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The projection from nucleus raphe magnus and other brainstem nuclei to the spinal cord in the rat: a study using the HRP blue-reaction
Neuroscienc~ Letters, 8 (1978) 119-~124 © Elsevier/North-Holland Scientific Publishers Ltd.
119
THE PI~,OJECTION FROM NUCLEUSRAPHE MAGNUS AND OTHER BHAINSTEM ~ C L E I T O THE SPINAL CORD Ut~THr~ RAT: A STUDY USING~THE HRP BL~.REACTIOI~: G;R, LEICHNETZ*, L. WATKINS, G. GRIFFIN, R. MURFIN and D.J. MAYER Departmentsof AMb0my* and Physiology, Medical College of Virginia, Health Sciences Diuision,Vi~inia Commonwealth UniwTsity, Richmond, Va. 23298 (U.S.A.) (R~.~eived August Ist, 1978) (Revised version received February 9th, 1978) (A¢icep~ed February 16th, 1978)
SUMMARY Horseradish peroxidase injections, or solid HRP placements, were made int.o the d o r s o l a ~ quadrant of the spinal cord in ten adult rats, processed according to the blue-reaction (benzidine dihydrochloride) protocol [ 6]. When the interver~tions involved the dorsal lateral funiculus (DLF)~ HRP-labelled somata were observed primarily in the ipsflateral nucleus raphe magnus (NRM), with an occasional cell in nucleus raphe obscurus (NRO) and l;~dlidus (NttP). Other brsinstem mphe nuclei consistently lacked labelled neurons. The red nucleus and p~demniscal reticular formation of the rostral ports also contained appreciable numbers of HRP back.filled perikarya. The direct NRM~pinal projection in the rat, involved in the modulation of pain transmission in the spinal cord dorsal horn, originates primarily from ~ e expanded region of the nucleus in the rostralmost medulla.
A considerable body' of evidence has developed over the past few years
~cating ~at the ~ m ! ~ o n
of nociceptive information through the spinal
corddo~al horn. is subjecti~ ~werful supraspm~ modulation by certain b r ~ s t e m nuclei [7], ~ 0 n g which are the mesencephalic periaqueductal gray matter (PAG)and nucleus :aphe magnus (NRM). While the precise origin, course ~ d ~ i n P t l o n of neural pathways connecting these structures with the spinal cord d0.mfl horn hu. only recently come under invesUgation, behaviora!and p h ~ l ~ c a l evidence indicates that the final pathway to the d 0 ~ horn d e ~ n d s ~ ~ e d o ~ ~ ~ funiculus (DLF) [ 1,7,8,10 ]. Anatomito the spinal LdKuypers and e mphe follow~ g spinal c o ~ lesions, and the latter made h o ~ m d i ~ peroxidase injections
129
in the cord and observed l~belled cells ~n this region. However, the large size cf the lesions or injections made it difficult to determine either the location ~ t h i n the cord of the descending projections, or specificity of their origin. On the other ]hand, an autoradiographic study in the cat [1 ] has demonstrated a direct spinal projection from NRM to the dorsal horn through the DLF, which was shown to terminate predominantly in laminae I and V, both of which laminae contain cells that prefe~mtially respond to noxious stimu~. J~lthough this work provides strong evidence for such a projection in the cat, the organization of spinal cord pathways can differ considerably in the rat (~.g. corticospinals), from which animal derives most of the behavioral evidence for centrifugal control of pain transmission. The present work attempted to verify the descending NRM projection in the rat, to establish the location and extent of mat,mus cell: that project to the spinal cord, and to examine the q~estion of a direct pathway from the PAG to the dorsal horn in the DLF. Horseradish peroxidase fluid injections (Sigma Type VI, 50% sol. dissolved in s;erile normal saline), or solid HRP placements, were made unilaterally in the dorsolateral quadrant of either the upper thoracic or cervical spinal cord in ten adult Sprague--Dawley rats. Three control placements, cases cR12, 16 and 17, ~ere purposely made in the ventrolateral quadrant of the cord for comparison. In injection cases (R1-R5), from 0.1--0.2/~1 was introduced through a fixed 30-gauge needle either into the substance of the spinal cord at the entrance zone of the dorsal roots, or directly into the wound following ]~esion of the DLF, since enhanced uptake of HRP by cut axons has been demonstrated [4]. Later it w ~ found that concentzated pellets of HRP could be placed directly into DLF wounds that had the advantage of limiting the widespread submeningeal and vascular diffusion of the enzyme which often occu:~s with even the smallest :fluid injection. Representative transverse spinal cord sections through each of the HRP interventions are illustrated in Fig. 1. Compare the injections (R1-RS) to placements (R8-R16; cR12, 16, 17). The animals each survived for 48 hrs. prior to sacrifice, peffusion, brain remove, and precessing according to the recent Mesulam [6] blue-reaction modification of the HRP procedure.
