Retrograde axonal transport of horseradish peroxidase in rat's forebrain

Brain Research, 67 (1974) 211-218

21 1

i:': ElsevierScientific Publishing Company, Amsterdam - Printed in The Netherlands

Research Reports

R E T R O G R A D E A X O N A L T R A N S P O R T OF H O R S E R A D I S H P E R O X I D A S E IN RAT'S F O R E B R A I N

H. G. J. M. KUYPERS, J. KIEVIT* AND A. C. GROEN-KLEVANT Department o[ 4natomy, Rotterdam Erasnm,~ UniversiO', Medical School, Rotterdam ( 77te Netherlands)

(Accepted August 29dl, 1973)

SUMMARY The use of retrograde axonal horseradish peroxidase (HRP) transport in determining cells of origin of central fiber systems has been explored in young and adult rats by injecting H R P in different portions of the forebrain. After H R P injection in different cortical areas, H R P reaction granules were found in the neuronal cell bodies of the corresponding thalamic nuclei, while after injection into the caudate putamen HRP-positive granules were found also in the cell bodies of the parafascicular nucleus and the substantia nigra. It is concluded that the H R P injection method represents a promising technique for determining remote ceils of origin of fiber systems in brains of young and adult rats and that it produces especially striking results when the material is examined under dark field illumination.

INTRODUCTION It has been demonstrated that especially in young animals horseradish peroxidase (HRP) can be transported in the retrograde direction through axons of the sciatic 7, hypoglossal s, cochlear 19 and optic nerves 9 to their parent cell bodies. This encouraged us to think that this method might also be effectively applied in determining the cells of origin of various fiber systems in the brain. Currently the cells of origin of central fiber system may be identified by interrupting the system and locating the affected parent cell bodies which may display lysis of the chromatin material (Nissl bodies) and eccentricity of the nucleus 2. However, since this chromatolytic reaction tends to be * J. Kievit, M.D., holds a FUNGO (Dutch Organization for Fundamental Research in Medicine) Fellowship.

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somewhat inconsistent t7, a technique based on retrograde axonal transport of material to the parent cell body could be a welcome complement or perhaps even a substitute for the chromatolytic reaction technique. In order to begin to explore the possible use of this method in the central nervous system we have studied retrograde H RP transport after injection of the enzyme in certain areas of the forebrain of rats, both young and adult. However, in order to become familiar with the application of this technique we first repeated the original experiments of Kristensson et al. s by injecting HRP in the tongues of young rats and studying the intracellular distribution of the enzyme in tile hypoglossal neurons. MATERIAL A N D M E T H O D S

Two young rats, 2 weeks of age, were anesthetized with ether and 0.1 ml 5 °;i H R P (Sigma type V1) was injected into the tongue. The animals were sacrificed after 2 days. In two additional young rats, 21 days of age, two adjacent injections of 0.4 ld 5 ~ H R P were made in the cortex of the frontal portion of the hemisphere I-2 mm from the midline. The enzyme appeared to be deposited mainly in the rostral part of the motor cortex and in the adjoining prefrontal area (gq refs. 6, 10 and 20). These animals were sacrificed after 1 and 2 days, respectively. Two adjacent cortical injections of 10 ~o H R P were also made in several adult rats. In 4 of these adult rats the injections were made in the frontal portion of the hemisphere, i.e. in approximately the same area as the injections in two of the young rats. The 4 adult rats were sacrificed after 2, 3, 4, and 5 days, respectively. The rats surviving 2, 3 and 5 days received 0.4 #1 1 0 ~ HRP per injection, while the animal surviving 4 days received 0.6 #1 [ 0 ~ HRP per injection. In addition, in this animal the enzyme was injected deeper into the brain than in the others and was deposited in the caudate putamen as well as in the cortex and the underlying white matter. This injection of HRP in the caudate putamen was then repeated in 4 adult rats which were allowed to survive for 4 days. In 2 of them, however, injections of 0.4 /~1 rather than 0.6 #1 10~o HRP were made. Finally in 2 adult rats 2 adjacent injections of 0 . 4 / d 1 0 ~ HRP were made in the cortex of the intermediate portion of the hemisphere and in I adult rat in the cortex nearer the caudal pole. These 3 animals were also allowed to survive for 4 days. After their survival periods the animals were deeply anesthetized with ether and perfused with a 4 ~o paraformaldehyde-5 ~ glutaraldehyde mixture 4. In the majority of the adult rats the perfusion with the paraformaldehyde-glutaraldehyde mixture was preceded by perfusion with 6 ~o dextran in order to remove as many erythrocytes from the brain as possible. Six hours after the perfusion the brains were removed from the skull. They were kept overnight in a cacodylate buffer 9 containing 5 ~ sucrose and then were cut transversely in 40 #m sections on the freezing microtome '~. The sections were incubated in a medium containing 3,3'-diaminobenzidine tetrahydrochloride and hydrogen peroxidO. 9, and then were mounted with gelatine, dehydrated and covered. Initially some sections were counterstained with cresyl violet. However, this

