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Species differences in the distribution and coexistence ratio of serotonin and substance P in the monkey, cat, rat and chick spinal cord
Neuroscience Letters, 132 (19913 155 158
155
~ 1991 Elsevier Scientific Publishers Ireland Ltd. 0304-3940/91/$ 03.50 ADONIS 030439409100632V NSL 08148
Species differences in the distribution and coexistence ratio of serotonin and substance P in the monkey, cat, rat and chick spinal cord N o b u o O k a d o 1, M u t s u m i M a t s u k a w a 1, S h i n o b u N o r i t a k e 1, Shigeru Ozaki 2, S h u n H a m a d a I , M i t s u k o Arita 1 and N o r i o K u d o 2 Departments g['l Anatomy and 2Physiology, Institute of Basic Medical Sciences, University of Tsukuba, Tsukuba, lbaraki (Japan) (Received 14 June 1991; Revised version received 26 July 1991; Accepted 26 July 1991)
Key words." lmmunohistochemistry: Motor neuron; Supraspinal input Serotoninergic raphe-spinal motor neuron projections exhibit wide species differences in both innervation pattern and coexistence of serotonin and substance P. The coexistence ratios vary widely ranging from more than 80% (rat) to less than 1% (chick). Serotonin and substance P positive fibers are also une,,enly distributed in the ventral horn of different species: dense clusters of serotonin and substance P positive fibers were preferentially located in the motor neuron pools of extensor muscles of the hip joint (chick) as well as antigravity muscles of the forelimb (cat and rat).
Since the initial reports that serotonin (5-HT) and substance P (SP) coexist in single neurons of the medullary raphe nucleus [3, 6], immunohistochemical evidence for the coexistence of several classical transmitters and peptides have been demonstrated in various parts of the central nervous system [5]. The highest ratio of coexistence of 5-HT and SP in single fibers was reported in the ventral horn of mammalian spinal cord [17, 19]: these raphe-spinal motor projections are considered as evidence against Dale's law [5]. Previous studies have suggested that large species differences exist in serotoninergic projections to spinal motor nuclei: in the chicken 5H T positive fibers are located densely in specific regions of the ventral horn (motoneuron pools innervating specific muscles) [7 9, 14], whereas in the rat 5-HT positive fibers cocontaining SP have not been reported to distribute preferentially in the specific regions of the ventral horn [19]; in the chick 5-HT and SP positive immunoreactivity is located in different terminals [1]; 5-HT positive fibers distribute mainly around the somal profiles of motoneurons in the chick [8] and monkey spinal cord [10], whereas 5-HT fibers were located in regions between motoneuron soma of the cat and rat spinal cord [10]. We were especially interested in the distribution as well as the coexistence of 5-HT and related peptides in different species, that may be related to characteristic motor Correspondence: N. Okado, Department of Anatomy, Institute of Basic Medical Sciences, University of Tsukuba, Tsukuba, Ibaraki 305, Japan.
