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The distribution of enkephalin-immunoreactive cell bodies in the rat central nervous system
Neuroscience Letters, 5 (1977) 25--31
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© Elsevier/North-Holland Scientific Publishers Ltd.
THE DISTRIBUTION OF ENKEPHALIN-IMMUNOREACTIVE CELL BODIES IN THE RAT CENTRAL NERVOUS SYSTEM*
TOMAS HOKFELT, ROBERT ELDE, OLLE JOHANSSON, LARS TERENIUS and LARRY STEIN
Department of Histology, Karolinska Institute, Stockholm, Department of Medical Pharmacology, Uppsala University, Uppsala (Sweden) and Wyeth Laboratories, Philadelphia, Pa. (U.S.A.) (Received March 7th, 1977) (Accepted March 7th, 1977)
SUMMARY
With the indirect immunofluorescence technique the distribution of methionine-enkephalin-immunoreactive cell bodies was studied in the central nervous system of rats pretreated with colchicine. The antiserum used did cross-react to 10% with leucine-enkephalin but to less than 0.1% with a-, ~-, and 7-endorphine. Cell bodies with a specific immunofluorescence were observed in the tel-, di-, mes- and rhombencephalon and in the spinal cord.
Recently two pentapeptides, methionine- and leucine-enkephalin, have been isolated from the central nervous system and identified [ 12]. They form together with other structurally related peptides the endorphines, a group of substances which may represent endogenous ligands for the opiate receptors [6,20,26,29,31]. In an earlier preliminary immunohistochemical study with an antiserum raised to Leu-enkephalin the gross distribution of nerve terminals containing enkephe2in-like immunoreactivity in the brain, spinal cord and intestinal wall of the rat was outlined [5]. However, no cel! bodies reacted [5]. In the present study rats were pretreated with colchicine, a mitosis inhibitor, known to arrest inti'aaxonal transport (see ref. 3). This experimental procedure has previously been used to increase cell body levels of catecholamines [ 3,7] and peptides [ 1,1(}]. Antiserum to Met-enkephalin was raised in rabbits as described elsewhere [ 5]. Characterization of the antiserum revealed about 10% cross-reaction with the Leu-enkephalin and less than 0.1% with ~-, ~-, and 7-endorphine, substance P and somatostatin. Because of these characteristics, and since Met-enkephalin levels are about 3 times Leu-enkephalin levels in rat brain [ 27], we denote the *Part of these results have been communicated at the ARNMD meeting, New York, December, 1976 [7 ] and the ACNP meeting, New Orleans, December 1976 [8 ].
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immunoreactive material Met-enkephalin-like immunoreactivity. Male albino rats (Sprague-Dawley, body weight 150 g) were used. Untreated rats, as well as rats receiving an intraventricular or intracisternal injection of colchicine (10 or 25 ul at 1 ~g/pl) 48 h before sacrifice were studied. Some rats received intraspinal injections (2 ~1 at 10 ~g/pl) at the level of the lumbar and sacral cord and were sacrificed 24 h after the injection. The rats were anaesthetized and perfused via the ascending aorta with ice-cold 4% formaldehyde in 0.1 M phosphate buffer [21] for 30 min. After dissection, the brain and spinal cord were immersed in the same fixative for 90 min, rinsed in 0.1 M phosphate buffer with sucrose added for at least 24 h and cut on a cryostat (Dittes, Heidelberg). The sections were incubated for 30 min in a humid atmosphere at +37°C with the antiserum diluted 1 : 10, rinsed with phosphate-buffered saline (PBS), further incubated as described above with fluorescein isothiocyanate conjugated antibodies (Statens Bakteriologiska Laboratorium, Stockholm, Sweden), diluted 1:4, rinsed in PBS, mounted in a glycerine :PBS (3 : 1 ~ mixture and finally examined in a Zeiss fluorescence microscope. For control of the specificity of the