R16
cR12
Fig. 1. Tracing of neutral red-stained transverse rat spired cord sectio:~s through the largest extent of each HRP intervention, reacted according to Mesulam [6 ] l)rotocol. R1--R3 upper thoracic injections; R4 and R5 cervical injections; R8--Rlt~ dorsal lateral cervical placements; cR 12-cR17 controls, ventral lateral cervical placements.
121
In every case where the D L F was successfullyinvolved, back-filledsomata containing prondnent blue H R P reaction granules were observed in the nucleus raphe :magnus. Case R8, a cervical H R P p~cement, best demonstrated the NRM-spinal projection (Fig. 2), which was essentiallyipsilateral.At the level
IC
D
HRP PLAGEMEN'r
Fig. 2. Tracing of n~utral red-stained transverse sections from case R8 (i~set, lower left) through A. Rostral midbrain B. Rostral pons-caudal midbrain C. Rostral medulla D. Caudal medulla indicatiag ~:he location of HRP-labelled cel~lsas small black dots. Each hbelled cell observed corresl~on0Js to a single dot except in A. where the ventral red nucleus contained more than fifty HR~ back-filled somata. Note the htrge numbers of labelled perikarya in NRM ipsilateral to the cervical spinal cord DLF placement in C. The nucleus raphe magnus is expanded laterally over the pyramids at this level, and its configuration appears to correspond to the catecholamine~ontaining cells of Palko~dts [9 ] located within and around the nucleus. Only an occasional labelled cell was observed in NRO, NRP or contralateral NRM. BC, Brachium conjunctivum, sup. cerebellar ped.; C, Nucleus cuneatus; CC, Crus cerebri; DCN, Dorsal cochlear nucleus; G, Nucleus gracilis; IC, Inferior colliculus; HI, Oculomotor nucleus; icp, Inferior cerebeilar peduncle; IN, Interpeduncular nucleus; io, Inferior olivary nucleus, TV, Trochlear nucleus; LL, Lateral lemniscus, MGN, Medial geniculate nucleus; ml, Medial lemniscus; mlf, Medial longitudinal fasciculus; nlo, Nucleus linearis oralis; NTD, Nucbms parabrachialis dorsalis; NPV, Nucleus parabrachialis ventralis; NRD, Nucleus raphe dorsalis; NRG, Nucleus reticularis gigantocellularis; ~rd, Nucleus reticularis medullae obio~/Jgatae, pars dorsalis; NRPO, Nucleus reticularis pontis oralis; nrv, Nucleus reticulari-'B medullae oblongatae, pars ventralis; NSTT, Descendin~I tract and nucleus of the trigemin~l nerve; nts, Nucleus tractus solitarii; p, Pyramidal tract; PN, Pontine nuclei; pr, Nucleu~ reticularis paramedianus; RL, Nucleus reticularis later~lis; RN, Red nucleus; RP, Nucleus raphe pontis; SC, Superior colliculus; SN, Substantia nigra; VC, Vestibular complex; VI~ Facial motor nucleus; X, Dorsal vagal nucleus; XII, Hypoglossal nucleus.
