Brain Research, 85 (1975) 307-312
© Elsevier ScientificPublishingCompany,Amsterdam- Printed in The Netherlands
307
THE ORTHOGRADE TRANSPORT OF HORSERADISH PEROXIDASE IN THE VISUAL SYSTEM JACQUES REPERANT Laboratoire de Neuromorphologie (U.106 I.N.S.E.R.M.), H6pital de Port-Royal, 75014 Paris, Laboratoire de Psychophysiologie sensorielle, 4 place Jussieu, Paris 75005, and Laboratoire d'Anatomie Compar~e, 55 rue de Buffon, Paris 75005 (France)
Though the phenomenon of retrograde intraaxonal transport of horseradish peroxidase (HRP) is by now well establishedl,a,4,6-11,1a, 14 the question of its orthograde axoplasmic active migration remains a subject of controversy1,4,11-13. In the rat visual system two recent studies demonstrated the extent to which opinion is divided on this subject. On the one hand, for Hansson 4, HRP may be taken up by the ganglion nerve cells and transported upwards to their synaptic endings. On the other hand, Bunt et al. 1 did not observe such phenomenon. In order to review this problem, HRP (Sigma type VI, 3-5 #1 of 20 ~ solution in saline) was injected into the vitreous humor of the eye in a series of young anesthetized albino rats 08-25 days postnatal). Controls were injected with the same volume of saline. From 24 to 72 h after injection the animals were perfused through the heart with 300 ml of 1 ~o paraformaldehyde and l ~ glutaraldehyde in 0.12 M phosphate buffer (pH 7.2). The brains were removed, washed in buffer-sucrose solution at 4 °C, cut in serial sections (40 #m) on a freezing microtome and incubated for 15 rain in a medium containing H202 and 3,3'-diaminobenzidine tetrahydrochlorideL For ultrastructural examination in some experiments the optic nerves and the brain slices containing the tractus opticus or primary optic centers were immersed overnight in the same buffer solution without sucrose and cut with a Vibratome in sections 30--80/~m thick. After peroxidase revelation proper in incubation medium~, these sections were postfixed in 2 ~ osmium tetroxide solution, dehydrated in ethanol and then embedded in Epon-Araldite mixture. Sections 0.2-0.1/~m were mounted on grids and observed without metal impregnation. Ultrathin sections were also prepared and stained with uranyl acetate and lead citrate. These sections were examined with a Siemens Elmiskop IA. In addition the rat visual projections were studied with Fink-Heimer and radioautographic methods. Primary optic fibers were also impregnated with the Golgi method. For comparison similar experiments were performed in pigeons. Besides the intraocular injection of HRP, the enzyme was also injected stereotaxically into the optic tract. This material was observed only at the light microscopical level. In only 6 0 ~ of the HRP experimental rats a peroxidase reaction (Fig. 1) can be observed 24 h after HRP intraocular injection, in the optic nerve, optic tracts and
O0
309 primary optic centers, as revealed by comparison with Fink-Heimer and radioautographic materials. In those visual centers the reddish brown peroxidase granulations are not accumulated in the neuronal perikarya but in the neuropil. This reaction is more or less intense depending on centers. On the side contralateral to the injection, it remains discrete in nucleus geniculatus lateralis and in the superficial layers of colliculus superior; however, it appears in higher intensity in pretectal nuclei (Fig. 1 ; nucleus of the optic tract, posterior pretectal nucleus and olivary pretectal nucleus of Scalia 15) and nuclei of the accessory optic fiber system (lateral and medial terminal nuclei of Hayhow et al.5). On the pretectal side of injection, only the olivary pretectal nucleus shows unquestioned marking. However, the suprachiasmatic nuclei were not labeled. In all of the studied sections no neuron was found to exhibit a retrograde peroxidase labeling. Further study of these positive neuropil i regions with the electron microscope shows that the peroxidase material is present only in axonal profiles and can be traced as far as the axon terminals (Figs. 7 and 8). This H R P reaction product appears as dense, oval-shaped bodies or, more often, is contained in tubular organelles which look like the cisterns of the smooth endoplasmic reticulum (Figs. 3 and 4). Thus, as in Hansson's observations 4, the present results show that in the primary visual system of the rat, H R P can be transported in the orthograde direction associated probably with the fast axonal flow as indicated by the speed of the migration of the enzyme. Nevertheless in 40 ~ of our experimental animals there was a complete absence of peroxidase reaction product in the primary visual system, explaining partially the recent results obtained by Bunt et al. 1. The non-reproducibility of the orthograde transport of peroxidase in the visual system has not yet a convincing explanation. Probably it is dependent