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Early development of rubrospinal and cerebellorubral projections in Xenopus laevis
Developmental Brain Research, 58 (1991) 297-300 Elsevier
297
BRESD 60392
Early development of rubrospinal and cerebellorubral projections in Xenopus laevis H.J. ten Donkelaar, R. de Boer-van Huizen and J.A.M. van der Linden Department of Anatomy and Embryology, University of Nijmegen, Nijmegen (The Netherlands) (Accepted 6 November 1990)
Key words: Rubrospinal tract; Cerebellorubral projection; Brachium conjunctivum; Anurans
In Xenopus laevis HRP was applied at the spinomedullary border at various stages of development. In these experiments labeled rubrospinal neurons were observed from stage 48 on. HRP applications to the mesencephalic tegmentum showed, from stage 49 on, retrogradely labeled neurons in the cerebellar nucleus, particularly contralaterally. These data suggest that anuran cerebellorubral projections arise early, well before the rubrospinal innervation of the spinal cord is complete.
In the clawed toad, Xenopus laevis, a developmental sequence was found in the formation of descending pathways to the spinal cord 11'13A5'19. Horseradish peroxidase (HRP) data 11-13"19 show that interstitiospinal, reticulospinal and vestibulospinal fibers innervate spinal segments very early in development, whereas the anuran red nucleus projects spinalwards definitely later in development 13-z5. This developmental sequence parallels the changes observed in locomotor pattern. Until stage ~° 58, locomotion (swimming) consists of coordinated, alternate contractions of the axial muscles on each side of the body. From stage 58 on swimming is gradually, from stage 63 on solely, accomplished with the hindlimbs. It is in this period that rubrospinal fibers innervate the lumbar spinal cord 13-15. Some recent data ~6 on the development of cerebellar efferents in Xenopus laevis seem to challenge the rather late outgrowth of rubrospinal fibers. H R P applications to the cerebellar anlage showed the formation of a small, strictly contralateral projection to the ventral mesencephalic tegmentum already at stage 4916, presumably arising in the single cerebellar nucleus present in amphibians t4. The anuran cerebellar nucleus gives rise to a small brachium conjunctivum projecting to the red nucleus 5-7'9. In this respect it is of interest to note that in the North American opossum in which a similar developmental sequence 2,3 in the formation of descending supraspinal pathways was found as in Xenopus laevis, it was observed that cerebeilorubral connections are present before rubrospinal innervation is complete s. There-
fore, the development of the rubrospinal tract in Xenopus laevis has been reinvestigated with retrograde and anterograde tract-tracing techniques. HRP, applied at the spinomedullary border, was used to study the early development of the rubrospinal tract. Furthermore, H R P as well as some other tracers (PHA-L, Dil) applied to the ventral part of the mesencephalic tegmentum including the red nucleus, are currently used to study the ingrowth of rubrospinal fibers into the spinal cord (ten Donkelaar and de Boer-van Huizen, in preparation). In the present study only the retrograde labeling of the cerebeUar nucleus will be discussed. In about 25 Xenopus laevis larvae from stage 41 until stage 66, H R P (Boehringer grade I), recrystallized from distilled water onto sharp tungsten or glass needles, was applied at the spinomeduilary border under tricaine methanesulphonate anesthesia (MS 222, Sandoz; 0.1 mg/ml tap water). In a similar way H R P was applied in 15 larvae to the ventral part of the mesencephalic tegmentum from stage 48 on. After appropriate survival times (several hours for the youngest stages, 24 h for the others), animals were reanesthetised with an overdose of MS 222 and perfused through the heart with 0.1 M phosphate buffer (pH 7.4) followed by a fixative containing 1.25% glutaraldehyde, 1% formaldehyde and 1% DMSO in phosphate buffer. The youngest animals were simply immersed in the fixative. During immersion fixation for one hour the brains and spinal cords were dissected out and subsequently washed overnight in phosphate buffer at 4 °C. They were then processed as a
Correspondence: H.J. ten Donkelaar, Department of Anatomy and Embryology, Faculty of Medicine, University of Nijmegen, P.O. Box 9101, 6500 HB Nijmegen, The Netherlands. 0165-3806/91/$03.50 © 1991 Elsevier Science Publishers B.V. (Biomedical Division)
298
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E Fig. 1. Retrogradely labeled neurons in the mesencephalon of Xenopus laevis after HRP applications at the spinomedullary border. A,B,C: drawings of representative experiments in stages 48 (A,B) and 53 (C). D,E: photomicrographs of labeled rubrospinal tract neurons in stage 48 (D, x154) and in stage 53 (E, x242). Modified DAB-staining technique. Iflm, interstitial nucleus of the tim; Rub, nucleus tuber.
