The Nucleus Corporis Pontobulbaris of the North American Opossum 1 G. F. MARTIN, M. LINAUTS AND J. M. WALKER Lkpartment ofAnatomy, The Ohio Stnte University,Colkge of Medicine, 1645 Neil Auenue, Columbus,Ohio43210
ABSTRACT The nucleus of the pontobulbar body (PBu) in the North American opossum is located, for the most part, adjacent to the motor root of the trigeminal nerve. Material prepared by degeneration and autoradiographic methods shows that the PBu receives projections from the facial motor-sensory cortex, red nucleus, spinal cord and cerebellum. The latter fibers probably take origin within the fastigial nucleus. Each of the afferent connections ends in a restricted part of the PBu, but there is considerable overlap. Use of the horseradish peroxidase technique reveals that the PBu projects to the spinal cerebellum (anterior lobe, pyramis and paramedian lobules), to visual-auditory areas of the vermis and to the lobus simplex as well as to crus I and I1 of the hemispheres. Although there is some topography to such projections, it is not sharply defined and many regions of the PBu contain labelled neurons after injections of horseradish peroxidase into widely separate areas of the cerebellar cortex. Because of its embryogenesis and position, the PBu is often considered part of the dorsolateral basilar pons. I t appears from our material, however, that the organization of PBu afferent and efferent connections is different from that of the adjacent basilar pons, and arguments for considering the PBu a separate precerebellar nucleus are presented. The corporis pontobulbaris was described by Essick (‘07) as a fibroganglionic mass in the human brain which extends from a position a t the lateral boundary of the fourth ventricle to the basilar pons. In a subsequent study, Essick (‘12)showed that the human basilar pons is formed by cells which course rostrally and ventrally from the germinal epithelium of the rhombic lip and that the corporis pontobulbaris in the adult is a remnant of that migration. Olszewski and Baxter (‘54) referred to the neurons contained within the human corporis pontobulbaris as the nucleus corporis pontobulbaris (PBu in this account) and that term has been used for apparently comparable cell groups in several species (e.g., the cat; Taber, ’611,including the American opossum (Oswaldo-Cruz and Rocha-Miranda, ‘68). Although information is available on the histogenesis of the PBu (Harkman, ’54a,b; Pierce, ,661, little is known about its connections. In normal human material both Swank (‘34) and Marburg (‘45) followed axons from J. COMP. NEUR., 175: 345-372.
the pyramidal tract which looped around the inferior olive and apparently ended within the PBu. Marburg (‘45) also suggested that the PBu projected to the cerebellum. The purposes of the present report are to present data concerning both afferent and efferent connections of the PBu in the North American opossum in order to extend observations made earlier (Martin e t al., ‘74, ’75) and to address the question of whether or not the PBu should be considered a separate precerebellar nucleus. MATERIALS AND METHODS
Observations on the organization and cytology of the PBu were made from adult opossum brains cut in different planes of section and stained for Nissl substance as well as from brains processed for either normal axons (silver carbonate technique of del Rio Hor!This investigation was supported by the United States Public Health Service, Grants NS-07410,NS-08798 and by funds from the Bremer Foundation, The Ohio State University. College of Medicine. * J. M. Walker is a graduate student in the Department of Psychology, The Ohio State University.
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tega, see Sharenberg and Liss, '69).or cholinesterase activity (Lewis, '61;modification of the Koelle technique, '50). Material impregnated by one of several Golgi techniques was made available from the laboratory of Doctor James S. King. Some of our results were taken from the brains of over 300 opossums which have been impregnated for degenerating axons following specific lesions. Survival times varied from two to fourteen days and several modifications of the Nauta-Gygax ('54) and Fink-Heimer ('67)methods were employed. In this study terminal degeneration refers to axonal debris which is either finely granular and/or randomly arranged in the PBu neuropil. Autoradiographic observations were made from brains in which SH-leucinewas injected into either the cerebral cortex, the brainstem or the cerebellum. The marker was deposited by means of a Hamilton syringe and #31 gauge needle attached to a stereotaxically guided microdrive. Quantities from 0.1-0.3plI injection were introduced into each animal (concentrated 50-100 pCi/pl) over periods from one-half to one hour and the needle was kept in the injection site for a t least ten minutes prior to and after the injection. After survival times of one to ten days the animals were anesthetized and sacrificed by intracardiac perfusion. Frozen sections were mounted and processed by standard autoradiographic procedures using a one month exposure time (Hendrickson, '75)and sections through the PBu were examined by light and dark field optics for concentrations of silver grains above background. Clumps of grains over axonal bundles were considered to indicate labelled axons in passage, whereas random labelling over the PBu neuropil was interpreted as terminal. The horseradish peroxidase (HRP) method was utilized to localize the PBu neurons which project to different cortical areas of the cerebellum. The enzyme was delivered to different parts of the cerebellar cortex as dep l of a 50% solution) scribed above (0.1-0.6 over a period varying from 45 minutes to over 1 hour. After approximately 24 hours the animals were anesthetized and perfused with a 1.0% paraformaldehyde, 1.0% glutaraldehyde solution in a 0.1 M phosphate buffer (pH = 7.4)containing 30% sucrose. Frozen sections were incubated according to the method of Graham and Karnovsky ('661, stained for
Nissl substance and examined for spread of the marker a t the injection site as well as for the presence of neurons containing yellowbrown granules of reaction product. RESULTS
Conformation and cytoarchitecture of the nuckus corporis pontobulbaris (PBu) As described by Oswaldo-Cruz and RochaMiranda ('681, the rostra1 extreme of the PBu in the opossum is squeezed between the latAbbreviations
aq, cerebral aqueduct CcD, dorsal cochlear nucleus Cn, culmen contra, side contralateral to the injection Crll, crus I1 ct, trapezoid body De, declive F1, flocculus Fol, folium IC, inferior colliculus IP, interpeduncular nucleus ipsi, side ipsilateral to the injection Lat, lateral Lg, lingula nlll, oculomotor nerve Nd, nodule ped, cerebral peduncle PFL, paraflocculus PM, paramedian lobule PrC, preculmen Pyc, pyramis RN, red nucleus rVm, major (sensory) root of trigeminal nerve rVn, motor root of trigeminal nerve Sx, lobus simplex trs, spinal trigeminal tract Uv, uvula Fig. 1 Dorsal view of the opossum brainstem with most of the cerebellum removed. The open block arrow points to the location of the stratum gliosum a t the lateral edge of the dorsal cochlear nucleus (CcD), the apparent origin of the pontobulbar migration. Magnification X 3.0. Fig. 2 Ventral view of the opossum brainstem. The open block arrow points to the position of the pontobulbar nucleus medial to the trigeminal nerve (rVm). Mag nification X 3.0. Fig. 3 Transversely cut, Nissl stained section through the largest part of the pontobulbar nucleus (arrow). The lateral aspect of the brainstem is on the reader's left and the ventral surface of the section is seen at the bottom of the figure. Both the motor rootlets of the trigeminal nerve (rVn) and the spinal trigeminal tract are labelled (trs). The bar indicates 440 pm. Fig. 4 Transversely cut, Nissl stained section through the most caudal part of the pontobulbar nucleus (arrow). The lateral part of the brainstem is on the reader's left and the ventral surface of the section is covered by the figure number. The motor roots of the trigeminal nerve and the spinal trigeminal tract are labelled and the insert shows the dendritic plexus of PBu neurons at this level (arrow). A motor fascicle of the trigeminal nerve is indicated (rVn). The magnification is the same as for figure 3.
