THE JOURNAL OF COMPARATIVE NEUROLOGY 296:130-158 (1990)

Fluorescent Histochemical Localization of Neutral Endopeptidase-24.11 (Enkephalinase)in the Rat Brainstem STEPHEN A. BACK AND CHARLES GORENSTEIN Department of Pharmacology, University of California, Irvine, California 92717

ABSTRACT Characterization of the distribution of the peptide-degrading enzyme neutral endopeptidase24.11 (E.C. 3.4.24.11; NEP; enkephalinase) in the rat brainstem was examined by means of a unique fluorescent histochemical method. Enzyme staining was completely blocked by three potent NEP inhibitors (thiorphan, phosphoramidon, and JHF-26) at a concentration of 50 nM, supporting the specificity of this method to visualize sites of NEP activity selectively. At all levels of the brainstem, NEP was localized to cell bodies, cell processes, or terminal-like fields and was localized to more than 90 distinct nuclei or subnuclei. In the mesencephalon these included the central gray, cuneiform n., dorsal and lateral tegmental n., inferior colliculus, interpeduncular n., lateral and medial geniculate n., central linear raphe n., mesencephalic n. of the trigeminal nerve, mammillary nuclei, occulomotor n., red n., superior colliculus, ventral n. of the lateral lemniscus, substantia nigra-ventral tegmental area, and the zona incerta. In the pons, NEP staining was restricted to fewer regions or nuclei, including the dorsal and ventral cochlear n., facial n., motor trigeminal n., principal sensory trigeminal n., parabrachial nuclei, pontine n., the oral and caudal pontine reticular n., pontine olivary nuclei, several pontine tegmental nuclei, pontine raphe nuclei, and the trapezoid n. In the cerebellum, staining was localized largely to the granule cell layer of the cerebellar cortex. Scattered staining was observed in the molecular cell layer. The medulla contained extensive NEP staining localized to nuclei that included the ambiguus n., dorsal motor n. of the vagus, hypoglossal n., inferior olivary n., prepositus hypoglossus n., solitary tract n., nuclei of the spinal tract of the trigeminal n., and the lateral, medial, and superior vestibular nuclei. Nuclei of the medullary reticular formation that were also richly stained for NEP included the raphe magnus n., raphe obscurus n., raphe pallidus n., dorsal, lateral, and ventral reticular nuclei of the medulla, and the gigantocellular, lateral paragigantocellular, linear, paramedian and parvicellular reticular nuclei. The widespread distribution of N E P in the brainstem suggests the existence of a number of functional systems, including the pathways involved in the mechanisms of pain and analgesia, which are potential targets of NEP inhibitors. In most regions, the distribution of NEP closely overlapped with that reported for the enkephalins, and showed a more restricted overlap with the reported distribution of substance P. Key words: histochemistry, enkephalins, substance P

A growing body of evidence indicates that the enzyme neutral endopeptidase-24.11 (NEP, E.C. 3.4.24.11, enkephalinase) may play a key role in regulating the action of peptides in a wide variety of neural (Hersh, '82; McKelvy and Blumberg, '86) and non-neural (Llorens and Schwartz, '81; Ronco et al., '88) tissues. NEP was first characterized as a membrane-bound metalloenzyme on the brush border of the proximal convoluted tubules of the kidney where it appears to play a role in the inactivation of circulating peptides (Kerr and Kenny, '74). The primary mechanism of o 1990 WILEY-LISS, INC.

inactivation of circulating atrial natriuretic peptide, for example, is via renal NEP, suggesting a role for NEP in blood volume and pressure regulation (Stephenson and Kenny, '87). NEP has been shown to cleave several peptides active in the immune system, including interleukin-lp (Pierart et al., '88) and the chemotactic factor, fMLF (f Met-LeuPhe) (Connelly et al., '85). It has recently been demonAccepted December 15,1989.

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Fig. 1. Low-power fluorescent photomicrographs of coronal sections from adjacent regions of the caudal medulla of a rat, at the level of the spinal nucleus of the trigeminal nerve (Sp5), demonstrating the specificity of the histochemical method. A: Typical distribution pattern of NEP reaction product in an untreated tissue section. Pa5 = paratrigeminal n. (arrow); LRt = lateral reticular n. B,C Absence of reaction

product in tissue incubated in histochemical staining solution containing 50 nM thiorphan (B) or 50 nM phosphoramidon (C). D: The appearance and distribution of NEP reaction product observed in tissue incubated in the presence of 300 pM captopril did not differ from untreated tissue (A). See Materials and Methods for details. See Table I for abbreviations. Bar in A-D = 300 pm.

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Figure 2

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strated that the gene for NEP is highly conserved and is identical to the gene for common acute lymphocytic leukemia antigen (CALLA), a primary marker for acute lymphocytic leukemia (Letarte et al., '88). The enhanced expression of NEP in a number of other malignancies including glioblastomas, melanoma, and colorectal cancer suggests that NEP may regulate the activity of peptides involved in tumor growth or, alternatively, modulate the action of immune system factors involved in the surveillance and response to tumor antigens (Jongeneel et al., '89). In the CNS, NEP appears to play a key role in regulating central peptidergic mechanisms of pain and analgesia (for review, see Hersh, '82; McKelvy and Blumberg, '86). A number of brainstem peptides including opiate peptides (Malfroy et al., '78; Gorenstein and Snyder, '80; Hersh, '84)' substance P (Matsas et al., '85), and neurotensin (Almenoff et al., '81) are cleaved by NEP and are present in ascending and descending pathways that converge in the spinal dorsal horn to integrate central responses to nociceptive stimuli (for review, see Basbaum and Fields, '84). A variety of studies indicate that the antinociceptive effects of NEP inhibitors may occur by centrally blocking the metabolism of peptides that promote analgesia. A number of these inhibitors potently inhibit the enzyme in vitro and in vivo and have significant naloxone-reversible antinociceptive effects (Roques et al., '80; Fulcher et al., '82; Yaksh and Harty, '82; Fournie-Zaluski et al., '83; Fournie-Zaluski et al., '84; Chipkin et al., '88). Other studies with NEP inhibitors suggest that the enzyme may mediate the metabolism of more than one peptide involved in promoting analgesia (Murthy et al., '84; Mendelsohn et al., '85). The interpretation of the antinociceptive effects of all these compounds is complicated by the fact that in most studies NEP inhibitors were administered intracerebroventricularly or intraperitoneally and, thereby, may be blocking the action of NEP a t multiple sites in the neuraxis. In the present study, the brainstem distribution of NEP was determined histochemically, verified by using three chemically distinct NEP inhibitors, and was compared to that reported for the enkephalins and substance P (SP). Neural systems whose function may be under the partial control of NEP were identified, including the brainstem circuitry involved in pain control systems.