on the difficulties in properly performing an eye injection in the rat. The failure to find peroxidase-positive neurons may indicate the absence of a centrifugal visual system in the albino rat. Although negative results are of difficult interpretation. As observed by LaVail and LaVail 1° in the chicken, intraocular injection of H R P in the pigeon produces, 24 h after injection, labeling of neurons of the nucleus isthmo-opticus (NIO), the site of origin of the centrifugal visual pathway, and no labeling of the primary visual system. Conversely, when the enzyme was injected into the optic tract a well-pronounced peroxidase reaction in the primary visual pathway was noted 24 h later. The comparison of this material with that obtained for the study of the visual projections in this species (Fink-Heimer, Golgi, radioautography) shows Fig. 1. Low power light micrograph of the pretectal region of a young albino rat 24 h after intraocular injection of 5/~1 of 20 % HRP solution in saline. Note the positive peroxidase reaction in the brachium of the superior colliculus (B) in the nucleus of the optic tract (NT) and in the olivary pretectal nucleus (NO). × 200. Fig. 2. Electron micrograph of the neuropile of the olivary pretectal nucleus from a rat similar to Fig. 1. The HRP reaction product occupies tubular and large vesicular profiles. Note that synaptic vesicles are free of labeling. × 30,000. Fig. 3. Electron micrograph of an optic axon terminal in the olivary pretectal nucleus. In the preterminal segment of the axon HRP reaction product (arrows) is contained in tubular formations which look like SER tubes. Synaptic vesicles are unlabeled. Same material as in Fig. 7. × 56,000.
310
311 that the heavily colored reddish-orange optic fibers are stained right up their terminal endings (Figs. 4-8). It appears throughout these experiments that H R P orthograde transport from ganglion nerve cells is species dependent. In fact, this process takes place in albino rat and fish (fresh-water species, Rep6rant et Lemire, unpublished observations) but is completely lacking in birds (chicken, pigeon) and snake (Vipera aspis, Rep6rant, unpublished observations). In birds the absence of any H R P marking of the primary visual system does not necessarily involve a problem of operative technique or the hypothetical existence of a barrier preventing penetration of H R P into the retina. In fact, labeling of the centrifugal optic neurons (NIO)indicates that H R P was indeed present in the inner plexiform layer of the retina, thus ganglion nerve cells were bathed in the H R P solution, but they could not pick up the enzyme or send it up towards the nerve endings. In the pigeon, the marking of centripetal visual fibers remains, nevertheless, possible when the enzyme is injected into the optic tract. In this traumatic condition H R P probably penetrates at the level of the lesioned axons and rapidly migrates or diffuses to the terminal endings. In conclusion, H R P injected into the eye was unequivocally demonstrated to be transported in the orthograde direction in the primary visual system of the rat; in different conditions such a transport was also visualized in the pigeon. In both cases neuroanatomists are provided with a new tool for the investigation of neuronal connectivity. Conversely, great care should now be taken in the interpretation of peroxidase experiments with regard to the perikaryal or peripheral location of the label. I am particulary grateful to Dr. C. Sotelo and Dr. Y. Galifret for their encouragement, and constructive criticisms. I would also like to thank Mr. D. Micelli for his generous cooperation and Mr. D. Le Cren for his skilfull photographical assistance. This study was partially supported by the Institut National de la Sant6 et de la Recherche M6dicale and the Centre National de la Recherche Scientifique.
1 BUNT, A. H., LUND, R. D., AND LUND, J. S., Retrograde axonal transport of horseradish perox-
idase by ganglion cells of the albino rat retina, Brain Research, 73 (1974) 215-228. 2 GRAHAM, R. C., JR., AND KARNOVSKY,M. J., The early stages of absorption of injected horseradish peroxidase in the proximal tubule of mouse kidney: ultrastructural cytochemistry by a new technique, J. Histochem. Cytochem., 14 (1966) 291-302. Figs. 4-6. Visual projections in the superficial layers of the tectum opticum of the pigeon (layers 1-7 of Cajal). Fig. 4. Autoradiogram after intravitreous injection with 100/*Ci of [3H]proline in the contralateral eye, 2 days before sacrifice, x 160. Fig. 5. Fink-Heimer stain 4 days after removal of the contralateral eye. × 140. Fig. 6. Peroxidase reaction after injection of 0.5/*1 of 20~ HRP solution in saline into the optic tract, 1 day before sacrifice, x 140. Figs. 7 and 8. Optic arborization (layer 5 of Cajal) stained by HRP reaction (Fig. 7), x 680, and by Golgi impregnation (Fig. 8), x 650.
