Brain Research Bulletin,Vol. 28, pp. 679-682, 1992 Printed in the USA. All rights reserved.
0361-9230/92 $5.00 + .OO Copyright 0 1992 Pergamon Press Ltd.
Efferent Projections of the Nucleus Raphe Magnus LAURA J. SIM AND SHIRLEY A. JOSEPH’
The Neuroendocrine Unit, University of Rochester School of Medicine and Dentistry, Rochester, NY 14642 Received 19 September 199 1 SlM, L. J. AND S. A. JOSEPH. E&rent projections ofthe nucleus raphe mugnus. BRAIN RES BULL 28(5) 679-682, 1992.This study was performed to identify supraspinal efferents of the nucleus raphe magnus (NRM) in the rat using the anterograde tracer phaseolus vulgaris leucoagglutinin (PHA-L). NRM-derived PHA-L-ir fibers, with putative terminals, were identified in regions including the lateral hypothalamus, parafascicular nucleus, ventral lateral periaqueductal gray (PAG), locus coeruleus, parabrachial nucleus, A7, A5, and nucleus tractus solitarius. Projections to the PAG demonstrate reciprocity in PAG-NRM connectivity that may modulate the PAG-NRM-spinal cord pathway. The NRM may contribute to supraspinal modulation of nociception by efferents identified in the PAG, as well as locus coeruleus, A7, and A5, which have been shown to project to the spinal cord dorsal horn. Our results provide neuroanatomical support for NRM involvement in supraspinal mechanism(s) for modulation of nociception. Antinociception
Phaseolus vulgaris leukoagglutinin
Periaqueductal gray
ELECTRICAL stimulation ( 15) or morphine microinjection (5) into the nucleus raphe magnus (NRM) elicits antinociception. This occurs, in part, via descending projections to the spinal cord dorsal horn (2) that may involve the release of serotonin (4,24) and opioid peptides (27) at the spinal level. Projections from the periaqueductal gray (PAG) to the NRM have also been identified anatomically and physiologically (1,3,6,8) that influence descending NRM projections. More recent studies also indicate that the NRM may influence nociception through supraspinal connectivity, particularly with catecholamine-containing nuclei ( 11,12). Our previous anatomical tracing studies showed that both the NRM and ventral aqueductal region are innervated by opiocortin-ir neurons in the arcuate nucleus ( 19,20) and may provide opioid modulation of nociception at both the mesencephalic and medullary levels of descending pathways that modulate nociception. The present study, which identifies the trajectory of NRM efferents in the brain, extends the importance of the NRM, as it may also contribute to modulation of nociception at the supraspinal level.
Brainstem
PA alternating positive current. After lo-14 d, animals were deeply anesthetized and perfused intracardially with saline, followed by 4% paraformaldehyde in 0.1 M acetate buffer (pH 6.5) and 4% paraformaldehyde in 0.1 Mborate buffer (pH 9.5). Brains were postfixed overnight in pH 9.5 fixative plus 10% sucrose. Fifty-micrometer sections were cut on a freezing sliding microtome and collected in 0.02 Mpotassium phosphate-buffered saline (KPBS), pH 7.4. The tissue was incubated for 48-60 h in rabbit-anti-PHA-L (Dakopatts) diluted 1:2500 in KPBS + 0.4% t&on X-100 + 1% bovine serum albumin. Sections were then rinsed and incubated in biotinylated goat-anti-rabbit (Vector) diluted 1:200 in KPBS with 0.04% triton X-100 + 1.5% normal goat serum. Sections were rinsed in KPBS and incubated for 1 h in avidin HRP (Vector), diluted 1:200 in KPBS, then rinsed in 0.1 M tris buffer, pH 7.4. The chromagen used was nickelenhanced 3,3’-diaminobenzidine tetrahydrochloride (DAB), consisting of 37.5 ml tris buffer, 50 ~1 H202, 0.187 g nickel ammonium sulfate, and 7.5 mg DAB. This chromagen produces a purple-black reaction product.
