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Localization of enkephalinase mRNA in rat brain by in situ hybridization: Comparison with immunohistochemical localization of the protein
Neuropeptides(1989) 14,7%83 0 Longman Group UK Ltd 1989
0143-4179/89/0014-0077/$10.00
Localization of Enkephalinase mRNA in Rat Brain by In Situ Hybridization: Comparison with lmmunohistochemical Localization of the Protein J. N. WILCOX, H. POLLARD*, J. MOREAU*, J. C. SCHWARTZ* and B. MALFROYt Departments of Molecular Biology and tPharmacological Sciences, Genetech, inc., 460 Point San Bruno Blvd., South San Francisco, CA 94080, USA. *Unitt+ de Neurobiologie et Pharmacologic (U. 109) DE L ‘INSERM, Centre Paul Broca, 2ter rue d’Al&ia, 75014 Paris, France. (Correspondence to BM)
Abstract-The messenger RNA (mRNA) encoding enkephalinase (EC. 3.4.24.11; neutral endopeptidase) has been localized in rat brain by in situ hybridization using 35S- or 32Plabelled cRNA probes. Hybridization was observed only in few brain areas, and was particularly strong in the striatum, olfactory bulb and pontine nuclei. The enkephalinase protein was also localized in brain sections using a radiolabelled monoclonal antibody. While some brain regions contained both the mRNA and its translation product, others, including in particular the substantia nigra, were rich in enkephalinase but did not contain any detectable amount of enkephalinase mRNA. Enkephalinase mRNA-containing cells could be identified in regions containing neurons known to project to the substantia nigra. The discrepancy between the mRNA and the protein labelling is likely to reflect the fact that the mRNA is exclusively located within the soma of the cells while the translated protein may be found anywhere along the axonal processes.
Introduction
elicit a variety of opioid-like, naloxone-reversible responses. In addition, the tripeptide Tyr-GlyGly, a characteristic enkephalin metabolite formed under the action of enkephalinase, was recently identified in brain, further indicating the role of this enzyme in the degradation of endogenous enkephalins (6,7). Enkephalinase, like most peptidases, displays a limited substrate specificity and its functional role is, therefore, defined not only by enzymological but also by topographical considerations. A precise localization of the peptidase in the CNS
Enkephalinase (EC 3.4.24.11, neutral endopeptidase) is a membrane-bound metallopeptidase involved in the inactivation of endogenous enkephalins and, possibly, other neuropeptides (reviewed in l-5). Its role in the inactivation of enkephalins was shown by the effects of inhibitors which protect, at least partially, the endogenous opioid pentapeptides from degradation and thus Date received 3 January 1989 Date accepted 9 March 1989 77
78
NEUROPEPTIDES
has been obtained by immunohistochemical studies using monoclonal antibodies (8-11) and by autoradiography technique using 3H-labelled inhibitors (10, 12, 13). Such studies have shown that the enzyme is mainly localized within brain areas enriched in enkephalins, which reinforces the idea that a major function of this enzyme in brain is to inactivate enkephalins. The recent cloning of the enkephalinase gene (14-16) has made possible the visualization of the mRNA by in situ hybridization. We now report results from such initial studies in which the mRNA and the translated protein were visualized at the cellular level using 32P- or 35S-labelled cRNA probes and a highly specific ‘251-labelled monoclonal antibody (mAb) (lo), respectively.
Materials and Methods Radioimmunohistochemical phalinase
visualization of enke-
Monoclonal antibody The monoclonal antibody (MAb 85 AZ), prepared by immunizing mice with tubular cells from rabbit renal cortex (17) was kindly supplied by Dr. P. Verroust and Dr. P. Ronco (Unite de NephrolU.64, de et pathologique, ogie normale I’INSERM, Paris, France). Its specificity was established by immunoprecipitation experiments (18). The monoclonal antibody was iodinated at a specific activity of 8uCi/mg using chloramine T (19) and [‘*‘I]INa (2OOOCilmmo1, Amersham). Tissue preparation
Male Wistar rats (180-200g) were killed, the brain rapidly removed, dropped into cold freon (-40°C monochlorodifluoro methane, Prestogaz) for a few minutes and kept frozen at -20°C until use. Frontal frozen sections (10-20).r,m thick) were cut on a cryostat (Ames) at -18”C, thaw-mounted onto gelatin-coated glass slides and stored at -20°C until used. Autoradiographic localization with [‘25Z]mAb 85A2
of enkephalinase
The sections were warmed to room temperature and dried for 30 min prior to incubation. They