299
Fig. 2. Labeled cerebellar nucleus neurons after H R P applications to the ventral mesencephalic tegmentum at stages 49, 53 and 57, plotted from several serial sections as viewed from a caudal position (left side of drawing is ipsilateral to the injection side). Cer, cerebellar nucleus.
whole with a modified ~9 cobalt intensified diaminobenzidine (DAB) staining technique 1. The smaller brains (up to stage 50) were osmicated for one hour (1% osmium tetroxide in 0.1 M phosphate buffer pH 7.4), dehydrated in graded ethanols, embedded in immersion oil, sectioned on a serial microtome at 20 ~m and mounted in Epon. The larger brains were embedded in polyacrylamide 4 and sectioned on a vibratome at 100/~m. These sections were stained once again with the cobalt/ DAB staining technique. They were then mounted directly in glycerin-gelatin or, alternatively, osmicated for one hour and embedded in Epon. In the series of H R P applications at the spinomedullary border in the youngest stages studied labeling in the mesencephalon was restricted to the interstitial nucleus of the tim, in keeping with other studies H'~9. But, in contrast to previous data Zg, at stage 48 the first retrogradely labeled neurons were observed in the red nucleus (Fig. 1A,B,D), their axons crossing the midline at the level of the rubrospinal tract neurons. The more ventral position, the smaller size of their somata and the crossing of their axons, makes it possible to distinguish rubrospinal neurons from the larger, more dorsally situated, ipsilaterally projecting interstitiospinal neurons. At stage 48 the rubrospinal neurons appear relatively immature with small, round to oval cell bodies and just the
beginning of a dendritic tree. The rubrospinal tract neurons extensively increase their dendritic trees as is exemplified in the experiment shown in Fig. 1C,E (stage 53). These data show that rubrospinal axons reach the spinomedullary border at stage 48. The negative findings in a previous study 19 most likely can be explained by the fact that the H R P applications in that study were made between the level of the 5th and 10th myotome, i.e. somewhat more caudally. HRP data on the ingrowth of rubrospinal fibers into the spinal cord (ten Donkelaar and de Boer-van Huizen, in preparation) also suggest the arrival of rubrospinal axons in the rostral spinal cord at stage 48. When H R P was applied to the mesencephalic tegmenturn, apart from the anterogradely labeled rubrospinal (and interstitiospinal) fibers, from stage 49 on, a group of small retrogradely labeled neurons was found, bilaterally, in the area directly below the cerebellar anlage, i.e. in the cerebellar nucleus (Fig. 2). In most cases it was not possible to restrict the H R P deposit to one side or, alternatively, crossing cerebellorubral axons were damaged or the tungsten needle with H R P invaded the contralateral tegmentum. When serial reconstructions were made of this cell group (Fig. 2), it was found that the most rostrally situated cells were also the most dorsomedially. Thus, when these reconstructions were viewed from a caudal position, the group of cells was seen to extend from ventrolateral to dorsomedial, or upwards toward the cerebellum. These data suggest that cerebeliorubral projections arise rather early in Xenopus laevis, and are present well before the rubrospinal innervation of the spinal cord is complete. Similar observations were made in the North American opossum 3"8. In contrast, in Xenopus laevis, cerebellovestibular projections are formed somewhat earlier than the cerebellorubral projection ~6'17, but innervate an already rather complete vestibulospinal system ~1,~9. Vestibulospinai projections in Xenopus laevis form very early in development ~'~2"~9, and so cerebellovestibular projections grow out when their targets are already well developed 16"17. [~H]Thymidine studies TM on the neurogenesis of brainstem neurons in Xenopus laevis show that neurons in the vestibular nuclear complex are 'born' very early in development, whereas neurons in the red nucleus undergo their final mitosis much later, but well before stage 50. The present study shows that rubrospinal tract neurons already start invading the spinal cord at stage 48 and receive cerebellorubral afferents by stage 49. Their role in the development of the control of hindlimb movements is the subject for further studies.