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technique reveals that the PBu receives a major contribution from the cerebral comz Degeneration is obtained within the PBu after lesions which are limited to the facial motor-sensory cortex Martin et al., ’751, but we have chosen to illustrate this connection by using a case subjected to unilateral decortication. Our intent is that the reader see the total cortical target of the PBu (fig. 7, left side). In rostral sections a few degenerating axons can be seen ipsilaterally in that part of the PBu located between the brachium pontis and the lateral surface of the pons. More caudally, however, they become more extensive (fig. 141, particularly laterally, and most evident around the emerging trigeminal fascicles (figs. 7A,B, left side). Fragmented axons can be traced into the nucleus from several directions, and some of them follow the dendritic bundles referred to previously (fig. 7B, left side). Axonal debris is located within comparable portions of the caudal PBu, as well as around and between motor fascicles of the trigeminal nerve Cfig. 7C). Although not as numerous, degenerating axons are present within the contralateral PBu. Evidence for a cortical-PBu projection is also available in the brains in which sH-leucine has been injected into facial motor-sensory cortex. After rubml lesions or lesions which undercut rubral axons, degeneration can be traced out of the crossed rubrobulbospinal bundles into the PBu (fig. 7, right side). Again we illustrate this connection from a case with a large lesion so that the “total” rubral domain can be appreciated. Few degenerating fibers enter rostral portions of the nucleus Cfig. 7A, right side), but distribute to its doraal and medial sectors more caudally (figs. 7B,C right side), as well as along the dendritic bundles in the reticular formation (figs. 7B,C, right side). As is the case after cortical lesione, degenerating axons also distribute between the motor trigeminal fascicles (fig. 7C, right side). Rubral lesions, large or small, necessarily interrupt axons from extra-rubral sources, making it difficult to determine the precise origin of degeneration within the PBu. In addition, degeneration methoda do not always reveal the complete distribution of axons even after careful attention to survival times. Because of these problems the autoradiographic method was employed on brains in which 91Afferentconnections of the nucleus corporis leucine was injected so as to label the red nupontobulbwis cleus and/or surrounding cells (e.g., figs. 18, Material processed by the Fink-Heimer 19). In all cases in which either the red nu-
era1 aspect of the pons and the fibers of the brachium pontis. At more caudal levels, however, it is more distinct and located adjacent to the motor root of the trigeminal nerve (figs. 2-4). In material processed for cholinesterase activity (fig. 6) the PBu stands out in relief from surrounding areas because of the intensity of the reaction product contained within it. Although the pontobulbar body is probably continuous with the stratum gliosum of Oswaldo-Cruz and Rocha-Miranda C68) (see also Voris and Hoerr, ’321, only a few maturelooking neurons are located at that level. The reader is referred to figure 1 where the gross position of the stratum gliosum is indicated. The largest part of the PBu appears on the ventral surface of the lateral pons just medial to the exiting fascicles of the minor (motor) root. of the trigeminal nerve (fig. 3). At that level it is generally wedge-shaped in transverse sections. The rounded, deeply staining neurons located dorsally and medially within the PBu measure from 11-22 p (figs. 3,311 and have dendrites which overlap with one anot.her. The dendrites of some of them extend out.of the nucleus, and a t some levels they can be followed into the reticular formation where they form bundles which parallel the course of penetrating blood vessels (fig. 5). Fusiformshaped neurons are also seen in such bundles and their dendrites contribute to them. In contrast, the neurons located ventrolaterally within the PBu tend to be small (10-16p ) and have relatively little cytoplasm (sgs. 3, 31). Their dendrites are so heavily impregnated in the available material that it is difficult to viwalize their geometry. In sections impregnated for normal axons a fairly rich afferent plexuR can be seen to pour into the dorsal medial part of the PBu, whereas impregnated axons are relatively sparse among the smaller neurons located ventrally and laterally (insert of fig. 15). At its caudal end the PBu becomes somewhat rectangular in transverse section and intimately related to the medial aspect of the motor trigeminal fascicles (fig. 4). Dendrites of the latter neurons are closely entangled and tend to be oriented from dorsal to ventral, although some of them extend laterally between the motor bundles of the trigeminal nerve (fig. 4, insert).
spinal cord WBS partially transected a t C-2 (P-320, insert on right side). Degenerating axons are drawn in each section as they appear in Fink-Heimer impregnated material.
(arrows) from a case subjected to a cerebellar split (arrow, P-208, insert on left side) and from another brain in which the
Fig. 8 A series of transverse sections through three levels (rostra1 A through caudal, C) of the pontobulbar nucleus
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cleus and/or the neurons dormmedial to it are well labelled (figs. 18, 191, the isotope was transported to precisely those regions of the PBu which contain degeneration after damage to the red nucleus or its axons (figs. 2022). The PBu is unlabelled or only equivocally labelled after injections of the superior colliculus and/or the interstitial tegmentum and after injections of more ventromedial nonrubral areas. Although we have numerous cases in which either 3H-leucineor proline has been injected into the pontine and medullary reticular formation, none of them shows definite evidence for transport to the PBu. The PBu also receives a small input from the cerebellum. Subsequent to a midline cut through the cerebellum which undercuts fastigial outflow (insert of fig. 8, left side), degenerating axons can be traced bilaterally around the brachium conjunctivum to a position between the brachium pontis and the lateral surface of the pons. Such fibers, plus others which take different routes, distribute to rostra1 parts of the PBu as well as to that part of the nucleus associated with the trigeminal nerve (fig. 8, left side). Although most of the injured axons lie medial and dorsal t o the PBu proper, some enter its dorsal extreme, particularly, caudally where they ramify in a terminal fashion (figs. 8A-C, left side and fig. 15’1.Although degenerating axons can be seen just dorsal to the PBu after lesions limited to the nucleus fastigius, only a very few actually enter the nucleus. We have cases in which 3H-leucinehas been deposited into the fastigial nucleus, but they contain little evidence for a PBu projection. No evidence for a projection to the PBu is preeent in cases with lesions of the interpositus and/or dentate nuclei or in brains in which sH-leucine was deposited within them. One of the major inputs to the PBu arises within the spinal cord. After a lesion which partially hemisects the cord a t high cervical level (insert, fig. 8, right side), degeneration is present bilaterally within the ventrolateral tracts which ascend within the brainstem (Hazlett et al., ’72). Degenerating bundles course dorsally from these tracts on their way to both the cerebellum (Hazlett et al., ’71) and the external inferior colliculus (Hazlett et al., ’72) and some of them pass through the matral PBu. At mid-rostra1 to caudal levels of the PBu there is a definite dorsomedialzone of terminal debris 6g. 8A, right side and %a. 16, 17) which gradually diminishes at more cau-
dal levels (6gs. 8B,C, right side). Degenerating axons also distribute between the motor fibersof the trigeminal nerve (figs. 8B,C, right side). A comparable distribution of degeneration is present in a case subjected to a large medullary lesion which undercut all ascending spinal fascicles. Degenerating axons are present within the PBu after spinal lesions a t either C-4 or midthoracic levels, but few can be discerned subsequent to lower lumbar or sacral transections.
A.siectione of the pontobulbar nucleua to the cerebellar cortex Neurons of the PBu contain reaction product after horseradish peroxidase injections of many areas of the cerebellar cortex. Subsequent to the medial anterior lobe placement shown in figure 23 neuronal labelling is present in medial and dorsal parts of the ipsilateral PBu at mid-rostra1 to caudal levels (figs. 9A-C, left side). Generally comparable results were obtained from the more lateral placement shown in figure 24 (figs. 9A-C, right side), although PBu neurons contained reaction product on both sides. As can be seen in figure 9, right side, some of the marked neurons are located in the medially oriented extensions of the PBu referred to previously. Only a very few of the smaller, more ventrolaterally situated neurons contain reaction product. In still another brain the enzyme was deposited a t a point intermediate between the injection sites illustrated, and similar results were obtained bilaterally. Injection of HRP into other “spinal areas” of the cerebellum (Hazlett et al., ’71) also results in labelling of PBu neurons. In the three brains available with injections of the paramedian lobule, PBu neurons contain reaction product on both sides, and most of these cells are aggregated within dorsal and medial parts of the nucleus. The placement site from one case is shown in figure 26 and the location of reactive neurons in the PBu is illustrated in figure 10 (top). In caudal sections (not shown in 10) backfilled neurons are located just medial to the PBu and apparently out of its confines. Although one of our injections of the medial pyramis failed to label PBu neurons, the one shown in figure 26 provided positive results. As can be seen in figure 10 (bottom) most of the reactive neurons are medially and dorsally situated. The available material provides evidence for a projection from the PBu to visual-audi-
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Fig. 10 Drawings of sections (rostral,A, caudr;l, Bj through the pontobulbar nucleus (arrows) on both sides of brains subjected to horseradish peroxidase injections of the paramedian lobule (arrow, P-459,insert on top) and pyramis (arrow, P-493,insert on bottom). Labelled neurons within the pontobulbar nucleus are drawn in each section.
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Fig. 11 In the upper part of the illustration sections are drawn from three levels kostral, A, to caudal, C) of the pontobulbar nucleus on both sides (arrows) from a brain subjected to a folium placement of horseradish peroxidase (arrow, P-497,insert on top). In the lower frame two sections (rostral, A, caudal, B) are drawn through the pontobulbar nucleus of both sides (arrows) from a brain in which horseradish peroxidase was deposited into the tuber of the cerebellar vermin (arrow, P-498,insert on bottom). In both cases labelled neurons within the pontobulbar are illustrated. Cells containing large amounts of reaction product are illustrated in solid black, whereas those which are lightly labelled are only outlined.
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Fig. 12 In the upper part of the illustration sections are drawn from two levels (rostral, A, caudal, B)of the pontobulbar nucleus (arrows) on both sides of a brain in which horseradish peroxidase was injected into crus I of the cerebellar hemisphere. As shown in the insert (P-473, arrow) the marker spread both rostrally and caudally from the injection site. In the lower part of the illustration two sections (rostral, A, caudal, B) are drawn through the pontobulbar nuclei (arrows) of both sides from a brain subjected to a placement of horseradish peroxidase into the lobus simplex (P-476, arrow, insert). In both cases reactive neurons are drawn where they appear in the sections.
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Fig. 13 At the top of the illustration two sections are drawn through the pontobulbar nucleus (arrows), bilaterally, from a case subjected to a placement of horseradish peroxidase into CNS I of the cerebellum with only minimal spread (arrow, P-508, insert). In the lower part of the illustration two sections are drawn through the pontobulbar nuclei of both sides (arrows) from a case in which horseradish peroxidase waa placed within crus I1 of the cerebellum (arrow, P-504, insert). In both cases reactive neurons are drawn where they appear in the sections.
