Ipsilaterally projecting rubrospinal neurons in adult and developing opossums.код для вставкиСкачать
THE ANATOMICAL RECORD 231538-547 (1991) lpsilaterally Projecting Rubrospinal Neurons in Adult and Developing Opossums XIAO MING XU AND G.F. MARTIN Department of Cell Biology, Neurobiology, and Anatomy, The Ohio State University College of Medicine, Colurnbus, Ohio ABSTRACT’ We have combined injections of Fast Blue with lesions of the rubrospinal tract rostra1 and contralateral to them to determine if an ipsilateral rubrospinal projection exists in adult or developing opossums and, if so, to characterize the neurons giving rise to it. Although the results indicate that some rubral neurons project ipsilaterally, they are very few in number. Using quantitative and image analysis techniques, we have shown that 0.6% of the rubral neurons that project to the lumbar cord in adult opossums do so ipsilaterally and that such neurons are comparable in location and size to those that project contralaterally. Similar results were obtained in developing opossums. Our results are discussed in light of rubrospinal development and ongoing experiments related to rubrospinal plasticity. The rubrospinal tract is generally considered to be crossed (see reviews by Kuypers, 1982; Walberg, 19821, but an ipsilateral component has been suggested for the hedgehog (Michaloudi et al., 1988) and documented for the rat (Shieh et al., 1983) and cat (Holstege and Kuypers, 1982; Holstege, 1987). One of the objectives of the present study was to establish whether an ipsilatera1 rubrospinal projection is present in the North American opposum, Didelphis virginiana, and, if so, to determine the location, number, and size of the neurons contributing to it. Previous studies suggested that such a projection exists (Martin and Dom, 1970; Martin et al., 1974; Cabana and Martin, 19861, but the results were inconclusive. To the best of our knowledge, ipsilaterally projecting rubrospinal neurons have not been characterized for any species. We have shown previously that the opposum’s rubrospinal tract develops postnatally (Cabana and Martin, 1986, Martin et al., 1986, 1988) rather than prenatally as in rats (Leong et al., 1984; Shieh et al., 1983) and cats (Bregman and Goldberger, 1982, 19831, making it possible to manipulate it without intrauterine surgery. In the experiments reported here, we took advantage of that fact to identify rubrospinal neurons that project ipsilaterally during selected stages of development. We paid particular attention to the critical period for rubrospinal plasticity, i.e., the period during which rubral axons can grow around a lesion of their spinal pathway (Martin and Xu, 1988; Xu and Martin 19891, because an earlier study suggested that a welldeveloped, ipsilateral pathway may exist during that stage of development (Cabana and Martin, 1986). If so, it would be relevant to the interpretation of ongoing experiments designed to determine whether rubrospinal plasticity results from regeneration of cut axons or new growth. Although our results suggest that an ipsilateral rubrospinal tract exists in both adult and developing opossums, the neurons that contribute to it are few in number. Using quantitative and image analysis techniques, we have shown that only 0.6% of the 0 1991 WILEY-LISS, INC. rubral neurons that project to the lumbar cord in adult opossums do so ipsilaterally and that such neurons cannot be distinguished from those that project contralaterally by their location or size. Similar results were obtained in developing opossums. METHODS Four adult female opossums were anesthetized with sodium pentobarbitol (40 mg/kg) for sterile surgery. The eighth or ninth segment of the thoracic cord was exposed first so that the rubrospinal tract could be transected on the right side. The first or second segment of the lumbar cord was then exposed for a 10 p1 injection of 3%Fast Blue (FBI on the left side. Since the rubrospinal tract was cut on the right, the side contralateral to the injection, we assumed that any neurons labeled in the left red nucleus projected ipsilaterally. Few, if any, rubral axons cross the midline at spinal levels (Martin and Dom, 1970; Martin et al., 1974; Cabana and Martin, 1986). After the injection, the soft tissues were sutured together in layers and the animals were returned to the vivarium under a veterinarian’s care, Seven days later they were given an overdose of the anesthetic and perfused