The lateral reticular nucleus of the opossum (Didelphis virginiana). II. Connections.

The Lateral Reticular Nucleus of the Opossum (Didelphis Virginiana) II. CONNECTIONS GEORGE F. MARTIN. J. ANDREZIK. K. CRUTCHER. M. LINAUTS AND M. PANNETON Department ofAnatomy, The Ohio State Uniuersity College of Medicine, Columbus, Ohio 43210

ABSTRACT Conformational, histochemical and histofluorescent studies reveal that the entire lateral reticular nucleus (LRN) of the Virginia opossum is positive for cholinesterase activity and that its rostra1 portion is rich in fluorescent varicosities of the catecholamine type. Although neocortical-LRN connections are relatively meagre, projections t o the LRN from the red nucleus are extensive and topographically organized. Rubral-LRN fibers arise from largemedium sized neurons and distribute t o the trigeminal division of the LRN as well as to specific portions of its external and internal divisions. Certain areas of the midbrain and pontine reticular formation, as well as the vestibular nuclei, project to the LRN and there is some evidence that reticular neurons adjacent to the LRN provide additional input. Although a relatively small fastigial-LRN projection exists, no evidence was found for a contribution from any of the other deep cerebellar nuclei. Spinal-LRN connections are extensive and topographically organized. Each of the inputs to the LRN have specific terminal targets, but there are varying degrees of overlap. Most of the LRN projects in an organized fashion to the anterior lobe of the spinal cerebellum, whereas only relatively restricted areas relay to the paramedian lobule and the pyramis. Lateral reticular axons distribute to specific longitudinal zones in such areas and the available material suggests that both convergence and divergence exist. The LRN also relays to the lobus simplex, and perhaps to crus I, as well as to visual-auditory areas of the cerebellar vermis. The distribution of the various afferent connections of the LRN is interpreted in light of LRN-cerebellar connections. Although we have described details that have not been elucidated in other species, where comparisons can be made it appears that the connectivity of the opossum LRN is comparable in most respects t o that of placental mammals. The lateral reticular nucleus (LRN) has been the subject of numerous studies in the cat, and in that species it receives a functionally complex and highly ordered input from the spinal cord (e.g., Brodal, '49; Oscarsson and Rosen, '66; Kitai e t al., '67; Rosen and Scheid, '72, '73a,b,c; Kiinzle, '73; Clendenin e t al., '74a,b,c; and Ekerot and Oscarsson, '75), a strong projection from the red nucleus (e.g., Walberg, '58; Courville, '66; Edwards, '72) as well as less extensive inputs from several sources, including the nucleus fastiguus (e.g., Thomas et al., '56; Cohen et al., '58; Walberg and Pompeiano, '60) and the cerebral cortex J. COMP. NEUR., 174: 151-186.

(Brodal e t al., '67; Kunzle and Wiesendanger, '74, among others). I t is of interest, however, that Mizuno e t al. ('75) claim that the known connections, particularly those from t h e spinal cord and red nucleus, account for only a small percentage of the terminals present in the feline LRN, indicating the presence of connections which have not been documented. In recent years our laboratory has directed considerable attention to the so-called precerebellar nuclei of the North American opossum (Yuen e t al., '74; Martin et al., '75; King et al., '75; Henkel et al., '75; Martin e t al., '76) as part of a program designed to pro-

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is either finely granular or randomly arranged in the LRN neuropil. The autoradiographic observations were made from brains in which either 3H-leucine or proline was injected into the cerebral cortex, brainstem, cerebellum or spinal cord. The amino acid was deposited by means of a Hamilton syringe and #31 gauge needle a t tached to a stereotaxically guided microdrive. Quantities from 0.1-0.3 Pl/injection were introduced into each animal (concentrated 50-100 yCilp1) over periods from one half to one hour and the needle was kept in the injection site for at 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 with MATERIALS AND METHODS either a formol-saline solution or with saline Observations relative to the interpretation followed by glutaraldehyde and paraforof LRN connectivity have been made from maldehyde. Frozen sections were mounted, brainstems prepared by one of several Golgi coated with Kodak NTB-2 liquid emulsion methods (laboratory of Doctor J a m e s S. diluted with an equal quantity of distilled King), sections impregnated for normal axons water and refrigerated for approximately four by a modification of del Rio Hortega's silver weeks. The slides were subsequently decarbonate method (Sharenberg and Liss, '69) veloped (D-19 high contrast developer), and material processed for cholinesterase ac- stained through the emulsion, coverslipped tivity by the Lewis ('61) modification of the and examined by light and dark field optics for Koelle technique (Koelle, '50). The location of the limits of the injection and for concentrabiogenic amines was determined from mate- tions of silver grains above background over rial prepared by the histofluorescent method the LRN. Silver grains clumped over axonal of Falck et al. ('62) with and without pretreat- bundles were considered to indicate axons en ment with a monoamine oxidase inhibitor. passage, whereas random labelling over the Briefly, the tissue blocks were removed while LRN neuropil was interpreted as terminal. the animal was deeply anesthetized and were The horseradish peroxidase (HRP) method frozen i n isopentane cooled with liquid was utilized in order to localize and characternitrogen. The tissue was placed in a Virtis ize neurons which project to the LRN and to lyophilizer for two weeks before being trans- plot the LRN neurons which relay to different ferred to an 80" oven in a n enclosed glass parts of the cerebellum. The enzyme was vessel containing formaldehyde (relative delivered to either the LRN or cerebellum as humidity = 70%; time = 1 hour). After par- described above ( 0 . 1 - 0 . 6 ~ 1of a 30-50% soluaffin embedding, t h e c u t sections were tion) over a period varying from 45 minutes to mounted with a non-fluorescent medium be- over one hour. Again, the needle was kept in fore being viewed and photographed through place for a t least ten minutes before and after a Leitz fluorescence microscope equipped with the injection was completed. After approxa K530 filter. For orientation every fifth sec- imately 24 hours the animals were anesthetion was stained for Nissl substance. tized and perfused with a 1-3%paraformaldeMany of the results reported herein were hyde, 1.0%glutaraldehyde solution in a 0.1 M taken from the brains of over 300 opossums phosphate buffer (pH = 7,4,24"C). The brains which have been impregnated for degenerat- were removed, immersed for six hours in the ing axons following specific lesions. The sur- perfusate and rinsed 24 hours in a 0.1 M phosvival times varied from 2 to 14 days and phate buffer (pH = 7.4) containing 30% several modifications of either the Nauta- sucrose. Frozen sections were incubated in a Gygax ('54) or Fink-Heimer method ('67) were medium containing hydrogen peroxide and employed. As used in this study, terminal de- 3,3'-diamino-benzidine tetrahydrochloride. generation refers to fine axonal debris which The sections were subsequently counter-

vide comparative information as well as baseline data for developmental studies in which we hope to take advantage of the opossum's unique embryology. In that spirit we have evaluated the connections of the opossum LRN using material processed by degeneration methods after appropriate lesions as well as by techniques which take advantage of axonal transport. Supplementary data has been obtained from sections processed by the Falck-Hillarp method for demonstrating biogenic monoamines. The terminology used for the opossum LRN is that which is described in P a r t I (Andrezik and King, '77) and for continuity periodic reference will be made to illustrations in that account.

OPOSSUM LATERAL RETICULAR NUCLEUS, CONNECTIONS

stained for Nissl substance and examined for spread of the marker at the injection site as well as for the presence of neurons containing granules of reaction product. RESULTS

In the following account selected observations from non-experimental material are presented first to provide a baseline for interpreting LRN connectivity. The results of experimental studies are described next in an order which allows the reader to see how some cases serve as partial controls for others, i.e., the projections from the cerebral cortex are dealt with first, followed in order by those from the brainstem, from rostral to caudal, the cerebellum and lastly, the spinal cord. Finally, the data concerning the projections of the LRN to the cerebellum are considered so that the pattern of LRN inputs can be discussed in terms of cerebellar circuitry.