METHOD
RESULTS
Phaseolus vulgaris leucoagglutinin
(PHA-L) was stereotaxically injected into the NRM of anesthetized (80 mg ketamine + 8 mg acepromazine maleate per kilogram body weight) male Sprague-Dawley rats according to the protocol previously described (20). A 2.5% solution of PHA-L (Vector) was delivered microiontophoretically via lo-15-pm glass micropipettes at 5
The results of this study are based upon seven brains in which PHA-L injections were placed in the caudal NRM, primarily at the level of the nucleus of the VII cranial nerve (Fig. 1). Injection sites often included diffusion of the tracer into the adjacent gigantocellular reticular nucleus pars alpha, and occasionally into nucleus raphe pallidus. Injections which included
’ Requests for reprints should be addressed to S. A. Joseph, Ph.D., The Neuroendocrine Unit, Box 609, University of Rochester School of Medicine and Dentistry, 60 I Elmwood Avenue, Rochester, NY 14642.
679
680
SIM AND JOSEPH
FIG. 1. Photomicrograph of a brainstem section showing a PHA-L injection site in the nucleus raphe magnus (bar = 250 pm).
have substantiated a role for the NRM in antinociception through a supraspinal mechanism involving the ventral PAG. Numerous anatomical and functional studies including our own (19,20), support the concept that the ventral PAG and adjacent dorsal raphe region is a pivotal region in opioid-mediated antinociceptive responses (26). A clear demarcation between the ventral PAG and DRN is not obvious based upon the cytoarchitectonic or neurochemical constitution of the perikarya in this region, therefore, we refer to this area as the ventral aqueductal region. Adding credence to this conclusion are functional studies that demonstrated that stimulation of the region ventral to the aqueduct elicits analgesia (7) and our neuroanatomical studies that demonstrate that the ventral PAG and the DRN share similar connectivity (2 I ). The results of the present study are of particular interest in regard to the anatomical and functional characterization of the ventral aqueductal region, because connectivity has been identified with a region critical in the modulation of nociception. In
diffusion into the gigantocellular or lateral paragigantocellular reticular nuclei were easily identified and brains in which these large injection sites were identified were not used in this analysis. PHA-L-ir fibers and terminals were seen primarily in brainstem nuclei after NRM injections (Fig. 2), although some PHA-L immunoreactivity was also identified in the lateral hypothalamus, periventricular gray, and parafascicular nucleus. PHA-Lir fibers with putative terminals were identified adjacent to the injection site in the NRM, gigantocellular reticular nucleus pars alpha and lateral paragigantocellular nucleus following NRM injections. NRM-derived PHA-L-ir fibers and terminals were identified in the lateral and ventral lateral PAG (Fig. 3) particularly at the level of the superior-inferior colhcular junction. PHAL-ir fibers were occasionally identified in the dorsal raphe nucleus and mesencephalic reticular formation. PHA-L-ir fibers and terminals were identified in the lateral parabrachial nucleus, with the most dense fiber accumulation in the ventral lateral parabrachial nucleus and in A7. Few PHA-L-ir fibers emanating from the NRM were identified in the nucleus of the VII cranial nerve, although PHA-L-ir fibers and terminals were found dorsal to this region in the parvocellular reticular nucleus. PHA-L-ir fibers and terminals were consistently identified in brainstemcatecholaminergic cell groups, including locus coeruleus (particularly at the level of Barrington’s nucleus), A7, AS, Cl, and nucleus of the solitary tract at C2, and less often at A2. PHAL-ir fibers and terminals were found in the trigeminal complex, particularly throughout the rostral-caudal extent of the spinal nucleus of the V cranial nerve. PHA-L-ir fibers and terminals were also scattered in the principal sensory nucleus of V, at its caudal extent, and in the motor nucleus of V. PHA-L-ir fibers and terminals were found throughout the brainstem reticular nuclei in caudal pontine reticular nucleus, parvocellular reticular nucleus, lateral reticular nucleus, and medullary reticular nucleus. DISCUSSION In this study, we have shown that neurons in the NRM project to brainstem regions which influence nociception. Fibers with putative terminals emanating from the NRM were localized in such regions as the PAG, locus coeruleus, A5, A7, and nuclei of the trigeminal complex. Although earlier studies elucidating pathways that modulate nociception focused on descending systems, particularly the PAG-NRM-dorsal horn axis (6,8,9), we
FIG. 2. Schematicdiagram depicting the distribution of PHA-L-ir fibers with putative terminals in the brainstem after PHA-L injections into the NRM. Abbreviations: Amb (nucleus ambiguus), cNTS (commissural nucleus tractus solitarius), LC (locus coeruleus), LPB (lateral parabrachial nucleus), LRt (lateral reticular nucleus), m5 (motor nucleus of the V cranial nerve), NTS (nucleus tractus solitarius), Pr5 (principle sensory nucleus of V cranial nerve), PrH (nucleus prepositus hypoglossi), s5 (sensory root of V cranial nerve), Sp5 (spinal nucleus of V cranial nerve), 7 (nucleus of the VII cranial nerve), 7n (VII cranial nerve), IO (dorsal motor nucleus of X cranial nerve).