hours. The injection coordinates were 1.0 mm posterior to bregma, 1.5 mm lateral to the sagittal suture, and 3.6 mm below the dura. Tissue fixed with 1.5% formaldehyde was prepared as previously described (Back and Gorenstein, '89a). Thirty-five to 50 ym-thick sections were cut with a Vibratome (Lancer). Free-floating sections were stained for NEP activity, as previously described (Back and Gorenstein, '89a), in a solution of 50 mM tris(hydroxymethy1)aminomethane (Tris)-HC1, pH 7.4, containing 0.5 mM glutarylala-ala-phe-4-methoxy-2-naphthylamide(GaapMNA; Enzyme Systems Products, Livermore, CA), 6 mM nitrosalicylaldehyde (NSA; Eastman-Kodak, Rochester, NY), and 20 yg/ml of endopeptidase-free aminopeptidase M (APM). Tissue sections were incubated at 37°C for 1-4 hours or a t 4°C for 1-3 days, and the reaction was terminated by rinsing the sections in ice-cold 50 mM Tris-HC1, pH 7.4. Sections were counterstained with a 1pg/ml aqueous stock solution of ethidium bromide prior to being coverslipped with spectroscopic grade glycerol diluted 1:6 with 50 mM phosphate buffered saline, pH 6.8. Histochemical controls were done as previously described (Back and Gorenstein, '89a). For each region of the brainstem examined, the specificity of the histochemical localization was examined by determining the lowest concentration of NEP inhibitors that completely blocked the appearance of enzyme reaction product. Briefly, tissue sections were preincubated for 45 min at 22OC in solutions of 50 mM Tris-HC1, pH 7.4 containing an NEP inhibitor thiorphan, phosphoramidon, or JHF-26 (1 x lo-' to 1 x M), or captopril (1 x lo-' to 3 x lo-* M). Tissue sections were stained a t 4°C or 37"C, as described above, in a solution containing the same respective concentration of drug as in the preincubation. NEP reaction product was photographed on a Leitz Dialux epifluorescence microscope fitted with a wide band violet (catecholamine) excitation filter (355-425 nm). Ethidium bromide fluorescence was visualized with a narrow band green (rhodamine) excitation filter (530-560 nm). With a wide band blue (FITC) excitation filter (390-490 nm), the NEP reaction product and the ethidium bromide counterstain could be viewed simultaneously. The distribution of reaction product was verified by the atlas of the rat brain of Paxinos and Watson ('86).

MATERIALS AND METHODS RESULTS Specificity of the histochemical localization

The distribution of NEP in the brainstem was analyzed by using 28 male Sprague-Dawley adult rats (8-12 weeks old). Eight of these animals under rompum-ketamine general anesthesia were injected into the lateral cerebral ventricle with 250 yg (10 pg/yl) of colchicine (Sigma, Saint Louis, MO) in 0.9% sodium chloride and allowed to survive 14-18

Figure 1 demonstrates the specificity of the staining method employed to localize NEP. A representative example of NEP staining in the brainstem is shown a t the level of

Fig. 2. A: Low-power fluorescent photomicrograph of a coronal section at the level of the mesodiencephalic border showing the appearance and distribution of NEP staining in the ventral medial geniculate n. (MGV) and the adjacent zona incerta (21).LT = lateral terminal n. of the accessory optic tract. B,C Fluorescent photomicrographs demonstrating the NEP staining in the dorsolateral geniculate n. (B) and the corresponding photomicrograph (C) of the same section as in B showing the distribution of cell bodies counterstained with ethidium bromide. Several examples of the codistribution of NEP reaction product (B) and

counterstained cell bodies are indicated (arrows). D. Low-power fluorescent photomicrograph of a coronal section of the rat mesencephalon showing the appearance and distribution of NEP staining in the substantia nigra, zona compacta (SNC) and zona reticulata (SNR). cp = cerebral peduncle. E,F High-power fluorescent photomicrographs of the identical area of a coronal section through the SNC showing the codistribution (arrows) of NEP reaction product (E)with SNC cell bodies (F), counterstained with ethidium biomide. Bar in A = 150 pm, bar in B,C = 100 pm; bar in D = 200 pm; bar in E,F = 50 pm.

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Fig. 3. Representative fluorescent photomicrographs showing the appearance and distribution of N E P staining in coronal sections through the superior colliculus (SC) (A-E) and the inferior colliculus (F). A: Rich NEP staining in the zonal (Zo) and superfical gray (SuG) layers of the SC and scattered staining in the intermediate gray layer (InG). Op = optic nerve layer; InWh = intermediate white layer. B Laminar distribution of ethidium bromide counterstained cell bodies in the medial part of the identical area shown in A. C Higher-power photomicrograph showing the appearance of the N E P reaction product in the SuG.

D,E High-power detail of the SuG showing the codistribution (arrows) of NEP reaction product (D) with medium-sized cell bodies (E), counterstained with ethidium bromide. F: Low-power photomicrograph showing the distribution of N E P staining in the inferior colliculus (IC) and the more ventrally situated sagulum (Sag). Two large brightly stained cell bodies of the mesencephalic n. of the trigeminal nerve (Me5) are seen a t lower left. Bar in A,B = 150 pm; bar in C = 30 pm; bar in D,E = 20 pm; bar in F = 300 Mm.

the spinal nucleus of the trigeminal nerve in the caudal medulla (Fig. 1A). In the presence of low concentrations of the NEP inhibitors thiorphan (50 nM, Fig. 1B) and phophoramidon (50 nM, Fig. 1C) all staining was blocked a t every level of the brainstem. The effective concentrations of these inhibitors are consistent with their reported Ki values when tested in vitro (Roques et al., '80; Almenoff and Orlowski, '84). The potent bidentate N E P inhibitor JHF-26 (Ki = 0.24 nM; Bernard Roques, personal communication)

blocked N E P staining a t a concentration as low as 10 nM (data not shown). By contrast, tissue stained in the presence of 300 pM captopril (Fig. lD), an angiotensin-converting enzyme inhibitor, did not differ from untreated tissue (Fig. 1A). In summary, three structurally and chemically distinct N E P inhibitors potently blocked all NEP activity in the brainstem indicating that the histochemical method employed localizes a single enzymatic activity corresponding to NEP.

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Fig. 4. Representative fluorescent photomicrographs showing the appearance and distribution of NEP staining in a coronal (A) and a sagittal section (B) of brainstem regions associated with the auditory system. A: Low-power photomicrograph showing the distribution of NEP staining in the anterior ventral cochlear n. (VCA). Note the minimal staining in the adjacent vestibulocochlear nerve root (8vn).B

Low-power sagittal section of the dorsal brainstem showing intense NEP staining in the dorsal cochlear n. (DCo). Several collections of cells are stained in the deep cerebellar nuclei (DCN) situated adjacent to one of the cerebellar folia, which displays intense staining over the granule cell layer (Gr); colchicine-treated. Bar in A and B = 200 pm.

General appearance of histochemically labelled cellular elements

combination of controlled staining a t 4"C, coupled with colchicine pretreatment, permitted the discrete localization of reaction product within some cell processes.

N E P staining was observed at all levels of the brainstem. Optimal staining was generally obtained in tissue sections incubated a t 4°C. At this low temperature, the reaction lead to the formation of a more discrete punctate reaction product than that obtained at 37°C. The quality of the staining was of three general types. The predominant type, seen in most regions, consisted of an evenly distributed collection of discrete punctate crystals which defined the general contours of cell somata. This type allowed visualization of cell bodies (ranging in size from about 5 to 30 pm in diameter) and sometimes the associated cellular processes (e.g., Fig. 2E,F). A second type of staining appeared as tightly organized arrays of crystalline reaction product which delineated the structure of processes associated with individual cell bodies or with fiber pathways (e.g., Figs. 11A,D; 12F,G). A third type of staining consisted of a very fine, evenly dispersed, precipitate which was localized within regional boundaries and which appeared to correspond to small cellular processes or terminals (e.g., Fig. 11F). In untreated animals, NEP staining was readily observed indicating that the levels of endogenous enzyme were sufficient to be detectable. The staining in many NEP-positive cell groups was enhanced in colchicine-treated animals, but, in general, the regional distribution of NEP staining did not change in response to colchicine. The most marked change in the amearance of NEP staining following colchicine treatmentwas the visualization of dense NEP-positive fiber plexuses in a number of brainstem nuclei. Hence, the

REGIONAL AND CELLULAR LOCALIZATION OF NEP IN THE BRAINSTEM Organization of the results Figures 2-14 are representative photomicrographs of NEP staining in the brainstem a t levels extending from the posterior commissure (the mesodiencephalic border) to the spinomedullary transition. Given that the NEP staining largely visualized cellular profiles, schematic maps are provided in Figures 13-24, which show the corresponding regional distribution of NEP-positive cell bodies and, where visualized, their associated processes. These morphological distinctions are discussed in the text and summarized in the alphabetical list of abbreviations (Table 1).The maps also provide an approximate indication of the relative density of NEP-stained cellular elements. Figures 13-24 and Table 1 have been organized so that the distribution of N E P may be directly compared with that previously obtained for the two putative endogenous substrates of NEP: the enkephalins (see Fallon and Leslie, '86) and substance P (see Ljungdahl et al., '78).