were then incubated for 3 h at 20°C with 100 ~1 of [‘251]mAb 85 A2 (5 x lo5 CPM/ml) dissolved in O.lM phosphate buffer saline (PBS) pH 7.2-7.4 containing 0.2% gelatin. Finally the slides were rinsed 4 times 2 min in PBS, dipped into distilled water to remove any salts remaining, immediately dried and apposed to 3H hyperfilms (Amersham) in X-ray cassettes. These films were developed l-3 days later. Localization of enkephalinase hybridization
mRNA
by in situ
cRNA probe
The probe used was a cRNA corresponding to the full length sequence of the rat enkephalinase mRNA (16), subcloned into SP64 recombinant plasmid and labelled with [32P]CTP or [35S]UTP (20). Tissue preparation
The brain was removed and immersed in freshly prepared 4% paraformaldehyde in O.lM sodium phosphate (pH 7.4). Tissues were fixed at 4°C for 3 hrs and then immersed in 15% sucrose-PBS overnight at 4°C to act as a cryoprotectant. Tissues were then embedded in OCT blocks and stored at -70°C. There was no loss of mRNA available for hybridization during this time. Tissues were sectioned at lO).rm thickness using a cryostat, thaw-mounted onto poly-lysine coated microscope slides and immediately refrozen and stored at -70°C with dessicant until hybridization. In situ hybridization
In situ hybridizations were carried out as described previously (21, 22). Prior to hybridization the sections were pretreated with paraformaldehyde (10 min), proteinase K (1 @ml) (10 min), and prehybridized for 1 to 2hrs, in 50~1 of prehybridization buffer (0.3M NaCl, 20mM Tris pH 8.0, 5mM EDTA, 1 x Denhardt’s solution, 10% dextran sulfate and 1OmM dithiothreitol). The hybridizations were started by adding 3OOOOOcpm of the 35S riboprobe in a small amount of prehybridization buffer and proceeded for 12-18 hrs at 55°C. After hybridization the sections were washed with 2 x SSC (2 x 10min) (1 x SSC =
LOCALIZATION OF ENKEPHALINASE mRNA IN RAT BRAIN
150mM NaCl, 15mM Na citrate, pH 7.0), treated with RNase (20t.rJm1, 30 min at room temperature), washed again in 2 x SSC (2 x 10 min), followed by a high stringency wash in 0.1 x SSC at 52 “C for 2 hrs. All SSC solutions up to this point of the procedure contained 10mM b-mercaptoethano1 and 1mM EDTA to help prevent non-specific binding of the probe. The sections were then washed in 0.5 x SSC without b-mercaptoethanol (2 x 10 min) and dehydrated by immersion in alochols containing 0.3 M ammonium acetate. The sections were dried and coated with NTB2 nuclear emulsion (Kodak) and exposed in the dark at 4°C for 4 to 8 weeks. After developing the sections were counterstained with hematoxylin and eosin. Some sections which were to be processed for film autoradiography were treated with a 32P-labelled riboprobe in the same conditions as described above and exposed for 2 weeks at room temperature to Kodak X-ray films prior to development.
79
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Results and Discussion The recent cloning of the enkephalinase gene has made possible the study of the localization of the enkephalinase mRNA by in situ hybridization, and its comparison to that of the translation product established by immunohistochemistry. Both the enkephalinase protein and the mRNA displayed a markedly heterogenous localization among various brain regions, as illustrated on a sagittal section (Fig 1). The localization of the enkephalinase protein was consistent with previous reports (9-11). However, the distribution of enkephalinase was more widespread than that of the mRNA, which was localized to restricted brain areas. This may reflect the fact that, while the mRNA is exclusively located within cell bodies, the translated protein may be found anywhere along the cells where it is synthetized, from the cell bodies to its processes. Although enkephalinase has been found to be expressed in a glioma cell line (23, 24), a large body of evidence suggests it has a predominantly neuronal localization in brain. In particular, the enzyme activity was decreased following treatment with neurotoxins (11, 25, 26), the enzyme was enriched in synaptosomal membranes (27,28)
Fig 1 Comparative localization of enkephalinase immunoreactivity and enkephalinase mRNA hybridization in rat brain sagittal sections. 1A: Autoradiographic visualization of enkephalinase using a ‘*%labeled monoclonal antibody. Sagittal sections (20uM thick) obtained at the level L = 1.40mm from the atlas of Paxinos and Watson (4.5). Exposure time was 3 days. 1B: Autoradiographic distribution of in situ cRNAmRNA hybridization using a 32P-labelled enkephalinase cRNA probe. Exposure time was 2 weeks. Abbreviations: Acb: accumbens nucleus; CAi, CA*, CAj: fields of Ammon’s horn; Cer: cerebellum; Ch: choroid plexus; CPU: caudate putamen; Cx: cerebral cortex; DG: dentate gyrus; LH: lateral hypothalamus, OB: olfactory bulb; Pn: pontine nucleus; SN: substantia nigra; TU: olfactory tubercle; VP: ventral pallidum.
