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Collateral projections of single neurons in the nucleus raphe magnus to both the sensory trigeminal nuclei and spinal cord in the rat
Brain Research, 602 (1993) 331-335 © 1993 Elsevier Science Publishers B.V. All rights reserved 0006-8993/93/$06.00
331
BRES 25492
Collateral projections of single neurons in the nucleus raphe magnus to both the sensory trigeminal nuclei and spinal cord in the rat Yun-Qing
L i *, M a s a h i k o
Takada,
Yasuhide
Shinonaga
and Noboru
Mizuno
Department of Morphological Brain Science, Faculty of Medicine, Kyoto Uni~'ersity, Kyoto (Japan)
(Accepted 6 October 1992)
Key words: Nucleus raphe magnus; Pain; Nociception; Serotonin; Sensory trigeminal nuclei; Spinal cord; Rat
After injecting Diamidino yellow and Fast blue respectively into the sensory trigeminal nuclei and spinal cord, we observed doubly labeled cells in the nucleus raphe magnus (NRM). Combining the fluorescent retrograde double labeling with serotonin (5-HT) immunofluorescence histochemistry, we further found that about 30% of the doubly labeled NRM neurons showed 5-HT-like immunoreactivity (5-HT-LI). Such 5-HT-LI NRM neurons may modulate nociceptive activities simultaneously in the sensory trigeminal nuclei and spinal cord by sending axon collaterals to these regions.
It appears to be established that the descending pain control from the periaqueductal gray ( P A G ) including the dorsal raphe nucleus is exerted mainly through serotonin (5-HT)-containing neurons in the subregions of the rostral ventral medulla, especially in the nucleus raphe magnus (NRM) (for reviews, cf. refs. 1,2,7,8,16). It has been reported that (1) the activities of nociceptive neurons in the spinal trigeminal nuclei and dorsal horn of the spinal cord are markedly suppressed by stimulation of the NRM 5'6'9'10'12'17, (2) lesions of the N R M interrupt the analgesic action of P A G stimulation 18, and (3) local anesthetic blockade of the N R M reversively eliminates the inhibitory action of P A G stimulation on dorsal horn neurons responding to noxious stimuli tt. The N R M has further been known to contain 5-HT-like immunoreactive (5-HT-LI) neurons projecting to the spinal trigeminal nuclei 3 and dorsal horn of the spinal cord 4A4'15. It is still unknown, however, whether or not single N R M neurons containing 5-HT send projection fibers to both the sensory trigeminal nuclei and spinal cord by way of axon collaterals. Thus, in the present study, we attempted to settle this problem by a fluorescent retrograde double-labeling
technique combined with immunofluorescence histochemistry for 5-HT. Adult male rats (Wistar) weighing 350-450 g were used. All surgical procedures were done under general anesthesia by intraperitoneal injection of sodium pentobarbital (40 m g / k g b.wt.). In each rat, stereotaxic injections with the fluorescent dyes Diamidino yellow (DY; Illing) and Fast blue (FB; Illing) were respectively made into a subdivision of the sensory trigeminal nuclei and the lumbar enlargement ( L 3 - L 6 ) of the spinal cord on one side of the central nervous system: 0.05-0.1 /zl of a 3% DY- or 5% FB-suspension in distilled water was injected by pressure over a period of 10-20 min through a fine needle which was attached to a 1-/xl Hamilton microsyringe mounted on a microdrive. After the injections, the rats were allowed to survive for 3 - 5 days, then re-anesthetized deeply, and perfused through the ascending aorta with 100 ml of 0.9% saline, followed with 500 ml of 10% formalin in 0.1 M phosphate buffer p H 7.3 (PB). After the perfusion, the brains and spinal cords were immediately removed, placed in the same fresh fixative for 4 - 6 h at 4°C, and then in a solution of 30% sucrose in PB overnight at
Correspondence: N. Mizuno, Department of Morphological Brain Science, Faculty of Medicine, Kyoto University, Kyoto 606-01, Japan. * On leave from Department of Anatomy, The Fourth Military Medical University, Xian, People's Republic of China.
332
R29
R~
Fig. 1. The sites of injections of DY (blackened areas) in the principal sensory trigeminal nucleus (Vp), and those of FB (striped areas) in the lumbar enlargement (L3-L6) of the spinal cord in 3 rats (Rg, R12, RI3). The sections of the lower brainstems are arranged rostrocaudally (left to right). M, motor trigeminal nucleus; p, pyramidal tract; t, spinal trigeminal tract.