functions of each species. To examine this possibility we have used an immunohistochemical double labelling technique [19] that reliably identifies different antigens in single axonal profiles. Adult Wistar strain rat (n=4), cat (n=3), chicken (n = 8) and monkey (n = 5) were used in this study. Prior to all the experimental procedures animals were deeply anesthetized, and animals were perfused with Zamboni solution. Ten-/tin thick free floating sections were sequentially treated with primary and secondary antibodies at 37°C. The combinations of primary antibodies were as follows: a polyclonal antibody against 5H T raised in the rabbit at a dilution of 1:10,000, and a monoclonal antibody against SP raised in rat (Sera Lab.) at a dilution of 1:2000, or a monoclonal antibody against enkephaline (Enk) raised in the mouse (Sera Lab.) at a dilution of 1:5000; a polyclonal antibody against Enk raised in the rabbit (UCB) at a dilution of 1:5000 and a monoclonal antibody against SP raised in the rat. An appropriate secondary antibody labelled with fluorescein isothiocyanate (FITC) or with tetramethyl rhodamine isothiocyanate (TRITC) were used. Species cross immunoreactivity was examined by testing all inappropriate combinations of primary and secondary antibodies, and no cross-reactions in the combinations used in the present study were found. For analysis of the size of varicosities of immunoreactive fibers another immunohistochemical technique was employed. Forty-/~m thick transverse frozen sections were processed with the ABC method using anti-5-HT,
156
anti-SP and anti-Enk antibodies. The cross-sectional area of varicosities in the dorsolateral part of the lateral motor column of the lumbar enlargement was measured from enlarged light microscopic photographs at a final magnification × 3000 with a semi-automatic measuring device. In the ventral horn of chick spinal cord, virtually none of the 5-HT positive profiles cocontained SP immunoreactivity (coexistence ratio, less than 1%) (Figs. 1 and 2). The number of SP positive fibers appears greater than
that of 5-HT positive fibers. Although 5-HT positive fibers were located mainly around motoneuron soma (Fig. 1), many SP positive fibers were found in the region between as well as around motoneuron soma (Fig. 2). The sizes of 5-HT and SP positive profiles were clearly different: the area (mean + S.D.) of varicosities of 5-HT and SP was 0.86 + 0.51/~m 2 and 2.11 :+_ 0.87 ltm:, respectively. With double labelling of SP and Enk, about 25% of SP positive varicosities cocontained Enk in the chick. The density of SP positive fibers and varicosities was highest in the dorsolateral part of the lateral motor column of lumbosacral segment 3 (Fig. 3), where 5-HT positive fibers were also densely located, and where the motor neuron pool of the posterior iliotibial muscle is found [12, 13]. Motor neuron pools with a dense accumulation of 5-HT positive fibers innervate extensor muscles of the hip joint [7, 13, 14] and also contain a high density of SP innervation. Another example with a low coexistence ratio of 5-HT and SP in the ventral horn was found in the monkey spinal cord (Figs. 4 and 5): 20% of 5 HT positive varicosities cocontained SP. 5-HT and SP positive fibers were evenly distributed in the ventral horn. The number of SP positive fibers was far less than 5-HT positive fibers. The immunoreactivities of 5-HT and SP were highly colocalized in single fibers of the ventral horn of both the rat (80%) and cat spinal cord: these data are consistent with those of previous studies [17, 19]. Although not so prominent as in the chick, 5-HT positive fibers were also densely distributed in the medial motor column as well as in the ventrolaterat part of the lateral motor column of the cervical enlargement of the cat (Fig. 6). In the lumbar enlargement 5-HT fibers were more evenly distriFigs. 1 and 2. Photographs taken from the motor neuron pool of posterior iliotibial muscle of the adult chicken. Asterisks show motoneuron somata. 5-HT (Fig. 1) and SP (Fig. 2) positive fibers and varieosities are located in a single section. Bars= 10 pro. Fig. 3. SP positive fibers in the lateral motor column (dotted line) of the chick lumbosacral spinal cord segment 3. A dense cluster of SP positive fibers is found at the motoneuron pool of the posterior iliotibial muscle. Bar = 100 gin. Figs. 4 and 5.5-HT (Fig. 4) and SP (Fig. 5) positive fibers in the lateral motor column of the lumbar segment 3 of the monkey spinal cord. Arrows show varicosities cocontaining 5-HT and SP. An asterisk in Fig. 4 indicates artefact, FITC was accumulated in the nucleus. Bars= l0 }tm. Fig. 6.5-HT positive fibers in the ventral horn of the cervical enlargement of the cat spinal cord. 5-HT positive fibers are densely distributed in the ventral part (large arrow) of the lateral motor column(encircled with dotted line) and in the medial motor column (small arrow). Bar=~ I00 ~lm.