transcardially with saline followed by a 0.1 M cirtate buffer-10% formaldehyde solution. The spinal cord and brain were dissected out and immersed in the same buffer with 30% sucrose for approximately 24 hours at 4°C.The brain was scored with a shallow cut on the side of the lesion so that laterality of the tissue sections could be determined after mounting. Frozen sections through the lesion, the injection, and the brainstem were cut in the corona1 plane at 40 pm. The Received October 26, 1991; accepted February 9, 1991. Address reprint requests to Dr. George F. Martin, Dept. of Cell Biology, Neurobiology, and Anatomy, The Ohio State University College of Medicine, 333 West 10th Ave., Columbus, OH 43210-1218. IPSILATERALLY PROJECTING RUBROSPINAL NEURONS sections were mounted immediately and coverslipped with Entellan (Merck) for viewing with a Leitz (Orthoplan) flurorescence microscope using the A cube of the Ploem illumination system (excitation wavelength = 340-380 nm). The positions of labeled neurons ipsilateral and contralateral to the injection were plotted and counted from every fifth section through the red nucleus using a n X-Y plotter attached to the microscope stage by position transducers. All labeled neurons were recorded, including those not sectioned through the nucleus, and selected fields were photographed. In each case, labeled neurons contralateral to the injection were drawn from two sections each through the rostral, middle, and caudal thirds of the red nucleus (about one out of 10 sections) using a drawing tube attached to the microscope. Since labeled neurons were sparse ipsilateral to the injection, all of them were drawn from every section. The areas of labeled neurons were determined with the aid of a n interactive computer-assisted image analysis system (Magiscan, NikoniJoyce Loebl). Drawings of the labeled neurons were fed into a black-and-white television camera (Doge-MtI series 68, Newvicon), then through a real-time video processor (Nippon Avionics, Model Image Sigma), and finally into a computer (Magiscan 2A, Joyce-Loebl) for digitization and analysis. The outline of each cell was filled in by the computer, which was instructed to separate dark areas (labeled cells) from light areas (noncells). Calibration was done so that the area of labeled cells was measured in pm2. Statistical analysis was accomplished by using the Results program supplied by Joyce-Loebl. The output from the computer provided histograms showing the size and frequency distribution of labeled neurons on each side. Using a comparable approach, one animal was subjected to a lesion of the right rubrospinal tract a t the third segment of the cervical cord and a n injection of FB on the left at the sixth cervical segment. Survival time, perfusion, tissue processing, and evaluation were accomplished a s described above. For the developmental studies, pouch-young opossums were employed at estimated postnatal day (EPD)20 (N = 41, 40 (N = 31, and 54 (N = 2). The developing animals were obtained from females captured in the wild so their snout-rump length (SRL) was measured by stretching them on a ruler to estimate age from the growth curve of Cutts e t al. (1978). The mother was anesthetized by a 1.2 ml intramuscular injection of Ketamine (100 mg/ml) followed by inhalation of Metofane and then placed on her back. During anesthesia the pouch sphincter relaxed, exposing the litter. The pouch-young, still attached to the nipples, were anesthetized individually by hypothermia or Metofane inhalation for lesions of the rubrospinal tract on the right side of the thoracic cord and FB injections into the left side of the lumbar cord as described for the adult animal. The incisions were closed and the operated animals returned with their mother to the vivarium. Seven days later, the pouch-young were removed, sacrificed by a n overdose of the anesthetic, and perfused through the heart with the same fixative used for the adult animal. The spinal cord and brain were removed, scored, sectioned, mounted, and examined as described above. In these cases, labeled neurons were 539 plotted and counted in one out of every three sections. Labeled neurons were not drawn and measured because the fluorescence fades too rapidly in young animals. RESULTS Studies on Adult Animals Figure 1provides photomicrographic documentation of the results obtained from one of the adult animals subjected to hemisection of the thoracic cord on the right and