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Electronmicroscopic detection of the axonal coexistence of serotonin and substance P in B1-B2 raphé cells transplanted into the transected spinal cord of adult rats
Brain Research, 542 (1991) 159-162
159
Elsevier BRES 24547
Electronmicroscopic detection of the axonal coexistence of serotonin and substance P in BI-B2 raph0 cells transplanted into the transected spinal cord of adult rats N. Rajaofetra 1, P. Kachidian 2, L. Marlier 1, P. Poulat 1, N. K6nig 1, M. Geffard 3 and A. Privat I ~INSERM, U-336, Ddveloppement, plasticit~ et vieiUissement du systdme nerveux, U.S.T.L. Montpellier (France), 2Laboratoire de neurobiologie, Equipe de neuromorphologie fonctionelle, CNRS, Marseille (France)and 3LB. C.N.-CRNS, Laboratoire de neuroimmunologie, Bordeaux (France)
(Accepted 13 November 1990) Key words: Serotonin; Substance P; Transplantation; Electron microscopy; Spinal cord; Dual immunocytochemistry; B1-B2; Rat
One week after a complete spinal cord transection at the thoracic (T8) level in adult rats, a suspension of rhombencephalic embryonic (day 14) cells containing the B1-B2 serotonergic groups was injected below the section. After a survival period of one month, the spinal cord was processed for an ultrastructural dual immunocytochemicaldetection of serotonin (5-HT) and substance P (SP). It was shown by ultrastructural dual immunolabeling that 5-HT and SP coexist in the same axon terminals of transplanted cells.
Studies using transplantation of embryonic rhombencephalon into the spinal cord, after drug-induced serotonin (5-HT) deprivation 5 or section of the cord 15'17-2°, have shown that serotonergic neurons were able to grow processes and innervate target regions. Moreover, K6nig et al. n found that, depending on the nucleus of origin of the grafted cells, the pattern of innervation of the cord was different, suggesting a high specificity in the phenotypic expression of these neurons. In rhombencephalic nuclei, a particularly interesting aspect of this phenotypic expression is the presence of several neurotransmitters and/or neuromodulators in the same neuron, as evidenced for the first time by Hfkfelt et al. 7'8 (see also refs. 2, 9, 10, 12, 14 and 22). However, limited data are presently available concerning intraneuronal coexistence after transplantation. In this regard, by using immunofluorescence, Foster et al. 5 showed that 5-HT co-exists with substance P (SP) or thyrotropinreleasing hormone (TRH) in rhombencephalic cells transplanted in rats pretreated with 5,7-dihydroxytryptamine. However, no demonstration for the coexistence of two neurotransmitters in the same axon terminal of transplanted neurons has been published as yet. In the present work, by using at the ultrastructural level a dual immunocytochemical detection of 5-HT and SP, we investigate the putative co-localization of these
two neurotransmitters in terminals of neurons from rhombencephalon, grafted below a complete transection of the spinal cord. Pieces of caudal rhombencephalon containing the anlagen of the B1-B2 groups were dissected from Sprague-Dawley rat embryos (IFFA-CREDO, France) at embryonic day 14 (for technical considerations, see ref. 11). The cells were mechanically dissociated and injected into adult rat spinal cord below a complete transection at the thoracic level (Ts). After a survival period of one month, the animals were deeply anaesthetized with pentobarbital and perfused transcardially with 300 ml glutaraldehyde 5% in a 50 mM sodium cacodylate 50 mM metabisulfite (SMB) buffer at pH 7.5. Spinal cords were removed and immersed overnight in the same fixative at 4 °C. For single labeling, vibratome sections were processed for the immunocytochemical detection of 5-HT according to the PAP method of Sternberger 21. For dual labeling, we used a technique modified from that of Vuillet et al. 23 which combines peroxidase labeling for the detection of substance P, and radioautographic detection of 5-HT using 125I-labeled protein A. Sections were then postfixed with osmium tetroxyde, block-stained with 2% uranylacetate and fiat embedded in Araldite. After polymerisation, sections were selected
embryonic(El4) caudal
Correspondence.. N. Rajaofetra, INSERM, U-336, D6veloppement, plasticit6 et vieillissement du syst~me nerveux, U.S.T.L., place Eug~.ne Bataillon, 34095 Montpellier Cedex 05, France.