157
buted. The distribution pattern of 5-HT positive fibers in the rat spinal cord was similar to that of the cat. Serotoninergic raphe-spinal motor projections have been primarily investigated in only a few mammalian species [17, 19] and are considered as a typical example of a dual messenger system. However, the present study shows that a wide variance in the coexistence ratio of 5HT and SP, as well as in the distribution pattern of these fibers, occurs between different species. In terms of 5-HT and SP fibers in raphe-spinal motoneuron projections, there appear to be two basic patterns: one is a dual neuromessenger system as found in the rat; the other is observed in the chick, in which 5-HT and SP are independently localized in different fibers ('independent system'). In other species of animals (cat and monkey), the serotoninergic raphe-spinal projection is made up of specific combinations of these two basic patterns. Namely, 80% and 20% of the serotoninergic raphe-spinal motoneuron projection of the monkey consist of the imlependent and dual neuromessenger systems, respectively. The number of 5-HT and SP positive fibers in the independent system varies widely between different species (for example, chick vs monkey). In contrast to the conventional idea that monoaminergic fibers innervate a non-specific target [15], 5-HT positive fibers were densely located in the motor neuron pools of extensor muscles of the hip joint (chick). Moreover, SP positive fibers were distributed in the same regions. A dense cluster of serotoninergic fibers was observed in the ventrolateral regions of lateral motor column of cervical enlargement in the rat and cat spinal cord. Because those animals use the forelimb more than the hindlimb for stance [18], and because the shoulder (girdle) muscles function during the extension (stance) phase [4], the girdle muscles appear to function as the antigravity muscles [11]. Therefore, serotoninergic fibers were densely localized in the motoneuron pools of antigravity muscles of the cat and rat spinal cord. 5-HT as well as SP are considered to mediate excitatory influences on spinal motoneurons [20]. In addition, recent studies showed that serotoninergic input has a direct excitatory effect on motoneurons [16] and a tonic excitatory effect on the monosynaptic reflex of extensor muscles [2]. Taken together, 5-HT and SP positive fibers may exert a major functional effect on postural control. These data suggest that species differences in raphe-spinal motoneuron projections may be associated with differences in animal posture or other motor behaviors between species. Although the biological significance of multiple neuromessenger systems has not been clarified, the selective activation of two kinds of synaptic vesicles has been postulated [5]: at low frequencies of nerve impulses
small vesicles release 5-HT, whereas at high frequencies large vesicles release both 5-HT and SP. Compared to this postulated mechanism for multiple neuromessengers, the localization of 5-HT and SP in different fibers appears to be precisely regulated. By various combinations of 5-HT and SP fibers, different neuronal systems could produce a wide range of intensity of excitation required for antigravity function or postural responses characteristic of a particular species (quadrupeds vs bipeds, for example). This study was supported by Grant-in-Aid (N.O.) for Scientific Research on Priority Area (cell death) from the Ministry of Education, Science and Culture, Japan. The authors are grateful to Prof. Ronald W. Oppenheim (Department of Neurobiology and Anatomy, BowmanGray School of Medicine) for his critical reading of the manuscript. I Atsumi, S., Sakamoto, H., Yokota, S. and Fujiwara, T., Substance P and 5-hydroxytryptamine immunoreactive presynaptic boutons on presumed :¢-motoneurons in the chicken ventral horn, Arch. Histol. Jpn., 48 (1985) 159 172. 