122 indicated in Fig. 2C in the rostral medulla twenty~ight labelled cells were observed on the side to the HRP placement in contrast to only two cells located just contralateml to the midline. At this level the labelled cells appeared to be spread laterally over the pyramids beyond what has been previously thought to be the limits of the NRM, based upon the work of Taber et al. [12] in the cat. We have considered that the majority of these cells belong to NRM and have held that its nuclear configuration in the rostralmost medulla corresponds with that described by Palkovits and Jacobowitz [9, Fig. 4c] for the rat, but which may also include some of the catecholamine-containing cells that they ilIustrate immediately around the nucleus. The HRP-labelled cells are not only continuous from the midline but are also morphologically sknilar to midline magnus neurons, i.e. medium ( ~ 2 5 um) and large ( ~ 5 0 ~m) multipolar polygonal and fusiform cells (Figs. 3a,b). The cells are clearly not part of the gigantocellular nucleus. In areas rostml or caudal to this level the NRM configuration was more vertically-oriented, ellipsoidal, and confined to the midline, slightly broader but similar to the nucleus raphe obscurus (NRO) dorsal, and nucleus raphe pallidus (N~.P) ventral. Only an occasional back-filled cell was observed in either NRO or NRP following ttRP placements in the DLF of cervical and upper thoracic cord segments. The nuclei raphe pontis, raphe dorsalis, and centralis superior consistently lacked HRP-labeUed somata. In case R8 only one small labelled neuron was cbserved in the mesencephalic periaqueductal gray, and in none of the cases was more "~han two labelled cells throughout the central gray. Our results appez' to indicate that the origin of the dh'ect raphe-signal projection in the rat is from the expanded configuration of NRM in the ipsilateral rostralmost medull~L, located dorsal to the pyramids, medial to the facial motor nuclei, and inferomedial to the ventral border of the nucleus reticularis gigantocelluht~l~. Large numbers of labelled cells(>50) were also observed in the contralateral red nucleus (Fig. 2A) in cases concentrated in the D L F (e.g. R8, 10, 11), confirming the findings of Waldron and G w y n [13] that the rubrospinal tract in the rat traverses the dorsal lateral funiculus. In the rostral midbrain the labelled perikarya occupied the ventral half of the nucleus, whereas more caudally cellsthroughout the nucleus were labelled,appearing to correspond to the magnocellular portion of the rat red nucleus as defined by Reid et al.
[II]. Also noteworthy in these cases were the appreciable numbers C> 15) of back-filled neurons in the contralater~ paralemniscal reticular formation of the rostralmost pons (Fig. 2B). Labelled somata were present in the nucleus p ~ a brach~alis ventral!is, paralemniscal gray just ventral (probably cell group A7 of Dahls~rom and F~uxe), and the l a t e ~ l m o s t nucleus reticularis pontis oralis. Occasionally labelled cells were found intermingled in the fascicles of the lateral lemniscus. In the caudal medulla (Fig. 2D) laLelled cells were dispersed in the contralateral medullary reticular formation. The nucleus reticularis medullae oblongatae pars ventralis contained the largest number, but HRP-containing cells were
t23
Fig. 3. (a) Dark-field photomicrograph of HRP-!abelled cells in the nucleus raphe magnus in rostral medulla (level of Fig. 2c}. Arrow (lower right) in ventral median fi~sure, oriented along midline. (10x obj.). (b) Dark-field photomicrograph of HRP-labelled cell in lateral N RM. Higher magnification of cells in box inset in a. (25x obj.}. (c) Dark4'ield photomicrograph of HRP-labelled cells in the ventral half of the rostral red nucleus. (10x obj.). (d) Dark-field photomicrograph of HRP-labelled cells in the nucleus parabrachialis ventralis and paralemniscal gray. Vertically-oriented fibers on right are lateral lemniscus. (10x obj.).
also present in the pars dorsalis of the nucleus reticularis medullae oblongatae, nucleus reticularis lateralis and cell group A1 [ 3]. In our control placements in the more ventral lateral funiculus, the nucleus raphe magnus, obscurus and paUidus lacked labelled neurons. In these cases negative results were also obtained in the red nucleus and paralemniscal gray of the midbrain. Thus this study provides strong anatomical evidence in support of considerable recent physiological data suggesting a direct projection, modulating pain transmission, from the nucleus raphe magnus to the dorsal horn of the spinal cord through the DLF in the rat. Specifically that expanded portion of NRM ha the rostralmost medulla appears to be the major contributor to the pathway.
