Three Bulbospinal Pathways from the Rostra1 Medulla of the Cat: An Autoradiographic Study of Pain Modulating Systems ALLAN I. BASBAUM, CHARLES H. CLANTON AND HOWARD L. FIELDS Department ofNeurology, Physiology and Anatomy, University ofCalifornia Sun Francisco, Sun Francisco, California 94143

ABSTRACT Small amounts of 3H-leucinewere injected into discrete regions in the rostral medulla of the cat. Descending projections from these sites were studied with autoradiographic methods. On the basis of differential projections t o the medulla and spinal cord, three distinct regions were delineated. Nucleus reticularis gigantocellularis (Rgc), located dorsally in the medullary reticular formation, projects primarily to "motor" related sites, including cranial motor nuclei VI, VII, XII, nucleus intercalatus, and a part of the ipsilateral medial accessory olive. The projection to the spinal cord is primarily via the ipsilateral ventrolateral and contralateral ventral funiculi. The Rgc terminal field is in lamina VII and VIII ipsilateral and lamina VIII contralateral t o the injection site. In contrast, nucleus raphe magnus, (NRM) located ventrally, in the midline of the rostral medulla projects primarily to structures with known nociceptive and/or visceral afferent input. These sites include the solitary nucleus, the dorsal motor nucleus (X) and the marginal and gelatinous layers of the spinal trigeminal nucleus caudalis. The projection to the spinal cord is bilateral, via the dorsolateral funiculus. Terminal fields are found in the marginal zone and the substantia gelatinosa of the dorsal horn, and more deeply in lamina V, medial VI and VII. Nucleus reticularis magnocellularis (Rmc), located lateral to NRM and ventral to Rgc, has an overlapping projection with NRM, but the projection is ipsilateral. This difference between Rmc and Rgc is correlated with cytoarchitectural features of the two regions. The possibility that the raphe-spinal pathway in the DLF mediates opiate and brain stimulation-produced analgesia is discussed. The functional importance of brainstem connections with the spinal cord is well established. Sherrington ('06) demonstrated that profound inhibition of spinal cutaneous reflexes arises from the brainstem in decerebrate cats. Later, Magoun and Rhines ('46) showed that stimulation of the medial medullary reticular formation can either facilitate or inhibit spinal monosynaptic reflexes depending on the region stimulated, and Hagbarth and Kerr ('54) demonstrated brainstem inhibition of ascending spinal pathways. These findings have been confirmed and extended by numerous workers (for review see Pompeiano, '73). Despite this long standing appreciation of the powerful brainstem influences on spinal cord function, the anatomical basis for this control has remained relatively obscure. The J. COMP. NEUR. (1978)178: 209-224.

inhibitory actions described by Magoun and Rhines ('46) are abolished by a lesion of the ventrolateral funiculus of the spinal cord. A pathway in the ventrolateral funiculus also mediates brainstem inhibition of primary afferent terminals (Jankowska et al., '68). Presumably, the anatomical substrate for this inhibition is a medullary reticulospinal projection in the ventrolateral funiculus of the cat which was demonstrated by retrograde chromatolytic (Torvik and Brodal, '57) and silver degeneration techniques (Nyberg-Hansen, '65; Petras, '67). Similar pathways have been described in the monkey (Kuypers e t al., '62) and in the opossum (Beran and Martin, '71; Martin and Dom, '71). Physiological and neurological studies in cat (Dougherty e t al., '70) and monkey (Chambers et al., '70) have shown that there are also

