective was to assess whether uninjured somas could take up the W G A - H R P and transport it in an anterograde fashion as avidly as cut axons in the cord. After the W G A - H R P was deposited on the DCN the wound was closed, and the animal placed back in the cage for 2 - 3 days. After this time the brains were again processed as described above. In 2 rats, 0.2/~1 of a 10% solution of H R P alone was placed on the DCN. The objective of this group of animals was to assess whether HRP alone could duplicate the anterograde transport observed when W G A - H R P was placed on the DCN. These animals had identical survival times and histochemical processing as the W G A - H R P animals. In 2 rats the spinal cord was exposed and 0.2 ~1 of 10% W G A - H R P was placed on the exposed spinal cord at upper lumbar levels. In this series of animals the objective was to assess if the conjugate could be taken up by uninjured fibers and anterogradely transported. After the W G A - H R P was placed on the cord the rats were handled in the same way as the W G A - H R P hemisected and injected animals. In 2 animals a sham operation was performed in which the cords were exposed, hemisected and reclosed. The rats survived for 2 days and processed using the same protocol as the hemisected and W G A - H R P injected animals. The objective of this series of animals was to see if any endogenous reaction product was present due to the surgical procedure employed.
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Results
WGA-HRP injections in the spinal cord A d a r k - b l u e g r a n u l a r reaction p r o d u c t m a r k e d the presence of H R P (Mesulam, 1978). In a n i m a l s injected with W G A - H R P we f o u n d that hemisection before injection a i d e d in labeling fibers. In hemisected W G A - H R P - i n j e c t e d animals labeled fibers could be o b s e r v e d in the spinal cord, a n d some of the fibers in the fasciculus gracilis a n d c u n e a t u s could be observed t e r m i n a t i n g in the dorsal c o l u m n nuclei ( D C N ) in a t o p o g r a p h i c a l pattern. In these W G A - H R P injected animals the a s c e n d i n g s p i n o t h a l a m i c tract (STT) fibers were heavily labeled a n d could be seen coursing t h r o u g h the medulla, p o n s a n d m e s e n c e p h a l o n (Fig. IA), usually c a p p i n g the medial lemniscus. A t the m e s o d i e n c e p h a l i c border, the s p i n o t h a l a m i c fibers w o u l d b e n d a w a y from the m a i n b o d y of the tract and end in dense puffs in discrete t h a l a m i c nuclei (Fig. 1B - F ) . The c o m p l e t e results of these STT studies in the rat, cat a n d m o n k e y will be p u b l i s h e d in s e p a r a t e papers. Both the fibers a n d the terminal fields could easily be viewed using b o t h light- (Fig. 1A, B, D and F) a n d d a r k - (Fig. I C a n d E) field optics; the N e u t r a l red stain p r o v i d e d g o o d c o n t r a s t for color p h o t o m i c r o g r a p h s u n d e r light-field optics. O t h e r a s c e n d i n g a n d d e s c e n d i n g tracts were also labeled including the spinoolivary, s p i n o - c e r e b e l l a r (Fig. 3A a n d B), spino-vestibular, a n d the spino-reticular. In general, the a n t e r o g r a d e labeling observed in these tracts and their terminal fields was at least as intense as that o b s e r v e d in the STT ( c o m p a r e Figs. 1A a n d 3B). F o r example, the s p i n o c e r e b e l l a r labeling was often so intense that i n d i v i d u a l m o s s y fibers could be seen t e r m i n a t i n g u p o n granule cells in the cerebellum (Fig. 3C a n d D). Small a m o u n t s of t r a n s n e u r o n a l labeling a p p e a r e d to occur after injection of W G A - H R P in cut axons. In a n i m a l s with injections of W G A - H R P in the spinal cord, light i n t r a s o m a t i c labeling of cerebellar granule cells a n d thalamic VPL n e u r o n s could be observed. In general, these t r a n s n e u r o n a l l y labeled neurons were o n l y lightly filled when c o m p a r e d to the intense labeling in the p r e s y n a p t i c fiber and the p o s t s y n a p t i c n e u r o n s p r o j e c t i o n fibers (the parallel fiber for the granule cell a n d
Fig. 1. A series of photomicrographs of coronal sections through the brain following hemisection and injection of WGA-HRP at C7-C8 in the spinal cord. A: this light-field photomicrograph shows labeled spino-thalamic tract (ST/') fibers in the monkey mesencephalon ipsilateral to the spinal injection site. Magnification: 100×. B. a light-field photomicrograph showing the dense anterograde labeling present in the caudal ventroposterior lateral nucleus (VPL) of the monkey ipsilateral to the injection site. Magnification: 100 x. C and D: a dark- and light-field photomicrograph, respectively, showing the intense anterograd¢ labeling present in the mid-VPL of the rat after an ipsilateral cervical injection. Note the absence of label in the adjacent ventroposterior medial nucleus (VPM). C is 100 p.m caudal to D. In C the anterograde labeling appears white; in D it appears black. The circular areas in VPL without label are probably bundles of unlabeled thalamo-cortical and cortico-thalamic fibers. Magnification: 70 ×. E and F: a dark- and light-field photomicrograph, respectively, showing the intense patchy labeling in the monkey VPL after an ipsilateral injection. E and F are from the same section. In E the anterograde labeling appears white while in F it is black, Magnification: 30 x.
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123 the thalamo-cortical fiber for VPL neurons) were never observed to be labeled. The m i n i m u m rate of anterograde axonal transport of the W G A - H R P could be estimated from these experiments. F r o m a n injection into the l u m b a r cord of a cat with a 1-day survival period, it appears that this transport travels at least 200 m m / d a y . This is based on 1-day survival a n d a distance of at least 200 m m from injection site to the thalamus in a cat. This rate is in the range of the 288 r a m / d a y estimated for the anterograde transport of H R P alone in the rat visual system ( M e s u l a m a n d Mufson, 1980). Since labeling of the STY was observed in all hemisected cases we can only determine the m i n i m u m speed. These results indicate that at least some of the anterograde transport of W G A - H R P is carried by fast axonal transport.