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siderable overlap of their territory as well as overlap in the dendritic domain of PBu neurons. Swank (‘34) suggested that the human PBu receives cortical input, but to our knowledge such a connection has not been verified by experimental techniques. Crossed rubral fibers have been traced to cell groups around the trigeminal nerve such as “Regio h,” “Regio M” and “Zellgruppe K ’ of Meesen and Olszewski (‘49) in the rabbit (Mizuno et al., ’73) and the lateral juxtatrigeminal nucleus of Riley in the monkey (Miller and Strom inger, ’73). Unfortunately, however, i t is not clear which, if any, of these areas correspond to the PBu of this account (Oswaldo-Cruz and Rocha-Miranda, ’68). Although fastigial axons have not been followed to an area identified as the PBu in any species, it is interesting that the uncrossed descending brachium conjunctivum of the rat distributes to an area around the emerging motor root of the trigeminal nerve (Mehler, ’67, ’69). Spinal fibers have not been described as projecting to the PBu either, but are reported (Mehler, ’69) to distribute within a n area near the trigeminal nerve in several species, including the opossum. Mehler identified the target of such fibers as the processus tegmentosus lateralis of the pontine grey after Olszewski and Baxter (‘54). I t can be seen from the above account that it is difficult to compare our results with those obtained from other species because the PBu is not identified as such in any of them. It appears, however, that many of the regions referred to in other species correspond to some part of the PBu as defined herein. Our material reveals that the PBu projects to the spinal cerebellum (i.e., the anterior lobe, the paramedian lobules and the pyramis, Hazlett et al., ’711, the visual-auditory region of the cerebellar vermis (the declive, the folium and the tuber, Snider and Stowell, ’44) as well as to several parts of the hemispheres (the lobus simplex, crus I and 11). No PBu neurons were labelled subsequent to HRP injections of the paraflocculus or uvula, and to date we have not successfully injected either the flocculus or the nodule. If the location of laDISCUSSION belled neurons is compared in the various The pontobulbar nucleus (PBu) of the cases, a rough topography appears to exist. It opossum receives afferent connections from is clear, however, that neurons in some areas the cerebral cortex, red nucleus, cerebellum project to widely separate parts of the cere(probably fastigial nucleus) and spinal cord. bellum. Although each of the afferent connections end The identification of afferent connections in a restricted part of the PBu, there is con- for any cell group becomes more meaningful
tory areas of the cerebellar vermis (Snider and Stowell, ’44). Separate HRP injections were made in the declive, the folium and the tuber, and the PBu contains reactive neurons in each case. The injection of the folium in P497 (fig. 27) produced labelling of a particularly large number of neurons, and the results are illustrated in figure 11 (top). It should be noted, however, that the needle penetrated into a subjacent folium, and very light contamination of that part of the anterior lobe just rostra1 to the primary fissure cannot be ruled out. As in the paramedian case described above, heavily reactive neurons are located just outside of the nucleus (open block arrow, right side of figure 11C, top). The tuber placement shown in figure 28 produced positive results in the caudal part of the nucleus, bilaterally (fig. 11, bottom). There is considerable evidence that the PBu projects to more lateral zones of the cerebellar hemisphere. The HRP injection in P-473 (fig. 12, top) was made in crus I, but the marker spread to the lobus simplex, crus I1 and the paramedian lobule. Although this case is of no use for localizing the precise targets of PBu neurons, i t does provide data relative to the extent of the PBu which projects to the cerebellar hemisphere. A large number of PBu neurons contain reaction product bilaterally, and the larger neurons are particularly well labelled (figs. 31-33). In contrast to the cases with injections of spinal or visual-auditory areas of the cerebellum, however, many of the small, ventrolaterally positioned neurons also contained reaction product (P-473, fig. 12A, ipsilateral side and arrows, figs. 31, 32). PBu neurons were labelled, mainly ipsilaterally, after the lobus simplex injection shown in figure 29 (fig. 12, bottom) and bilaterally after injections of crus I and 11. The results from a large crus I injection are plotted in figure 13 (top) and those obtained from a smaller crus I1 injection (fig. 30) are illustrated in the bottom of figure 13. In cases where the injection was limited to either the paraflocculus or the uvula there was no evidence of enzyme incorporation by PBu neurons.
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when interpreted in light of its efferent projections. By comparing the distribution of the fiber systems which penetrate the PBu with the location of labelled neurons in the HRP experiments, at least two tentative conclusions can be drawn. First, it appears that fibers from the red nucleus, the fastigial nucleus and the spinal cord seek out neurons of the PBu which project to the spinal cerebellum, to visual-auditory regions of the vermis and to parts of the hemisphere. Secondly, cortical fibers appear to concentrate on those neurons which project heavily to the hemispheres. I t should be emphasized that cortical contacts with neurons projecting to nonhemisphere areas cannot be ruled out and that all these conclusions must await better resolution by other methods. For example, fibers distributing to an area of the PBu containing neurons which project to more than one region of the cerebellum may contact cells projecting to only one of the several areas. An additional complication is the degree of divergence which may exist. I t is certainly possible that many PBu neurons have axons which branch and distribute to widely separate areas of the cerebellum. If such is the case, afferent discharge through any one system may activate multiple zones of the cerebellar cortex. The reader is referred to Armstrong et al. (‘74) for a discussion of this topic as it relates to the inferior olivary nucleus. As mentioned previously, the PBu often is not identified as a separate nucleus, but included within the basilar pons. There are several reasons for this categorization, but perhaps the most significant is that the PBu is considered simply a remnant of the migration which forms the pontine protuberance (see discussions by Olszewski and Baxter, ’54 and Berman, ’68). We would present several arguments, however, which favor considering the PBu as a separate precerebellar nucleus. First, examination of pouch young material from our collection shows that neurons of the PBu separate from the pontobulbar migration and begin to increase their cytoplasmichuclear ratio at about the same time as those cells which continue to the base of the pons. When the PBu can first be identified in Nissl preparations of pouch young opossums, the basilar pons is also just beginning to appear. This simultaneity suggests that the PBu is “predestined” for its definitive position, and does not simply assume it because there is no other place left for it. The reader is referred to
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Pierce (‘66) for a description of the PBu and pontine histogenesis in the mouse. Second, the PBu receives afferent connections somewhat different than those classically considered to distribute to the basilar pons. Third, the PBu seems to have its own diversified and extensive complement of projections to the cerebellar cortex. If the PBu is simply a “misplaced” part of the dorsolateral pons, i t would seem reasonable that it would project to the same areah) of the cerebellum reached by fibers from the most closely adjacent part of the pons. Such is not the case (unpublished results). Separately none of the above arguments are definitive and, in fact, some might be considered circular. Taken together, however, and compared with those which might be offered for considering either the reticuloteg mental or lateral reticular nuclei as autonomous, we find them attractive and sensible. Certainly, the fact that the PBu is derived from the same migration as the basilar pons is no argument against considering it as a separate nuclear group. Several questions remain to be answered. Obviously the comparable cell group needs to be identified in other species on the basis of connections, not topographic position. In addition, the mode of termination within the cerebellar cortex (mossy or climbing fibers) needs to be elucidated as well as whether there are projections to the cerebellar nuclei. Even without such information, however, we conclude that the PBu can be considered a separate precerebellar nucleus and that its circuitry is sufficiently distinctive to warrant its inclusion in models of cerebellar function. ACKNOWLEDGMENTS
The authors wish to thank Mrs. Nann Patterson for technical help, Ma. Malinda Amspaugh for typing the manuscript and Mr. Gabriel Palkuti for photographic help. LITERATURE CITED Armstrong, D. M., R. J. Harvey and R. F. Schild 1974 Topographical localization in the olivo-cerebellar projection: An electrophysiological study in the cat. J. Comp. Neur., 164: 287-302. Berman, A. L. 1968 The Brain Stem of the Cat. A Cytoarchitectonic Atlas with Steraotaxic Coordinates. The University of Wisconsin Press, Madison, Wisconsin. Essick, C. R. 1907 The corpus ponto-bulbare - A hitherto undescribed nuclear mass in the human hindbrain. J. Comp. Neur., 7: 119-137. 1912 The development of the nuclei pontis and the nucleus arcuatus in man. J. Comp. Neur., 13: 26-64.
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Fink, R.P..and L Heimer 1967 Twomethod. for selective silver impregnation ofdemerating axom and their synaptic endings in the central nervous system. Brain Ren., 4: 369-374. Graham, R. C.. and M. J. K a r m k y lQ66 Glomerular permeability. Ultrastructural cytochemical studies using pemxidalles as protein tracers. J. Exp. Med., 184: 11231134. Harkman, W. l9Me Cell migrations from the rhombic lip to the inferior olive, the nucleua raphe and the pons. A morphological and experimental investigation on chick em-. J. Comp. Neur., 1W: 116-210. 1954b The rlunnbic lip and its derivations in relation to the theory of neurobiotaxia In: Ampecta ofCerebellar Anatomy. J. J a ~ e nand A. Brodal, edn. Johan Grudt. Tanum-Forlag, Oslo, pp. 264-384. Hazlett, J., 0.F.Martin and R. Dom 1971 Spino-cerebellar fibers of the opowum, Didelphio manup& uiginiMcl Brain Res.. 33: 257-271. Hazlett, J., R. Dom and 0. F. Martin 1972 Spino-bulbar, wino-thalamic and medial lemniacal conneetiom of the American opaasum mlphb mamupi& viginiana). J. a m p . Neur., 146: 96-118. Hendrickson. A. 1976 Tracing neumnal connections with radioisotopee applied extracellularly. Federation Proceedings. 34: 1612-1616. Koelle. G. B. 1960 The histoehemid differentiation of typee of cholineutexasea and their location in tissues of the cat. J. Pharmaeol., 100: 168-179. Lewis,P. R. 1961 The dect of varying the conditions in the Koelle technique. Bibl. Anat. (Bad), 2: 11-20. Marburg. 0. 1946 Nucleus eminentine teretis, cmpus puntobulbare and their fiber connections. J. Neumpath. and Exp Neur.. 4: 196-216. Martin, G. F., J. C. Bresnehan, C. K. Heukel and D. in the North AmeriMegirian 1976 Corticobulbarfih can opaasum (LJidelphb marsupicrlk uiginiculal with notes on the Tasmanian kush-tailed opoauum t l k i c h urua uulpecule)and other mamupinla. J. Anat. Uandon), 120: 433-438. Martin, G. F..R. Dom,8. Rats and J. 8. King 1974 The orpnisation of projection neurons in the opossum red nucleus. Brain Rw., 78: 17-34.