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bulbospinal inhibitory pathways in the dorsolateral funiculus (DLF). Lundberg and his colleagues studied the effects of ventromedial medullary reticular and midline raphe stimulation on transmission from high threshold afferents (flexor reflex afferents or FRA's). Stimulation of the serotonin-rich raphe nuclei exerts an inhibitory action upon FRA transmission which is abolished by bilateral DLF section (Holmqvist and Lundberg, '59; Engberg et al., '68b,c). Recent studies also indicate that descending pathways in the DLF are required for both opiate and stimulation produced analgesia (Basbaum e t al., '76b, '77; Murfin et al., '76; Price et al., '76b). The serotonin containing raphe-spinal fibers (Dahlstrom and Fuxe, '65) undoubtedly mediate part of the monoaminergic inhibition of FRA transmission (Engberg e t al., '68a). Increased 5HT fluorescence is seen in raphe neurons after spinal lesions, (Dahlstrom and Fuxe, '65) and after DLF lesions retrograde chromatolysis is seen in raphe neurons (Brodal et al., '60). However, except for a brief description of Marchi degeneration in the DLF after reticular lesions (Kuru et al., '591, no anatomical substrate for the dorsal reticulospinal system has been found; for this reason it was assumed to be a polysynaptic system (Engberg e t al., '68b,c). Although the physiological separation of these three descending systems is clear, their precise origin within the brainstem is uncertain. The ventromedial reticular region may contribute to the dorsolateral funiculus (DLF) pathway but i t is not known whether or to what extent more dorsal reticular structures, e.g., nucleus reticularis gigantocellularis (Rgc) also contribute to the DLF. Conversely, it is not known to what extent ventromedial regions contribute to the ventral quadrant reticulospinal pathway. Cytoarchitectural analysis does not reveal major differences between the dorsal and ventromedial regions(Taber e t al., '60; Taber, '61). Furthermore, recent studies using the techniques of retrograde transport of horseradish peroxidase (HRP) (Kuypers and Maisky, '75) and autoradiographic analysis of the anterograde transport of labeled amino acids (Bobillier et al., '76) have failed to distinguish the efferent connections of these regions. Because of the functional importance of the medial medullary reticular formation and raphe nuclei and the possibility of more accurately tracing efferent connections using

autoradiographic techniques (Cowan et al., '72; Edwards, '75) we have reexamined the ascending, descending and local connections of this region. This report analyzes differential descending projections of the nucleus reticularis gigantocellularis (Rgc) located dorsally, nucleus reticularis magnocellularis (Rmc) located ventromedially and the midline nucleus raphe magnus (NRM). A preliminary report of some of this work has been published (Basbaum et al., '76b). METHOD

Sixteen cats were used in this study. One hundred microcuries L--(4,53H)-1eucinespecific activity 30-55 Ci/mmole) was desiccated under vacuum and reconstituted with 0.9% saline to yield a concentration of 200 pCi/bl. A 10-p1 Hamilton syringe, with its plunger fixed to a Kopf hydraulic microdrive, was used for intracranial injections. Injections were made by passing the needle through the cerebellum at an angle of approximately 30-40" from the vertical. The medullary targets were taken from the stereotaxic atlas of Berman ('68). The target for NRM was frontal plane P6.0-7.0, V-8.0, LO.0; for Rmc P7.0, V-7.5, L1.5; for Rgc P7.0, V-6.0, L1.5. In this report nucleus reticularis gigantocellularis, Rgc, and nucleus reticularis magnocellularis, Rmc, are equivalent to the gigantocellular and magnocellular tegmental fields of Berman ('68) respectively. Injections of 0.1-0.5 jd were made over a period of 45 minutes to l hour. The syringe was left in place for 30 minutes after injections, at which time the plunger was retracted to aspirate excess fluid and the needle was removed from the brain. Both short (24 hour and 48 hour) and long (11 day) survival times were used. This allows visualization of both tracts and terminal fields. Even the shortest survival times (24 hours), however, reveal considerable labelling of fiber tracts. After the appropriate surival period, the animals were anesthetized with Nembutal (60 mg/kg) and perfused intracardially with an 0.1 M phosphate buffered isotonic saline wash followed by fixation with 2 1 of 4%paraformaldehyde and 0.5%glutaraldehyde in 0.1 M phosphate buffer a t 5°C. The brainstem and spinal cord were blocked, placed in 30% phosphate buffered sucrose, sectioned serially at 30 p on a freezing microtome, mounted on gelatinchrome-alum slides and defatted in xylene. The slides were coated with Kodak NTB-2

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Fig. 1 Examples of 3H-leucine injection sites. A, nucleus reticularis gigantocellularis (Rgc); B, Nucleus reticularis magnocellularis (Rmcf ;C,Bilaterally in nucleus raphe magnus (NRM) and unilaterally in rostra1 Rmc. Duration of exposure of injection sites was two months. The same exposure time was used for the projection sites. x 6.75.

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emulsion (1:l with water), dried and exposed in the dark a t 4°C for periods of three weeks to three months. At the appropriate time the slides were developed in full strength D-19, for two and one-half minutes a t 19OC and fixed in Rapid Fix without hardener. Alternate sections were lightly stained with neutral red. Sections were examined with dark and bright field microscopy. RESULTS

