Neuroscience Letters, 14 (1979) 55--60 © Elsevier/North-Holland Scientific Publishers Ltd.
55
EVIDENCE F O R ENKEPHALIN IMMUNOREACTIVE N E U R O N S IN THE M E D U L L A O B L O N G A T A P R O J E C T I N G TO THE SPINAL CORD
T. HOKFELT, L. TERENIUS, H.G.J.M. KUYPERS and O. DANN
Department of Histology, Karolinska Institutet, Stockholm, Department of Pharmacology, Uppsala University (Sweden), Department of Anatomy, E ~ m u s University Medical School, Rotterdam (The Netherlands) and Institute of Pharmacy and Food Chemistry, Friedrich Alexander University, Erlangen (F.R.G.) (Received June 7th, 1979) (Accepted June 7th, 1979)
SUMMARY
In the present study a m e t h o d is used, with which retrograde tracing with the fluorescent d y e 'True Blue' is combined with the indirect immunofluorescence technique for visualizing enkephalin-like immunoreactivity. Evidence is presented for the existence of an enkephalin immunoreactive system originating in the lower medulla oblongata and projecting to the spinal cord.
The enkephalin pentapeptides [15] have a wide-spread distribution in the central nervous system. Immunocytochemical methods have shown enkephalin immunoreactive nerve terminals in the central and dorsal horns of the spinal cord and a particular dense network in laminae I and II [4,9,10,13,27, 28,30,31]. In the dorsal horn there are also enkephalin immunoreactive cell bodies [10,13,30]. Previous attempts to elucidate the origin of the enkephalin immunoreactive fibers in the gray matter of the spinal cord. Since no dorsal rhizotomy as well as total transection of the spinal cord. Since no distinct effects could be observed after either operation, we concluded that the fibers are intrinsic to the cord [ 1 3 ] . Quantitative estimations on the basis of immunocytochemistry are, however, difficult to perform. Thus, a decrease in the number of immunoreactive fibers, particularly in the densely innervated laminae I and II, might be difficult to detect. We have therefore reinvestigated this problem utilizing a different approach, b y which retrograde tracing principles [16,19] were combined with immunohistochemistry ([ 12] ; see also ref. 22). For retrograde tracing a fluorescent marker was used [2] and enkephalin-like material was demonstrated with the indirect immunofluorescence technique [6,9,10]. We n o w present evidence that enkephalin immunoreactive cell bodies located in the lower medulla oblongata project to the spinal cord.
56 Male albino rats (Sprague--Dawley; body wt 150 g) were used. Under halothane anaesthesia, 1 pl of a 10% solution of the dye 'True Blue' (code number 150/129; trans-l,2-bis(5.amidino-2-benzofuranyl) ethylene 2 HC1) [ 7 ] in distilled water was injected bilaterally at two levels of lower thoracicupper lumbar spinal cord with a Hamilton syringe [2]. Six days later 25 pl colchicine (concentration 3 pg/ul), dissolved in 0.9% sodium chloride, were injected into the lateral ventricle. After 24 h the rats were perfused with 4% ice cold formaldehyde for 30 min and the brain and spinal cord were removed, immersed in the same fixative for 90 min, rinsed for 24 h in 0.1 M phosphate buffer with 5% sucrose added and cut on a cryostat. Sections from the lower medulla oblongata were examined in a Zeiss fluorescence microscope and fluorescent cells were photographed. After removal of the cover slip, the sections were transferred to a buffer and processed for the indirect technique of Coons and collaborators (see ref. 6) as described previously [11]. Briefly, the sections were incubated at 4°C with antiserum to met-enkephalin diluted 1 : 160 for 16--20 h, rinsed, incubated with fluorescein isothiocyanate-conjugated sheep antirabbit antibodies (SBL, Stockholm, Sweden}, mounted, examined in the same fluorescence microscope and fluorescent cells were photographed. As described in detail in a parallel paper [12], the 'True Blue' fluorescence disappears almost completely after the processing for immunohistochemistry. Thus, the green fluorescent cells and fibers seen after the immunohistochemical procedure represent enkephalin immunoreactive structures. It should also be emphasized that, with the filter combinations used (excitation filter: either Schott B912 or KP500; stopfilter: either Zeiss 50 or LP520), the green-yellow 'True Blue' colour can be distinguished clearly from that of the FITC induced green fluorescence. Seven days after injection of the dye into the spinal cord numerous greenyellow fluorescent cells were observed in the lower medulla oblongata These cells were located in several nuclei, particularly in the midline raphe nuclei, the ventral part of the reticular nucleus and on the gigantocellular