Neuroscience Research, 15 (1992) 273-280 © 1992 Elsevier Science Publishers Ireland, Ltd. All rights reserved 0168-0102/92/$05.00
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Spinal cord cells innervating the bilateral parabrachial nuclei in the rat A retrograde fluorescent double-labeling study Jinzo Y a m a d a and Taiko Kitamura Department of Anatomy, Tokyo Medical College, Tokyo, Japan (Received 16 July 1992; accepted 29 September 1992)
Key words: Parabrachial nucleus; Internal lateral subnucleus; Spinal cord cell; Collateral; Retrograde transport; Rat Summary The internal lateral nucleus (IL) of the parabrachial nucleus receives information from the spinal cord. The IL perhaps relays nociceptive signals to the intralaminar nuclei of the thalamus, apparently being implicated in the motivational-affective component of pain reactions. However, cells of origin of spinal fibers to the IL have not been investigated enough. We intended to clarify these cells, as well as their shapes, by retrograde double-labeling techniques. Fast blue and diamidino yellow dyes were injected, respectively, into the left and right ILs. The distribution of double-labeled cells was almost the same as that of single-labeled cells on both sides of the spinal cord. The total number of bilateral double-labeled cells was highest in the dorsolateral part of the lateral funiculus (DL), followed, in order, by lamina I, the dorsomedial part of the lateral funiculus (DM), lamina V and lamina VII. A few double-labeled cells were seen in laminae II-IV, VI, VIII and X. The ratio of the total number of bilateral double-labeled cells to the total number of bilateral single-labeled cells through the spinal cord was 43% in the DL, 37% in the DM, 28% in lamina V and 24% in lamina I. The ratio was 10% or less in the other remaining laminae. No marked differences were observed between the shapes of double- and single-labeled cells.
Introduction The parabrachial nucleus is a major relay for ascending gustatory, visceral and nociceptive afferent pathways, located around the brachium conjunctivum in the caudal midbrain (Saper and Loewy, 1980; Fulwiler and Saper, 1984; Milner et al., 1984; Cechetto et al., 1985; Milner and Pickel, 1986a,b; Herbert et al., 1990; Men6trey and de Pommery, 1991; Men6trey et al., 1992a,b). However, the parabrachial nucleus is a complex consisting of the medial nucleus, lateral nucleus and K611iker-Fuse nucleus. The lateral nucleus in Correspondence to: Dr. Jinzo Yamada, Department of Anatomy, Tokyo Medical College, 6-1-1 Shinjuku, Shinjuku-ku, Tokyo 160, Japan.
the rat is further divided into seven subnuclei, i.e., the internal lateral (IL), dorsal, central and superior (DCS) lateral and other subnuclei. Furthermore, afferents of varied origins converge onto the parabrachial nucleus, in particular from the solitary nucleus and the spinal cord. Precise anatomical knowledge is of essential importance in order to uncover specific structural-functional relationships for the parabrachial nucleus. We have been paying special attention to the IL which receives perhaps nociceptive signals from the spinal cord (Cechetto et al., 1985; Men6trey et al., 1992b) and which projects almost entirely to the intralaminar nucleus of the thalamus (Fulwiler and Saper, 1984). Responses of cells in the intralaminar nucleus are related to reward and escape behavior (Keene, 1973), and it is suggested that the intralaminar nucleus
274 is involved in the motivational-affective component of pain reaction (Yokota et al., 1991; Nishikawa et al., 1992). In a previous work by Cechetto et al. (1985), injection of wheat germ agglutinin-conjugated horseradish peroxidase (WGA-HRP) into the parabrachial nucleus on one side retrogradely labeled a large number of lamina I cells in the dorsal horn throughout the entire length of the spinal cord, bilaterally but with an ipsilateral predominance. In this study, however, the injected tracer covered the DCS lateral subnuclei, but not sufficiently in the IL. When we sufficiently covered the IL with W G A - H R P or fast blue (Kitamura et al., in course of publication), many retrogradely labeled cells were found bilaterally in laminae 1, V and VII of the dorsal horn as well as in the dorsolateral (DL) and dorsomedial (DM) parts of the lateral funiculus. These findings are similar to those reported for the lower thoracic to upper sacral segments by Men,trey and de Pommery (1991), even though the degree of coverage L
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of the 1L with the tracer was unclear in their experiment. In the present study, after injecting fast blue (FB) and diamidino yellow (DY) dyes into the left and right ILs of the rat, respectively, we investigated cells showing single and double retrograde labeling in the spinal cord, calculated the ratio of thc double-labeled cells to that of single-labeled cells and identified the shape of these labeled cells.
Material and methods
Adult female rats (Wistar), weighing 190-210 g, were operated on under sodium pentobarbital anesthesia (45 m g / k g body wt., i.p.). FB (Sigma) and DY (Sigma) were dissolved in distilled water or dimethyl sulfoxide. 10-31/ nl 2% ( w / v ) FB and 20-5(} nl 2% (w/v) DY were injected into the internal lateral subnuclei and their adjacent regions on the left side and the
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Fig. 1. Drawings showing the extent (shown by dashed lines) of in,iection of fast bluc on the left side (1,) and diamidino yellow on the right side (R). A - F : frontal sections of the brainstem are drawn at 400 # m intervals, but at 200 # m between C and D in rostrocaudal sequence. The numerals 82, 85 and 92 indicate the experimental rats. Each tracer covers the IL (dark circles) and its adjacent areas. AO: aqueductus ccrchrl, BC: brachium conjunctivum, CE: cerebellar cortex, CI: inferior colliculus, CU: nucleus cuneit~rmis, DLL: dorsal nucleus of the lateral lemniscu~,, FV: fourth ventricle, KF: K611iker-Fuse nucleus, LC: locus ceruleus, ME: mesenccphalic nucleus of the trigemimd nerve, MO: motor nucleus of the trigeminal nerve, PB: parabrachial nucleus, PCM: medial cerebellar peduncle, PS: principal sensory nucleus of the trigeminat nerve, 11= internal lateral subnucleus of the parabrachial nucleus. S('T; spinocerebellar tract, TR: trochlear ne~wc.