Nl ‘CLEUS RAPHE MAGNUS
681
EFFERENTS
FIG. 3. (A and B) High-power photomicrographs of PHA-L-ir fibers with putative terminals (indicated by arrows)in the VL-PAG after PHAL injection into the NRM (bar = 11 pm).
addition, the demonstration of reciprocity in the connectivity between the NRM and PAG indicates that medullary input to the ventral aqueductal region may contribute to integration of the overall response to nociception. This region of the mesencephalon also receives afferents from the spinal cord via the spinomesencephalic tract, which transmits information regarding
nociception. The connectivity identified between the ventral aqueductal region and the NRM supports the hypothesis that this region is a critical component in antinociception, and further demonstrates the importance of this mesencephalic region in the integration of forebrain and brainstem activity. In addition to the importance ofthe PAG-NRM-spinal cord pathway, recent evidence indicates that parallel bulbospinal pathways exist that may also modulate nociception. The output of the descending PAG-NRM-dorsal horn system is the modulation of spinal neuronal activity (8) that is elicited in part by release of S-hydroxytryptamine (SHT) (4,24,25). However, stimulation of the NRM results in the release of both 5HT and norepinephrine (NE) from terminals in the spinal cord (11) and pharmacological manipulation of either 5HT or NE interferes with NRM antinociception (12). The connectivity identified between the NRM and brainstem catecholaminergic groups in this study may provide the neuroanatomical substrate by which NRM stimulation elicits NE release. Stimulation of the locus coeruleus (13,18) or ventral lateral pontine tegmentum in the region of A5 and A7 (14) elicits antinociception and intrathecal NE produces dose-dependent antinociception ( 16). Neurons in A5, A6, and A7 have been shown to project to the spinal cord, where noradrenergic fibers and terminals have been identified in regions including lamina I and II (22), as well as contacting spinothalamic projection neurons in lamina I and V (23). NRM projections to brainstem catecholaminergic nuclei may modulate the activity of bulbospinal projection neurons. These results demonstrate connectivity that may result in modulation of nociception by NRM neurons at the supraspinal level. Putative terminal fields identified in the PAG, locus coeruleus, A5, A7, and nuclei of the trigeminal complex may be particularly important in such a system. ACKNOWLEDGEMENTS
This work was supported by NIH No. NS2 1323 (S. A. J.) and USPHS No. DA07232 (L. J. S.).
REFERENCES 1. Abols, I. A.; Basbaum, A. I. Afferent connections of the rostra1 medulla of the cat: A neural substrate for midbrain-medullary interactions in the modulation of pain. J. Comp. Neural. 201:285-297; 1981. 2. Basbaum, A. 1.; Clanton, C. H.; Fields, H. L. Opiate and stimulus produced analgesia: Functional anatomy of a medullospinal pathway. Proc. Natl. Acad. Sci. USA 73:4685&88; 1976. 3. Basbaum. A. I.: Ma&v. N. J. E.: O’Keefe. J.: Clanton. C. H. Reversal of morphine and stim&produced analgesia by subtotal spinal cord lesions. Pain 3:43-56; 1977. 4. Bowker, R. M.; Westlund, K. N.; Coulter, J. D. Origins of serotonergic projections to the spinal cord in rat: An immunocytochemical-retrograde transport study. Brain Res. 226:187-199; 1981. 5. Dickenson, A. H.; Oliveras, J. L.; Besson, J. M. Role of the nucleus raphe magnus in opiate analgesia as studies by the microinjection technique in the rat. Brain Res. 170:95-l 11; 1979. 6. Fardin, V.; Oliveras, J. L.; Besson, J. M. Projections from the periaqueductal gray matter to the B3 cellular area (nucleus raphe magnus and nucleus reticularis paragigantocellulatis) as revealed by the retrograde transport horseradish peroxidase in the rat. J. Comp. Neural. 223:483-500; 1984a. 7. Fardin, V.; Oliveras, J. L.; Besson, J. M. A reinvestigation of the analgesic effects induced by stimulation of the periaqueductal gray matter in the rat: I. The production of behavioral side effects together with analgesia. Brain Res. 306:105-123; 1984b.