reticular nucleus. Labelled cells were also seen in the dorsal aspects of the lateral reticular nucleus and dorsal and lateral to the inferior olive, i.e., in the paragigantocellular nucleus and in the pars a of the gigantocellular reticular nucleus [32]. In the latter area the fluorescent cells were often found in small groups with up to 10 cells per section and side (Fig. 1A}. These results are in general agreement with earlier results on efferent connections of the lower medulla oblongata and t h e spinal cord [1,5,18,20,24]. After labelling for the indirect immunofluorescence technique with enkephalin antiserum, the labelling pattern was markedly different. The fluorescent cells described above were no longer detectable, except for some very weakly yellow fluorescentcells. On the other hand, green fluorescent enkephalin immunoreactive cells were observed in several nuclei with a distribution similar to that described previously [9,27,30], that is, for example, in the superficial layers of the spinal trigeminal nucleus, the nucleus tractus solitarii,
57
Fig. 1. Fluorescence micrographs of the lower medulla oblongata of the same section showing the area immediately dorsal to the inferior olive (IO). In (A) numerous greenyellow fluorescent cell bodies are seen, which have been retrogradely labelled with 'True Blue' after spinal injections of the dye. In (B) the same area has been photographed after processing of the section for indirect immunofluorescence histochemistry using an antiserum to met-enkephalin. A small group of cells (arrows), which in A contains 'True Blue', is in B shown to be enkephalin immunoreactive. Note that a very weak (yellow) fluorescence, clearly different from the FITC induced fluorescence, remains in some cells after processing for immunohistochemistry (arrow heads). Bars indicate 50 ~m.
58
the medulla~v raphe nuclei and adjacent areas, in the gigantocellular reticular nucleus, including its pars a (Fig. 1B) and in the nucleus paragigantocellularis. Interestingly, in at least one area there was a clear overlap between 'True Blue' positive and enkephalin immunoreactive cells. Thus, a close comparison between photographs taken after the t w o procedures, respectively, revealed that, immediately dorsal to the inferior olive, presumably in the pars a of the gigantocellular reticular nucleus, a group of enkephalin immunoreactive cell bodies had previously been retrogradely 'stained' with 'True Blue' injected into the spinal cord (Fig. 1A,B). We previously suggested that enkephalin immunoreactive fibers in the spinal cord are intrinsic, since neither total transection of the cord, nor dorsal rhizotomy appeared to reduce the number of fibers, while for instance, the same experiments revealed a decrease in substance P immunoreactive fibers [ 13 ]. The conclusion that enkephalin fibers of the spinal cord are mainly intrinsic is therefore still valid and the nerve terminals of descending fibers probably only constitute a small fraction of the total number. It is of course also possible that enkephalin cells in other brain stem areas, e.g., the nucleus raphe magnus give rise to descending projections. This possibility is presently under investigation. Supraspinal descending systems have been implicated in stimulation produced analgesia [1,3,21,23,25,26], possibly via activation o-f enkephalin immunoreactive interneurons ([ 13] ; see also ref. 1). For instance, descending 5-hydroxytryptamine neurons, originally described by DahlstrSm and Fuxe [7], have been considered important (see ref. 25). Since a substance P-like peptide is present in at least a proportion of the 5-HT neurons [ 1 4 ] , this peptide may also be involved in stimulation produced analgesia. Whether the present descending ENK immunoreactive neurons are involved in this p h e n o m e n o n remains to be elucidated. It is interesting that areas adjacent to the nucleus raphe magnus also have been related to stimulation produced analgesia [1,29]. ACKNOWLEDGEMENTS
This work was supported by grants from Karolinska Institutet, the Swedish Medical Research Council (04X-2887; 25X-03766), Magnus Bergvalls Stiftelse, Knut and Alice Wallenbergs Stiftelse and Claes Groschinskys Stiftelse, The skilful technical assistance of Mrs W. Hiort and Miss Ulla Lindefeldt is gratefully acknowledged. REFERENCES 1 Basbaum, A.I. and Fields, H.L., Endogenous pain control mechanisms: review and hypothesis, Ann. Neurol., 4 (1978) 451--462. 2 Bentivoglio, M., Kuypers, H.G.J.M., Catsman-Berrevoets, C.E. and Dann, O., Fluorescent retrograde neuronal labeling in rat by means of substances binding specifically to adenine-thymine rich DNA, Neurosci. Lett., 12 (1979) 235--240.