a lumbar injection of FB on the left. The photomicrographs were taken from one out of every 10 sections through the red nucleus from its rostral (top) to caudal (bottom) ends. On the side contralateral to the injection (right column, Fig. l ) , labeled neurons were numerous throughout the length of the nucleus. In rostral sections, most of them were found ventrally (Fig. lB,D), whereas more caudally (Fig. lF,H,J,L), they were more evenly dispersed dorsoventrally. In caudal sections, there was a tendency for labeled neurons to be most numerous laterally. On the side ipsilateral to the injection, only one labeled neuron can be observed (arrow, Fig. 1C). Figure 2 contains a plot of the labeling present in one out of five sections from the same case and a histogram of the quantitative results. In the four cases studies, a n average of 431.2 neurons was counted in one out of five sections on the side contralateral to the injection, whereas only 2.7 were counted ipsilaterally. The paired t test showed that the difference between the two groups was statistically significant (P < 0.01). The histograms in Figure 3 show the frequency of labeled neurons according to size in the rostral (top), middle (middle), and caudal (bottom) thirds of the red nucleus contralateral and ipsilateral to the injection. Because of their sparsity, all of the labeled neurons were counted and measured ipsilateral to the injection, whereas only those in two sections from each third of the nucleus (about one out of 10 sections) were evaluated on the contralateral side. The results suggest that neurons projecting ipsilaterally do not constitute a separate subset based on size. The locations of all labeled neurons ipsilateral to the injection in the four lumbar cases are plotted on the right in Figure 3. The top drawing depicts the locations of such neurons in the rostral 1/3 of the red nucleus, and i t can be seen that most of them were located ventrally. Those in the middle and caudal thirds of the nucleus were found dorsally and ventrally. In general, the ipsilaterally projecting neurons were found in areas that also project contralaterally. Figure 4 illustrates the rubral neurons labeled contralateral and ipsilateral to the injection in the case subjected to a lesion of the rubrospinal tract on the right a t the third cervical segment and a n injection of FB on the left three segments caudal to the lesion. On the side contralateral to the injection, labeled neurons were numerous throughout the length of the red nucleus (right column, Fig. 4). Rostrally, more of them were labeled in the dorsal part of the nucleus than after lumbar injections (compare Figs. 1D and 4D) and, caudally, neurons were labeled dorsomedially where they were not labeled after lumbar injections (compare Figs. 1J and 4J).On the side ipsilateral to the injection (left column Fig. 41, only three neurons were labeled (ar- 540 X.M. XU A N D G.F. MARTIN Fig. 1, Fluorescence photomicrographs of labeled neurons in one out of every 10 sections from the rostra1 (top) to caudal (bottom) poles of the red nucleus contralateral (right column) and ipsilateral (left column) to the injection in a n adult opossum subjected to a lesion of the right rubrospinal tract a t the eighth thoracic segment and a lumbar injection of Fast Blue on the left. The arrow in C points to a neuron labeled ipsilateral to the injection. IPSILATERALLY PROJECTING RUBROSPINAL NEURONS 54 1 Fig. 2. Plot showing the locations of labeled neurons in one out of five sections through the red nucleus from the case documented in Figure 1. The arrow indicates the only neuron labeled ipsilateral to the injection in these sections. The histogram gives the average number of neurons labeled contralateral and ipsilateral to the injection in the four adult cases subjected to lumbar injections rows, Fig. 4A,C,G). Counts of labeled neurons on the two sides show that 472 were present contralaterally, whereas only 8 were present ipsilaterally. The photomicrographs in Figure 5 show that the neurons labeled ipsilateral to the injection (Fig. 5C) were comparable in size, shape, and labeling intensity t o those labeled contralaterally (Fig. 5B,D). The same was true for the neurons labeled after lumbar injections. Studies on Developing Animals At the ages studies (PD20, 40 and 541, the results were comparable to those obtained in the adult animals, Figure 6 illustrates the labeling produced in one of the cases operated a t PD20, which