immunoreaction a consecutive section at each level was incubated with antiserum blocked by Metenkephalin (50 ~g/ml antiserum, diluted 1:10). The anatomical nomenclature is according to Jacobowitz and Palkovits [ 13] and Palkovits and Jacobowitz [ 19]. Met-enkephalin-immunoreactive cell bodies {Figs. 1A---C, 2A) o~ varying size (ranging between 15 and 40 pm) and numbers and with a varying intensity of the immunofluorescence were observed in the tel-, di-, mes-, and rhombencephalon and in the spinal cord. Mostly they were present in more or less well-defined groups. They almost always made up only a minor fraction of the total number of cell bodies within the various nuclei. None of the areas described below contained immunofluorescent cell bodies after incubation with control serum (Figs. 1D, 2B). Immunoreactive cell bodies were observed in the caudate nucleus, the caudal ventromedial part of the lateral septal nucleus, the nucleus interstitialis striae terminalis (lateral part), the nucleus preopticus periventricularis, the nucleus preopticus medialis, the paraventricular nucleus, the perifornical area (Fig. 1A), the nucleus arcuatus, the ventromedial nucleus, the nucleus prernami!laris dorsalis and ventralis extending laterally into the medial forebrain bundle, the mesencephalic periaqueductal central grey (ventrolateral parts), in several layers of the superior colliculus, in the most dorsolateral parts of the substantia nigra, within the lemniscus lateralis, the nuclei parabrachialis
Fig. 1. Immunofluorescence micrographs of the hypothalamic perifornical area (A), the nucleus raphe magnus of the medulla oblongata (B--D) after incubation with antiserum to Met-enkephalin (A--C) or control serum (D). Numerous strongly fluorescent cell bodies are seen around the fornix, F (A) as well as in the raphe magnus nucleus (B and C). Note fluorescent nerve terminals in the latter nucleus (arrows in C). No fluorescent cell bodies or terminals are seen after incubation with control serum (D). P = pyramidal tract. Magnification x 120 (A and B) and × 300 (C and D).
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Fig. 2. Immunofluorescence micrographs of spinal cord (sacral level) after incubation with antiserum to Met-enkephalin (A) or control serum (B). Several cell bodies (arrows) are seen dorsolaterally of the central canal (asterisk) (A). No specific immunoreactive structures are observed after incubation with control serum (B). Arrowheads in B point to unspecific fluorescence. DF = dorsal funiculus, x 120.
ventralis and dorsalis, the nucleus vestibularis medialis, the nucleus raphe magnus (Fig. 1B, C) extending laterally over the tractus corticospinalis, the nucleus cochlearis dorsalis, the ventromedial parts of the nucleus reticularis gigantocellularis and ventrally of this nucleus, the nucleus tractus solitarii, the nucleus raphe pallidus, the nucleus reticularis paramedianus, the marginal layer of the nucleus tractus spinalis nervi trigemini, the substantia gelatinosa trigemini and in the dorsal horn of the spinal cord (Fig. 2A). It must be emphasized that there may be still more enkephalin-immunoreactive cells in the rat nervous system than described in this preliminary study. For instance, intraventricularly or intracistemally administered co!chicine may not reach all areas of the brain equally well. Many cells therefore 'escape' the axonal transport-inhibiting effect of this drug and thus remain 'undetectable'. The marked variations in the intensity of the immunoreaction between various enkephalin-positive cell body groups may indicate that they have been affected differently by the colchicine treatment. Other