Mehler, W. R. 1967 Double dpseending pthwnya m&inating from the superior cerebellar peduncle. An exampla of neural species diffemncea. Anat. Rec., 167: 374.
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pomziori Ann. N. Y. Aead &i 16Z ., 424-468. Miller,R. A,and N. L-S 1973 Efferent amnections of the red nucleus in the brainstam and spinal cord of the rheaus monkey. J. Comp. Neur., 162: 327.348. Yisuno, N., M. Kaori, A. Chihim, M. Ryotam and Y. Naknmura 1873 Rubrobulbnr projection6 in the rabbit. A light and electron mimmcopic study. J. Comp. Neur., 147: 267-260. Nauta, W.J. H.,and P. A. Gygax 1964 Silver impregnation of degenerating amrm in the central nervous system: a modified technic. Stain Teeh., 89: 91-93. O l d i , J., and D. Baxtar 1964 Cgtoarehitecture of the human brain stem. 8. Karp, New York, Bnwl. Os~aldo-cru~, E.,and C. E. Roeha-Miranda 1The brain of the opowum UMelphio mcvsypdalial. Imtituto de Bio6siciea.Unimmidade, Federal do Ri de Janeiro, Rio de Janeiro, 99 pp. Reme, E. T. lseS Histogenenis of the nuclei griwum pontis, corpus pontobulbaris and reticularia tegmenti pontio (Becht8rew) in the mouse. J. Camp. New., 126: 21s240. S h m b e r g , C., and L. Lisa 1969 Techniques for silver carbonate impregnation of uervou tissue. In: Neuroee-
todennalTumorsofCentralandP~pherdN~System. Williams and Wilkem, Baltimore, pp 218228. Snider, R. S., and A. Stows11 1944 Receiving arena of the tactile, auditmy andvisual systems in the cerebellum. J. Neunphyniol., 7: 931-367. Swank, R. L. 1934 The relatimuhip between the circumolivarypyramidal fascicles and the pontobulbar body in man. J. Camp. Neur., 60: 308317. Taber, E. 1961 The cytoarehiteeture of the brain stem of the cat. I. Brain stem nuclei of the cat. J. Comp. Neur.,
116: 27-70.
Voris, € C.I ,. and N. L. Hoerr 1932 The hindbrain of the opoauum, Didelphir viginiMcl J. Camp. New., 64: 277366.
PLATES
17 High power photomicrograph of the axonal degeneration in the dorsomedial part of the pontobulbar nucleus produced by a C-2 spinal lesion. This photomicrograph was taken from the section shown in figure 16 and the arrow points to the blood vessel indicated in figure 16. The magnification is the same as that in figure 14.
cent motor trigeminal rootlet is labelled for reference. The small arrow within the nucleus points to the blood vessel similarly indicated in figure 17.The bar indicator is 300 pm.
16 Low power photomicrograph through the pontobulbar nucleus on the side of a C-2 spinal lesion. An adja-
elicited by splitting the cerebellum. The small arrows outline the ventral limits of the degeneration, whereas the larger arrow points to a medial extension of the nucleus comparable to that indicated in figures 5 and 6.The insert is a photomicrograph of a section through the pontobulbar nucleus which is impregnated for normal axons. Most of them ramify in dorsomedial portions of the nucleus and relatively few penetrate its ventrolateral extreme (arrow). A motor trigeminal rootlet is labelled (rVn) for reference. The magnification for figure 15 is the same as that in figure 14 and that for the insert is comparable to that in figure 16.
15 High power photomicrograph of the sparse degeneration in the dorsal part of the pontobulbar nucleus
14 High power photomicrograph of axonal degeneration within the lateral part of the pontobulbar nucleus subsequent to removal of all neocortex on the same side. The bar is 44 pm.
EXPLANATION OF FIGURES
PLATE 1
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NUCLEUS OF THE PONTOBULBAR BODY C. F. Martin, M. Linauta and J. M. Walker
PLATE 1
(RN),
22 Darkfield photomicrograph showing terminal label which is restricted to the dorsomedial part of the pontobulbar nucleus (arrow) on the side opposite the placement shown in figure 19. This section is caudal to that illustrated in figures 20 and 21. Motor trigeminal (rVn) and spinal trigeminal (trs) fascicles are labelled for reference and the magnification is the same as that for figure 20.
and the magnification is the same. Motor trigeminal (rVn) and spinal trigeminal (trs) fascicles are indicated.
21 Darkfield photomicrograph showing terminal label in the dorsomedial part of the pontobulbar nucleus (arrow) contralateral to the injection shown in figure 19. This section is caudal to that shown in figure 20
which enter the pontobulbar nucleus by coursing along dendritic bundles comparable to those shown in figure 5. Motor trigeminal kVn) and spinal trigeminal (trs) fascicles are indicated. The bar equals 300 pm.
20 Darkfield photomicrograph showing terminal label (dark arrow) in the dorsal part of the pntobulbar nucleus contralateral to the injection shown in figure 19. The white, open arrow points to labelled axons
the neurons in the interstitial area dorsomedial to it and the cells of the ventral periaqueductal grey. The survival time was ten days and the exposure time four weeks. Results from this case are illustrated in figures 20 to 22. The cerebral aqueduct (aq), the interpeduncular nucleus (IP)and the cerebral peduncle (ped) are labelled. The magnification is the same as for figure 18.
19 Autoradiogram showing a placement (arrow) which labels the dorsomedial cells of the red nucleus
and the exposure time was four weeks. The oculomotor nerve (nIII) is indicated.
18 Autoradiogram showing the placement site of a brain in which the red nucleus (RN)was injected with SHleucine. The bar equals 2 mm. The insert shows an injection site from another brain which labelled the medial part of the red nucleus and adjacent areas bilaterally. In both cases the survival time was ten days
EXPLANATION OF FIGURES
PLATE 2
NUCLEUS OF THE PONTOBULBAR BODY G. F. Martin, M. Linauts and J. M. Walker
PLATE 2
26 Photomicrograph through the horseradish peroxidase injection of the pyramis and uvula in P-493 (fig. 10, bottom). The arrow points to the needle tract and the uvula is labelled for reference (Uv). The bar equals 2 mm.
25 Photomicrograph through the horseradish peroxidase injection site in P-459 (fig. 10, top). The arrow points to the needle tract in the paramedian lobule (PM). The bar equals 2 mm.
24 Photomicrograph through the horseradish peroxidase injection site of P-487 (fig. 9, right side). The arrow points to the needle tract and several areas of the cerebellum are labelled for reference. The bar equals 2.5 mm.
23 Photomicrograph of a section through the horseradish peroxidase injection site of P-485 (fig. 9, left side). The arrow points to the needle tract and several areas of the cerebellum are labelled. The bar equals 2.5 mm.
EXPLANATION OF FIGURES
PLATE 3
NUCLEUS OF THE PONTOBULBAR BODY G . F. Martin, M. Linauta and J. M. Walker PLATE 3
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30 Photomicrograph of the horseradish peroxidase injection site (arrow) of P-504 (fig. 13,bottom). The lingula (Lg) and crus I1 (CrII) are labelled and the bar equals 1.4 mm.
29 Photomicrograph of the horseradish peroxidase injection site (arrow) of P-476 (fig. 12, bottom). Various portions of the cerebellum are labelled for reference. The bar equals 2.5 mm.
28 Photomicrograph of a sagittal section through the horseradish peroxidase injection site from P-498 (fig. 11, bottom). The entrance point of the needle is indicated by the arrow and various divisions of the vermis are labelled. The bar equals 4.5 mm.
27 Photomicrograph through the horseradish peroxidase injection site of P-497 (fig. 11,top). The arrow points to the injection site and several regions of the cerebellum are labelled for reference. The bar indicates 2.5 mm.
EXPLANATION OF FIGURES
PLATE 4
NUCLEUS OF THE PONTOBULBAR BODY I2 P Martin M. I.inanta and .I M Walkrr PLATE 4
0
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PLATE 5
33 Darkfield photomicrograph of a caudal section through the pontobulbar nucleus on the side of the placement in P-473(fig.12,top). One of the motor trigeminal rootlets is labelled GVn)and the magnification is the same as that for figure 31.
readily Been and a small labelled neuron is indicated by the arrow. The magnification m i the same as that for figure 31.
32 Darkfield photomicrograph of the section shown in figure 31,oriented a little differently. Neurons within the pontobulbar nucleus which contain reaction product after the injection shown in figure 12 (top) are
31 Light field photomicrograph through the pontobulbar nucleus on the aide of the horaeradish peroxidase placement of P-473 (fig. 12, top). A motor fascicle of the trigeminal nerve is labelled for reference (rVn) and a small neuron which contains reaction product is indicated (arrow). The reaction product can be seen with darkfield optice (fig. 32,arrow). The small bar equals 42 pm.
