Proc. Natl. Acad. Sci. USA Vol. 83, pp. 1523-1527, March 1986 Neurobiology Autoradiographic comparison of the distribution of the neutral "" and of ja and 6 receptors in rat brain (enkephailnase inhibitor/) GILLES WAKSMAN*, EDITH HAMELt, MARIE-CLAUDE FOURNI&-ZALUSKI*, AND BERNARD P. ROQUES*t *DJpartement de Chimie Organique, U 266 Institut National de la Santd et de la Recherche Mddicale, UA 498 Centre National de la Recherche Scientifique, UER des Sciences Pharmaceutiques et Biologiques, 4, avenue de l'Observatoire, 75006 Paris, France; and tCerebral Circulation and Metabolism Group, Laboratoire d'Etudes et de Recherches Synthdlabo, 92220 Bagneux, France Communicated by Jean-Pierre Changeux, October 7, 1985

ABSTRACT The neutral endopeptidase EC 3.4.24.11, also inhibitors. Although an operational re-uptake mechanism designated enkephalinase, has been visualized by in vitro cannot be definitively excluded, it is generally accepted that autoradiography using the tritiated inhibitor [3H]-N-[(2RS)-3- extracellular hydrolysis of by the endopeptidase hydroxyaminocarbonyl-2-benzyl-1-oxopropyl]glycine, EC 3.4.24.11 represents a major mechanism for terminating ([3H]HACBO-Gly). Specific binding of [3H]HACBO-Gly (Kd = the enkephalinergic signal (8-10). However, the ability of 0.4 ± 0.05 nM) corresponding to 85% of the total binding to purified enkephalinase to degrade a variety of neuropeptides brain slices was inhibited by 1 IAM , a selective in vitro, including substance P, cholecystokinin-(26-33) [sul- inhibitor of enkephalinase, but remained unchanged in the fated cholecystokinin (CCK)-octapeptide], and neurotensin presence of captopril, a selective inhibitor of angiotensin- (reviewed in ref. 11), raises questions concerning the selec- converting enzyme. Very high levels of [3H]HACBO-Gly bind- ing were found in the choroid plexus and the substantia nigra. tive involvement of the neutral endopeptidase in enkephalin High levels were present in the caudate putamen, globus metabolism in vivo. A plausible hypothesis is that the pallidus, nucleus accumbens, olfactory tubercle, and in the specificity of enkephalinase action in vivo is determined by substantia gelatinosa of the spinal cord. Moderate densities the selective association of the enzyme with enkephalinergic were found in parts of the amygdala, the periaqueductal gray neuronal systems. matter, the interpeduncular nucleus, and the molecular layer To test this hypothesis and to obtain a better understanding of the cerebellum. The distribution of enkephalinase was of enkephalinergic function, we have compared the regional compared to that of ,u and 6 opioid receptors, selectively distributions of opioid receptors and enkephalinase in rat labeled with [3H]Tyr-D-Ala-Gly-MePhe-glycinol and [3H]Tyr- brain by autoradiography. Selective labeling of A and 8 D-Thr-Gly-Phe-Leu-Thr, respectively. In the caudate puta- receptor types (12-14) was achieved by use ofthe appropriate men, [3H]HACBO-Gly binding overlapped the clustered IA sites radiolabeled probes, [3H]DAMGE and [3H]DTLET, respec- but appeared more closely related to the diffusely distributed tively (15, 16). The enkephalinase was selectively visualized 6 sites. High levels of enkephalinase and ,u opioid binding sites with the recently described (17) potent and tritiated inhibitor were present at the level of the periaqueductal gray matter and [3H]-N-[(2RS)-3-hydroxyaminocarbonyl-2-benzyl-1-oxopro- in the substantia gelatinosa of the spinal cord, regions where pyliglycine ([3H]HACBO-Gly). The autoradiographic analy- only sparse 6 opioid receptors could be detected. The associ- sis of the neutral endopeptidase's distribution in many brain ation of enkephalinase with 6 and ,u opioid receptors in these regions is consistent with a selective role of enkephalinase in areas is consistent with the observed role of the enzyme in the termination of the enkephalinergic signal. regulating the effects of opioid in striatal dopamine release and analgesia, respectively. Except for the choroid plexus and the cerebellum, the close similarity observed in MATERIALS