Nucleus reticularis gigantocellularis (Rgc) All injections of Rgc were unilateral. If the injected area does not include either the vestibular nuclei or the ventral nucleus reticularis magnocellularis, there is little variation in the descending projections of the nucleus reticularis gigantocellularis (Rgc). For this reason one representative case will be described in detail. The injection site from this animal is illustrated in figure l a . Descending tracts Fibers radiate from the injection site in all directions; however two distinct fiber systems are formed. One of these derives from fibers which leave the rostral part of Rgc, course dorsomedially to the floor of the fourth ventricle and cross the midline (figs. 2, 3A). In Golgi preparations Cajal (‘52) described axons of gigantocellular neurons which take the same trajectory. Some of these axons turn rostrally and ascend to midbrain and thalamus (Basbaum e t al., ’76a) however, the majority turn caudally and form a large descending bundle in the contralateral medial longitudinal fasciculus (MLF) (fig. 3A). In its descent through the medulla, this tract is displaced laterally by the decussating pyramidal tract fibers, but maintains a position in the dorsal part of the MLF, just medial to the hypoglossal nucleus, to which reticular fibers are bilaterally distributed (figs. 3B,D). At the spinal cord, this bundle is located in the contralateral ventral funiculus (VF), and is seen a t all spinal levels. A few fibers are displaced laterally and continue in the contralateral ventral part of the lateral funiculus of the spinal cord. This crossed pathway is presumably the source of the fiber degeneration in the ventral funiculus described by Nyberg-Hansen (‘65) after medullary reticular lesions. He attributed this to cut fibers of passage originating in the pontine reticular formation. A similar tract

has been described by Busch (’64) and Petras (’67). The second major descending system is formed ipsilateral to the injection site. Axons leave the injection site and course ventromedially to form a bundle lying just off the midline and ventral to the descending bundle in the contralateral MLF. I t is situated in the ventromedial MLF, just dorsolateral to the inferior olive (fig. 3C). In the caudal medulla, some of the fibers shift laterally to form a large ipsilateral ventrolateral component separated from a smaller ventral component by the roots of the twelfth nerve. This arrangement persists a t all spinal levels, resulting in a large fiber tract in the ventral part of the lateral funiculus and a considerably smaller tract lying in the most ventral part of the ipsilateral ventral funiculus. No projection to the spinal cord via the dorsal part of the lateral funiculus was found. The grain density and thus presumably, the number of reticulospinal fibers in the VF contralateral to the injection site is much greater than it is in the ipsilatera1 VF. The large ipsilateral VF pathway described by Petras (‘67) is probably the result of cutting the predominantly ipsilateral pontospinal fibers (Torvik and Brodal, ’57).

Terminal distribution within the medulla and spinal cord In general, the terminals of Rgc efferent fibers are concentrated within “motor” components of the medulla and spinal cord, bilaterally, with a larger ipsilateral component (fig. 2). Particularly dense projections are seen in several cranial motor nuclei, V, VI, VII and XII. The projection is dense bilaterally, except that the abducens nucleus receives a much heavier contralateral projection. Bilateral projections are found in the nucleus praepositus hypoglossi, nucleus intercalatus, and nucleus X of Brodal and Pompeiano (’57). There is a dense projection to the ipsilateral medial accessory olive which is restricted to its caudal, dorsal aspect (fig. 3 0 . Smaller projections are found to the nucleus raphe magnus of the rostral medulla, the ipsilateral paramedian reticular nucleus, the lateral vestibular nucleus of Deiters, and to the asomatotopic component of the dorsal column nuclei located ventral to the cell cluster region (Kuypers and Tuerk, ’64; Millar and Basbaum, ’75). There is a diffuse projection throughout the

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A bbreuiations ap, area postrema rp, nucleus raphe pallidus cr, restiform body vde, inferior vestibular nucleus ecn, external cuneate nucleus vl, lateral vestibular nucleus g, genuof VII vm, medial vestibular nucleus vs, superior vestibular nucleus IOm/iom, medial accessory olive lcn, lateral cuneate nucleus ts, solitary tract lrn, lateral reticular nucleus x, nucleus x nc, cuneate nucleus Vc, spinal trigeminal nucleus caudalis ng, gracile nucleus Vip, spinal trigeminal nucleus interpolaris npr, nucleus praepositus hypoglossi Vo, spinal trigeminal nucleus oralis NRMhm, nucleus raphe magnus Vpr, principal sensory nucleus pc, nucleus reticularis parvocellularis VI, abducens nucleus Rgc/gc, nucleus reticularis gigantocellularis VII, facial nucleus X, dorsal motor nucleus Rmc, nucleus reticularis magnocellularis XII, hypoglossal nucleus

Fig. 2 Drawings of representative transverse sections through the medulla and spinal cord, illustrating autoradiograms of the descending projections originating from an injection into Rgc. Large dots represent single or multiple fibers and fiber bundles cut in cross-section; lines represent fibers coursing in the plane of the section; small dots represent probable sites of terminatioe.