is during the critical period for rubrospinal plasticity. Labeling in the red nucleus contralateral to the injection was extensive and present in all of the areas labeled in adult animals (right column, Fig. 6). As in the adult animal, that on the ipsilateral side was sparse (arrows, Fig. 6A, K). Figure 7 shows a plot of the labeling obtained in one out of three sections from the same case. In the four cases studies, the average number of labeled neurons contralateral to the injection (one out of three sections) was 227.8, whereas on the ipsilateral side it was 3.0. The paired t test showed that the difference between the two groups was statistically significant (P< 0.01). DISCUSSION In degeneration studies, there was often evidence for axonal degeneration in the opposum’srubrospinal tract ipsilateral as well as contralateral to lesions of the red nucleus (Martin and Dom, 1970; Martin et al., 1974). Such degeneration could not be taken as definitive evidence for an ipsilateral rubrospinal tract, however, since the lesions may have damaged rubral axons from the contralateral side. Although the orthograde transport of wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP) also provided evidence for an ipsilateral projection (Cabana and Martin, 19861, the injections may have produced “injury label” of ax- 542 X.M. X U AND G.F. MARTIN Area of Labeled Neurons n Plot of Ipsi. Proi. Neurons Latera i Medial Rostra1 Area ( p m ' ) x 0 C 0 3 U Q) r= "I .I n Middle x 0 C 0) 3 U f! LL 8 I 488 888 I( Ho 1 I 148 1U8 UI Caudal Fig. 3. Histograms showing the frequency of labeled neurons in different size ranges from the rostral (top), middle (middle), and caudal (lower) thirds of the red nucleus ipsilateral and contralateral to lumbar injections. The labeled neurons contralateral to the injection were measured from only two sections in each third of the nucleus (about one out of each 10 sections), whereas all of the neurons labeled on the ipsilateral side were measured. The positions of the labeled neurons in the rostral, middle, and caudal thirds of the red nucleus ipsilateral to the injections are plotted on the right from all the lumbar cases. ons from the contralateral red nucleus. The results of the present study clearly document the existence of ipsilaterally projecting rubrospinal neurons, however, and show that they are comparable in location and size to those that project contralaterally. The existence of such neurons can be inferred in the hedgehog (Michaloudi et al., 1988) and their presence has been documented in the rat (Shieh et al., 1983) and cat (Hol- IPSILATERALLY PROJECTING RUBROSPINAL NEURONS Fig. 4. Fluorescence photomicrographs of labeled neurons in one out of every 10 sections from the rostra1 (top) to the caudal (bottom) end of the red nucleus contralateral (right column) and ipsilateral (left column) to the injection in a case subjected to a lesion of the right 543 rubrospinal tract a t the third cervical segment and an injection of Fast Blue into the sixth cervical segment on the left. The arrows in A, C, and G point to neurons labeled ipsilateral to the injection. 544 X.M. XU AND G.F. MARTIN Fig. 5.High power fluorescence photomicrographs of sections through the red nucleus contralateral (B, D) and ipsilateral (A, C) to the injection from the case referred to in Figure 4. stege and Kuypers, 1982; Holstege, 1987). The present study is the first, however, to characterize them as to location and size. In a previous orthograde transport study, we observed substantial labeling in the area of the rubrospinal tract ipsilateral as well as contralateral to injections of WGA-HRP into the red nucleus of developing opossums (Fig. 2, Cabana and Martin, 1986). With increasing age, the ipsilateral labeling became less obvious (Cabana and Martin, 1986), suggesting that the ipsilateral pathway decreased in size due to axonal degeneration andlor retraction. Evidence for loss of ipsilateral projections in the development of the predominantly crossed corticospinal (Cabana and Martin, 1985; Theriault and Tatton, 1989) and retinocollicular projections (Jeffrey and Perry, 1982; Martin et al., 1983; Insausti et al., 1984; Jacobs et al., 1984) has been reported previously, so it was reasonable t o assume that the same phenomenon might occur in rubrospinal development. The present results indicate that rubral neurons that project ipsilaterally are almost as sparse during development as in the adult animal, however, arguing