29 explanations are also possible such as differences in rates of synthesis or of transport of the peptide. In a previous preliminary study, extensive networks of enkephalinimmunoreactive nerve terminals were observed in the central nervous system and in the intestinal wall of the rat [ 5]. In that study an antiserum raised to Leu-enkephalin was used. However, subsequent characterization (Terenius, unpublished) revealed that the Leu-enkephalin antibodies cross-react with Metenkephalin to about 20% (but to less than 0.1% .with a-,/]-, and 7-endorphin, substance P and somatostatin). In view of the facts that (a) there is considerably more Met- than Leu-enkephalin in the rat brain [ 27] and (b) our present antiserum to Met-enkephalin (which cross-reacts to 10% with Leu-enkephalin) reveals an immunofluorescence distribution pattern similar to the °he found with the Leu-enkephalin antiserum (to be published), the possibility has to be considered that the nerve terminals previously described [ 4] as well as the cell bodies observed in this paper mainly contain Met-enkephalin. Simantov et al. [25] have recently used antisera to Leu- and Met-enkephalin in immunohistochemical studies. They described distribution patterns for nerve terminals similar to those observed by us [ 5]. No remarks on a differential distribution of Leu- versus Met-enkephalin were made. Clearly, a detailed analysis of 'staining' patterns of Met- and Leu-enkephalin antisera is needed. The use of colchicine treatment of the rats permitted the detection of enkephalin-positive cell bodies which was not possible previously in untreated rats [ 5]. Colchicine has been used in many earlier studies to increase cell body levels of putative transmitters [3], enzymes [ 14] and peptides [ 1,10]. It is assumed that it arrests a.xonal (and also intrasomal) transport by an action on microtubules [3,23]. Since synthesis appears to be unaffected, accumulation of substances produced in the cell bodies will occur. The observation of enkephalin-positive staining in the cell bodies indicates that they possess all the necessary synthetic pathways. Whether biosynthesis is confined to cell bodies will require further in~,estigation. Met-enkephalin-immunoreactive cell bodies were observed in many parts of the central nervous system although so far not in the cerebral and cerebellar cortices. They constitute mostly rather small populations of the cell bodies within the different brain nuclei. The number of enkephalin cell groups appears to be rather large, at the present time exceeding 20. It should be remembered that there are about 15 catecholamine cell groups in the rat brain [4]. The projections of the enkephalin-immunoreactive cells are unknown. It may, however, be mentioned that Met-enkephalin-positive nerve terminals in the spinal cord [5], including the dorsal horn, do not disappear after a total transection of the cord or dorsal rhizotomy [9]. As shown in this study Met. enkephalin-positive cell bodies are present in the dorsal horn of the spinal cord. Enkephalin neurones here~ and perhaps in other areas, may therefore represent local intemeurones. The possible involvement of such hypothetical enkephalin intemeurones in the phenomenon of morphine-induced and stimulation-induced analgesia [16---I8,22 ] will be discussed in a separate paper [11 ].
30
It must, however, be pointed o u t t h a t the a p p a r e n t l y very wide distribution o f t h e enkephalin systems indicate functional roles in the nervous system b e y o n d those related to pain and analgesia. One e x a m p l e of this is t h e release o f prolactin p r o d u c e d b y enkephalin [15]. The existence of n u m e r o u s enkephalin nerve endings i n the external layer o f t h e median em inence [ 8 ] and n u m e r o u s h y p o t h a l a m i c enkephalin-immunoreactive cell bodies indicates t h a t there m a y exist t u b ero i n fu n d i b u lar [ 28] enkephalin neurones involved in the h y p o t h a l a m i c control of pituitary h o r m o n e secretion.