Architectural, histochemical and histofluorescent observations Although the organization and nomenclature of the opossum LRN is dealt with in Part I (Andrezik and King, '77), certain features deserve emphasis. First, all techniques reveal that much of the internal division is truely reticular in character (e.g., fig. 5: Andrezik and King, '77). In such areas the dendrites of several neurons collect in bundles, oriented in the transverse plane, which either extend from medial to lateral for some distance or surround axonal fascicles coursing through the complex (arrows: fig. 1).It should be noted that LRN dendrites also spread to some extent in the rostral-caudal axis (horizontal sections). In experimental material, as well as in Golgi preparations, it appears that the axons which leave major pathways follow the dendritic bundles. Sections processed for cholinersterase (Che) activity reveal that the entire LRN is positive for the enzyme. As can be seen in figure 2 the reaction product outlines the nerve cell bodies and dendritic bundles (arrow) making them stand out from the non-reactive axons which traverse the nucleus. In contrast, only rostral portions of the complex are rich in presumptive monoamine terminals (figs. 3, 4). Such areas include the trigeminal and external divisions (figs. 3, 41, as well as portions of the internal division. Catecholamine containing neurons are located just dorsal and lateral t o

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the caudal LRN and dorsal to the nucleus at all levels. It should be pointed out that figures 3 and 4 were taken from cases which were not treated with nialimide prior t o sacrifice and in no instance was there evidence for substantial fluorescence of the serotonergic type.

Afferent connections of the lateral reticular nucleus Connections from the cerebral cortex Although evidence for a projection to the LRN is present in brains with small lesions of t h e motor-somatosensory cortex (Lende, '63a,b), it is by no means remarkable. Consequently, several brains were used in which most of the neocortex had been removed, hopefully producing degeneration of all the fibers in question. In one particularly good case fragmented axons can be traced laterally from the degenerating pyramidal tract and into the rostral one-half or more of the LRN (fig. 5). Most of the fibers distribute ipsilaterally and are dispersed along the neuronal strands a t the periphery of the internal division (arrows, figs. 5, B-D). There is some spillover into adjacent portions of the internal and external LRN, but only a few degenerating axons are present in the trigeminal division and few appear to end caudal to the level shown in figure 5 of Andrezik and King, '77 (fig. 5E, present study). Although several cases are available with 3H-leucine placements in small areas of the motor-sensory cortex, they show little evidence for a projection to the LRN. Connections from the brainstem Degeneration within the LRN after brainstem lesions may result from interruption of axons arising a t sites distal to the lesion as well as from the nerve cells which are damaged directly. For that reason we will describe the results obtained from brains with rostral lesions first so that they can be used to interpret those derived from cases with damage to more caudal areas. Since it is often impossible to control for axonal undercutting we have also utilized autoradiography. Lesions limited to either the superior colliculus or the underlying midbrain tegmentum produce little degeneration within the LRN of either side. Similar negative results were obtained from several autoradiographic experiments even though survival times of 10 days were employed in order to accentuate

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axonal labelling. As expected, however, lesions of the red nucleus, particularly its rostral two-thirds, result in extensive degeneration within the contralateral LRN. Although degenerating axons are often present in the ipsilateral LRN after such lesions, the presence of exclusively contralateral label in companion autoradiographic experiments suggest that they result from undercutting of rubral fibers from the opposite side. Since in degeneration experiments the full projection of a fiber system cannot always be deduced by adding the results of small lesions we intentionally undercut the entire crossed rubral projection in one case (insert, fig. 6). In that brain extensive degeneration is present within the trigeminal portion of the LRN, within rostral and generally lateral parts of its external division, as well as within adjacent portions of the internal division (figs. 6AD, 8 ) . Degeneration within the external and internal divisions is particularly extensive a t the level shown in fig. 6D. In sections immediately caudal t o the midpoint of the LRN de-

generating axons reach rostral and lateral extremes of the compact portion of the internal division (fig. 6D: arrow), but terminal debris is not seen at more caudal levels (fig. 6E). The rubral origin of the fibers in question as well as their distribution was substantiated by the autoradiographic technique (figs. 9, 14, 15). Large ventromedial midbrain lesions (e.g., fig. 7) induce a small amount of additional degeneration in the medial part of the internal LRN of the same side as well as bilateral degeneration within the rubral targets just described. Comparison of different cases leads to the conclusion that the rubral-LRN projection is topographically organized. For example, when lesions or 3H-leucine placements are limited to the caudal medial extreme of the red nucleus (fig. 10) there is little evidence for a projection t o the trigeminal LRN. The labelled neurons of the red nucleus in P-417 are restricted to its dorsomedial sector (insert, fig. 11) and although silver grains are dispersed in a terminal fashion over several areas of the LRN, they are very sparse over the trigeminal

A bbreuintions a, subnucleus a of the medial accessory olive APo, area postrema b, subnucleus b of the medial accessory olive bc, brachium conjunctivum c, subnucleus c of the medial accessory olive C-2, second cervical cord level CcD, dorsal cochlear nucleus CeS, superior central nucleus CI, inferior colliculus Cn, culmen Coe, locus coeruleus cr, restiform body CS, superior colliculus CuL, lateral cuneate nucleus CUM, medial cuneate nucleus d, dorsal accessory olivary nucleus De, declive EXT, external division of lateral reticular nucleus Fac, motor facial nucleus Fast, fastigial nucleus GM, medial geniculate nucleus Hg, hypoglossal nucleus InP, interpositus complex

IP, interpeduncular nucleus INT, internal division of lateral reticular nucleus LES, lesion Lg, lingula of the cerebellum LRN, lateral reticular nucleus LUMB, lumbar Nd, nodule 01, inferior olivary nucleus PM, paramedian lobule pr, principal olivary nucleus PrC, preculmen of cerebellum PyC, pyramis of cerebellum pyr, pyramidal tract RN, red nucleus rVII, facial nerve sac, sacral TrMo, motor trigeminal nucleus trs, spinal trigeminal tract Tz, nucleus of trapezoid body Uv, uvula VstM, medial vestibular nucleus X, dorsal vagal nucleus 111, oculomotor nucleus

Fig. 1 Transverse section of a Golgi impregnated section through about the midlevel of the lateral reticular nucleus. The small arrow on the left points to a bundle of dendrites which help to surround a group of axons coursing longitudinally through the nucleus. The larger arrow to the right indicates a n aggregate of dendrites oriented in the transverse plane. The size indication is the same for figures 1-4. Fig. 2 Transverse section through the caudal LRN showing both the external (EXT) and internal (INT) divisions after being reacted for cholinesterase activity by the Koelle technique. The arrow indicates a dendritic bundle interconnecting the two divisions. Fig. 3 Transverse section through the rostral lateral part of t h e external LRN as i t appears in material processed by the Falck-Hillarp technique without the use of a monoamine oxidase inhibitor. The arrow points to one of the dendritic bundles outlined by green varicosities. Fig. 4 Transverse section through the medial part of the rostral LRN in a section processed by the Falck-Hillarp technique. No monoamine oxidase inhibitor has been used. The arrow points to a non-fluorescent neuron surrounded by greenish varicosities and the insert shows a comparably positioned neuron (arrow) from a directly adjacent section.