59 3 Besson, J.M., Dickenson, A.H., Le Bars, D. and Oliveras, J.L., Opiate analgesia: The physiology and pharmacology of spinal pain systems, Advance. Pharmacol. Ther., 5 (1978) 61--81. 4 Bloom, F., Battenberg, E., Rossier, J., Ling, N. and Guillemin, R., Neurons containing 5-endorphin in rat brain exist separately from those containing enkephalin: Immunocytochemical studies, Proc. nat. Acad. Sci. (Wash.), 75 (1978) 1591--1595. 5 Brodal, A., Taber, E. and Walberg, F., The raphe nuclei of the brain stem in the cat. III. Efferent connections, J. comp. Neurol., 114 (1960) 239--259. 6 Coons, A.H., Fluorescent antibody methods. In J.F. Danielli (Ed.), General Cytochemical Methods, Academic Press, New York, pp. 399--422. 7 Dahlstr6m, A. and Fuxe, K., Evidence for the existence of monoamine containing neurones'in the central nervous system: I. Demonstration of monoamines in the cell bodies of brain stem neurons. Acts physiol, scand., (Suppl. 232) 62 (1964) 1--55. 8 Dann, O., Volz, G., Demant, E., Pfeifer, W., Bergen, G., Fick, H. and Walkenhorst, E., Trypanocide Diamidine mit vier Ringen in einem oder zwei Ringssystemen, Liebigs Ann. Chem., (1973) 1112--1140. 9 Elde, R., H6kfelt, T., Johansson, O. and Terenius, L., Immunohistochemical studies using antibodies to leucine-enkephalin: Initial observations on the nervous system of the rat, Neuroscience, 1 (1976) 349--351. 10 HSkfelt, T., Elde, R., Johansson, O., Terenius, L. and Stein, L., The distribution of enkephalin-immunoreactive cell bodies in the rat central nervous system, Neurosci. Lett., 5 (1977) 25--31. 11 H6kfelt, T., Fuxe, K., Goldstein, M. and Joh, T., Immunohistochemical localization of three catecholamine synthesizing enzymes: Aspects on Methodology, Histochemie, 33 (1973) 231--254. 12 HSkfelt, T., Kuypers, H.G.J.M., Bentivoglio, M., Catsman-Berrevoets, C.E., Van der Kooy, D., Dann, O., Philipson, O., Goldstein, M., Steinbusch, H., Verhofstad, A. and Nilsson, G., A combined method for transmitter histochemical identification of retrogradely traced neurons: Descending spinal systems characterized by immunohistochemistry using antisera to catecholamine synthesizing enzymes, serotonin and substance P, Neuroscience, in press. 13 HSkfelt, T., Ljungdahl, ~ . , Terenius, L., Elde, R.P. and Nilsson, G., Immunohistochemical analysis of peptide pathways possibly related to pain and analgesia: Enkephalin and substance P, Proc. nat. Acad. Sci. (Wash.), 74 (1977) 3081--3085. 14 HSkfelt, T., Ljungdahl, .~., Steinbusch, H., Verhofstad, A., Nilsson, G., Brodin, E., Pernow, B. and Goldstein, M., Immunohistochemical evidence of substance P-like immunoreactivity in some 5-hydroxytryptamine-containing neurons in the rat central nervous system. Neuroscience, 3 (1978) 517--538. 15 Hughes, J., Smith, T.W., Kosterlitz, H.W., Fothergill, L.H., Morgan, B.A. and Morris, H.R., Identification of two related pentapeptides from the brain with potent opiate agonist activity, Nature (Lond.), 258 (1975) 577--579. 