2 Crone, C., Hultborn, H., Kien, O., Mazieres, L. and Wingslrom, H., Maintained changes in motoneuronal excitability by short-lasting synaptic inputs in the decerebrate cat, J. Physiol.. 405 (1988I 321 343, 3 Chan-Play, V., Combined immunocytochemistry and autoradiography after in vivo injections of monoclonal antibody to substance P and )H-serotonin: coexistence of two putative transmitters in single raphe cells and fiber plexuses, Anat. Embryol., 156 (1979) 241 254. 4 English, A.W., An electromyographic analysis of forelimb muscles during overground stepping in the cat, J. Exp. Biol., 76 (1978) 105 122. 5 H6kfeli, T., Holets, V.R., Staines, W., Meister, B., Melander, T., Schalling, M., Schultzberg, M., Freedman, J., Bj6rklund. H., Olson. L., Lindh, B., Elfvin, L.-G., Lundberg, J.M., Lindgren, J.A., Samuelsson, B., Pernow, B,, Terenius, L. Post, C., Everin, B. and Goldstein, M., Coexistence of neuronal messengers an overview. In T, H6kfelt, K. Fuxe and B. Pernow (Eds.), Coexistence of Neuronal Messengers: A New Principle in Chemical Transmission, Progress in Brain Research, Vol. 68, Elsevier, Amsterdam, 1986, pp. 33-70. 6 H6kfelt. T., Ljungdahl, A., Steinbusch, H., Verhofstad, A., Nilsson, G., Brodin, E., Pernow, B. and Goldstein, M., Immunohistochemical evidence of substance P-like immunoreactivity in some 5hydroxytryptamine-containing neurons in the rat central nervous system, Neuroscience, 3 (1978) 517 538. 7 Homma, S., Kohno, K. and Okado, N., Selective localization of serotonin positive fibers in motor neuron pools of the lumbosacral spinal cord of the chicken, Biomed. Res., 10 (Suppl. 3) (1989) 233 239. 8 Homma, S., Sako, H., Kohno. K. and Okado, N., The pattern of distribution of serotoninergic fibers in the anterior horn of the chick spinal cord, Anat. Embryol., 179 (I988) 25 31. 9 Kojima, T., Homma, S., Sako, H., Shimizu, I., Okada, A. and Okado, N., Developmental changes in density and distribution of serotoninergic fibers in the chick spinal cord, J. Comp. Neurol., 267 (1988) 580 589.
158 10 Kojima, M. and Sano, Y., The organization of serotonin fibers in the anterior column of the mammalian spinal cord. An immunohistochemical study, Anat. Embryol., 167 (1983) I 11. 11 Kuypers, H.G.J.M., A new look at the organization of the motor system, In H.G.J.M. Kuypers and G.F. Martin (Eds.), Anatomy of Descending Pathways to the Spinal Cord, Progress in Brain Research, Vol. 57, Elsevier, Amsterdam, 1982, pp. 381 403. 12 Okado, N., Hayashi, H., Hosoya, Y. and Matsukawa, M., Retrograde neuronal labelling by E. coli enterotoxin subunit B, Neurosci. Lett., 120 (1990) 263-266. 13 Okado, N., Homma, S., Ishihara, R. and Kohno, K., Distribution patterns of dendrites in motor neuron pools of lumbosacral spinal cord of the chicken, Anat. Embryol., 182 (1990) I13--121. 14 Okado, N., Homma, S., Ishihara, R., Sako, H. and Kohno, K., Differential innervation of specific motor neuron pools by serotoninergic fibers in the chick spinal cord, Neurosci. Lett., 94 (1988) 29 -~ 32. 15 Parnavelas, J.G. and Papadopoulos, G.C., The monoaminergic
16 17
18
19
20
innervation of the cerebral cortex is not ditfuse and nonspecitic. Trends Neurosci., 12 (1989) 315 319. Takahashi, T. and Berger, A., Direct excitation of rat spinal moloneurons by serotonin, J. Physiol., 423 (1990i 63 76r Tashiro, T. and Ruda, M.A., Immunocytochemical identification of axons containing coexistent serotonin and substance P in the cat lumbar spinal cord, Peptides, 9 (1988) 383 391. Tokuriki, M., Electromyographic and joinl-mechanical studies in quadrupedal locomotion. 1. Walk, Jpn. J. Vet. Sci., 35 (1973)433 446. Wessendorf, M.W. and Elde, R., The coexistence of serotonin- and substance P-like immunoreactivity in the spinal cord of the rat as shown by immunofluorescent double labeling, J. Neurosci., 7 (1987) 2352-.2363. White, S.R., A comparison of the effects of serotonin, substance P and thyrotropine-releasing hormone on excitability of rat spinal motoneurons in vivo, Brain Res., 335 (1985) 63 -70.