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Fig. 6 Drawings of transverse section through the LRN (rostral, A through caudal, E) on the side of a lesion (insert) which undercuts all of the crossed descending axons of the red nucleus, but which spares most of the pons. Degenerating axon terminals are drawn in the LRN a s they appear after impregnation by the FinkHeimer method. The arrow in section D points to the small focus of degeneration which reaches the internal division.

the red nucleus is also heavily labelled in P418 (insert, fig. 12), but in addition neurons are moderately to lightly labelled more laterally. In that brain grains are extensive over the trigeminal LRN and present over

most areas of the external and internal divisions shown to be targets of rubral axons in other experiments. It is noteworthy, however, that labelling is sparse in the ventral extreme of the external division a t the level shown in

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figure 12C. Evidence for a strong projection to the latter area is present in P-282 (arrow: fig. 13C) and in a n autoradiographic case with a generally comparable placement. Several attempts were made to limit injections of horseradish peroxidase to the rubral targets of the LRN. However, the opossum brain is relatively small and most cases show spread of the marker to regions dorsal to the LRN and along axon bundles coursing through it. “Injury” label of the latter axons is also apparent. After such placements both the amount of reaction product visible in rubral neurons and their exact location varies, possibly reflecting differences in t h e area of the LRN most heavily injected and/or the amount of contamination present. Of particular note is the fact that the marker spread to the “hilum” of the dorsal column nuclei in each brain, an area which is also a major terminal site for rubral fibers (Martin and Dom, ’70; Martin et al., ’74). In all cases, however, labelled neurons are present mainly in the rostral two-thirds of the red nucleus and fall within the large-medium size range (25-40 p , see Martin et al., ’74). No giant neurons (4570 p ) contain reaction product. Lesions of the rostral pontine reticular formation (including both the nucleus pontis centralis oralis and the nucleus subcoeruleus) elicit degeneration of axons within both the paramedian and ventrolateral medulla. Again we have taken the view that the results of small lesions and placements can be interpreted best in light of the total projection from a particular area. On that basis we hemisected the pons in one animal (insert of fig. 18) producing degeneration in rubral targets of the ipsilateral LRN (figs. 18A-D), in nonrubral areas of the external division (solid arrows: figs. 18B,C), and within medial portions of the internal division (open block arrows: figs. 18C-El. The degeneration in the latter two areas can be traced from bundles dorsal and medial to the LRN. In spite of the size of the lesion few degenerating axons terminate within the compact area of the internal LRN (e.g., fig. ME). In brains with 3H-leucine injections of the rostral pontine reticular formation (e.g., fig. 22 which shows P-444 of our collection) labelled axons are numerous in the paramedian medulla, particularly after 10-day survivals, but grain dispersion is light over the LRN and present mainly in those regions of the internal division which contain degeneration after lesions of the same area (open arrows: figs.

18C-E). Cases with placements smaller than that in figure 22 are available, but they show little label over the LRN. In one brain 3H-leucine was placed in that portion of the reticular formation medial to the ventral nucleus of the lateral lemniscus (not shown). In contrast to the other cases, axonal bundles are labelled in the lateral medulla of the opposite side as well as in paramedian regions (mainly ipsilateral). However, evidence for input to the LRN is still limited to the medial part of its internal division, mainly on the side of the placement. In still another case, the motor trigeminal nucleus, the oral portion of the spinal trigeminal complex, the adjacent parvocellular reticular formation, and even some of the caudal parabrachial complex contain labelled neurons at the injection site and evidence is present for a small projection to the trigeminal division of the opposite LRN. The origin of this projection is not known, however, since i t is not present in any of the brains with small injections.’ Lesions placed in the caudal pons also produce degeneration within the internal LRN, but again not in its compact portion. In companion autoradiographic experiments (e.g., figs. 23, 24, which show the injection sites in P-404 and P-446 of our collection) silver grains are clumped over axonal bundles in the paramedian medulla as well as dispersed over

’ See note added in proof, page 185. Fig. 7 Nissl stained section through a portion of a large lesion of the ventromedial midbrain. This lesion extends rostrally and caudally for a considerable distance and undercuts axons from the entire rostral two-thirds of the red nucleus. Fig. 8 Axonal degeneration in the LRN produced by the lesion illustrated in figure 7. The arrow points to a blood vessel shown a t higher power in the insert. For comparison with autoradiographic results a t approximately the same level the reader is referred to figure 15.Fink-Heimer technique. Fig. 9 Nissl stained, autoradiographically processed section from P-469 showinga large 3H-leucine placement in the red nucleus and adjacent tegmentum (large arrow). The lightly stained red nucleus is indicated on the opposite side (RN) and the oculomotor rootlets (111) are labelled. The photomicrograph in the insert shows the placement site in another case (P-467) in which the marker was introduced into the midline, labelling medial rubral neurons on one side (large arrow). The lightly stained red nucleus and the oculomotor nerve are indicated. In both cases the survival time was ten days. Fig. 10 Nissl stained section from a brain with a lesion (arrow) limited to the caudal medial third of the red nucleus. The insert shows the placement site from an autoradiographic case (P-409) in which the red nucleus labelling (arrow) is restricted to its caudal, medial third. The survival time was 24 hours.


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Fig. 11 Drawings of transverse sections through the LRN (rostra1 A, to caudal, E) on the side opposite the Wleucine placement schematically illustrated in the insert. Labelled neurons are indicated by the crosses as they appear after a 10 day survival. The terminal labelling in the LRN is illustrated (dots) as it appears in the emulsion over the sections. Labelled non-terminal axons are not illustrated.

the medial part of the internal division, particularly dorsally. The placement shown in figure 23 produced labelling over fiber bundles which was mainly ipsilateral and grain dis-

persion over the LRN was very light. The results from P-446 (fig. 24) were particularly conclusive, however. Labelling over axonal bundles in the paramedian medulla was ex-


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Fig. 12 Drawings of transverse sections through the LRN (rostra1 A, to caudal E) on the side opposite the 3H-leucine placement shown in the insert. Again, the labelled neurons are illustrated as crosses (10day survival). Only the terminal dispersion of silver grains over the LRN is plotted on the sections.

tensive, bilaterally, (left insert, fig. 24) and well dispersed over medial areas of the internal LRN (right insert, fig. 24).Light label is also present over axons in more lateral areas

of the internal division with some evidence for terminal dispersion. One case is available with a lesion of the parvocellular reticular formation caudal to

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Fig. 13 Drawings of transverse sections through the LRN (rostral, A through caudal, E) on the side opposite the lesion shown in the insert. Only terminal degeneration is plotted (arrows).

the facial nucleus (P-116, fig. 19). Axonal degeneration is extensive in the lateral medulla, but terminal debris within the LRN is present only within those areas which contain degeneration after rubral lesions (figs. 19A-D). A suggestion that not all such degeneration resulted from undercutting of rubral fibers is found in one of our autoradiographic cases (P401, fig. 19).

After lesions which involve both the lateral and medial vestibular nuclei (P-177,fig. 20) degenerating fibers can be seen to outline some of the more peripheral strands of the internal LRN on the same side (P-177,figs. 20A-C) and a few of them extend into adjacent portions of both the external and internal divisions. Additional degeneration distributes within the internal division proper (P-177, figs. 20B-


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Fig. 14 Nissl stained, autoradiographically processed section through the LRN contralateral to the rubral placement in P-418(insert, fig. 12).The section was photographed through light fieldoptics and the arrow indicates a cluster of blood vessels similarly shown in figure 15. The size indicator is the same for figures 14-17. Fig. 15 Darkfield photomicrograph of the section shown in figure 14.The arrow points to the blood vessels similarly indicated in figure 14. Fig. 16 Nissl stained, autoradiographically processed section through the internal LRN on the side of a large W l e u cine placement in the vestibular complex (P-386, fig. 20).The section was photographed through light field optics and the arrow points to a neuron similarly indicated in figure 17. Fig. 17 Darkfield photomicrograph of t h e section shown in figure 16. The arrow points to the neuron indicated in figure 16.

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Fig. 18 Drawings of transverse section through the LRN on the side of the lesion (insert). The most rostra1 section (A) is in the upper left hand corner, whereas the most caudal (E) is a t the lower right. Degenerating axons a r e plotted in the LRN as they appear in Fink-Heimer impregnated material.