against the existence of a large ipsilateral pathway that diminishes with age. It is likely that the ipsilateral labeling observed in our previous study resulted from incorporation of the marker by axons from the contralateral red nucleus and/or spread of the injection into the dorsolateral pons (Martin et al., 1979). During development, rubrospinal axons have to choose whether they will cross a t the ventral tegmental decussation or remain ipsilateral. Although our results speak only to the development of laterality in the lumbar cord, they suggest that rubrospinal axons make the correct choice and that laterality is established early in development. Laterality of reticulospinal and vestibulospinal projections is already established in the 11-day chicken embryo (Glover and Petursdottir, 1988), but that of the rubrospinal tract has not been reported. Retina1 axons also make a choice at the optic chiasm, and it has been shown in the mouse that most of them make the correct one (Sretavan, 1990). We have shown that rubral axons grow around a lesion of their spinal pathway at about postnatal day 20 in the opossum, although relatively few rubrospinal neurons survive axotomy (Martin and Xu, 1988; Xu and Martin, 1989). From these observations we hypothesized that rubrospinal plasticity results primarily from new growth, not true regeneration. In an attempt to test that hypothesis, we injected FB into the spinal cord at about postnatal day 18 to label rubral neurons that innervate that level. Four days later the axons of such neurons were cut in a second surgery. The animals were allowed to survive for about 30 days, after which another fluorescent marker, Diamidino Yellow (DY),was injected between the first injection and the lesion. The intent of the second injection was to label IPSILATERALLY PROJECTING RUBROSPINAL NEURONS Fig. 6. Fluorescence photomicrographs of labeled neurons in one out of three sections from the rostra1 (top) to the caudal (bottom) end of the red nucleus contralateral (right column) and ipsilateral (left column) to the injection in a n animal subjected to a thoracic lesion of the 545 rubrospinal tract on the right and a n injection of Fast Blue into the lumbar cord on the left a t postnatal day 20. The arrows in A and K indicate the neurons labeled on the ipsilateral side. 546 L X.M. XU AND G.F. MARTIN 300 3 2 71 200 QJ d QJ Q d d 100 6- 0 6 0 Z CAUDAL 1 mm Fig. 7. Plot showing the locations of labeled neurons in one out of every three sections through the red nucleus from the pouch-young opossium referred to in Figure 6. The arrow points to labeled neurons ipsilateral to the injection. The histogram gives the average number of neurons labeled contralateral and ipsilateral to the injection in the four cases studies at this age. rubral neurons whose axon had grown around the lesion during the 30 day survival. Upon microscopic examination, relatively few neurons in the contralateral red nucleus were labeled by FB, but many were labeled by DY. Some were labeled by both markers, however, suggesting that they survived axotomy and t h a t their axons grew around the lesion to incorporate the second marker. Alternative explanations include the possibility that the double-labeled neurons projected ipsilaterally and t h a t they incorporated both markers because of bilateral spread at the injection sites. The results of the present study suggest t h a t is not likely, however, because so few rubrospinal neurons project ipsilaterally. ACKNOWLEDGMENTS The authors thank Ms. Mary Ann Jarrell for surgical assistance, tissue processing, and typing of the manuscript; Mr. Karl Rubin for photographic help; and Mr. Michael Pindzola for helping with the computer counts and measurements. We are also grateful to Dr. Michael Beattie for providing helpful comments on the manuscript. This study was supported by USPHS grants NS25095 and NS-10165 as well a s a n Academic Challenge Award to the Neuroscience Program from the State of Ohio. LITERATURE CITED Bregman, B.S., and M.E. Goldberger 1982 Anatomical plasticity and sparing of function after spinal cord damage in neonatal cats. Science, 21 7553-555. Bregman, B.S., and M.E. Goldberger 1983 Infant lesion effect: 111. Anatomical correlates of sparing and recovery of function after spinal cord damage in newborn and adult cats. Dev. Brain Res., 9t137-154. Cabana, T., and G.F. Martin 1985 Corticospinal development in the North American opossum: Evidence for a