16 Kristensson, K., Olsson, Y. and Sj6strand, J., Axonal uptake and retrograde transport of exogenous proteins in the hypoglossal nerve, Brain Res., 32 (1971) 399--406. 17 Kuypers, H.G.J.M., Catsman-Berrevoets, C.A. and Padt, R.E., Retrograde axonal transport of fluorescent substances in the rat's forebrain, Neurosci. Lett., 6 (1977) 127-135. 18 Kuypers, H.G.J.M. and Maisky, V.A., Retrograde axonal transport of horseradish peroxidase from spinal cord to brain stem cell groups in the cat, Neurosci. Lett., 1 (1975) 9--14. 19 LaVail, J.H. and LaVail, M., Retrograde axonal transport in the central nervous system, Science, 176 (1972) 1416--1417. 20 Leichnitz, G.R., Watkins, L., Griffin, G., Muffin, R. and Mayer, D.J., The projection from nucleus raphe magnus and other brainstem nuclei to the spinal cord in the rat: a study using the HRP blue-reaction, Neurosci. Lett., 8 (1978) 119--124.
60 21 Liebeskind, J.C., Mayer, D.J. and Akil, H., Central mechanisms of pain inhibition: studies of analgesia from focal brain stimulation. In J.J. Bonica (Ed.), Advances in Neurology, Volume 4, Raven Press, New York, 1974, pp. 261--268. 22 Ljungdahl,/~., H6kfelt, T., Goldstein, M. and Park, D., Retrograde peroxidase tracing of neurons combined with transmitter histochemistry, Brain Res., 84 (1975) 313-319. 23 Mayer, D.J. and Price, D.D., Central nervous system mechanisms of analgesia, Pain, 2 (1976) 379--404. 24 Martin, R.F., Jordan, L.M. and Willis, W.D., Differential projections of cat medullar raphe neurons demonstrated by retrograde labeling following spinal cord lesions, J. comp. Neurol., 182 (1978) 77--88. 25 Messing, R.B. and Lytle, L.D., Serotonin containing neurons: their possible role in pain and analgesia, Pain, 4 (1977) 1--21. 26 Reynolds, D.V., Surgery in the rat during electrical analgesia induced by focal brain stimulation, Science, 164 (1969) 444---445. 27 Sar, M., Stumpf, W.E., Miller, R.J., Chang, K.-J. and Cuatrecasas, P., Immunohistochemical localization of enkephalin in rat brain and spinal cord, J. comp. Neurol., 182 (1978) 17--37. 28 Simantov, R., Kuhar, M.J., Uhl, G.R. and Snyder, S.H., Opioid peptide enkephalin: immunohistochemical mapping in the rat central nervous system, Proc. nat. Acad. Sci. (Wash.), 74 (1977) 2167--2171. 29 Takagi, H., Doi, T. and Kawasaki, K., Effects of morphine, L-dopa and tetrabenazine on the lamina V cells of spinal dorsal horn, Life Sci., 17 (1975) 67. 30 Uhl, G., Goodman, R.R., Kuhar, N.J., Childers, S.R. and Snyder, S.H., Immunohistochemical mapping of enkephalin containing cell bodies, fibers and nerve terminals in the brain stem of the rat, Brain Res., 166 (1979) 75--94. 31 Watson, S.J., Akil~ H., Sullivan, S. and Barchas, J.O., Immunocytochemical localization of methionine enkephalin: preliminary observations, Life Sci., 21 (1977) 733-738. 32 W~nscher, W., Schober, W. and Werner, L., Architektonischer Atlas vom Hirnstamm der Ratte, S. Hirzel, Leipzig, 1965.