El. In spite of the fact that a somewhat comparable display of degeneration is present after destruction of the nucleus fastigius or its efferent axons, and such axons are neces-

sarily interrupted by the above lesions, the existence of vestibular-LRN connections is verified by the presence of light labelling over the internal LRN after 3H-leucine injections


Fig. 19 Drawings of transverse sections through the LRN (rostral, A through caudal E) on the side of the lesion shown in the insert (P-116). Degenerating axons are illustrated within the nucleus as they appear in Fink-Heimer impregnated material. In the upper right hand corner silver grains are illustrated (arrows) over the LRN in two sections from P-401(autoradiographic material). One section through the placement site is and for clarity labelled axons in passage are not shown. drawn in t h e smallest insert (P-401)

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Fig. 20 Drawings of transverse sections through the LRN (rostral, A through caudal, E) in P-177. The lesion is illustrated in the small box and degenerating axons within the LRN are drawn as they appear in the Fink-Heimer impregnated sections. In t h e two sections illustrated for P-386 (upper right) silver grains are plotted over axon bundles as well as over terminals in the LRN. One section through the placement site (3H-leucine) is shown in the smallest insert (P-386).

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of the vestibular complex (P-386, fig. 20 and figs. 16, 17). In P-106 (fig. 21) the lesion is limited mainly to the nucleus interfascicularis of the medullary reticular formation as well as the lateral part of the rostral inferior olive. Although the lesion obviously undercuts fascicles from more rostral areas, degeneration is relatively sparse in the LRN and limited to its internal division (figs. 21, P-106, A-C). As in the pontine cases, no obvious degeneration is present in the compact part of the internal division. Although the lesion in P-111 (fig. 21) is more laterally situated, it is still the medial part of the internal LRN which contains most of the degeneration. In contrast to P-106, however, degenerating axons can be traced into rostral areas of the external LRN and into ventrolatera1 regions of the internal division (fig. 21, P111,A,B). Since degeneration material is obviously difficult to interpret with respect to LRN connections from the medulla, the autoradiographic technique was attempted in 13 animals. In contrast to the approach used with midbrain and certain pontine placements, short survival times were employed in an a t tempt to maximize definition of the injection site. Unfortunately, however, most of the injections spread to the LRN and in those which did not, neurons in more than one nucleus were labelled a t the injection site making it impossible to determine the origin of labelled axons in the LRN. Such problems are a result of the small size of the medulla in the opossum, and t h e tendency of injected markers to spread, particularly in the reticular formation. In spite of the major problems referred to above, several observations deserve mention. First, some of the medullary cases were negative for transport to the LRN even though the injections were large and labelled axons could be traced to other areas. The injection site from one such case is illustrated in figure 25. Secondly, in several instances there was only light spillover into the rostral part of the LRN, but grains were heavily dispersed over the compact part of the internal division caudally -a region which apparently receives little, if any, input from either the midbrain or pons. Thirdly, in one case the caudal LRN was injected, with some spillover to adjacent reticular areas, producing apparently terminal label in the rostral LRN. Such label was

limited to those areas failing to receive rubral input. The available degeneration and autoradiographic (fig. 25) material indicates that few fibers from the caudal nucleus gracilis and cuneatus project to the LRN. However, in one brain with damage to both the rostral cuneate and accessory cuneate nuclei (P-312, not shown) degenerating internal arcuate fibers can be seen to traverse the internal LRN as well as reach the external division by skirting the spinal trigeminal tract. Although terminal degeneration, as used in the usual sense, is questionable, degenerating axons traverse the LRN in a fashion compatible with en passage endings.

Connections from the cerebellum Large lesions including both the dentate and interpositus nuclei of the cerebellum produce sparse but definite degeneration in the ipsilateral LRN (figs. 26A-El. The axonal debris becomes sparse in caudal sections and none is located in the compact portion of the LRN (fig. 26E). I t appears, however, that most of the fibers which are damaged in such cases arise within the fastigial nuclei since: (1) they degenerate bilaterally when the nucleus fastigius is included in the lesion, (2) comparable degeneration can be induced by lesions which are limited to the nucleus fastigius and (3) they filter out of the reticular formation rather than course through the descending limb of the brachium conjunctivum. No evidence for a projection to the LRN is available in the brains of ten oppossums subjected to one or more injections of 3H-leucine into either the interpositus or Fig. 22 Nissl stained autoradiogram of the placement site (arrow) in a brain with a %leucine injection of the rostral pontine reticular formation (P-444).The survival time was ten days and t h e exposure time was four weeks. Fig. 23 Nissl stained autoradiogram through the injection site (arrow) of P-404. The survival time was 24 hours and the exposure time was four weeks. Fig. 24 Nissl stained autoradiogram through the injection site (arrow) of P-446. The insert on the left shows axonal labelling in the paramedian medulla, whereas the insert on the right illustrates clumped (arrow) and dispersed label over the medial LRN. The survival time was ten days and the exposure was four weeks. Fig. 25 Nissl stained autoradiogram through the injection site (arrow) from an animal with a placement which labelled the dorsal column nuclei and the underlying reticular formation (P-359). The nucleus of the tractus solitarius and hypoglossal nucleus are also labelled. The survival time was 24 hours and the exposure time was four weeks.


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Fig. 26 Drawings of transverse sections (rostral, A through caudal, E) through the LRN on the side of the cerebellar lesion shown in the insert (P-291).The degenerating (fastigial) axons (see text) in the LRN are illustrated as they appear in the Fink-Heimer impregnated sections.

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Fig. 27 Drawings of transverse sections (rostral, A through caudal, E) of the LRN on the side of the lumbar The degenerating axons within the LRN are illustrated as cord lesion illustrated in the small insert (P-325). they appear in Fink-Heimer impregnated sections and the area referred to as the compact part of the internal LRN is marked with a n “X” (sections D and El. I n the larger insert a section through t h e medulla is shown from a case with a complete sacral transection. The degeneration in t h e LRN is indicated (open arrow).

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the fact that a large number of degenerating fibers traverse it. In contrast, most of the compact area, particularly its ventrolateral secConnections from the spinal cord tor, is packed with terminal degeneration, as Lesions of the rostral spinal cord necessari- well as non-terminal debris, in another case ly interrupt fibers arising more caudally. For with a lesion of the fourth cervical segment. The most rostral lesions in our collection that reason the results obtained from sacral transactions will be described first followed by are a t C-2. There is some ventral funiculus those derived from cases subjected to pro- sparing in all of them, but swollen and obgressively more rostral lesions. Although de- viously pathological axons are present indigeneration is often bilateral, most of it is pres- cating some involvement. Such lesions elicit degeneration within the same areas which ent ipsilaterally. After complete sacral transection a small contain debris after more caudal trauma (figs. amount of terminal degeneration is located 28C-E),although it appears that degeneration within the external LRN (P-326, fig. 27). I t is is particularly abundant in the dorsomedial present rostral to the level shown in figure 4 portion of the compact area and the region of of Andrezik and King, ('77) and extends some- the internal division adjacent to it. As in the what past the level illustrated in figure 5 of other cases, degenerating axons are randomly the same communication. Degenerating axons arranged within the medial part of the interare much more numerous after lumbar hemi- nal LRN (figs. 28C-El. The above degenerasections (P-325, fig. 27). In such cases termi- tion becomes relatively sparse in more rostral nal degeneration begins at the most caudal sections so that the ventromedial part of the level of the external division (see fig. 4 of internal division and much of the adjacent Andrezik and King, '77) and is particularly external division contain little debris (fig. dense dorsally and laterally. In progressively 28A-C). Only a few abnormal axons can be more rostral sections the dorsolateral area is seen within the trigeminal division (fig. 28A). I t would appear from the above account packed with terminal debris and additional degeneration is present within ventral and that the compact part of the internal LRN remedial parts of the external division (figs. ceives fibers from the cervical cord, but not 27C-E, 30). Fragmented axons also course from more caudal levels (compare figs. 27D,E within the internal LRN in a fashion which is with figs. 28D,E). One case with a C-2 lesion is compatible with en passage terminals (figs. cut in the sagittal plane (animal pH-10) and 27C-E) although none distribute to the com- the region in question stands out from the repact region ("X" in figs. 27D,E). The dense de- mainder of the LRN because of the density of generation outlining the dorsolateral part of the degeneration contained within it (fig. 31). the external LRN can be traced to the level Since degeneration is present in the caudal shown in figure 5 of Andrezik and King, ('771, part of the external LRN after lumbosacral whereas that present ventrally and medially lesions, that present in the same area after in the same division can be followed to more cervical lesions could result from undercutrostral levels. Degeneration within the inter- ting axons arising more caudally. In an atnal LRN also is found more rostrally than tempt to determine if any such fibers arise that located dorsally and laterally, but i t is within the cervical cord itself one animal was not present in sections as far rostral as that subjected to four injections of 3H-leucineinto shown in figure 6 of Andrezik and King, ('77). the cervical enlargement (50 FCihnjection). Although the results obtained by thoracic The injections were made 0.5 cm apart with transections are comparable to those just de- one each in C-4 through C-8. In order to allow scribed, lesions of the cervical cord produce a for build-up of the amino acid the animal was slightly different picture. After a C-8 hemisec- maintained for eight days before sacrifice. tion degenerating axons are present in the The developed autoradiographs show good external reticulated zone of the internal divi- neuronal labelling of the spinal grey a t all sion as well as in those regions which contain levels on the side of the placements and, as degeneration after lumbrosacral lesions. The might be expected from the survival time, lacompart part of the internal division still belling over axonal bundles within the LRN. appears to be free of terminal debris in spite of However, the only terminal dispersion of dentate nuclei after survival times varying from one to ten days.