Mesodiencephalic border The brainstem distribution of N E P in this study begins with the mesodiencephalic border at the level of the poste-

Figure 5

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rior commissure, an area of the brainstem rich in NEP (Fig. 13). Included in this region are a number of NEP-rich thalamic, subthalamic, hypothalamic, and mesencephalic structures which extend from the diencephalon into the mesencephalon. These regions are included in this study because of their functional relationship to structures in the mesencephalic brainstem. Although scattered cells were seen within the mesencephalic core at this level, the majority of NEP-positive cells were concentrated in various laterally and ventrally situated nuclei. Geniculate and pretectal nuclei (Figs. 2, 13). The staining of numerous small round or fusiform cell bodies in the zona incerta merged medially with light staining of many small round cells localized to all subdivisions of the medial geniculate nucleus (Fig. 2A). Within visual areas, scattered NEP stained cells were observed throughout the pretectal nuclei and in the adjacent more medial areas dorsal to the posterior commissure (Fig. 13). Many small cells were stained in the dorsolateral geniculate nucleus (Fig. 2B,C). Within the ventral lateral geniculate nucleus, rich staining was observed in both subdivisions, as well as in the lateral terminal nucleus of the accessory optic tract. Substantia nigra, ventral tegmental area, mammilary, and interpeduncular nuclei (Figs. 2 , 1 3 - 15). In the zona incerta, NEP staining merged ventrolaterally with that of the substantia nigra (Fig. 2D). Numerous cell bodies and associated proximal processes stained for NEP in the pars compacta (Fig. 2E,F), and scattered cell bodies were also observed in the pars reticulata. Adjacent to the nigra, the ventral tegmental area (VTA) was also richly stained for NEP. The staining in the VTA merged dorsolaterally with that of the zona incerta and substantia nigra and ventrally with the mammillary nuclei (Fig. 13). Scattered cells were seen throughout the mammillary nuclei with the greatest concentration observed within the medial mammillary nucleus. More caudally, numerous small scattered NEPstained cells were observed throughout all subdivisions of the interpeduncular nucleus (Figs. 14,15). The staining was uniform with no particular nucleus staining more prominently. Ventral to the interpeduncular nuclei, the pontine nuclei displayed rich staining of many small evenly dispersed cell bodies (Fig. 15).

A t the lateral border of the central gray the large principal cell bodies of the mesencephalic nucleus of the trigeminal nerve were intensely stained for NEP, and were visualized throughout the full extent of this nucleus (see also, Fig. 5A). Staining was also observed in the nuclei of the oculomotor complex ventral to the central gray. Intense staining was observed to overlie all the large motor neurons of the oculomotor nucleus (Figs. 8A,B, 14, 15). In addition, light staining was seen more rostrally in the nucleus of Darkschewitsch (Fig. 13) and the Edinger-Westphal nucleus (Figs. 14,15). In the rostral ventral mesencephalon, scattered NEP stained cells were present in the prerubral (Fig. 13) and retrorubral fields (Fig. 15). A number of prominently stained cell bodies were usually observed in both the magnocellular and parvocellular divisions of the red nucleus (Fig. 14). Superior colliculus (Figs. 3 , 14, 15). A laminar distribution of NEP reaction product was observed in the superior colliculus (Fig. 3A,B). The densest staining was situated in the outer layers, particularly in the superficial gray layer (Figs. 3A, 14, 15). The other layers, contained scattered NEP-positive cells in no particular pattern. A discrete subset of ovoid small and medium-sized cells stained for the enzyme (Fig. 3C-E). Inferior colliculus and other auditory relay nuclei (Figs. 3, 4, 16-19). The inferior colliculus was one of several relay nuclei of the brainstem auditory system which stained richly for NEP (Figs. 16-18). The external cortex of the inferior colliculus contained numerous cell bodies of varying size and morphology (Figs. 3F, 16-18), as did the central nucleus. Ventral to the inferior colliculus, scattered stained small cells were observed in the sagulum (Figs. 3F, 16). NEP-positive cell bodies were also visualized in other auditory nuclei including the nuclei of the lateral lemniscus (Fig. 16), the nucleus of the trapezoid body (Figs. 16-18) and the ventral (Figs. 4A, 18, 19) and dorsal (Figs. 4B, 19) cochlear nuclei. Raphe nuclei (Figs. 14- 16) and mesencephalic reticular nuclei (Figs. 14- 17). Occasional NEP-positive cells were observed in the raphe nuclei. These nuclei included the rostral linear n., the caudal linear n. (Figs. 14,15), and the dorsal raphe n. (Fig. 16). Scattered NEP stained cells were seen throughout the deep mesencephalic nucleus (DpMe; Figs. 14, 15) and rostral mesencephalic reticular formation with a higher concentration of cells at the mesodiencephalic border. More medially, numerous small and medium-sized cells were stained for NEP in the cuneiform nucleus (Figs. 16, 17). The staining in this nucleus merged with staining in the adjacent lateral parabrachial nucleus and the dorsal nucleus of the lateral lemniscus (Fig. 17).

Mesencephalon Central g r a y and adjacent nuclei (Figs. 14-16). At all levels of the mesencephalic central gray numerous small stained cell bodies were localized. The staining was most heavily concentrated in the dorsal and lateral central gray, with fewer stained cells scattered medially, ventrally, and adjacent to the cerebral aqueduct. In the medial central gray small cell bodies were occasionally seen demarcating the lateral border of the ependymal cell layer of the cerebral aqueduct. In general, a unique subset of small caliber cells were stained in each region of the central gray. No rostralcaudal differences in the distribution of NEP staining were detected.

Fig. 5. Representative appearance and distribution of NEP staining in nuclei of the trigeminal nerve. A Fluorescent photomontage of the dorsal pons at the level of the motor trigeminal n. (Mo5) showing intense NEP staining localized over the posterodorsal tegmental n. (PDTg) and the mesencephalic n. of the trigeminal nerve (Me5). The arrows indicate the pial surface. 4V = fourth ventricle; LC = locus coeruleus. B-E: High-power fluorescent photomicrographs showing the codistribution of NEP reaction product (arrows) with ethidium bromide-counterstained

Pons Tegmental nuclei (Figs. 5 , 15-18). Several of the pontine tegmental nuclei had distinct NEP staining including the pedunculopontine tegmental n. (Fig. 15), the ventral

small cells of the PDTg ( C ) and large cells of the Me5 (E).F Fluorescent photomicrograph of a sagittal section through the brainstem showing intensely stained cell bodies and processes in the Mo5 and the lateral vestibular n (LVe); colchicine-treated. G Higher-power detail of F showing the intense staining of the Mo5 and light staining over the more rostrally situated dorsal subcoeruleus n. (SubCD). Bar in A = 150 pm; bar in B-E = 50 pm; bar in F = 200 pm; bar in G = 100 pm.