124
The finding that the nucleus raphe obscurus and pallidus apparently only possess a fe~v cells which project through the DLF could indicate that their spinal projections also traverse regions of the spinal cord white matter vental to the placements made in this study. Further histochemistry is necessary to determine whether a l l o f the neurons of the 'spinal,projecting' expanded NRM configuration zctually synthesize serotonin, and thus constitute the source of this neurotransmitter to the spinal cord. Since NRM, but not periaqueductal gray, projects si~o~nificantly to the cord, i t i s likely that, as Basbaum [1] suggested, the central gray stimulation-produced analgesia is mediated through the nucleus raphe magnus. ACKNOWLEDGEMENT
This study was supported in part by USPHS Grant DA00576 to D.J.M. REFERENCES 1 Basbaum, A.I., Clanton, C.H. and Field, H.L., Opiate and stimulus-produced analgesia: functional anatomy of a medullospinal pathway, Proc. nat. Acad. Sci. (Wash.), 73 (1976) 4685--4688. 2 Brodal, A., Taber, E. and Walberg, F., The raphe nuclei of the brain stem in the cat. II. Efferent connections, J. comp. Neuro|., 114 (1960) 239--260. 3 Dahlstrom, A. and Fuxe, K., Evidence for the existence of monoamine-containing neurons in the central nervous system, Acta. physiol scand. 62 Suppl. 232 (1964) 1--55.
4 Kristensson, K. and Oisson, Y., Retrograde transporl~ of horseradish peroxidase in transected axons. I. Time relationships between transport and induction of chromatolysi~, Brain Res., 79 (1974) 101--109. 5 Kuypers, H.G.J.M. and Maisky, V.A., Retrograde a~:onal transport of horseradish peroxidase from spinal cord to brain stem cell group~ in the cat, Neuroscience Letters, 1 (1975) 9--14. t3 Mesulam, M.-M., The blue-reaction" product in horseradish peroxidase neurohistochemistry: incubation parameters and visibility, J. Histochem. Cytochem., 24 (].976) 1273--1280. 7 Mayer, D.J. and Price, D.D., Central nervous system mechanisms of analgesia, Pain, 2 (1976) 379--404. 8 Murfin, R., Bennett, G.J. and Ma]=er, D.J., The effect of dorsolateral spinal cord lesions on analgesia from morphine microinjection into the periaqueductal gray matter of the rat, Soc. Neuroscience, No. 1365, p. ~46, 1976. ~, Palkovits, M., and Jacobowitz, D.M., Topographical atlas of catecholamine and acetyl~:holinesterase-containing neurons in the rat brain. II. Hindbrain (Mesencephalon, l~hombencephalon), J. comp. Neurol. 157 (1974) 29--42. 10 Hayes, R.L., Price, D.D., Bennett, G.J., Wilcox, G.L. and Mayer, D.J., Differential effects of spinal cord lesions on narcotic and non-narcotic suppression of nociceptive reflexes: further evide~lce for the physiologic multiplicity of pain modulation. Brain Res., (1978) in press. 11 Reid, J.M., Gwyn, D.G. and Flumerfelt, B.A., A cytoarchietectonic vnd Go!gi study of the red nucleus in the rat, J. comp. Neurol., 162 (1975) 337--362. 12 Taber, E., Brodal, A. and Walberg, F., The raphe nuclei of the br~n stem in the cst. ~. Normal topography and cytoarchietecl;ure and general discussion, J. comp. Neurcl., 114 (1960) 161--188. 13 Waldron, H.A. and Gwyn, D.G., Descending trac~s in the spinal cord of the rat. I~ Fibers from ~he midbrain, J. comp. Neurol., 137 (1969) 143--154.
119
THE PI~,OJECTION FROM NUCLEUSRAPHE MAGNUS AND OTHER BHAINSTEM ~ C L E I T O THE SPINAL CORD Ut~THr~ RAT: A STUDY USING~THE HRP BL~.REACTIOI~: G;R, LEICHNETZ*, L. WATKINS, G. GRIFFIN, R. MURFIN and D.J. MAYER Departmentsof AMb0my* and Physiology, Medical College of Virginia, Health Sciences Diuision,Vi~inia Commonwealth UniwTsity, Richmond, Va. 23298 (U.S.A.) (R~.~eived August Ist, 1978) (Revised version received February 9th, 1978) (A¢icep~ed February 16th, 1978)
SUMMARY Horseradish peroxidase injections, or solid HRP placements, were made int.o the d o r s o l a ~ quadrant of the spinal cord in ten adult rats, processed according to the blue-reaction (benzidine dihydrochloride) protocol [ 6]. When the interver~tions involved the dorsal lateral funiculus (DLF)~ HRP-labelled somata were observed primarily in the ipsflateral nucleus raphe magnus (NRM), with an occasional cell in nucleus raphe obscurus (NRO) and l;~dlidus (NttP). Other brsinstem mphe nuclei consistently lacked labelled neurons. The red nucleus and p~demniscal reticular formation of the rostral ports also contained appreciable numbers of HRP back.filled perikarya. The direct NRM~pinal projection in the rat, involved in the modulation of pain transmission in the spinal cord dorsal horn, originates primarily from ~ e expanded region of the nucleus in the rostralmost medulla.