t \

Fig 28 Drawings of transverse sections (rostra1 A caudal E) through the LRN on the side of the C-2 lesion shown in the insert. The degeneration within the LRN is plotted as it appears in Fink-Heimer impregnated sec tions

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ularly numerous laterally. Evidence for a projection to areas of the vermis between the primary fissure and the pyramis is equivocal, but cannot be ruled out. Terminal fields in all of the areas just described are characterized by clumps of grains over glomeruli in the granular layer. Autoradiographic evidence for an LRN projection to the deep cerebellar nuclei is present, but not remarkable, and labelled axons can be seen to cross the midline of the cerebellum. Horseradish peroxidase was injected into each of the cerebellar targets of the LRN in Efferent projections of the lateral order to localize and characterize the neurons reticular nucleus which project to them. In three cases, large After lesions of the lateral reticular nucleus amounts of HRP were injected in order to ladegenerating axons can be followed bilateral- bel as many of the neurons projecting to the ly into the granular layer of the anterior lobe anterior lobe as possible. Such injections (mainly ipsilaterally) as well as into the spread to most areas of the anterior lobe ipsilateral paramedian lobule, pyramis and except the lingula (fig. 33) and neurons are lauvula (personal communication, Doctor belled in some part of all subdivisions of the Richard Dom). In such material degeneration LRN (dots in fig. 37). Reactive neurons are is also present in the interposed and lateral sparse, however, in the trigeminal division nuclei of the cerebellum. Since LRN lesions and in the rostromedial part of the external potentially undercut axons from paramedian division (fig. 37A). The backfilled neurons areas which also project to the cerebellum (left insert, fig. 36) range in size from 13-35p. (unpublished results), we prepared two cases Although the LRN labelling is bilateral and for autoradiography. In both of them the there is spread of the enzyme to both sides of heaviest neuronal labelling is present in the the cerebellar midline (fig. 331, it is obvious internal LRN with relative sparing of its t h a t the majority of reactive neurons are external and trigeminal divisions; and, as re- located on the side of the placement. In one ported by Kunzle and Cuenod (’731, there are case the marker fills the lingula and preculapparently unlabelled cells even a t the center men with relative sparing of the culmen. In of the injection site. Although most of the la- that brain neurons are positive for reaction belled fibers reach the cerebellum via the product throughout most of the LRN (mainly ipsilateral restiform body, some cross the mid- ipsilaterally), except for its trigeminal diviline and traverse the comparable bundle on sion. In the caudal spinal zone, however, the the opposite side. In the ipsilateral preculmen compact part of the internal division is relaand culmen labelled axons distribute in a nonuniform manner, extending rostrally and cauFig. 29 Nissl stained section through the caudal LRN. dally in a series of three or more bands per A portion of t h e region referred to in the text as the comside. This is particularly obvious with dark pact area of the internal LRN is labelled with a n “ X ’ in field illumination. Evidence for dense termi- both figures 29 and 30. The area of the external LRN which contains degeneration in figure 30 (arrow) is also indicated. nals is also present within pars lateralis of the The section shown in figure 30 is from approximately the culmen and the adjacent medial bank of the same level. Fig. 30 Fink-Heimer impregnated section through the lobus simplex. Labelling in the lingula is light, possibly reflecting the relative sparing caudal LRN on the side of a lumbar lesion. Although terdebris is present dorsolaterally in the external diviof the external division. Labelling of the minal sion (arrow) only degenerating fibers of passage are seen paramedian lobule is also heavy, particularly within the area marked “X”. in the superior folium, and arranged in bands Fig. 31 Degeneration in area “ X ’ of an animal subwith at least two in each folium. In the jected to a C-2 spinal lesion. The section is cut in the sagitinferior folium the medial band is the wider of tal plane and processed by the Fink-Heimer technique. 32 Darkfield photomicrograph of axonal labelling the two. The arrangement of silver grains in Fig. the compact part of the internal LRN (“X’) subsequent over the pyramis is only slightly suggestive of to multiple 3H-leucine injections of the cervical enlargecompartmentalization, but they are partic- ment %day survival).

grains over the LRN is found in the compact part of its internal division (“x” in fig. 32) and in that portion of the internal division dorsomedial to it. Cases cut in either the horizontal or sagittal plane prior to processing by the FinkHeimer method are particularly good for revealing the caudal to rostra1 extent of spinal degeneration within the LRN. The available material indicates t h a t spinal fibers distribute to some extent throughout the caudal three-quarters of the complex.


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tively free of reactive neurons when compared to other areas of the nucleus at the same level. In other brains smaller anterior lobe injections were accomplished (0.1 to 0.3 p l h j e c tion). In one the dark area around the needle tract is limited to a zone just off the midline, but extending throughout most of the culmen (lower left insert, fig. 33). In that brain reactive neurons are present bilaterally and are most numerous in the medial part of the internal division. No labelling is present within the trigeminal area and only a very few neurons contain reaction product in either the rostral portion of the external LRN or the compact part of the internal division. A second case was prepared in which the injection was limited to the lateral culmen, missing only its ventrolateral extreme (right insert, fig. 33). In contrast t o the brain with the medial placement, the most numerous and heavily labelled neurons are located in rostral and lateral parts of the external division and in adjacent lateral portions of the internal division. A few cells are lightly stippled in the trigeminal division. Although labelled neurons are present bilaterally in the internal division they are seen only ipsilaterally in either the rostral external division or the compact portion of the internal division. In still another case (not shown) the bulk of the injection is intermediate to those just described and a few reactive neurons are found (bilaterally) in some part of all divisions except for the trigeminal. The heaviest labelling is in the lateral extreme of the internal division, however, about midway through the nucleus. In three brains HRP was introduced into the paramedian lobule. In one of them the marker was injected so as to label as many LRN neurons as possible. The injection site from that brain (P-459) is shown in figure 34 and the retrogradely labelled neurons are plotted as circles in figure 37). In spite of the size of the injection, relatively few LRN neurons contain reaction product and most of them are ipsilateral to the placement. In rostral sections such neurons are located within the external division, particularly laterally, and in the strands connecting the external and trigeminal divisions (circles in fig. 37A). More caudally labelled neurons begin to appear in the internal LRN and a t the levels shown in figures 37B and C they are most numerous dorsally and laterally. Heavi-