The authors would like to thank Mr. H. Eikholt for the breeding and taking care of the animals and Mrs. C. Udo for typing the manuscript.
300
1 Adams, J.C., Heavy metal intensification of DAB-based HRP reaction product, J. Histochem. Cytochem., 29 (198l) 775. 2 Cabana, T. and Martin, G.E, Developmental sequence in the origin of descending spinal pathways. Studies using retrograde transport techniques in the North American Opossum (Didelphis virginiana), Dev. Brain Res., 15 (1984) 247-263. 3 Cabana, T. and Martin, G.F., The adult organization and development of the rubrospinal tract. An experimental study using the orthograde transport of WGA-HRP in the NorthAmerican opossum, Dev. Brain Res., 30 (1986) 1-11. 4 de Boer-Van Huizen, R., Polyacrylamide als inbedmedium voor vries- en vibratoom-coupes, Histotechniek, 8 (1989) 148-152. 5 Gonzalez, A., ten Donkelaar, H.J. and de Boer-van Huizen, R., Cerebeilar connections in Xenopus laevis. An HRP study, Anat. Embryol., 169 (1984) 167-176. 6 Grover, B.G., Topographic organization of cerebellar efferents in the frog (Rana esculenta) as revealed by retrograde transport of wheat germ agglutinin-conjugated horseradish peroxidase, Neurosci. Lett. Suppl., 14 (1983) S 146. 7 Larson-Prior, L. and Cruce, W.L.R., A reciprocal connection between cerebellum and nucleus ruber in a frog (Rana pipiens), Anat. Rec., 208 (1984) 101 A. 8 Martin, G.F., Cabana, T., Hazlett, J.C., Ho, R. and Waltzer, R., Development of brainstem and cerebellar projections to the diencephalon with notes on thalamocortical projections: studies in the North American opossum, J. Comp. Neurol., 260 (1987) 186--200. 9 Montgomery, N., Projections of the vestibular and cerebellar nuclei in Rana pipiens, Brain Behav. Evol., 31 (1988) 82-95. 10 Nieuwkoop, P.D. and Faber, J., Normal Table ofXenopus laevis (Daudin), 2nd edn., North-Holland Publishing Co., Amsterdam, 1967.
11 Nordlander, R., Bader, S.T. and Ryba, T., Development of early brainstem projections to the tail spinal cord of Xenopuz, J. Comp. Neurol., 231 (1985) 519-529. 12' Roberts, A. and Clarke, J.D.W., The neuroanatomy of an amphibian embryo spinal cord, Phil. Trans. R. Soc. Lond. B. 296 (1982) 195-212. 13 Ten Donkelaar, H.J., Organization of descending pathways to the spinal cord in amphibians and reptiles. In H.G.J.M. Kuypers and G.E Martin (Eds.), Descending Pathways to the Spinal Cord, Progress in Brain Research, Vol. 57, Elsevier, Amsterdam, 1982, pp. 25-67. 14 Ten Donkelaar, H.J., Evolution of the red nucleus and rubrospinal tract, Behav, Brain Res., 28 (1988) 9-20. 15 Ten Donkelaar, H.J, and de Boer-van Huizen, R., Observations on the development of descending pathways from the brainstem to the spinal cord in the clawed toad, Xenopus laevis, Anat. Embryol., 163 (1982) 461-473. 16 Van der Linden, J.A.M., The Development of Cerebellar Connections in the Clawed Toad, Xenopus laevis, Thesis, University of Nijmegen, 1990. 17 Van der Linden, J.A.M. and ten Donkelaar, H,J., Morphological evidence for a monosynaptic connection between cerebellar Purkinje cells and vestibulospinal tract neurons in the larval clawed toad, Xenopus laevis, Neurosci. Lett., 112 (1990) 121-126. 18 Van Mier, P., The Development of the Motor System in the Clawed Toad, Xenopus laevis, Thesis, University of Nijmegen, 1986. 19 Van Mier, P. and ten Donkelaar, H.J., Early development of descending pathways from the brain stem to the spinal cord in Xenopus laevis, Anat. Embryol., 170 (1984) 295-306.