ACKNOWLEDGEMENTS This work was supported by grants from the Swedish Medical Research Council (04X-2887; 25X-5063), Magnus BergvaUs Stiftelse, Sven och EbbaChristina Hagbergs Stiftelse, Harald Jeanssons Stiftelse and Harald och Greta Jeanssons Stiftelse. The skilful technical assistance o f Miss A. N y g ~ d is gratefully acknowledged. REFERENCES 1 Barry, J., Dubois, M.P. and Poulain, P., LRF producing cells of the mammalian hypothalamus. A fluorescent antibody study, Z. Zellforsch., 146 (1973) 351--366. 2 Coons, A.H., Fluorescent antibody methods. In J.F. Danielli (Ed.), General Cytochemical Methods, Academic Press, New York, 1958, pp. 399--422. 3 DahlstrSm, A., The effects of drugs on axonal transport of amine storage granules. In H.J. Schiimann and G. Kroneberg (Eds.), New Aspects of Storage and Release Mechanisms of Catecholamines, Bayer Symposium II, Springer, Berlin~ 1970, pp. 20--26. 4 Dahlstr6m, A. and Fuxe, K., Evidence for the existence of monoamine containing neurons in the central nervous system. I. Demonstration of monoamines in the cell bodies of brain stem neuron, Acta physiol, scand., 62, Suppl. 232 (1964) 1--55. 5 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. 6 Guillemin, R., Ling, N, et Burgus, R., Endorphines, peptides, d'origine hypothalamique et neurohypophysaire fi activit~ morphinomim~tique. Isolement et structure mol~cuhire de l'~-endorphine, C.R. Acad. Sci. (Paris), D, 282 (1976) 783--785. 7 H~kfelt, T. and DahlstrSm, A., Effects of two mitosis inhibitors (colchicine and vinblastine) on the distribution and axonal transport of noradrenaline storage particles, studied by fluorescence and electron microscopy, Z. Zellforsch., 119 (1971) 460--482. 8 HSkfelt, T., Elde, R., Fuxe, K., Johansson, O., Ljungdahl, A., Goldstein, M., Luft, R., Efendi~, S., Nilsson, G., Terenius, L., Ganten, D., Jeffcoate, S.L., Rehfeld, J., Said, S., Perez de la Mora~ M., Possani, L., Tapia, R., Teran, L. and Palacios, R., Aminergic and peptidergic pathways in the nervous system with special reference to the hypothalamus. In S. Reichlin and ]~. Baldessarini (Eds.), The Hypothalamus, Raven Press, New York, in press. 9 HSkfelt, T,, Elde, R,, Johansson, O., Ljungdahl, A., Schultzberg, M., Fuxe, K., Goldstein, M., Nilsson, G., Pernow, B., Terenius, L., Ganten, D., Jeffcoate, S.L., Rehfeld, J. and Said, S., The distribution of peptide containing neurons in the nervous system, In M. Lipton, G. Aghajanian and F. Bloom (Eds.), American College of Neuropsychopharmacology, Raven Press, New York, in press.
31 10 HSkfelt, T., Kellerth, J.-O., Nilsson, G. and Pernow, B., Experimental immunohistochemical studies on the localization and distribution of substance P in cat primary sensory neurons, Brain Res., 100 (1975) 235--252. 11 HSkfe~t, T., Ljungdahl, A., Elde, R.~ Nilsson, G. and Terenius, L., Histochemical studies on central nervous system regions possibly related to stimulation produced analgesia: distrib~tion of enkephalin- and substance P-immunoreactive neurons, in preparation. 12 Hughes, J., Smith, T.W., Kosterlitz, H.W., Fothergill, L.A., 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. 13 Jacobowitz, D. and Palkovits, M., Topographic atlas of catecholamine and acetylcholinesterase-containing neurons in the rat brain. I. Forebrain (telencephalon, diencephalon), J. comp. Neurol., 157 (1974) 13--28. 14 Kreutzberg, G., Neuronal dynamics and flow. IV. Blockage of intra-axonal enzyme transport by colchicine, Proc. nat. Acad. Sci. (Wash.), 62 (1969), 722--728. 15 Lien, E.Lo, Fenschel, R.L., Garsky, V., Sarantakis, D. and Grant, N.H., Enkephalinstimulated prolactin release, Life Sci., 19 (1976) 837--840. 