A considerable body' of evidence has developed over the past few years
~cating ~at the ~ m ! ~ o n
of nociceptive information through the spinal
corddo~al horn. is subjecti~ ~werful supraspm~ modulation by certain b r ~ s t e m nuclei [7], ~ 0 n g which are the mesencephalic periaqueductal gray matter (PAG)and nucleus :aphe magnus (NRM). While the precise origin, course ~ d ~ i n P t l o n of neural pathways connecting these structures with the spinal cord d0.mfl horn hu. only recently come under invesUgation, behaviora!and p h ~ l ~ c a l evidence indicates that the final pathway to the d 0 ~ horn d e ~ n d s ~ ~ e d o ~ ~ ~ funiculus (DLF) [ 1,7,8,10 ]. Anatomito the spinal LdKuypers and e mphe follow~ g spinal c o ~ lesions, and the latter made h o ~ m d i ~ peroxidase injections
129
in the cord and observed l~belled cells ~n this region. However, the large size cf the lesions or injections made it difficult to determine either the location ~ t h i n the cord of the descending projections, or specificity of their origin. On the other ]hand, an autoradiographic study in the cat [1 ] has demonstrated a direct spinal projection from NRM to the dorsal horn through the DLF, which was shown to terminate predominantly in laminae I and V, both of which laminae contain cells that prefe~mtially respond to noxious stimu~. J~lthough this work provides strong evidence for such a projection in the cat, the organization of spinal cord pathways can differ considerably in the rat (~.g. corticospinals), from which animal derives most of the behavioral evidence for centrifugal control of pain transmission. The present work attempted to verify the descending NRM projection in the rat, to establish the location and extent of mat,mus cell: that project to the spinal cord, and to examine the q~estion of a direct pathway from the PAG to the dorsal horn in the DLF. Horseradish peroxidase fluid injections (Sigma Type VI, 50% sol. dissolved in s;erile normal saline), or solid HRP placements, were made unilaterally in the dorsolateral quadrant of either the upper thoracic or cervical spinal cord in ten adult Sprague--Dawley rats. Three control placements, cases cR12, 16 and 17, ~ere purposely made in the ventrolateral quadrant of the cord for comparison. In injection cases (R1-R5), from 0.1--0.2/~1 was introduced through a fixed 30-gauge needle either into the substance of the spinal cord at the entrance zone of the dorsal roots, or directly into the wound following ]~esion of the DLF, since enhanced uptake of HRP by cut axons has been demonstrated [4]. Later it w ~ found that concentzated pellets of HRP could be placed directly into DLF wounds that had the advantage of limiting the widespread submeningeal and vascular diffusion of the enzyme which often occu:~s with even the smallest :fluid injection. Representative transverse spinal cord sections through each of the HRP interventions are illustrated in Fig. 1. Compare the injections (R1-RS) to placements (R8-R16; cR12, 16, 17). The animals each survived for 48 hrs. prior to sacrifice, peffusion, brain remove, and precessing according to the recent Mesulam [6] blue-reaction modification of the HRP procedure.
R16
cR12
Fig. 1. Tracing of neutral red-stained transverse rat spired cord sectio:~s through the largest extent of each HRP intervention, reacted according to Mesulam [6 ] l)rotocol. R1--R3 upper thoracic injections; R4 and R5 cervical injections; R8--Rlt~ dorsal lateral cervical placements; cR 12-cR17 controls, ventral lateral cervical placements.