EXPLANATION OF FIGURES
S3Nfl3IB A 0 NOlLVNV’ldX3
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NUCLEUS OF THE PONTOBULBAR BODY G. F. Martin, M. Linauta and J. M. Walker PLATE 5
ABSTRACT The nucleus of the pontobulbar body (PBu) in the North American opossum is located, for the most part, adjacent to the motor root of the trigeminal nerve. Material prepared by degeneration and autoradiographic methods shows that the PBu receives projections from the facial motor-sensory cortex, red nucleus, spinal cord and cerebellum. The latter fibers probably take origin within the fastigial nucleus. Each of the afferent connections ends in a restricted part of the PBu, but there is considerable overlap. Use of the horseradish peroxidase technique reveals that the PBu projects to the spinal cerebellum (anterior lobe, pyramis and paramedian lobules), to visual-auditory areas of the vermis and to the lobus simplex as well as to crus I and I1 of the hemispheres. Although there is some topography to such projections, it is not sharply defined and many regions of the PBu contain labelled neurons after injections of horseradish peroxidase into widely separate areas of the cerebellar cortex. Because of its embryogenesis and position, the PBu is often considered part of the dorsolateral basilar pons. I t appears from our material, however, that the organization of PBu afferent and efferent connections is different from that of the adjacent basilar pons, and arguments for considering the PBu a separate precerebellar nucleus are presented. The corporis pontobulbaris was described by Essick (‘07) as a fibroganglionic mass in the human brain which extends from a position a t the lateral boundary of the fourth ventricle to the basilar pons. In a subsequent study, Essick (‘12)showed that the human basilar pons is formed by cells which course rostrally and ventrally from the germinal epithelium of the rhombic lip and that the corporis pontobulbaris in the adult is a remnant of that migration. Olszewski and Baxter (‘54) referred to the neurons contained within the human corporis pontobulbaris as the nucleus corporis pontobulbaris (PBu in this account) and that term has been used for apparently comparable cell groups in several species (e.g., the cat; Taber, ’611,including the American opossum (Oswaldo-Cruz and Rocha-Miranda, ‘68). Although information is available on the histogenesis of the PBu (Harkman, ’54a,b; Pierce, ,661, little is known about its connections. In normal human material both Swank (‘34) and Marburg (‘45) followed axons from J. COMP. NEUR., 175: 345-372.
the pyramidal tract which looped around the inferior olive and apparently ended within the PBu. Marburg (‘45) also suggested that the PBu projected to the cerebellum. The purposes of the present report are to present data concerning both afferent and efferent connections of the PBu in the North American opossum in order to extend observations made earlier (Martin e t al., ‘74, ’75) and to address the question of whether or not the PBu should be considered a separate precerebellar nucleus. MATERIALS AND METHODS
Observations on the organization and cytology of the PBu were made from adult opossum brains cut in different planes of section and stained for Nissl substance as well as from brains processed for either normal axons (silver carbonate technique of del Rio Hor!This investigation was supported by the United States Public Health Service, Grants NS-07410,NS-08798 and by funds from the Bremer Foundation, The Ohio State University. College of Medicine. * J. M. Walker is a graduate student in the Department of Psychology, The Ohio State University.
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tega, see Sharenberg and Liss, '69).or cholinesterase activity (Lewis, '61;modification of the Koelle technique, '50). Material impregnated by one of several Golgi techniques was made available from the laboratory of Doctor James S. King. Some of our results were taken from the brains of over 300 opossums which have been impregnated for degenerating axons following specific lesions. Survival times varied from two to fourteen days and several modifications of the Nauta-Gygax ('54) and Fink-Heimer ('67)methods were employed. In this study terminal degeneration refers to axonal debris which is either finely granular and/or randomly arranged in the PBu neuropil. Autoradiographic observations were made from brains in which SH-leucinewas injected into either the cerebral cortex, the brainstem or the cerebellum. The marker was deposited by means of a Hamilton syringe and #31 gauge needle attached to a stereotaxically guided microdrive. Quantities from 0.1-0.3plI injection were introduced into each animal (concentrated 50-100 pCi/pl) over periods from one-half to one hour and the needle was kept in the injection site for a t least ten minutes prior to and after the injection. After survival times of one to ten days the animals were anesthetized and sacrificed by intracardiac perfusion. Frozen sections were mounted and processed by standard autoradiographic procedures using a one month exposure time (Hendrickson, '75)and sections through the PBu were examined by light and dark field optics for concentrations of silver grains above background. Clumps of grains over axonal bundles were considered to indicate labelled axons in passage, whereas random labelling over the PBu neuropil was interpreted as terminal. The horseradish peroxidase (HRP) method was utilized to localize the PBu neurons which project to different cortical areas of the cerebellum. The enzyme was delivered to different parts of the cerebellar cortex as dep l of a 50% solution) scribed above (0.1-0.6 over a period varying from 45 minutes to over 1 hour. After approximately 24 hours the animals were anesthetized and perfused with a 1.0% paraformaldehyde, 1.0% glutaraldehyde solution in a 0.1 M phosphate buffer (pH = 7.4)containing 30% sucrose. Frozen sections were incubated according to the method of Graham and Karnovsky ('661, stained for
Nissl substance and examined for spread of the marker a t the injection site as well as for the presence of neurons containing yellowbrown granules of reaction product. RESULTS
Conformation and cytoarchitecture of the nuckus corporis pontobulbaris (PBu) As described by Oswaldo-Cruz and RochaMiranda ('681, the rostra1 extreme of the PBu in the opossum is squeezed between the latAbbreviations
aq, cerebral aqueduct CcD, dorsal cochlear nucleus Cn, culmen contra, side contralateral to the injection Crll, crus I1 ct, trapezoid body De, declive F1, flocculus Fol, folium IC, inferior colliculus IP, interpeduncular nucleus ipsi, side ipsilateral to the injection Lat, lateral Lg, lingula nlll, oculomotor nerve Nd, nodule ped, cerebral peduncle PFL, paraflocculus PM, paramedian lobule PrC, preculmen Pyc, pyramis RN, red nucleus rVm, major (sensory) root of trigeminal nerve rVn, motor root of trigeminal nerve Sx, lobus simplex trs, spinal trigeminal tract Uv, uvula Fig. 1 Dorsal view of the opossum brainstem with most of the cerebellum removed. The open block arrow points to the location of the stratum gliosum a t the lateral edge of the dorsal cochlear nucleus (CcD), the apparent origin of the pontobulbar migration. Magnification X 3.0. Fig. 2 Ventral view of the opossum brainstem. The open block arrow points to the position of the pontobulbar nucleus medial to the trigeminal nerve (rVm). Mag nification X 3.0. Fig. 3 Transversely cut, Nissl stained section through the largest part of the pontobulbar nucleus (arrow). The lateral aspect of the brainstem is on the reader's left and the ventral surface of the section is seen at the bottom of the figure. Both the motor rootlets of the trigeminal nerve (rVn) and the spinal trigeminal tract are labelled (trs). The bar indicates 440 pm. Fig. 4 Transversely cut, Nissl stained section through the most caudal part of the pontobulbar nucleus (arrow). The lateral part of the brainstem is on the reader's left and the ventral surface of the section is covered by the figure number. The motor roots of the trigeminal nerve and the spinal trigeminal tract are labelled and the insert shows the dendritic plexus of PBu neurons at this level (arrow). A motor fascicle of the trigeminal nerve is indicated (rVn). The magnification is the same as for figure 3.
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technique reveals that the PBu receives a major contribution from the cerebral comz Degeneration is obtained within the PBu after lesions which are limited to the facial motor-sensory cortex Martin et al., ’751, but we have chosen to illustrate this connection by using a case subjected to unilateral decortication. Our intent is that the reader see the total cortical target of the PBu (fig. 7, left side). In rostral sections a few degenerating axons can be seen ipsilaterally in that part of the PBu located between the brachium pontis and the lateral surface of the pons. More caudally, however, they become more extensive (fig. 141, particularly laterally, and most evident around the emerging trigeminal fascicles (figs. 7A,B, left side). Fragmented axons can be traced into the nucleus from several directions, and some of them follow the dendritic bundles referred to previously (fig. 7B, left side). Axonal debris is located within comparable portions of the caudal PBu, as well as around and between motor fascicles of the trigeminal nerve Cfig. 7C). Although not as numerous, degenerating axons are present within the contralateral PBu. Evidence for a cortical-PBu projection is also available in the brains in which sH-leucine has been injected into facial motor-sensory cortex. After rubml lesions or lesions which undercut rubral axons, degeneration can be traced out of the crossed rubrobulbospinal bundles into the PBu (fig. 7, right side). Again we illustrate this connection from a case with a large lesion so that the “total” rubral domain can be appreciated. Few degenerating fibers enter rostral portions of the nucleus Cfig. 7A, right side), but distribute to its doraal and medial sectors more caudally (figs. 7B,C right side), as well as along the dendritic bundles in the reticular formation (figs. 7B,C, right side). As is the case after cortical lesione, degenerating axons also distribute between the motor trigeminal fascicles (fig. 7C, right side). Rubral lesions, large or small, necessarily interrupt axons from extra-rubral sources, making it difficult to determine the precise origin of degeneration within the PBu. In addition, degeneration methoda do not always reveal the complete distribution of axons even after careful attention to survival times. Because of these problems the autoradiographic method was employed on brains in which 91Afferentconnections of the nucleus corporis leucine was injected so as to label the red nupontobulbwis cleus and/or surrounding cells (e.g., figs. 18, Material processed by the Fink-Heimer 19). In all cases in which either the red nu-
era1 aspect of the pons and the fibers of the brachium pontis. At more caudal levels, however, it is more distinct and located adjacent to the motor root of the trigeminal nerve (figs. 2-4). In material processed for cholinesterase activity (fig. 6) the PBu stands out in relief from surrounding areas because of the intensity of the reaction product contained within it. Although the pontobulbar body is probably continuous with the stratum gliosum of Oswaldo-Cruz and Rocha-Miranda C68) (see also Voris and Hoerr, ’321, only a few maturelooking neurons are located at that level. The reader is referred to figure 1 where the gross position of the stratum gliosum is indicated. The largest part of the PBu appears on the ventral surface of the lateral pons just medial to the exiting fascicles of the minor (motor) root. of the trigeminal nerve (fig. 3). At that level it is generally wedge-shaped in transverse sections. The rounded, deeply staining neurons located dorsally and medially within the PBu measure from 11-22 p (figs. 3,311 and have dendrites which overlap with one anot.her. The dendrites of some of them extend out.of the nucleus, and a t some levels they can be followed into the reticular formation where they form bundles which parallel the course of penetrating blood vessels (fig. 5). Fusiformshaped neurons are also seen in such bundles and their dendrites contribute to them. In contrast, the neurons located ventrolaterally within the PBu tend to be small (10-16p ) and have relatively little cytoplasm (sgs. 3, 31). Their dendrites are so heavily impregnated in the available material that it is difficult to viwalize their geometry. In sections impregnated for normal axons a fairly rich afferent plexuR can be seen to pour into the dorsal medial part of the PBu, whereas impregnated axons are relatively sparse among the smaller neurons located ventrally and laterally (insert of fig. 15). At its caudal end the PBu becomes somewhat rectangular in transverse section and intimately related to the medial aspect of the motor trigeminal fascicles (fig. 4). Dendrites of the latter neurons are closely entangled and tend to be oriented from dorsal to ventral, although some of them extend laterally between the motor bundles of the trigeminal nerve (fig. 4, insert).