275 right side, respectively. The tracer was injected into these regions dorsoventrally with a glass capillary tube (30/~m or less in outer diameter) attached to a nanoliter p u m p (WPI, Model 1400) held on a stereotaxic apparatus. After a survival time of 10 to 15 days, the animals were deeply anesthetized and perfused transcardially with physiological saline, followed by 4% paraformaldehyde in phosphate buffer (pH 7.4). The brains and spinal cords were removed, stored overnight in the same fixative containing 30% sucrose and cut into 40-/~m thick frontal sections. The sections were examined with a Nikon fluorescent microscope equipped with U V (wavelength; 365 nm) and G (546 nm wavelength) filter assemblies. Although D Y in the nuclei of the cells and some elements of blood cells fluoresced yellow under the U V filter, blood components fluoresced red and D Y did not fluoresce at all under the G filter. We were thus easily able to differentiate retrogradely DY-labeled nuclei of the cells from blood cell components using this U V and G filter system. However, it was sometimes difficult to distinguish yellow-fluorescing glial cell bodies from nuclei of DY-labeled cells. Retrogradely FB-labeled cells were drawn under a camera lucida (Nikon), and the drawings were used to observe the shapes of labeled cells and to calculate their numbers. For comparison of the number of labeled cells among cases given different injections and among different segments of the spinal cord, the average cell number in a 1-mm thickness of each segment was calculated unilaterally for each cell group in frontal sections through the entire spinal cord. In the present study, the sum of the average cell number in each segment of the spinal cord was expressed as the total number of labeled cells. The total number of ipsilateral single-labeled cells (IPS) was that of FB-labeled cells on the side ipsilateral to the FB injection site, and that of DY-labeled cells on the side ipsilateral to the D Y injection site. On the other hand, the total number of contralateral single-labeled cells (CON) was that of FB-labeled cells on the side contralateral to the FB injection site, and that of DY-labeled cells on the side contralateral to the D Y injection site. The number of yellow-illuminated cell nuclei was counted to obtain the number of DY-labeled cells. A double-labeled cell displayed a blue-fluorescent cytoplasm and a yellowfluorescent nucleus. In every case, we rounded off the fractions to the first decimal place. According to fluorescent microscopy, the FB or DY fluorescent injection areas consisted of two concentric fluorescent zones (inner, Z o n e I; outer, Zone II) (Huisman et al., 1983). When only Z o n e II covered regions receiving
spinoparabrachial fibers, labeled cells were evident only sparsely in the spinal cord. From this, Zone I was identified as the effective extent of injected FB or DY.
Results
We found three ideal cases (rat82, rat85, rat92) in which injected FB and injected D Y covered the left and the right ILs, respectively (82, 85, 92 in Fig. 1). When the tracer covered only the DCS lateral subnuclei, labeled cells were found bilaterally in lamina I with an ipsilateral predominance (Cechetto et al., 1985; our previous study submitted elsewhere). On the other hand, when the tracer covered mainly the IL and small parts of the DCS lateral subnuclei, labeled cells were found bilaterally with a contralateral predominance (our previous experiments submitted elsewhere and present study). From these results, cases in which the n u m b e r of cells labeled by FB a n d / o r DY was greater ipsilaterally than contralaterally were not included among these ideal cases. In these 3 cases, the distribution of FB-labeled cells was similar to that of DYlabeled cells in the spinal cord. However, the number of DY-labeled cells was higher (approximately 10%) than that of FB-labeled cells. Because the size of glial cell bodies was similar to that of nuclei of DY-labeled cells, some yellow-fluorescing glial cell bodies may have been included in the count of DY-labeled cells. The outline of FB-labeled cells, even though they were
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Fig. 2. Distribution of the spinoparabrachial fiber cells in rat85 (see Fig. 1; 85). A-F: sections of the spinal cord at the indicated segmental levels. DL and DM: dorsolateral and dorsomedial parts of the lateral funiculus. IBN: internal basilary nucleus.
276 small, was clearly recognizable. Therefore, from the a b o v e - m e n t i o n e d results, FB-labeled cells were shown as single-labeled cells in the present study. T h e total n u m b e r of bilateral single-labeled cells t h r o u g h the spinal cord was highest in rat85 (rat82, n = 7893; rat85, n = 8391; rat92, n = 8164). In rat85, the total n u m b e r of double-labeled cells on the left side (n = 1100) was
close to that on the right side (n = 1056). Hence, the results described here are those for rat85. Singlelabeled cells in lamina I were seen bilaterally with an ipsilateral p r e d o m i n a n c e in C1 and C2 and with a contralateral p r e d o m i n a n c e in the other spinal segments (Figs. 2 and 5). T h e m e a n n u m b e r of bilateral single-labeled cells per segment in C1 and C2 was
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FiB. 3. Pholon~iclographs showing retrogradely labeled cell,', after fast bluc (FB) and diamidmo yellow ( D Y ) mjectlO11:,, in I:.d~5 ( i c l c l Io t'ig. 1: 8,5). f, y and d: FB-labeled cells, DY-labeled cells and FB and D Y double-labeled cells. FB accumulating in the cytoplasm and D Y accumulating in the nucleus o f the cell are illuminated blue and yellow, respectively, under a U V (wavelength; 365 nm) filter. A, B and C: laminae 1 and V of the first cervical segments ( e l ) and lamina V I I at C2 on the left side (ipsilateral to the FB injection site). D, E and F: lamina X at CI on the right side (contralateral to the FB injection site), D L at C I and D M C3 on the left side. The asterisk in D shows a dendrite crossing the midline dorsal to the c e n t r a l canal. B a r = 50/.tin.