8. Fields, H. L.; Anderson, S. D. Evidence that raphe-spinal neurons mediate opiate and midbrain stimulation-produced analgesias. Pain 5:333-349; 1978. 9. Fields, H. L.; Basbaum, A. I. Brainstem control of spinal pain-transmission neurons. Ann. Rev. Phvsiol. 40~217-248: 1978. 10. Hammond, D. L.; Levy, R. A.; P&d&, H. K. HypoaIgesia folIowing microinjection of noradrenergic antagonists in the nucleus raphe magnus. Pain 9:85-101; 1980. Il. Hammond, D. L.; Tyce, G. M.; Yaksh, T. L. Efflux of 5-hydroxytryptamine and noradrenaline into spinal superfusates during stimulation of the rat medulla. J. Physioi. 359: 15I- 162; 1985. _ 12. Jensen. T. S.: Yaksh. T. L. II. Examination of spinal monoamine receptors through which brainstem opiate-sensitive svstems act in the rat. Brain Res. 363:114-127; 1986. 13. Jones. S. L.: Gebhart. G. F. Inhibition of soinal nociceotive transmission from the midbrain, pons and medulla in the rat: Activation of descending inhibition by morphine, glutamate and electrical stimulation. Brain Res. 460:281-296; 1988. 14. Miller, J. F.; Proudfit, H. K. Antagonism of stimulation-produced antinociception from ventrolateral pontine sites by intrathecal administration of alpha-adrenergic antagonists and naloxone. Brain Res. 530:20-34; 1990. 15. Oliveras, J. L.; Guilbaud, G.; Besson, J. M. A map of serotonergic structures involved in stimulation-produced analgesia in unrestrained freely moving cats. Brain Res. 164317-322; 1979. 16. Reddy, S. V. R.; Yaksh, T. L. Spinal noradrenergic terminal system mediates antinociception. Brain Res. 189:39 l-40 1; 1980.
682
17. Sagen, J.; Proudfit, H. K. Alterations in nociception following lesions of the A5 catecholamine nucleus. Brain Res. 370:93-101; 1986. 18. SegaJ, M.; Sandberg, D. Analgesia produced by electrical stimulation of catecholamine nuclei in the rat brain. Brain Res. 123:369-372: 1977. 19. Sim, L. J.; Joseph, S. A. Opiocortin and catecholamine projections to raphe nuclei. Pentides 10:1019-1025: 1989. 20. Sim,.L. J.; Joseph,-S. A. Arcuate nucleus projections to brainstem regions which modulate nociception. J. Chem. Neuroanat. 4:97109; 1991. 2 I. Sim, L. J.; Joseph, S. A. Dorsal raphe nucleus efferents and putative contacts with peptidergic neurons. Peptides (in press). 22. Westlund, K. N.; Bowker, R. M.; Ziegler, M. G.; Coulter, J. D. Noradrenergic projections to the spinal cord of the rat. Brain Res. 263:15-31; 1983.
SIM AND JOSEPH 23. Westlund, K. N.; Carbon, S. M.; Zhang, D.; Willis, W. D. Direct catecholaminergic innervation of primate spinothalamic tract neurons. J. Comp. Neurol. 299: 178-l 86; 1990. 24. Yaksh, T. L. Direct evidence that spinal serotonin and nomdrenaline terminals mediate the spinal antinociceptive effects of morphine in the oeriaaueductal erav. Brain Res. 160:180- 185; 1979. 25. Yaksh, T: L.; Ty& d. M. Microinjection of morphine into the periaqueductal gray evokes the release of serotonin from spinal cord. Brain Res. 171:176-181; 1979. 26. Yaksh, T. L.; Yeung, J. C.; Rudy, T. A. Systematic examination in the rat of brain sites sensitive to the direct application of morphine: Observation of differential effects within the periaqueductal gray. Brain Res. Il4:83-103; 1976. 27. Zorman, G.; Belcher, G.; Adams, J. E.; Fields, H. L. Lumbar intrathecal naloxone blocks analgesia produced by microstimulation of the ventromedial medulla in the rat. Brain Res. 236:77-84: 1982.