155
~ 1991 Elsevier Scientific Publishers Ireland Ltd. 0304-3940/91/$ 03.50 ADONIS 030439409100632V NSL 08148
Species differences in the distribution and coexistence ratio of serotonin and substance P in the monkey, cat, rat and chick spinal cord N o b u o O k a d o 1, M u t s u m i M a t s u k a w a 1, S h i n o b u N o r i t a k e 1, Shigeru Ozaki 2, S h u n H a m a d a I , M i t s u k o Arita 1 and N o r i o K u d o 2 Departments g['l Anatomy and 2Physiology, Institute of Basic Medical Sciences, University of Tsukuba, Tsukuba, lbaraki (Japan) (Received 14 June 1991; Revised version received 26 July 1991; Accepted 26 July 1991)
Key words." lmmunohistochemistry: Motor neuron; Supraspinal input Serotoninergic raphe-spinal motor neuron projections exhibit wide species differences in both innervation pattern and coexistence of serotonin and substance P. The coexistence ratios vary widely ranging from more than 80% (rat) to less than 1% (chick). Serotonin and substance P positive fibers are also une,,enly distributed in the ventral horn of different species: dense clusters of serotonin and substance P positive fibers were preferentially located in the motor neuron pools of extensor muscles of the hip joint (chick) as well as antigravity muscles of the forelimb (cat and rat).
Since the initial reports that serotonin (5-HT) and substance P (SP) coexist in single neurons of the medullary raphe nucleus [3, 6], immunohistochemical evidence for the coexistence of several classical transmitters and peptides have been demonstrated in various parts of the central nervous system [5]. The highest ratio of coexistence of 5-HT and SP in single fibers was reported in the ventral horn of mammalian spinal cord [17, 19]: these raphe-spinal motor projections are considered as evidence against Dale's law [5]. Previous studies have suggested that large species differences exist in serotoninergic projections to spinal motor nuclei: in the chicken 5H T positive fibers are located densely in specific regions of the ventral horn (motoneuron pools innervating specific muscles) [7 9, 14], whereas in the rat 5-HT positive fibers cocontaining SP have not been reported to distribute preferentially in the specific regions of the ventral horn [19]; in the chick 5-HT and SP positive immunoreactivity is located in different terminals [1]; 5-HT positive fibers distribute mainly around the somal profiles of motoneurons in the chick [8] and monkey spinal cord [10], whereas 5-HT fibers were located in regions between motoneuron soma of the cat and rat spinal cord [10]. We were especially interested in the distribution as well as the coexistence of 5-HT and related peptides in different species, that may be related to characteristic motor Correspondence: N. Okado, Department of Anatomy, Institute of Basic Medical Sciences, University of Tsukuba, Tsukuba, Ibaraki 305, Japan.