121
In every case where the D L F was successfullyinvolved, back-filledsomata containing prondnent blue H R P reaction granules were observed in the nucleus raphe :magnus. Case R8, a cervical H R P p~cement, best demonstrated the NRM-spinal projection (Fig. 2), which was essentiallyipsilateral.At the level
IC
D
HRP PLAGEMEN'r
Fig. 2. Tracing of n~utral red-stained transverse sections from case R8 (i~set, lower left) through A. Rostral midbrain B. Rostral pons-caudal midbrain C. Rostral medulla D. Caudal medulla indicatiag ~:he location of HRP-labelled cel~lsas small black dots. Each hbelled cell observed corresl~on0Js to a single dot except in A. where the ventral red nucleus contained more than fifty HR~ back-filled somata. Note the htrge numbers of labelled perikarya in NRM ipsilateral to the cervical spinal cord DLF placement in C. The nucleus raphe magnus is expanded laterally over the pyramids at this level, and its configuration appears to correspond to the catecholamine~ontaining cells of Palko~dts [9 ] located within and around the nucleus. Only an occasional labelled cell was observed in NRO, NRP or contralateral NRM. BC, Brachium conjunctivum, sup. cerebellar ped.; C, Nucleus cuneatus; CC, Crus cerebri; DCN, Dorsal cochlear nucleus; G, Nucleus gracilis; IC, Inferior colliculus; HI, Oculomotor nucleus; icp, Inferior cerebeilar peduncle; IN, Interpeduncular nucleus; io, Inferior olivary nucleus, TV, Trochlear nucleus; LL, Lateral lemniscus, MGN, Medial geniculate nucleus; ml, Medial lemniscus; mlf, Medial longitudinal fasciculus; nlo, Nucleus linearis oralis; NTD, Nucbms parabrachialis dorsalis; NPV, Nucleus parabrachialis ventralis; NRD, Nucleus raphe dorsalis; NRG, Nucleus reticularis gigantocellularis; ~rd, Nucleus reticularis medullae obio~/Jgatae, pars dorsalis; NRPO, Nucleus reticularis pontis oralis; nrv, Nucleus reticulari-'B medullae oblongatae, pars ventralis; NSTT, Descendin~I tract and nucleus of the trigemin~l nerve; nts, Nucleus tractus solitarii; p, Pyramidal tract; PN, Pontine nuclei; pr, Nucleu~ reticularis paramedianus; RL, Nucleus reticularis later~lis; RN, Red nucleus; RP, Nucleus raphe pontis; SC, Superior colliculus; SN, Substantia nigra; VC, Vestibular complex; VI~ Facial motor nucleus; X, Dorsal vagal nucleus; XII, Hypoglossal nucleus.
122 indicated in Fig. 2C in the rostral medulla twenty~ight labelled cells were observed on the side to the HRP placement in contrast to only two cells located just contralateml to the midline. At this level the labelled cells appeared to be spread laterally over the pyramids beyond what has been previously thought to be the limits of the NRM, based upon the work of Taber et al. [12] in the cat. We have considered that the majority of these cells belong to NRM and have held that its nuclear configuration in the rostralmost medulla corresponds with that described by Palkovits and Jacobowitz [9, Fig. 4c] for the rat, but which may also include some of the catecholamine-containing cells that they ilIustrate immediately around the nucleus. The HRP-labelled cells are not only continuous from the midline but are also morphologically sknilar to midline magnus neurons, i.e. medium ( ~ 2 5 um) and large ( ~ 5 0 ~m) multipolar polygonal and fusiform cells (Figs. 3a,b). The cells are clearly not part of the gigantocellular nucleus. In areas rostml or caudal to this level the NRM configuration was more vertically-oriented, ellipsoidal, and confined to the midline, slightly broader but similar to the nucleus raphe obscurus (NRO) dorsal, and nucleus raphe pallidus (N~.P) ventral. Only an occasional back-filled cell was observed in either NRO or NRP following ttRP placements in the DLF of cervical and upper thoracic cord segments. The nuclei raphe pontis, raphe dorsalis, and centralis superior consistently lacked HRP-labeUed somata. In case R8 only one small labelled neuron was cbserved in the mesencephalic periaqueductal gray, and in none of the cases was more "~han two labelled cells throughout the central gray. Our results appez' to indicate that the origin of the dh'ect raphe-signal projection in the rat is from the expanded configuration of NRM in the ipsilateral rostralmost medull~L, located dorsal to the pyramids, medial to the facial motor nuclei, and inferomedial to the ventral border of the nucleus reticularis gigantocelluht~l~. Large numbers of labelled cells(>50) were also observed in the contralateral red nucleus (Fig. 2A) in cases concentrated in the D L F (e.g. R8, 10, 11), confirming the findings of Waldron and G w y n [13] that the rubrospinal tract in the rat traverses the dorsal lateral funiculus. In the rostral midbrain the labelled perikarya occupied the ventral half of the nucleus, whereas more caudally cellsthroughout the nucleus were labelled,appearing to correspond to the magnocellular portion of the rat red nucleus as defined by Reid et al.