277 markedly higher than the mean number in each of all other spinal segments. For convenience, the number of labeled cells in each of the laminae and regions of the spinal cord was described in five divisions of the spinal cord: (1) first 2 cervical segments (C1 and C2), (2) third to eighth cervical segments (C3-C8), (3) thoracic segments (T1-T13), (4) lumbar segments (L1-L6), (5) slacro-coccygial segments (S1-CO). Labeled cells tended to be concentrated along the curvature of the dorsal horn (lamina I), and sometimes were scattered in Lissauer's tract and clustered in small groups of two to four cells in these areas (Fig. 2). Most of the double-labeled cells in lamina I were round and fusiform (Fig. 3A). In the reticular part of lamina V, which is characterized by strands of gray matter and bundles of fibers coursing longitudinally through the spinal cord, the majority of double-labeled cells were fusiform and triangular (Fig. 3B). In the medial part of lamina V just ventral to the posterior funiculus (i.e., the internal basilary nucleus: IBN; refer to Figs. 2B and 4B), which is characterized by scarce myelinated fibers, only a few labeled cells were seen (Figs. 2 and 4). Labeled ceils tended to be concentrated mainly in the central and lateral parts of lamina Vll (Fig. 2). Labeled cells in the central part were polygonal through all spinal segments, and those in the lateral part were triangular in C1 and C2, and polygonal in the other spinal segments. However, doublelabeled cells in lamina VII were mainly triangular in C1 and C2 and polygonal in the other spinal segments (Fig. 3C). Labeled cells in lamina X ventral to the posterior funiculus were fusiform, whereas those in the regions closely surrounding the central canal were oval and fusiform. Their dendrites occasionally crossed the midline dorsal to the central canal (Fig. 3D). Labeled cells tended to be concentrated in the dorsolateral part of the lateral funiculus ventral to lamina I (DL), and in the dorsomedial part of the lateral funiculus and the lateral marginal zone of lamina V adjacent to the lateral funiculus (DM) (Figs. 2 and 4). The DL is identified as the lateral nuclei by Giesler and Elde (1985). Double-labeled cells were fusiform, oval and triangular in the D L (Fig. 3E) and triangular and fusiform in the DM (Fig. 3F). The sizes of single- and double-labeled cells were essentially the same in every case. The number of bilateral single-labeled ceils was greatest in lamina I, followed, in order, by the DL and DM, in lamina VII and lamina V. A relatively large number of labeled cells were seen in laminae II-IV, VI and X at C1 and C2, and lamina VIII from the cervical to thoracic segments (Figs. 2 and 5). The total number
of bilateral double-labeled cells was highest in the DL, followed, in order, by lamina I, the DM, lamina V and lamina VII. Only a few double-labeled cells were seen in laminae II-IV, VI, VIII and X (Figs. 4 and 5). The ratio of the total number of bilateral double-labeled cells to the total number of bilateral single-labeled cells was 43% in the DL, 37% in the DM, 28% in lamina V, and 24% in lamina I. The ratio was 10% or less in laminae II-IV, V I - V I I I and X.
Discussion In the present study, we applied retrograde doublelabeling techniques in order to reveal further details of cells of spinal fibers to the IL. A major finding thus obtained is that a substantial population of spinal cord cells projected to the IL bilaterally. These cells were similar in morphology to those projecting to the IL unilaterally. The distribution of the spinoparabrachial cells described in this study is similar to that of the following ascending tract cells in rats. The spinothalamic fibers arise from cells in laminae V and X in the cervical segments are related to somatic nociception and influenced directly by the vagus nerve (Chandler et al., 1991); spinal fibers projecting directly to the hypothalamus and telencephalon arise from cells in laminae I, V and X (Burstein et al., 1990; M e n , t r e y and de Pommery, 1991) and the DL and DM (Men6trey and
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Fig. 4. Drawings showing spinoparabrachial fiber double-labeled cells in rat85 (refer to Fig. 1: 85). The right and the left sides of each section correspond to the FB and the DY injection sites, respectively. Illustrated similar to Fig. 2. O n e dot indicates one doublelabeled cell.
278 de Pommery, 1991) and are related to somatic and autonomic nociception; some components of propriospinal cells in these laminae and regions in the cervical segments have collaterals to the medulla, tecturn or thalamus (Verburgh et al., 1990). The IL presently defined is equivalent to the structure previously attached in the other names: magnocellular part (Yamada and Otani, 1979), dorso-medial
cluster (Voshart and Kooy, 1981), round area (Kitamura et al., 1989), perhaps medial aspect of the dorsal PB (Zemlan et al., 1978) and dorsal cap of the external lateral nucleus (Ward, 1989), in the rat, and to a limited area of the lateral PB in the cat (McBride and Sutin, 1976; Nomura et al., 1979). The present observation in rats may apply also to other animal species, when one considers the following data in the literature.
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Fig. 5. ttistograms showing the number of single- and double-labeled cells in rat85 (refer to Fig. 1; 85). Solid bars, the total number of FB- and DY-labeled cells and cells clear bars, that of FB and DY double-labeled cells. A 1: laminae I, I I - I V , V, V1, VII, VII1 and X, DL and DM. The numerals on the abscissae show the number of single- and double-labeled cells. IPS, total number of FB+labeled cells on the side ipsilateral to the FB injection site, and that of DY-labeled cells on the side ipsilateral to the DY injection site. CON, the total number of FB-labeled cells on the side contralateral to the FB injection site and that of DY-labeled cells on the side contralateral to the DY injection site. C, CO, L, S and T: cervical, coccygial, lumbar, sacral and thoracic segments.
279 The IL and/or the DCS lateral subnuclei receive a large number of spinal fibers in the rat (Blomqvist et al., 1989; C e c h e t t o e t al., 1985; F u l w i l e r a n d S a p e r , 1984; K i t a m u r a e t al., 1989; M e h l e r , 1969; Y a m a d a a n d O ~ a n i , 1979; Z e m l a n e t al., 1978), a n d also in t h e o p o s s u m ( H a z l e t t e t al., 1972), c a t ( H y l d e n e t al., 1985; M a e t al., 1989; M e h l e r , 1969; P a n n e t o n a n d B u r t o n , 1985; W i b e r g and Blomqvist, 1984), t r e e s h r e w ( S c h r o e d e r a n d J a n e , 1971) a n d m o n k e y ( G i r a r d o t e t al., 1987; M e h l e r , 1969). F u r t h e r m o r e , the cells of origin of the spinoparabrachial fibers are located bilate r a l l y in l a m i n a I o f t h e s p i n a l c o r d i n t h e c a t ( H y l d e n e t al., 1 9 8 5 ) a n d m o n k e y ( G i r a r d o t e t al., 1987), a n d also, s o m e c o m p o n e n t s o f t h e f i b e r s o c c u r b i l a t e r a l l y in l a m i n a V i n t h e r a t ( C e c h e t t o e t al., 1 9 8 5 ) a n d l a m i n a e I V a n d V I I i n t h e m o n k e y ( G i r a r d o t e t al., 1987).