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medullary reticular formation, again bilaterally, but heavier ipsilaterally. Of particular interest is a dense projection to the contralatera1 part of the reticular formation which is the mirror image of the injection site. As Walberg ('74) demonstrated, this projection derives from axons which course directly across the midline from the injection site. The following regions do not receive a projection from Rgc: the solitary nucleus, the dorsal motor nucleus of the vagus, the spinal trigeminal nucleus, the medial and descending vestibular nuclei, the dorsal accessory and principal olive and the external cuneate nucleus. Projections to the spinal gray matter are in agreement with the findings of both Petras ('67) and Nyberg-Hansen ('65). Except for a small terminal field in the lateral aspects of lamina V of the dorsal horn, which was described by Petras ('671, the projection from Rgc is bilaterally to ventral horn. The major ipsilateral projection is to lamina VII of Rexed ('52).A smaller distribution is seen in lamina VIII. Although some evidence for a terminal field in lamina IX is present, the silver grain pattern indicates that it is primarily axons coursing through lamina IX. Contralatera1 to the injection, the projection is primarily to lamina VIII, with a smaller projection to lamina VII. Some fibers which distribute to contralateral lamina VIII arise from axons crossing in the anterior commissure of the cord, but the majority derive from the descending tract in the contralateral ventral funiculus.

spinal V receives a heavy projection from fibers which course dorsolaterally, sweeping through and possibly terminating in the dorsaImost part of the facial nucleus. A small component courses directly dorsalward to the floor of the fourth ventricle but so few silver grains are present that a terminal field is difficult to distinguish. Some fibers turn caudally in the MLF, but few can be traced caudal to the hypoglossal nuclei. The primary descending bundle is located just lateral to NRM. This position is maintained to the level of the inferior olive. The descending fibers intercalate with the cells of the olive but do not terminate within it. The fibers gradually assume a more lateral position, just ventral to the spinal trigeminal nucleus caudalis. At the level of the inferior olive a branch of the main descending bundle peels off and courses dorsolaterally through the caudal medullary reticular formation to the solitary nucleus. The terminal field of these fibers is scattered throughout the medial medullary reticular formation, but is most concentrated within the solitary nucleus. Both magnocellular and parvocellular subcomponents of the solitary nucleus receive a heavy projection from NRm (figs. 5A,B). In contrast, the gelatinous subdivision located rostrally (Messen and Olszewski, '49) is distinctly clear of grains (fig. 5A). Subpostrema receives a projection as does the dorsal motor nucleus of the vagus. Caudal to the obex, a dense terminal field is found in the midline commissural nucleus (which probably constitutes the caudal extent of the solitary nucleus [Torvik, '561).

Nucleus raphe magnus and nucleus reticularis magnocellularis Injections directed a t the nucleus raphe magnus (NRM) were always bilateral, and to different degrees, spread into the adjacent reticular formation (Rmc). Other injections were focused in Rmc and did not spread into NRM. By comparing the projections deriving from combined injections of Rmc/NRM vs. Rmc alone, the differential projections from NRM and Rmc can be discerned. Figure 1C illustrates a n injection which completely fills NRM and spreads heavily into Rmc. In general the descending projection is bilateral with some exceptions attributable to the predominantly unilateral involvement of Rmc. Figure 4A illustrates the primary descending trajectory of efferent fibers from the injection site of figure 1C. The oral nucleus of

Fig. 3 Darkfield photomicrographs showing the medullary course and terminal fields of Rgc efferent fibers. A A section taken just caudal to the injection site in Rgc. Axons course dorsomedially from the site of injection and cross beneath the floor of the fourth ventricle to form a large fiber bundle in the contralateral MLF. A small part of the ipsilateral descendingfiber bundle can be seen in the lower left. x 160. B This fiber bundle can be traced caudally to the level of the hypoglossal nuclei. A dense terminal field is found bilaterally in XII. X 70. C Photomicrograph illustrating a portion of the ipsilateral descending fiber bundle originating from Rgc. Note absence of label over the twelfth nerve (NXII), despite heavy label surrounding the cell bodies of XI1 (fig. 3B). A dense terminal field overlying a restricted part of the medial accessory olive (IOm) is also present. X 70. D Higher magnification of a selected region of the contralateral descending fiber bundle in the MLF (inset fig. 3B). The profile running in the plane of section may represent one or more axons, in a field of axons cut in cross section. Some of the profiles cut in cross section measure 2030 A./ in diameter. X 770.