0361-9230/92 $5.00 + .OO Copyright 0 1992 Pergamon Press Ltd.
Efferent Projections of the Nucleus Raphe Magnus LAURA J. SIM AND SHIRLEY A. JOSEPH’
The Neuroendocrine Unit, University of Rochester School of Medicine and Dentistry, Rochester, NY 14642 Received 19 September 199 1 SlM, L. J. AND S. A. JOSEPH. E&rent projections ofthe nucleus raphe mugnus. BRAIN RES BULL 28(5) 679-682, 1992.This study was performed to identify supraspinal efferents of the nucleus raphe magnus (NRM) in the rat using the anterograde tracer phaseolus vulgaris leucoagglutinin (PHA-L). NRM-derived PHA-L-ir fibers, with putative terminals, were identified in regions including the lateral hypothalamus, parafascicular nucleus, ventral lateral periaqueductal gray (PAG), locus coeruleus, parabrachial nucleus, A7, A5, and nucleus tractus solitarius. Projections to the PAG demonstrate reciprocity in PAG-NRM connectivity that may modulate the PAG-NRM-spinal cord pathway. The NRM may contribute to supraspinal modulation of nociception by efferents identified in the PAG, as well as locus coeruleus, A7, and A5, which have been shown to project to the spinal cord dorsal horn. Our results provide neuroanatomical support for NRM involvement in supraspinal mechanism(s) for modulation of nociception. Antinociception
Phaseolus vulgaris leukoagglutinin
Periaqueductal gray
ELECTRICAL stimulation ( 15) or morphine microinjection (5) into the nucleus raphe magnus (NRM) elicits antinociception. This occurs, in part, via descending projections to the spinal cord dorsal horn (2) that may involve the release of serotonin (4,24) and opioid peptides (27) at the spinal level. Projections from the periaqueductal gray (PAG) to the NRM have also been identified anatomically and physiologically (1,3,6,8) that influence descending NRM projections. More recent studies also indicate that the NRM may influence nociception through supraspinal connectivity, particularly with catecholamine-containing nuclei ( 11,12). Our previous anatomical tracing studies showed that both the NRM and ventral aqueductal region are innervated by opiocortin-ir neurons in the arcuate nucleus ( 19,20) and may provide opioid modulation of nociception at both the mesencephalic and medullary levels of descending pathways that modulate nociception. The present study, which identifies the trajectory of NRM efferents in the brain, extends the importance of the NRM, as it may also contribute to modulation of nociception at the supraspinal level.
Brainstem
PA alternating positive current. After lo-14 d, animals were deeply anesthetized and perfused intracardially with saline, followed by 4% paraformaldehyde in 0.1 M acetate buffer (pH 6.5) and 4% paraformaldehyde in 0.1 Mborate buffer (pH 9.5). Brains were postfixed overnight in pH 9.5 fixative plus 10% sucrose. Fifty-micrometer sections were cut on a freezing sliding microtome and collected in 0.02 Mpotassium phosphate-buffered saline (KPBS), pH 7.4. The tissue was incubated for 48-60 h in rabbit-anti-PHA-L (Dakopatts) diluted 1:2500 in KPBS + 0.4% t&on X-100 + 1% bovine serum albumin. Sections were then rinsed and incubated in biotinylated goat-anti-rabbit (Vector) diluted 1:200 in KPBS with 0.04% triton X-100 + 1.5% normal goat serum. Sections were rinsed in KPBS and incubated for 1 h in avidin HRP (Vector), diluted 1:200 in KPBS, then rinsed in 0.1 M tris buffer, pH 7.4. The chromagen used was nickelenhanced 3,3’-diaminobenzidine tetrahydrochloride (DAB), consisting of 37.5 ml tris buffer, 50 ~1 H202, 0.187 g nickel ammonium sulfate, and 7.5 mg DAB. This chromagen produces a purple-black reaction product.