Control experiments In agreement with previous studies, occasional anterograde label was observed after injection of H R P alone. However, the a m o u n t s of H R P needed to be injected into the rat cervical cord (1 ~1 of 10% solution) to see a n y anterograde labeling in the t h a l a m u s also produced an artifactual 'edge effect' a r o u n d the lateral a n d dorsal borders of the m e d u l l a a n d pons. This effect is p r o b a b l y due to the leakage and spread of the H R P by capillary action along the meninges up to these rostral regions. This rostral spread of H R P can seriously c o m p r o m i s e the interpretations of the results o b t a i n e d in the spinal injections due to u n w a n t e d somatic pickup by the dorsal c o l u m n nuclei somas (see below). Thus intense anterograde labeling in the b r a i n s t e m a n d thalamus was never observed in the hemisected rats injected with c o m p a r a b l e a m o u n t s of H R P alone. Other ascending systems including the spinocerebellar tract were also rarely labeled after H R P injection a n d when present the label was observed in fewer areas, a n d had a greatly diminished intensity as c o m p a r e d with the labeling seen after similar a m o u n t s of W G A - H R P were injected. In the rats in which W G A - H R P was placed on the DCN, a striking a n d well delineated anterograde labeling was apparent. In these animals the cell bodies which Fig. 2. This series of photomicrographs shows the anterograde transport of WGA-HRP after 0.2/~1 of 10% solution of the conjugate was placed on the dorsal column nuclei (DCN) of the rat, so as to eliminate the possibility of any mechanical damage to the cell bodies or axons. A: this light-field photomicrograph shows the extent of the spread of WGA-HRP in the DCN. Note the absence of label ventral to the nucleus gracilis (GR) and fasciculus cuneatus (FC). The unlabeled oval structure at the lower left is the central canal. Magnification 50 x. B: this light-field photomicrograph is a higher magnification of A and shows the cell bodies in the GR which are black and have apparently taken up the WGA-HRP. Magnification: 170x. C: this dark-field photomicrograph shows the labeled fibers in the medial lemniscus (LM) of the rat after WGA-HRP was placed on the DCN. Magnification: 85 x. D-F: this series of photomicrographs from the same section shows the terminations of the rat DCN projections in the VPL contralateral to the labeled DCN. D is a low-powerdark-field photomicrograph showing that the pattern of labeling is quite similar to that seen in Fig. 1C after an STT injection. Magnification 65 x. E: a light-field photomicrograph showing the dense anterograde labeling in the VPL. The white unlabeled areas in E are probably unlabeled bundles of thalamo-cortical and cortico-thalamic fibers. Magnification: 130x. F: a dark-field photomicrograph of E. Again note the lack of any labeling in the internal capsule (IC) where the thalamo-cortical fibers would be expected to be traveling. Magnification: 130 ×.
125 were exposed to the W G A - H R P (Fig. 2A) were densely filled with reaction product (Fig. 2B). A n t e r o g r a d e l y labeled fibers could be traced out of the D C N . These traveled in the medial lemniscus in distinct fiber b u n d l e s (Fig. 2C) a n d t e r m i n a t e d in the ventroposterior lateral nucleus of the thalamus (Fig. 2 D - F ) . This type of a n t e r o g r a d e fiber a n d terminal labeling in the VPL was similar to that seen with spinal injections (compare Figs. 1C a n d 2D) a n d is consistent with studies d e m o n strating that n e u r o n a l damage is not necessary for anterograde axonal transport of various tracers such as H R P alone. Small a m o u n t s of t r a n s n e u r o n a l labeling also appeared in the thalamic VPL n e u r o n s in the a n i m a l s injected with W G A - H R P in the D C N . This t r a n s n e u r o n a l labeling was similar to that observed after injections into cut spinal axons where the i n t r a s o m a t i c labeling of the thalamic n e u r o n was light compared to the intense labeling in the presynaptic fibers. In the rats in which similar a m o u n t s of H R P alone were placed on the D C N , only little anterograde labeling was evident in either the medial lemniscus or the VPL. The animals which had W G A - H R P placed on their spinal cords showed no signs of anterograde transport. A p p a r e n t l y the reason that the u n d a m a g e d spinal cord n e u r o n s did not transport W G A - H R P , but D C N n e u r o n s did, is due to the fact that the spinal cord n e u r o n s are u n s h e a t h e d by white matter while D C N n e u r o n s in the rat are at the dorsal-most extent of the caudal medulla where the W G A - H R P can easily penetrate. N o reaction product was observed in the dorsal thalamus in the group of s h a m - o p e r a t e d animals. Two areas which did display an e n d o g e n o u s reaction p r o d u c t when reacted with the T M B protocol were the arcuate nucleus of the h y p o t h a l a m u s a n d the i n t e r p e d u n c u l a r nucleus of the mesencephalon, the same areas that d e m o n s t r a t e an e n d o g e n o u s reaction p r o d u c t when reacted with dia m i n o b e n z i d i n e (Wong-Riley, 1976). Fig. 3. A: a light-field photomicrogrzph showing labeled spinocerebellar fibers in the monkey restiform body after an ipsilateral hemisection and injection of WGA-HRP at C7. Note the darkly labeled fibers in the restiform body and the darkly stained cells in the granule layer. Magnification: 50 ×. B: this light-field photomicrograph shows at higher magnification the intense spino-cerebellar fiber labeling obtained using this technique after an ipsilateral hemisection and injection of WGA-HRP at C7 in the monkey. Magnification: 130 ×. C: a light-field photomicrograph demonstrating the possible transneuronal labeling of granule neurons of the monkey cerebellum after the same injection shown in A and B. Magnification: 170 x. D: this light-field photomicrograph depicts at a higher magnification labeled granule cells in the monkey cerebellum after an injection in the monkey spinal cord at C7. Note that the reaction product appears to be inside the cell body, suggesting transneuronal transport. Magnification: 240x. E: a light-field photomicrograph which shows some of the monkey Purkinje cells occasionally observed surrounded by reaction product after an injection of WGA-HRP at C7. Since no direct spino-cerebellar projection to the Purkinje cells is known, this labeling possibly represents the projections of a granule cell upon a Purkinje cell. If true, this demonstrates that: (1) transneuronal transport after injection of WGA-HRP does occur; (2) the postsynaptic cell can also anterogradely transport the transneuronally transported material. Magnification: 120 x. F: a light-field photomicrograph showing the distribution of reaction product in the rat VPL after a hemisection and injection of WGA-HRP in the spinal cord at C7. Note the fine granular label (probably representing terminals and fibers) and the occasional dark aggregates of reaction product resembling cell bodies, possibly due to transneuronal transport into the VPL neurons. Magnification: 170 X.