and its developmental pattern during ontogenesis paralleled synapse formation (29). More convincingly, enkephalinase immunoreactivity was found to be associated with fibre bundles linking the striatum to the entopeduncular nucleus and substantia nigra, and was eliminated in the substantia nigra following ablation of the corresponding perikarya in striatum (11). These observations strongly suggest a localization of enkephalinase within a striatonigral neuronal pathway. The presence of the enkephalinase mRNA in the caudate
80
NEUROPEPTIDES
of periglomerular cells expresses the enkephalinase and proenkephalinase A genes, in the same manner as the same cells sometimes express both acetylcholinesterase and the acetylcholine-synthetizing enzyme choline acetyltransferase (39). The pontine nuclei were also among the areas with both high hybridization and immunoreactivity. The pontine nuclei are the source of mossy fibres projecting to most regions of the cerebellar
Fig 2 Cellular distribution of silver grains in the rat caudate putamen after in situ cRNA-mRNA hybridization using a 35S-labelled enkephalinase cRNA probe and nuclear emulsion coating. After developing, the sections were counterstained with hematoxylin and easin. Exposure time was 6 weeks. The photograph was taken using a combination of polarized light epiluminescence and bright field illumination so that the silver grains appear white. Note the tight localization of the silver grains over in situ positive cells. In contrast, no labelling is seen in the adjacent lateral septum. A
putamen, but its apparent absence from the entopeduncular nucleus or substantia nigra (Figs 1 and 2) are fully consistent with a high expression of the enzyme in cells of this striatonigral pathway. A minor association of the enzyme with nigrostriatal neurons was suggested by the effects of the neurotoxin 6-hydroxydopamine (25), and the lack of detectable enkephalinase mRNA in the substantia nigra (Fig 1) might be due to low abundance of the mRNA which may hinder its detection. In the globus pallidus, which contains the highest enkephalinase activity (30) and immunoreactivity in brain (Fig l), no enkephalinase mRNA could be detected. This is again consistent with a massive association of the peptidase with a striatopallidal pathway (11, 31), which has previously been shown to contain enkephalins (32-36). In the olfactory bulb, both enkephalinase immunoreactivity and mRNA hybridization were detected at the level of the glomerular layer; in addition, the mRNA was more specifically associated with periglomerular cells (Fig 3). A fraction of these cells has been shown to express enkephalins (33,37-38). It will be interesting to further assess whether the same neuronal subpopulation
Fig 3 Comparative localization of enkephalinase immunoreactivity and enkephalinase mRNA hybridization in rat olfactory bulb. Section A was processed for enkephalinase immunohistochemistry using a ‘2SI-labelled monoclonal antibody (right). The left picture shows the Nissl-stained tissue section from which the autoradiogram was generated. Note the selective association of the immunoreactivity with the glomerular layer. Section B was processed for in situ hybridization using a 35S-labelled probe and was photographed as described in legend of Fig. 2. Note the tight localization of the silver grains over positive periglomerular cells. Abbreviations: AOB: accessory olfactory bulb; E: ependyma and subependymal layer; EPl: external plexiform layer; Gl: glomerular layer; GrA: granular cell layer; IGr: internal granular layer.
LOCALIZATION
OF ENKEPHALINASE
81
mRNA IN RAT BRAIN
Fig 4 Comparative localization of enkephalinase itnmunoreactivity and enkephalinase mRNA hybridization in rat hippofor enkephahnase campus. Section A was processed immunohistochemistry using a “SI-labelled monoclonal antibody. Section B was processed for in situ hybridization using a “5S-labelled cRNA probe and was photographed as described in legend of Fig. 2. Note the tight localization of the silver grains over positive pyramidal cells (CA2-CA3 area). Abbreviations: CAr. CA;?, CA_+ fields of Ammon’s horn; DG: dentate gyrus; LMol: lacunosum molecular layer; MoL: molecular layer; Or: oriens layer; PoDG: polymorph layer of the dentate gyrus; Py: pyramidal cell layer.
pyramidal layer of Ammon’s horn, is likely to correspond to the same cells (Fig 4). It seems well established that the pyramidal cell layer contains enkephalinergic interneurons (40). In addition, it also contains opiate receptors (26, 41-43), mainly of the lo,subtype, which are presumably located on the pyramidal cells themselves (44). Expression of enkephalinase in these cells could thus be related to the inactivation of enkephalins on these target cells. The apparent parallelism between enkephalinase and proenkephalin A gene expression suggested by our data in the striatopallidal pathway, the olfactory bulb and the hippocampal formation lends further support to the parallelism between enkephalinase activity and enkephalin immunoreactivity in some brain areas reported earlier (30). In conclusion, the comparison of the distribution of cells containing enkephalinase mRNA by in situ hybridization, and the translated protein, by radioimmunohistochemistry, has provided important information regarding the types of cells and the origin of the pathways expressing this neuropeptidase (1). Clearly each technique provides complementary results, with in situ hybridization labelling the soma of the cells and radioimmunohistochemistry labelling the translated protein where it is transported. As shown in this study, the use of both techniques is particularly advantageous in brain, since neurons extend long processes so that one can in fact expect the localization of proteins to be different from that of their mRNA. The comparison of the localizations of mRNAs and the corresponding translated proteins could prove helpful to unravel novel neuronal pathways in the brain.
References cortex. In the cerebellum, a rather dense immunoreactivity could be detected in the molecular layer, and mRNA hybridization was detected there as well. In the hippocampal formation, high enkephalinase mRNA signal was detected in the pyramidal cells of the CA*-CA3 fields. Enkephalinase immunoreactivity, present all along the cell-dense
Schwartz, J. C., Malfroy, B. and De la Baume, S. (1981). Biological inactivation of enkephalins and the role of enkephalindipeptidyl-carboxypeptidase (enkephalinase) as neuropeptidase. Life Sci., 29: 17151740. Lynch, D. R. and Snyder, S. H. (1986). Neuropeptides: muhiple molecular forms, metabolic pathways and receptors. Ann. Rev. Biochem. 5.5: 773-799. McKelvy, J. F. and Blumberg, S. (1986). Inactivation and metabolism of neuropeptides. Ann. Rev. Neurosci., 9: 415-434.