0006-8993/91/$03.50 (~ 1991 Elsevier Science Publishers B.V. (Biomedical Division)
160
Fig. 1. Electron micrograph of a 5-HT positive radioimmunocytochemically labeled neuron. Bar = 1 pm.
under light microscopy for satisfactory peroxidase SP staining and glued on top of prepolymerized Araldite blocks. Ultrathin sections were then processed for radioautography 3 and examined with a Jeol 2000 electron microscope. Examination of control vibratome sections treated with the PAP method for immunocytochemical detection of 5-HT showed the presence of a well developed transplant located in the dorsal funiculi and neighboring grey matter. Sertotonergic fibers were detected at a distance of the transplant, i.e. in the ventral horn and the intermediolateral cell column. At the electronmicroscopic level, radioimmunolabeled perikarya, showing a typical accumulation of silver grains, were found in the transplant region (Fig. 1). Numerous silver grains were present throughout the cytoplasm, contrasting with a very low background. None of the radioimmunolabeled somata exhibited peroxidase reaction, suggesting that in our technical conditions the 5-HT/SP coexistence is not detectable in nerve cell bodies. Outside the transplant, peroxidase-labeling for SP, radioimmunolabeling for 5-HT, and 5-HT/SP dually labeled profiles were revealed throughout the neuropil of the ventral horn and the intermediolateral cell column. Some of these dually labeled profiles appeared as large varicosities containing spherical and pleomorphic vesi-
cles. In one instance (Figs. 2-4) such a varicosity could be found over consecutive sections, and it was not facing a postsynaptic density in any of these sections. In other instances, large varicosities were facing postsynaptic densities (Figs. 5, 6) located on an unlabeled profile, most often a small dendrite or a dendritic spine. The present study using a novel dual immunocytochemical technique has shown that transplanted serotonergic immunoreactive neurons from B1-B2 groups can also express SP in their axon terminals. In perikarya, only 5-HT was demonstrated due to the inability to detect peptides in nerve cell bodies, without any colchicine treatment. In order to avoid excess radiolabeling, which would have obscured the pictures, the duration of the radioautographic exposure and the development conditions were adapted to obtain a light radiolabeling. However, the use of serial sections (see Figs. 2-4) ascertained that the radioautographic labeling was specific. Moreover, the absence of systematic dual labeling confirms the absence of any detectable crossreaction during the immunocytochemical procedure. We noticed that only some labeled varicosities made typical synapses, as it has already been shown by others for 5-HT boutons in different locations 1' 4. That such is also the case in the spinal cord has been shown in situ by Maxwell et al. 13 for the dorsal horn. Grafted neurons apparently develop a phenotype similar in this respect to their in situ counterpart 17-19. The coexistence of 5-HT and SP in the same bouton was shown in situ in the spinal cord by Pelletier et al. 16, and confirmed by Wessendorf and Elde 24. In addition, Foster et al. s, using immunofluorescence, demonstrated that grafted serotonergic cells could express other transmitters, such as SP or T R H . This would indicate that the expression of these phenotypic characters is independent of the tissular environment of serotonergic neurons, as it may occur when these neurons are transplanted to an ectopic location s. This expression may be either strictly under the dependence of an intrinsic program, or, conversely, dependent on the nature of the target. We have previously shown ~1'~8that transplanted serotonergic neurons exhibit a great specificity in the orientation of their growth and in the recognition of their target. It can be hypothesized that this target is able to induce, by retrograde signaling, the synthesis of neurotransmitters relevant to the corresponding receptors. Future work with transplantations at other ectopic sites, either constituting a target or not for serotonergic neurons, will contribute to elucidate this point. This work was supported by grants from IRME, D. Heumann Fund and AFM. We wish to thank J.R. Teilhac for excellent photographical assistance.
161
Fig. 2-4. Non-consecutive serial sections through a dually labeled 5-HT/SP terminal. Fig. 5. Double labeled 5-HT/SP varicosity in synaptic contact with small dendrites (arrows). Fig. 6. Double labeled 5-HT/SP varicosities in synaptic contact with dendritic spines (arrows). The lower profile is in fact made of two separate processes (arrowheads). Bar = 1/~m.