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Discussion The retrograde transport of HRP, first introduced by Kristensson et al. (1971) in the peripheral nervous system and by LaVail and LaVail (1972) in the central nervous system has been used in a wide variety of neuroanatomical studies. Recently, Mesulam (1978) introduced the use of tetramethyl benzidine (TMB) as a chromagen. This has produced significant increases in sensitivity as compared with previously used chromagens such as diaminobenzidine (Mesulam and Rosene, 1979). The use of H R P as an anterograde tracer has proven difficult, for although it has been clearly shown that anterograde transport of H R P does occur (Mesulam and Mufson, 1980; Craig and Burton, 1981), the amounts of H R P needed to be injected to demonstrate this anterograde transport appear to be relatively high for use in restricted areas of the central nervous system. The present studies have introduced evidence that there is a quantitative difference in the amount of anterograde labeling obtained after injection of W G A - H R P and H R P alone in the central nervous system. These results are in agreement with previous studies which have shown that W G A alone and W G A - H R P are avidly transported in the anterograde direction in the chick (Margolis et al., 1981) and rat (Trojanowski et al., 1981) retino-tectal pathway. However, it is noteworthy that the rate of anterograde transport of W G A - H R P ( > 200 m m / d a y ) is much faster than the 30 m m / d a y which was reported for the anterograde transport of [~251]WGA in the chick visual system (Margolis and LaVail, 1981). Thus, the speed of anterograde transport of W G A - H R P is closer to that estimated for H R P alone (Mesulam and Mufson, 1980) than that estimated for W G A alone (Margolis and LaVail, 1981). In our studies we have not attempted to study the specific mechanisms of uptake of W G A - H R P or to quantitatively define the exact rate of anterograde transport after injection of W G A - H R P . Rather we have tested the feasibility of W G A - H R P for the anterograde tracing of neuronal connections in two anatomical systems, both of which have rather long projections: spino-thalamic tract (STT) and the dorsal column nuclei (DCN). The spinothalamic tract was chosen for two reasons: (a) its efferent projections have proved to be extremely difficult to demonstrate with silver degeneration techniques because its axons are relatively small and poorly myelinated (Mountcastle, 1974) and therefore difficult to stain with silver degeneration methods. Autoradiographic tracing methods proved to be impractical because the cell bodies of STT neurons are spread along the whole length of the cord (Mountcastle, 1974); (b) it is a convenient system for examining the question of whether W G A - H R P can be taken up either by intact fibers, cut fibers of passage, or both. The D C N was chosen as a model to investigate whether uninjured cell bodies could equally well take up and anterogradely transport the conjugate. We also tried several experimental species to determine if any of the properties of anterograde transport of W G A - H R P were species-specific, From these studies it appears that both cut axons and undamaged somas can avidly take up and anterogradely transport the W G A - H R P conjugate over long distances. It is noteworthy that in many of the hemisected rat spinal cords in which H R P alone was injected, good retrograde transport of H R P could be observed while
127 there was no evidence of anterograde labeling. In general, after W G A - H R P injections both the n u m b e r and intensity of retrogradely labeled cells appeared to be significantly greater than after injections of H R P alone. It has been suggested that W G A and W G A - H R P may be transneuronally transported (Ruda, 1981). We also found suggestive evidence that transneuronal transport does occur after injection of W G A - H R P in both cut axons and intact somas. In the hemisected animals injected with W G A - H R P , lightly filled neurons could be seen both in the ventrobasal complex (Fig. 3F) and in the granule cells of the cerebellum (Fig. 3C and D). Similarly, lightly labeled VB cells were also observed after W G A - H R P was placed on the D C N . It is of interest, however, that the intensity of labeling in these postsynaptic neurons was generally weak compared to the presynaptic fibers and their projection fibers (i.e. thalamo--, corticals or D C N ---, thalamic) were never labeled. In some animals Purkinje cells appeared to be surrounded by reaction product (Fig. 3E). Purkinje cells do not receive an input from the spino-cerebellar fibers (Carpenter, 1976). It is possible that this stain reflects an unreported input from the spinocerebellar fibers or that significant transneuronal transport from the granule cells a n d / o r from olivo-cerebellar climbing fibers had occurred. The present experiments suggest that the W G A - H R P is transported in both an anterograde and retrograde direction much more avidly than H R P alone and that W G A - H R P appears to be taken up by both u n d a m a g e d cell bodies and cut axons alike. Transneuronal transport of the W G A - H R P conjugate was observed at the light level but final resolution of the mechanism and amounts transferred must await ultrastructural analysis. Thus, when properly employed, W G A - H R P appears to be an extremely effective tracer for both anterograde and retrograde axonal transport studies.