Fig. 3. The sites of injections of DY (blackened areas) in the interpolar division of the spinal trigeminal nucleus (Vi), and those of FB (striped areas) in the lumbar enlargement (L3-L6) of the spinal cord in 3 rats (R24, R29, R32). The sections of the lower brainstems are arranged rostrocaudally (left to right). Abbreviations are as in Fig. 1.
overnight at 4°C. Subsequently, the sections were incu4°C. Subsequently, the lower b r a i n s t e m s a n d l u m b a r on a freezing microtome.
b a t e d with b i o t i n - c o n j u g a t e d goat a n t i - r a b b i t I g G (Vector; 1 : 150 dilution) for 3 h at room t e m p e r a t u r e , a n d finally with a v i d i n - c o n j u g a t e d Texas red (Vector; 1 : 200
T h e sections of the lower b r a i n s t e m s and spinal cords were collected consecutively in two dishes con-
were then m o u n t e d onto clean glass slides.
cord s e g m e n t s were cut transversely at 40 ~ m thickness
t a i n i n g PB; each dish c o n t a i n e d every second section. T h e sections of the lower b r a i n s t e m s a n d spinal cords from the first dish were m o u n t e d onto clean glass slides. T h e sections of the lower b r a i n s t e m s from the second dish were processed for 5 - H T i m m u n o f l u o r e s cence histochemistry according to the A B C methodl3: They were i n c u b a t e d with rabbit polyclonal a n t i - s e r u m against 5 - H T (Incstar; 1 : 3000 dilution with the i n c u b a tion m e d i u m c o n t a i n i n g 2.5% n o r m a l goat serum)
~
dilution) for I h at room t e m p e r a t u r e . T h e sections T h e slides were observed with a Zeiss epifluorescence microscope. A n ultraviolet filter providing excitation light of a b o u t 360 n m wave-length was used to e x a m i n e yellow-emitting DY-positive n e u r o n s a n d b l u e - e m i t t i n g FB-positive ones, while a g r e e n filter providing excitation light of a b o u t 550 n m wave-length was used to view r e d - e m i t t i n g 5 - H T - L I n e u r o n s . A total of 34 rats injected with D Y a n d FB respectively into the sensory t r i g e m i n a l nuclei a n d l u m b a r
L4 L5
1.5
Fig. 2. The sites of injections of DY (blackened areas) in the oral division of the spinal trigeminal nucleus (Vo), and those of FB (striped areas) in the lumbar enlargement (L4-L6) of the spinal cord in 3 rats (R15, R16, RI9). The sections of the lower brainstems are arranged rostrocaudally (left to right). F, facial nucleus; g, genu of the exiting root of the facial nerve. Other abbreviations are as in Fig. 1.
Fig. 4. The sites of injections of DY (blackened areas) in the caudal division of the spinal trigeminal nucleus (Vc), and those of FB (striped areas) in the lumbar enlargement (L3-L6) of the spinal cord in 3 rats (R3, R5, R6). The sections of the lower brainstems are arranged rostrocaudally (left to right). Abbreviations are as in Fig. 1.
333
Fig. 5. Dark-field photomicrographs of retrogradely labeled neurons in the N R M (a) and nucleus reticularis gigantocellularis pars alpha (b), seen in a rat (R5) after DY and FB injections respectively into the Vc and lumbar enlargement (L5 and L6) of the spinal cord (Fig. 4). Neurons doubly labeled with D Y and FB are indicated by double arrowheads. Neurons singly labeled with DY are indicated by arrowheads, and those singly labeled with FB are pointed with arrows. Scale bar 50 = / x m .
enlargement of the spinal cord. In 12 of them, the site of DY injection in the sensory trigeminal nuclei was centered on the principal sensory trigeminal (Vp), oral spinal trigeminal (Vo), interpolar spinal trigeminal (Vi), or caudal spinal trigeminal nucleus (Vc), ipsilateral to the FB injection into the lumbar enlargement of the spinal cord (Figs. 1-4). In these rats, neuronal cell
bodies retrogradely labeled with DY or/and FB were examined at the pontine and medullary levels of the lower brainstem. Retrogradely labeled neuronal cell bodies were distributed mainly in the pontine and medullary reticular formation and raphe nuclei, especially in the rostromediai part of the medullary reticular formation and the raphe nuclei in the rostroventral
i
O Fig. 6. a and b: labeling with DY and FB in the N R M (a) or nucleus raphe obscurus (b). a' and b': 5-HT-LI in the same field as shown respectively in a and b. Neurons labeled doubly with DY and FB (arrowheads in a and b) display 5-HT-LI (arrowheads in a' and b'). Scale bar 25 =/~m.