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s e e m e d e i t h e r to o b s c u r e o r to r e m o v e s o m e o f the H R P r e a c t i o n p r o d u c t s . T h e r e f o r e , c o u n t e r s t a i n i n g was a b a n d o n e d and the sections were studied u n d e r dark-field illumin a t i o n , which was f o u n d to be p a r t i c u l a r l y effective in d e m o n s t r a t i n g the H R P reaction p r o d u c t s . RI~SULIS in the t w o y o u n g rats (2 weeks o f age) in w h i c h 0.1 ml 5~', H R P had been inj e c t e d into the t o n g u e ,

H R P - p o s i t i v e g r a n u l e s were a b u n d a n t

in the h y p o g l o s s a l

n e u r o n s , T h e s e g r a n u l e s were located in the cell body, a r o u n d the nucleus, and in the p r o x i m a l part o f the dendrites, as d e s c r i b e d by K r i s t e n s s o n et al. ~.

Fig. I. Distributions of neuronal cell bodies containing horseradish peroxidase (H R P) reaction products (ell Fig. 2D and text) in 2 adult rats after HRP injections in forebrain. On left, distribution after HRP injection in cerebral cortex (3 days survival) and on right after HRP injection through cortex and white matter in caudate pLitamen (C.-P.) (4 days survival). Note that in case on left HRP-positive neurons are present contralaterally in a cortical area homologotls to the injection site and ipsilaterally in thalamus, mainly in ventralis lateralis (VL) nucleus, which sends fibers to the frontal cortex, and in inlralaminar nuclei (IL.N.), Note that on the right side such neurons are present also in ipsilateral parafascicular nucleus (PF.) and substantia nigra (S.N.) which send fibers to the caudate putamen (C.~P.t.C.G.M., medial geniculate body; F,R., fasciculus retroflextls: I,.D., nucleus lateralis dorsaIts: M.L., medial lemniscus: S.. septum: V.B., ventrobasal complex.