Acknowledgements The authors are indebted to Dr. H.J. Ralston III for some of the material support, to Dr. A.I. Basbaum and Dr. W.R. Mehler for their valuable criticisms and comments, to A. Schilling and K. Kimura for typing the manuscript and to Dave Akers for his excellent photographic assistance. P.W.M. is an National Research Council Fellow.
References Bull, M.S., Mitchell, S.K. and Berkley, K.J. (1981) Independent projections from the dorsal column nuclei to the tectal area, pretectal area, and thalamus in cats, Soc. Neurosci. Abstr., 7: 394. Carpenter, M.C. (1976) In Human Neuroanatomy, Williams and Wilkins, Baltimore, p. 411. Craig, A.D.and Burton, H. (1981) Spinal and medullary lamina I project to the nucleus submedius in medial thalamus: a possible pain center, J. Neurophysiol., 45: 443-466. Gonatas, N.K., Harper, C., Mizutani, T. and Gonatas, J.O. (1979) Superior sensitivity of conjugates to horseradish peroxidase with wheat germ agglutinin for studies of retrograde axonal transport, J. Histochem. Cytochem., 27: 728-734.
128 Kristensson, K., Olsson, Y. and Sjostrand, J. (1971) Axonal uptake and retrograde transport of exogenous proteins in the hypoglossal nerve, Brain Res., 32, 399-406. LaVail, J.H. and LaVail, M.M. (1972) Retrograde axonal transport of horseradish peroxidase in the central nervous system, Science, 176: 1416-1417. Margolis, T.P., Marchand, C.M.F., Kistler, H.B. and LaVail, J.H. (1981) Uptake and anterograde axonal transport of wheat germ agglutinin from retina to optic tectum in the chick, J. Cell Biol., 89: 152-156. Margolis, T.P. and LaVail, J.H. (1981) Rate of anterograde axonal transport of [l:~l]wheat germ agglutinin from retina to optic rectum in the chick, Brain Res., 229: 218-224. Mesulam, M.-M. (1978) Tetramethyl benzidine for horseradish peroxidase neurohistochemistry: a noncarcinogenic blue reaction-product with superior sensitivity for visualizing neural afferents and efferents, J. Histochem. Cytochem., 26: 106-117. Mesulam, M.-M. and Rosene, D.L. (1979) Sensitivity in horseradish peroxidase neurohistochemistry: a comparative and quantitative analysis of nine methods, J. Histochem. Cytochem., 27: 763-773. Mesulam, M.-M. and Mufson, E.J. (1980) The rapid anterograde transport of horseradish peroxidase, Neurosci., 5: 1277-1286. Mitchell, S.K,, Bull, M.S. and Berkley, K.J. (1981) A somatic sensory projection system involving the dorsal column nucleus, pretectal area and inferior olive as visualized with three different bidirectional tracers in cats, Soc. Neurosci. Abstr., 7: 395. Mountcastle, V.B. (1974) Pain and temperature sensibilities. In Mountcastle (Ed.), Medical Physiolog), Mosby, St, Louis, pp. 348-381. Ruda, M.A. (1981) Lectins, cytochemical markers for nervous system carbohydrates, Neurosci. Abstr., 7: 1. Ruda, M.A. and Coulter, J.D. (1980) Lectins as markers of axoplasmic transport in the nervous system, J. Histochem. Cytochem., 28: 607. Saper, C.B. (1981) Brainstem and basal forebrain projections to the cerebral cortex in the rat, Soc. Neurosci. Abstr., 7: 658. Schwab, M.E., Javoy-Agid, F. and Agid, Y. (1978) Labeled wheat germ agglutinin (WGA) as a new, highly sensitive retrograde tracer in rat brain hippocampal system, Brain Res., 152: 145-150. Staines, W.A., Kimura, H., Fibiger, H.C. and McGeer, E.G. (1980) Peroxidase-labeled lectin as a neurochemical tracer: evaluation in a CNS pathway, Brain Res., 197: 485-490. Steindler, D.A. and Deniau, J.M. (1980) Anatomical evidence for collateral branching of substantia nigra neurons: a combined horseradish peroxidase and (3 H) wheat germ agglutinin axonal transport study in the rat, Brain Res.. 196: 228-236, Streit, P. (1980) Selective retrograde labeling indicating the transmitter of neuronal pathways, J. comp. Neurol., 191: 429-465. Trojanowski, J.Q., Gonatas, J.O. and Gonatas, N.K. (1981) Conjugates of horseradish peroxidase (HRP) with cholera toxin and wheat germ agglutinin are superior to free H R P as orthogradely transported markers, Brain Res., 223: 381-385. Wong-Riley, M.T.T. (1976) Endogenous peroxidatic activity in brainstem neurons as demonstrated by their staining with diaminobenzidine in normal squirrel monkeys, Brain Res., 108, 257-277.
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The use of wheat germ agglutinin-horseradish peroxidase conjugates for studies of anterograde axonal transport Patrick W. Mantyh I and Marc Peschanski (P. blLM. and M.P.) Department of A naton~v, Universi O' of Cahfornia, San Francisco, San Francis~'o. ('A 94143. (P. IJLM.) Biomedical Research Division, Ames Research Center, NASA, Moffett Field. CA 94035 (IZ S.A.), and (M. P,) Unite de Recherches de Neurophysiologie Pharma~'ologique, I N S E R M U161, 2 rue d'Alesia. Paris, (France) (Received June 14th, 1982) (Accepted June 16th, 1982)
Keywords: wheat germ a g g l u t i n i n - H R P - - a n t e r o g r a d e transport--transneuronal transport--spinothalamic tract--dorsal column nuclei Injection of wheat germ agglutinin conjugated to horseradish peroxidase ( W G A - H R P ) into the hemisected spinal cord of the rat, cat and monkey consistently resulted in the intense anterograde labeling of ascending spinal projections such as the spinothalamic tract and spinocerebellar tracts and theiT terminal fields. Injections of W G A - H R P in the dorsal column nuclei resulted in the anterograde labeling of the medial lemniscus and its terminal fields in the thalamus. Injection of similar amounts of horseradish peroxidase alone (HRP) in hemisected animals or the dorsal column nuclei resulted in little anterograde labeling. The rate of the anterograde transport of W G A - H R P in cut axons appears to be greater than 200 r a m / d a y . Small amounts of transneuronal labeling appeared to occur after iniection of W G A - H R P in both cut axons and undamaged cell bodies. These results suggest that the amount of anterograde labeling observed after injection of W G A - H R P into both cut axons and undamaged cell bodies is significantly greater than the anterograde labeling observed after injections of H R P alone. Therefore, in the central nervous system W G A - H R P appears to be a far more effective anterograde tracer than H R P alone.