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4. Schwartz, J. C. (1988). Enkephalinase
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inhibitors in drugs. In “Design of enzyme inhibitors as drugs”. Edited by M. Sandler and J. Smith. Oxford University Press, Oxford, 2: 206: 226. Malfroy, B., Giros, B., Gros, C., Llorens-Cortes, C. and Schwartz, J. C. (1989). Metabolism of enkephalins in the central nervous system. Opioid Peptides, CRC Press, Orlando, Florida, Vol. IV, (in press). Llorens-Cortes, C., Gras, C. and Schwartz, J. C. (1985) Study of endogenous Tyr-Gly-Gly, a putative enkephalin metabolite in mouse brain: validation of radioimmunoassay, localization and effects of peptidase inhibitors. Eur. J. Pharmacol., 119: 183-191. Llorens, C.. Schwartz, J. C. and Gros, C. (1985). Detection of the tripeptide Tyr-Gly-Gly, a putative enkephalin metabolite in brain, using a sensitive radioimmunoassay. FEBS Lett., 189: 325-328. Gras, C., Giros, B., Llorens, C., Malfroy, B., Pollard, H., Pachot, I., Schwartz, J. C. and Mazie, J. C. (1985). “Enkephalinase” and “aminopeptidase M” as enkephalininactivating peptidases and their inhibition. Transact. Biochem. Sot., 13: 47-50. Matsas, R., Kenny, A. J. and Turner, A. J. (1986). An immunohistochemical study of endopeptidase 24.11 (enkephalinase) in the pig nervous system. Neurosci., 18: 991-1012. Pollard, H., De la Baume, S., Bouthenet, M. L., Schwartz, J. C., Ronco, P. and Verroust, P. (1987). Characterization of two probes for the localization of enkephalinase in rat brain: [3H]thiorphan and A 1251-labelled monoclonal antibody. Eur. J. Pharmacol., 133: 155-164. Pollard, H., Llorens-Cortes, C., Couraud, J. Y., Ronco, P., Verroust, P. and Schwartz, J. C. (1987). Enkephalinase (EC 3.4.24.11) is highly localized to a striatonigral pathway in rat brain. Neurosci. Lett. 77: 267-271. Waksman, G., Hamel, E., Bouboutou, R., Besselievre, R., Fournie-Zaluski, M. C. and Roques, B. P. (1984). Distribution regionale de I’enktphalinase dans le cerveau du rat par autoradiographie. C. R. Acad. Sci. Paris, 229: 613-615. Waksman, G., Hamel, E., Fournit-Zaluski, M. C. and Roques, B. P. (1986). Autoradiographic comparison of the distribution of the neutral endopeptidase “enkephalinase” and of u and opioid receptors in rat brain. Proc. Natl. Acad. Sci. USA, 83: 1523-1527. Devault, A., Lazure, C., Nault, C., Le Moual, H., Seidah, N. G., Chretien, M., Kahn, P., Powell, J., Mallet, J., Beaumont, A., Roques, B. P., Crine, P. and Boileau, G. (1987). Amino acid sequence of rabbit kidney neutral endopeptidase 24.11 (enkephalinase) deduced from a complementary DNA. Embo. J., 6: 1317-1322. Malfroy, B., Kuang, W. J., Seeburg, P. H., Mason, A. J. and Schofield, P. S. (1988) Molecular cloning and amino acid sequence of human enkephalinase (neutral endopeptidase). FEBS Lett., 229: 206-210. Malfroy, B., Schofield, P. R., Kuang, W. J., Seeburg, P. H., Mason, A. J. and Henzel, W. J. (1987). Molecular cloning and amino acid sequence of rat enkephalinase. Biochem. Biophys. Res. Corn., 144: 59-66.
17. Ronco, P., Geniteau. M., Poujeol, P., Melcion, C., Verroust , P. and Vandewalle , A. (1986). Characterization of monoclonal antibodies to rabbit renal cortical cells. Am. J. Physiol., 250L C506-C516. 18. Ronco, P., Pollard, H., Galleran, M., Delauche, M., Schwartz, J. C. and Verroust, P. (1988). Distribution of enkephalinase (membrane metalloendopeptidase EC 3.4.24.11) in rat organs: detection using a monoclonal antibody. Jab. Invest., 58: 210-217. 19. McConahey and Dixon, F. J. (1966). A method of trace iodination of proteins for immunologic studies. Int. Arch. Allergy Appl. Immunol., 29: 185-191. 20. Melton, D. A., Krieg, P. A., Rebagliati, M. R., Maniatis, T., Zinn, K. and Green, M. R. (1984). Efficient in vitro synthesis of biologically active RNA hybridization probes from plasmid containing a bacteriophage. Nucleic Acids Res., 12: 7053-7056. 21. Rosenthal, A., Chan, S. Y., Henzel, W., Haskell, C., Kuang, W. J.. Chen, E., Wilcox, J. N., Ullrich, A., Goeddel, D. V. and Routtenberg, A. (1987). Primary structure and mRNA localization of protein Fl, a growth related protein kinase C substrate associated with synaptic plasticity. The Embo J., 6: 2641-3646. 22. Wilcox, J. N., Gee, C. E. and Roberts, J. L. (1986). In situ cDNA: mRNA hybridization: development of a technique to measure mRNA levels in individual cells. Methods Enzymol., 124: 510-533. 23. Lentzen, H. and Palenker, T. (1983). Localization of the thiorphan sensitive endopeptidase, termed enkephalinase A on glial cells. FEBS. Lett., 153: 93-97. 