sequence in the growth of cortical axons in the spinal cord and for transient projections. Dev. Brain Res., 23r69-80. Cabana, T., and G.F. Martin 1986 The development of the rubrospinal tract. An experimental study using the orthograde transport of WGA-HRP in the North American opossum. Dev. Brain Res., 3O:l-11. Cutts, J.H., W.J. Krause, and C.R. Leeson 1978 General observations on the growth and development of the pouch young opossum, Didelphzs uirginiana. Biol. Neonate, 33:264-272. Glover, J.C., and C. Petursdottir 1988 Pathway specificity of reticulospinal and vestibulospinal projections in the 1 1-day chicken embryo. J . Comp. Neurol., 270:25-38. Holstege, G. 1987 Anatomical evidence for a n ipsilateral rubrospinal pathway and for direct rubrospinal projections to motoneurons in the cat. Neurosci. Lett. 74t269-274. Holstege, G., and H.G.J.M. Kuypers 1982 The anatomy of brain stem IPSILATERALLY PROJECTING RUBROSPINAL NEURONS pathways to the spinal cord in cat. A labeled amino acid tracing study. In: Descending Pathways to the Spinal Cord, Progress in Brain Research, Vol. 57. H.G.J.M. Kuypers and G.F. Martin, eds. Elsevier, Amsterdam, pp. 145-175. Insausti, R., C. Blakemore, and W.M. Cowan 1984 Ganglion cell death during development of ipsilateral retino-collicular projection in golden hamster. Nature, 308:362-365. Jacobs, D.S., V.H. Perry, and M.J. Hawken 1984 The postnatal reduction of the uncrossed projection from the nasal retina in the cat. J . Neurosci., 4.2425-2433. Jeffrey, G., and V.H. Perry 1982 Evidence for ganglion cell death during development of the ipsilateral retinal projection in the rat. Dev. Brain Res., 2:176-180. Kuypers, H.G.J.M. 1982 A new look at the organization of the motor system. In: Descending Pathways to the Spinal Cord, Progress in Brain Research, Vol. 57. H.G.J.M. Kuypers and G.F. Martin, eds., Elsevier, Amsterdam, pp. 381-403. Leong, S.K., J.Y. Shieh, and W.C. Wong 1984 Localizing spinal-cordprojecting neurons in neonatal and immature albino rats. J . Comp. Neurol., 228.18-23. Martin, G.F., and R. Dom 1970 The rubro-spinal tract of the opossum (Didelphis uirginiana). J . Comp. Neurol., 138:19-30. Martin, G.F., and X.M. Xu 1988 Evidence for developmental plasticity of the rubrospinal tract. Studies using the North American opossum. Dev. Brain Res., 39:303-308. Martin, G.F., R. Dom, S.Katz, and J.S. King 1974 The organization of projection neurons in the opossum red nucleus. Brain Res., 78: 17-34. Martin, G.F., A.O. Humbertson, and G.F. Martin 1979 Spinal projections from the mesencephalic and pontine reticular formation in the North American opossum. A study using axonal transport techniques. J. Comp. Neurol., 187:373-400. Martin, P., R.A.J. Sefton, and B. Dreher 1983 The retinal location and 547 fate of ganglion cells which project to the ipsilateral superior colliculus in neonatal albino and hooded rats. Neurosci. Lett., 41 :219-226. Martin, G.F., T. Cabana, and J.C. Hazlett 1986 The development of rubrospinal, cerebellorubral and corticorubral connections in the North American opossum. Evidence for asynchronism. Neurochem. Pathol., 5221-236. Martin, G.F., T. Cabana, and J.C. Hazlett 1988 The development of selected rubral connections in the North American opossum. Behav. Brain Res. 28t21-28. Michaloudi, J., A. Dinopoulos, A.N. Karamanlidis, G.C. Papadopoulos, and J . Antonopoulos 1988 Cortical and brain stem projections to the spinal cord of the hedgehog (Erinaceus europaeus). Anat. Embryol., 178.259-270, Shieh, J.Y., S.K. Leong, and W.C. Wong 1983 Origin of the rubrospinal tract in neonatal, developing, and mature rats. J. Comp. Neurol., 214:79-86. Sretavan, D.W. 1990 Specific routing of retinal ganglion cell axons a t the mammalian optic chiasm during embryonic development. J . Neurosci. 10:1995-2007. Theriault, E., and W.G. Tatton 1989 Postnatal redistribution of pericruciate motor cortical projections within the kitten spinal cord. Dev. Brain Res., 45:219-237. Walberg, F. 1982 Paths descending from the brain stem-An overview. In: Brain Stem Control of Spinal Mechanisms. Fernstrom Foundation Series, Vol. 3. B. Sjolund and A. Bjorklund, eds. Elsevier, Amsterdam, pp. 1-27. Xu, X.M., and G.F. Martin 1989 Developmental plasticity of the rubrospinal tract. Studies using the North American opossum. J . Comp. Neurol., 279:368-387. Xu, X.M., and G.F. Martin 1990 The response of rubrospinal neurons to axotomy in the adult opossum, Didelphis uirginiana. Exp. Neurol., 108r46-54.