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Fig. 4A Drawings of representative transverse sections through the medulla and spinal cord, illustrating autoradiograms of descending projections originating from an injection into NRM and Rmc. This injection site corresponds to t h a t of figure 1C. B Descending projections from an injection concentrated in Rmc, with no involvement of NRM. This injection site corresponds to t hat of figure 1B. Symbols as in figure 2.

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Fig. 5 Darkfield photomicrographs and corresponding Nissl sections, illustrating terminal field pattern at two transverse planes in the solitary nucleus. The injection site included NRM and Rmc. A Note dense projection to the parvocellular (NSpc) and magnocellular (NSmc) components and absence of a projection to the gelatinous component (GEL) and the solitary tract (TS).x 80. B Transverse section caudal to A a t level of area postrema (AP).Note dense projection as in A; but absence of a projection to the hypoglossal nucleus (XII). X 80.

Crossing fibers are found in the commissural nucleus just dorsal to the central canal. A dense terminal field is present in both the marginal and gelatinous layers of the spinal trigeminal nucleus caudalis and the adjacent reticular formation, ventral to the magnocellular layer. In contrast, the magnocellular layer itself contains very few grains (fig. 6A). Predominantly unilateral projections are found in the nucleus praepositus hypoglossi, the medial part of the lateral reticular nucleus, the “ventral horn” of the caudal medulla, and very lightly in XI1 and nucleus inter-

calatus. These unilateral fields are located ipsilateral to the Rmc injection. Many sites which receive a projection from Rgc receive no input from Rmc/NRM: this includes cranial nerve VI, most of VII, the medial accessory olive and the paramedian reticular formation. The projection to the spinal cord from this injection is similarly distinct from the projection from Rgc. The fibers distribute to the cord via the dorsal part of the lateral funiculus bilaterally, and the ventral part of the lateral funiculus, on one side (fig. 4A). At C1, the

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fibers in the DLF form a crescent, just lateral to the cortieospinal tract and medial to (or partially intermingling with) ascending dorsal spinocerebellar t r a c t fibers (fig. 4A: Basbaum et al., '76b). These bundles continue caudally through the spinal cord but become less compact. The terminal distribution is concentrated bilaterally in the dorsal horn and intermediate gray. The heaviest projection is to lamina I (marginal) and I1 (substantia gelatinosa). This projection is most prominent a t cervical and lumbar enlargements. Dense clusters of grains are also found in lamina V, medial VI and VII (fig. 6B). Some fibers cross in the posterior commissure of the cord. Lamina IV receives a much smaller projection. This spinal dorsal horn distribution is thus analogous to that found in the spinal trigeminal nucleus caudalis, its medullary homologue. At both thoracic and lumbar segments, the intermediolateral column (containing preganglionic cell bodies) also receives a bilateral projection. Lamina VII and VIII, ipsilateral to the Rmc injection, also receive a projection, presumably from fibers in the VLF. There is no projection to the lateral cervical nucleus. An injection into Rmc (fig. lB), which does not spread into NRM, produces a similar picture, except that the projection is predominantly ipsilateral (fig. 4B). Since Rmc does not project bilaterally, the heavy bilateral label found after combined NRM/Rmc injections (e.g., fig. 1C) must originate in NRM. Thus, NRM and Rmc are independent sources of afferents to the spinal trigeminal nucleus, solitary nucleus and, by way of the DLF, to spinal dorsal horn. Rmc has additional projections, to the nucleus praepositus hypoglossi, the lateral reticular nucleus and by way of the VLF to lamina VII and VIII of the spinal grey. Rmc also projects to the nucleus raphe magnus and pallidus; the latter projection is particularly dense (fig. 4B). DISCUSSION

Cytoarchitectural and retrograde cell degeneration studies have described three major cell groups in the medullary reticular formation: nucleus reticularis gigantocellularis, and its caudal continuation, nucleus reticularis ventralis; nucleus reticularis parvocellularis and nucleus reticularis paramedianus (Meesen and Olszewski, '49; Brodal, '57). Nucleus reticularis gigantocellularis forms the bulk of the medullary reticular formation