31.2 3 GRAYBIEL, A. M., nr~l:) DEVOR, M., A microelectrophoretic delivery technique tbr use with horseradish peroxidase, Brain Research, 68 (1974) 167-173. 4 HANSSON, H . A . , Uptake and intracellular bidirectional transport of horseradish peroxidase in retinal ganglion cells, Exp. Eye Res., 16 (1973) 377-388. 5 HAYHOW, W. R., WEBB, C., AND JERWE, A., The accessory optic fiber system in the rat, J. temp. Neurol., 115 (1960) 187-215. 6 JONES, E. G., AND LEAVITT, R . Y . , Demonstration of thalamo-cortical connectivity in the cat somato-sensory system by retrograde axonal transport of horseradish peroxidase, Brain Research, 63 (1973) 414-418. 7 KRISTENSSON, K., AND OLSSON, Y., Uptake and retrograde axonal transport of peroxidase in hypoglossal neurones; electron microscopical localization in the neuronal perikaryon, Aeta neuropath. (Berl.), 19 (1971) 1 9. 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 KUYPERS, H. G. J. M., K~EV1T, J., AND GROEN-KLEVANT, E. C., Retrograde axonal transport of horseradish peroxidase in rat's forebrain, Brain Research, 67 (1974) 211-218. 10 LAVAIL, J. H., AND LAVAIL, M. M., Retrograde axonal transport in the central nervous system, Seience, 176 (1972) 1416-1417. 11 LAVA1L,J. H., WJNSTON, K. R., AND TISH, 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)470477. 12 LYNCH, G., GALL, C., ME,SAn, P., AND COTMAN, C. W., Horseradish peroxidase histochemistry : a new method for tracing efferent projections in the central nervous system, Brain Research, 65 (1974) 373 380. 13 NAUTA, H. J. W., PmTZ, M. B., A~D LASEK, R . J . , Afferents to the rat caudoputamen studied with horseradish peroxidase. An evaluation of a retrograde neuroanatemical research method, Brain Research, 67 (1974) 219-238. 14 RALSTON, H. J., 111, AND SHARP, P. V., The identification of thalamocortical relay cells in the adult cat by means of retrograde axonal transport of horseradish peroxidase, Brain Research, 62 (1973) 273-278. 15 SCALJA, F., The termination of retinal axons in the pretectal region of mamzrals, J. comp. Neurol., 145 (1972) 223-258.
© Elsevier ScientificPublishingCompany,Amsterdam- Printed in The Netherlands
307
THE ORTHOGRADE TRANSPORT OF HORSERADISH PEROXIDASE IN THE VISUAL SYSTEM JACQUES REPERANT Laboratoire de Neuromorphologie (U.106 I.N.S.E.R.M.), H6pital de Port-Royal, 75014 Paris, Laboratoire de Psychophysiologie sensorielle, 4 place Jussieu, Paris 75005, and Laboratoire d'Anatomie Compar~e, 55 rue de Buffon, Paris 75005 (France)
Though the phenomenon of retrograde intraaxonal transport of horseradish peroxidase (HRP) is by now well establishedl,a,4,6-11,1a, 14 the question of its orthograde axoplasmic active migration remains a subject of controversy1,4,11-13. In the rat visual system two recent studies demonstrated the extent to which opinion is divided on this subject. On the one hand, for Hansson 4, HRP may be taken up by the ganglion nerve cells and transported upwards to their synaptic endings. On the other hand, Bunt et al. 1 did not observe such phenomenon. In order to review this problem, HRP (Sigma type VI, 3-5 #1 of 20 ~ solution in saline) was injected into the vitreous humor of the eye in a series of young anesthetized albino rats 08-25 days postnatal). Controls were injected with the same volume of saline. From 24 to 72 h after injection the animals were perfused through the heart with 300 ml of 1 ~o paraformaldehyde and l ~ glutaraldehyde in 0.12 M phosphate buffer (pH 7.2). The brains were removed, washed in buffer-sucrose solution at 4 °C, cut in serial sections (40 #m) on a freezing microtome and incubated for 15 rain in a medium containing H202 and 3,3'-diaminobenzidine tetrahydrochlorideL For ultrastructural examination in some experiments the optic nerves and the brain slices containing the tractus opticus or primary optic centers were immersed overnight in the same buffer solution without sucrose and cut with a Vibratome in sections 30--80/~m thick. After peroxidase revelation proper in incubation medium~, these sections were postfixed in 2 ~ osmium tetroxide solution, dehydrated in ethanol and then embedded in Epon-Araldite mixture. Sections 0.2-0.1/~m were mounted on grids and observed without metal impregnation. Ultrathin sections were also prepared and stained with uranyl acetate and lead citrate. These sections were examined with a Siemens Elmiskop IA. In addition the rat visual projections were studied with Fink-Heimer and radioautographic methods. Primary optic fibers were also impregnated with the Golgi method. For comparison similar experiments were performed in pigeons. Besides the intraocular injection of HRP, the enzyme was also injected stereotaxically into the optic tract. This material was observed only at the light microscopical level. In only 6 0 ~ of the HRP experimental rats a peroxidase reaction (Fig. 1) can be observed 24 h after HRP intraocular injection, in the optic nerve, optic tracts and