334 TABLE I Labeled N R M neurons in rats injected with D Y (into the Vp, Vo, Vi or Vc) and FB (into the lumbar cord: L) Injection sites and Rat No.
Number o f labeled cells
Vp+ L R9 R12 RI3
237 194 170
Total
Labeled cells with FB (%)
Labeled cells with D Y + FB (%)
75 (31.6) 53 (27.3) 56 (32.9)
147 (62.0) 128 (66.0) 105 (61.8)
15 (6.3) 13 (6.7) 9 (5.3)
601
184 (30.6)
380 (63.2)
37 (6.2)
Vo+ L R15 RI6 RI9
196 207 227
48 (24.4) 42 (20.3) 53 (23.3)
137 (70.0) 156 (75.4) 162 (71.4)
11 (5.6) 9 (4.3) 12 (5.3)
Total
630
143 (22.7)
455 (72.2)
32 (5.1)
178 202 187
38 (21.3) 43 (21.3) 36 (19.3)
135 (75.8) 152 (75.2) 147 (78.6)
5 (2.8) 7 (3.5) 4 (2.1)
567
117 (20.6)
434 (76.5)
16 (2.8)
234 261 230
83 (35.5) 97 (37.1) 82 (35.7)
124 (53.0) 133 (51.0) 118 (51.3)
27 (11.5) 31 (11.9) 30 (13.0)
725
262 (36.1)
375 (51.7)
88 (12.1)
Vi+ L R24 R29 R32
Total Vc+ L R3 R5 R6
Total
Labeled cells with D Y (%)
medulla. Labeling with DY o r / a n d FB was recognized by yellow fluorescence in cellular nuclei, o r / a n d by blue fluorescence in perikarya, respectively (Fig. 5). Neuronal cell bodies doubly labeled with DY and FB were seen most frequently in the NRM (Fig. 5a); they were distributed throughout the entire rostrocaudal extent of the NRM. They were fusiform, triangular or multipolar in shape; the maximal diameters of the cell bodies ranged 15-50 /xm. Although no particular differences were observed in the distribution pattern of the doubly labeled neurons in the NRM among the rats examined in the present study, NRM neurons were doubly labeled most frequently in the rats injected with DY into the Vc: Out of DY o r / a n d FB labeled NRM neurons, 11.5-13.0% (27-31 cells) were
TABLE II 5HT-LI in N R M neurons doubly labeled with D Y and FB injected respectit,ely into the Vc and lumbar cord Rat No.