AND METHODS numerous rat brain areas between the distribution of Chemicals. enkephalinase and that of it and/or 6 opioid binding sites could [3,5-3H]Tyr-D-Ala-Gly-MePhe-glycinol account for most of the pharmacological effects elicited by ([3H]DAMGE, 60 Ci/mmol; 1 Ci = 37 GBq) was purchased enkephalinase inhibitors. from Amersham. [3 ,5-3H]Tyr-D-Thr-Gly-Phe-Leu-Thr ([3H]DTLET, 45 Ci/mmol) (16) and [3H]HACBO-Gly were The endogenous opioid enkephalins are chiefly and synthesized in our laboratory and tritiated by reduction, with rapidly metabolized in the brain (1) by both an aminopepti- 3H2, of the benzylidene precursor (17) at the Commissariat a dase activity (2, 3) and enkephalinase (4), a membrane-bound l'Energie Atomique (Saclay, France). Thiorphan was from enzyme identical to the neutral EC our laboratory (6). was a gift from Hoffman-La 3.4.24.11 (5). The physiological relevance of brain enkepha- Roche Laboratories (Basel, Switzerland). linase is supported by the naxolone-reversible decrease in the Binding Procedures. Male Sprague-Dawley rats (150-200 g) were decapitated, and their brains, including the upper responsiveness to nociceptive stimuli - elicited by intracerebral administration of highly potent enkephalinase segments of the cervical spinal cord, were rapidly removed inhibitors such as thiorphan {N-[(2RS)-2-mercaptomethyl-1- and frozen in isopentane at -45°C. Coronal sections (20 ,tm oxo-3-phenylpropyl]glycine} (6) or {N-[(2R)-3- thick) were cut on a cryostat at -17°C, thaw-mounted onto hydroxyaminocarbonyl-2-benzyl-1-oxopropyl]-L-alanine} gelatin-coated slides, and stored at -80'C until used. All (7), a compound belonging to a new series of bidentate Abbreviations: CCK, cholecystokinin; DAMGE, [D-Ala2,N-methyl- Phe4,glycinol5]enkephalin; DTLET, Tyr-D-Thr-Gly-Phe-Leu-Thr The publication costs of this article were defrayed in part by page charge ([D-Thr2,Leu5]enkephalin-Thr6); HACBO-Gly, N-[(2RS)-3-hydroxy- payment. This article must therefore be hereby marked "advertisement" aminocarbonyl-2-benzyl-1-oxopropyl]glycine. in accordance with 18 U.S.C. §1734 solely to indicate this fact. tTo whom reprint requests should be addressed. 1523 Downloaded by guest on September 27, 2021 1524 Neurobiology: Waksman et al. Proc. Natl. Acad. Sci. USA 83 (1986) sections were warmed to room temperature just prior to As shown in Fig. LA, binding of [3H]HACBO-Gly to rat incubation for binding as described below. brain tissue sections was highly specific; the nonspecific For selective labeling of g opioid binding sites, the sections binding determined in the presence of 1 ,uM thiorphan, a were incubated with 4 nM [3H]DAMGE for 30 min at room highly potent inhibitor of enkephalinase, was 12-15% oftotal temperature in 50 mM Tris HCl (pH 7.4) as described (15). binding (Fig. 1B). In contrast, the potent and selective The 8 opioid sites were selectively labeled with 3 nM inhibitor of angiotensin-converting enzyme, captopril (19), [3H]DTLET (18). At the end of the incubation, the sections was unable to modify the specific binding of [3H]HACBO- were washed twice for 10 min in fresh buffer at 0-40C, Gly (Fig. 1C). followed by a rapid rinse in ice-cold distilled water, and then Moreover, HACBO-Gly was shown to be a selective dried under a stream of cold air. For nonspecific binding, the inhibitor of enkephalinase with a Ki of approximately 0.4 nM sections were incubated as above with either [3H]DAMGE or but greater than 30 nM for the dipeptidyl and [3H]DTLET but in the presence of 10 1LM levorphanol. In 1 uM for other brain metallopeptidases, such as aminopep- both cases, nonspecific binding accounted for <10%1 of total tidase M and angiotensin-converting enzyme (17). binding in all brain regions studied. Autoradiographic Localization of [H]HACBO-Gly Binding The enkephalinase was labeled with 3 nM [3H]HACBO- Sites in Rat Brain. [3H]HACBO-Gly binding sites were found Gly for 60 min at room temperature in 50 mM Tris HCl (pH to be discretely distributed in rat brain (Fig. 1A and Table 1), 7.4). At the end of the incubation, the sections were washed with the