55
EVIDENCE F O R ENKEPHALIN IMMUNOREACTIVE N E U R O N S IN THE M E D U L L A O B L O N G A T A P R O J E C T I N G TO THE SPINAL CORD
T. HOKFELT, L. TERENIUS, H.G.J.M. KUYPERS and O. DANN
Department of Histology, Karolinska Institutet, Stockholm, Department of Pharmacology, Uppsala University (Sweden), Department of Anatomy, E ~ m u s University Medical School, Rotterdam (The Netherlands) and Institute of Pharmacy and Food Chemistry, Friedrich Alexander University, Erlangen (F.R.G.) (Received June 7th, 1979) (Accepted June 7th, 1979)
SUMMARY
In the present study a m e t h o d is used, with which retrograde tracing with the fluorescent d y e 'True Blue' is combined with the indirect immunofluorescence technique for visualizing enkephalin-like immunoreactivity. Evidence is presented for the existence of an enkephalin immunoreactive system originating in the lower medulla oblongata and projecting to the spinal cord.
The enkephalin pentapeptides [15] have a wide-spread distribution in the central nervous system. Immunocytochemical methods have shown enkephalin immunoreactive nerve terminals in the central and dorsal horns of the spinal cord and a particular dense network in laminae I and II [4,9,10,13,27, 28,30,31]. In the dorsal horn there are also enkephalin immunoreactive cell bodies [10,13,30]. Previous attempts to elucidate the origin of the enkephalin immunoreactive fibers in the gray matter of the spinal cord. Since no dorsal rhizotomy as well as total transection of the spinal cord. Since no distinct effects could be observed after either operation, we concluded that the fibers are intrinsic to the cord [ 1 3 ] . Quantitative estimations on the basis of immunocytochemistry are, however, difficult to perform. Thus, a decrease in the number of immunoreactive fibers, particularly in the densely innervated laminae I and II, might be difficult to detect. We have therefore reinvestigated this problem utilizing a different approach, b y which retrograde tracing principles [16,19] were combined with immunohistochemistry ([ 12] ; see also ref. 22). For retrograde tracing a fluorescent marker was used [2] and enkephalin-like material was demonstrated with the indirect immunofluorescence technique [6,9,10]. We n o w present evidence that enkephalin immunoreactive cell bodies located in the lower medulla oblongata project to the spinal cord.
56 Male albino rats (Sprague--Dawley; body wt 150 g) were used. Under halothane anaesthesia, 1 pl of a 10% solution of the dye 'True Blue' (code number 150/129; trans-l,2-bis(5.amidino-2-benzofuranyl) ethylene 2 HC1) [ 7 ] in distilled water was injected bilaterally at two levels of lower thoracicupper lumbar spinal cord with a Hamilton syringe [2]. Six days later 25 pl colchicine (concentration 3 pg/ul), dissolved in 0.9% sodium chloride, were injected into the lateral ventricle. After 24 h the rats were perfused with 4% ice cold formaldehyde for 30 min and the brain and spinal cord were removed, immersed in the same fixative for 90 min, rinsed for 24 h in 0.1 M phosphate buffer with 5% sucrose added and cut on a cryostat. Sections from the lower medulla oblongata were examined in a Zeiss fluorescence microscope and fluorescent cells were photographed. After removal of the cover slip, the sections were transferred to a buffer and processed for the indirect technique of Coons and collaborators (see ref. 6) as described previously [11]. Briefly, the sections were incubated at 4°C with antiserum to met-enkephalin diluted 1 : 160 for 16--20 h, rinsed, incubated with fluorescein isothiocyanate-conjugated sheep antirabbit antibodies (SBL, Stockholm, Sweden}, mounted, examined in the same fluorescence microscope and fluorescent cells were photographed. As described in detail in a parallel paper [12], the 'True Blue' fluorescence disappears almost completely after the processing for immunohistochemistry. Thus, the green fluorescent cells and fibers seen after the immunohistochemical procedure represent enkephalin immunoreactive structures. It should also be emphasized that, with the filter combinations used (excitation filter: either Schott B912 or KP500; stopfilter: either Zeiss 50 or LP520), the green-yellow 'True Blue' colour can be distinguished clearly from that of the FITC induced green fluorescence. Seven days after injection of the dye into the spinal cord numerous greenyellow fluorescent cells were observed in the lower medulla oblongata These cells were located in several nuclei, particularly in the midline raphe nuclei, the ventral part of the reticular nucleus and on the gigantocellular reticular nucleus. Labelled cells were also seen