297
BRESD 60392
Early development of rubrospinal and cerebellorubral projections in Xenopus laevis H.J. ten Donkelaar, R. de Boer-van Huizen and J.A.M. van der Linden Department of Anatomy and Embryology, University of Nijmegen, Nijmegen (The Netherlands) (Accepted 6 November 1990)
Key words: Rubrospinal tract; Cerebellorubral projection; Brachium conjunctivum; Anurans
In Xenopus laevis HRP was applied at the spinomedullary border at various stages of development. In these experiments labeled rubrospinal neurons were observed from stage 48 on. HRP applications to the mesencephalic tegmentum showed, from stage 49 on, retrogradely labeled neurons in the cerebellar nucleus, particularly contralaterally. These data suggest that anuran cerebellorubral projections arise early, well before the rubrospinal innervation of the spinal cord is complete.
In the clawed toad, Xenopus laevis, a developmental sequence was found in the formation of descending pathways to the spinal cord 11'13A5'19. Horseradish peroxidase (HRP) data 11-13"19 show that interstitiospinal, reticulospinal and vestibulospinal fibers innervate spinal segments very early in development, whereas the anuran red nucleus projects spinalwards definitely later in development 13-z5. This developmental sequence parallels the changes observed in locomotor pattern. Until stage ~° 58, locomotion (swimming) consists of coordinated, alternate contractions of the axial muscles on each side of the body. From stage 58 on swimming is gradually, from stage 63 on solely, accomplished with the hindlimbs. It is in this period that rubrospinal fibers innervate the lumbar spinal cord 13-15. Some recent data ~6 on the development of cerebellar efferents in Xenopus laevis seem to challenge the rather late outgrowth of rubrospinal fibers. H R P applications to the cerebellar anlage showed the formation of a small, strictly contralateral projection to the ventral mesencephalic tegmentum already at stage 4916, presumably arising in the single cerebellar nucleus present in amphibians t4. The anuran cerebellar nucleus gives rise to a small brachium conjunctivum projecting to the red nucleus 5-7'9. In this respect it is of interest to note that in the North American opossum in which a similar developmental sequence 2,3 in the formation of descending supraspinal pathways was found as in Xenopus laevis, it was observed that cerebeilorubral connections are present before rubrospinal innervation is complete s. There-
fore, the development of the rubrospinal tract in Xenopus laevis has been reinvestigated with retrograde and anterograde tract-tracing techniques. HRP, applied at the spinomedullary border, was used to study the early development of the rubrospinal tract. Furthermore, H R P as well as some other tracers (PHA-L, Dil) applied to the ventral part of the mesencephalic tegmentum including the red nucleus, are currently used to study the ingrowth of rubrospinal fibers into the spinal cord (ten Donkelaar and de Boer-van Huizen, in preparation). In the present study only the retrograde labeling of the cerebeUar nucleus will be discussed. In about 25 Xenopus laevis larvae from stage 41 until stage 66, H R P (Boehringer grade I), recrystallized from distilled water onto sharp tungsten or glass needles, was applied at the spinomeduilary border under tricaine methanesulphonate anesthesia (MS 222, Sandoz; 0.1 mg/ml tap water). In a similar way H R P was applied in 15 larvae to the ventral part of the mesencephalic tegmentum from stage 48 on. After appropriate survival times (several hours for the youngest stages, 24 h for the others), animals were reanesthetised with an overdose of MS 222 and perfused through the heart with 0.1 M phosphate buffer (pH 7.4) followed by a fixative containing 1.25% glutaraldehyde, 1% formaldehyde and 1% DMSO in phosphate buffer. The youngest animals were simply immersed in the fixative. During immersion fixation for one hour the brains and spinal cords were dissected out and subsequently washed overnight in phosphate buffer at 4 °C. They were then processed as a
Correspondence: H.J. ten Donkelaar, Department of Anatomy and Embryology, Faculty of Medicine, University of Nijmegen, P.O. Box 9101, 6500 HB Nijmegen, The Netherlands. 0165-3806/91/$03.50 © 1991 Elsevier Science Publishers B.V. (Biomedical Division)
298
/
/
i J
C
\
~
/
~ ~ ~O
i X,
J i!//
QI
E Fig. 1. Retrogradely labeled neurons in the mesencephalon of Xenopus laevis after HRP applications at the spinomedullary border. A,B,C: drawings of representative experiments in stages 48 (A,B) and 53 (C). D,E: photomicrographs of labeled rubrospinal tract neurons in stage 48 (D, x154) and in stage 53 (E, x242). Modified DAB-staining technique. Iflm, interstitial nucleus of the tim; Rub, nucleus tuber.