162
1 Beaudet, A. and Descarries, L., The monoamine innervation of rat cerebral cortex: synaptic and nonsynaptic axon terminals, Neuroscience, 8 (1979) 851-860. 2 Chan-Palay, V., Jonsson, G. and Palay, S.L., Serotonin and substance P coexist in neurons of rat's central nervous system, Proc. Natl. Acad. Sci. U.S.A., 75 (1978) 1582-1586. 3 Descarries, L. and Beaudet, A., The use of radioautography for investigating transmitter-specific neurons. In A. Bj6rklund and T. HOkfelt (Eds.), Handbook of Chemical Neuroanatomy, Vol. 1, Elsevier, Amsterdam, 1983, pp. 286-364. 4 Descarries, L., Beaudet, A. and Watkins, K.C., Serotonin nerve terminals in adult rat neocortex, Brain Research, I00 (1975) 563-588. 5 Foster, G.A., Schultzberg, M., Gage, EH., Bj6rklund, A., H6kfelt, T., Nornes, H., Cuello, A.C., Verhofstad, A.A.J. and Visser, T.J., Transmitter expression and morphological development of embryonic medullary and mesencephalic raph6 neurones after transplantation to the adult rat central nervous system. I. Grafts to the spinal cord, Exp. Brain Res., 60 (1985) 427-444. 6 Geffard, M., Henrick-Rock, A.M., Dulluc, J. and Seguela, P., Antisera against small neurotransmitter-like molecules, Neurochem. Int., 7 (1985) 403-413. 7 H6kfelt, T., Fuxe, K., Johansson, O., Jeffcoate, S. and White, N., Thyrotropin releasing hormone (TRH)-containing nerve terminals in certain brain stem nuclei and in the spinal cord, Neurosci. Len., 1 (1975) 133-139. 8 H6kfelt, T., Ljungdahl, A., Steinbusch, H., Verhofstad, A., Nilsson, G., Brodin, E., Pernow, B. and Goldstein, M., lmmunohistochemical evidence of substance P-like immunoreactivity in some 5-hydroxytryptamine-containing neurons in the rat central nervous system, Neuroscience, 3 (1978) 517-538. 9 Holets, V.R., Hfkfelt, T., Ude, J., Eckert, M., Penzlin, H., Verhofstad, A. and Visser, T.J., A comparative study of the immunohistochemical localization of a presumptive proctolinlike peptide, thyrotropin-releasing hormone and 5-hydroxytryptamine in the rat central nervous system, Brain Research, 408 (1987) 141-153. 10 Johansson, O., H6kfelt, T., Pernow, B., Jeffcoate, S.L., White, N., Steinbusch, H.W.M., Verhofstad, A.A.J., Emson, P.C. and Spindel, E., Immunohistochemical support for three putative transmitters in one neuron: coexistence of 5-hydroxytryptamine, substance P- and thyrotropin-releasing hormone-like immunoreactivity in medullary neurons projecting to the spinal cord, Neuroscience, 6 (1981) 1857-1981. 11 K6nig, N., Rajaofetra, N., Sandillon, E, Drian, M.J., Fuentes, C., Favier, E and Privat, A., Serotonin-expressing cells from different microregions of the embryonic rat rhombencephalon: behaviour in cell culture and in transplantation to the adult spinal cord. In F. Gage, A. Privat and Y. Christen (Eds.),
1PSEN Monographies Series, Neuronal Grafting and Alzheimer's Disease, Springer, Berlin, 1989, pp. 150-166. 12 Lechan, R.M., Molitch, M.E. and Jackson, M.D., Distribution of immunoreactive human growth hormone-like material and thyrotropin-releasing hormone in the rat central nervous system: evidence for their coexistence in the same neurons, Endocrinology, 112 (1983) 877-884. 13 Maxwell, D.J., Leranth, C.S. and Verhofstad, A.A.J., Fine structure of serotonin-containing axons in the marginal zone of the spinal cord, Brain Research, 266 (1983) 253-259. 