16 Mayer, D. and Price, D., Central nervous system mechanisms of analgesia, Pain, 2 (1976) 379--404. 17 Mayer, D.J., Wolfle, T.L., Akil, H., Carder, B. and Liebeskind, J.C., Analgesia from electrical stimulation in the brainstem of the rat, Science, 1974 (1971 ) 13 51--1354. 18 Oliveras, J.L., Besson, J.M., Guilbaud, G. and Liebeskind, J.C., Behavioral and electrophysiological evidence of pain inhibition from midbrain stimulation in the cat, Exp. Brain Res., 20 (1974) 32--44. 19 Palkovits, M. and Jacobowitz, D., Topographic atlas of catecholamine and acetylcholinesterase-containing neurons in the rat brain. II. Hindbrain (mesencephalon, rhombencephalon), J. comp. Neurol., 157 (1974) 29--42. 20 Pasternak, G.E., Goodman, R. and Snyder, S.H., An endogenous morphine-like factor in mammalian brain, Life Sci., 16 (1975) 1765--1769. 21 Pease, D.C., Buffered formaldehyde as a killing agent and primary fixative for electron microscopy, Anat. Res., 142 (1962) 342. 22 Reynolds, D.V., Surgery in the rat during electrical analgesia fLnduced by focal brain stimulation, Science, 164 (1969)444--445. 23 Schmitt, F.O., The molecular biology of neural fibrous proteins, Neurosci. Res. Progr. Bull., 6 (1968) 119--144. 24 Simantov, R., Kuhar, M.J., Pasternak, G.W. and Snyder, S.H., The regional distributiow of morphine-like factor enkephalin in monkey brain, Brain Res., 106 (1976) 189--197. 25 Simantov, R., Kuhar, M.J., D'hl, G.R. and Snyder, S.H., Opioid peptide enkephalin: immunohistochemical mapping in the rat central nervous system. Proc. nat. Acad. Sci. (Wash.), in press. 26 Simantov, R. and Snyder, S., Isolation and structure identification of a morphine-like peptide 'enkephalin' in bovine brain, Life Sci., 18 (1976) 781--788. 27 Smith, T.W., Hughes, J., Kosterlitz, H.W. and Sosa, R.P., Enkephalins: isolation, distribution and function. In H.W. Kosterlitz (Ed.), Opiates and Endogenous Opioid Peptides, North-Holland, Amsterdam, 1977, pp. 57--62. 28 Szentagothai, J., Flerkb, B.~ Mess, B. and Hal~sz, B., Hypothalamic Control of the Anterior Pituitary, Akademia Kiadb, Budapest, 1962. 29 Terenius, L. and WahlstrSm, A., Inhibitor(s) of narcotic receptor binding in brain extracts and cerebrospinal fluid, Acta pharmacol. (Kbh.), 35, Suppl. I (1974 ) 55. 30 Terenius, L. and WahlstrSm, A., Search for an endogenous ligand for the opiate receptor, Acta physiol, scand., 94 (1975) 74--81. 31 Teschemacher, H., Opheim, K.E., Cox, B.M. and Goldstein, A., A peptide-~ike substance from pituitary that acts like morphine. 1. Isolation, Life Sci., 16 (1975) 1771--1776.
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© Elsevier/North-Holland Scientific Publishers Ltd.
THE DISTRIBUTION OF ENKEPHALIN-IMMUNOREACTIVE CELL BODIES IN THE RAT CENTRAL NERVOUS SYSTEM*
TOMAS HOKFELT, ROBERT ELDE, OLLE JOHANSSON, LARS TERENIUS and LARRY STEIN
Department of Histology, Karolinska Institute, Stockholm, Department of Medical Pharmacology, Uppsala University, Uppsala (Sweden) and Wyeth Laboratories, Philadelphia, Pa. (U.S.A.) (Received March 7th, 1977) (Accepted March 7th, 1977)
SUMMARY
With the indirect immunofluorescence technique the distribution of methionine-enkephalin-immunoreactive cell bodies was studied in the central nervous system of rats pretreated with colchicine. The antiserum used did cross-react to 10% with leucine-enkephalin but to less than 0.1% with a-, ~-, and 7-endorphine. Cell bodies with a specific immunofluorescence were observed in the tel-, di-, mes- and rhombencephalon and in the spinal cord.