and is the source of long ascending and descending axon systems, (Torvik and Brodal, '57). According to Taber ('611, Rgc contains four cell types, giant, large, medium and small. The giant cells, which give the region its name, are located dorsally, while the large cells are found ventrally. This difference in the distribution of the giant and large cells was observed in the rat, (Valverde, '61, '62). The same distribution in the cat prompted Berman ('68) to differentiate between a dorsal gigantocellular and a ventral magnocellular tegmental field. The midline medullary raphe nuclei are distinct from the adjacent reticular formation (Taber et al., '60). However, the nucleus raphe magnus (NRM), which extends from the rostral pole of the inferior olive to the rostral pole of the superior olive has cytoarchitectural features in common with Rgc (Taber, '61). Furthermore, NRM and Rgc receive afferents via the spinal ventral quadrant (Mehler, '69) and both give rise to medullospinal fibers. Because of these anatomical similarities it has been suggested that Rgc and NRM are functionally related (Petras, '67). In contrast, the present results demonstrate that the dorsomedial (Rgc), ventromedial (Rmc) and midline (NRM) regions of the rostral medulla give rise to anatomically distinct descending pathways. Taken together with pharmacological and physiological studies, these results suggest important functional differences between these three regions. Our autoradiographic analysis of descending projections of Rgc of the cat is in general agreement with previous studies using silver degeneration techniques (Nyberg-Hansen, '65; Petras, '67). Although Nyberg-Hansen ('65) attributed degeneration in the ventral funiculus after lesions in Rgc to cut ponto-spinal fibers, it is now certain that Rgc is a source of axons in both the ventrolateral and the ventral funiculus, confirming physiological observations (It0 e t al., '70). In contrast, the more ventrally situated nucleus reticularis magnocellularis (Rmc) and the midline nucleus raphe magnus (NRM) are a major medullary source of fibers in the dorsolateral funiculus. NRM projects chiefly to "sensory" nuclei of the medulla, including the solitary nucleus and the spinal trigeminal nucleus caudalis, and via the DLF to the spinal dorsal horn and intermediolateral column. The projection from Rmc overlaps with that from NRM (on one side) and also extends into

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Fig. 6 Darkfield photomicrographs illustrating terminal field pattern in the medulla and spinal cord after injection in NRM/Rmc. A In the spinal trigeminal nucleus caudalis the silver grains are concentrated over the marginal and outer gelatinous layers, but are much less dense in the magnocellular layer (mag). X 60. B In the cervical dorsal horn the silver grains are similarly concentrated over lamina I and I1 (substantia gelatinosa). Descending tract of V in A, and primary afferent fibers traversing the dorsal horn in B are unlabelled. Arrows point to a second dense projection to the reticular formation deep to the magnocellular layer (A), and to the analogous region in the dorsal horn, lamina V, medial VI and VII (B). DC denotes dorsal columns. x 75.

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some "motor" regions of the medulla and spinal cord. Both Nyberg-Hansen ('65) and Petras ('67) made large lesions of the reticular formation of the medulla, including Rmc and Rgc. Some of their lesions were, in fact, centered in Rmc, not Rgc. Their lesions did not encroach on NRM, but some of the descending axons of NRM as well as Rrnc must have been severed. Despite this, only ventral reticulospinal pathways are described and except for some sparse degeneration in lateral lamina VI (Petras, '671, there is no description of degeneration in the DLF or in the dorsal horn. The failure of reduced silver techniques to stain small myelinated and unmyelinated axons may partly explain these results. I t is possible that degeneration seen in the DLF was ignored because of the difficulty in distinguishing degeneration resulting from damage to corticospinal or rubrospinal axons of passage from that produced by a lesion to ventromedial reticular structures. A recent autoradiographic study of the descending projections from NRM described pathways in the DLF, VLF and ventral funiculus (Bobillier e t al., '76). The presence of ventral pathways attributed to NRM was no doubt due to the large injections of C14 leucine which included Rgc and Rmc as well as the midline raphe. Our results are, however, supported by the electron microscopic demonstration of degenerating axons in the DLF and terminals in lamina I and substantia gelatinosa after raphe magnus lesions in the opossum (Goode, '76). The distinction between the descending projection of the dorsal Rgc and those of the ventromedial NRM/Rmc region is thus firmly established. Although NRM and Rmc have overlapping descending projections, several lines of evidence suggest that Rmc is not merely a lateral extension of NRM. Since enhanced 5HT fluorescence is observed in NRM, but not Rmc, after spinal section (Dahlstrom and Fuxe, '65) bulbospinal Rmc neurons probably are not serotonergic. Furthermore, physiological studies have demonstrated two distinct ventromedial medullospinal inhibitory systems t h a t descend in the DLF (Holmqvist and Lundberg, '59; Engberg et al., '68b,c). One originates in the midline raphe (NRM) and is monoaminergic (Engberg et al., '68a); a second, the dorsal reticulospinal system, originates in Rmc, survives large lesions of the