O0
309 primary optic centers, as revealed by comparison with Fink-Heimer and radioautographic materials. In those visual centers the reddish brown peroxidase granulations are not accumulated in the neuronal perikarya but in the neuropil. This reaction is more or less intense depending on centers. On the side contralateral to the injection, it remains discrete in nucleus geniculatus lateralis and in the superficial layers of colliculus superior; however, it appears in higher intensity in pretectal nuclei (Fig. 1 ; nucleus of the optic tract, posterior pretectal nucleus and olivary pretectal nucleus of Scalia 15) and nuclei of the accessory optic fiber system (lateral and medial terminal nuclei of Hayhow et al.5). On the pretectal side of injection, only the olivary pretectal nucleus shows unquestioned marking. However, the suprachiasmatic nuclei were not labeled. In all of the studied sections no neuron was found to exhibit a retrograde peroxidase labeling. Further study of these positive neuropil i regions with the electron microscope shows that the peroxidase material is present only in axonal profiles and can be traced as far as the axon terminals (Figs. 7 and 8). This H R P reaction product appears as dense, oval-shaped bodies or, more often, is contained in tubular organelles which look like the cisterns of the smooth endoplasmic reticulum (Figs. 3 and 4). Thus, as in Hansson's observations 4, the present results show that in the primary visual system of the rat, H R P can be transported in the orthograde direction associated probably with the fast axonal flow as indicated by the speed of the migration of the enzyme. Nevertheless in 40 ~ of our experimental animals there was a complete absence of peroxidase reaction product in the primary visual system, explaining partially the recent results obtained by Bunt et al. 1. The non-reproducibility of the orthograde transport of peroxidase in the visual system has not yet a convincing explanation. Probably it is dependent on the difficulties in properly performing an eye injection in the rat. The failure to find peroxidase-positive neurons may indicate the absence of a centrifugal visual system in the albino rat. Although negative results are of difficult interpretation. As observed by LaVail and LaVail 1° in the chicken, intraocular injection of H R P in the pigeon produces, 24 h after injection, labeling of neurons of the nucleus isthmo-opticus (NIO), the site of origin of the centrifugal visual pathway, and no labeling of the primary visual system. Conversely, when the enzyme was injected into the optic tract a well-pronounced peroxidase reaction in the primary visual pathway was noted 24 h later. The comparison of this material with that obtained for the study of the visual projections in this species (Fink-Heimer, Golgi, radioautography) shows Fig. 1. Low power light micrograph of the pretectal region of a young albino rat 24 h after intraocular injection of 5/~1 of 20 % HRP solution in saline. Note the positive peroxidase reaction in the brachium of the superior colliculus (B) in the nucleus of the optic tract (NT) and in the olivary pretectal nucleus (NO). × 200. Fig. 2. Electron micrograph of the neuropile of the olivary pretectal nucleus from a rat similar to Fig. 1. The HRP reaction product occupies tubular and large vesicular profiles. Note that synaptic vesicles are free of labeling. × 30,000. Fig. 3. Electron micrograph of an optic axon terminal in the olivary pretectal nucleus. In the preterminal segment of the axon HRP reaction product (arrows) is contained in tubular formations which look like SER tubes. Synaptic vesicles are unlabeled. Same material as in Fig. 7. × 56,000.