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In the two young rats (21 days of age) which received two adjacent cortical injections of 0.4 t~l 5 °~i HRP in the rostral portion of one hemisphere and which survived for I and 2 days, respectively, the following findings were obtained. At the cortical injection sites in both animals brown HRP granules were present in a cylindrical region of approximately 300 # m diameter around the needle tract. The HRP reaction products were located in neuropil, glial cells, neurons and cells lining the blood vessels in this area and other parts of the brain l. From the injection site brown HRP-positive axons were traced into the white matter of the hemisphere, some of the fiber bundles of the internal capsule and the corpus callosum. In the animal which survived 2 days, the brown axons could be traced into the diencephalon. Around the injection site in the cerebral cortex many neurons contained HRP granules (@ Fig. I, left). They showed the same intracellular distribution as seen in the hypoglossal neurons after HRP injection in the tongue 8, but were slightly less numerous. In addition, many such HRP-positive neurons were present in the cortex of the medial surface of the hemisphere at the level of the injection and a few HRPpositive cells were present in the contralateral cerebral cortex, in the area homologous to the injection site. HRP granules were also present in the most medial portion of the caudate putamen and in the septum, but it was difficult to ascertain whether these granules were all located intracellularly. In the animal which survived I day, virtually no HRP-positive neurons were found in the diencephalon. However, in the animal which survived 2 days many such neurons were found in the rostral part of the ipsilateral thalamus, mainly in the VL nucleus and in the lateral portion of the DM nucleus (nomenclature as in refs. I 1 and 12). Some HRP-positive neurons were present also in the intralaminar nuclei (c£ ref. 14). In view of the findings in the forebrain of these 2 young rats, 2 adjacent cortical injections of 0.4 #l 10 °/o H R P were made in the rostral portion of the hemisphere of 4 adult rats which survived for 2, 3.4 and 5 days, respectively. The findings (Figs. 1, left and 2A) in the adult rats which survived 2, 3 and 5 days, were similar to those in the young ones, but the optimal survival time was slightly longer. For example, brown HRP-positive axons were present in the medial portion of the cerebral peduncle but only after a survival of 3 or more days, and such survival times were also required to label a large population of neurons in the thalamus. Different findings, however, were obtained in the adult rat which survived 4 days and in which injections of 0.6 #I 10°//o HRP had been made through the cortex and the underlying white matter into the caudate putamen (Fig. 1, right). In this animal many HRP-positive cells were present in the rostral portion of the caudate putamen and throughout the cerebral cortex on both sides but mainly ipsilaterally. HRP-positive neurons were present in the ipsilateral thalamus (Fig. 2D), mainly in the VL nucleus (Fig. 2B, C), the rostral part of the VB complex, the paralaminar part of the LP nucleus and the lateral part of the DM nucleus (nomenclature as in refs. l I and 12). Many HRP-positive neurons were also present in the intralaminar nuclei (Figs. 1, right and 2C). In addition, contrary to the findings in the other rats, many HRPpositive neurons occurred in the ipsilateral parafascicular nucleus (Figs. I, right and 3A, B) and substantia nigra, especially its pars compacta (Figs. 1, right and 3C, D).

P,|~TROGRADE AXONAL TRANSPORT ()F- H R P

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Fig. 2. Photomicrographs (dark-field illumination) showing neuronal cell bodies containing HRP reaction products, A: HRP-positive neurons (magnification, 70) in VL nucleus and intralaminar nuclei after HRP injection in frontal cortex of adult rat (3 days survival ). Arrow points to intralaminar nuclei. B: HRP-positive neurons (magnification, 300) in the VL nucleus shown in C. C: HRPpositive neurons (magnification, < 70) in left VL nucleus and intralaminar nuclei (IL.N) after HRP injection through cortex and cerebral white matter in left caudate putamen (C.P.) of adult rat (4 days survival ). D: absence of such neurons in the right thalamic nuclei of this animal (magnification, 70).

Prompted by these findings, the injections of 0.6/~l 10 ~,; H R P through the cortex in the caudate putamen were repeated in 2 adult animals. In these animals, which also survived for 4 days, precisely the same findings were obtained. The same holds true for the 2 other adult animals in which injections of 0.4 #l 10~% HRP were made in the caudate putamen. However, in the latter 2 animals fewer HRP-positive neurons were present in the substantia nigra than in the former. In the 3 adult rats which received two adjacent cortical injections of 0.4 /d HRP in the intermediate and caudal portions of the hemisphere respectively and

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Fig. 3. Photomicrographs (dark-field illumination) showing neuronal cell bodies containing HRP reaction produc~s. A: HRP-positive neurons (magnification, ,~ 70) in left parafascictflar nucleus (PF.N) and C shox~s such neurons in left substantia nigra (SN) pars compacta after HRP injection in lef~ cauda~,e putamen (CPt of adult rat (4 days survival). B: tile absence of such neurons (magnification, 7(1t in right parafascicular nucleus (PF.Nt and D in right substantia nigra ISN) of the same animal. which survived for 4 days, the H R P - p o s i t i v e neurons were located only in the caudal p a r t o f the thalamus. In the 2 animals which had cortical injections in the intermediate p o r t i o n of the hemisphere, H R P - p o s i t i v e neurons were present in the caudal p a r t o f the L D nucleus and in the nucleus p u l v i n a r anterior ( n o m e n c l a t u r e as in retd. 1l and 12). In the third animal in which the cortical injections were made nearer the caudal pole of the hemisphere the H R P - p o s i t i v e n e u r o n s were present mainly in the area o f the geniculate p o r t i o n o f the nucleus pulvinar and in the lateral geniculate b o d y ( n o m e n c l a t u r e as in refs. I1 a n d 12).