METHOD
RESULTS
Phaseolus vulgaris leucoagglutinin
(PHA-L) was stereotaxically injected into the NRM of anesthetized (80 mg ketamine + 8 mg acepromazine maleate per kilogram body weight) male Sprague-Dawley rats according to the protocol previously described (20). A 2.5% solution of PHA-L (Vector) was delivered microiontophoretically via lo-15-pm glass micropipettes at 5
The results of this study are based upon seven brains in which PHA-L injections were placed in the caudal NRM, primarily at the level of the nucleus of the VII cranial nerve (Fig. 1). Injection sites often included diffusion of the tracer into the adjacent gigantocellular reticular nucleus pars alpha, and occasionally into nucleus raphe pallidus. Injections which included
’ Requests for reprints should be addressed to S. A. Joseph, Ph.D., The Neuroendocrine Unit, Box 609, University of Rochester School of Medicine and Dentistry, 60 I Elmwood Avenue, Rochester, NY 14642.
679
680
SIM AND JOSEPH
FIG. 1. Photomicrograph of a brainstem section showing a PHA-L injection site in the nucleus raphe magnus (bar = 250 pm).
have substantiated a role for the NRM in antinociception through a supraspinal mechanism involving the ventral PAG. Numerous anatomical and functional studies including our own (19,20), support the concept that the ventral PAG and adjacent dorsal raphe region is a pivotal region in opioid-mediated antinociceptive responses (26). A clear demarcation between the ventral PAG and DRN is not obvious based upon the cytoarchitectonic or neurochemical constitution of the perikarya in this region, therefore, we refer to this area as the ventral aqueductal region. Adding credence to this conclusion are functional studies that demonstrated that stimulation of the region ventral to the aqueduct elicits analgesia (7) and our neuroanatomical studies that demonstrate that the ventral PAG and the DRN share similar connectivity (2 I ). The results of the present study are of particular interest in regard to the anatomical and functional characterization of the ventral aqueductal region, because connectivity has been identified with a region critical in the modulation of nociception. In
diffusion into the gigantocellular or lateral paragigantocellular reticular nuclei were easily identified and brains in which these large injection sites were identified were not used in this analysis. PHA-L-ir fibers and terminals were seen primarily in brainstem nuclei after NRM injections (Fig. 2), although some PHA-L immunoreactivity was also identified in the lateral hypothalamus, periventricular gray, and parafascicular nucleus. PHA-Lir fibers with putative terminals were identified adjacent to the injection site in the NRM, gigantocellular reticular nucleus pars alpha and lateral paragigantocellular nucleus following NRM injections. NRM-derived PHA-L-ir fibers and terminals were identified in the lateral and ventral lateral PAG (Fig. 3) particularly at the level of the superior-inferior colhcular junction. PHAL-ir fibers were occasionally identified in the dorsal raphe nucleus and mesencephalic reticular formation. PHA-L-ir fibers and terminals were identified in the lateral parabrachial nucleus, with the most dense fiber accumulation in the ventral lateral parabrachial nucleus and in A7. Few PHA-L-ir fibers emanating from the NRM were identified in the nucleus of the VII cranial nerve, although PHA-L-ir fibers and terminals were found dorsal to this region in the parvocellular reticular nucleus. PHA-L-ir fibers and terminals were consistently identified in brainstemcatecholaminergic cell groups, including locus coeruleus (particularly at the level of Barrington’s nucleus), A7, AS, Cl, and nucleus of the solitary tract at C2, and less often at A2. PHAL-ir fibers and terminals were found in the trigeminal complex, particularly throughout the rostral-caudal extent of the spinal nucleus of the V cranial nerve. PHA-L-ir fibers and terminals were also scattered in the principal sensory nucleus of V, at its caudal extent, and in the motor nucleus of V. PHA-L-ir fibers and terminals were found throughout the brainstem reticular nuclei in caudal pontine reticular nucleus, parvocellular reticular nucleus, lateral reticular nucleus, and medullary reticular nucleus. DISCUSSION In this study, we have shown that neurons in the NRM project to brainstem regions which influence nociception. Fibers with putative terminals emanating from the NRM were localized in such regions as the PAG, locus coeruleus, A5, A7, and nuclei of the trigeminal complex. Although earlier studies elucidating pathways that modulate nociception focused on descending systems, particularly the PAG-NRM-dorsal horn axis (6,8,9), we
FIG. 2. Schematicdiagram depicting the distribution of PHA-L-ir fibers with putative terminals in the brainstem after PHA-L injections into the NRM. Abbreviations: Amb (nucleus ambiguus), cNTS (commissural nucleus tractus solitarius), LC (locus coeruleus), LPB (lateral parabrachial nucleus), LRt (lateral reticular nucleus), m5 (motor nucleus of the V cranial nerve), NTS (nucleus tractus solitarius), Pr5 (principle sensory nucleus of V cranial nerve), PrH (nucleus prepositus hypoglossi), s5 (sensory root of V cranial nerve), Sp5 (spinal nucleus of V cranial nerve), 7 (nucleus of the VII cranial nerve), 7n (VII cranial nerve), IO (dorsal motor nucleus of X cranial nerve).