raphe and is not modified by monoaminergic blocking agents (Engberg e t al., '68a). The two systems have also been distinguished by the latency of their inhibitory action on spinal cord interneurons. Conduction velocities for the dorsal reticulospinal system and the raphe spinal pathway were estimated to be 20 m/sec and 1 m/sec, respectively (Engberg e t al., '68a). The latency data are consistent with the assumption t h a t the raphe-spinal projection is unmyelinated (Dahlstrom and Fuxe, '65). However, Willis e t al. ('771, have questioned this assumption and we have been unable to find slowly conducting raphe magnus fibers using antidromic activation from cervical cord (Anderson e t al., '77). Finally, we have recently demonstrated that the retrograde transport of horseradish peroxidase from lumbosacral cord to neurons in Rmc and NRM is preserved after large lesions of the thoracic cord which spare the DLF. However, lateral hemisection of the cord blocks transport to ipsilateral Rmc neurons. In contrast, dorsal hemisection (sparing the ventral cord) blocks all transport to NRM, but some label in Rmc is maintained (Basbaum and Fields, '77). This is consistent with the autoradiographic studies showing that Rmc has primarily a n ipsilateral projection, to the spinal cord via the DLF and VLF, and that NRM projects bilaterally via the DLF. There are thus two distinct bulbospinal pathways in the DLF. We suggest that the DLF projection originating in Rmc is the nonaminergic dorsal reticulospinal system described by Engberg e t al. ('68b,c). Our preliminary HRP studies have also demonstrated that the efferent fibers from nucleus raphe pallidus descend in both the dorsal and the ventral quadrant. Nucleus raphe pallidus is thus a possible source of ventral quadrant bulbospinal serotonergic inhibition of spinal monosynaptic reflexes (Clineschmidt and Anderson, '70).The inhibition of the monosynaptic reflex which is abolished by ventral cord lesions (Magoun and Rhines, '46) may originate in Rgc, Rmc, or both. In summary, the present studies indicate t h a t there are three spinofugal systems arising in the rostra1 medulla. One pathway descends in the DLF bilaterally and terminates in sensory and autonomic structures. A second, from Rmc, descends in the ipsilateral DLF and VLF. The third pathway arises from the more dorsal Rgc, descends in the ipsilat-

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B

A Fig 7 Summary diagram of the origm and course of t h e bulbospinal pathways from rostral, medial medulla A The origin of the three bulbospinal pathways is illustrated on a transverse section of the rostral me dulla taken from the atlas of Berman ('68). Although not indicated, there is presumably overlap in the cells of origin of the pathways. B Approximate location of the three bulbospinal pathways in the cervical enlargement. Rgc (dots) projects primarily via the ipsilateral ventrolateral and the contralateral ventral funiculus. Rmc (vertical lines) projects via t h e ipsilateral dorsolateral and ventrolateral funiculi. NRM (horizontal lines) projects bilaterally via the dorsolateral funiculus. Arrows are directed toward t h e regions of densest termination within the gray matter.

era1 VLF and contralateral VF and terminates primarily in motor nuclei. Figure 7 schematically summarizes the three bulbospinal pathways and their origin in the medulla. This anatomical distinction is supported by behavioral and physiological data. Casey ('71) has demonstrated that stimulation in Rgc elicits escape behaviour in cats. In contrast, NRM stimulation produces profound analgesia in cats (Oliveras e t al., '75) and rats (Oleson and Liebeskind, '75; Proudfit and Anderson, '75). The analgesia produced by activation of NRM is probably mediated by a n inhibitory action of NRM on selected neurons of the trigeminal nucleus caudalis and the spinal dorsal horn. Unit recordings in nucleus caudalis have shown that cells with nociceptive inputs are concentrated in the marginal layer and a t

the border between the magnocellular layer and the adjacent reticular formation (Mosso and Kruger, '73; Yokota, '75; Price e t al., '76b) -precisely those sites where NRM projects most densely. Similarly, in the spinal dorsal horn, the NRM projection overlaps regions where units responsive to noxious inputs are concentrated; namely lamina I (Christensen and Perl, '70) and lamina V (Wall, '67). In contrast, the magnocellular part of trigeminal nucleus caudalis and its dorsal horn analogue, lamina IV, which contain neurons predominantly responsive to innocuous stimulation (Wall, '67; Price e t al., '76) receive a much smaller projection from Rmc/NRM. Electrical stimulation of NRM strongly inhibits the discharge of the nociceptive lamina I and V neurons in cat dorsal horn (Le Bars et al., '74; Basbaum et al., '76b; Fields e t al., '77).