functions of each species. To examine this possibility we have used an immunohistochemical double labelling technique [19] that reliably identifies different antigens in single axonal profiles. Adult Wistar strain rat (n=4), cat (n=3), chicken (n = 8) and monkey (n = 5) were used in this study. Prior to all the experimental procedures animals were deeply anesthetized, and animals were perfused with Zamboni solution. Ten-/tin thick free floating sections were sequentially treated with primary and secondary antibodies at 37°C. The combinations of primary antibodies were as follows: a polyclonal antibody against 5H T raised in the rabbit at a dilution of 1:10,000, and a monoclonal antibody against SP raised in rat (Sera Lab.) at a dilution of 1:2000, or a monoclonal antibody against enkephaline (Enk) raised in the mouse (Sera Lab.) at a dilution of 1:5000; a polyclonal antibody against Enk raised in the rabbit (UCB) at a dilution of 1:5000 and a monoclonal antibody against SP raised in the rat. An appropriate secondary antibody labelled with fluorescein isothiocyanate (FITC) or with tetramethyl rhodamine isothiocyanate (TRITC) were used. Species cross immunoreactivity was examined by testing all inappropriate combinations of primary and secondary antibodies, and no cross-reactions in the combinations used in the present study were found. For analysis of the size of varicosities of immunoreactive fibers another immunohistochemical technique was employed. Forty-/~m thick transverse frozen sections were processed with the ABC method using anti-5-HT,
156
anti-SP and anti-Enk antibodies. The cross-sectional area of varicosities in the dorsolateral part of the lateral motor column of the lumbar enlargement was measured from enlarged light microscopic photographs at a final magnification × 3000 with a semi-automatic measuring device. In the ventral horn of chick spinal cord, virtually none of the 5-HT positive profiles cocontained SP immunoreactivity (coexistence ratio, less than 1%) (Figs. 1 and 2). The number of SP positive fibers appears greater than
that of 5-HT positive fibers. Although 5-HT positive fibers were located mainly around motoneuron soma (Fig. 1), many SP positive fibers were found in the region between as well as around motoneuron soma (Fig. 2). The sizes of 5-HT and SP positive profiles were clearly different: the area (mean + S.D.) of varicosities of 5-HT and SP was 0.86 + 0.51/~m 2 and 2.11 :+_ 0.87 ltm:, respectively. With double labelling of SP and Enk, about 25% of SP positive varicosities cocontained Enk in the chick. The density of SP positive fibers and varicosities was highest in the dorsolateral part of the lateral motor column of lumbosacral segment 3 (Fig. 3), where 5-HT positive fibers were also densely located, and where the motor neuron pool of the posterior iliotibial muscle is found [12, 13]. Motor neuron pools with a dense accumulation of 5-HT positive fibers innervate extensor muscles of the hip joint [7, 13, 14] and also contain a high density of SP innervation. Another example with a low coexistence ratio of 5-HT and SP in the ventral horn was found in the monkey spinal cord (Figs. 4 and 5): 20% of 5 HT positive varicosities cocontained SP. 5-HT and SP positive fibers were evenly distributed in the ventral horn. The number of SP positive fibers was far less than 5-HT positive fibers. The immunoreactivities of 5-HT and SP were highly colocalized in single fibers of the ventral horn of both the rat (80%) and cat spinal cord: these data are consistent with those of previous studies [17, 19]. Although not so prominent as in the chick, 5-HT positive fibers were also densely distributed in the medial motor column as well as in the ventrolaterat part of the lateral motor column of the cervical enlargement of the cat (Fig. 6). In the lumbar enlargement 5-HT fibers were more evenly distriFigs. 1 and 2. Photographs taken from the motor neuron pool of posterior iliotibial muscle of the adult chicken. Asterisks show motoneuron somata. 5-HT (Fig. 1) and SP (Fig. 2) positive fibers and varieosities are located in a single section. Bars= 10 pro. Fig. 3. SP positive fibers in the lateral motor column (dotted line) of the chick lumbosacral spinal cord segment 3. A dense cluster of SP positive fibers is found at the motoneuron pool of the posterior iliotibial muscle. Bar = 100 gin. Figs. 4 and 5.5-HT (Fig. 4) and SP (Fig. 5) positive fibers in the lateral motor column of the lumbar segment 3 of the monkey spinal cord. Arrows show varicosities cocontaining 5-HT and SP. An asterisk in Fig. 4 indicates artefact, FITC was accumulated in the nucleus. Bars= l0 }tm. Fig. 6.5-HT positive fibers in the ventral horn of the cervical enlargement of the cat spinal cord. 5-HT positive fibers are densely distributed in the ventral part (large arrow) of the lateral motor column(encircled with dotted line) and in the medial motor column (small arrow). Bar=~ I00 ~lm.