References Blomqvist, A., Ma, W. and Berkley, K.J. (1989) Spinal input to the parabrachial nucleus in the cat. Brain Res., 480: 29-36. Burstein, R., Dado, R.J. and Giesler, G.J., Jr. (1990) The cells of origin of the spinohypothalamic tract of the rat: a quantitative reexamination. Brain Res., 551: 329-337. Cechetto, D.F., Standaert, D.G. and Saper, C.G. (1985) Spinal trigeminal dorsal horn projections to the parabrachial nucleus in the rat. J. Comp. Neurol., 240: 153-160. Chandler, M.J., Hobbs, S.F., Bolser, D.C. and Foreman, R.D. (1991) Effects of vagal afferent stimulation on cervical spinothalamic tract neurons in monkeys. Pain, 44:81-87. Fulwiler, C.E. and Saper, C.B. (1984) Subnuclear organization of the efferent connections of the parabrachial nucleus in the rat. Brain Res. Rev., 7: 229-259. Giesler, G.J., Jr. and Elde, R.P. (1985) Immunocytochemical studies of the peptidergic content of fibers and terminals within the lateral spinal and lateral cervical nuclei. J. Neurosci., 5: 18331841. Girardot, M.-N., Brennan, T.J., Martindale, M.E. and Foreman, R.D. (1987) Effects of stimulating the subcoeruleus-parabrachial region on the non-noxious and noxious responses of T1-T5 spinothalamic tract neurons in the primate. Brain Res. 409: 19-30. Hazlett, J.C., Don, R. and Martin, G.F. (1972) Spino-bulbar, spinothalamic and medial lemniscal connections in the American opossum, Didelphis rnarsupialis verginiana. J. Comp. Neurol., 146: 95-118. Herbert, H., Moga, M.M. and Saper, C.B. (1990) Connections of the parabrachial nucleus with the nucleus of the solitary tract and medullary reticular formation in the rat. J. Comp. Neurol., 293: 5540-580. Huisman, A.M., Kuypers, H.G.J.M., Condr~, F. and Keizer, K. (1983) Collaterals of rubrospinal neurons to the cerebellum in rat. A retrograde fluorescence double labeling study. Brain Res., 264: 181-196. Hylden, L.J.K., Hayashi, H., Bennett, G.J. and Dubner, R. (1985)
Spinal lamina I neurons projecting to the parabrachial area of the cat midbrain. Brain Res., 336: 195-198. Keene, J.J (1973) Reward-associated inhibition and pain-associated excitation lasting seconds in single intralaminar thalamic units. Brain Res., 64: 211-224. Kitamura, T., Yamada, J. and Sato, H. (1989) Axon collaterals of spinocerebellar fibers terminate in the parabrachial nucleus of the rat. Neurosci. Lett., 99: 24-29. Ma, W., Blomqvist, A. and Berkley, K.J. (1989) Spino-diencephalic relays through the parabrachial nucleus in the rat. Brain Res., 480: 37-50. McBride, R.L. and Sutin, J. (1976) Projections of the locus coeruleus and adjacent pontine tegmentum in the cat. J. Comp. Neurol., 165: 265-284. Mehler, W.R. (1976) Some neurological species differences - a posteriori. Ann. N.Y. Acad. Sci., 167: 424-468. Men,trey, D., and de Pommery, J. 11976) Origins of spinal ascending pathways that reach central areas involved in visceroception and visceronociception in the rat. Eur. J. Neurosci., 3: 249-259. Men~,trey, D., de Pommery, J., Baimbridge, K.G. and Thomasset, M. (1992a) Calbindin-D28K (CaBP28k)-like immunoreactivity in ascending projections. I. Trigeminal nucleus caudalis and dorsal vagal complex projections. Eur. J. Neurosci., 4: 61-69. Men6trey, D., de Pommery, J. Thomasset, M. and Baimbridge, K.G. (1992b) Calbindin-D28K (CaBP28k)-like immunoreactivity in ascending projections. II. Spinal projections to the brain stem and mesencephalic areas. Eur. J. Neurosci., 4: 70-76. Milner, M.-M. and Pickel, V.M. (1986a) Neurotensin in the rat parabrachiat region: Ultrastructural localization and extrinsic sources of immunoreactivity. J. Comp. Neurol., 247: 326-343. Milner, M.-M. and Pickel, V.M. (1986b) Ultrastructural localization and afferent sources of substance P in the rat parabrachial region. Neuroscience, 17: 687-707. Milner, M.-M., Joh, T.H., Miller, R.J. and Pickel, V.M. (1984) Substance P, neurotensin, enkephalin, and cathecholaminesynthesizing enzymes: light microscopic localizations compared with autoradiographic label in solitary efferents to the rat parabrachial region. J. Comp. Neurol., 226: 434-447. Nishikawa, Y., Koyama, N. and Yokota, Y. 11992) Ascending inhibition of nociceptive neurons in the thalamic intralaminar nuclei following conditioning stimulation of the mesencephalic periaqueductal gray. Pain Res., 7: 81-91. Nomura, S., Mizuno, N., Itoh, K., Matsuda, T., Sugimoto, T. and Nakamura, Y. (1979) Localization of parabrachial nucleus neurons projecting to the thalamus or the amygdala in the cat using horseradish peroxidase. Exp. Neurol., 64: 375-385. Panneton, W.M. and Burton, H. (1985) Projections from the paratrigeminal nucleus and medullary and spinal dorsal horns to the parabrachial area in the cat. Neuroscience, 15: 779-797. Saper, C.B. and Loewy, A.D. (1980) Efferent connections of the parabrachial nucleus in the rat. Brain Res., 197: 291-317. Schroeder, D.M. and Jane, J.A. (1971) Projection of dorsal column nuclei and spinal cord to brainstem and thalamus in the tree shrew, Tupaia glis. J. Comp. Neurol., 142: 3119-350. Verburgh, C.A., Voogd, J., Kuypers, H.G.J.M. and Stevens, H.P.J.D. (1990) Propriospinal neurons with ascending collaterals to the dorsal medulla, the thalamus and the tectum: a retrograde fluorescence double-labeling study of the cervical cord of the rat. Exp. Brain Res., 80:577 591). Voshart, K. and Von der Kooy, D. (1981) The organization of the efferent projections of the parabrachial nucleus to the forebrain
280 in the rat: A retrograde fluorescence double-labeling study. Brain Res., 212:271 286. Ward, D.G. (1989) Neurons in the parabrachial nuclei respond to hemorrhage. Brain Res., 491: 80-92. Wiberg, M. and Blomqvist, A. (1984) The spinomesencephalic tract in the cat: Its cells of origin and termination pattern as demonstrated by the intraaxonal transport method. Brain Res., 291: 1 28. Yamada, J. and Otani, K. (1979) Subunits of the nucleus
parabrachialis and their fiber connections in the rat. Neurosci. Lett., Suppl. 2: $23. Yokota, T., Koyama, N., Nishikawa, Y. and Hasegawa A. (1991) Trigeminal nociceptive neurons in the subnucleus reticularis ventralis. II. Ascending projection. Neurosci. Res., 11: 18-27. Zemlan, F.P., Leonard, C.M., Kow, L.-K. and Pfaff, D.W. (1978) Ascending tracts of the lateral columns of the rat spinal cord: A study using the silver impregnation and horseradish peroxidase techniques. Exp. Neurol., 62: 298-334.