R36 R37 R38 Total
Number of cells doubly labeled with D Y + FB
Number o f 5HT-LI cells doubly labeled with D Y + FB (%)
32 28 34
10 (31.3) 9 (32.1) 12 (35.3)
94
31 (33.0)
doubly labeled in the rats injected with DY into the Vc, whereas 2.1-6.7% (4-15 cells) were doubly labeled in the rats injected with DY into the Vp, Vo or Vi (Table I). A few doubly labeled neurons were further distributed in other regions of the medullary reticular formation, especially in the nucleus reticularis gigantocellularis pars alpha (Fig. 5b), nucleus raphe obscurus, and nucleus raphe pallidus. Neuronal cell bodies showing 5-HT-LI were recognized by red fluorescence in perikarya (Fig. 6a',b'). About 30% of the NRM neurons doubly labeled with DY and FB showed 5-HT-LI in the rats injected with DY into the Vc (Fig. 6a,a') (Table II). The nucleus reticularis gigantocellularis pars alpha, nucleus raphe obscurus and nucleus raphe pallidus also contained a few 5-HT-LI neurons labeled doubly with DY and FB (Fig. 6b,b'). In the other rats injected with DY into the Vp, Vo or Vi, only less than 5% of the NRM neurons doubly labeled with DY and FB exhibited 5-HT-LI; the nucleus reticularis gigantocellularis pars alpha, nucleus raphe obscurus and nucleus raphe pallidus rarely contained 5-HT-LI neurons labeled doubly with DY and FB. It has been reported that the NRM sends projection fibers to the spinal trigeminal nuclei 3 and spinal c o r d 4'14"15. The present results indicate that the NRM of the rat contains neurons projecting directly by way of axon collaterals to both the sensory trigeminal nuclei (not only spinal trigeminal nuclei but also Vp) and spinal cord, that the axon collaterals projecting to the sensory trigeminal nuclei terminate most frequently in the Vc, the main target of nociceptive primary afferent fibers in the trigeminal, facial, glossopharyngeal and vagus nerves, and that at least 30% of the NRM neurons sending their axon collaterals to both the Vc and spinal cord contain 5-HT. It has been known that the NRM is a key structure in the descending pain control system (for reviews, cf. refs. 1,2,5-12,16-18), and that NRM neurons projecting to the spinal trigeminal nuclei and spinal cord contain 5 - H T 3'4'14'15. Thus, the 5-HT-LI NRM neurons projecting directly to both the sensory trigeminal nuclei and spinal cord by way of axon collaterals may be capable to exert control influences simultaneously upon the nociceptive neuronal activities in the lower brainstem and spinal cord. The authors are grateful for the photographic help of Mr. Akira Uesugi, and for the support of Drs. Ryosuke Fujimori, Satoru Fukuchi, Toshio Fukuda, Ritsu Hayashi, Sozaburo Hayashi, Mizuho Katsurada, Yutaka Kitani, Keiko Kumagai, Hiroshi Kuroda, Toshihiko Kuroda, Hiroshi Matsubara, Hiroshi Matsushima, Chisato Minakuchi, Masatoshi Nishio, Gonpei Niwa, Hajime Oda, Masahiko Ohbayashi, Sei-ichi Ohbayashi, Hiroyasu Ohtsuka, Shigeo Tamaki, Eizo Watanabe, Kazuo Yoshino, and Toshiaki Yoshino. This work
335 was supported in part by Grants-in-Aid for Special Research on Priority Areas (04255204 and 04246106) and Scientific Research (B) (02454113) from the Ministry of Education, Science and Culture of Japan.
1 Basbaum, A.I. and Fields, H.L., Endogenous pain control mechanisms: review and hypothesis, Ann. Neurol., 4 (1978)451-462. 2 Basbaum, A.I. and Fields, H.L., Endogenous pain control systems: brainstem spinal pathways and endorphin circuitry, Annu. Rev. Neurosci., 7 (1984) 309-338. 3 Beitz, A.J., The nuclei of origin of brainstem serotonergic projections to the rodent spinal trigeminal nucleus, Neurosci. Lett., 32 (1982) 223-228. 4 Bowker, R.M., Westlund, K.N. and Coulter, J.D., Origins of serotonergic projections to the spinal cord in rat: an immunocytochemical-retrograde transport study, Brain Res., 226 (1981) 187199. 5 Dickenson, A.H., Oliveras, J.-L. and Besson, J.