157
buted. The distribution pattern of 5-HT positive fibers in the rat spinal cord was similar to that of the cat. Serotoninergic raphe-spinal motor projections have been primarily investigated in only a few mammalian species [17, 19] and are considered as a typical example of a dual messenger system. However, the present study shows that a wide variance in the coexistence ratio of 5HT and SP, as well as in the distribution pattern of these fibers, occurs between different species. In terms of 5-HT and SP fibers in raphe-spinal motoneuron projections, there appear to be two basic patterns: one is a dual neuromessenger system as found in the rat; the other is observed in the chick, in which 5-HT and SP are independently localized in different fibers ('independent system'). In other species of animals (cat and monkey), the serotoninergic raphe-spinal projection is made up of specific combinations of these two basic patterns. Namely, 80% and 20% of the serotoninergic raphe-spinal motoneuron projection of the monkey consist of the imlependent and dual neuromessenger systems, respectively. The number of 5-HT and SP positive fibers in the independent system varies widely between different species (for example, chick vs monkey). In contrast to the conventional idea that monoaminergic fibers innervate a non-specific target [15], 5-HT positive fibers were densely located in the motor neuron pools of extensor muscles of the hip joint (chick). Moreover, SP positive fibers were distributed in the same regions. A dense cluster of serotoninergic fibers was observed in the ventrolateral regions of lateral motor column of cervical enlargement in the rat and cat spinal cord. Because those animals use the forelimb more than the hindlimb for stance [18], and because the shoulder (girdle) muscles function during the extension (stance) phase [4], the girdle muscles appear to function as the antigravity muscles [11]. Therefore, serotoninergic fibers were densely localized in the motoneuron pools of antigravity muscles of the cat and rat spinal cord. 5-HT as well as SP are considered to mediate excitatory influences on spinal motoneurons [20]. In addition, recent studies showed that serotoninergic input has a direct excitatory effect on motoneurons [16] and a tonic excitatory effect on the monosynaptic reflex of extensor muscles [2]. Taken together, 5-HT and SP positive fibers may exert a major functional effect on postural control. These data suggest that species differences in raphe-spinal motoneuron projections may be associated with differences in animal posture or other motor behaviors between species. Although the biological significance of multiple neuromessenger systems has not been clarified, the selective activation of two kinds of synaptic vesicles has been postulated [5]: at low frequencies of nerve impulses
small vesicles release 5-HT, whereas at high frequencies large vesicles release both 5-HT and SP. Compared to this postulated mechanism for multiple neuromessengers, the localization of 5-HT and SP in different fibers appears to be precisely regulated. By various combinations of 5-HT and SP fibers, different neuronal systems could produce a wide range of intensity of excitation required for antigravity function or postural responses characteristic of a particular species (quadrupeds vs bipeds, for example). This study was supported by Grant-in-Aid (N.O.) for Scientific Research on Priority Area (cell death) from the Ministry of Education, Science and Culture, Japan. The authors are grateful to Prof. Ronald W. Oppenheim (Department of Neurobiology and Anatomy, BowmanGray School of Medicine) for his critical reading of the manuscript. I Atsumi, S., Sakamoto, H., Yokota, S. and Fujiwara, T., Substance P and 5-hydroxytryptamine immunoreactive presynaptic boutons on presumed :¢-motoneurons in the chicken ventral horn, Arch. Histol. Jpn., 48 (1985) 159 172. 