Figure 6

BRAINSTEM ENKEPHALINASE LOCALIZATION tegmental n. (Figs. 16, 17), the reticulotegmental n. (Fig. 16), the laterodorsal tegmental n. (Fig. 17), the pericentral dorsal tegmental n. (Fig. 17), and the posterodorsal tegmental n. of Gudden (Fig. 18). The staining was often quite striking as was observed in the caudal pons in the posterodorsal tegmental n. (Fig. 5A-C). In this nucleus a discrete subset of small cells stained for N E P (Fig. 5B,C). In colchicine-treated animals a very fine network of fibers was observed in sagittal sections through the pedunculopontine tegmental nucleus which merged rostrally with NEP staining in the cuneiform n. Trigeminal nuclei (Figs. 5, 15- 18). Rich NEP staining was observed in many of the sensory and motor nuclei of the trigeminal system. The majority of the large neurons of the mesencephalic n. of the trigeminal nerve were intensely stained (Fig. 5D,E). Relatively few stained cells were scattered throughout the principal sensory trigeminal nucleus (Figs. 17, 18). The staining in this nucleus merged more caudally with staining of a similar nature in the spinal trigeminal n., oralis (Fig. 19). Within the motor trigeminal n., a few small-sized cells, as well as large motor neurons, stained lightly for NEP (Figs. 17-18). In colchicine-treated animals, staining was markedly increased. Intensely labelled large cell bodies and processes were visualized coursing obliquely through the nucleus in a dorsoventral orientation (Fig. 5F,G). Pontine olivary nuclei (Figs. 6, 16-18). In the ventral pons, rich NEP staining delineated several distinct cell types in the pontine olivary nuclei (Fig. 6A). In the superior paraolivary n. numerous intensely stained small to medium-sized round cells were seen (Fig. 6B,C). More laterally, the lateral superior olivary n. (LSO), showed a distinctly different staining pattern where many small fusiform cells stained within a network of fine associated processes (Fig. 6D,E). The staining in the rostral periolivary region (RPO) and the LSO merged ventrally with similar staining in the medial and lateral ventral periolivary complex (Fig. 6F).

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Cerebellum (Figs. 7, 18). The cerebellum showed a laminar distribution pattern of rich NEP staining a t all cerebellar levels. The most intense staining was observed overlying the granule cell layer (Figs. 7A-C, 18).Due to the densely packed nature of the granule cells it was often difficult to discern whether the N E P staining corresponded to distinct cellular elements, although apparent labeling of granule cells was suggested (Fig. 7B,C). A significant but lesser amount of NEP staining was seen over the molecular cell layer, where it was possible to discern labeling over discrete cellular elements and, on occasion, their associated processes (Fig. 7D,E). No N E P staining was observed in the Purkinje cell layer.

Medulla

Raphe nuclei and pontine reticular formation (Figs. Three pontine raphe nuclei, the raphe pontis n. (Fig. 17), the nucleus pallidus (Fig. 18), and the nucleus raphe magnus (Figs. 18, 19), all had moderate scattered staining. In the raphe magnus, scattered stained cells were most prominently seen a t the level of the facial n. in the rostral ventral medulla (Fig. 19). The N E P staining in the pontine reticular formation was light to moderate in the oral and caudal parts (Figs. 16-18) and no distinct populations of cellular elements were observed. Parabrachial nuclei, locus coeruleus, and subcoer uleus n. (Figs. 5, 17, 18). A moderate number of scattered NEP-positive cells were observed in the lateral and medial parabrachial nuclei (Figs. 17, 18). Only occasional cells were stained in the locus coeruleus. Scattered light NEP staining was also seen ventral to the parabrachial nuclei in the Kolliker-Fuse n. and in the subcoeruleus nucleus (Fig. 5G).

Cranial nerve nuclei (Figs. 8, 14, 15, 19-23). Within the brainstem, NEP was localized to all the cranial nerve nuclei. Staining of the nuclei innervating the extraocular eye muscles included the nuclei of the occulomotor complex (Figs. 8A,B, 14, 15) and the abducens n. (Fig. 19). The facial motor n. showed light-to-moderate staining of scattered large cell bodies in untreated animals. In colchicinetreated animals, numerous large cells bodies and an extensive network of processes stained for NEP (Figs. 6F, 19). As discussed above, N E P was also richly localized to both the dorsal and ventral cochlear nuclei (Fig. 4A,B). The NEP staining of a small collection of medium-sized cells in the nucleus ambiguus merged with that of the adjacent parvocellular reticular n. (Figs. 8C,D, 20-21). Light to moderate staining was observed over the cell bodies of the dorsal motor nucleus of the vagus (Figs. 8E,F, 21-23). As previously described (Back et al., '89), N E P also richly localized to the motor neurons of the hypoglossal nucleus (Figs. 8G,H, 21-23). Spinal trigeminal nuclei and adjacent nuclei (Figs. 1, 19-23). All subdivisions of the spinal nucleus of the trigeminal nerve contained extensive populations of N E P stained cell bodies and processes (Figs. 19-23). The staining in all subdivisions was similar in intensity, extent, and distribution and included dense staining in the marginal zone (Fig. lA,D). Many cells of the paratrigeminal n. (Fig. 21) and some displaced cells and intercalated cell clusters in the spinal tract of the trigeminal nerve also stained for NEP. Occassional NEP-positive cells were also seen in the cuneate and external cuneate nuclei (Figs. 20-24). In the caudal medulla within the nucleus gracilis, a small number of medium-sized elongated cells stained for NEP. Characteristically, these cells were evenly spaced and oriented in serial arrays that were distributed in an oblique dorsal-ventral orientation (Figs. 22-23). These groups of stained cells extended into the gracile tail n. in the spinomedullary transition (Fig. 24), as previously described (Back and Gorenstein, '89a). Solitary nucleus (Figs. 9, 20-23). The solitary n. showed rich NEP staining throughout its rostral-to-caudal extent. NEP localized to numerous small and medium-sized

Fig. 6. Representative appearance and distribution of NEP staining in the nuclei of the superior olivary complex. A Fluorescent photomontage showing rich bilateral staining of the nuclei of the superior olivary complex at the level of the caudal extent of the mesencephalic central gray. Note the autofluoresence of the white matter of the pyramidal tracts (py) M-LVPO = medial and lateral ventral periolivary nuclei; RPO = rostral periolivary region. B-E High-power fluorescent photomicrographs showing the codistribution of NEP reaction product (arrows)

with ethidium bromide-counterstained cells of the superior paraolivary n. (SPO) in C and the lateral superior olivary n (LSO) in E. F: Fluorescent photomontage of a sagittal section through the ventral brainstem showing the distribution of NEP staining in several nuclei of the superior olivary complex relative to a more caudally situated collection of intensely stained cell bodies and processes in the facial motor n. (7); colchicine-treated. The arrows indicate the pial surface. Bar in A = 300 pm; bar in B-E = 50 pm; bar in F = 200 pm.

16-19).