273
NEURES 00592
Spinal cord cells innervating the bilateral parabrachial nuclei in the rat A retrograde fluorescent double-labeling study Jinzo Y a m a d a and Taiko Kitamura Department of Anatomy, Tokyo Medical College, Tokyo, Japan (Received 16 July 1992; accepted 29 September 1992)
Key words: Parabrachial nucleus; Internal lateral subnucleus; Spinal cord cell; Collateral; Retrograde transport; Rat Summary The internal lateral nucleus (IL) of the parabrachial nucleus receives information from the spinal cord. The IL perhaps relays nociceptive signals to the intralaminar nuclei of the thalamus, apparently being implicated in the motivational-affective component of pain reactions. However, cells of origin of spinal fibers to the IL have not been investigated enough. We intended to clarify these cells, as well as their shapes, by retrograde double-labeling techniques. Fast blue and diamidino yellow dyes were injected, respectively, into the left and right ILs. The distribution of double-labeled cells was almost the same as that of single-labeled cells on both sides of the spinal cord. The total number of bilateral double-labeled cells was highest in the dorsolateral part of the lateral funiculus (DL), followed, in order, by lamina I, the dorsomedial part of the lateral funiculus (DM), lamina V and lamina VII. A few double-labeled cells were seen in laminae II-IV, VI, VIII and X. The ratio of the total number of bilateral double-labeled cells to the total number of bilateral single-labeled cells through the spinal cord was 43% in the DL, 37% in the DM, 28% in lamina V and 24% in lamina I. The ratio was 10% or less in the other remaining laminae. No marked differences were observed between the shapes of double- and single-labeled cells.
Introduction The parabrachial nucleus is a major relay for ascending gustatory, visceral and nociceptive afferent pathways, located around the brachium conjunctivum in the caudal midbrain (Saper and Loewy, 1980; Fulwiler and Saper, 1984; Milner et al., 1984; Cechetto et al., 1985; Milner and Pickel, 1986a,b; Herbert et al., 1990; Men6trey and de Pommery, 1991; Men6trey et al., 1992a,b). However, the parabrachial nucleus is a complex consisting of the medial nucleus, lateral nucleus and K611iker-Fuse nucleus. The lateral nucleus in Correspondence to: Dr. Jinzo Yamada, Department of Anatomy, Tokyo Medical College, 6-1-1 Shinjuku, Shinjuku-ku, Tokyo 160, Japan.
the rat is further divided into seven subnuclei, i.e., the internal lateral (IL), dorsal, central and superior (DCS) lateral and other subnuclei. Furthermore, afferents of varied origins converge onto the parabrachial nucleus, in particular from the solitary nucleus and the spinal cord. Precise anatomical knowledge is of essential importance in order to uncover specific structural-functional relationships for the parabrachial nucleus. We have been paying special attention to the IL which receives perhaps nociceptive signals from the spinal cord (Cechetto et al., 1985; Men6trey et al., 1992b) and which projects almost entirely to the intralaminar nucleus of the thalamus (Fulwiler and Saper, 1984). Responses of cells in the intralaminar nucleus are related to reward and escape behavior (Keene, 1973), and it is suggested that the intralaminar nucleus
274 is involved in the motivational-affective component of pain reaction (Yokota et al., 1991; Nishikawa et al., 1992). In a previous work by Cechetto et al. (1985), injection of wheat germ agglutinin-conjugated horseradish peroxidase (WGA-HRP) into the parabrachial nucleus on one side retrogradely labeled a large number of lamina I cells in the dorsal horn throughout the entire length of the spinal cord, bilaterally but with an ipsilateral predominance. In this study, however, the injected tracer covered the DCS lateral subnuclei, but not sufficiently in the IL. When we sufficiently covered the IL with W G A - H R P or fast blue (Kitamura et al., in course of publication), many retrogradely labeled cells were found bilaterally in laminae 1, V and VII of the dorsal horn as well as in the dorsolateral (DL) and dorsomedial (DM) parts of the lateral funiculus. These findings are similar to those reported for the lower thoracic to upper sacral segments by Men,trey and de Pommery (1991), even though the degree of coverage L
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of the 1L with the tracer was unclear in their experiment. In the present study, after injecting fast blue (FB) and diamidino yellow (DY) dyes into the left and right ILs of the rat, respectively, we investigated cells showing single and double retrograde labeling in the spinal cord, calculated the ratio of thc double-labeled cells to that of single-labeled cells and identified the shape of these labeled cells.
Material and methods
Adult female rats (Wistar), weighing 190-210 g, were operated on under sodium pentobarbital anesthesia (45 m g / k g body wt., i.p.). FB (Sigma) and DY (Sigma) were dissolved in distilled water or dimethyl sulfoxide. 10-31/ nl 2% ( w / v ) FB and 20-5(} nl 2% (w/v) DY were injected into the internal lateral subnuclei and their adjacent regions on the left side and the
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Fig. 1. Drawings showing the extent (shown by dashed lines) of in,iection of fast bluc on the left side (1,) and diamidino yellow on the right side (R). A - F : frontal sections of the brainstem are drawn at 400 # m intervals, but at 200 # m between C and D in rostrocaudal sequence. The numerals 82, 85 and 92 indicate the experimental rats. Each tracer covers the IL (dark circles) and its adjacent areas. AO: aqueductus ccrchrl, BC: brachium conjunctivum, CE: cerebellar cortex, CI: inferior colliculus, CU: nucleus cuneit~rmis, DLL: dorsal nucleus of the lateral lemniscu~,, FV: fourth ventricle, KF: K611iker-Fuse nucleus, LC: locus ceruleus, ME: mesenccphalic nucleus of the trigemimd nerve, MO: motor nucleus of the trigeminal nerve, PB: parabrachial nucleus, PCM: medial cerebellar peduncle, PS: principal sensory nucleus of the trigeminat nerve, 11= internal lateral subnucleus of the parabrachial nucleus. S('T; spinocerebellar tract, TR: trochlear ne~wc.