ly reactive neurons are abundant in dorsal and lateral areas throughout most of the internal division, including its compact area, and a few lightly labelled neurons can be seen in caudal parts of the external division. Relatively few neurons in the medial part of the internal LRN contain reaction product at any level, but particularly those shown in figures 37B and C. Reactive cells are not present in the trigeminal division of the case illustrated in figures 34 and 37, but a few are seen in another brain with a comparably large injection. In one brain, a small amount of HRP was deposited in the medial part of the inferior folium (lower left insert, fig. 34). In spite of the small size of the injection, lightly labelled neurons are present in the lateral part of the external LRN, rostrally as well as caudally, and reactive neurons can be seen in the lateral extreme of the internal division caudally. Neurons containing reaction product are notably absent in the compact part of the internal division, however. In two cases HRP was injected into the pyramis. After the placement shown in figure 35, labelled neurons (crosses in fig. 37) were limited to the ipsilateral LRN. Only a very few could be found within either the trigeminal division or the adjacent rostral part Fig. 33 Low power photomicrograph of the injection site in a case in which a large amount of horseradish peroxidase was injected into t h e anterior lobe of the cerebellum, sparing only the lingula. The case was prepared in order to determine the full expanse of LRN giving rise to anterior lobe projections and the results are plotted in figure 37 (dots). The insert a t the lower left shows a smaller paramidline placement in the anterior lobe and the insert a t the upper right shows a smaller lateral placement in the same lobe. The results from the smaller cases are described in the text. Fig. 34 Low power photomicrograph from a case in which horseradish peroxidase was injected so as to fill the paramedian lobule. The large placement was made in order to label as many of the LRN neurons projecting to the paramedian lobe as possible (circles, fig. 37). The insert a t the lower left shows a smaller placement in the medial part of the inferior paramedian folium. Fig. 35 Low power photomicrograph of a case in which horseradish peroxidase was injected into the pyramis (PyC) and uvula (Uv). The section is lightly stained for Nissl substance. The insert was taken from another brain in which HRP was deposited in t h e lateral extreme of the pyramis with spread into t h e uvula. A control placement limited to the uvula produced no labelling of the LRN. Fig. 36 Sagittal section of the cerebellum from a case in which HRP has been injected into the tuber of the cerebellar vermis. The insert a t the upper left illustrates HRP labelled neurons in the LRN after a n anterior lobe placement, whereas t h e insert on the right shows a placement which was limited to the lobus simplex.


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of the external division (not illustrated in fig. was injected into crus Zof the hemisphere. 37A), although in progressively more caudal However, subsequent examination of t h e sections they became apparent in the periph- reacted sections revealed that the marker eral strands of the internal LRN and in adja- spread to the lobus simplex and included the cent portions of its external division. At levels area known to receive LRN input from the decomparable to that shown in figure 37B, generation and autoradiographic cases. The labelled neurons are located in the dorsolat- results from one of the cases are plotted in eral extreme of the external LRN (relatively figure 37. Of particular note is the presence of abundant), as well as within the internal divi- numerous, heavily labelled neurons in the trision, particularly dorsally and laterally. A geminal division of the LRN, (as well as somewhat comparable arrangement is present throughout much of the adjacent subnucleus in more caudal sections (crosses: fig. 3 7 0 , interpolaris of the trigeminal complex) and where reactive neurons can be seen in the within rostral, medial portions of its external compact portion of the internal LRN as well division (triangles in fig. 37A). Such regions as in its external division. It is apparent that are either unlabelled or only lightly labelled the lateral zone of reactive neurons in the after injections of spinal areas of the cerebelexternal division extends for some distance lum. Labelled neurons are present in other throughout the nucleus. No labelled neurons areas of the LRN although few can be found are present a t the caudal pole of the LRN and in the medial part of the internal division they are very sparse in the medial part of the (figs. 37B, C) and none are located at its most internal division a t all levels (figs. 37B,C). caudal end. As illustrated in figure 37C reacThe second pyramis placement was made tive neurons are located in the dorsolateral more lateral than the first (left insert, fig. 25) part of the internal division, but mainly dorand although the results were generally com- sal and medial to the compact area. Most of parable to those just described, reactive neu- the labelled neurons are located on the side of rons were less abundant in the lateral ex- the placement, although some can be seen treme of the external division. No LRN neu- contralaterally, particularly in rostral secrons are labelled in a control case in which the tions. Generally comparable results were seen injection was limited to the uvula suggesting in another brain in which the marker was rethat the spread to that region illustrated in stricted mainly to the dorsal half of the lobus simplex (right insert, fig. 36). The number of figure 30 was not significant. Reactive neurons are also present in the labelled neurons was greatly reduced, howLRN after injections of non-spinal areas of ever. Trigeminal neurons are labelled in still the cerebellar vermis. Small injections of the another brain in which HRP was deposited declive elicit labelling of a few neurons in into crus I1 with only very light spread to crus each division, but they are suspect because of I and no involvement of the lobus simplex. No possible anterior lobe contamination. In spite other portion of the LRN is labelled. As might of the relatively small size of one folium injec- be expected no LRN neurons contain reaction tion numerous neurons are heavily labelled in product after injections of the paraflocculus. all divisions of the LRN, bilaterally. Such laDISCUSSION belling extends throughout rostral to caudal levels of the nucleus. Again, however, there is I t is apparent from our material that the the possibility that some of the marker spread LRN receives inputs from multiple sources to that part of the anterior lobe just rostral and in the following account the data will be to the primary fissure. The sagittal section discussed and compared with that available shown in figure 36 illustrates the proximity of for other species. Since the benefits and the declive and folium to the caudal anterior limitations of the hodological methods used in lobe. Fortunately, however, the tuber injec- this study have been extensively reviewed tion illustrated in figure 36 is unequivocally elsewhere (e.g., Hendrickson, '75; LaVail, '75; clean and resulted in labelling of the rostral Kim and Strick, '76), they will not be distrigeminal division, mainly ipsilaterally, and cussed in detail. in some labelling of the rostral internal diviOur material verifies the existence of a relasion. No reactive neurons were observed a t tively sparse cortical projection to the ipsilatintermediate or caudal levels of the nucleus. eral LRN in the opossum and indicates that it In cases prepared for another study HRP arises within the motor somatosensory cortex


Fig. 37 Drawings of sections through rostral, middle and caudal levels of the LRN on which neurons t h a t are labelled after different HRP injections of the cerebellar cortex are plotted. The dots represent t h e neurons labelled after the large anterior lobe injection shown in figure 33, the circles indicate those labelled in the paramedian case shown in figure 34 and the crosses show t h e position of reactive neurons in the pyramis-uvula case illustrated in figure 35. The triangles demarcate the position of labelled neurons in another case in which a large injection was made in crus I of the cerebellum with spread rostrally into the lobus simplex and caudally as far as the rostral paramedian lobule.