Nl ‘CLEUS RAPHE MAGNUS
681
EFFERENTS
FIG. 3. (A and B) High-power photomicrographs of PHA-L-ir fibers with putative terminals (indicated by arrows)in the VL-PAG after PHAL injection into the NRM (bar = 11 pm).
addition, the demonstration of reciprocity in the connectivity between the NRM and PAG indicates that medullary input to the ventral aqueductal region may contribute to integration of the overall response to nociception. This region of the mesencephalon also receives afferents from the spinal cord via the spinomesencephalic tract, which transmits information regarding
nociception. The connectivity identified between the ventral aqueductal region and the NRM supports the hypothesis that this region is a critical component in antinociception, and further demonstrates the importance of this mesencephalic region in the integration of forebrain and brainstem activity. In addition to the importance ofthe PAG-NRM-spinal cord pathway, recent evidence indicates that parallel bulbospinal pathways exist that may also modulate nociception. The output of the descending PAG-NRM-dorsal horn system is the modulation of spinal neuronal activity (8) that is elicited in part by release of S-hydroxytryptamine (SHT) (4,24,25). However, stimulation of the NRM results in the release of both 5HT and norepinephrine (NE) from terminals in the spinal cord (11) and pharmacological manipulation of either 5HT or NE interferes with NRM antinociception (12). The connectivity identified between the NRM and brainstem catecholaminergic groups in this study may provide the neuroanatomical substrate by which NRM stimulation elicits NE release. Stimulation of the locus coeruleus (13,18) or ventral lateral pontine tegmentum in the region of A5 and A7 (14) elicits antinociception and intrathecal NE produces dose-dependent antinociception ( 16). Neurons in A5, A6, and A7 have been shown to project to the spinal cord, where noradrenergic fibers and terminals have been identified in regions including lamina I and II (22), as well as contacting spinothalamic projection neurons in lamina I and V (23). NRM projections to brainstem catecholaminergic nuclei may modulate the activity of bulbospinal projection neurons. These results demonstrate connectivity that may result in modulation of nociception by NRM neurons at the supraspinal level. Putative terminal fields identified in the PAG, locus coeruleus, A5, A7, and nuclei of the trigeminal complex may be particularly important in such a system. ACKNOWLEDGEMENTS
This work was supported by NIH No. NS2 1323 (S. A. J.) and USPHS No. DA07232 (L. J. S.).
REFERENCES 1. Abols, I. A.; Basbaum, A. I. Afferent connections of the rostra1 medulla of the cat: A neural substrate for midbrain-medullary interactions in the modulation of pain. J. Comp. Neural. 201:285-297; 1981. 2. Basbaum, A. 1.; Clanton, C. H.; Fields, H. L. Opiate and stimulus produced analgesia: Functional anatomy of a medullospinal pathway. Proc. Natl. Acad. Sci. USA 73:4685&88; 1976. 3. Basbaum. A. I.: Ma&v. N. J. E.: O’Keefe. J.: Clanton. C. H. Reversal of morphine and stim&produced analgesia by subtotal spinal cord lesions. Pain 3:43-56; 1977. 4. Bowker, R. M.; Westlund, K. N.; Coulter, J. D. Origins of serotonergic projections to the spinal cord in rat: An immunocytochemical-retrograde transport study. Brain Res. 226:187-199; 1981. 5. Dickenson, A. H.; Oliveras, J. L.; Besson, J. M. Role of the nucleus raphe magnus in opiate analgesia as studies by the microinjection technique in the rat. Brain Res. 170:95-l 11; 1979. 6. Fardin, V.; Oliveras, J. L.; Besson, J. M. Projections from the periaqueductal gray matter to the B3 cellular area (nucleus raphe magnus and nucleus reticularis paragigantocellulatis) as revealed by the retrograde transport horseradish peroxidase in the rat. J. Comp. Neural. 223:483-500; 1984a. 7. Fardin, V.; Oliveras, J. L.; Besson, J. M. A reinvestigation of the analgesic effects induced by stimulation of the periaqueductal gray matter in the rat: I. The production of behavioral side effects together with analgesia. Brain Res. 306:105-123; 1984b.