24. Palenker, J., Lentzen, H. and Brandt, V. (1984). Enkephahn degradation by enkephalinergic neuroblastoma cells. Involvement of angiotensin converting enzyme. Naunyn. Schmiedb. Arch. Neural., 325: 214-217. 25. Malfroy, B., Swerts, J. P., Llorens, C. and Schwartz, J. C. (1979). Regional distribution of a high affinity enkephalin degrading peptidase (enkephalinase) and effects of lesions suggest localization in the vicinity of opiate receptors in brain. Neurosci. Lett., 11: 329-334. 26. Waksman, G., Hamel, E., Delay-Goyet, P. and Roques, B. P. (1987). Neutral endopeptidase 24.11, u and opioid receptors after selective brain lesions: an autoradiographic study. Brain Res., 436: 205-216. 27. Altstein, M. and Vogel, Z. (1980). On the inactivation of enkephalin by enkephalinase. In “Neurotransmitters and their receptors”, edited by.V. Z. Littauer (John Wiley, New-York), 497-507. 28. De la Baume, A., Patey, G. and Schwartz, J. C. (1981). Subcellular distribution of enkephalin-dipeptidyl carboxypeptidase (enkephalinase) in rat brain. Neurosci., 6: 315-321. 29. Patey, G., De ia Baume, S., Gros, C. and Schwartz, J. C. (1980). Ontogenesis of enkephalinergic systems in rat brain: postnatal changes in enkephalin levels, receptors, and degrading enzyme activity. Life Sci., 27: 245-252. 30. Llorens-Cortes, C., Malfroy, B., Schwartz, J. C., Gacel, G., Roques, B. P., Roy, J., Morgat, J. L., Javoy-Agid, F. and Agid, Y. (1982). Enkephalin dipeptidyl carboxypeptidase (enkephalinase) activity: selective radioassay
mRNA IN RAT BRAIN
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properties and regional distribution in human brain. J. Neurochem., 39: 1081-1089. Pollard, H., Bouthenet, M. L., Moreau, J., Souil, E., Vcrroust, P., Ronco, P. and Schwartz, J. C. (submitted) Detailed immunoautoradiographic mapping of enkephalinase (EC 3.4.24.11) in rat central nervous system: comparison with enkephalins and substance P. Cuello, A, C. and Paxinos, G. (1978). Evidence for a long leu-enkephalin striatopallidal pathway in rat brain. Nature, 271: 178-180. Del Fiacco, M., Paxinos, G. and Cuello, A. C. (1982). Neostriatal enkephalin-immunoreactive neurons project to the globus pallidus. Brain Res., 231: 1-17. Haber, S. N. and Nauta, W. J. H. (1983). Ramifications of the globus pallidus in the rat as indicated by patterns of immunohistochemistry. Neurosci., 9: 245260. McLean, S., Skirboll, L. R. and Pert, C. B. (1985). Comparison of substance P and enkephalin distribution in rat brain: an overview using radioimmunocytochemistry. Neurosci., 14: 837-852. Paxinos, G., Staines, W. A., Hokfelt, W. A., Oertel, W. and Terenius, L. (1984). Enkephalin (EK) dynorphin (Dyn), Substance P (SP) and glutamic acid decarboxylase (GAD) in striatal efferents. Sot. Neurosci. Abst., 10: 516. Davis, B. J., Burd, G. D. and Macrides, F. (1982). Localization of Methionine-enkephalin, substance P, and somatostatin immunoreactivities in the main olfactory bulb of the hamster. J. Comp. Neurol., 204: 377-383. Merchenthaler, I., Maderdrut, J. L., Altschuler, R. A. and Petrusz, P. (1986). Immunocytochemical localization of proenkephalin-derived peptides in the central nervous system of the rat. Neurosci., 17: 325-348.
39. Mesulam, M. M., Mufson, E. J., Levey, A. I. and Wainer, B. H. (1984). Atlas of choline@ neurons in the forebrain and upper brainstem of the macaque based on monoclonal choline acetyl-transferase immunohistochemistry and acetylcholinesterase histochemistry. Neurosci., 12: 669-686. 40. Gall, C., Brecha, N., Karten, H. J. and Chang, K. I. (1981). Localization of enkephalin-like immunoreactivity to identified axonal and neuronal populations of the rat hippocampus. J. Comp. Neurol., 198: 225-250. 41. Crain, B. J., Chang, C. J. and McNamara, J. 0. (1986). Quantitative autoradiographic analysis of mu and delta opioid binding sites in the rat hippocampal formation. J. Comp. Neural., 246: 170-181. 42. Mansour, A., Lewis, M. E., Khachaturian H., Akil, H. and Watson, S. J. (1986). Pharmacological and anatomical evidence of selective )I and S opioid receptor binding in rat brain. Brain Res., 399: 69-79. 43. McLean, S., Rothmann, R. 8. and Herkenham, M. (1986). Autoradiographic localization of u and opiate receptors in the forebrain of the rat. Brain Res., 378,49&I. 44. Swanson, L. W., Kohler, C. and Bjorklud (1987). The limbic region. I: The septohippocampal system. In “Handbook of Chemical neuroanatomy”. Vol. 5: integrated systems of the CNS, Part I, hypothalamus, hippocampus, Amygdala, Retina. Edited by A. Bjorklund, T., Hdkfelt and Swanson L. W. Elsevier Science Publishers B. V.. 125-255. 45. Paxinos. G. and Watson, C. (1986). The rat brain in stereotaxic coordinate. 2nd Edition. Academic Press, Sidney.
LOCALIZATION
31.
32.
33.
34.
35.
36.
37.
38.