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cells (Fig. 9A,B) distributed throughout all portions of the nucleus (Figs. 20-23). Laterally and ventrally the staining merged with staining in the surrounding cell groups of the reticular formation. Vestibular nucleus and prepositus hypoglossus n. (Figs. 9, 19,20). The lateral, medial, superior and spinal vestibular nuclei each displayed a rich distinct staining pattern for NEP. At the rostral extent of the medulla a collection of small and medium-sized cell bodies and their associated processes was stained in the superior vestibular n. (Fig. 19). The lateral vestibular n. (Dieter’s nucleus; Fig. 5F) displayed richly stained small and medium-sized cell bodies and processes. At the level of the prepositus hypoglossus nucleus, which contained large NEP stained cell bodies (Figs. 9A,B, 20), rich NEP staining was observed in the medial and spinal vestibular nuclei. NEP staining was seen in numerous small and medium-sized cells in the spinal vestibular nucleus (Fig. 9A,B). Colchicine treatment resulted in the visualization of an extensive network of NEP-stained processes in the spinal vestibular n. (Fig. 9C). By contrast, no NEP stained processes were observed in the medial vestibular nucleus, but numerous small to medium-sized round cell bodies were stained (Fig. 9D,E). Raphe nuclei (Figs. 10, 19-23). The medullary raphe nuclei were richly stained for NEP. The rich NEP staining in the raphe obscurus and paramedian reticular nuclei (Figs. 19-23) visualized many of the morphological features of the cells in these nuclei. The staining in this region was continuous ventrally with the staining in the inferior olivary nuclei and laterally with the staining in the adjacent reticular nuclei (Fig. 10A). The cells stained were oriented in vertical arrays parallel to the midline and showed staining which decreased over the region of the cell nucleus but which clearly delineated the processes of these cells (Fig. 10B,C). Inferior olivary nucleus (Figs. 10, 11,20-23). The inferior olivary nucleus contained numerous NEP stained cell bodies throughout its rostral to caudal extent (Figs. 10A, 20-23). The staining in this nucleus was clearly distinct from that of the surrounding reticular formation (Fig. 11C,E). These cells were uniformly shaped, medium-sized, and were localized in aggregates separated by largely unstained regions (Fig. 11F,G). In the area immediately surrounding these cells was a finely precipitated reaction product which was suggestive of staining in fine processes or terminals. Medullary reticular formation and adjacent nuclei (Figs. 11, 19-23). One of the most striking localizations of NEP in the CNS was seen in the medullary reticular formation, The endogenous levels of NEP in the reticular formation were sufficient to visualize numerous cell bodies and, often, associated proximal cell processes (Fig. 11A-E). For example, Figure 11C shows the varied morphology of

the cells that were visualized by NEP staining in the gigantocellular reticular n. of the medulla (Figs. 19-21). Large triangular-shaped multipolar cells were strikingly stained in this nucleus (Fig. 11D). The cellular staining in this and other reticular nuclei (e.g., the lateral paragigantocellular reticular n., Fig. 11E)demarcated the boundaries of the cells and was often markedly reduced over the region of the cell corresponding to the cell nucleus. In general, there was not a clear demarcation in the staining among adjacent reticular nuclei. For example, the staining in the parvocellular reticular n. merged with the distinct staining of the linear reticular n. and of cells of the reticular formation adjacent to the nucleus ambiguus (Figs. 19-21). In the caudal medulla, NEP also localized richly to cells of both the dorsal and ventral reticular nuclei of the medulla (Figs. 22, 23). The staining in these regions merged laterally with that of the medial border of the caudal spinal trigeminal n. Ventrally, a distinct population of cells in the lateral reticular n. was also richly stained (Figs. 21-23). Spinomedullary transition (Figs. 12,24). The rich staining in the caudal medulla was continuous with extensive NEP staining in the high cervical cord. At this level, the staining in the spinomedullary transition (Fig. 24) was characterized by the presence of a rich collection of medium and large-sized round and fusiform cell bodies which were concentrated around the central canal (Fig. 12A,B,E) in lamina X. More caudally, these cells were continuous with NEP stained cells in the central cervical nucleus and in lamina X, as previously reported (Back and Gorenstein, ’89a). Situated between these lamina X cells and the adjacent reticular formation were numerous labelled fusiform cells contained into two arcs which spanned the white matter (Fig. 12A,C). Some of the cells had a long stained process radiating away from the nucleus (Fig. 12D). In colchicine-treated animals, numerous apparent fine NEPpositive fibers were visualized which streamed laterally or ventrally (Fig. 12F). This fiber-like staining appeared to be localized in the white matter between rays of serially oriented glial cells (Fig. 12G,H).

Fig. 7. Representative fluorescent photomicrographs showing the appearance and distribution of NEP staining in coronal sections through the rat cerebellum. A: Fluorescent photomontage of a coronal section through the second cerebellar lobule a t about the level of the pons shown in Fig. 6A. There is extensive NEP staining of the granule cell layer (Gr) with scattered staining over the molecular cell layer (Mol). B,C: Higher-power detail of the NEP staining in the Gr (B) and the corresponding ethidium bromide fluorescent counterstain (C) of the

identical region shown in B suggests that a subset of the numerous granule cells in C may express the enzyme. D,E: Higher-power detail of the NEP staining in the Mol (D) and the corresponding ethidium bromide counterstain (E) of the identical region shown in D shows the presence of several NEP-positive medium-sized cell bodies with a single radially oriented process. Note that several of these cells are situated at the Gr/Mol border. No staining was observed over the Purkinje cell layer (P).Bar in A = 200 pm; bar in B-E = 30 pm.

DISCUSSION Specificity of the histochemical method In order to examine the role of NEP in the regulation of opiate and nonopiate peptides, this study has characterized the cellular elements in the rat brainstem which contain NEP activity and has provided the first detailed maps of the enzyme’s distribution. Evidence that NEP was the only enzymatic activity localized in the tissue was supported by the use of three chemically distinct NEP inhibitors. At a concentration of 50 nM, all three inhibitors caused a similar, potent inhibition of all histochemical staining detected in the brainstem which was in close agreement with the

Figure 8

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reported Ki of these compounds: thiorphan (4.7 nM; Roques et al., '80); phosphoramidon (12 nM; Almenoff and Orlowski, '84), and JHF-26 (0.24 nM, Bernard Roques, personal communication). All three inhibitors, thus, appear to act at identical sites in the rat brainstem. In addition, the selectivity of the method for NEP is further supported by the lack of effect of the thiorphan-like angiotensin converting-enzyme inhibitor, captopril, when tested at concentrations up to 300 pM. Hence, the anatomical distribution of NEP reported in this study reflects the target sites of action of these NEP inhibitors and may be useful to predict the physiological effects of these drugs and their potential side effects. Several studies have examined the anatomical distribution of NEP in the brainstem using autoradiography. Using tritiated HACBO-Gly, Waksman et al. ('86) autoradiographically localized NEP to the mesencephalon and the cerebellum, but did not report on the localization in the pons or medulla. In the mesencephalon, NEP was localized to the superior colliculus, inferior colliculus, medial geniculate nucleus, interpeduncular nucleus, periaqueductal gray, and substantia nigra, in agreement with the present study. A recent exhaustive study by Pollard et al. ('89) reported a widespread distribution for NEP in the brainstem which is in very close agreement with the distribution of histochemically labeled cellular elements observed in the present study. In contrast to the autoradiographic localization of NEP, immunocytochemical localization of NEP did not detect the enzyme in any area of the brainstem except the substantia nigra and the interpeduncular nucleus (Matsas et al., '86). This difference in localization may be due to the sensitivity to formaldehyde fixation of NEP epitopes detected by the antibodies employed.