Introduction Since the introduction of wheat germ agglutinin (Schwab et al., 1978) (WGA) and wheat germ agglutinin conjugated to horseradish peroxidase (Gonatas et al., 1979) (WGA-HRP) as a neuroanatomical tracer, several authors have successfully applied these techniques to studies of retrograde axonal transport (Ruda and Coulter, 1980;
i To whom correspondence should be addressed at: M R C Neurochemical Pharmacology Unit, Medical Research Council Centre, Medical School, Hills Road, Cambridge CB2 2AH, U.K. 0165-0270/83/0000-0000/$03.00 ~ 1983 Elsevier Biomedical Press
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Staines et al., 1980; Steindler and Deniau, 1980; Streit, 1980; Bull et al.. 1981, Saper, 1981). Recently Gonatas et al. (1979) have reported that the retrograde transport of the W G A - H R P conjugate is up to 40 × as avid as H R P alone. In some of these published reports using W G A - H R P or W G A alone for retrograde tracing, the authors have incidentally commented that W G A - H R P a n d / o r W G A is also transported in an anterograde direction (Ruda and Coulter, 1980; Staines et al., 1980: Steindler and Deniau, 1980; Streit, 1980; Bull et al., 1981). Mesulam and Mufson (1980) have previously demonstrated that H R P is transported in an anterograde direction in the rat retino-tectal pathway. Craig and Burton (1981) have confirmed this finding by showing that anterograde transport of H R P can also take place in the cat spinal cord. However, in both studies the amounts of H R P used to accomplish the necessary labeling seem quite large if one were to apply this H R P anterograde technique to the various regions of the central nervous system (CNS). Recently, it has been reported that [~25I]WGA (Margolis et al., 1981) and H R P conjugates of cholera toxin and W G A (Trojanowski et al., 1981) appear to be transported in an anterograde direction in the chick (Margolis et al., 1981) and rat (Trojanowski et al., 1981) retino-tectal pathway respectively. These results suggest the possibility that W G A - H R P might be superior to H R P alone for neuroanatomical studies which utilize anterograde axonal transport. We have therefore investigated the anterograde transport of W G A - H R P in the central nervous system of the rat, cat and monkey. The present series of experiments were designed to test whether W G A - H R P could be a more effective tracer for anterograde connections in the CNS than H R P alone,whether this anterograde transport of W G A - H R P could occur from cut axons a n d / o r intact cell bodies and whether transneuronal transport of the tracer occurs.
Materials and Methods
Forty-one rats, 5 cats and 5 monkeys were used in these experiments.
W G A - H R P injections in the spinal cord Twenty-six rats, 5 cats and 5 monkeys received W G A - H R P injections into their spinal cords after hemisection. The rats were anesthetized with chloral hydrate (400 m g / k g ) while the cats and monkeys were first tranquilized with ketamine and then anesthetized with pentobarbital (40 mg/kg). The spinal column was exposed at either C7-C8 or L2-L3. A laminectomy was then performed, the dura reflected and the cord hemisected. A solution of 10% W G A - H R P (Sigma) in saline was injected into the spinal cord 1 m m rostral to the hemisection. In the rat 0.02-1.0 /zl was injected while in the cat and monkey 1-5 ~1 was injected. By hemisecting the cord and then injecting, we attempted to ensure that all cut fibers in the cord at the level of the hemisection would be exposed to the W G A - H R P . One to five clays after the injection of W G A - H R P into the cord, the animals were anesthetized and perfused transcardially with a 0.1 M phosphate-buffered (pH 7.4) saline solution followed by a 0.1 M phosphate-buffered solution (pH 7.4) containing
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2% glutaraldehyde and 1% paraformaldehyde fixative. After perfusion with the fixative a solution of 0.1 M phosphate-buffered solution (pH 7.4) containing 5% sucrose was perfused through the vasculature to remove any excess fixative (Mesulam, 1978). The brain and spinal cord were then removed and carried through a series of graded sucrose solutions to cryoprotect the brain. Serial 50 #m frozen sections were cut, collected, and every other section was reacted for the presence of W G A - H R P using tetramethyl benzidine (TMB) as the chromagen (Mesulam, 1978). Every other reacted section was counterstained with Neutral red. All sections were examined under both light- and dark-field optics and all areas containing W G A - H R P in fibers or terminals were plotted onto a drawing of the section that had been made with the aid of an overhead projector.