275 right side, respectively. The tracer was injected into these regions dorsoventrally with a glass capillary tube (30/~m or less in outer diameter) attached to a nanoliter p u m p (WPI, Model 1400) held on a stereotaxic apparatus. After a survival time of 10 to 15 days, the animals were deeply anesthetized and perfused transcardially with physiological saline, followed by 4% paraformaldehyde in phosphate buffer (pH 7.4). The brains and spinal cords were removed, stored overnight in the same fixative containing 30% sucrose and cut into 40-/~m thick frontal sections. The sections were examined with a Nikon fluorescent microscope equipped with U V (wavelength; 365 nm) and G (546 nm wavelength) filter assemblies. Although D Y in the nuclei of the cells and some elements of blood cells fluoresced yellow under the U V filter, blood components fluoresced red and D Y did not fluoresce at all under the G filter. We were thus easily able to differentiate retrogradely DY-labeled nuclei of the cells from blood cell components using this U V and G filter system. However, it was sometimes difficult to distinguish yellow-fluorescing glial cell bodies from nuclei of DY-labeled cells. Retrogradely FB-labeled cells were drawn under a camera lucida (Nikon), and the drawings were used to observe the shapes of labeled cells and to calculate their numbers. For comparison of the number of labeled cells among cases given different injections and among different segments of the spinal cord, the average cell number in a 1-mm thickness of each segment was calculated unilaterally for each cell group in frontal sections through the entire spinal cord. In the present study, the sum of the average cell number in each segment of the spinal cord was expressed as the total number of labeled cells. The total number of ipsilateral single-labeled cells (IPS) was that of FB-labeled cells on the side ipsilateral to the FB injection site, and that of DY-labeled cells on the side ipsilateral to the D Y injection site. On the other hand, the total number of contralateral single-labeled cells (CON) was that of FB-labeled cells on the side contralateral to the FB injection site, and that of DY-labeled cells on the side contralateral to the D Y injection site. The number of yellow-illuminated cell nuclei was counted to obtain the number of DY-labeled cells. A double-labeled cell displayed a blue-fluorescent cytoplasm and a yellowfluorescent nucleus. In every case, we rounded off the fractions to the first decimal place. According to fluorescent microscopy, the FB or DY fluorescent injection areas consisted of two concentric fluorescent zones (inner, Z o n e I; outer, Zone II) (Huisman et al., 1983). When only Z o n e II covered regions receiving
spinoparabrachial fibers, labeled cells were evident only sparsely in the spinal cord. From this, Zone I was identified as the effective extent of injected FB or DY.
Results
We found three ideal cases (rat82, rat85, rat92) in which injected FB and injected D Y covered the left and the right ILs, respectively (82, 85, 92 in Fig. 1). When the tracer covered only the DCS lateral subnuclei, labeled cells were found bilaterally in lamina I with an ipsilateral predominance (Cechetto et al., 1985; our previous study submitted elsewhere). On the other hand, when the tracer covered mainly the IL and small parts of the DCS lateral subnuclei, labeled cells were found bilaterally with a contralateral predominance (our previous experiments submitted elsewhere and present study). From these results, cases in which the n u m b e r of cells labeled by FB a n d / o r DY was greater ipsilaterally than contralaterally were not included among these ideal cases. In these 3 cases, the distribution of FB-labeled cells was similar to that of DYlabeled cells in the spinal cord. However, the number of DY-labeled cells was higher (approximately 10%) than that of FB-labeled cells. Because the size of glial cell bodies was similar to that of nuclei of DY-labeled cells, some yellow-fluorescing glial cell bodies may have been included in the count of DY-labeled cells. The outline of FB-labeled cells, even though they were
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Fig. 2. Distribution of the spinoparabrachial fiber cells in rat85 (see Fig. 1; 85). A-F: sections of the spinal cord at the indicated segmental levels. DL and DM: dorsolateral and dorsomedial parts of the lateral funiculus. IBN: internal basilary nucleus.
276 small, was clearly recognizable. Therefore, from the a b o v e - m e n t i o n e d results, FB-labeled cells were shown as single-labeled cells in the present study. T h e total n u m b e r of bilateral single-labeled cells t h r o u g h the spinal cord was highest in rat85 (rat82, n = 7893; rat85, n = 8391; rat92, n = 8164). In rat85, the total n u m b e r of double-labeled cells on the left side (n = 1100) was
close to that on the right side (n = 1056). Hence, the results described here are those for rat85. Singlelabeled cells in lamina I were seen bilaterally with an ipsilateral p r e d o m i n a n c e in C1 and C2 and with a contralateral p r e d o m i n a n c e in the other spinal segments (Figs. 2 and 5). T h e m e a n n u m b e r of bilateral single-labeled cells per segment in C1 and C2 was
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FiB. 3. Pholon~iclographs showing retrogradely labeled cell,', after fast bluc (FB) and diamidmo yellow ( D Y ) mjectlO11:,, in I:.d~5 ( i c l c l Io t'ig. 1: 8,5). f, y and d: FB-labeled cells, DY-labeled cells and FB and D Y double-labeled cells. FB accumulating in the cytoplasm and D Y accumulating in the nucleus o f the cell are illuminated blue and yellow, respectively, under a U V (wavelength; 365 nm) filter. A, B and C: laminae 1 and V of the first cervical segments ( e l ) and lamina V I I at C2 on the left side (ipsilateral to the FB injection site). D, E and F: lamina X at CI on the right side (contralateral to the FB injection site), D L at C I and D M C3 on the left side. The asterisk in D shows a dendrite crossing the midline dorsal to the c e n t r a l canal. B a r = 50/.tin.