highest concentrations in the choroid plexus, in fresh ice-cold buffer for 1 min followed by two 5-min substantia nigra, caudate putamen, globus pallidus, olfactory rinses, rapidly washed in ice-cold distilled water, and imme- tubercle, nucleus accumbens, and the substantia gelatinosa diately dried. Nonspecific binding was determined in sections of the spinal cord. A second group of structures containing incubated as above but in the presence of 1 AM thiorphan and moderate levels of [3H]HACBO-Gly binding included parts accounted for <15% of the total binding. of the amygdala, the interpeduncular nucleus, the molecular Autoradiography. After being carefully dried, all labeled layer of the cerebellum, the periaqueductal gray matter, and sections were mounted with eight calibrated tritium stan- the hippocampus. dards and closely apposed to sheets of tritium-sensitive Interestingly, an accumulation of silver grains, intermin- Ultrofilm (LKB, Fisher) inside x-ray cassettes. The films gled with streaks of nonlabeled zones corresponding to the were exposed for 8-12 weeks at 4°C and then developed in presence of white matter tracts, was observed in the region Kodak LX-24 developer for 1.5 min at 18°C, fixed in Kodak connecting the substantia nigra and the caudate putamen L-4 fixative for 5 min, washed for 30 min under running tap (Fig. LA); this labeling was particularly dense in the area of water, and dried. the entopeduncular nucleus. Such a pattern of labeling has Quantitation of Binding Sites by Densitometry. All films been reported at the level of the nigrostriatal pathway for were analyzed by spot densitometry using a Quantimet 720 angiotensin-converting enzyme visualized with [3Hicaptopril (Cambridge Instrument Ltd., Cambridge, England). Optical (20). The quantitative evaluation ofspecific [3H]HACBO-Gly densities observed in the selected regions were compared to binding in various rat brain regions obtained by microdensi- those of the calibrated tritium standards and to film back- tometry is shown in Table 1. These results allow an accurate ground. Using the standards, the total amount of ligand comparison of the enzyme distribution with those of ,t and 8 bound to each region could be calculated from the specific opioid binding sites labeled with [3H]DAMGE and activity of the ligand and the amount of radioactivity in the [3H]DTLET, respectively. A general agreement with previ- tissue. The specific binding in each area was calculated by ously published autoradiographic distributions of ,u and 8 subtracting from the total binding the amount ofligand bound opioid receptors was found (18, 21). to the corresponding region in sections incubated for non- In the telencephalon (striatum, olfactory tubercle, nucleus specific labeling (see above). accumbens, cerebral cortex), the distribution ofthe peptidase (Fig. 2A) seemed to be more closely related to that of 8 (Fig. 2C) than ,u opioid binding sites (Fig. 2B). In the caudate RESULTS putamen (Fig. 2A), however, the intense accumulation of Binding of [3H]HACBO-Gly to Slices of Rat Brain Tissue. silver grains associated with [3H]HACBO-Gly binding over- Experimental conditions for the autoradiographic visualiza- lapped both the patchy ji sites and the diffusely labeled 8 tion of [3H]HACBO-Gly binding were essentially based on receptors (Fig. 2 B and C, respectively), two populations of those determined in tissue-homogenate studies (17). Prelim- opioid binding sites reported to belong to different compart- inary experiments carried out on tissue sections for two ments with distinctive neuronal connections (22). different inhibitor concentrations at 30- and 60-min incuba- Among limbic cortical areas, dense labeling of a and 8 tion times closely agreed, in terms of proportion of nonspe- binding sites was observed in the posteromedial cortical cific binding and amount of [3H]HACBO-Gly bound to the amygdaloid nucleus, whereas ,u binding sites were more tissue, with those obtained in homogenates. abundant than 8 sites in the hippocampus (Fig. 2 E and F,

-19 f" I~ ~ ~ ~ ~ ~ ~~ ,1 1. . c 1. ..:,...f .' .401, "I'.