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DISCUSSION

The present findings indicate tile existence of a retrograde, as well as anterograde H R P transport in axons of neurons in the forebrains of both young and adult rats. The existence of an anterograde transport, which has been reported recently by others la, is concluded from the appearance of H R P in the frontal cortical fibers in the medial portion of the cerebral peduncle. The accumulation of H R P granules in the so-called HRP-positive neurons in the thalamus, the parafascicular nucleus and tile substantia nigra, is taken to indicate the existence of a retrograde transport. This conclusion is based on the following considerations. The HRP granules in these cells were distributed in tile same fashion as in tile hypoglossal neurons after H R P injection in tile tongue s. In addition, after injections restricted to cerebral cortex, HRP-positive neurons occurred only in those diencephalic nuclei which are known to send fibers to the injected cortical areasa,~4, as. In contrast, after injection of H R P into tile caudate putamen, such neurons were present also in the parafascicular nucleus and tile substantia nigra (pars compacta), which are known to send fibers to tile caudate putanlenS,15,16

The above findings clearly demonstrate the occurrence of retrograde HRP transport to distant cell bodies. However, it is difficult to determine whether the H R P granules in neurons of the cerebral cortex and caudate putamen after injection in these structures reflect local diffusion or retrograde axonal transport. In this respect it has been pointed out that the diffusion of H R P may render this technique unsuitable as a neuroanatomical research tooP. However, in our cases which involve long-distance retrograde transport, local diffusion of HRP did not seem to be a very confounding factor since after injection of HRP in different areas of the cerebral cortex HRPpositive neurons were present in different parts of the thalamus. Moreover, HRP was transported to the parafascicular nucleus and tile substantia nigra only when the enzyme had been deposited in the caudate putamen and not when tile injection was restricted to tile cerebral cortex. To improve further the resolution in interpreting the results obtained by this method it would be of importance to ascertain whether absorption of HRP in amounts sufficient for its demonstration in distant cell bodies is effected by intact nerve terminals, damaged ones or both, Moreover, it should be determined whether such absorption also occurs at the cut ends of severed axons. Regardless, our present findings in the rat allow us to conclude that the H R P injection method: (a) represents a promising technique for determining tile remote ceils of origin of central, as well as peripheral, fiber systems: (b) is equally effective in the central nervous systems of both young and adult rats: and (c) produces particularly striking results when the material is examined under dark-field illumination. A('KNOWLEDGEMENTS

The authors thank Mr. W. van den Oudenalder and Miss P. Delfos for their help with the photography, Dr. D. Cohen for reading tile manuscript and Miss A. Wessels for typing it. This study was supported in part by Grant 13-31-12 of the F U N G O (Dutch

H . G . J . M . KUYPERS eta[.