299
Fig. 2. Labeled cerebellar nucleus neurons after H R P applications to the ventral mesencephalic tegmentum at stages 49, 53 and 57, plotted from several serial sections as viewed from a caudal position (left side of drawing is ipsilateral to the injection side). Cer, cerebellar nucleus.
whole with a modified ~9 cobalt intensified diaminobenzidine (DAB) staining technique 1. The smaller brains (up to stage 50) were osmicated for one hour (1% osmium tetroxide in 0.1 M phosphate buffer pH 7.4), dehydrated in graded ethanols, embedded in immersion oil, sectioned on a serial microtome at 20 ~m and mounted in Epon. The larger brains were embedded in polyacrylamide 4 and sectioned on a vibratome at 100/~m. These sections were stained once again with the cobalt/ DAB staining technique. They were then mounted directly in glycerin-gelatin or, alternatively, osmicated for one hour and embedded in Epon. In the series of H R P applications at the spinomedullary border in the youngest stages studied labeling in the mesencephalon was restricted to the interstitial nucleus of the tim, in keeping with other studies H'~9. But, in contrast to previous data Zg, at stage 48 the first retrogradely labeled neurons were observed in the red nucleus (Fig. 1A,B,D), their axons crossing the midline at the level of the rubrospinal tract neurons. The more ventral position, the smaller size of their somata and the crossing of their axons, makes it possible to distinguish rubrospinal neurons from the larger, more dorsally situated, ipsilaterally projecting interstitiospinal neurons. At stage 48 the rubrospinal neurons appear relatively immature with small, round to oval cell bodies and just the
beginning of a dendritic tree. The rubrospinal tract neurons extensively increase their dendritic trees as is exemplified in the experiment shown in Fig. 1C,E (stage 53). These data show that rubrospinal axons reach the spinomedullary border at stage 48. The negative findings in a previous study 19 most likely can be explained by the fact that the H R P applications in that study were made between the level of the 5th and 10th myotome, i.e. somewhat more caudally. HRP data on the ingrowth of rubrospinal fibers into the spinal cord (ten Donkelaar and de Boer-van Huizen, in preparation) also suggest the arrival of rubrospinal axons in the rostral spinal cord at stage 48. When H R P was applied to the mesencephalic tegmenturn, apart from the anterogradely labeled rubrospinal (and interstitiospinal) fibers, from stage 49 on, a group of small retrogradely labeled neurons was found, bilaterally, in the area directly below the cerebellar anlage, i.e. in the cerebellar nucleus (Fig. 2). In most cases it was not possible to restrict the H R P deposit to one side or, alternatively, crossing cerebellorubral axons were damaged or the tungsten needle with H R P invaded the contralateral tegmentum. When serial reconstructions were made of this cell group (Fig. 2), it was found that the most rostrally situated cells were also the most dorsomedially. Thus, when these reconstructions were viewed from a caudal position, the group of cells was seen to extend from ventrolateral to dorsomedial, or upwards toward the cerebellum. These data suggest that cerebeliorubral projections arise rather early in Xenopus laevis, and are present well before the rubrospinal innervation of the spinal cord is complete. Similar observations were made in the North American opossum 3"8. In contrast, in Xenopus laevis, cerebellovestibular projections are formed somewhat earlier than the cerebellorubral projection ~6'17, but innervate an already rather complete vestibulospinal system ~1,~9. Vestibulospinai projections in Xenopus laevis form very early in development ~'~2"~9, and so cerebellovestibular projections grow out when their targets are already well developed 16"17. [~H]Thymidine studies TM on the neurogenesis of brainstem neurons in Xenopus laevis show that neurons in the vestibular nuclear complex are 'born' very early in development, whereas neurons in the red nucleus undergo their final mitosis much later, but well before stage 50. The present study shows that rubrospinal tract neurons already start invading the spinal cord at stage 48 and receive cerebellorubral afferents by stage 49. Their role in the development of the control of hindlimb movements is the subject for further studies.