Control experiments Fifteen rats were used for various control experiments as described below. In six rats, 0.1-1.0 ~1 of 10% solution of H R P alone (Sigma type VI) was injected into the cervical cord (3 rats) or the lumbar cord (3 rats) after hemisection. The objective was to compare the anterograde transport of H R P to that of WGA-HRP. In these experiments all aspects of the surgery, amounts injected, recovery period and histochemical processing were identical to the W G A - H R P injected material outlined above. In 3 rats the cisterna magna was opened, the dura retracted and 0.2/LI of 10% W G A - H R P was placed on the dorsal column nuclei (DCN). The objective was to assess whether uninjured somas could take up the W G A - H R P and transport it in an anterograde fashion as avidly as cut axons in the cord. After the W G A - H R P was deposited on the DCN the wound was closed, and the animal placed back in the cage for 2 - 3 days. After this time the brains were again processed as described above. In 2 rats, 0.2/~1 of a 10% solution of H R P alone was placed on the DCN. The objective of this group of animals was to assess whether HRP alone could duplicate the anterograde transport observed when W G A - H R P was placed on the DCN. These animals had identical survival times and histochemical processing as the W G A - H R P animals. In 2 rats the spinal cord was exposed and 0.2 ~1 of 10% W G A - H R P was placed on the exposed spinal cord at upper lumbar levels. In this series of animals the objective was to assess if the conjugate could be taken up by uninjured fibers and anterogradely transported. After the W G A - H R P was placed on the cord the rats were handled in the same way as the W G A - H R P hemisected and injected animals. In 2 animals a sham operation was performed in which the cords were exposed, hemisected and reclosed. The rats survived for 2 days and processed using the same protocol as the hemisected and W G A - H R P injected animals. The objective of this series of animals was to see if any endogenous reaction product was present due to the surgical procedure employed.
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Results
WGA-HRP injections in the spinal cord A d a r k - b l u e g r a n u l a r reaction p r o d u c t m a r k e d the presence of H R P (Mesulam, 1978). In a n i m a l s injected with W G A - H R P we f o u n d that hemisection before injection a i d e d in labeling fibers. In hemisected W G A - H R P - i n j e c t e d animals labeled fibers could be o b s e r v e d in the spinal cord, a n d some of the fibers in the fasciculus gracilis a n d c u n e a t u s could be observed t e r m i n a t i n g in the dorsal c o l u m n nuclei ( D C N ) in a t o p o g r a p h i c a l pattern. In these W G A - H R P injected animals the a s c e n d i n g s p i n o t h a l a m i c tract (STT) fibers were heavily labeled a n d could be seen coursing t h r o u g h the medulla, p o n s a n d m e s e n c e p h a l o n (Fig. IA), usually c a p p i n g the medial lemniscus. A t the m e s o d i e n c e p h a l i c border, the s p i n o t h a l a m i c fibers w o u l d b e n d a w a y from the m a i n b o d y of the tract and end in dense puffs in discrete t h a l a m i c nuclei (Fig. 1B - F ) . The c o m p l e t e results of these STT studies in the rat, cat a n d m o n k e y will be p u b l i s h e d in s e p a r a t e papers. Both the fibers a n d the terminal fields could easily be viewed using b o t h light- (Fig. 1A, B, D and F) a n d d a r k - (Fig. I C a n d E) field optics; the N e u t r a l red stain p r o v i d e d g o o d c o n t r a s t for color p h o t o m i c r o g r a p h s u n d e r light-field optics. O t h e r a s c e n d i n g a n d d e s c e n d i n g tracts were also labeled including the spinoolivary, s p i n o - c e r e b e l l a r (Fig. 3A a n d B), spino-vestibular, a n d the spino-reticular. In general, the a n t e r o g r a d e labeling observed in these tracts and their terminal fields was at least as intense as that o b s e r v e d in the STT ( c o m p a r e Figs. 1A a n d 3B). F o r example, the s p i n o c e r e b e l l a r labeling was often so intense that i n d i v i d u a l m o s s y fibers could be seen t e r m i n a t i n g u p o n granule cells in the cerebellum (Fig. 3C a n d D). Small a m o u n t s of t r a n s n e u r o n a l labeling a p p e a r e d to occur after injection of W G A - H R P in cut axons. In a n i m a l s with injections of W G A - H R P in the spinal cord, light i n t r a s o m a t i c labeling of cerebellar granule cells a n d thalamic VPL n e u r o n s could be observed. In general, these t r a n s n e u r o n a l l y labeled neurons were o n l y lightly filled when c o m p a r e d to the intense labeling in the p r e s y n a p t i c fiber and the p o s t s y n a p t i c n e u r o n s p r o j e c t i o n fibers (the parallel fiber for the granule cell a n d
Fig. 1. A series of photomicrographs of coronal sections through the brain following hemisection and injection of WGA-HRP at C7-C8 in the spinal cord. A: this light-field photomicrograph shows labeled spino-thalamic tract (ST/') fibers in the monkey mesencephalon ipsilateral to the spinal injection site. Magnification: 100×. B. a light-field photomicrograph showing the dense anterograde labeling present in the caudal ventroposterior lateral nucleus (VPL) of the monkey ipsilateral to the injection site. Magnification: 100 x. C and D: a dark- and light-field photomicrograph, respectively, showing the intense anterograd¢ labeling present in the mid-VPL of the rat after an ipsilateral cervical injection. Note the absence of label in the adjacent ventroposterior medial nucleus (VPM). C is 100 p.m caudal to D. In C the anterograde labeling appears white; in D it appears black. The circular areas in VPL without label are probably bundles of unlabeled thalamo-cortical and cortico-thalamic fibers. Magnification: 70 ×. E and F: a dark- and light-field photomicrograph, respectively, showing the intense patchy labeling in the monkey VPL after an ipsilateral injection. E and F are from the same section. In E the anterograde labeling appears white while in F it is black, Magnification: 30 x.
i .....
123 the thalamo-cortical fiber for VPL neurons) were never observed to be labeled. The m i n i m u m rate of anterograde axonal transport of the W G A - H R P could be estimated from these experiments. F r o m a n injection into the l u m b a r cord of a cat with a 1-day survival period, it appears that this transport travels at least 200 m m / d a y . This is based on 1-day survival a n d a distance of at least 200 m m from injection site to the thalamus in a cat. This rate is in the range of the 288 r a m / d a y estimated for the anterograde transport of H R P alone in the rat visual system ( M e s u l a m a n d Mufson, 1980). Since labeling of the STY was observed in all hemisected cases we can only determine the m i n i m u m speed. These results indicate that at least some of the anterograde transport of W G A - H R P is carried by fast axonal transport.