in the dorsal aspects of the lateral reticular nucleus and dorsal and lateral to the inferior olive, i.e., in the paragigantocellular nucleus and in the pars a of the gigantocellular reticular nucleus [32]. In the latter area the fluorescent cells were often found in small groups with up to 10 cells per section and side (Fig. 1A}. These results are in general agreement with earlier results on efferent connections of the lower medulla oblongata and t h e spinal cord [1,5,18,20,24]. After labelling for the indirect immunofluorescence technique with enkephalin antiserum, the labelling pattern was markedly different. The fluorescent cells described above were no longer detectable, except for some very weakly yellow fluorescentcells. On the other hand, green fluorescent enkephalin immunoreactive cells were observed in several nuclei with a distribution similar to that described previously [9,27,30], that is, for example, in the superficial layers of the spinal trigeminal nucleus, the nucleus tractus solitarii,
57
Fig. 1. Fluorescence micrographs of the lower medulla oblongata of the same section showing the area immediately dorsal to the inferior olive (IO). In (A) numerous greenyellow fluorescent cell bodies are seen, which have been retrogradely labelled with 'True Blue' after spinal injections of the dye. In (B) the same area has been photographed after processing of the section for indirect immunofluorescence histochemistry using an antiserum to met-enkephalin. A small group of cells (arrows), which in A contains 'True Blue', is in B shown to be enkephalin immunoreactive. Note that a very weak (yellow) fluorescence, clearly different from the FITC induced fluorescence, remains in some cells after processing for immunohistochemistry (arrow heads). Bars indicate 50 ~m.
58
the medulla~v raphe nuclei and adjacent areas, in the gigantocellular reticular nucleus, including its pars a (Fig. 1B) and in the nucleus paragigantocellularis. Interestingly, in at least one area there was a clear overlap between 'True Blue' positive and enkephalin immunoreactive cells. Thus, a close comparison between photographs taken after the t w o procedures, respectively, revealed that, immediately dorsal to the inferior olive, presumably in the pars a of the gigantocellular reticular nucleus, a group of enkephalin immunoreactive cell bodies had previously been retrogradely 'stained' with 'True Blue' injected into the spinal cord (Fig. 1A,B). We previously suggested that enkephalin immunoreactive fibers in the spinal cord are intrinsic, since neither total transection of the cord, nor dorsal rhizotomy appeared to reduce the number of fibers, while for instance, the same experiments revealed a decrease in substance P immunoreactive fibers [ 13 ]. The conclusion that enkephalin fibers of the spinal cord are mainly intrinsic is therefore still valid and the nerve terminals of descending fibers probably only constitute a small fraction of the total number. It is of course also possible that enkephalin cells in other brain stem areas, e.g., the nucleus raphe magnus give rise to descending projections. This possibility is presently under investigation. Supraspinal descending systems have been implicated in stimulation produced analgesia [1,3,21,23,25,26], possibly via activation o-f enkephalin immunoreactive interneurons ([ 13] ; see also ref. 1). For instance, descending 5-hydroxytryptamine neurons, originally described by DahlstrSm and Fuxe [7], have been considered important (see ref. 25). Since a substance P-like peptide is present in at least a proportion of the 5-HT neurons [ 1 4 ] , this peptide may also be involved in stimulation produced analgesia. Whether the present descending ENK immunoreactive neurons are involved in this p h e n o m e n o n remains to be elucidated. It is interesting that areas adjacent to the nucleus raphe magnus also have been related to stimulation produced analgesia [1,29]. ACKNOWLEDGEMENTS
This work was supported by grants from Karolinska Institutet, the Swedish Medical Research Council (04X-2887; 25X-03766), Magnus Bergvalls Stiftelse, Knut and Alice Wallenbergs Stiftelse and Claes Groschinskys Stiftelse, The skilful technical assistance of Mrs W. Hiort and Miss Ulla Lindefeldt is gratefully acknowledged. REFERENCES 1 Basbaum, A.I. and Fields, H.L., Endogenous pain control mechanisms: review and hypothesis, Ann. Neurol., 4 (1978) 451--462. 2 Bentivoglio, M., Kuypers, H.G.J.M., Catsman-Berrevoets, C.E. and Dann, O., Fluorescent retrograde neuronal labeling in rat by means of substances binding specifically to adenine-thymine rich DNA, Neurosci. Lett., 12 (1979) 235--240.