degree of variability in the relative amounts of enzyme and either peptide that might exist in a given region. Brainstem motor systems. NEP was richly localized to large apparent motor neurons in a number of cranial nerve motor nuclei including the occulomotor n., the motor trigeminal n., the abducens n., the facial motor n., and the hypoglossal n.. All of these nuclei receive fibers or terminals immunoreactive for Enk (Simantov et al., '77; Uhl et al., '79; Finley et al., '81; Sar et al., '82; Khachaturian et al., '83; Senba and Tohyama, '83; Petrusz et al., '85; Fallon and Leslie, '86) or SP (Ljungdahl et al., '78), but no EnkIR or SPIR cell bodies have been identified in any of these nuclei, In rats subjected to hypoglossal nerve axotomy, we have previously shown that NEP is synthesized in hypoglossal motor neurons (Back et al., '89b) which are known to receive both enkephalinergic axodendritic synapses and SP axosomatic synapses (Connaughton et al., '86). We have also localized NEP activity to motor neurons of the ventral horn of the spinal cord (Back and Gorenstein, '89a) which also have EnkIR and SPIR fibers and varicosities in the surrounding neuropil (Bresnahan et al., '84). Hence, throughout the brainstem populations of NEP-positive motor neurons appear to be closely associated with converging EnkIR or SPIR inputs. This generalization also appears to hold true for the extrapyramidal motor system, which contains several regions rich in NEP-positive cell bodies. We have previously visualized an extensive population of NEP-positive cell bodies in the globus pallidus (Back and Gorenstein, '89b), an area also known to receive a dense enkephalinergic innervation but which contains no EnkIR cell bodies (Palkovits et al., '81; Del Fiacco et al., '82). NEP-labeled cells and processes were also extensively localized to the pedunculopontine tegmental n., which projects to several areas of the basal ganglia including the globus pallidus (Jackson and Crossman, '83). In agreement with previous studies (Waksman et al., '87; Pollard et al., '87, '89), we found rich NEP staining in the substantia nigra (SN). The majority of the NEP stained cells visualized in the SN localized to the pars compacta (SNC), which has been shown to receive rich Enk immunoreactive inputs (Simantov et al., '77; Uhl et al., '79; Finley et al., '81; Sar et al., '82; Petrusz et al., '85), and lesser amounts of the dynorphins (Fallon et al., '85; Fallon and Leslie, '86) and SP (Brownstein et al., '76; Kanazawa and Jessel, '76; Ljungdahl et al., '78; Paxinos et al., '78). Some neurons of the SNC have also been shown to be EnkIR (Petrusz et al., '85). NEP also localized to scattered cells of the substantia nigra, pars reticulata (SNR), which have been shown to receive largely dynorphinergic (Fallon et al., '85) and SPIR projections. The distribution of NEP in the SN, thus, parallels that of the enkephalins (Fallon et al., '85), and shows a differential localization largely to the SNC. Given the rich collection of dopaminergic neurons in the SNC

Anatomical relationship of NEP to the enkephalins,other opiate peptides and substance P, and functional correlations As summarized in Table 1, the common feature of all NEP-positive regions in the brainstem was the visualization of cell bodies. In some regions, NEP-positive processes or terminal-like fields were also seen in association with a given population of labeled cells. When the distribution of NEPpositive cellular elements was compared with that reported for the enkephalins (see Table l ) , all NEP-containing regions were also found to contain enkephalin immunoreactivity (EnkIR). While the majority of NEP-positive regions had both EnkIR cell bodies and fibers, a number of regions had only EnkIR fibers. Many, but not all, NEP-positive regions have previously been reported to contain SPIR. In contrast to the enkephalins, most regions containing NEPpositive cell populations contained SPIR fibers or terminal fields. As discussed below, there appeared to be a large

Fig. 8. Representative fluorescent photomicrographs from coronal sections through four nuclei contributing to brainstem cranial nerves showing the codistribution (arrows) of NEP reaction product (A,C,E,G) with cell bodies, visualized in the respective identical areas of each nucleus with ethidium bromide fluorescent counterstain (B,D,F,H), A , B Intensely stained cell bodies and processes of the occulomotor n. DR = dorsal raphe. C,D: NEP staining visualized round large-sized cell bodies of the ambiguus n. (Amb) (arrows) and medium-sized fusiform cell bodies of the adjacent parvicellular reticular n. (PCRt). E,F Light

NEP staining (E) over the cell bodies (F)of the dorsal motor nucleus of the vagus (10). Note that often the fine precipitate of reaction product was largely visualized around the perimeter of these large cells. 12 = hypoglossal nucleus. G , H Rich distribution of reaction product ( G ) associated both with cell bodies and the surrounding neuropil of the hypoglossal nucleus (H). Note that often the fine precipitate of reaction product was largely visualized around the perimeter of these large cells. 4V = fourth ventricle. Bar in A,B = 100 pm; bar in C-H = 50 pm.

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Fig. 9. A , B Low-power fluorescent photomontages showing the extensive distribution of NEP staining (A) in the dorsal medulla at the level of the prepositus n. (PrH) and the corresponding distribution of cell bodies, in the identical area as in A, demonstrated by ethidium bromide fluorescent counterstain (B). Both the medial vestibular n. (MVe) and the spinal vestibular n. (SpVe) are stained for NEP. Ventral to these nuclei there is extensive staining in the nucleus of the solitary tract (Sol), which merges with staining in the adjacent reticular forma-

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tion. C: Higher-power photomicrograph of NEP staining in the SpVe from a colchicine-treated animal revealed the presence of intensely labelled processes and scattered cell bodies. The arrows indicate the pial surface. D,E: High-power detail of NEP staining in the MVe (D) and the corresponding ethidium bromide fluorescent counterstain (E), which demonstrate the presence of a discrete subset of cells in the nucleus which express the enzyme (arrows).Bar in A,B = 150 pm; bar in C = 100 pm; bar in D,E = 40 pm.

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receiving opiate peptide inputs (Johnson et al., '80), NEP may colocalize to some of these neurons and, thus, be postsynaptically situated relative to the sites of peptide release. Several biochemical and physiological studies support the localization of NEP to the SN. Inhibitors of NEP have been shown to increase the spontaneous activity of nigral cells exhibiting excitatory responses to striatal stimulation (Bier et al., '87). The degradation of SP by NEP was supported by studies using synaptic membranes prepared from rat SN (Oblin et al., '88). Potassium-evoked release of SP-like IR from slices of rat SN is selectively enhanced by NEP inhibitors but not by inhibitors of angiotensin-converting enzyme (Mauborgne et al., '87). Brainstem circuitry mediatingpain and analgesia. NEP-positive cell bodies were richly localized to brainstem nuclei known to be involved in the descending and ascending circuitry mediating mechanisms of pain and analgesia. In the rostra1 ventral medulla, numerous NEP-positive cells localize to nuclei which receive rich EnkIR (Hokfelt et al., '77b; Simantov et al., '77; Uhl et al., '79; Finley et al., '81; Glazer et al., '81; Beitz, '82; Conrath-Verrier et al., '83; Petrusz et al., '85; Zamir et al., '85; Fallon and Leslie, '86; Millhorn et al., '87; Back et al., '90) and SPIR (Cuello and Kanazawa, '78; Hokfelt et al., '78; Ljungdahl et al., '78). Many of these regions are also rich in mu-opioid receptors (Bodnar et al., '88; Bowker and Dilts, '88). These nuclei include the raphe magnus n., the gigantocellular reticular n., pars alpha, the paragigantocellular reticular n., and the lateral paragigantocellular reticular n. which largely compose the descending pathways which control spinal nociceptive neurons (see Basbaum and Fields, '84, for review). The rich localization of NEP to pain control pathways is consistent with the fact that NEP inhibitors have potent, centrally-mediated, naloxone-reversible, antinociceptive effects in rats and mice (Roques et al., '80; Fulcher et al., '82; Yaksh and Harty, '82; Fournie-Zaluski et al., '83, '84; Chipkin et al., '88). In addition, other data indicate that NEP may mediate the metabolism of more than one peptide involved in promoting analgesia (Murthy et al., '84; Mendelsohn et al., '85). It is, thus possible that the antinociceptive effects of NEP inhibitors could be mediated through decreased NEP metabolism of both the enkephalins and SP. Given that SP produces a naloxone-reversible analgesia when injected into the PAG (Mohrland and Gephart, '79), the extensive distribution of NEP in cells of the PAG may serve to inactivate both enkephalins released from inhibitory interneurons acting on mu-opioid receptors (Chaillet et al., '84; Goodman and Pasternak, '85; Bodnar et al., '87; Pasternak, '88), as well as SP released from terminal fields in the PAG. Hence, NEP inhibition might result in elevation

Fig. 10. A Low-power fluorescent photomontage showing the extensive distribution of NEP staining in the midrostral medulla at the level of the prepositus n. (PrH). The NEP staining in the raphe obscurus n. (Rob) merges with the surrounding reticular formation including the paramedian reticular n. (PMn) and the gigantocellular reticular n (Gi). At the bottom of the figure, extensive staining of the inferior olivary nuclei (10)is seen. 4V = fourth ventricle. B,C:High-power detail of the NEP staining in the Rob (B) and the corresponding ethidium bromide fluorescent counterstain (C),of the identical region shown in B. Note that the fine precipitate of reaction product visualized both the large cell bodies and a dense plexus of associated cell processes (arrows). The arrow in B indicates the midline. Bar in A = 200 pm; in C,D = 50 pm.