A B C

FIG. 1. Autoradiographic visualization of enkephalinase in rat brain with 3 nM [3H]HACBO-Gly. (A) Total binding of [3H]HACBO-Gly. (B) [3H]HACBO-Gly binding in the presence of 1 /LM thiorphan (nonspecific binding). (C) [3H]HACBO-Gly binding in the presence of 1 ,uM captopril. Labeling is particularly dense at the level ofthe choroid plexus, substantia nigra, caudate putamen, globus pallidus, olfactory tubercle, parts of the amygdala, and the molecular layer of the cerebellum. Downloaded by guest on September 27, 2021 Neurobiology: Waksman et al. Proc. Natl. Acad. Sci. USA 83 (1986) 1525

Table 1. Comparative distribution of enkephalinase (EKase) and regions, 8 opioid sites were rather sparse but exhibited a of .t and 8 opioid receptors in rat brain pattern of labeling similar to that of At binding sites (Fig. 2F). EKase 8A5 The distribution ofenkephalinase was closely correlated with that of .u opioid receptors in more caudal regions [substantia Telencephalon nigra, periaqueductal gray matter, and interpeduncular nu- Olfactory tubercle ++ cleus 2 G and H and Table Cortex I and IV ++ ++ (Fig. 1, respectively)]. layers + In the spinal cord (Fig. 2 J-L), enkephalinase and A binding Cortex II and V +++ ++ layers ++ ++ sites were highly concentrated in the substantia gelatinosa. Cortex layer VI 0o Although sparse 8 binding sites were detected at the level of Nucleus accumbens ++ ++ the spinal cord (Fig. 2L), their concentrations were consid- Caudate putamen erably lower than those of binding sites (Table 1). Globus pallidus g Hippocampus CA1 + ++ + Hippocampus CA3 + ++ + DISCUSSION Dentate gyrus ++ +++ + The use of [3H]HACBO-Gly, a compound belonging to a new Lateral septum + + + + series of inhibitors of enkephalin-degrading enzymes (7, 24), Cortical amygdaloid nucleus* + + + + + ++ + + permitted a detailed analysis of the distribution of enkepha- Diencephalon linase in rat brain. The specificity of HACBO-Gly for Medial habenula ++ +++++ ++ enkephalinase is supported by previous demonstrations of a Mediodorsal thalamic nucleus + ++++ ++ close correlation in many brain regions between the levels of Ventroposterior thalamic [3H]HACBO-Gly binding and enkephalinase activity (17) and nucleus + +++ + between the potencies of various peptidase inhibitors to Entopeduncular nucleus + + +++ + displace [3H]HACBO-Gly binding and their efficiencies in Ventromedial hypothalamic inhibiting enkephalinase activity (17). The present finding nucleus + +++ + that [3H]HACBO-Gly binding to rat brain sections was Arcuate hypothalamic abolished by the highly specific nucleus + + 0 thiorphan but unaffected by the angiotensin-converting en- Mammillary bodies + + + + + zyme inhibitor captopril, further demonstrates that the Choroid plexus +++++ 0 0 enkephalinase was selectively labeled in these studies. Mesencephalon The autoradiographic analysis of [3H]HACBO-Gly bind- Superior colliculus ing, described in a preliminary study (25) and presented in Superficial gray layer ++ ++ ++ detail here, confirms a previous report (10) that the striatum Intermediate gray layer + + + + and substantia nigra are particularly enriched in enkephalin- Inferior colliculus + ++++ ++ ase. In addition, the present findings demonstrate the pres- Medial geniculate nucleus + ++++ ++ ence of enkephalinase in the hippocampus, thalamus, mo- Interpeduncular nucleus ++ ++++ + lecular layer of the cerebellum, periaqueductal gray matter, Periacqueductal gray matter + + ++++ + and substantia gelatinosa of the spinal cord. Substantia nigra ++++ ++++ ++ In general, a good correspondence between the localiza- Metencephalon tion of [3H]HACBO-Gly binding and the distributions of j Cerebellum and 8 opioid binding sites was observed. With the exception Molecular layer ++ 0 0 of the cerebellum and choroid plexus, labeling with Granular layer 0 0 0 [3H]DAMGE and/or [3H]DTLET correlated with the pres- Spinal cord ence of