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(Lende, '63a,b and Pubols e t al., '76). It would appear that the neurons giving rise to cortical-LRN axons are relatively dispersed since the connection is difficult to demonstrate in cases with either small lesions or small injections of 3H-amino acids. Generally comparable connections have been detailed in the cat (e.g., Rossi and Brodal, '56; Kuypers, '58; Nimi et al., '63; Kunzle and Wiesendanger, '741, although in that species most of the fibers are said to arise within the contralateral motor-sensory (pericruciate) cortex and to distribute more extensively within the internal LRN (compare fig. 5 of the present study with fig. 1of Kiinzle and Wiesendanger, '74). Our material clearly shows that most cortical-LRN fibers issue from the pyramidal tract, confirming the similar observation of Kunzle and Wiesendanger ('741, and it is of interest t h a t they do not penetrate the caudal LRN in either species. Since cortical fibers distribute to parts of the LRN which are labelled after HRP injections of the anterior lobe and, to a lesser extent, the paramedian lobule, it appears that cortical discharge potentially affects both regions. Cortical influence over the pyramis and non-spinal portions of the vermis is not as clear, but cannot be negated. With regard to potential indirect influences of the cortex over the LRN, i t should be recalled that in the opossum, as in other species (see Allen and Tsukahara, '74 for review), the motor-sensory cortex projects strongly to the red nucleus (Martin, '68; King et al., '72; Martin et al., '75), particularly to those areas which relay to the LRN (see Martin e t al., '74 and present study), and to portions of the reticular formation (Martin e t al., '75) which provide additional input to the LRN (see below). For a review of the physiological literature on cortical-LRN connections, the reader is referred to Kunzle and Wiesendanger, '74, as well as to Allen and Tsukahara, '74. One of the better known projections to the LRN arises within the red nucleus, (e.g., Walberg, '58; Hinman and Carpenter, '59; Courville, '66; Martin and Dom, '70; Edwards, '72; Miller and Strominger, '73; Mizuno e t al., '73) and the present data reinforces oui- previous conclusion t h a t most rubral-LRN take origin from large-medium sized neurons (Martin e t al., '74). Although there is some confusion in the literature regarding the precise LRN

targets of rubral fibers, it is probably a result of atlas semantics. In the opossum, rubralLRN fibers distribute to the trigeminal division, to certain areas of the external division as well as to restricted portions of the internal division. Our material indicates that rubral-LRN fibers are topographically organized, a finding not previously reported to our knowledge. For example, axons arising from more laterally situated cells project to the ventral edge of the external LRN at the level shown in figure 5 of Andrezik and King, ('771, whereas those issuing from medial regions tend to distribute more dorsally a t comparable levels. Rubral axons which end within the trigeminal division also appear to arise from generally lateral and rostral parts of the nucleus. Although most of the rubral zones of the LRN are rostral and lateral to those which receive spinal input, there appears to be some overlap a t levels generally comparable to that shown in figure 5 of Andrezik and King, ('77). Correlation of our data on the LRN targets of the red nucleus with t h a t related to the location of LRN neurons projecting to different parts of the cerebellum (HRP studies, see below) suggests that the red nucleus influences the lateral part of the anterior lobe and adjacent areas (the lobus simplex and perhaps crus I), the paramedian lobule, the pyramis and nonspinal areas of the vermis. Apparently reticular inputs to the LRN from the midbrain and pons have not been defined previously. Subtraction techniques would allow us to suggest the existence of such connections from degeneration material, but it is the autoradiographic cases which supply definitive data. I t was interesting to note that in some areas amino acid injections had to be fairly large and concentrated to produce positive LRN labelling, a finding which may reflect the relative sparseness of the projection. The available material indicates that the paramedian portion of the caudal pons (the nucleus gigantocellularis) projects quite strongly to the LRN. When the results derived from both methods are compared it can be seen that fibers from the midbrain and pontine reticular formation distribute mainly to medial areas of the internal LRN. Such fibers overlap to some degree with comparably diffuse projections from the spinal cord and vestibular nuclei and seem to prefer those regions which relay to medial zones of the


anterior lobe and possibly to non-spinal areas of the vermis. In addition, there is a suggestion that the caudal parvo-cellular reticular formation and/or closely adjacent areas distribute fibers to portions of the LRN which also receive rubral inputs and there is evidence that neurons in the lateral pons a t the level of the motor trigeminal nucleus provide a small input to the opposite trigeminal LRN. The material processed for formaldehyde induced fluorescence indicates that catecholamine terminals are extensive within the rostral external and trigeminal divisions of the LRN. A portion of this area receives rubral input as well as overlapping projections from the lateral pons (P-401). Although fluorescent neurons are located around the opossum red nucleus (Crutcher and Humbertson, unpublished) and may have been included in some of our rubral lesions and/or placements, few, are present within the confines of the injection site in P-401 (fig. 19). It is possible that some of the catecholamine containing processes within the medial, nonrubral part of the external division take origin within the rostral pons since degeneration is present in the appropriate areas after large pontine lesions, but not after lesions of the red nucleus (compare figs. 6 and 18).2I t should be noted also that extensive label is present in the same medial area of the rostral LRN in a case in which 3H-leucine was placed within the caudal part of the LRN. Since catecholamine containing neurons are located just dorsal to the LRN at all levels, some of them may contribute to the fluorescent varicosities in question. I t is difficult to make injections of 3Hmarkers into the medullary reticular formation without labelling adjacent nuclei, including the LRN itself. Even in cases where the LRN is not visibly involved, it is possible that grains over its neuropil simply reflect dendritic labelling or direct spread through the extra-cellular space. However, the available data can be interpreted as suggesting that a projection to the LRN arises from closely adjacent reticular areas andlor from neurons within its own confines. It is particularly noteworthy that some of the cases with medullary injections provide the only evidence for a substantial non-spinal input to the compact portion of the internal division. The possibility of a major LRN projection from local sources is supported by the finding that only a

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relatively small percentage of synaptic terminals within the LRN degenerate after undercutting of its “major” distal inputs (see Andrezik and King, ’77 and Mizuno e t al., ’75 for comparable results in the cat), Our material contains some evidence for a small LRN projection from the rostral dorsal column nuclei. Although LRN neurons of the cat can be activated by paths ascending through the dorsal funiculus and by trigeminal afferents, most of them are influenced indirectly (Clendenin et al., ’75). I t has been proposed that the relay occurs in “brainstem centers” interposed between the dorsal column nuclei and the LRN, a suggestion further supporting the existence of LRN projections from closely adjacent reticular neurons. It should be noted that LRN activation by such neurons would not necessitate conventional synapses since i t might occur through dendritic interaction. The present report documents the existence of projections to the LRN from the vestibular complex (see Ladpli and Brodal, ’68 for a report of a similar projection in the cat) and the nucleus fastigius (Thomas e t al., ’56; Cohen et al., ’58; Walberg and Pompeiano, ’60).Although vestibular fibers distribute to regions of the internal LRN which are apparently comparable to those illustrated for the cat (fig. 14 of Brodal, ’72) comparison of fastigialLRN targets in the two species is not as clear. I t should be noted, however, that in both animals fastigial axons distribute mainly to the rostral half of the nucleus. Vestibular and fastigial fibers appear to project to parts of the LRN which relay to medial areas of the anterior lobe and perhaps to visual-auditory areas of the vermis. Historically, the best known projection to the LRN is from the spinal cord. Such connections have been studied most extensively in the cat (see Kiinzle, ’73 for review), although they have been reported for other mammals (e.g., Mehler, ’691, including the opossum (Hazlett et al., ’71). Our material indicates that spino-LRN fibers probably arise from all cord levels in the opossum, that they traverse the lateral and ventrolateral funiculi and that they are organized in a fashion generally comparable to that described for the cat (Kiinzle, ’73).The latter author discusses the problems inherent in the interpretation of See note added in proof, page 185.