Recently two pentapeptides, methionine- and leucine-enkephalin, have been isolated from the central nervous system and identified [ 12]. They form together with other structurally related peptides the endorphines, a group of substances which may represent endogenous ligands for the opiate receptors [6,20,26,29,31]. In an earlier preliminary immunohistochemical study with an antiserum raised to Leu-enkephalin the gross distribution of nerve terminals containing enkephe2in-like immunoreactivity in the brain, spinal cord and intestinal wall of the rat was outlined [5]. However, no cel! bodies reacted [5]. In the present study rats were pretreated with colchicine, a mitosis inhibitor, known to arrest inti'aaxonal transport (see ref. 3). This experimental procedure has previously been used to increase cell body levels of catecholamines [ 3,7] and peptides [ 1,1(}]. Antiserum to Met-enkephalin was raised in rabbits as described elsewhere [ 5]. Characterization of the antiserum revealed about 10% cross-reaction with the Leu-enkephalin and less than 0.1% with ~-, ~-, and 7-endorphine, substance P and somatostatin. Because of these characteristics, and since Met-enkephalin levels are about 3 times Leu-enkephalin levels in rat brain [ 27], we denote the *Part of these results have been communicated at the ARNMD meeting, New York, December, 1976 [7 ] and the ACNP meeting, New Orleans, December 1976 [8 ].
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immunoreactive material Met-enkephalin-like immunoreactivity. Male albino rats (Sprague-Dawley, body weight 150 g) were used. Untreated rats, as well as rats receiving an intraventricular or intracisternal injection of colchicine (10 or 25 ul at 1 ~g/pl) 48 h before sacrifice were studied. Some rats received intraspinal injections (2 ~1 at 10 ~g/pl) at the level of the lumbar and sacral cord and were sacrificed 24 h after the injection. The rats were anaesthetized and perfused via the ascending aorta with ice-cold 4% formaldehyde in 0.1 M phosphate buffer [21] for 30 min. After dissection, the brain and spinal cord were immersed in the same fixative for 90 min, rinsed in 0.1 M phosphate buffer with sucrose added for at least 24 h and cut on a cryostat (Dittes, Heidelberg). The sections were incubated for 30 min in a humid atmosphere at +37°C with the antiserum diluted 1 : 10, rinsed with phosphate-buffered saline (PBS), further incubated as described above with fluorescein isothiocyanate conjugated antibodies (Statens Bakteriologiska Laboratorium, Stockholm, Sweden), diluted 1:4, rinsed in PBS, mounted in a glycerine :PBS (3 : 1 ~ mixture and finally examined in a Zeiss fluorescence microscope. For control of the specificity of the immunoreaction a consecutive section at each level was incubated with antiserum blocked by Metenkephalin (50 ~g/ml antiserum, diluted 1:10). The anatomical nomenclature is according to Jacobowitz and Palkovits [ 13] and Palkovits and Jacobowitz [ 19]. Met-enkephalin-immunoreactive cell bodies {Figs. 1A---C, 2A) o~ varying size (ranging between 15 and 40 pm) and numbers and with a varying intensity of the immunofluorescence were observed in the tel-, di-, mes-, and rhombencephalon and in the spinal cord. Mostly they were present in more or less well-defined groups. They almost always made up only a minor fraction of the total number of cell bodies within the various nuclei. None of the areas described below contained immunofluorescent cell bodies after incubation with control serum (Figs. 1D, 2B). Immunoreactive cell bodies were observed in the caudate nucleus, the caudal ventromedial part of the lateral septal nucleus, the nucleus interstitialis striae terminalis (lateral part), the nucleus preopticus periventricularis, the nucleus preopticus medialis, the paraventricular nucleus, the perifornical area (Fig. 1A), the nucleus arcuatus, the ventromedial nucleus, the nucleus prernami!laris dorsalis and ventralis extending laterally into the medial forebrain bundle, the mesencephalic periaqueductal central grey (ventrolateral parts), in several layers of the superior colliculus, in the most dorsolateral parts of the substantia nigra, within the lemniscus lateralis, the nuclei parabrachialis
Fig. 1. Immunofluorescence micrographs of the hypothalamic perifornical area (A), the nucleus raphe magnus of the medulla oblongata (B--D) after incubation with antiserum to Met-enkephalin (A--C) or control serum (D). Numerous strongly fluorescent cell bodies are seen around the fornix, F (A) as well as in the raphe magnus nucleus (B and C). Note fluorescent nerve terminals in the latter nucleus (arrows in C). No fluorescent cell bodies or terminals are seen after incubation with control serum (D). P = pyramidal tract. Magnification x 120 (A and B) and × 300 (C and D).