spinal cord WBS partially transected a t C-2 (P-320, insert on right side). Degenerating axons are drawn in each section as they appear in Fink-Heimer impregnated material.
(arrows) from a case subjected to a cerebellar split (arrow, P-208, insert on left side) and from another brain in which the
Fig. 8 A series of transverse sections through three levels (rostra1 A through caudal, C) of the pontobulbar nucleus
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cleus and/or the neurons dormmedial to it are well labelled (figs. 18, 191, the isotope was transported to precisely those regions of the PBu which contain degeneration after damage to the red nucleus or its axons (figs. 2022). The PBu is unlabelled or only equivocally labelled after injections of the superior colliculus and/or the interstitial tegmentum and after injections of more ventromedial nonrubral areas. Although we have numerous cases in which either 3H-leucineor proline has been injected into the pontine and medullary reticular formation, none of them shows definite evidence for transport to the PBu. The PBu also receives a small input from the cerebellum. Subsequent to a midline cut through the cerebellum which undercuts fastigial outflow (insert of fig. 8, left side), degenerating axons can be traced bilaterally around the brachium conjunctivum to a position between the brachium pontis and the lateral surface of the pons. Such fibers, plus others which take different routes, distribute to rostra1 parts of the PBu as well as to that part of the nucleus associated with the trigeminal nerve (fig. 8, left side). Although most of the injured axons lie medial and dorsal t o the PBu proper, some enter its dorsal extreme, particularly, caudally where they ramify in a terminal fashion (figs. 8A-C, left side and fig. 15’1.Although degenerating axons can be seen just dorsal to the PBu after lesions limited to the nucleus fastigius, only a very few actually enter the nucleus. We have cases in which 3H-leucinehas been deposited into the fastigial nucleus, but they contain little evidence for a PBu projection. No evidence for a projection to the PBu is preeent in cases with lesions of the interpositus and/or dentate nuclei or in brains in which sH-leucine was deposited within them. One of the major inputs to the PBu arises within the spinal cord. After a lesion which partially hemisects the cord a t high cervical level (insert, fig. 8, right side), degeneration is present bilaterally within the ventrolateral tracts which ascend within the brainstem (Hazlett et al., ’72). Degenerating bundles course dorsally from these tracts on their way to both the cerebellum (Hazlett et al., ’71) and the external inferior colliculus (Hazlett et al., ’72) and some of them pass through the matral PBu. At mid-rostra1 to caudal levels of the PBu there is a definite dorsomedialzone of terminal debris 6g. 8A, right side and %a. 16, 17) which gradually diminishes at more cau-
dal levels (6gs. 8B,C, right side). Degenerating axons also distribute between the motor fibersof the trigeminal nerve (figs. 8B,C, right side). A comparable distribution of degeneration is present in a case subjected to a large medullary lesion which undercut all ascending spinal fascicles. Degenerating axons are present within the PBu after spinal lesions a t either C-4 or midthoracic levels, but few can be discerned subsequent to lower lumbar or sacral transections.
A.siectione of the pontobulbar nucleua to the cerebellar cortex Neurons of the PBu contain reaction product after horseradish peroxidase injections of many areas of the cerebellar cortex. Subsequent to the medial anterior lobe placement shown in figure 23 neuronal labelling is present in medial and dorsal parts of the ipsilateral PBu at mid-rostra1 to caudal levels (figs. 9A-C, left side). Generally comparable results were obtained from the more lateral placement shown in figure 24 (figs. 9A-C, right side), although PBu neurons contained reaction product on both sides. As can be seen in figure 9, right side, some of the marked neurons are located in the medially oriented extensions of the PBu referred to previously. Only a very few of the smaller, more ventrolaterally situated neurons contain reaction product. In still another brain the enzyme was deposited a t a point intermediate between the injection sites illustrated, and similar results were obtained bilaterally. Injection of HRP into other “spinal areas” of the cerebellum (Hazlett et al., ’71) also results in labelling of PBu neurons. In the three brains available with injections of the paramedian lobule, PBu neurons contain reaction product on both sides, and most of these cells are aggregated within dorsal and medial parts of the nucleus. The placement site from one case is shown in figure 26 and the location of reactive neurons in the PBu is illustrated in figure 10 (top). In caudal sections (not shown in 10) backfilled neurons are located just medial to the PBu and apparently out of its confines. Although one of our injections of the medial pyramis failed to label PBu neurons, the one shown in figure 26 provided positive results. As can be seen in figure 10 (bottom) most of the reactive neurons are medially and dorsally situated. The available material provides evidence for a projection from the PBu to visual-audi-
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Fig. 10 Drawings of sections (rostral,A, caudr;l, Bj through the pontobulbar nucleus (arrows) on both sides of brains subjected to horseradish peroxidase injections of the paramedian lobule (arrow, P-459,insert on top) and pyramis (arrow, P-493,insert on bottom). Labelled neurons within the pontobulbar nucleus are drawn in each section.
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Fig. 11 In the upper part of the illustration sections are drawn from three levels kostral, A, to caudal, C) of the pontobulbar nucleus on both sides (arrows) from a brain subjected to a folium placement of horseradish peroxidase (arrow, P-497,insert on top). In the lower frame two sections (rostral, A, caudal, B) are drawn through the pontobulbar nucleus of both sides (arrows) from a brain in which horseradish peroxidase was deposited into the tuber of the cerebellar vermin (arrow, P-498,insert on bottom). In both cases labelled neurons within the pontobulbar are illustrated. Cells containing large amounts of reaction product are illustrated in solid black, whereas those which are lightly labelled are only outlined.
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Fig. 12 In the upper part of the illustration sections are drawn from two levels (rostral, A, caudal, B)of the pontobulbar nucleus (arrows) on both sides of a brain in which horseradish peroxidase was injected into crus I of the cerebellar hemisphere. As shown in the insert (P-473, arrow) the marker spread both rostrally and caudally from the injection site. In the lower part of the illustration two sections (rostral, A, caudal, B) are drawn through the pontobulbar nuclei (arrows) of both sides from a brain subjected to a placement of horseradish peroxidase into the lobus simplex (P-476, arrow, insert). In both cases reactive neurons are drawn where they appear in the sections.
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Fig. 13 At the top of the illustration two sections are drawn through the pontobulbar nucleus (arrows), bilaterally, from a case subjected to a placement of horseradish peroxidase into CNS I of the cerebellum with only minimal spread (arrow, P-508, insert). In the lower part of the illustration two sections are drawn through the pontobulbar nuclei of both sides (arrows) from a case in which horseradish peroxidase waa placed within crus I1 of the cerebellum (arrow, P-504, insert). In both cases reactive neurons are drawn where they appear in the sections.