14 Menetrey, D. and Basbaum, A.I., The distribution of substance P-, enkephalin- and dynorphin-immunoreactive neurons in the medulla of the rat and their contribution to buibospinal pathways, Neuroscience, 23 (1987) 173-187. 15 Nygren, L.G., Olson, L. and Seiger, A., Monoaminergic reinnervation of the transected spinal cord by homologous fetal brain grafts, Brain Research, 129 (1977) 225-235. 16 Pelletier, G., Steinbusch, H.W. and Verhofstad, A., Immunoreactive substance P and serotonin present in the same densecore vesicles, Nature, 293 (1981) 71-72. 17 Privat, A., Mansour, H., Geffard, M. and Sandillon, F., Transplantation of dissociated fetal serotonin neurons into the transected spinal cord of adult rats, Neurosci. Lett., 66 (1986) 61-66. 18 Privat, A., Mansour, H. and Geffard, M., Transplantation of fetal serotonin neurons into the transected spinal cord of adult rats: morphological development and functional influence, Prog. Brain Res., 78 (1988) 155-166. 19 Privat, A., Mansour, H., Rajaofetra, N. and Geffard, M., Intraspinal transplants of serotonergic neurons, Brain Res. Bull., 22 (1989) 123-129. 20 Rajaofetra, N., Poulat, P., Marlier, L., Proust, E, Drian, M.J., Passagia, J.G., Verschuere, B., K6nig, N., Gouy, D. and Privat, A., First transplantation of embryonic serotonergic neurons in a primate spinal cord, C.R. Acad. Sci., 310 (1990) 81-88. 21 Sternberger, L.A., The unlabeled antibody enzyme method of immunohistochemistry, J. Histochem. Cytochem., 8 (1970) 315-325. 22 Tsuruo, Y., H6kfeit, T. and Visser, T., Thyrotropin-releasing hormone (TRH)-immunoreactive cell groups in the rat central nervous system, Exp. Brain Res., 68 (1987) 213-217. 23 Vuillet, J., Kerkerian, L., Kachidian, P., Bosler, O. and NieouUon, A., Ultrastructural correlates of functional relationships between nigral dopaminergic or cortical afferent fibers and neuropeptide Y-containing neurons in the rat striatum, Neurosci. Lett., 100 (1989) 99-104. 24 Wessendorf, M.W. and Elde, T., The coexistence of serotoninand substance P-like immunoreactivity in the spinal cord of the rat as shown by immunofluorescent double labeling, J. Neurosci., 7 (1987) 2352-2363.
159
Elsevier BRES 24547
Electronmicroscopic detection of the axonal coexistence of serotonin and substance P in BI-B2 raph0 cells transplanted into the transected spinal cord of adult rats N. Rajaofetra 1, P. Kachidian 2, L. Marlier 1, P. Poulat 1, N. K6nig 1, M. Geffard 3 and A. Privat I ~INSERM, U-336, Ddveloppement, plasticit~ et vieiUissement du systdme nerveux, U.S.T.L. Montpellier (France), 2Laboratoire de neurobiologie, Equipe de neuromorphologie fonctionelle, CNRS, Marseille (France)and 3LB. C.N.-CRNS, Laboratoire de neuroimmunologie, Bordeaux (France)
(Accepted 13 November 1990) Key words: Serotonin; Substance P; Transplantation; Electron microscopy; Spinal cord; Dual immunocytochemistry; B1-B2; Rat
One week after a complete spinal cord transection at the thoracic (T8) level in adult rats, a suspension of rhombencephalic embryonic (day 14) cells containing the B1-B2 serotonergic groups was injected below the section. After a survival period of one month, the spinal cord was processed for an ultrastructural dual immunocytochemicaldetection of serotonin (5-HT) and substance P (SP). It was shown by ultrastructural dual immunolabeling that 5-HT and SP coexist in the same axon terminals of transplanted cells.