OF ENKEPHALINASE
0143-4179/89/0014-0077/$10.00
Localization of Enkephalinase mRNA in Rat Brain by In Situ Hybridization: Comparison with lmmunohistochemical Localization of the Protein J. N. WILCOX, H. POLLARD*, J. MOREAU*, J. C. SCHWARTZ* and B. MALFROYt Departments of Molecular Biology and tPharmacological Sciences, Genetech, inc., 460 Point San Bruno Blvd., South San Francisco, CA 94080, USA. *Unitt+ de Neurobiologie et Pharmacologic (U. 109) DE L ‘INSERM, Centre Paul Broca, 2ter rue d’Al&ia, 75014 Paris, France. (Correspondence to BM)
Abstract-The messenger RNA (mRNA) encoding enkephalinase (EC. 3.4.24.11; neutral endopeptidase) has been localized in rat brain by in situ hybridization using 35S- or 32Plabelled cRNA probes. Hybridization was observed only in few brain areas, and was particularly strong in the striatum, olfactory bulb and pontine nuclei. The enkephalinase protein was also localized in brain sections using a radiolabelled monoclonal antibody. While some brain regions contained both the mRNA and its translation product, others, including in particular the substantia nigra, were rich in enkephalinase but did not contain any detectable amount of enkephalinase mRNA. Enkephalinase mRNA-containing cells could be identified in regions containing neurons known to project to the substantia nigra. The discrepancy between the mRNA and the protein labelling is likely to reflect the fact that the mRNA is exclusively located within the soma of the cells while the translated protein may be found anywhere along the axonal processes.
Introduction
elicit a variety of opioid-like, naloxone-reversible responses. In addition, the tripeptide Tyr-GlyGly, a characteristic enkephalin metabolite formed under the action of enkephalinase, was recently identified in brain, further indicating the role of this enzyme in the degradation of endogenous enkephalins (6,7). Enkephalinase, like most peptidases, displays a limited substrate specificity and its functional role is, therefore, defined not only by enzymological but also by topographical considerations. A precise localization of the peptidase in the CNS
Enkephalinase (EC 3.4.24.11, neutral endopeptidase) is a membrane-bound metallopeptidase involved in the inactivation of endogenous enkephalins and, possibly, other neuropeptides (reviewed in l-5). Its role in the inactivation of enkephalins was shown by the effects of inhibitors which protect, at least partially, the endogenous opioid pentapeptides from degradation and thus Date received 3 January 1989 Date accepted 9 March 1989 77
78
NEUROPEPTIDES
has been obtained by immunohistochemical studies using monoclonal antibodies (8-11) and by autoradiography technique using 3H-labelled inhibitors (10, 12, 13). Such studies have shown that the enzyme is mainly localized within brain areas enriched in enkephalins, which reinforces the idea that a major function of this enzyme in brain is to inactivate enkephalins. The recent cloning of the enkephalinase gene (14-16) has made possible the visualization of the mRNA by in situ hybridization. We now report results from such initial studies in which the mRNA and the translated protein were visualized at the cellular level using 32P- or 35S-labelled cRNA probes and a highly specific ‘251-labelled monoclonal antibody (mAb) (lo), respectively.
Materials and Methods Radioimmunohistochemical phalinase
visualization of enke-
Monoclonal antibody The monoclonal antibody (MAb 85 AZ), prepared by immunizing mice with tubular cells from rabbit renal cortex (17) was kindly supplied by Dr. P. Verroust and Dr. P. Ronco (Unite de NephrolU.64, de et pathologique, ogie normale I’INSERM, Paris, France). Its specificity was established by immunoprecipitation experiments (18). The monoclonal antibody was iodinated at a specific activity of 8uCi/mg using chloramine T (19) and [‘*‘I]INa (2OOOCilmmo1, Amersham). Tissue preparation
Male Wistar rats (180-200g) were killed, the brain rapidly removed, dropped into cold freon (-40°C monochlorodifluoro methane, Prestogaz) for a few minutes and kept frozen at -20°C until use. Frontal frozen sections (10-20).r,m thick) were cut on a cryostat (Ames) at -18”C, thaw-mounted onto gelatin-coated glass slides and stored at -20°C until used. Autoradiographic localization with [‘25Z]mAb 85A2
of enkephalinase
The sections were warmed to room temperature and dried for 30 min prior to incubation. They
were then incubated for 3 h at 20°C with 100 ~1 of [‘251]mAb 85 A2 (5 x lo5 CPM/ml) dissolved in O.lM phosphate buffer saline (PBS) pH 7.2-7.4 containing 0.2% gelatin. Finally the slides were rinsed 4 times 2 min in PBS, dipped into distilled water to remove any salts remaining, immediately dried and apposed to 3H hyperfilms (Amersham) in X-ray cassettes. These films were developed l-3 days later. Localization of enkephalinase hybridization
mRNA
by in situ
cRNA probe
The probe used was a cRNA corresponding to the full length sequence of the rat enkephalinase mRNA (16), subcloned into SP64 recombinant plasmid and labelled with [32P]CTP or [35S]UTP (20). Tissue preparation
The brain was removed and immersed in freshly prepared 4% paraformaldehyde in O.lM sodium phosphate (pH 7.4). Tissues were fixed at 4°C for 3 hrs and then immersed in 15% sucrose-PBS overnight at 4°C to act as a cryoprotectant. Tissues were then embedded in OCT blocks and stored at -70°C. There was no loss of mRNA available for hybridization during this time. Tissues were sectioned at lO).rm thickness using a cryostat, thaw-mounted onto poly-lysine coated microscope slides and immediately refrozen and stored at -70°C with dessicant until hybridization. In situ hybridization
In situ hybridizations were carried out as described previously (21, 22). Prior to hybridization the sections were pretreated with paraformaldehyde (10 min), proteinase K (1 @ml) (10 min), and prehybridized for 1 to 2hrs, in 50~1 of prehybridization buffer (0.3M NaCl, 20mM Tris pH 8.0, 5mM EDTA, 1 x Denhardt’s solution, 10% dextran sulfate and 1OmM dithiothreitol). The hybridizations were started by adding 3OOOOOcpm of the 35S riboprobe in a small amount of prehybridization buffer and proceeded for 12-18 hrs at 55°C. After hybridization the sections were washed with 2 x SSC (2 x 10min) (1 x SSC =
LOCALIZATION OF ENKEPHALINASE mRNA IN RAT BRAIN
150mM NaCl, 15mM Na citrate, pH 7.0), treated with RNase (20t.rJm1, 30 min at room temperature), washed again in 2 x SSC (2 x 10 min), followed by a high stringency wash in 0.1 x SSC at 52 “C for 2 hrs. All SSC solutions up to this point of the procedure contained 10mM b-mercaptoethano1 and 1mM EDTA to help prevent non-specific binding of the probe. The sections were then washed in 0.5 x SSC without b-mercaptoethanol (2 x 10 min) and dehydrated by immersion in alochols containing 0.3 M ammonium acetate. The sections were dried and coated with NTB2 nuclear emulsion (Kodak) and exposed in the dark at 4°C for 4 to 8 weeks. After developing the sections were counterstained with hematoxylin and eosin. Some sections which were to be processed for film autoradiography were treated with a 32P-labelled riboprobe in the same conditions as described above and exposed for 2 weeks at room temperature to Kodak X-ray films prior to development.