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G. F. MARTIN. M. LINAUTS AND J. M. WALKER
siderable overlap of their territory as well as overlap in the dendritic domain of PBu neurons. Swank (‘34) suggested that the human PBu receives cortical input, but to our knowledge such a connection has not been verified by experimental techniques. Crossed rubral fibers have been traced to cell groups around the trigeminal nerve such as “Regio h,” “Regio M” and “Zellgruppe K ’ of Meesen and Olszewski (‘49) in the rabbit (Mizuno et al., ’73) and the lateral juxtatrigeminal nucleus of Riley in the monkey (Miller and Strom inger, ’73). Unfortunately, however, i t is not clear which, if any, of these areas correspond to the PBu of this account (Oswaldo-Cruz and Rocha-Miranda, ’68). Although fastigial axons have not been followed to an area identified as the PBu in any species, it is interesting that the uncrossed descending brachium conjunctivum of the rat distributes to an area around the emerging motor root of the trigeminal nerve (Mehler, ’67, ’69). Spinal fibers have not been described as projecting to the PBu either, but are reported (Mehler, ’69) to distribute within a n area near the trigeminal nerve in several species, including the opossum. Mehler identified the target of such fibers as the processus tegmentosus lateralis of the pontine grey after Olszewski and Baxter (‘54). I t can be seen from the above account that it is difficult to compare our results with those obtained from other species because the PBu is not identified as such in any of them. It appears, however, that many of the regions referred to in other species correspond to some part of the PBu as defined herein. Our material reveals that the PBu projects to the spinal cerebellum (i.e., the anterior lobe, the paramedian lobules and the pyramis, Hazlett et al., ’711, the visual-auditory region of the cerebellar vermis (the declive, the folium and the tuber, Snider and Stowell, ’44) as well as to several parts of the hemispheres (the lobus simplex, crus I and 11). No PBu neurons were labelled subsequent to HRP injections of the paraflocculus or uvula, and to date we have not successfully injected either the flocculus or the nodule. If the location of laDISCUSSION belled neurons is compared in the various The pontobulbar nucleus (PBu) of the cases, a rough topography appears to exist. It opossum receives afferent connections from is clear, however, that neurons in some areas the cerebral cortex, red nucleus, cerebellum project to widely separate parts of the cere(probably fastigial nucleus) and spinal cord. bellum. Although each of the afferent connections end The identification of afferent connections in a restricted part of the PBu, there is con- for any cell group becomes more meaningful
tory areas of the cerebellar vermis (Snider and Stowell, ’44). Separate HRP injections were made in the declive, the folium and the tuber, and the PBu contains reactive neurons in each case. The injection of the folium in P497 (fig. 27) produced labelling of a particularly large number of neurons, and the results are illustrated in figure 11 (top). It should be noted, however, that the needle penetrated into a subjacent folium, and very light contamination of that part of the anterior lobe just rostra1 to the primary fissure cannot be ruled out. As in the paramedian case described above, heavily reactive neurons are located just outside of the nucleus (open block arrow, right side of figure 11C, top). The tuber placement shown in figure 28 produced positive results in the caudal part of the nucleus, bilaterally (fig. 11, bottom). There is considerable evidence that the PBu projects to more lateral zones of the cerebellar hemisphere. The HRP injection in P-473 (fig. 12, top) was made in crus I, but the marker spread to the lobus simplex, crus I1 and the paramedian lobule. Although this case is of no use for localizing the precise targets of PBu neurons, i t does provide data relative to the extent of the PBu which projects to the cerebellar hemisphere. A large number of PBu neurons contain reaction product bilaterally, and the larger neurons are particularly well labelled (figs. 31-33). In contrast to the cases with injections of spinal or visual-auditory areas of the cerebellum, however, many of the small, ventrolaterally positioned neurons also contained reaction product (P-473, fig. 12A, ipsilateral side and arrows, figs. 31, 32). PBu neurons were labelled, mainly ipsilaterally, after the lobus simplex injection shown in figure 29 (fig. 12, bottom) and bilaterally after injections of crus I and 11. The results from a large crus I injection are plotted in figure 13 (top) and those obtained from a smaller crus I1 injection (fig. 30) are illustrated in the bottom of figure 13. In cases where the injection was limited to either the paraflocculus or the uvula there was no evidence of enzyme incorporation by PBu neurons.
NUCLEUS OF THE PONTOBULBAR BODY
when interpreted in light of its efferent projections. By comparing the distribution of the fiber systems which penetrate the PBu with the location of labelled neurons in the HRP experiments, at least two tentative conclusions can be drawn. First, it appears that fibers from the red nucleus, the fastigial nucleus and the spinal cord seek out neurons of the PBu which project to the spinal cerebellum, to visual-auditory regions of the vermis and to parts of the hemisphere. Secondly, cortical fibers appear to concentrate on those neurons which project heavily to the hemispheres. I t should be emphasized that cortical contacts with neurons projecting to nonhemisphere areas cannot be ruled out and that all these conclusions must await better resolution by other methods. For example, fibers distributing to an area of the PBu containing neurons which project to more than one region of the cerebellum may contact cells projecting to only one of the several areas. An additional complication is the degree of divergence which may exist. I t is certainly possible that many PBu neurons have axons which branch and distribute to widely separate areas of the cerebellum. If such is the case, afferent discharge through any one system may activate multiple zones of the cerebellar cortex. The reader is referred to Armstrong et al. (‘74) for a discussion of this topic as it relates to the inferior olivary nucleus. As mentioned previously, the PBu often is not identified as a separate nucleus, but included within the basilar pons. There are several reasons for this categorization, but perhaps the most significant is that the PBu is considered simply a remnant of the migration which forms the pontine protuberance (see discussions by Olszewski and Baxter, ’54 and Berman, ’68). We would present several arguments, however, which favor considering the PBu as a separate precerebellar nucleus. First, examination of pouch young material from our collection shows that neurons of the PBu separate from the pontobulbar migration and begin to increase their cytoplasmichuclear ratio at about the same time as those cells which continue to the base of the pons. When the PBu can first be identified in Nissl preparations of pouch young opossums, the basilar pons is also just beginning to appear. This simultaneity suggests that the PBu is “predestined” for its definitive position, and does not simply assume it because there is no other place left for it. The reader is referred to
359
Pierce (‘66) for a description of the PBu and pontine histogenesis in the mouse. Second, the PBu receives afferent connections somewhat different than those classically considered to distribute to the basilar pons. Third, the PBu seems to have its own diversified and extensive complement of projections to the cerebellar cortex. If the PBu is simply a “misplaced” part of the dorsolateral pons, i t would seem reasonable that it would project to the same areah) of the cerebellum reached by fibers from the most closely adjacent part of the pons. Such is not the case (unpublished results). Separately none of the above arguments are definitive and, in fact, some might be considered circular. Taken together, however, and compared with those which might be offered for considering either the reticuloteg mental or lateral reticular nuclei as autonomous, we find them attractive and sensible. Certainly, the fact that the PBu is derived from the same migration as the basilar pons is no argument against considering it as a separate nuclear group. Several questions remain to be answered. Obviously the comparable cell group needs to be identified in other species on the basis of connections, not topographic position. In addition, the mode of termination within the cerebellar cortex (mossy or climbing fibers) needs to be elucidated as well as whether there are projections to the cerebellar nuclei. Even without such information, however, we conclude that the PBu can be considered a separate precerebellar nucleus and that its circuitry is sufficiently distinctive to warrant its inclusion in models of cerebellar function. ACKNOWLEDGMENTS
The authors wish to thank Mrs. Nann Patterson for technical help, Ma. Malinda Amspaugh for typing the manuscript and Mr. Gabriel Palkuti for photographic help. LITERATURE CITED Armstrong, D. M., R. J. Harvey and R. F. Schild 1974 Topographical localization in the olivo-cerebellar projection: An electrophysiological study in the cat. J. Comp. Neur., 164: 287-302. Berman, A. L. 1968 The Brain Stem of the Cat. A Cytoarchitectonic Atlas with Steraotaxic Coordinates. The University of Wisconsin Press, Madison, Wisconsin. Essick, C. R. 1907 The corpus ponto-bulbare - A hitherto undescribed nuclear mass in the human hindbrain. J. Comp. Neur., 7: 119-137. 1912 The development of the nuclei pontis and the nucleus arcuatus in man. J. Comp. Neur., 13: 26-64.
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Fink, R.P..and L Heimer 1967 Twomethod. for selective silver impregnation ofdemerating axom and their synaptic endings in the central nervous system. Brain Ren., 4: 369-374. Graham, R. C.. and M. J. K a r m k y lQ66 Glomerular permeability. Ultrastructural cytochemical studies using pemxidalles as protein tracers. J. Exp. Med., 184: 11231134. Harkman, W. l9Me Cell migrations from the rhombic lip to the inferior olive, the nucleua raphe and the pons. A morphological and experimental investigation on chick em-. J. Comp. Neur., 1W: 116-210. 1954b The rlunnbic lip and its derivations in relation to the theory of neurobiotaxia In: Ampecta ofCerebellar Anatomy. J. J a ~ e nand A. Brodal, edn. Johan Grudt. Tanum-Forlag, Oslo, pp. 264-384. Hazlett, J., 0.F.Martin and R. Dom 1971 Spino-cerebellar fibers of the opowum, Didelphio manup& uiginiMcl Brain Res.. 33: 257-271. Hazlett, J., R. Dom and 0. F. Martin 1972 Spino-bulbar, wino-thalamic and medial lemniacal conneetiom of the American opaasum mlphb mamupi& viginiana). J. a m p . Neur., 146: 96-118. Hendrickson. A. 1976 Tracing neumnal connections with radioisotopee applied extracellularly. Federation Proceedings. 34: 1612-1616. Koelle. G. B. 1960 The histoehemid differentiation of typee of cholineutexasea and their location in tissues of the cat. J. Pharmaeol., 100: 168-179. Lewis,P. R. 1961 The dect of varying the conditions in the Koelle technique. Bibl. Anat. (Bad), 2: 11-20. Marburg. 0. 1946 Nucleus eminentine teretis, cmpus puntobulbare and their fiber connections. J. Neumpath. and Exp Neur.. 4: 196-216. Martin, G. F., J. C. Bresnehan, C. K. Heukel and D. in the North AmeriMegirian 1976 Corticobulbarfih can opaasum (LJidelphb marsupicrlk uiginiculal with notes on the Tasmanian kush-tailed opoauum t l k i c h urua uulpecule)and other mamupinla. J. Anat. Uandon), 120: 433-438. Martin, G. F..R. Dom,8. Rats and J. 8. King 1974 The orpnisation of projection neurons in the opossum red nucleus. Brain Rw., 78: 17-34.