-M., Role of the nucleus raphe magnus in opiate analgesia as studied by the microinjection technique in the rat, Brain Res., 170 (1979) 95-111. 6 Dostrovsky, J.O., Shah, Y. and Gray, B.G., Descending inhibitory influences from periaqueductal gray, nucleus raphe magnus, and adjacent reticular formation. II. Effects on medullary dorsal horn nociceptive and nonnociceptive neurons, J. Neurophysiol., 49 (1983) 948-960. 7 Dubner, R. and Bennett, G.J., Spinal and trigeminal mechanisms of nociception, Annu. Rev. Neurosci., 6 (1983) 381-418. 8 Fields, H.L. and Basbaum, A.I., Brainsiem control of spinal pain-transmission neurons, Annu. Rev. Physiol., 40 (1978)217248. 9 Fields, H.L., Basbaum, A.I., Clanton, C.H. and Anderson, S.D., Nucleus raphe magnus inhibition of spinal cord dorsal horn neurons, Brain Res., 126 (1977) 441-453. 10 Gebhart, G.F., Sandkiihler, J., Thalhammer, J.G. and Zimmermann, M., Quantitative comparison of inhibition in spinal cord of
11
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nociceptive information by stimulation in periaqueductal gray or nucleus raphe magnus of the cat, J. Neurophysiol., 50 (1983) 1433-1445. Gebhart, G.F., Sandkiihler, J., Thalhammer, J.G. and Zimmermann, M., Inhibition of spinal nociceptive information by stimulation in midbrain of the cat is blocked by lidocaine microinjected in nucleus raphe magnus and medullary reticular formation, J. Neurophysiol., 50 (1983) 1446-1459. Gray, B.G. and Dostrovsky, J.O., Descending inhibitory influences from periaqueductal gray, nucleus raphe magnus, and adjacent reticular formation. I. Effects on lumbar spinal cord nociceptive and nonnociceptive neurons, J. NeurophysioL, 49 (1983) 932-947. Hsu, S.-M., Raine, L. and Fanger, H., Use of avidin-biotin-peroxidase complex (ABC) in immunoperoxidase techniques: a comparison between ABC and unlabeled antibody (PAP) procedures, J. Histochern. Cytochem., 29 (1981) 577-580. Jones, S.L. and Light, A.R., Serotoninergic medullary raphespinal projection to the lumbar spinal cord in the rat: a retrograde immunohistochemical study, J. Cornp. Neurol., 322 (1992) 599610. Kwiat, G.C. and Basbaum, A.I., The origin of brainstem noradrenergic and serotonergic projections to the spinal cord dorsal horn in the rat, Somatosens. Motor Res., 9 (1992) 157-174. Lakos, S. and Basbaum, A.I., An ultrastructural study of the projections from the midbrain periaqueductal gray to spinally projecting, serotonin-immunoreactive neurons of the medullary nucleus raphe magnus in the rat, Brain Res., 443 (1988) 383-388. Lovick, T.A. and Wolstencroft, J.H., Inhibitory effects of nucleus raphe magnus on neuronal responses in the spinal trigeminal nucleus to nociceptive compared with nonnociceptive inputs, Pain, 7 (1979) 135-145. Prieto, G.J., Cannon, J.T. and Liebeskind, J.C., N. raphe magnus lesions disrupt stimulation-produced analgesia from ventral but not dorsal midbrain areas in the rat, Brain Res., 261 (1983) 53-57.
331
BRES 25492
Collateral projections of single neurons in the nucleus raphe magnus to both the sensory trigeminal nuclei and spinal cord in the rat Yun-Qing
L i *, M a s a h i k o
Takada,
Yasuhide
Shinonaga
and Noboru
Mizuno
Department of Morphological Brain Science, Faculty of Medicine, Kyoto Uni~'ersity, Kyoto (Japan)
(Accepted 6 October 1992)
Key words: Nucleus raphe magnus; Pain; Nociception; Serotonin; Sensory trigeminal nuclei; Spinal cord; Rat
After injecting Diamidino yellow and Fast blue respectively into the sensory trigeminal nuclei and spinal cord, we observed doubly labeled cells in the nucleus raphe magnus (NRM). Combining the fluorescent retrograde double labeling with serotonin (5-HT) immunofluorescence histochemistry, we further found that about 30% of the doubly labeled NRM neurons showed 5-HT-like immunoreactivity (5-HT-LI). Such 5-HT-LI NRM neurons may modulate nociceptive activities simultaneously in the sensory trigeminal nuclei and spinal cord by sending axon collaterals to these regions.