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spinal-LRN somatotopy and suggests that the lack of a comparable organization as revealed by physiological techniques (e.g., Oscarsson et al., '66; Rosen et al., '731, rather than being incompatible with the anatomical results, is suggestive of a considerable degree of convergence a t spinal levels. Oscarsson ('73) has suggested that spinal-LRN pathways monitor the activity of interneurons involved in reflexes, an activity which is influenced by descending, local and peripheral inputs. Spinal fibers distribute to portions of the opossum LRN which relay to the anterior lobe, the paramedian lobule and the pyramis-areas which also receive direct spinal input (Hazlett et al., '71). The excitation-inhibition evoked in various LRN neurons by different inputs has been reported by numerous authors (the cat, Oscarsson and Rosen, '66; Crichlow and Kennedy, '67; Kitai et al., '67; Bruckmoser et al., '70a,b; Rosen and Scheid, '73a,b; Zangger and Wiesendanger, '73) and the effect of such activity on the cerebellar cortex has been most recently studied by Clendenin et al., ('74a). Clendenin et al. ('74a), Kunzle ('75) and Matsushita and Ikeda ('76) have suggested that the cerebellar target of LRN axons correspond mainly to those areas which also receive direct spinal connections. Such is generally the case in the opossum (compare present results with those of Hazlett et al., '71), but our material also indicates that the LRN projects to the lobus simplex, and crus I as well as to nonspinal areas of the vermis. As reported by Kunzle ('75) for the cat, LRN fibers distribute in a series of longitudinal rows within both the anterior lobe and the paramedian lobule. The segregation of LRN fibers into zones is not as apparent in the pyramis, however, nor in the "non-spinal" areas referred to above. Although evidence for a projection from the LRN to the deep cerebellar nuclei is present in degeneration material (see also Matsushita and Ikeda, '76), it is not as clear in the autoradiographic preparations (see also Kunzle, '75). Studies in the cat (Brodal, '75) and opossum (present study) indicate that most areas of the LRN project to the anterior lobe. However, after even large injections of HRP into the anterior lobe, relatively few neurons are labelled in the trigeminal division and there is often a rostra1 medial portion of the external division which is unlabelled. There is evidence for a subdued somatotopy regarding the

anterior lobe projection from those parts of the LRN which receive lumbosacral versus cervical spinal inputs. In essence LRN regions receiving cervical fibers relay most heavily to presumptive forelimb parts of the anterior lobe, whereas those regions receiving projections from the lumbosacral cord project more to probable hindlimb regions. Our material also shows that the LRN-anterior lobe projection is organized in the medial to lateral dimensions, i.e., neurons in the medial internal division relay most heavily to medial areas of the anterior lobe, whereas those more externally situated, particularly rostrally, tend to distribute to lateral zones. In contrast, the paramedian lobule and pyramis receive connections from more restricted regions of the LRN. As regards the laterality and topography of LRN projections to the paramedian lobule, our findings are generally comparable to those of Brodal ('75). It is possible to interpret our data as providing evidence for a topographically organized projection to the pyramis, but details of the organization are fuzzy. Although evidence for such projections was not clear in either degeneration or autoradiographic material, the HRP experiments indicate that the LRN relays to cerebellar regions generally considered to respond to visual and auditory stimuli (e.g., Snider and Stowell, '44). The apparent negative results obtained from the orthograde tracing experiments might be explained by failure to destroy or label the appropriate neurons. Since small HRP injections of the cerebellar cortex often result in labelling throughout much of the LRN, a marked degree of convergence must exist. On the other hand, there are some regions of the LRN which are labelled after HRP injections into widely separate cerebellar cortical areas suggesting that a t least some LRN neurons have axons which branch within the cerebellum, distributing to more than one cortical zone. Similar conclusions have been suggested for the cat by Brodal ('75). Neurons of the trigeminal LRN are extensively labelled after HRP is introduced into the lobus simplex or when the marker spreads into that region, and it is of interest that Clendenin et al. ('75) reports the comparable region can be activated by trigeminal stimulation. It should be noted, however, that after lobus simplex injections neurons are labelled in non-trigeminal areas of the LRN as well,


perhaps correlating with the finding that some indirect spinal information reaches the same area of the cerebellum through the LRN (Clendenin et al., '75). The opossum LRN is extremely complex both in the organization of its inputs and in its projections to the cerebellum. Each of its afferent projections have specific terminal zones, but they overlap to varying degrees with connections from different areas of the neural axis. Unlike the basilar pons and inferior olive (unpublished data), the opossum LRN does not project to all major areas of the cerebellar cortex. In addition to confirming the inputs to the opossum LRN which have been reported for certain placental species, we have provided for the first time: (1)direct evidence that the rubral+LRNprojection is topographically organized, (2) evidence for a LRN projection from the reticular formation and (3) the relation of many LRN inputs with the projections of the same nucleus to the cerebellum. Although we do not intend to imply that the opossum LRN is completely comparable to that of divergent and highly specialized placental mammals (e.g., the cat), the available data suggests that the similarities outweigh the differences. ACKNOWLEDGMENTS

This investigation was supported by the United States Public Health Service, Grants NS-07410 to Doctor Martin and NS-08798to Doctor James S. King as well as by the Bremer Foundation Fund, "he Ohio State University, College of Medicine. The authors wish to thank Mrs. Nan Patterson for excellent technical help, Ms. Malinda Amspaugh for typing the manuscript and putting up with the senior author and Mr. Gabriel Palkuti whose photographic expertise is greatly appreciated by the Department of Anatomy. In addition, some of the horseradish peroxidase cases were prepared for another study by Doctor Martine RoBards during her postdoctoral training a t The University of Virginia, Charlottesville. We are indebted for the opportunity to use her material. The senior author also extends his thanks to Doctor Richard Dom of The Medical University of South Carolina for sharing the results from some of his cases. LITERATURE CITED Allen, G. I., and N. Tsukahara 1974 Cerebrocerebellar communication systems. Physiol. Rev., 54:957-1006.

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Note added in proof: In a recent experiment the locus coeruleus, pars a (nucleus subcoeruleus) and the adjacent reticular formation were labelled with 3H-leucine. In t h a t case labelled axons could be followed to rostra1 levels of the external LRN and to most of the internal division more caudally. The caudal part of the internal division is traversed by numerous labelled bundles, particularly ipsilaterally; but terminal label does not appear to be present in its compact portion. Terminal label is most extensive in the reticular formation just dorsal to the LRN.

The lateral reticular nucleus of the opossum (Didelphis virginiana). I. Conformation, cytology and synaptology.

The inferior olivary nucleus of the opossum (Didelphis marsupialis virginiana), its organization and connections.

The vestibular complex of the American opossum didelphis virginiana. II. Afferent and efferent connections.

Development of the neonate opossum (Didelphis virginiana).

Megakaryocytopoiesis in the liver of the developing opossum (Didelphis virginiana).

Lung lesions in an opossum (Didelphis virginiana) associated with Capillaria didelphis.

The response of rubrospinal neurons to axotomy in the adult opossum, Didelphis virginiana.

General observations on the growth and development of the young pouch opossum, Didelphis virginiana.

Response to cryptorchidism of the testis and epididymis of the opossum (Didelphis virginiana).

Morphological observations on the metanephros in the postnatal opossum, Didelphis virginiana.

Cellular mechanisms involved in cyclic stromal renewal of the uterus. I. The opossum, Didelphis virginiana.

Development of catecholaminergic projections to the spinal cord in the North American opossum, Didelphis virginiana.

Laterality and topography of cerebellar cortical efferents in the opossum (Didelphis marsupialis virginiana).

Spectrophotometric measurement of hemoglobin oxygen saturation in the opossum Didelphis virginiana.

Morphological observations on the mesonephros in the postnatal opossum, Didelphis virginiana.

Efferent connections of the caudate nucleus in the Virginia opossum.

Physiological interactions between enkephalin and excitatory amino acids in the cerebellar cortex of the opossum Didelphis marsupialis virginiana.

CUTANEOUS EPITHELIOTROPIC T-CELL LYMPHOMA WITH METASTASES IN A VIRGINIA OPOSSUM (DIDELPHIS VIRGINIANA).

Aerobic bacteria cultured from the mouth of the American opossum (Didelphis virginiana) with reference to bacteria associated with bite infections.

Organization of midbrain catecholamine-containing nuclei and their projections to the striatum in the North American opossum, Didelphis virginiana.

West Nile virus isolated from a Virginia opossum (Didelphis virginiana) in northwestern Missouri, USA, 2012.

Postnatal development of the epidermis in a marsupial, Didelphis virginiana.

Inhibitory spinal paths to the lateral reticular nucleus.

Connections of the parabrachial nucleus with the nucleus of the solitary tract and the medullary reticular formation in the rat.