59 3 Besson, J.M., Dickenson, A.H., Le Bars, D. and Oliveras, J.L., Opiate analgesia: The physiology and pharmacology of spinal pain systems, Advance. Pharmacol. Ther., 5 (1978) 61--81. 4 Bloom, F., Battenberg, E., Rossier, J., Ling, N. and Guillemin, R., Neurons containing 5-endorphin in rat brain exist separately from those containing enkephalin: Immunocytochemical studies, Proc. nat. Acad. Sci. (Wash.), 75 (1978) 1591--1595. 5 Brodal, A., Taber, E. and Walberg, F., The raphe nuclei of the brain stem in the cat. III. Efferent connections, J. comp. Neurol., 114 (1960) 239--259. 6 Coons, A.H., Fluorescent antibody methods. In J.F. Danielli (Ed.), General Cytochemical Methods, Academic Press, New York, pp. 399--422. 7 Dahlstr6m, A. and Fuxe, K., Evidence for the existence of monoamine containing neurones'in the central nervous system: I. Demonstration of monoamines in the cell bodies of brain stem neurons. Acts physiol, scand., (Suppl. 232) 62 (1964) 1--55. 8 Dann, O., Volz, G., Demant, E., Pfeifer, W., Bergen, G., Fick, H. and Walkenhorst, E., Trypanocide Diamidine mit vier Ringen in einem oder zwei Ringssystemen, Liebigs Ann. Chem., (1973) 1112--1140. 9 Elde, R., H6kfelt, T., Johansson, O. and Terenius, L., Immunohistochemical studies using antibodies to leucine-enkephalin: Initial observations on the nervous system of the rat, Neuroscience, 1 (1976) 349--351. 10 HSkfelt, T., Elde, R., Johansson, O., Terenius, L. and Stein, L., The distribution of enkephalin-immunoreactive cell bodies in the rat central nervous system, Neurosci. Lett., 5 (1977) 25--31. 11 H6kfelt, T., Fuxe, K., Goldstein, M. and Joh, T., Immunohistochemical localization of three catecholamine synthesizing enzymes: Aspects on Methodology, Histochemie, 33 (1973) 231--254. 12 HSkfelt, T., Kuypers, H.G.J.M., Bentivoglio, M., Catsman-Berrevoets, C.E., Van der Kooy, D., Dann, O., Philipson, O., Goldstein, M., Steinbusch, H., Verhofstad, A. and Nilsson, G., A combined method for transmitter histochemical identification of retrogradely traced neurons: Descending spinal systems characterized by immunohistochemistry using antisera to catecholamine synthesizing enzymes, serotonin and substance P, Neuroscience, in press. 13 HSkfelt, T., Ljungdahl, ~ . , Terenius, L., Elde, R.P. and Nilsson, G., Immunohistochemical analysis of peptide pathways possibly related to pain and analgesia: Enkephalin and substance P, Proc. nat. Acad. Sci. (Wash.), 74 (1977) 3081--3085. 14 HSkfelt, T., Ljungdahl, .~., Steinbusch, H., Verhofstad, A., Nilsson, G., Brodin, E., Pernow, B. and Goldstein, M., Immunohistochemical evidence of substance P-like immunoreactivity in some 5-hydroxytryptamine-containing neurons in the rat central nervous system. Neuroscience, 3 (1978) 517--538. 15 Hughes, J., Smith, T.W., Kosterlitz, H.W., Fothergill, L.H., Morgan, B.A. and Morris, H.R., Identification of two related pentapeptides from the brain with potent opiate agonist activity, Nature (Lond.), 258 (1975) 577--579. 