79
‘
Results and Discussion The recent cloning of the enkephalinase gene has made possible the study of the localization of the enkephalinase mRNA by in situ hybridization, and its comparison to that of the translation product established by immunohistochemistry. Both the enkephalinase protein and the mRNA displayed a markedly heterogenous localization among various brain regions, as illustrated on a sagittal section (Fig 1). The localization of the enkephalinase protein was consistent with previous reports (9-11). However, the distribution of enkephalinase was more widespread than that of the mRNA, which was localized to restricted brain areas. This may reflect the fact that, while the mRNA is exclusively located within cell bodies, the translated protein may be found anywhere along the cells where it is synthetized, from the cell bodies to its processes. Although enkephalinase has been found to be expressed in a glioma cell line (23, 24), a large body of evidence suggests it has a predominantly neuronal localization in brain. In particular, the enzyme activity was decreased following treatment with neurotoxins (11, 25, 26), the enzyme was enriched in synaptosomal membranes (27,28)
Fig 1 Comparative localization of enkephalinase immunoreactivity and enkephalinase mRNA hybridization in rat brain sagittal sections. 1A: Autoradiographic visualization of enkephalinase using a ‘*%labeled monoclonal antibody. Sagittal sections (20uM thick) obtained at the level L = 1.40mm from the atlas of Paxinos and Watson (4.5). Exposure time was 3 days. 1B: Autoradiographic distribution of in situ cRNAmRNA hybridization using a 32P-labelled enkephalinase cRNA probe. Exposure time was 2 weeks. Abbreviations: Acb: accumbens nucleus; CAi, CA*, CAj: fields of Ammon’s horn; Cer: cerebellum; Ch: choroid plexus; CPU: caudate putamen; Cx: cerebral cortex; DG: dentate gyrus; LH: lateral hypothalamus, OB: olfactory bulb; Pn: pontine nucleus; SN: substantia nigra; TU: olfactory tubercle; VP: ventral pallidum.
and its developmental pattern during ontogenesis paralleled synapse formation (29). More convincingly, enkephalinase immunoreactivity was found to be associated with fibre bundles linking the striatum to the entopeduncular nucleus and substantia nigra, and was eliminated in the substantia nigra following ablation of the corresponding perikarya in striatum (11). These observations strongly suggest a localization of enkephalinase within a striatonigral neuronal pathway. The presence of the enkephalinase mRNA in the caudate
80
NEUROPEPTIDES
of periglomerular cells expresses the enkephalinase and proenkephalinase A genes, in the same manner as the same cells sometimes express both acetylcholinesterase and the acetylcholine-synthetizing enzyme choline acetyltransferase (39). The pontine nuclei were also among the areas with both high hybridization and immunoreactivity. The pontine nuclei are the source of mossy fibres projecting to most regions of the cerebellar
Fig 2 Cellular distribution of silver grains in the rat caudate putamen after in situ cRNA-mRNA hybridization using a 35S-labelled enkephalinase cRNA probe and nuclear emulsion coating. After developing, the sections were counterstained with hematoxylin and easin. Exposure time was 6 weeks. The photograph was taken using a combination of polarized light epiluminescence and bright field illumination so that the silver grains appear white. Note the tight localization of the silver grains over in situ positive cells. In contrast, no labelling is seen in the adjacent lateral septum. A
putamen, but its apparent absence from the entopeduncular nucleus or substantia nigra (Figs 1 and 2) are fully consistent with a high expression of the enzyme in cells of this striatonigral pathway. A minor association of the enzyme with nigrostriatal neurons was suggested by the effects of the neurotoxin 6-hydroxydopamine (25), and the lack of detectable enkephalinase mRNA in the substantia nigra (Fig 1) might be due to low abundance of the mRNA which may hinder its detection. In the globus pallidus, which contains the highest enkephalinase activity (30) and immunoreactivity in brain (Fig l), no enkephalinase mRNA could be detected. This is again consistent with a massive association of the peptidase with a striatopallidal pathway (11, 31), which has previously been shown to contain enkephalins (32-36). In the olfactory bulb, both enkephalinase immunoreactivity and mRNA hybridization were detected at the level of the glomerular layer; in addition, the mRNA was more specifically associated with periglomerular cells (Fig 3). A fraction of these cells has been shown to express enkephalins (33,37-38). It will be interesting to further assess whether the same neuronal subpopulation
Fig 3 Comparative localization of enkephalinase immunoreactivity and enkephalinase mRNA hybridization in rat olfactory bulb. Section A was processed for enkephalinase immunohistochemistry using a ‘2SI-labelled monoclonal antibody (right). The left picture shows the Nissl-stained tissue section from which the autoradiogram was generated. Note the selective association of the immunoreactivity with the glomerular layer. Section B was processed for in situ hybridization using a 35S-labelled probe and was photographed as described in legend of Fig. 2. Note the tight localization of the silver grains over positive periglomerular cells. Abbreviations: AOB: accessory olfactory bulb; E: ependyma and subependymal layer; EPl: external plexiform layer; Gl: glomerular layer; GrA: granular cell layer; IGr: internal granular layer.