Studies using transplantation of embryonic rhombencephalon into the spinal cord, after drug-induced serotonin (5-HT) deprivation 5 or section of the cord 15'17-2°, have shown that serotonergic neurons were able to grow processes and innervate target regions. Moreover, K6nig et al. n found that, depending on the nucleus of origin of the grafted cells, the pattern of innervation of the cord was different, suggesting a high specificity in the phenotypic expression of these neurons. In rhombencephalic nuclei, a particularly interesting aspect of this phenotypic expression is the presence of several neurotransmitters and/or neuromodulators in the same neuron, as evidenced for the first time by Hfkfelt et al. 7'8 (see also refs. 2, 9, 10, 12, 14 and 22). However, limited data are presently available concerning intraneuronal coexistence after transplantation. In this regard, by using immunofluorescence, Foster et al. 5 showed that 5-HT co-exists with substance P (SP) or thyrotropinreleasing hormone (TRH) in rhombencephalic cells transplanted in rats pretreated with 5,7-dihydroxytryptamine. However, no demonstration for the coexistence of two neurotransmitters in the same axon terminal of transplanted neurons has been published as yet. In the present work, by using at the ultrastructural level a dual immunocytochemical detection of 5-HT and SP, we investigate the putative co-localization of these
two neurotransmitters in terminals of neurons from rhombencephalon, grafted below a complete transection of the spinal cord. Pieces of caudal rhombencephalon containing the anlagen of the B1-B2 groups were dissected from Sprague-Dawley rat embryos (IFFA-CREDO, France) at embryonic day 14 (for technical considerations, see ref. 11). The cells were mechanically dissociated and injected into adult rat spinal cord below a complete transection at the thoracic level (Ts). After a survival period of one month, the animals were deeply anaesthetized with pentobarbital and perfused transcardially with 300 ml glutaraldehyde 5% in a 50 mM sodium cacodylate 50 mM metabisulfite (SMB) buffer at pH 7.5. Spinal cords were removed and immersed overnight in the same fixative at 4 °C. For single labeling, vibratome sections were processed for the immunocytochemical detection of 5-HT according to the PAP method of Sternberger 21. For dual labeling, we used a technique modified from that of Vuillet et al. 23 which combines peroxidase labeling for the detection of substance P, and radioautographic detection of 5-HT using 125I-labeled protein A. Sections were then postfixed with osmium tetroxyde, block-stained with 2% uranylacetate and fiat embedded in Araldite. After polymerisation, sections were selected
embryonic(El4) caudal
Correspondence.. N. Rajaofetra, INSERM, U-336, D6veloppement, plasticit6 et vieillissement du syst~me nerveux, U.S.T.L., place Eug~.ne Bataillon, 34095 Montpellier Cedex 05, France.
0006-8993/91/$03.50 (~ 1991 Elsevier Science Publishers B.V. (Biomedical Division)
160
Fig. 1. Electron micrograph of a 5-HT positive radioimmunocytochemically labeled neuron. Bar = 1 pm.
under light microscopy for satisfactory peroxidase SP staining and glued on top of prepolymerized Araldite blocks. Ultrathin sections were then processed for radioautography 3 and examined with a Jeol 2000 electron microscope. Examination of control vibratome sections treated with the PAP method for immunocytochemical detection of 5-HT showed the presence of a well developed transplant located in the dorsal funiculi and neighboring grey matter. Sertotonergic fibers were detected at a distance of the transplant, i.e. in the ventral horn and the intermediolateral cell column. At the electronmicroscopic level, radioimmunolabeled perikarya, showing a typical accumulation of silver grains, were found in the transplant region (Fig. 1). Numerous silver grains were present throughout the cytoplasm, contrasting with a very low background. None of the radioimmunolabeled somata exhibited peroxidase reaction, suggesting that in our technical conditions the 5-HT/SP coexistence is not detectable in nerve cell bodies. Outside the transplant, peroxidase-labeling for SP, radioimmunolabeling for 5-HT, and 5-HT/SP dually labeled profiles were revealed throughout the neuropil of the ventral horn and the intermediolateral cell column. Some of these dually labeled profiles appeared as large varicosities containing spherical and pleomorphic vesi-