Control experiments In agreement with previous studies, occasional anterograde label was observed after injection of H R P alone. However, the a m o u n t s of H R P needed to be injected into the rat cervical cord (1 ~1 of 10% solution) to see a n y anterograde labeling in the t h a l a m u s also produced an artifactual 'edge effect' a r o u n d the lateral a n d dorsal borders of the m e d u l l a a n d pons. This effect is p r o b a b l y due to the leakage and spread of the H R P by capillary action along the meninges up to these rostral regions. This rostral spread of H R P can seriously c o m p r o m i s e the interpretations of the results o b t a i n e d in the spinal injections due to u n w a n t e d somatic pickup by the dorsal c o l u m n nuclei somas (see below). Thus intense anterograde labeling in the b r a i n s t e m a n d thalamus was never observed in the hemisected rats injected with c o m p a r a b l e a m o u n t s of H R P alone. Other ascending systems including the spinocerebellar tract were also rarely labeled after H R P injection a n d when present the label was observed in fewer areas, a n d had a greatly diminished intensity as c o m p a r e d with the labeling seen after similar a m o u n t s of W G A - H R P were injected. In the rats in which W G A - H R P was placed on the DCN, a striking a n d well delineated anterograde labeling was apparent. In these animals the cell bodies which Fig. 2. This series of photomicrographs shows the anterograde transport of WGA-HRP after 0.2/~1 of 10% solution of the conjugate was placed on the dorsal column nuclei (DCN) of the rat, so as to eliminate the possibility of any mechanical damage to the cell bodies or axons. A: this light-field photomicrograph shows the extent of the spread of WGA-HRP in the DCN. Note the absence of label ventral to the nucleus gracilis (GR) and fasciculus cuneatus (FC). The unlabeled oval structure at the lower left is the central canal. Magnification 50 x. B: this light-field photomicrograph is a higher magnification of A and shows the cell bodies in the GR which are black and have apparently taken up the WGA-HRP. Magnification: 170x. C: this dark-field photomicrograph shows the labeled fibers in the medial lemniscus (LM) of the rat after WGA-HRP was placed on the DCN. Magnification: 85 x. D-F: this series of photomicrographs from the same section shows the terminations of the rat DCN projections in the VPL contralateral to the labeled DCN. D is a low-powerdark-field photomicrograph showing that the pattern of labeling is quite similar to that seen in Fig. 1C after an STT injection. Magnification 65 x. E: a light-field photomicrograph showing the dense anterograde labeling in the VPL. The white unlabeled areas in E are probably unlabeled bundles of thalamo-cortical and cortico-thalamic fibers. Magnification: 130x. F: a dark-field photomicrograph of E. Again note the lack of any labeling in the internal capsule (IC) where the thalamo-cortical fibers would be expected to be traveling. Magnification: 130 ×.
125 were exposed to the W G A - H R P (Fig. 2A) were densely filled with reaction product (Fig. 2B). A n t e r o g r a d e l y labeled fibers could be traced out of the D C N . These traveled in the medial lemniscus in distinct fiber b u n d l e s (Fig. 2C) a n d t e r m i n a t e d in the ventroposterior lateral nucleus of the thalamus (Fig. 2 D - F ) . This type of a n t e r o g r a d e fiber a n d terminal labeling in the VPL was similar to that seen with spinal injections (compare Figs. 1C a n d 2D) a n d is consistent with studies d e m o n strating that n e u r o n a l damage is not necessary for anterograde axonal transport of various tracers such as H R P alone. Small a m o u n t s of t r a n s n e u r o n a l labeling also appeared in the thalamic VPL n e u r o n s in the a n i m a l s injected with W G A - H R P in the D C N . This t r a n s n e u r o n a l labeling was similar to that observed after injections into cut spinal axons where the i n t r a s o m a t i c labeling of the thalamic n e u r o n was light compared to the intense labeling in the presynaptic fibers. In the rats in which similar a m o u n t s of H R P alone were placed on the D C N , only little anterograde labeling was evident in either the medial lemniscus or the VPL. The animals which had W G A - H R P placed on their spinal cords showed no signs of anterograde transport. A p p a r e n t l y the reason that the u n d a m a g e d spinal cord n e u r o n s did not transport W G A - H R P , but D C N n e u r o n s did, is due to the fact that the spinal cord n e u r o n s are u n s h e a t h e d by white matter while D C N n e u r o n s in the rat are at the dorsal-most extent of the caudal medulla where the W G A - H R P can easily penetrate. N o reaction product was observed in the dorsal thalamus in the group of s h a m - o p e r a t e d animals. Two areas which did display an e n d o g e n o u s reaction p r o d u c t when reacted with the T M B protocol were the arcuate nucleus of the h y p o t h a l a m u s a n d the i n t e r p e d u n c u l a r nucleus of the mesencephalon, the same areas that d e m o n s t r a t e an e n d o g e n o u s reaction p r o d u c t when reacted with dia m i n o b e n z i d i n e (Wong-Riley, 1976). Fig. 3. A: a light-field photomicrogrzph showing labeled spinocerebellar fibers in the monkey restiform body after an ipsilateral hemisection and injection of WGA-HRP at C7. Note the darkly labeled fibers in the restiform body and the darkly stained cells in the granule layer. Magnification: 50 ×. B: this light-field photomicrograph shows at higher magnification the intense spino-cerebellar fiber labeling obtained using this technique after an ipsilateral hemisection and injection of WGA-HRP at C7 in the monkey. Magnification: 130 ×. C: a light-field photomicrograph demonstrating the possible transneuronal labeling of granule neurons of the monkey cerebellum after the same injection shown in A and B. Magnification: 170 x. D: this light-field photomicrograph depicts at a higher magnification labeled granule cells in the monkey cerebellum after an injection in the monkey spinal cord at C7. Note that the reaction product appears to be inside the cell body, suggesting transneuronal transport. Magnification: 240x. E: a light-field photomicrograph which shows some of the monkey Purkinje cells occasionally observed surrounded by reaction product after an injection of WGA-HRP at C7. Since no direct spino-cerebellar projection to the Purkinje cells is known, this labeling possibly represents the projections of a granule cell upon a Purkinje cell. If true, this demonstrates that: (1) transneuronal transport after injection of WGA-HRP does occur; (2) the postsynaptic cell can also anterogradely transport the transneuronally transported material. Magnification: 120 x. F: a light-field photomicrograph showing the distribution of reaction product in the rat VPL after a hemisection and injection of WGA-HRP in the spinal cord at C7. Note the fine granular label (probably representing terminals and fibers) and the occasional dark aggregates of reaction product resembling cell bodies, possibly due to transneuronal transport into the VPL neurons. Magnification: 170 X.