LOCALIZATION
OF ENKEPHALINASE
81
mRNA IN RAT BRAIN
Fig 4 Comparative localization of enkephalinase itnmunoreactivity and enkephalinase mRNA hybridization in rat hippofor enkephahnase campus. Section A was processed immunohistochemistry using a “SI-labelled monoclonal antibody. Section B was processed for in situ hybridization using a “5S-labelled cRNA probe and was photographed as described in legend of Fig. 2. Note the tight localization of the silver grains over positive pyramidal cells (CA2-CA3 area). Abbreviations: CAr. CA;?, CA_+ fields of Ammon’s horn; DG: dentate gyrus; LMol: lacunosum molecular layer; MoL: molecular layer; Or: oriens layer; PoDG: polymorph layer of the dentate gyrus; Py: pyramidal cell layer.
pyramidal layer of Ammon’s horn, is likely to correspond to the same cells (Fig 4). It seems well established that the pyramidal cell layer contains enkephalinergic interneurons (40). In addition, it also contains opiate receptors (26, 41-43), mainly of the lo,subtype, which are presumably located on the pyramidal cells themselves (44). Expression of enkephalinase in these cells could thus be related to the inactivation of enkephalins on these target cells. The apparent parallelism between enkephalinase and proenkephalin A gene expression suggested by our data in the striatopallidal pathway, the olfactory bulb and the hippocampal formation lends further support to the parallelism between enkephalinase activity and enkephalin immunoreactivity in some brain areas reported earlier (30). In conclusion, the comparison of the distribution of cells containing enkephalinase mRNA by in situ hybridization, and the translated protein, by radioimmunohistochemistry, has provided important information regarding the types of cells and the origin of the pathways expressing this neuropeptidase (1). Clearly each technique provides complementary results, with in situ hybridization labelling the soma of the cells and radioimmunohistochemistry labelling the translated protein where it is transported. As shown in this study, the use of both techniques is particularly advantageous in brain, since neurons extend long processes so that one can in fact expect the localization of proteins to be different from that of their mRNA. The comparison of the localizations of mRNAs and the corresponding translated proteins could prove helpful to unravel novel neuronal pathways in the brain.
References cortex. In the cerebellum, a rather dense immunoreactivity could be detected in the molecular layer, and mRNA hybridization was detected there as well. In the hippocampal formation, high enkephalinase mRNA signal was detected in the pyramidal cells of the CA*-CA3 fields. Enkephalinase immunoreactivity, present all along the cell-dense
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39. Mesulam, M. M., Mufson, E. J., Levey, A. I. and Wainer, B. H. (1984). Atlas of choline@ neurons in the forebrain and upper brainstem of the macaque based on monoclonal choline acetyl-transferase immunohistochemistry and acetylcholinesterase histochemistry. Neurosci., 12: 669-686. 40. Gall, C., Brecha, N., Karten, H. J. and Chang, K. I. (1981). Localization of enkephalin-like immunoreactivity to identified axonal and neuronal populations of the rat hippocampus. J. Comp. Neurol., 198: 225-250. 41. Crain, B. J., Chang, C. J. and McNamara, J. 0. (1986). Quantitative autoradiographic analysis of mu and delta opioid binding sites in the rat hippocampal formation. J. Comp. Neural., 246: 170-181. 42. Mansour, A., Lewis, M. E., Khachaturian H., Akil, H. and Watson, S. J. (1986). Pharmacological and anatomical evidence of selective )I and S opioid receptor binding in rat brain. Brain Res., 399: 69-79. 43. McLean, S., Rothmann, R. 8. and Herkenham, M. (1986). Autoradiographic localization of u and opiate receptors in the forebrain of the rat. Brain Res., 378,49&I. 44. Swanson, L. W., Kohler, C. and Bjorklud (1987). The limbic region. I: The septohippocampal system. In “Handbook of Chemical neuroanatomy”. Vol. 5: integrated systems of the CNS, Part I, hypothalamus, hippocampus, Amygdala, Retina. Edited by A. Bjorklund, T., Hdkfelt and Swanson L. W. Elsevier Science Publishers B. V.. 125-255. 45. Paxinos. G. and Watson, C. (1986). The rat brain in stereotaxic coordinate. 2nd Edition. Academic Press, Sidney.
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31.
32.
33.
34.
35.
36.
37.
38.
OF ENKEPHALINASE