277 markedly higher than the mean number in each of all other spinal segments. For convenience, the number of labeled cells in each of the laminae and regions of the spinal cord was described in five divisions of the spinal cord: (1) first 2 cervical segments (C1 and C2), (2) third to eighth cervical segments (C3-C8), (3) thoracic segments (T1-T13), (4) lumbar segments (L1-L6), (5) slacro-coccygial segments (S1-CO). Labeled cells tended to be concentrated along the curvature of the dorsal horn (lamina I), and sometimes were scattered in Lissauer's tract and clustered in small groups of two to four cells in these areas (Fig. 2). Most of the double-labeled cells in lamina I were round and fusiform (Fig. 3A). In the reticular part of lamina V, which is characterized by strands of gray matter and bundles of fibers coursing longitudinally through the spinal cord, the majority of double-labeled cells were fusiform and triangular (Fig. 3B). In the medial part of lamina V just ventral to the posterior funiculus (i.e., the internal basilary nucleus: IBN; refer to Figs. 2B and 4B), which is characterized by scarce myelinated fibers, only a few labeled cells were seen (Figs. 2 and 4). Labeled ceils tended to be concentrated mainly in the central and lateral parts of lamina Vll (Fig. 2). Labeled cells in the central part were polygonal through all spinal segments, and those in the lateral part were triangular in C1 and C2, and polygonal in the other spinal segments. However, doublelabeled cells in lamina VII were mainly triangular in C1 and C2 and polygonal in the other spinal segments (Fig. 3C). Labeled cells in lamina X ventral to the posterior funiculus were fusiform, whereas those in the regions closely surrounding the central canal were oval and fusiform. Their dendrites occasionally crossed the midline dorsal to the central canal (Fig. 3D). Labeled cells tended to be concentrated in the dorsolateral part of the lateral funiculus ventral to lamina I (DL), and in the dorsomedial part of the lateral funiculus and the lateral marginal zone of lamina V adjacent to the lateral funiculus (DM) (Figs. 2 and 4). The DL is identified as the lateral nuclei by Giesler and Elde (1985). Double-labeled cells were fusiform, oval and triangular in the D L (Fig. 3E) and triangular and fusiform in the DM (Fig. 3F). The sizes of single- and double-labeled cells were essentially the same in every case. The number of bilateral single-labeled ceils was greatest in lamina I, followed, in order, by the DL and DM, in lamina VII and lamina V. A relatively large number of labeled cells were seen in laminae II-IV, VI and X at C1 and C2, and lamina VIII from the cervical to thoracic segments (Figs. 2 and 5). The total number
of bilateral double-labeled cells was highest in the DL, followed, in order, by lamina I, the DM, lamina V and lamina VII. Only a few double-labeled cells were seen in laminae II-IV, VI, VIII and X (Figs. 4 and 5). The ratio of the total number of bilateral double-labeled cells to the total number of bilateral single-labeled cells was 43% in the DL, 37% in the DM, 28% in lamina V, and 24% in lamina I. The ratio was 10% or less in laminae II-IV, V I - V I I I and X.
Discussion In the present study, we applied retrograde doublelabeling techniques in order to reveal further details of cells of spinal fibers to the IL. A major finding thus obtained is that a substantial population of spinal cord cells projected to the IL bilaterally. These cells were similar in morphology to those projecting to the IL unilaterally. The distribution of the spinoparabrachial cells described in this study is similar to that of the following ascending tract cells in rats. The spinothalamic fibers arise from cells in laminae V and X in the cervical segments are related to somatic nociception and influenced directly by the vagus nerve (Chandler et al., 1991); spinal fibers projecting directly to the hypothalamus and telencephalon arise from cells in laminae I, V and X (Burstein et al., 1990; M e n , t r e y and de Pommery, 1991) and the DL and DM (Men6trey and
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Fig. 4. Drawings showing spinoparabrachial fiber double-labeled cells in rat85 (refer to Fig. 1: 85). The right and the left sides of each section correspond to the FB and the DY injection sites, respectively. Illustrated similar to Fig. 2. O n e dot indicates one doublelabeled cell.
278 de Pommery, 1991) and are related to somatic and autonomic nociception; some components of propriospinal cells in these laminae and regions in the cervical segments have collaterals to the medulla, tecturn or thalamus (Verburgh et al., 1990). The IL presently defined is equivalent to the structure previously attached in the other names: magnocellular part (Yamada and Otani, 1979), dorso-medial
cluster (Voshart and Kooy, 1981), round area (Kitamura et al., 1989), perhaps medial aspect of the dorsal PB (Zemlan et al., 1978) and dorsal cap of the external lateral nucleus (Ward, 1989), in the rat, and to a limited area of the lateral PB in the cat (McBride and Sutin, 1976; Nomura et al., 1979). The present observation in rats may apply also to other animal species, when one considers the following data in the literature.
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Fig. 5. ttistograms showing the number of single- and double-labeled cells in rat85 (refer to Fig. 1; 85). Solid bars, the total number of FB- and DY-labeled cells and cells clear bars, that of FB and DY double-labeled cells. A 1: laminae I, I I - I V , V, V1, VII, VII1 and X, DL and DM. The numerals on the abscissae show the number of single- and double-labeled cells. IPS, total number of FB+labeled cells on the side ipsilateral to the FB injection site, and that of DY-labeled cells on the side ipsilateral to the DY injection site. CON, the total number of FB-labeled cells on the side contralateral to the FB injection site and that of DY-labeled cells on the side contralateral to the DY injection site. C, CO, L, S and T: cervical, coccygial, lumbar, sacral and thoracic segments.
279 The IL and/or the DCS lateral subnuclei receive a large number of spinal fibers in the rat (Blomqvist et al., 1989; C e c h e t t o e t al., 1985; F u l w i l e r a n d S a p e r , 1984; K i t a m u r a e t al., 1989; M e h l e r , 1969; Y a m a d a a n d O ~ a n i , 1979; Z e m l a n e t al., 1978), a n d also in t h e o p o s s u m ( H a z l e t t e t al., 1972), c a t ( H y l d e n e t al., 1985; M a e t al., 1989; M e h l e r , 1969; P a n n e t o n a n d B u r t o n , 1985; W i b e r g and Blomqvist, 1984), t r e e s h r e w ( S c h r o e d e r a n d J a n e , 1971) a n d m o n k e y ( G i r a r d o t e t al., 1987; M e h l e r , 1969). F u r t h e r m o r e , the cells of origin of the spinoparabrachial fibers are located bilate r a l l y in l a m i n a I o f t h e s p i n a l c o r d i n t h e c a t ( H y l d e n e t al., 1 9 8 5 ) a n d m o n k e y ( G i r a r d o t e t al., 1987), a n d also, s o m e c o m p o n e n t s o f t h e f i b e r s o c c u r b i l a t e r a l l y in l a m i n a V i n t h e r a t ( C e c h e t t o e t al., 1 9 8 5 ) a n d l a m i n a e I V a n d V I I i n t h e m o n k e y ( G i r a r d o t e t al., 1987).