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Discussion The retrograde transport of HRP, first introduced by Kristensson et al. (1971) in the peripheral nervous system and by LaVail and LaVail (1972) in the central nervous system has been used in a wide variety of neuroanatomical studies. Recently, Mesulam (1978) introduced the use of tetramethyl benzidine (TMB) as a chromagen. This has produced significant increases in sensitivity as compared with previously used chromagens such as diaminobenzidine (Mesulam and Rosene, 1979). The use of H R P as an anterograde tracer has proven difficult, for although it has been clearly shown that anterograde transport of H R P does occur (Mesulam and Mufson, 1980; Craig and Burton, 1981), the amounts of H R P needed to be injected to demonstrate this anterograde transport appear to be relatively high for use in restricted areas of the central nervous system. The present studies have introduced evidence that there is a quantitative difference in the amount of anterograde labeling obtained after injection of W G A - H R P and H R P alone in the central nervous system. These results are in agreement with previous studies which have shown that W G A alone and W G A - H R P are avidly transported in the anterograde direction in the chick (Margolis et al., 1981) and rat (Trojanowski et al., 1981) retino-tectal pathway. However, it is noteworthy that the rate of anterograde transport of W G A - H R P ( > 200 m m / d a y ) is much faster than the 30 m m / d a y which was reported for the anterograde transport of [~251]WGA in the chick visual system (Margolis and LaVail, 1981). Thus, the speed of anterograde transport of W G A - H R P is closer to that estimated for H R P alone (Mesulam and Mufson, 1980) than that estimated for W G A alone (Margolis and LaVail, 1981). In our studies we have not attempted to study the specific mechanisms of uptake of W G A - H R P or to quantitatively define the exact rate of anterograde transport after injection of W G A - H R P . Rather we have tested the feasibility of W G A - H R P for the anterograde tracing of neuronal connections in two anatomical systems, both of which have rather long projections: spino-thalamic tract (STT) and the dorsal column nuclei (DCN). The spinothalamic tract was chosen for two reasons: (a) its efferent projections have proved to be extremely difficult to demonstrate with silver degeneration techniques because its axons are relatively small and poorly myelinated (Mountcastle, 1974) and therefore difficult to stain with silver degeneration methods. Autoradiographic tracing methods proved to be impractical because the cell bodies of STT neurons are spread along the whole length of the cord (Mountcastle, 1974); (b) it is a convenient system for examining the question of whether W G A - H R P can be taken up either by intact fibers, cut fibers of passage, or both. The D C N was chosen as a model to investigate whether uninjured cell bodies could equally well take up and anterogradely transport the conjugate. We also tried several experimental species to determine if any of the properties of anterograde transport of W G A - H R P were species-specific, From these studies it appears that both cut axons and undamaged somas can avidly take up and anterogradely transport the W G A - H R P conjugate over long distances. It is noteworthy that in many of the hemisected rat spinal cords in which H R P alone was injected, good retrograde transport of H R P could be observed while
127 there was no evidence of anterograde labeling. In general, after W G A - H R P injections both the n u m b e r and intensity of retrogradely labeled cells appeared to be significantly greater than after injections of H R P alone. It has been suggested that W G A and W G A - H R P may be transneuronally transported (Ruda, 1981). We also found suggestive evidence that transneuronal transport does occur after injection of W G A - H R P in both cut axons and intact somas. In the hemisected animals injected with W G A - H R P , lightly filled neurons could be seen both in the ventrobasal complex (Fig. 3F) and in the granule cells of the cerebellum (Fig. 3C and D). Similarly, lightly labeled VB cells were also observed after W G A - H R P was placed on the D C N . It is of interest, however, that the intensity of labeling in these postsynaptic neurons was generally weak compared to the presynaptic fibers and their projection fibers (i.e. thalamo--, corticals or D C N ---, thalamic) were never labeled. In some animals Purkinje cells appeared to be surrounded by reaction product (Fig. 3E). Purkinje cells do not receive an input from the spino-cerebellar fibers (Carpenter, 1976). It is possible that this stain reflects an unreported input from the spinocerebellar fibers or that significant transneuronal transport from the granule cells a n d / o r from olivo-cerebellar climbing fibers had occurred. The present experiments suggest that the W G A - H R P is transported in both an anterograde and retrograde direction much more avidly than H R P alone and that W G A - H R P appears to be taken up by both u n d a m a g e d cell bodies and cut axons alike. Transneuronal transport of the W G A - H R P conjugate was observed at the light level but final resolution of the mechanism and amounts transferred must await ultrastructural analysis. Thus, when properly employed, W G A - H R P appears to be an extremely effective tracer for both anterograde and retrograde axonal transport studies.
Acknowledgements The authors are indebted to Dr. H.J. Ralston III for some of the material support, to Dr. A.I. Basbaum and Dr. W.R. Mehler for their valuable criticisms and comments, to A. Schilling and K. Kimura for typing the manuscript and to Dave Akers for his excellent photographic assistance. P.W.M. is an National Research Council Fellow.
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