enkephalinase. Substantia gelatinosa +++ +++ + In some brain regions, such as the striatum, olfactory Sections were incubated with 3 rnM [3H]HACBO-Gly (EKase), 4 tubercle, and cerebral cortex, the distribution of enkepha- nm [3H]DAMGE (,u), or 4 nM [3H]r)TLET (8). Optical densities and linase resembled more closely that of 8 rather than ,uopioid corresponding amounts of ligands bound were determined as de- receptors. In the caudate putamen, for example, the scribed in Materials and Method.Is. ++++ +, >401 fmol/mg of enkephalinase and 8 opioid receptors showed similar uniform protein; + + + +, 201-400 fmol/mgr;++++, 101-200 fmol/mg; ++, distributions and higher concentrations in the ventropos- 51-100 fmol/mg; +, 26-50 fmol/mlg; 0, <25 fmol/mg. terior part of the putamen. This pattern of distribution has *Posteromedial nucleus. also been found for the striatal D2 dopamine receptors (26). These findings could provide a basis for the reported increase respectively) and septum (Fig. 2 B and C, respectively). The in striatal dopamine release induced by selective 8 agonists distribution of enkephalinase vvas moderate in the cortical (27) and by the potent inhibitor of enkephalin metabolism, amygdaloid nucleus and sparse ini the septum (Table 1), while kelatorphan (28). This latter result suggests the presence of a a moderate labeling, organized iin laminae, was present in the phasic enkephalinergic modulation ofnigrostriatal dopamine, hippocampus (Fig. 2D). Labelling of [3H]HACBO-Gly in the which is also supported by the potency ofthiorphan to induce hippocampus paralleled that of,£ and 8 opioid receptors in the a conditioned reward after injection into the rat ventral pyramidal cells CA1 and C)A3 regions. However, the midbrain (29). enkephalinase appeared more densely distributed in the The limbic seizures selectively induced by enkephalin dentate gyrus (Fig. 2D and TabliLe 1) and the outer parts ofthe analogs (30) have been attributed to their interaction with 8 stratum moleculare, two areas c:orresponding to the terminal opioid receptors located in the hippocampus (31). As shown fields of excitatory fiber tracts and known to contain high by the present findings, this region also contains moderate levels of enkephalin-like immutnoreactivity (23). levels of [3H]HACBO-Gly binding sites that could be in- [3H]HACBO-Gly binding was low in the thalamus, some- volved in the proconvulsant effect of enkephalinase inhibi- what higher in the hypothalamius, and showed moderately tors such as phosphoramidon (32). high density at the level of the entopeduncular nucleus (Fig. The distribution of enkephalinase in the substantia nigra, 2D). The ,uopioid binding sites, on the other hand, were very periaqueductal gray matter, and substantia gelatinosa of the dense in specific thalamic nucllei (Fig. 2E and Table 1) but spinal cord, on the other hand, correlated with the presence moderate in the entopeduncular nucleus (Table 1). In these of A rather than 8 opioid receptors. The high concentrations Downloaded by guest on September 27, 2021 1526 Neurobiology: Waksman et al. Proc. Natl. Acad Sci. USA 83 (1986)

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*AN INN FIG. 2. Autoradiograms showing the relationship be- tween the distribution ofenkephalinase (Left:A, D, G, and Ag. K. J) and that of A (Center: B, E, H, and K) and 8 (Right: C, 4L' MR F, I, and L) subtypes at different levels of G H I the rat brain. Tissue sections were incubated with 3 nM [3H]HACBO-Gly (Left), 4 nM [3H]DAMGE (Center), and 4 nM [3H]DTLET (Right), respectively, and apposed to LKB Ultrofilm for 2-3 months. (A-C) Caudate-putamen (.. ON, level. (D-F) Thalamus-hypothalamus level. (G-I) Substantia nigra level. (J-L) Spinal cord level. Optimal photographic representation of individual autoradiograms appears in this figure. For a real comparison of the levels of enkephalinase and of a and 8 opioid binding sites, see J K L Table 1.