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Cells in the region of lamina IV which do not have nociceptive inputs are unaffected (Fields e t al., '77). In the primate, spinothalamic tract neurons with nociceptive inputs are also inhibited by NRM stimulation (Beall et al., '77; Willis e t al., '77). In both c a t ' a n d monkey, NRM induced inhibition of nociceptive dorsal horn neurons is reduced by lesions of the DLF (Fields et al., '77; Willis et al., '77) suggesting that the inhibition is mediated by axons from NRM which terminate near neurons with nociceptive inputs. There is also a dense projection from NRM to the substantia gelatinosa. Some neurons in this region respond to input from small diameter peripheral afferents but probably do not contribute to ascending pathways (Kumazawa and Perl, '76). This may indicate that part of the inhibition exerted by NRM is mediated by interneurons in the substantia. The projection from NRM to the solitary nucleus, dorsal motor nucleus (X) and to the intermediolateral column is presumably a substrate for the modulation of autonomic function by raphe stimulation (Adair e t al., '77). Physiological evidence for raphe connections to the intermediolateral column has been described (Henry and Calaresu, '74). Recent studies have suggested that NRM is also a link in the analgesia produced by midbrain electrical and opiate stimulation (Basbaum e t al., '76b; Mayer and Price, '76). Pharmacological (Vogt, '73) or electrolytic lesions of NRM (Proudfit and Anderson, '75) antagonize opiate analgesia. Lesions of the DLF in the rat abolish the analgesic effect of periaqueductal gray stimulation and systemic morphine administration (Basbaum e t al., '76a, '77). The analgesia produced by microinjection of opiates into periaqueductal gray is also abolished by lesions of the DLF (Murfin et al., '76). On the basis of these studies, it has been suggested that lesions of the DLF block analgesia by disrupting the NRM inhibition of spinal pain transmission neurons (Basbaum et al., '76; Mayer and Price, '76). The demonstration that systemic opiate administration excites NRM neurons (Anderson e t al., '77; Deakin et al., '77; Oleson et al., '77) including some that project to spinal cord (Anderson e t al., '771, is consistent with the hypothesis that opiate analgesia is, a t least, partially mediated by NRM projections to spinal cord. In summary, the present autoradiographic studies provide a clear separation of three

major bulbospinal pathways, one originating in the dorsal Rgc; a second in Rmc and a third in NRM. Additional raphe spinal pathways, from raphe obscurus, and raphe pallidus will be examined in a subsequent paper. Although pharmacologically distinct, NRM and Rmc have considerable overlap in the course and distribution of their medullary and spinal projections. The descending projections of Rgc are apparently involved with traditional elements of the motor system (e.g., Nakamura et al., '761, while those of NRM/Rmc are clearly involved in autonomic control and in the modulation of sensory transmission. Of particular interest is the possibility that the descending projection of NRM and perhaps Rmc is the anatomical substrate of a bulbospinal pathway mediating both opiate and stimulation-produced analgesia. That NRM is a significant link in an endogenous analgesiaproducing system is also supported by the close correspondence of its projection with the anatomical distribution of opiate receptor (Pert e t al., '76) and endogenous morphinelike compounds (Elde et al., '76), particularly in the solitary nucleus and in the marginal and gelatinous layers of spinal trigeminal nucleus caudalis and the dorsal horn. The recent immunohistochemical demonstration of leuenkephalin the DLF (Simantov et al., '77) raises the possibility that the brainstem modulation underlying opiate and brain stimulation produced analgesia is mediated via bulbospinal enkephalin-containing axons. ACKNOWLEDGMENTS

We thank Doctors H. J. Ralston and S. D. Anderson for their thoughts and suggestions concerning the manuscript, Ms. M. Liu and Mr. D. Akers, for histological and photographic assistance and Mrs. S. Bornstein for typing the manuscript. This study was supported by NS70777, NS11529, NS05272 and NS11614. LITERATURE CITED Adair, J. R., B. L. Hamilton, K. A. Scappaticci, C. J. Helke and R. A. Gillis 1977 Cardiovascular responses to electrical stimulation of the medullary raphe area of the cat. Brain Res., 128: 141-145. Anderson, S. D., A. I. Basbaum and H. L. Fields 1977 Response of medullary raphe neurons to peripheral stimulation and to systemic opiates. Brain Res., 123: 363-368. Basbaum, A. I., C. H. Clanton and H. L. Fields 1976a Ascending projections of nucleus gigantocellularis and nucleus raphe magnus in the cat. An autoradiographic study. Anat. Rec., 184: 354. 1976b Opiate and stimulus-produced analgesia:

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