It appears to be established that the descending pain control from the periaqueductal gray ( P A G ) including the dorsal raphe nucleus is exerted mainly through serotonin (5-HT)-containing neurons in the subregions of the rostral ventral medulla, especially in the nucleus raphe magnus (NRM) (for reviews, cf. refs. 1,2,7,8,16). It has been reported that (1) the activities of nociceptive neurons in the spinal trigeminal nuclei and dorsal horn of the spinal cord are markedly suppressed by stimulation of the NRM 5'6'9'10'12'17, (2) lesions of the N R M interrupt the analgesic action of P A G stimulation 18, and (3) local anesthetic blockade of the N R M reversively eliminates the inhibitory action of P A G stimulation on dorsal horn neurons responding to noxious stimuli tt. The N R M has further been known to contain 5-HT-like immunoreactive (5-HT-LI) neurons projecting to the spinal trigeminal nuclei 3 and dorsal horn of the spinal cord 4A4'15. It is still unknown, however, whether or not single N R M neurons containing 5-HT send projection fibers to both the sensory trigeminal nuclei and spinal cord by way of axon collaterals. Thus, in the present study, we attempted to settle this problem by a fluorescent retrograde double-labeling
technique combined with immunofluorescence histochemistry for 5-HT. Adult male rats (Wistar) weighing 350-450 g were used. All surgical procedures were done under general anesthesia by intraperitoneal injection of sodium pentobarbital (40 m g / k g b.wt.). In each rat, stereotaxic injections with the fluorescent dyes Diamidino yellow (DY; Illing) and Fast blue (FB; Illing) were respectively made into a subdivision of the sensory trigeminal nuclei and the lumbar enlargement ( L 3 - L 6 ) of the spinal cord on one side of the central nervous system: 0.05-0.1 /zl of a 3% DY- or 5% FB-suspension in distilled water was injected by pressure over a period of 10-20 min through a fine needle which was attached to a 1-/xl Hamilton microsyringe mounted on a microdrive. After the injections, the rats were allowed to survive for 3 - 5 days, then re-anesthetized deeply, and perfused through the ascending aorta with 100 ml of 0.9% saline, followed with 500 ml of 10% formalin in 0.1 M phosphate buffer p H 7.3 (PB). After the perfusion, the brains and spinal cords were immediately removed, placed in the same fresh fixative for 4 - 6 h at 4°C, and then in a solution of 30% sucrose in PB overnight at
Correspondence: N. Mizuno, Department of Morphological Brain Science, Faculty of Medicine, Kyoto University, Kyoto 606-01, Japan. * On leave from Department of Anatomy, The Fourth Military Medical University, Xian, People's Republic of China.
332
R29
R~
Fig. 1. The sites of injections of DY (blackened areas) in the principal sensory trigeminal nucleus (Vp), and those of FB (striped areas) in the lumbar enlargement (L3-L6) of the spinal cord in 3 rats (Rg, R12, RI3). The sections of the lower brainstems are arranged rostrocaudally (left to right). M, motor trigeminal nucleus; p, pyramidal tract; t, spinal trigeminal tract.
Fig. 3. The sites of injections of DY (blackened areas) in the interpolar division of the spinal trigeminal nucleus (Vi), and those of FB (striped areas) in the lumbar enlargement (L3-L6) of the spinal cord in 3 rats (R24, R29, R32). The sections of the lower brainstems are arranged rostrocaudally (left to right). Abbreviations are as in Fig. 1.
overnight at 4°C. Subsequently, the sections were incu4°C. Subsequently, the lower b r a i n s t e m s a n d l u m b a r on a freezing microtome.
b a t e d with b i o t i n - c o n j u g a t e d goat a n t i - r a b b i t I g G (Vector; 1 : 150 dilution) for 3 h at room t e m p e r a t u r e , a n d finally with a v i d i n - c o n j u g a t e d Texas red (Vector; 1 : 200
T h e sections of the lower b r a i n s t e m s and spinal cords were collected consecutively in two dishes con-
were then m o u n t e d onto clean glass slides.
cord s e g m e n t s were cut transversely at 40 ~ m thickness
t a i n i n g PB; each dish c o n t a i n e d every second section. T h e sections of the lower b r a i n s t e m s a n d spinal cords from the first dish were m o u n t e d onto clean glass slides. T h e sections of the lower b r a i n s t e m s from the second dish were processed for 5 - H T i m m u n o f l u o r e s cence histochemistry according to the A B C methodl3: They were i n c u b a t e d with rabbit polyclonal a n t i - s e r u m against 5 - H T (Incstar; 1 : 3000 dilution with the i n c u b a tion m e d i u m c o n t a i n i n g 2.5% n o r m a l goat serum)
~
dilution) for I h at room t e m p e r a t u r e . T h e sections T h e slides were observed with a Zeiss epifluorescence microscope. A n ultraviolet filter providing excitation light of a b o u t 360 n m wave-length was used to e x a m i n e yellow-emitting DY-positive n e u r o n s a n d b l u e - e m i t t i n g FB-positive ones, while a g r e e n filter providing excitation light of a b o u t 550 n m wave-length was used to view r e d - e m i t t i n g 5 - H T - L I n e u r o n s . A total of 34 rats injected with D Y a n d FB respectively into the sensory t r i g e m i n a l nuclei a n d l u m b a r
L4 L5
1.5
Fig. 2. The sites of injections of DY (blackened areas) in the oral division of the spinal trigeminal nucleus (Vo), and those of FB (striped areas) in the lumbar enlargement (L4-L6) of the spinal cord in 3 rats (R15, R16, RI9). The sections of the lower brainstems are arranged rostrocaudally (left to right). F, facial nucleus; g, genu of the exiting root of the facial nerve. Other abbreviations are as in Fig. 1.