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Fig. 2. Immunofluorescence micrographs of spinal cord (sacral level) after incubation with antiserum to Met-enkephalin (A) or control serum (B). Several cell bodies (arrows) are seen dorsolaterally of the central canal (asterisk) (A). No specific immunoreactive structures are observed after incubation with control serum (B). Arrowheads in B point to unspecific fluorescence. DF = dorsal funiculus, x 120.
ventralis and dorsalis, the nucleus vestibularis medialis, the nucleus raphe magnus (Fig. 1B, C) extending laterally over the tractus corticospinalis, the nucleus cochlearis dorsalis, the ventromedial parts of the nucleus reticularis gigantocellularis and ventrally of this nucleus, the nucleus tractus solitarii, the nucleus raphe pallidus, the nucleus reticularis paramedianus, the marginal layer of the nucleus tractus spinalis nervi trigemini, the substantia gelatinosa trigemini and in the dorsal horn of the spinal cord (Fig. 2A). It must be emphasized that there may be still more enkephalin-immunoreactive cells in the rat nervous system than described in this preliminary study. For instance, intraventricularly or intracistemally administered co!chicine may not reach all areas of the brain equally well. Many cells therefore 'escape' the axonal transport-inhibiting effect of this drug and thus remain 'undetectable'. The marked variations in the intensity of the immunoreaction between various enkephalin-positive cell body groups may indicate that they have been affected differently by the colchicine treatment. Other
29 explanations are also possible such as differences in rates of synthesis or of transport of the peptide. In a previous preliminary study, extensive networks of enkephalinimmunoreactive nerve terminals were observed in the central nervous system and in the intestinal wall of the rat [ 5]. In that study an antiserum raised to Leu-enkephalin was used. However, subsequent characterization (Terenius, unpublished) revealed that the Leu-enkephalin antibodies cross-react with Metenkephalin to about 20% (but to less than 0.1% .with a-,/]-, and 7-endorphin, substance P and somatostatin). In view of the facts that (a) there is considerably more Met- than Leu-enkephalin in the rat brain [ 27] and (b) our present antiserum to Met-enkephalin (which cross-reacts to 10% with Leu-enkephalin) reveals an immunofluorescence distribution pattern similar to the °he found with the Leu-enkephalin antiserum (to be published), the possibility has to be considered that the nerve terminals previously described [ 4] as well as the cell bodies observed in this paper mainly contain Met-enkephalin. Simantov et al. [25] have recently used antisera to Leu- and Met-enkephalin in immunohistochemical studies. They described distribution patterns for nerve terminals similar to those observed by us [ 5]. No remarks on a differential distribution of Leu- versus Met-enkephalin were made. Clearly, a detailed analysis of 'staining' patterns of Met- and Leu-enkephalin antisera is needed. The use of colchicine treatment of the rats permitted the detection of enkephalin-positive cell bodies which was not possible previously in untreated rats [ 5]. Colchicine has been used in many earlier studies to increase cell body levels of putative transmitters [3], enzymes [ 14] and peptides [ 1,10]. It is assumed that it arrests a.xonal (and also intrasomal) transport by an action on microtubules [3,23]. Since synthesis appears to be unaffected, accumulation of substances produced in the cell bodies will occur. The observation of enkephalin-positive staining in the cell bodies indicates that they possess all the necessary synthetic pathways. Whether biosynthesis is confined to cell bodies will require further in~,estigation. Met-enkephalin-immunoreactive cell bodies were observed in many parts of the central nervous system although so far not in the cerebral and cerebellar cortices. They constitute mostly rather small populations of the cell bodies within the different brain nuclei. The number of enkephalin cell groups appears to be rather large, at the present time exceeding 20. It should be remembered that there are about 15 catecholamine cell groups in the rat brain [4]. The projections of the enkephalin-immunoreactive cells are unknown. It may, however, be mentioned that Met-enkephalin-positive nerve terminals in the spinal cord [5], including the dorsal horn, do not disappear after a total transection of the cord or dorsal rhizotomy [9]. As shown in this study Met. enkephalin-positive cell bodies are present in the dorsal horn of the spinal cord. Enkephalin neurones here~ and perhaps in other areas, may therefore represent local intemeurones. The possible involvement of such hypothetical enkephalin intemeurones in the phenomenon of morphine-induced and stimulation-induced analgesia [16---I8,22 ] will be discussed in a separate paper [11 ].