218 Organization for Fundamental

R e s e a r c h in Medicine[) a n d b y a D u t c h I n t e r d e p a r t -

mental Government Science Grant. NOTE ADDED IN PROOF After submitting this paper to Brain Research, two other papers on the subject have appeared : LAVML, J. lq..., W~NSTON, K. R., AND TIsn, A., A method based on retrograde intraaxonal transport of protein for identification of cell bodies of origin of axons terminating within the CNS, Brain Research, 58 (1973) 470-477. RALSTON, 111, H. R., AND SHARe, P. V., The identification of thalamocortical relay cells in the adult cat by means of retrograde axonal transport of horseradish peroxidase, Brain Research, 62 11973) 273-278. REFERENCES 1 BECKER, N. H., HIRANO, A., AND ZIMMERMAN, H. M., Observations of the distribution of exogenous peroxidase in the rat cerebrum, J. Neuropath. exp. Neurol., 27 (1968) 439-452. 2 BRODAL, A., The Reticular Formation of the Brain Stem. Anatomical Aspects and Functional Correlations, Henderson Trust Lectures, Oliver and Boyd, Edinburgh, 1957. 3 CASTRO, A. J., The effects of cortical ablations on digital usage in the rat, Brain Reseca-ctl, 37 (1972) 173-185. 4 GRAHAM, R. C., AND KARNOVSKY, M. J., Glomerular permeability. Ultrastructural cytochemical studies using peroxidases as protein tracers, J. exp, Med., 124 (1966) 1123-1133. 5 HEDRE~N, J. C., AND CrtALMERS, J. P., Neuronal degeneration in rat brain induced by 6-hydroxydopamine; a histological and biochemical study, Brain Research, 47 (1972) 1-36. 6 KRIeG, W. J. S., Connections of the cerebral cortex. I. The albino rat. A. Topography of the cortical areas, J. comp. Neurol., 84 (1946) 221-275. 7 KRISTENSSON, K., AND OLSSON, Y., Retrograde axonal transport of protein, Brain Research, 29 (1971) 363-365. 8 KRISTENSSON, K., OLSSON, Y., AND SJOSTRAND, J., Axonal uptake and retrograde transport of exogenous proteins in the hypoglossal nerve, Brain Research, 32 (1971) 399-406. 9 LAVAtL, J. H., ANt) LAVAIL, M. M., Retrograde axonal transport in the central nervous system, Science, 176 (1972) 1416-1417. 10 LEONARD, C. M., The prefrontal cortex of the rat. I. Cortical projection of the mediodorsal nucleus, lI. Efferent connections, Brain Research, 12 (1969) 321-343. 11 Lt~ND, R. D., AND WEBSTER, K. E., Thalamic afferents from the spinal cord and trigeminal nuclei. An experimental anatomical study in the rat, J. comp. Neurol., 130 (1967) 313-328. 12 Lt3ND~ R. D., Ar~o WEBSTER, K. E., Thalamic afferents from the dorsal column nuclei. An experimental anatomical study in the rat, ,L camp. Neurol., 130 (1967) 301-312. 13 LYNCH, G., SMITH, R. L., MENSAH, P., AND COTMAN, C., Tracing the dentate gyrus mossy fiber system with horseradish peroxidase histochemistry, Exp. Nearol., 40 (1973) 516-524. 14 MACCHI, G., QUATTRlr~I, A., CrUNZAR1, P., E CAPOCCm, G., Valutazione quantitativa delle perdite cellulari net nuclei intralaminari del talamo dopo lesioni corticali e sottocorticali, Boll. Soc. ital. Biol. sper., 46 (1970) 218-221. 15 MEttLER, W. R., Further notes on the center median nucleus of Luys. In D. P. PURPt3RA AND M. D. YArtR (Eds.), The Thalamus, Columbia Univ. Press, New York, 1966, pp. 109-122. 16 FOWELL, T. P. S., AND COWAN, W. M., A study of thalamo-striate relations in the monkey, Brain, 79 (1956) 364-390. 17 STERHNG, P., AND KUYPERS, H. G. J. M., Anatomical organization of the brachial spinal cord of the cat. 111. The propriospinal connections, Brain Research, 7 (1968) 419--443. 18 WALLER, W. H., Topographical relations of cortical lesions to thalamic nuclei in the albino rat, J. comp. Neurol., 60 (1934)237-269. 19 WA/~R, W. B., Localization of olivocochlear neurons by means of retrograde axonal transport of horseradish peroxidase, Anat. Rec., 175 (1973) 464. 20 WOOLSEY, C. N., Organization of somatic sensory and motor areas of the cerebral cortex. In H. F. HARLOW AND C. N. WOOLSEY (Eds.), Biological and Biochemical Bases oJ Behavior, Univ. Wisconsin Press, Madison, Wisc., 1958, pp. 63-81.