310
311 that the heavily colored reddish-orange optic fibers are stained right up their terminal endings (Figs. 4-8). It appears throughout these experiments that H R P orthograde transport from ganglion nerve cells is species dependent. In fact, this process takes place in albino rat and fish (fresh-water species, Rep6rant et Lemire, unpublished observations) but is completely lacking in birds (chicken, pigeon) and snake (Vipera aspis, Rep6rant, unpublished observations). In birds the absence of any H R P marking of the primary visual system does not necessarily involve a problem of operative technique or the hypothetical existence of a barrier preventing penetration of H R P into the retina. In fact, labeling of the centrifugal optic neurons (NIO)indicates that H R P was indeed present in the inner plexiform layer of the retina, thus ganglion nerve cells were bathed in the H R P solution, but they could not pick up the enzyme or send it up towards the nerve endings. In the pigeon, the marking of centripetal visual fibers remains, nevertheless, possible when the enzyme is injected into the optic tract. In this traumatic condition H R P probably penetrates at the level of the lesioned axons and rapidly migrates or diffuses to the terminal endings. In conclusion, H R P injected into the eye was unequivocally demonstrated to be transported in the orthograde direction in the primary visual system of the rat; in different conditions such a transport was also visualized in the pigeon. In both cases neuroanatomists are provided with a new tool for the investigation of neuronal connectivity. Conversely, great care should now be taken in the interpretation of peroxidase experiments with regard to the perikaryal or peripheral location of the label. I am particulary grateful to Dr. C. Sotelo and Dr. Y. Galifret for their encouragement, and constructive criticisms. I would also like to thank Mr. D. Micelli for his generous cooperation and Mr. D. Le Cren for his skilfull photographical assistance. This study was partially supported by the Institut National de la Sant6 et de la Recherche M6dicale and the Centre National de la Recherche Scientifique.
1 BUNT, A. H., LUND, R. D., AND LUND, J. S., Retrograde axonal transport of horseradish perox-
idase by ganglion cells of the albino rat retina, Brain Research, 73 (1974) 215-228. 2 GRAHAM, R. C., JR., AND KARNOVSKY,M. J., The early stages of absorption of injected horseradish peroxidase in the proximal tubule of mouse kidney: ultrastructural cytochemistry by a new technique, J. Histochem. Cytochem., 14 (1966) 291-302. Figs. 4-6. Visual projections in the superficial layers of the tectum opticum of the pigeon (layers 1-7 of Cajal). Fig. 4. Autoradiogram after intravitreous injection with 100/*Ci of [3H]proline in the contralateral eye, 2 days before sacrifice, x 160. Fig. 5. Fink-Heimer stain 4 days after removal of the contralateral eye. × 140. Fig. 6. Peroxidase reaction after injection of 0.5/*1 of 20~ HRP solution in saline into the optic tract, 1 day before sacrifice, x 140. Figs. 7 and 8. Optic arborization (layer 5 of Cajal) stained by HRP reaction (Fig. 7), x 680, and by Golgi impregnation (Fig. 8), x 650.
31.2 3 GRAYBIEL, A. M., nr~l:) DEVOR, M., A microelectrophoretic delivery technique tbr use with horseradish peroxidase, Brain Research, 68 (1974) 167-173. 4 HANSSON, H . A . , Uptake and intracellular bidirectional transport of horseradish peroxidase in retinal ganglion cells, Exp. Eye Res., 16 (1973) 377-388. 5 HAYHOW, W. R., WEBB, C., AND JERWE, A., The accessory optic fiber system in the rat, J. temp. Neurol., 115 (1960) 187-215. 6 JONES, E. G., AND LEAVITT, R . Y . , Demonstration of thalamo-cortical connectivity in the cat somato-sensory system by retrograde axonal transport of horseradish peroxidase, Brain Research, 63 (1973) 414-418. 7 KRISTENSSON, K., AND OLSSON, Y., Uptake and retrograde axonal transport of peroxidase in hypoglossal neurones; electron microscopical localization in the neuronal perikaryon, Aeta neuropath. (Berl.), 19 (1971) 1 9. 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 KUYPERS, H. G. J. M., K~EV1T, J., AND GROEN-KLEVANT, E. C., Retrograde axonal transport of horseradish peroxidase in rat's forebrain, Brain Research, 67 (1974) 211-218. 10 LAVAIL, J. H., AND LAVAIL, M. M., Retrograde axonal transport in the central nervous system, Seience, 176 (1972) 1416-1417. 11 LAVA1L,J. H., WJNSTON, K. R., AND TISH, 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)470477. 12 LYNCH, G., GALL, C., ME,SAn, P., AND COTMAN, C. W., Horseradish peroxidase histochemistry : a new method for tracing efferent projections in the central nervous system, Brain Research, 65 (1974) 373 380. 13 NAUTA, H. J. W., PmTZ, M. B., A~D LASEK, R . J . , Afferents to the rat caudoputamen studied with horseradish peroxidase. An evaluation of a retrograde neuroanatemical research method, Brain Research, 67 (1974) 219-238. 14 RALSTON, H. J., 111, AND SHARP, P. V., The identification of thalamocortical relay cells in the adult cat by means of retrograde axonal transport of horseradish peroxidase, Brain Research, 62 (1973) 273-278. 15 SCALJA, F., The termination of retinal axons in the pretectal region of mamzrals, J. comp. Neurol., 145 (1972) 223-258.