30
It must, however, be pointed o u t t h a t the a p p a r e n t l y very wide distribution o f t h e enkephalin systems indicate functional roles in the nervous system b e y o n d those related to pain and analgesia. One e x a m p l e of this is t h e release o f prolactin p r o d u c e d b y enkephalin [15]. The existence of n u m e r o u s enkephalin nerve endings i n the external layer o f t h e median em inence [ 8 ] and n u m e r o u s h y p o t h a l a m i c enkephalin-immunoreactive cell bodies indicates t h a t there m a y exist t u b ero i n fu n d i b u lar [ 28] enkephalin neurones involved in the h y p o t h a l a m i c control of pituitary h o r m o n e secretion.
ACKNOWLEDGEMENTS This work was supported by grants from the Swedish Medical Research Council (04X-2887; 25X-5063), Magnus BergvaUs Stiftelse, Sven och EbbaChristina Hagbergs Stiftelse, Harald Jeanssons Stiftelse and Harald och Greta Jeanssons Stiftelse. The skilful technical assistance o f Miss A. N y g ~ d is gratefully acknowledged. REFERENCES 1 Barry, J., Dubois, M.P. and Poulain, P., LRF producing cells of the mammalian hypothalamus. A fluorescent antibody study, Z. Zellforsch., 146 (1973) 351--366. 2 Coons, A.H., Fluorescent antibody methods. In J.F. Danielli (Ed.), General Cytochemical Methods, Academic Press, New York, 1958, pp. 399--422. 3 DahlstrSm, A., The effects of drugs on axonal transport of amine storage granules. In H.J. Schiimann and G. Kroneberg (Eds.), New Aspects of Storage and Release Mechanisms of Catecholamines, Bayer Symposium II, Springer, Berlin~ 1970, pp. 20--26. 4 Dahlstr6m, A. and Fuxe, K., Evidence for the existence of monoamine containing neurons in the central nervous system. I. Demonstration of monoamines in the cell bodies of brain stem neuron, Acta physiol, scand., 62, Suppl. 232 (1964) 1--55. 5 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. 6 Guillemin, R., Ling, N, et Burgus, R., Endorphines, peptides, d'origine hypothalamique et neurohypophysaire fi activit~ morphinomim~tique. Isolement et structure mol~cuhire de l'~-endorphine, C.R. Acad. Sci. (Paris), D, 282 (1976) 783--785. 7 H~kfelt, T. and DahlstrSm, A., Effects of two mitosis inhibitors (colchicine and vinblastine) on the distribution and axonal transport of noradrenaline storage particles, studied by fluorescence and electron microscopy, Z. Zellforsch., 119 (1971) 460--482. 8 HSkfelt, T., Elde, R., Fuxe, K., Johansson, O., Ljungdahl, A., Goldstein, M., Luft, R., Efendi~, S., Nilsson, G., Terenius, L., Ganten, D., Jeffcoate, S.L., Rehfeld, J., Said, S., Perez de la Mora~ M., Possani, L., Tapia, R., Teran, L. and Palacios, R., Aminergic and peptidergic pathways in the nervous system with special reference to the hypothalamus. In S. Reichlin and ]~. Baldessarini (Eds.), The Hypothalamus, Raven Press, New York, in press. 9 HSkfelt, T,, Elde, R,, Johansson, O., Ljungdahl, A., Schultzberg, M., Fuxe, K., Goldstein, M., Nilsson, G., Pernow, B., Terenius, L., Ganten, D., Jeffcoate, S.L., Rehfeld, J. and Said, S., The distribution of peptide containing neurons in the nervous system, In M. Lipton, G. Aghajanian and F. Bloom (Eds.), American College of Neuropsychopharmacology, Raven Press, New York, in press.
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