cles. In one instance (Figs. 2-4) such a varicosity could be found over consecutive sections, and it was not facing a postsynaptic density in any of these sections. In other instances, large varicosities were facing postsynaptic densities (Figs. 5, 6) located on an unlabeled profile, most often a small dendrite or a dendritic spine. The present study using a novel dual immunocytochemical technique has shown that transplanted serotonergic immunoreactive neurons from B1-B2 groups can also express SP in their axon terminals. In perikarya, only 5-HT was demonstrated due to the inability to detect peptides in nerve cell bodies, without any colchicine treatment. In order to avoid excess radiolabeling, which would have obscured the pictures, the duration of the radioautographic exposure and the development conditions were adapted to obtain a light radiolabeling. However, the use of serial sections (see Figs. 2-4) ascertained that the radioautographic labeling was specific. Moreover, the absence of systematic dual labeling confirms the absence of any detectable crossreaction during the immunocytochemical procedure. We noticed that only some labeled varicosities made typical synapses, as it has already been shown by others for 5-HT boutons in different locations 1' 4. That such is also the case in the spinal cord has been shown in situ by Maxwell et al. 13 for the dorsal horn. Grafted neurons apparently develop a phenotype similar in this respect to their in situ counterpart 17-19. The coexistence of 5-HT and SP in the same bouton was shown in situ in the spinal cord by Pelletier et al. 16, and confirmed by Wessendorf and Elde 24. In addition, Foster et al. s, using immunofluorescence, demonstrated that grafted serotonergic cells could express other transmitters, such as SP or T R H . This would indicate that the expression of these phenotypic characters is independent of the tissular environment of serotonergic neurons, as it may occur when these neurons are transplanted to an ectopic location s. This expression may be either strictly under the dependence of an intrinsic program, or, conversely, dependent on the nature of the target. We have previously shown ~1'~8that transplanted serotonergic neurons exhibit a great specificity in the orientation of their growth and in the recognition of their target. It can be hypothesized that this target is able to induce, by retrograde signaling, the synthesis of neurotransmitters relevant to the corresponding receptors. Future work with transplantations at other ectopic sites, either constituting a target or not for serotonergic neurons, will contribute to elucidate this point. This work was supported by grants from IRME, D. Heumann Fund and AFM. We wish to thank J.R. Teilhac for excellent photographical assistance.
161
Fig. 2-4. Non-consecutive serial sections through a dually labeled 5-HT/SP terminal. Fig. 5. Double labeled 5-HT/SP varicosity in synaptic contact with small dendrites (arrows). Fig. 6. Double labeled 5-HT/SP varicosities in synaptic contact with dendritic spines (arrows). The lower profile is in fact made of two separate processes (arrowheads). Bar = 1/~m.
162
1 Beaudet, A. and Descarries, L., The monoamine innervation of rat cerebral cortex: synaptic and nonsynaptic axon terminals, Neuroscience, 8 (1979) 851-860. 2 Chan-Palay, V., Jonsson, G. and Palay, S.L., Serotonin and substance P coexist in neurons of rat's central nervous system, Proc. Natl. Acad. Sci. U.S.A., 75 (1978) 1582-1586. 3 Descarries, L. and Beaudet, A., The use of radioautography for investigating transmitter-specific neurons. In A. Bj6rklund and T. HOkfelt (Eds.), Handbook of Chemical Neuroanatomy, Vol. 1, Elsevier, Amsterdam, 1983, pp. 286-364. 4 Descarries, L., Beaudet, A. and Watkins, K.C., Serotonin nerve terminals in adult rat neocortex, Brain Research, I00 (1975) 563-588. 5 Foster, G.A., Schultzberg, M., Gage, EH., Bj6rklund, A., H6kfelt, T., Nornes, H., Cuello, A.C., Verhofstad, A.A.J. and Visser, T.J., Transmitter expression and morphological development of embryonic medullary and mesencephalic raph6 neurones after transplantation to the adult rat central nervous system. I. Grafts to the spinal cord, Exp. Brain Res., 60 (1985) 427-444. 