8. Fields, H. L.; Anderson, S. D. Evidence that raphe-spinal neurons mediate opiate and midbrain stimulation-produced analgesias. Pain 5:333-349; 1978. 9. Fields, H. L.; Basbaum, A. I. Brainstem control of spinal pain-transmission neurons. Ann. Rev. Phvsiol. 40~217-248: 1978. 10. Hammond, D. L.; Levy, R. A.; P&d&, H. K. HypoaIgesia folIowing microinjection of noradrenergic antagonists in the nucleus raphe magnus. Pain 9:85-101; 1980. Il. Hammond, D. L.; Tyce, G. M.; Yaksh, T. L. Efflux of 5-hydroxytryptamine and noradrenaline into spinal superfusates during stimulation of the rat medulla. J. Physioi. 359: 15I- 162; 1985. _ 12. Jensen. T. S.: Yaksh. T. L. II. Examination of spinal monoamine receptors through which brainstem opiate-sensitive svstems act in the rat. Brain Res. 363:114-127; 1986. 13. Jones. S. L.: Gebhart. G. F. Inhibition of soinal nociceotive transmission from the midbrain, pons and medulla in the rat: Activation of descending inhibition by morphine, glutamate and electrical stimulation. Brain Res. 460:281-296; 1988. 14. Miller, J. F.; Proudfit, H. K. Antagonism of stimulation-produced antinociception from ventrolateral pontine sites by intrathecal administration of alpha-adrenergic antagonists and naloxone. Brain Res. 530:20-34; 1990. 15. Oliveras, J. L.; Guilbaud, G.; Besson, J. M. A map of serotonergic structures involved in stimulation-produced analgesia in unrestrained freely moving cats. Brain Res. 164317-322; 1979. 16. Reddy, S. V. R.; Yaksh, T. L. Spinal noradrenergic terminal system mediates antinociception. Brain Res. 189:39 l-40 1; 1980.
682
17. Sagen, J.; Proudfit, H. K. Alterations in nociception following lesions of the A5 catecholamine nucleus. Brain Res. 370:93-101; 1986. 18. SegaJ, M.; Sandberg, D. Analgesia produced by electrical stimulation of catecholamine nuclei in the rat brain. Brain Res. 123:369-372: 1977. 19. Sim, L. J.; Joseph, S. A. Opiocortin and catecholamine projections to raphe nuclei. Pentides 10:1019-1025: 1989. 20. Sim,.L. J.; Joseph,-S. A. Arcuate nucleus projections to brainstem regions which modulate nociception. J. Chem. Neuroanat. 4:97109; 1991. 2 I. Sim, L. J.; Joseph, S. A. Dorsal raphe nucleus efferents and putative contacts with peptidergic neurons. Peptides (in press). 22. Westlund, K. N.; Bowker, R. M.; Ziegler, M. G.; Coulter, J. D. Noradrenergic projections to the spinal cord of the rat. Brain Res. 263:15-31; 1983.
SIM AND JOSEPH 23. Westlund, K. N.; Carbon, S. M.; Zhang, D.; Willis, W. D. Direct catecholaminergic innervation of primate spinothalamic tract neurons. J. Comp. Neurol. 299: 178-l 86; 1990. 24. Yaksh, T. L. Direct evidence that spinal serotonin and nomdrenaline terminals mediate the spinal antinociceptive effects of morphine in the oeriaaueductal erav. Brain Res. 160:180- 185; 1979. 25. Yaksh, T: L.; Ty& d. M. Microinjection of morphine into the periaqueductal gray evokes the release of serotonin from spinal cord. Brain Res. 171:176-181; 1979. 26. Yaksh, T. L.; Yeung, J. C.; Rudy, T. A. Systematic examination in the rat of brain sites sensitive to the direct application of morphine: Observation of differential effects within the periaqueductal gray. Brain Res. Il4:83-103; 1976. 27. Zorman, G.; Belcher, G.; Adams, J. E.; Fields, H. L. Lumbar intrathecal naloxone blocks analgesia produced by microstimulation of the ventromedial medulla in the rat. Brain Res. 236:77-84: 1982.