of both enkephalinase and ,u receptors in areas implicated in linase present in the cerebellum serves to degrade other pain perception and analgesia (periaqueductal gray matter peptide substrates must await further experimentation. and substantia gelatinosa) is particularly significant, consid- The striatonigral system contains high levels of both ering the demonstrated central (6, 33) and spinal (34) anti- angiotensin-converting enzyme and enkephalinase, with the nociceptive effects of enkephalinase inhibitors and the pref- former enzyme being about 10 times more concentrated (20). erential involvement of a opioid receptors in the control of Several neuropeptides (enkephalins, substance P, and CCK- pain (33, 35). The low but measurable levels of 8 opioid octapeptide) are abundant in this brain area and could serve binding sites in the substantia gelatinosa could suggest a as substrates for both peptidases. However, degradation possible additional involvement of this type of opioid recep- studies on rat striatal slices and pharmacological experiments tors in spinal analgesia (34). using selective inhibitors (captopril for angiotensin-convert- In general, the distribution ofenkephalinase in rat brain did ing enzyme and thiorphan for enkephalinase) strongly sug- not correlate selectively with that of a particular opioid gest that substance P is preferentially cleaved by angiotensin- receptor subtype but overlapped the localizations of both 8 converting enzyme (42, 43), whereas the enkephalins are and 1L opioid receptors. The codistribution of enkephalinase metabolized by enkephalinase (6, 10). In the striatum, neither and opioid binding sites, along with the physiological effects of these enzymes appears to be involved in the inactivation of enkephalinase inhibitors, strongly supports the view that of CCK-octapeptide (44). enkephalinase is mainly involved in terminating the The concentration of enkephalinase calculated from quan- enkephalinergic signal. Nevertheless, owing to the broad titative autoradiography was found to be about one-fifth to specificity of enkephalinase (11), one cannot exclude that one-tenth that of opioid receptors. The relative affinities of neuropeptides other than the enkephalins could be cleaved by [Met5]enkephalin for enkephalinase and opioid receptors are the peptidase in some brain regions. Comparison of the in the micromolar (Km 20 ,uM) and nanomolar range (Kd = distribution of enkephalinase with those of enkephalinase- 1-10 nM), respectively. An efficient degradation by susceptible peptides such as substance P (36) and CCK- enkephalinase therefore requires that, at least during the (26-33) (37) and their respective receptors [substance P (38) nerve impulse, a micromolar concentration of the peptide and CCK-octapeptide (39)], however, indicates that should be present in the synaptic area. This implies that the [3H]HACBO-Gly binding follows more closely the localiza- physiologically active receptors should also exhibit a Kd tion of opioid binding sites than those of other neuropeptide value in the micromolar range, in order to ensure rapid and receptors. In the choroid plexus and cerebellum, the high repetitive synaptic communication (45). The low affinities amounts of enkephalinase contrast with the sparse distribu- exhibited by opioid receptors in binding experiments per- tion of both enkephalins and opioid receptors (40). Since the formed in vivo or in artificial physiological medium (46) are choroid plexus is also enriched in angiotensin-converting consistent with this hypothesis. enzyme (20), peptidases located at this level could regulate Another possible explanation could reside in the occur- the passage of various peptides at the brain-ventricles inter- rence for neuropeptides (40, 47, 48), as well as for other face. Despite the presence ofpresynaptic opioid receptors on neurotransmitters (49, 50), of chemical nonsynaptic trans- y-aminobutyric acid-activated terminals in rat cerebellum mission over relatively large distances (51). Both the long (41), the low concentrations of both enkephalin peptides and response latencies elicited by various peptides (52) and the opioid receptors in this region (40) contrast with the relatively ultrastructural visualization of nonsynaptic opioid binding high levels of enkephalinase. The possibility that enkepha- sites (53) might be compatible with the nanomolar affinity of Downloaded by guest on September 27, 2021 Neurobiology: Waksman et A Proc. Natl. Acad. Sci. USA 83 (1986) 1527 the peptide for its receptors. Accordingly, a decreased 20. Strittmatter, S. M., Lo, M. M. S., Javitch, J. A. & Snyder, S. H. concentration of the peptide at the receptor level could occur (1984) Proc. NatI. Acad. Sci. USA 81, 1599-1603. via as 21. Goodman, R. R., Snyder, S. H., Kuhar, M. J. & Young, W. S., III degrading enzymes (such enkephalinase) located closer (1980) Proc. Natl. Acad. Sci. USA 77, 6239-6243. to the site of release than to the site of action. 22. Gerfen, C. R. (1984) Nature (London) 311, 461-464. The present studies show a good correspondence between 23. Hong, J. S. & Schmid, R. (1981) Brain Res. 205, 415-418. the regional distributions of enkephalinase and 8 and ,u opioid 24. Fourni&Zaluski, M. C., Chaillet, P., Bouboutou, R., Coulaud, A., receptors in rat brain and support a selective role for Chdrot, P., Waksman, G., Costentin, J. & Roques, B. P. (1984) in transmission. As Eur. J. Pharmacol. 102, 525-528. enkephalinase regulating enkephalinergic 25. Waksman, G., Hamel, E., Bouboutou, R., Besselievre, R., evidenced by this study and by reports on the distribution of Fourni-Zaluski, M. C. & Roques, B. P. (1984) C. R. Acad. Sci. angiotensin-converting enzyme (20) and enkephalin conver- Ser. 3 299, 613-615. tase (54), the use of radiolabeled enzyme inhibitors as 26. Martres, M. P., Bouthenet, M. L., Sales, N., Sokoloff, P. & autoradiographic probes offers a highly efficient method for Schwartz, J. C. (1985) Science 228, 752-755. the regional localization of brain enzymes. Finally, quanti- 27. Chesselet, M. F., Cheramy, A., Reisine, T. D., Lubetzki, J., tative measurements of Glowinski, J., Fourni-Zaluski, M. C. & Roques, B. P. (1982) Life autoradiographic opioid receptors Sci. 31, 2291-2294. and enkephalinase levels in human brain could be used to 28. Petit, F., Hamon, M., Fourni-Zaluski, M. C., Roques, B. P. & investigate possible defects in the enkephalinergic system Glowinski, J. (1986) Eur. J. Pharmacol., in press. that have been associated with endogeneous depression and 29. Glimcher, P. W., Giovino, A. A., Margolin, D. H. & Hoebel, psychiatric disorders (55). B. G. (1984) Behav. Neurosci. 98, 262-268. 30. Haffmans, J., Blankwater, Y. J., Ukponmwam, 0. E., Zijlstra, We are grateful to Dr. Michael Dennis for reviewing the manu- F. J., Vincent, J. E., Hespe, W. & Dzol ic, M. R. (1983) script, Dr. Nicole Sallds for helpful discussion, and M.-L. Pernelet Neuropharmacology 22, 1021-1028. for typing. This work was supported by funds from the Fondation 31. Zieglgansberger, W., French, E. D., Siggins, G. R. & Bloom, F. 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