2 Crone, C., Hultborn, H., Kien, O., Mazieres, L. and Wingslrom, H., Maintained changes in motoneuronal excitability by short-lasting synaptic inputs in the decerebrate cat, J. Physiol.. 405 (1988I 321 343, 3 Chan-Play, V., Combined immunocytochemistry and autoradiography after in vivo injections of monoclonal antibody to substance P and )H-serotonin: coexistence of two putative transmitters in single raphe cells and fiber plexuses, Anat. Embryol., 156 (1979) 241 254. 4 English, A.W., An electromyographic analysis of forelimb muscles during overground stepping in the cat, J. Exp. Biol., 76 (1978) 105 122. 5 H6kfeli, T., Holets, V.R., Staines, W., Meister, B., Melander, T., Schalling, M., Schultzberg, M., Freedman, J., Bj6rklund. H., Olson. L., Lindh, B., Elfvin, L.-G., Lundberg, J.M., Lindgren, J.A., Samuelsson, B., Pernow, B,, Terenius, L. Post, C., Everin, B. and Goldstein, M., Coexistence of neuronal messengers an overview. In T, H6kfelt, K. Fuxe and B. Pernow (Eds.), Coexistence of Neuronal Messengers: A New Principle in Chemical Transmission, Progress in Brain Research, Vol. 68, Elsevier, Amsterdam, 1986, pp. 33-70. 6 H6kfelt. T., Ljungdahl, A., Steinbusch, H., Verhofstad, A., Nilsson, G., Brodin, E., Pernow, B. and Goldstein, M., Immunohistochemical evidence of substance P-like immunoreactivity in some 5hydroxytryptamine-containing neurons in the rat central nervous system, Neuroscience, 3 (1978) 517 538. 7 Homma, S., Kohno, K. and Okado, N., Selective localization of serotonin positive fibers in motor neuron pools of the lumbosacral spinal cord of the chicken, Biomed. Res., 10 (Suppl. 3) (1989) 233 239. 8 Homma, S., Sako, H., Kohno. K. and Okado, N., The pattern of distribution of serotoninergic fibers in the anterior horn of the chick spinal cord, Anat. Embryol., 179 (I988) 25 31. 9 Kojima, T., Homma, S., Sako, H., Shimizu, I., Okada, A. and Okado, N., Developmental changes in density and distribution of serotoninergic fibers in the chick spinal cord, J. Comp. Neurol., 267 (1988) 580 589.
158 10 Kojima, M. and Sano, Y., The organization of serotonin fibers in the anterior column of the mammalian spinal cord. An immunohistochemical study, Anat. Embryol., 167 (1983) I 11. 11 Kuypers, H.G.J.M., A new look at the organization of the motor system, In H.G.J.M. Kuypers and G.F. Martin (Eds.), Anatomy of Descending Pathways to the Spinal Cord, Progress in Brain Research, Vol. 57, Elsevier, Amsterdam, 1982, pp. 381 403. 12 Okado, N., Hayashi, H., Hosoya, Y. and Matsukawa, M., Retrograde neuronal labelling by E. coli enterotoxin subunit B, Neurosci. Lett., 120 (1990) 263-266. 13 Okado, N., Homma, S., Ishihara, R. and Kohno, K., Distribution patterns of dendrites in motor neuron pools of lumbosacral spinal cord of the chicken, Anat. Embryol., 182 (1990) I13--121. 14 Okado, N., Homma, S., Ishihara, R., Sako, H. and Kohno, K., Differential innervation of specific motor neuron pools by serotoninergic fibers in the chick spinal cord, Neurosci. Lett., 94 (1988) 29 -~ 32. 15 Parnavelas, J.G. and Papadopoulos, G.C., The monoaminergic
16 17
18
19
20
innervation of the cerebral cortex is not ditfuse and nonspecitic. Trends Neurosci., 12 (1989) 315 319. Takahashi, T. and Berger, A., Direct excitation of rat spinal moloneurons by serotonin, J. Physiol., 423 (1990i 63 76r Tashiro, T. and Ruda, M.A., Immunocytochemical identification of axons containing coexistent serotonin and substance P in the cat lumbar spinal cord, Peptides, 9 (1988) 383 391. Tokuriki, M., Electromyographic and joinl-mechanical studies in quadrupedal locomotion. 1. Walk, Jpn. J. Vet. Sci., 35 (1973)433 446. Wessendorf, M.W. and Elde, R., The coexistence of serotonin- and substance P-like immunoreactivity in the spinal cord of the rat as shown by immunofluorescent double labeling, J. Neurosci., 7 (1987) 2352-.2363. White, S.R., A comparison of the effects of serotonin, substance P and thyrotropine-releasing hormone on excitability of rat spinal motoneurons in vivo, Brain Res., 335 (1985) 63 -70.