16 Kristensson, K., Olsson, Y. and Sj6strand, J., Axonal uptake and retrograde transport of exogenous proteins in the hypoglossal nerve, Brain Res., 32 (1971) 399--406. 17 Kuypers, H.G.J.M., Catsman-Berrevoets, C.A. and Padt, R.E., Retrograde axonal transport of fluorescent substances in the rat's forebrain, Neurosci. Lett., 6 (1977) 127-135. 18 Kuypers, H.G.J.M. and Maisky, V.A., Retrograde axonal transport of horseradish peroxidase from spinal cord to brain stem cell groups in the cat, Neurosci. Lett., 1 (1975) 9--14. 19 LaVail, J.H. and LaVail, M., Retrograde axonal transport in the central nervous system, Science, 176 (1972) 1416--1417. 20 Leichnitz, G.R., Watkins, L., Griffin, G., Muffin, R. and Mayer, D.J., The projection from nucleus raphe magnus and other brainstem nuclei to the spinal cord in the rat: a study using the HRP blue-reaction, Neurosci. Lett., 8 (1978) 119--124.
60 21 Liebeskind, J.C., Mayer, D.J. and Akil, H., Central mechanisms of pain inhibition: studies of analgesia from focal brain stimulation. In J.J. Bonica (Ed.), Advances in Neurology, Volume 4, Raven Press, New York, 1974, pp. 261--268. 22 Ljungdahl,/~., H6kfelt, T., Goldstein, M. and Park, D., Retrograde peroxidase tracing of neurons combined with transmitter histochemistry, Brain Res., 84 (1975) 313-319. 23 Mayer, D.J. and Price, D.D., Central nervous system mechanisms of analgesia, Pain, 2 (1976) 379--404. 24 Martin, R.F., Jordan, L.M. and Willis, W.D., Differential projections of cat medullar raphe neurons demonstrated by retrograde labeling following spinal cord lesions, J. comp. Neurol., 182 (1978) 77--88. 25 Messing, R.B. and Lytle, L.D., Serotonin containing neurons: their possible role in pain and analgesia, Pain, 4 (1977) 1--21. 26 Reynolds, D.V., Surgery in the rat during electrical analgesia induced by focal brain stimulation, Science, 164 (1969) 444---445. 27 Sar, M., Stumpf, W.E., Miller, R.J., Chang, K.-J. and Cuatrecasas, P., Immunohistochemical localization of enkephalin in rat brain and spinal cord, J. comp. Neurol., 182 (1978) 17--37. 28 Simantov, R., Kuhar, M.J., Uhl, G.R. and Snyder, S.H., Opioid peptide enkephalin: immunohistochemical mapping in the rat central nervous system, Proc. nat. Acad. Sci. (Wash.), 74 (1977) 2167--2171. 29 Takagi, H., Doi, T. and Kawasaki, K., Effects of morphine, L-dopa and tetrabenazine on the lamina V cells of spinal dorsal horn, Life Sci., 17 (1975) 67. 30 Uhl, G., Goodman, R.R., Kuhar, N.J., Childers, S.R. and Snyder, S.H., Immunohistochemical mapping of enkephalin containing cell bodies, fibers and nerve terminals in the brain stem of the rat, Brain Res., 166 (1979) 75--94. 31 Watson, S.J., Akil~ H., Sullivan, S. and Barchas, J.O., Immunocytochemical localization of methionine enkephalin: preliminary observations, Life Sci., 21 (1977) 733-738. 32 W~nscher, W., Schober, W. and Werner, L., Architektonischer Atlas vom Hirnstamm der Ratte, S. Hirzel, Leipzig, 1965.