Fig. 4. The sites of injections of DY (blackened areas) in the caudal division of the spinal trigeminal nucleus (Vc), and those of FB (striped areas) in the lumbar enlargement (L3-L6) of the spinal cord in 3 rats (R3, R5, R6). The sections of the lower brainstems are arranged rostrocaudally (left to right). Abbreviations are as in Fig. 1.
333
Fig. 5. Dark-field photomicrographs of retrogradely labeled neurons in the N R M (a) and nucleus reticularis gigantocellularis pars alpha (b), seen in a rat (R5) after DY and FB injections respectively into the Vc and lumbar enlargement (L5 and L6) of the spinal cord (Fig. 4). Neurons doubly labeled with D Y and FB are indicated by double arrowheads. Neurons singly labeled with DY are indicated by arrowheads, and those singly labeled with FB are pointed with arrows. Scale bar 50 = / x m .
enlargement of the spinal cord. In 12 of them, the site of DY injection in the sensory trigeminal nuclei was centered on the principal sensory trigeminal (Vp), oral spinal trigeminal (Vo), interpolar spinal trigeminal (Vi), or caudal spinal trigeminal nucleus (Vc), ipsilateral to the FB injection into the lumbar enlargement of the spinal cord (Figs. 1-4). In these rats, neuronal cell
bodies retrogradely labeled with DY or/and FB were examined at the pontine and medullary levels of the lower brainstem. Retrogradely labeled neuronal cell bodies were distributed mainly in the pontine and medullary reticular formation and raphe nuclei, especially in the rostromediai part of the medullary reticular formation and the raphe nuclei in the rostroventral
i
O Fig. 6. a and b: labeling with DY and FB in the N R M (a) or nucleus raphe obscurus (b). a' and b': 5-HT-LI in the same field as shown respectively in a and b. Neurons labeled doubly with DY and FB (arrowheads in a and b) display 5-HT-LI (arrowheads in a' and b'). Scale bar 25 =/~m.
334 TABLE I Labeled N R M neurons in rats injected with D Y (into the Vp, Vo, Vi or Vc) and FB (into the lumbar cord: L) Injection sites and Rat No.
Number o f labeled cells
Vp+ L R9 R12 RI3
237 194 170
Total
Labeled cells with FB (%)
Labeled cells with D Y + FB (%)
75 (31.6) 53 (27.3) 56 (32.9)
147 (62.0) 128 (66.0) 105 (61.8)
15 (6.3) 13 (6.7) 9 (5.3)
601
184 (30.6)
380 (63.2)
37 (6.2)
Vo+ L R15 RI6 RI9
196 207 227
48 (24.4) 42 (20.3) 53 (23.3)
137 (70.0) 156 (75.4) 162 (71.4)
11 (5.6) 9 (4.3) 12 (5.3)
Total
630
143 (22.7)
455 (72.2)
32 (5.1)
178 202 187
38 (21.3) 43 (21.3) 36 (19.3)
135 (75.8) 152 (75.2) 147 (78.6)
5 (2.8) 7 (3.5) 4 (2.1)
567
117 (20.6)
434 (76.5)
16 (2.8)
234 261 230
83 (35.5) 97 (37.1) 82 (35.7)
124 (53.0) 133 (51.0) 118 (51.3)
27 (11.5) 31 (11.9) 30 (13.0)
725
262 (36.1)
375 (51.7)
88 (12.1)
Vi+ L R24 R29 R32
Total Vc+ L R3 R5 R6
Total
Labeled cells with D Y (%)
medulla. Labeling with DY o r / a n d FB was recognized by yellow fluorescence in cellular nuclei, o r / a n d by blue fluorescence in perikarya, respectively (Fig. 5). Neuronal cell bodies doubly labeled with DY and FB were seen most frequently in the NRM (Fig. 5a); they were distributed throughout the entire rostrocaudal extent of the NRM. They were fusiform, triangular or multipolar in shape; the maximal diameters of the cell bodies ranged 15-50 /xm. Although no particular differences were observed in the distribution pattern of the doubly labeled neurons in the NRM among the rats examined in the present study, NRM neurons were doubly labeled most frequently in the rats injected with DY into the Vc: Out of DY o r / a n d FB labeled NRM neurons, 11.5-13.0% (27-31 cells) were
TABLE II 5HT-LI in N R M neurons doubly labeled with D Y and FB injected respectit,ely into the Vc and lumbar cord Rat No.