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Figure 11

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of SP levels in the central gray which may reinforce the antinociceptive effects of the enkephalins.

Visual. EnkIR and SPIR cells and/or fibers have been visualized in the rat anterior pretectal area, the ventral n. of the lateral geniculate body, the superior colliculus, the terminal nuclei of the accessory optic tract, the occulomotor n., and the hypoglossal prepositus n. (Hokfelt et al., '77a; Finley et al., '81; Petrusz et al., '85; Fallon and Leslie, '86; Miguel-Hidalgo et al., '89). As discussed below, all these regions of the visual system also contain NEP-positive cell bodies. In the rat superior colliculus, we observed NEP to be densely localized in the zonal and superfical gray layers (Fig. 3A) where SPIR neurons and fibers are also densely localized (Miguel-Hidalgo et al., '89). The mu and kappa opiate receptors are also enriched in the superficial gray layer of the rat superior colliculus (Morris and Herz, '86; c.f. Fig. 25, K, Kornblum et al., '87), whereas EnkIR fibers show only a scattered distribution (Miguel-Hidalgo et al., '89). Deeper layers of the SC contained only scattered NEP stained cellular elements, as is also true for the enkephalins. By contrast, SPIR patches have been detected in the rat (Miguel-Hidalgo et al., '89); and, in the cat, EnkIR is organized into a series of patches (Graybiel et al., '84). More dense EnkIR has been observed in the intermediate gray layer (Miguel-Hidalgo et al., '89) where NEP staining is also higher. It also noteworthy that the terminal nuclei of the accessory optic tract are rich in NEP-positive cells. These nuclei contain high opiate receptor binding (Herkenham and Pert, '82) and dense SPIR (Ljungdahl et al., '78). NEP is extensively localized to cholinergic regions which serve to coordinate eye movements and reflex movements involving the head and eyes. NEP localizes to the pretectal area and the Edinger-Westphaal n. involved in pathways subserving the direct and consensual light reflexes. The EW n. gives rise to parasympathetic innervation of the ciliary ganglion and is both cholinergic and NEP-positive, as are the n. of Darkschewitsch and interstitial n. of Cajal. Parallel innervation of the NEP-positive abducens and hypoglossal prepositus nuclei appears to coordinate the integration of signals which control the velocity of eye movement (Belknap and McCrea, '88). The widespread presence of NEP in regions involved with the coordination of eye movements suggests that coordinated gaze and visual tracking may be coupled by certain broadly expressed peptidergic systems. Auditory. Collections of NEP-positive cell bodies were represented in essentially every nucleus of the brainstem

auditory relay system: the dorsal and ventral cochlear nuclei, the periolivary nuclei, the nuclei of the trapezoid body, the nuclei of the lateral lemniscus, the inferior colliculus and the medial geniculate nucleus. EnkIR cell bodies or fibers are most extensively seen in the marginal layer of the medial geniculate n., the external n. of the inferior colliculus, the periolivary nuclei, the ventral trapezoid n., and the dorsal and ventral cochlear nuclei (Simantov et al., '77; Uhl et al., '79; Finley et al., '81; Petrusz et al., '85; Fallon and Leslie, '86). Scattered EnkIR cells and fibers have also been seen in the lateral trapezoid n. and the nuclei of the lateral leminscus, regions which also have less pronounced NEP staining. SPIR is generally low throughout the auditory system except for the marginal nucleus of the medial geniculate n. and the most ventral part of the inferior colliculus (Ljungdahl et al., '78). Numerous Dyn 1-8 and MERGL cell bodies are present in periolivary regions and the ventral trapezoid n. (Fallon and Leslie, '86) and may contribute to the descending olivocochlear bundle. Colocalization of Enk and dynorphin together or with choline acetyltransferase-IR has been observed in olivocochlear efferents of the lateral superior olivary n. (White and Warr, '83; Altschuler et al., '84; Abou-Madi et al., '87; Altschuler et al., '88). Hence, the enriched levels of NEP in cell bodies of the LSO may serve to metabolize one or more opiate peptides released in the organ of Corti. The extensive localization of NEP to the external n. of the inferior colliculus suggests that the enzyme may metabolize other peptides in this region which is low in opiate peptides and SP. One candidate is cholecystokinin octapeptide (CCK8), which is cleaved by NEP in vitro and by brain synaptosomes (Deschodt-Lanckman, '85; Zuzel et al., '85), although less actively than Enk or SP (Turner et al., '85). CCK-8 fibers and terminals localize throughout the medial geniculate and the inferior colliculus and CCK-8 olivocollicular and lemnisco-collicular afferents also exist (Adams and Mugnaini, '85; Fallon and Seroogy, '85). Vestibular and proprioceptive. Collections of NEPpositive cell bodies were localized in multiple nuclei of vestibular and proprioceptive systems including: the red n., the medial, lateral, superior and spinal vestibular nuclei, the inferior olivary n., the lateral reticular n., the paramedian reticular n., and cells of the granule and molecular cell layers of the cerebellum, the flocculus, and the paraflocculus. The localization of NEP to vestibular nuclei correlates well with that of the enkephalins, whereas little SPIR is seen in these nuclei (Ljungdahl et al., '78). EnkIR cells and fibers have been localized to all the vestibular nuclei (Simantov et

Fig. 11. Fluorescent photomicrographs demonstrating various morphological features of cell types visualized by NEP histochemistry in the midrostral medulla. A: High-power photomicrograph showing the localization of NEP reaction product within a single cell in the raphe obscurus nucleus. Note that the reaction product appears to distribute along the perimeter of the cell, and the region of the cell corresponding to the cell nucleus is largely devoid of reaction product. The arrows indicate the presence of puncta of reaction product in the proximal processes of the cell. B Corresponding ethidium bromide fluorescent counterstain of the same cell as in A. The fluorescence of several puncta of reaction product was bright enough to visualize together with the counterstain (arrows),thus demonstrating the presence of the reaction product within the proximal cell process. Note, at left, the presence of several fluorescent profiles not within the plane of focus. C: Low-power fluorescent photomicrograph demonstrating the visualization of numer-

ous cell bodies of varied size and morphology in the gigantocellular reticular n. of the medulla stained by NEP histochemistry. Cell bodies of the inferior olivary n. (10) are stained for NEP at the bottom of the figure. D,E High-power fluorescent photomicrographs of cells stained for NEP in the gigantocellular reticular n. (D) and the paragigantocellular reticular n. (E).Note the presence in E of staining of cell bodies in the dorsal inferior olivary n. (10). F,G Higher-power detail of the NEP staining in the inferior olivary n (F) and the corresponding ethidium bromide fluorescent counterstain (G) of the identical region shown in F. Several examples of the codistribution of NEP reaction product with olivary cell bodies are indicated (arrows). Note the presence of a finely dispersed reaction product in the neuropil, closely associated with the cell bodies, whereas the region between the subnuclei is essentially devoid of staining. Bar in A,B = 20 Fm; bar in C = 50 Gm; bar in D-G = 30 Gm.