[II]. Also noteworthy in these cases were the appreciable numbers C> 15) of back-filled neurons in the contralater~ paralemniscal reticular formation of the rostralmost pons (Fig. 2B). Labelled somata were present in the nucleus p ~ a brach~alis ventral!is, paralemniscal gray just ventral (probably cell group A7 of Dahls~rom and F~uxe), and the l a t e ~ l m o s t nucleus reticularis pontis oralis. Occasionally labelled cells were found intermingled in the fascicles of the lateral lemniscus. In the caudal medulla (Fig. 2D) laLelled cells were dispersed in the contralateral medullary reticular formation. The nucleus reticularis medullae oblongatae pars ventralis contained the largest number, but HRP-containing cells were
t23
Fig. 3. (a) Dark-field photomicrograph of HRP-!abelled cells in the nucleus raphe magnus in rostral medulla (level of Fig. 2c}. Arrow (lower right) in ventral median fi~sure, oriented along midline. (10x obj.). (b) Dark-field photomicrograph of HRP-labelled cell in lateral N RM. Higher magnification of cells in box inset in a. (25x obj.}. (c) Dark4'ield photomicrograph of HRP-labelled cells in the ventral half of the rostral red nucleus. (10x obj.). (d) Dark-field photomicrograph of HRP-labelled cells in the nucleus parabrachialis ventralis and paralemniscal gray. Vertically-oriented fibers on right are lateral lemniscus. (10x obj.).
also present in the pars dorsalis of the nucleus reticularis medullae oblongatae, nucleus reticularis lateralis and cell group A1 [ 3]. In our control placements in the more ventral lateral funiculus, the nucleus raphe magnus, obscurus and paUidus lacked labelled neurons. In these cases negative results were also obtained in the red nucleus and paralemniscal gray of the midbrain. Thus this study provides strong anatomical evidence in support of considerable recent physiological data suggesting a direct projection, modulating pain transmission, from the nucleus raphe magnus to the dorsal horn of the spinal cord through the DLF in the rat. Specifically that expanded portion of NRM ha the rostralmost medulla appears to be the major contributor to the pathway.
124
The finding that the nucleus raphe obscurus and pallidus apparently only possess a fe~v cells which project through the DLF could indicate that their spinal projections also traverse regions of the spinal cord white matter vental to the placements made in this study. Further histochemistry is necessary to determine whether a l l o f the neurons of the 'spinal,projecting' expanded NRM configuration zctually synthesize serotonin, and thus constitute the source of this neurotransmitter to the spinal cord. Since NRM, but not periaqueductal gray, projects si~o~nificantly to the cord, i t i s likely that, as Basbaum [1] suggested, the central gray stimulation-produced analgesia is mediated through the nucleus raphe magnus. ACKNOWLEDGEMENT
This study was supported in part by USPHS Grant DA00576 to D.J.M. REFERENCES 1 Basbaum, A.I., Clanton, C.H. and Field, H.L., Opiate and stimulus-produced analgesia: functional anatomy of a medullospinal pathway, Proc. nat. Acad. Sci. (Wash.), 73 (1976) 4685--4688. 2 Brodal, A., Taber, E. and Walberg, F., The raphe nuclei of the brain stem in the cat. II. Efferent connections, J. comp. Neuro|., 114 (1960) 239--260. 3 Dahlstrom, A. and Fuxe, K., Evidence for the existence of monoamine-containing neurons in the central nervous system, Acta. physiol scand. 62 Suppl. 232 (1964) 1--55.
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