The authors would like to thank Mr. H. Eikholt for the breeding and taking care of the animals and Mrs. C. Udo for typing the manuscript.
300
1 Adams, J.C., Heavy metal intensification of DAB-based HRP reaction product, J. Histochem. Cytochem., 29 (198l) 775. 2 Cabana, T. and Martin, G.E, Developmental sequence in the origin of descending spinal pathways. Studies using retrograde transport techniques in the North American Opossum (Didelphis virginiana), Dev. Brain Res., 15 (1984) 247-263. 3 Cabana, T. and Martin, G.F., The adult organization and development of the rubrospinal tract. An experimental study using the orthograde transport of WGA-HRP in the NorthAmerican opossum, Dev. Brain Res., 30 (1986) 1-11. 4 de Boer-Van Huizen, R., Polyacrylamide als inbedmedium voor vries- en vibratoom-coupes, Histotechniek, 8 (1989) 148-152. 5 Gonzalez, A., ten Donkelaar, H.J. and de Boer-van Huizen, R., Cerebeilar connections in Xenopus laevis. An HRP study, Anat. Embryol., 169 (1984) 167-176. 6 Grover, B.G., Topographic organization of cerebellar efferents in the frog (Rana esculenta) as revealed by retrograde transport of wheat germ agglutinin-conjugated horseradish peroxidase, Neurosci. Lett. Suppl., 14 (1983) S 146. 7 Larson-Prior, L. and Cruce, W.L.R., A reciprocal connection between cerebellum and nucleus ruber in a frog (Rana pipiens), Anat. Rec., 208 (1984) 101 A. 8 Martin, G.F., Cabana, T., Hazlett, J.C., Ho, R. and Waltzer, R., Development of brainstem and cerebellar projections to the diencephalon with notes on thalamocortical projections: studies in the North American opossum, J. Comp. Neurol., 260 (1987) 186--200. 9 Montgomery, N., Projections of the vestibular and cerebellar nuclei in Rana pipiens, Brain Behav. Evol., 31 (1988) 82-95. 10 Nieuwkoop, P.D. and Faber, J., Normal Table ofXenopus laevis (Daudin), 2nd edn., North-Holland Publishing Co., Amsterdam, 1967.
11 Nordlander, R., Bader, S.T. and Ryba, T., Development of early brainstem projections to the tail spinal cord of Xenopuz, J. Comp. Neurol., 231 (1985) 519-529. 12' Roberts, A. and Clarke, J.D.W., The neuroanatomy of an amphibian embryo spinal cord, Phil. Trans. R. Soc. Lond. B. 296 (1982) 195-212. 13 Ten Donkelaar, H.J., Organization of descending pathways to the spinal cord in amphibians and reptiles. In H.G.J.M. Kuypers and G.E Martin (Eds.), Descending Pathways to the Spinal Cord, Progress in Brain Research, Vol. 57, Elsevier, Amsterdam, 1982, pp. 25-67. 14 Ten Donkelaar, H.J., Evolution of the red nucleus and rubrospinal tract, Behav, Brain Res., 28 (1988) 9-20. 15 Ten Donkelaar, H.J, and de Boer-van Huizen, R., Observations on the development of descending pathways from the brainstem to the spinal cord in the clawed toad, Xenopus laevis, Anat. Embryol., 163 (1982) 461-473. 16 Van der Linden, J.A.M., The Development of Cerebellar Connections in the Clawed Toad, Xenopus laevis, Thesis, University of Nijmegen, 1990. 17 Van der Linden, J.A.M. and ten Donkelaar, H,J., Morphological evidence for a monosynaptic connection between cerebellar Purkinje cells and vestibulospinal tract neurons in the larval clawed toad, Xenopus laevis, Neurosci. Lett., 112 (1990) 121-126. 18 Van Mier, P., The Development of the Motor System in the Clawed Toad, Xenopus laevis, Thesis, University of Nijmegen, 1986. 19 Van Mier, P. and ten Donkelaar, H.J., Early development of descending pathways from the brain stem to the spinal cord in Xenopus laevis, Anat. Embryol., 170 (1984) 295-306.