R36 R37 R38 Total
Number of cells doubly labeled with D Y + FB
Number o f 5HT-LI cells doubly labeled with D Y + FB (%)
32 28 34
10 (31.3) 9 (32.1) 12 (35.3)
94
31 (33.0)
doubly labeled in the rats injected with DY into the Vc, whereas 2.1-6.7% (4-15 cells) were doubly labeled in the rats injected with DY into the Vp, Vo or Vi (Table I). A few doubly labeled neurons were further distributed in other regions of the medullary reticular formation, especially in the nucleus reticularis gigantocellularis pars alpha (Fig. 5b), nucleus raphe obscurus, and nucleus raphe pallidus. Neuronal cell bodies showing 5-HT-LI were recognized by red fluorescence in perikarya (Fig. 6a',b'). About 30% of the NRM neurons doubly labeled with DY and FB showed 5-HT-LI in the rats injected with DY into the Vc (Fig. 6a,a') (Table II). The nucleus reticularis gigantocellularis pars alpha, nucleus raphe obscurus and nucleus raphe pallidus also contained a few 5-HT-LI neurons labeled doubly with DY and FB (Fig. 6b,b'). In the other rats injected with DY into the Vp, Vo or Vi, only less than 5% of the NRM neurons doubly labeled with DY and FB exhibited 5-HT-LI; the nucleus reticularis gigantocellularis pars alpha, nucleus raphe obscurus and nucleus raphe pallidus rarely contained 5-HT-LI neurons labeled doubly with DY and FB. It has been reported that the NRM sends projection fibers to the spinal trigeminal nuclei 3 and spinal c o r d 4'14"15. The present results indicate that the NRM of the rat contains neurons projecting directly by way of axon collaterals to both the sensory trigeminal nuclei (not only spinal trigeminal nuclei but also Vp) and spinal cord, that the axon collaterals projecting to the sensory trigeminal nuclei terminate most frequently in the Vc, the main target of nociceptive primary afferent fibers in the trigeminal, facial, glossopharyngeal and vagus nerves, and that at least 30% of the NRM neurons sending their axon collaterals to both the Vc and spinal cord contain 5-HT. It has been known that the NRM is a key structure in the descending pain control system (for reviews, cf. refs. 1,2,5-12,16-18), and that NRM neurons projecting to the spinal trigeminal nuclei and spinal cord contain 5 - H T 3'4'14'15. Thus, the 5-HT-LI NRM neurons projecting directly to both the sensory trigeminal nuclei and spinal cord by way of axon collaterals may be capable to exert control influences simultaneously upon the nociceptive neuronal activities in the lower brainstem and spinal cord. The authors are grateful for the photographic help of Mr. Akira Uesugi, and for the support of Drs. Ryosuke Fujimori, Satoru Fukuchi, Toshio Fukuda, Ritsu Hayashi, Sozaburo Hayashi, Mizuho Katsurada, Yutaka Kitani, Keiko Kumagai, Hiroshi Kuroda, Toshihiko Kuroda, Hiroshi Matsubara, Hiroshi Matsushima, Chisato Minakuchi, Masatoshi Nishio, Gonpei Niwa, Hajime Oda, Masahiko Ohbayashi, Sei-ichi Ohbayashi, Hiroyasu Ohtsuka, Shigeo Tamaki, Eizo Watanabe, Kazuo Yoshino, and Toshiaki Yoshino. This work
335 was supported in part by Grants-in-Aid for Special Research on Priority Areas (04255204 and 04246106) and Scientific Research (B) (02454113) from the Ministry of Education, Science and Culture of Japan.
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