References Blomqvist, A., Ma, W. and Berkley, K.J. (1989) Spinal input to the parabrachial nucleus in the cat. Brain Res., 480: 29-36. Burstein, R., Dado, R.J. and Giesler, G.J., Jr. (1990) The cells of origin of the spinohypothalamic tract of the rat: a quantitative reexamination. Brain Res., 551: 329-337. Cechetto, D.F., Standaert, D.G. and Saper, C.G. (1985) Spinal trigeminal dorsal horn projections to the parabrachial nucleus in the rat. J. Comp. Neurol., 240: 153-160. Chandler, M.J., Hobbs, S.F., Bolser, D.C. and Foreman, R.D. (1991) Effects of vagal afferent stimulation on cervical spinothalamic tract neurons in monkeys. Pain, 44:81-87. Fulwiler, C.E. and Saper, C.B. (1984) Subnuclear organization of the efferent connections of the parabrachial nucleus in the rat. Brain Res. Rev., 7: 229-259. Giesler, G.J., Jr. and Elde, R.P. (1985) Immunocytochemical studies of the peptidergic content of fibers and terminals within the lateral spinal and lateral cervical nuclei. J. Neurosci., 5: 18331841. Girardot, M.-N., Brennan, T.J., Martindale, M.E. and Foreman, R.D. (1987) Effects of stimulating the subcoeruleus-parabrachial region on the non-noxious and noxious responses of T1-T5 spinothalamic tract neurons in the primate. Brain Res. 409: 19-30. Hazlett, J.C., Don, R. and Martin, G.F. (1972) Spino-bulbar, spinothalamic and medial lemniscal connections in the American opossum, Didelphis rnarsupialis verginiana. J. Comp. Neurol., 146: 95-118. Herbert, H., Moga, M.M. and Saper, C.B. (1990) Connections of the parabrachial nucleus with the nucleus of the solitary tract and medullary reticular formation in the rat. J. Comp. Neurol., 293: 5540-580. Huisman, A.M., Kuypers, H.G.J.M., Condr~, F. and Keizer, K. (1983) Collaterals of rubrospinal neurons to the cerebellum in rat. A retrograde fluorescence double labeling study. Brain Res., 264: 181-196. Hylden, L.J.K., Hayashi, H., Bennett, G.J. and Dubner, R. (1985)
Spinal lamina I neurons projecting to the parabrachial area of the cat midbrain. Brain Res., 336: 195-198. Keene, J.J (1973) Reward-associated inhibition and pain-associated excitation lasting seconds in single intralaminar thalamic units. Brain Res., 64: 211-224. Kitamura, T., Yamada, J. and Sato, H. (1989) Axon collaterals of spinocerebellar fibers terminate in the parabrachial nucleus of the rat. Neurosci. Lett., 99: 24-29. Ma, W., Blomqvist, A. and Berkley, K.J. (1989) Spino-diencephalic relays through the parabrachial nucleus in the rat. Brain Res., 480: 37-50. McBride, R.L. and Sutin, J. (1976) Projections of the locus coeruleus and adjacent pontine tegmentum in the cat. J. Comp. Neurol., 165: 265-284. Mehler, W.R. (1976) Some neurological species differences - a posteriori. Ann. N.Y. Acad. Sci., 167: 424-468. Men,trey, D., and de Pommery, J. 11976) Origins of spinal ascending pathways that reach central areas involved in visceroception and visceronociception in the rat. Eur. J. Neurosci., 3: 249-259. Men~,trey, D., de Pommery, J., Baimbridge, K.G. and Thomasset, M. (1992a) Calbindin-D28K (CaBP28k)-like immunoreactivity in ascending projections. I. Trigeminal nucleus caudalis and dorsal vagal complex projections. Eur. J. Neurosci., 4: 61-69. Men6trey, D., de Pommery, J. Thomasset, M. and Baimbridge, K.G. (1992b) Calbindin-D28K (CaBP28k)-like immunoreactivity in ascending projections. II. Spinal projections to the brain stem and mesencephalic areas. Eur. J. Neurosci., 4: 70-76. Milner, M.-M. and Pickel, V.M. (1986a) Neurotensin in the rat parabrachiat region: Ultrastructural localization and extrinsic sources of immunoreactivity. J. Comp. Neurol., 247: 326-343. Milner, M.-M. and Pickel, V.M. (1986b) Ultrastructural localization and afferent sources of substance P in the rat parabrachial region. Neuroscience, 17: 687-707. Milner, M.-M., Joh, T.H., Miller, R.J. and Pickel, V.M. (1984) Substance P, neurotensin, enkephalin, and cathecholaminesynthesizing enzymes: light microscopic localizations compared with autoradiographic label in solitary efferents to the rat parabrachial region. J. Comp. Neurol., 226: 434-447. Nishikawa, Y., Koyama, N. and Yokota, Y. 11992) Ascending inhibition of nociceptive neurons in the thalamic intralaminar nuclei following conditioning stimulation of the mesencephalic periaqueductal gray. Pain Res., 7: 81-91. Nomura, S., Mizuno, N., Itoh, K., Matsuda, T., Sugimoto, T. and Nakamura, Y. (1979) Localization of parabrachial nucleus neurons projecting to the thalamus or the amygdala in the cat using horseradish peroxidase. Exp. Neurol., 64: 375-385. Panneton, W.M. and Burton, H. (1985) Projections from the paratrigeminal nucleus and medullary and spinal dorsal horns to the parabrachial area in the cat. Neuroscience, 15: 779-797. Saper, C.B. and Loewy, A.D. (1980) Efferent connections of the parabrachial nucleus in the rat. Brain Res., 197: 291-317. Schroeder, D.M. and Jane, J.A. (1971) Projection of dorsal column nuclei and spinal cord to brainstem and thalamus in the tree shrew, Tupaia glis. J. Comp. Neurol., 142: 3119-350. Verburgh, C.A., Voogd, J., Kuypers, H.G.J.M. and Stevens, H.P.J.D. (1990) Propriospinal neurons with ascending collaterals to the dorsal medulla, the thalamus and the tectum: a retrograde fluorescence double-labeling study of the cervical cord of the rat. Exp. Brain Res., 80:577 591). Voshart, K. and Von der Kooy, D. (1981) The organization of the efferent projections of the parabrachial nucleus to the forebrain
280 in the rat: A retrograde fluorescence double-labeling study. Brain Res., 212:271 286. Ward, D.G. (1989) Neurons in the parabrachial nuclei respond to hemorrhage. Brain Res., 491: 80-92. Wiberg, M. and Blomqvist, A. (1984) The spinomesencephalic tract in the cat: Its cells of origin and termination pattern as demonstrated by the intraaxonal transport method. Brain Res., 291: 1 28. Yamada, J. and Otani, K. (1979) Subunits of the nucleus
parabrachialis and their fiber connections in the rat. Neurosci. Lett., Suppl. 2: $23. Yokota, T., Koyama, N., Nishikawa, Y. and Hasegawa A. (1991) Trigeminal nociceptive neurons in the subnucleus reticularis ventralis. II. Ascending projection. Neurosci. Res., 11: 18-27. Zemlan, F.P., Leonard, C.M., Kow, L.-K. and Pfaff, D.W. (1978) Ascending tracts of the lateral columns of the rat spinal cord: A study using the silver impregnation and horseradish peroxidase techniques. Exp. Neurol., 62: 298-334.