Mehler, W. R. 1967 Double dpseending pthwnya m&inating from the superior cerebellar peduncle. An exampla of neural species diffemncea. Anat. Rec., 167: 374.
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pomziori Ann. N. Y. Aead &i 16Z ., 424-468. Miller,R. A,and N. L-S 1973 Efferent amnections of the red nucleus in the brainstam and spinal cord of the rheaus monkey. J. Comp. Neur., 162: 327.348. Yisuno, N., M. Kaori, A. Chihim, M. Ryotam and Y. Naknmura 1873 Rubrobulbnr projection6 in the rabbit. A light and electron mimmcopic study. J. Comp. Neur., 147: 267-260. Nauta, W.J. H.,and P. A. Gygax 1964 Silver impregnation of degenerating amrm in the central nervous system: a modified technic. Stain Teeh., 89: 91-93. O l d i , J., and D. Baxtar 1964 Cgtoarehitecture of the human brain stem. 8. Karp, New York, Bnwl. Os~aldo-cru~, E.,and C. E. Roeha-Miranda 1The brain of the opowum UMelphio mcvsypdalial. Imtituto de Bio6siciea.Unimmidade, Federal do Ri de Janeiro, Rio de Janeiro, 99 pp. Reme, E. T. lseS Histogenenis of the nuclei griwum pontis, corpus pontobulbaris and reticularia tegmenti pontio (Becht8rew) in the mouse. J. Camp. New., 126: 21s240. S h m b e r g , C., and L. Lisa 1969 Techniques for silver carbonate impregnation of uervou tissue. In: Neuroee-
todennalTumorsofCentralandP~pherdN~System. Williams and Wilkem, Baltimore, pp 218228. Snider, R. S., and A. Stows11 1944 Receiving arena of the tactile, auditmy andvisual systems in the cerebellum. J. Neunphyniol., 7: 931-367. Swank, R. L. 1934 The relatimuhip between the circumolivarypyramidal fascicles and the pontobulbar body in man. J. Camp. Neur., 60: 308317. Taber, E. 1961 The cytoarehiteeture of the brain stem of the cat. I. Brain stem nuclei of the cat. J. Comp. Neur.,
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Voris, € C.I ,. and N. L. Hoerr 1932 The hindbrain of the opoauum, Didelphir viginiMcl J. Camp. New., 64: 277366.
PLATES
17 High power photomicrograph of the axonal degeneration in the dorsomedial part of the pontobulbar nucleus produced by a C-2 spinal lesion. This photomicrograph was taken from the section shown in figure 16 and the arrow points to the blood vessel indicated in figure 16. The magnification is the same as that in figure 14.
cent motor trigeminal rootlet is labelled for reference. The small arrow within the nucleus points to the blood vessel similarly indicated in figure 17.The bar indicator is 300 pm.
16 Low power photomicrograph through the pontobulbar nucleus on the side of a C-2 spinal lesion. An adja-
elicited by splitting the cerebellum. The small arrows outline the ventral limits of the degeneration, whereas the larger arrow points to a medial extension of the nucleus comparable to that indicated in figures 5 and 6.The insert is a photomicrograph of a section through the pontobulbar nucleus which is impregnated for normal axons. Most of them ramify in dorsomedial portions of the nucleus and relatively few penetrate its ventrolateral extreme (arrow). A motor trigeminal rootlet is labelled (rVn) for reference. The magnification for figure 15 is the same as that in figure 14 and that for the insert is comparable to that in figure 16.
15 High power photomicrograph of the sparse degeneration in the dorsal part of the pontobulbar nucleus
14 High power photomicrograph of axonal degeneration within the lateral part of the pontobulbar nucleus subsequent to removal of all neocortex on the same side. The bar is 44 pm.
EXPLANATION OF FIGURES
PLATE 1
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NUCLEUS OF THE PONTOBULBAR BODY C. F. Martin, M. Linauta and J. M. Walker
PLATE 1
(RN),
22 Darkfield photomicrograph showing terminal label which is restricted to the dorsomedial part of the pontobulbar nucleus (arrow) on the side opposite the placement shown in figure 19. This section is caudal to that illustrated in figures 20 and 21. Motor trigeminal (rVn) and spinal trigeminal (trs) fascicles are labelled for reference and the magnification is the same as that for figure 20.
and the magnification is the same. Motor trigeminal (rVn) and spinal trigeminal (trs) fascicles are indicated.
21 Darkfield photomicrograph showing terminal label in the dorsomedial part of the pontobulbar nucleus (arrow) contralateral to the injection shown in figure 19. This section is caudal to that shown in figure 20
which enter the pontobulbar nucleus by coursing along dendritic bundles comparable to those shown in figure 5. Motor trigeminal kVn) and spinal trigeminal (trs) fascicles are indicated. The bar equals 300 pm.
20 Darkfield photomicrograph showing terminal label (dark arrow) in the dorsal part of the pntobulbar nucleus contralateral to the injection shown in figure 19. The white, open arrow points to labelled axons
the neurons in the interstitial area dorsomedial to it and the cells of the ventral periaqueductal grey. The survival time was ten days and the exposure time four weeks. Results from this case are illustrated in figures 20 to 22. The cerebral aqueduct (aq), the interpeduncular nucleus (IP)and the cerebral peduncle (ped) are labelled. The magnification is the same as for figure 18.
19 Autoradiogram showing a placement (arrow) which labels the dorsomedial cells of the red nucleus
and the exposure time was four weeks. The oculomotor nerve (nIII) is indicated.
18 Autoradiogram showing the placement site of a brain in which the red nucleus (RN)was injected with SHleucine. The bar equals 2 mm. The insert shows an injection site from another brain which labelled the medial part of the red nucleus and adjacent areas bilaterally. In both cases the survival time was ten days
EXPLANATION OF FIGURES
PLATE 2
NUCLEUS OF THE PONTOBULBAR BODY G. F. Martin, M. Linauts and J. M. Walker
PLATE 2
26 Photomicrograph through the horseradish peroxidase injection of the pyramis and uvula in P-493 (fig. 10, bottom). The arrow points to the needle tract and the uvula is labelled for reference (Uv). The bar equals 2 mm.
25 Photomicrograph through the horseradish peroxidase injection site in P-459 (fig. 10, top). The arrow points to the needle tract in the paramedian lobule (PM). The bar equals 2 mm.
24 Photomicrograph through the horseradish peroxidase injection site of P-487 (fig. 9, right side). The arrow points to the needle tract and several areas of the cerebellum are labelled for reference. The bar equals 2.5 mm.
23 Photomicrograph of a section through the horseradish peroxidase injection site of P-485 (fig. 9, left side). The arrow points to the needle tract and several areas of the cerebellum are labelled. The bar equals 2.5 mm.
EXPLANATION OF FIGURES
PLATE 3
NUCLEUS OF THE PONTOBULBAR BODY G . F. Martin, M. Linauta and J. M. Walker PLATE 3
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30 Photomicrograph of the horseradish peroxidase injection site (arrow) of P-504 (fig. 13,bottom). The lingula (Lg) and crus I1 (CrII) are labelled and the bar equals 1.4 mm.
29 Photomicrograph of the horseradish peroxidase injection site (arrow) of P-476 (fig. 12, bottom). Various portions of the cerebellum are labelled for reference. The bar equals 2.5 mm.
28 Photomicrograph of a sagittal section through the horseradish peroxidase injection site from P-498 (fig. 11, bottom). The entrance point of the needle is indicated by the arrow and various divisions of the vermis are labelled. The bar equals 4.5 mm.
27 Photomicrograph through the horseradish peroxidase injection site of P-497 (fig. 11,top). The arrow points to the injection site and several regions of the cerebellum are labelled for reference. The bar indicates 2.5 mm.
EXPLANATION OF FIGURES
PLATE 4
NUCLEUS OF THE PONTOBULBAR BODY I2 P Martin M. I.inanta and .I M Walkrr PLATE 4
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PLATE 5
33 Darkfield photomicrograph of a caudal section through the pontobulbar nucleus on the side of the placement in P-473(fig.12,top). One of the motor trigeminal rootlets is labelled GVn)and the magnification is the same as that for figure 31.
readily Been and a small labelled neuron is indicated by the arrow. The magnification m i the same as that for figure 31.
32 Darkfield photomicrograph of the section shown in figure 31,oriented a little differently. Neurons within the pontobulbar nucleus which contain reaction product after the injection shown in figure 12 (top) are
31 Light field photomicrograph through the pontobulbar nucleus on the aide of the horaeradish peroxidase placement of P-473 (fig. 12, top). A motor fascicle of the trigeminal nerve is labelled for reference (rVn) and a small neuron which contains reaction product is indicated (arrow). The reaction product can be seen with darkfield optice (fig. 32,arrow). The small bar equals 42 pm.
EXPLANATION OF FIGURES
S3Nfl3IB A 0 NOlLVNV’ldX3
53
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NUCLEUS OF THE PONTOBULBAR BODY G. F. Martin, M. Linauta and J. M. Walker PLATE 5