6 Geffard, M., Henrick-Rock, A.M., Dulluc, J. and Seguela, P., Antisera against small neurotransmitter-like molecules, Neurochem. Int., 7 (1985) 403-413. 7 H6kfelt, T., Fuxe, K., Johansson, O., Jeffcoate, S. and White, N., Thyrotropin releasing hormone (TRH)-containing nerve terminals in certain brain stem nuclei and in the spinal cord, Neurosci. Len., 1 (1975) 133-139. 8 H6kfelt, T., Ljungdahl, A., Steinbusch, H., Verhofstad, A., Nilsson, G., Brodin, E., Pernow, B. and Goldstein, M., lmmunohistochemical evidence of substance P-like immunoreactivity in some 5-hydroxytryptamine-containing neurons in the rat central nervous system, Neuroscience, 3 (1978) 517-538. 9 Holets, V.R., Hfkfelt, T., Ude, J., Eckert, M., Penzlin, H., Verhofstad, A. and Visser, T.J., A comparative study of the immunohistochemical localization of a presumptive proctolinlike peptide, thyrotropin-releasing hormone and 5-hydroxytryptamine in the rat central nervous system, Brain Research, 408 (1987) 141-153. 10 Johansson, O., H6kfelt, T., Pernow, B., Jeffcoate, S.L., White, N., Steinbusch, H.W.M., Verhofstad, A.A.J., Emson, P.C. and Spindel, E., Immunohistochemical support for three putative transmitters in one neuron: coexistence of 5-hydroxytryptamine, substance P- and thyrotropin-releasing hormone-like immunoreactivity in medullary neurons projecting to the spinal cord, Neuroscience, 6 (1981) 1857-1981. 11 K6nig, N., Rajaofetra, N., Sandillon, E, Drian, M.J., Fuentes, C., Favier, E and Privat, A., Serotonin-expressing cells from different microregions of the embryonic rat rhombencephalon: behaviour in cell culture and in transplantation to the adult spinal cord. In F. Gage, A. Privat and Y. Christen (Eds.),
1PSEN Monographies Series, Neuronal Grafting and Alzheimer's Disease, Springer, Berlin, 1989, pp. 150-166. 12 Lechan, R.M., Molitch, M.E. and Jackson, M.D., Distribution of immunoreactive human growth hormone-like material and thyrotropin-releasing hormone in the rat central nervous system: evidence for their coexistence in the same neurons, Endocrinology, 112 (1983) 877-884. 13 Maxwell, D.J., Leranth, C.S. and Verhofstad, A.A.J., Fine structure of serotonin-containing axons in the marginal zone of the spinal cord, Brain Research, 266 (1983) 253-259. 14 Menetrey, D. and Basbaum, A.I., The distribution of substance P-, enkephalin- and dynorphin-immunoreactive neurons in the medulla of the rat and their contribution to buibospinal pathways, Neuroscience, 23 (1987) 173-187. 15 Nygren, L.G., Olson, L. and Seiger, A., Monoaminergic reinnervation of the transected spinal cord by homologous fetal brain grafts, Brain Research, 129 (1977) 225-235. 16 Pelletier, G., Steinbusch, H.W. and Verhofstad, A., Immunoreactive substance P and serotonin present in the same densecore vesicles, Nature, 293 (1981) 71-72. 17 Privat, A., Mansour, H., Geffard, M. and Sandillon, F., Transplantation of dissociated fetal serotonin neurons into the transected spinal cord of adult rats, Neurosci. Lett., 66 (1986) 61-66. 18 Privat, A., Mansour, H. and Geffard, M., Transplantation of fetal serotonin neurons into the transected spinal cord of adult rats: morphological development and functional influence, Prog. Brain Res., 78 (1988) 155-166. 19 Privat, A., Mansour, H., Rajaofetra, N. and Geffard, M., Intraspinal transplants of serotonergic neurons, Brain Res. Bull., 22 (1989) 123-129. 20 Rajaofetra, N., Poulat, P., Marlier, L., Proust, E, Drian, M.J., Passagia, J.G., Verschuere, B., K6nig, N., Gouy, D. and Privat, A., First transplantation of embryonic serotonergic neurons in a primate spinal cord, C.R. Acad. Sci., 310 (1990) 81-88. 21 Sternberger, L.A., The unlabeled antibody enzyme method of immunohistochemistry, J. Histochem. Cytochem., 8 (1970) 315-325. 22 Tsuruo, Y., H6kfeit, T. and Visser, T., Thyrotropin-releasing hormone (TRH)-immunoreactive cell groups in the rat central nervous system, Exp. Brain Res., 68 (1987) 213-217. 23 Vuillet, J., Kerkerian, L., Kachidian, P., Bosler, O. and NieouUon, A., Ultrastructural correlates of functional relationships between nigral dopaminergic or cortical afferent fibers and neuropeptide Y-containing neurons in the rat striatum, Neurosci. Lett., 100 (1989) 99-104. 24 Wessendorf, M.W. and Elde, T., The coexistence of serotoninand substance P-like immunoreactivity in the spinal cord of the rat as shown by immunofluorescent double labeling, J. Neurosci., 7 (1987) 2352-2363.