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Figures 13-14

Fig. 12. Fluorescent photomicrographs demonstrating the appearance and distribution of NEP staining in coronal sections through various regions of the spinomedullary transition. A , B Fluorescent photomontages of the NEP staining in the region of the central canal (CC) and, in B, the respective identical area as in A showing the corresponding distribution of cell bodies counterstained with ethidium bromide. The arrows indicate several examples of the codistribution of reaction product with cells in this region. C Low-power photomicrograph of the NEP staining in the region surrounding the central canal and the adjacent reticular formation. D Higher-power detail of the region indicated in C showing a collection of laterally situated medium

and large-sized cell bodies. The arrows indicate a fine precipitate of NEP reaction product within two long processes of a single large cell. E Higher-power detail of the region indicated in C showing the visualization of several different morphological cell types. F: Colchicine treatment resulted in the apparent visualization of many finely stained processes streaming from the region of lamina X. G , H Higher-power detail (G) of the NEP staining in F and the corresponding distribution of cell bodies counterstained with ethidium bromide (H). The extensive fiber-like staining appears to be localized in the neuropil in the areas between the tiny cell bodies. Bar in A,B,F = 100 wm; bar in C = 150 wm; bar in D,E,G,H = 50 gm.

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al., '77; Uhl et al., '79; Finley et al., '81; Sar et al., '82; Petrusz et al., '85; Fallon and Leslie, '86), and the action of Enk in the MVe is mediated through mu- and delta-opiate receptors (Iasnetsov and Pravdivtsev, '86). EnkIR fibers extend throughout the inferior olivary complex (Finley et al., '81; Sar et al., '82; Petrusz et al., '85; Fallon and Leslie, '86). SPIR is enriched in the most lateral parts of the inferior olivary complex (Ljungdahl et al., '78; Bishop and Ho, '84). The localization of NEP in the cerebellum is consistent with the finding of both preproenkephalin mRNA and EnkIR in neurons of the granule cell layer of the cerebellum and scattered EnkIR fibers in the molecular cell layer (Petrusz et al., '85; Bloch et al., '86; Harlan et al., '87). The distribution of proenkephalin A is consistent with the distribution of kappa opioid binding sites in the cerebellum (Castanas et al., '86; Kornblum et al., '87). Although much more NEP appears to be present in the cerebellum than opiate peptides, Pollard et al. ('89) have also observed rich binding to N E P sites in the cerebellum. However, in contrast to the present study, they observed N E P to be more extensively localized to the molecular cell layer than the granule cell layer.

Orofacial and visceral somatosensory and motor systems NEP staining was visualized in regions involved in visceral sensory and motor functions related to the face, nasal, and oral cavities, and the gut including the trigeminal nuclei (the mesencephalic n. of the trigeminal nerve, the principal sensory n. of the trigeminal nerve, the motor n. of the trigeminal nerve and the paratrigeminal n.), the facial motor n., the n. ambiguus, the n. of the solitary tract, the area postrema, the region of the salivatory n., the dorsal motor n. of the vagus, and the hypoglossal nucleus. All these regions contain EnkIR fibers or perikarya (Simantov et al., '77; Uhl et al., '79; Finley et al., '81; Sar et al., '82; Petrusz et al., '85; Fallon and Leslie, '86) and SPIR fibers (Ljungdahl et al., '78) and may constitute a widespread primitive system which modulates and integrates the complex sensory, proprioceptive, and motor pathways involved in tasting, mastication, swallowing, and the gag reflex. A number of these regions are special visceral efferent cholinergic motor nuclei which innervate branchial skeletal musculature of the face, larynx or pharynx (i.e. the facial motor n., the n. ambiguus, the dorsal motor n of the vagus, and hypoglossal n.) supporting the apparent association between cholinergic nuclei and NEP in brainstem regions which receive Enk and SP inputs.

Cardiovascular and respiratory centers The epinephrine-rich C1 area of the rostroventrolateral medulla is an important region for central mechanisms of

Figs. 13-24. Plots of NEP reaction product distribution visualized in association with cell bodies (black dots) and cell processes (short black lines) in coronal sections of the albino rat brain. Figures 13-24 were redrawn from the stereotaxic atlas of Paxinos and Watson ('86). The stereotaxic levels correspond to interaural f4.20 (Fig. 13), +2.96 (Fig. 14), +2.28 (Fig. 15), +0.70 (Fig. 16), +0.20 (Fig. 17), -0.80 (Fig. 18), -1.52 (Fig. 19), -2.80 (Fig. 20), -4.24 (Fig. 21), -4.80 (Fig. 22), -5.30 (Fig. 23). Figure 24 is redrawn from a plot of NEP reaction product distribution observed in the spinomedullary transition prepared with the aid of an Olympus drawing tube attached to a Leitz Dialux Epifluorescent microscope. The regional boundaries were determined by using an ethidium bromide counterstain. See Table 1for the list of abbreviations.

Figures 15-16

cardiovascular regulation via tonic vasomotor control and the baroreceptor reflex (Reis et al., '87). Neurons of the C1 area are intermixed with EnkIR and SPIR neurons (Hokfelt et al., '77b; Lorenz et al., '85). Given the rich distribution of NEP-positive cells in the rostroventrolateral medulla, NEP may function in regulating peptidergic actions in this area. The activity of the C1 area is further regulated via connections with a number of Enk/SP/NEP-containing regions including the parabrachial nuclei, the dorsal motor n. of the vagus, the caudal n. of the solitary tract, and the paramedian reticular n. (Reis et al., '87). The medial and lateral nuclei of the solitary tract (NTS) are associated with cardiovascular and respiratory control, respectively. EnkIR cells and terminals are concentrated in

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Figures 22-24

association with the medial NTS, whereas the lateral NTS displays predominantly Dyn B labeling (Lee and Basbaum, '84). Mu agonists acting in the NTS elicit an increase in mean arterial pressure or heart rate mediated through increased sympathetic activity (Appel et al., '86; Hassen and Feuerstein, '87), whereas SP mainly affects respiratory rate (Carter and Lightman, '85). The uniform distribution of NEP within the NTS would be consistent with a role for NEP in the inactivation of both opiate peptides and SP. In addition, the parabrachial nuclei also constitute an important part of the brainstem cardiovascular system, and all contain extensive NEP, Enk, SP, and catecholamine-rich neuronal elements (Armstrong et al., '81; Milner et al., '84). In summary, the extensive localization of NEP in regions involved with cardiovascular control suggests that the ac-

tions of Enk or SP on catecholamine neurons may be partly regulated by NEP.

Functional considerations:NEP-peptide mismatch? Both the present study and studies localizing NEP autoradiographically have found that NEP showed an extensive localization in a number of areas of the brainstem. It was a common finding in the present study for the majority of the cells in many nuclei to stain for NEP. The question thus arises whether NEP may be more widely distributed than any single one of its putative peptide substrates. In the spinal cord we have observed NEP to be extensively localized to the majority of cells in many regions of the cord (Back and Gorenstein, '89a). This finding is compatible with

S.A. BACK A N D C. GORENSTEIN

154

TABLE 1. Regional Distribution of Cells (C) and Fibers/Terminal-like Fields (F) i n the Rat Brainstem for NEP, the Enkephalins, and SubstanceP' Abbreviation 3 6 7 7n 10 12 A& Amb

AP

APTD APTV BIC CeC CG CGD CGM cic CIC CLi CnF

CP cu DCo Dk DLG DLL DpMe DR DTgP ECIC ECu Eth EW F1 fr g7 GiA Gi Gr Gr(Cb) IC icp ILL InG InWh I0 IPA IPC IPI IPL IPR IRt KF LC LIm Li 11 LM LP LPB LPGi LRt LRtF'C LSO LT LVe LWO MdV MdD Me5 MGD MGV ML m rnlf MM MMN MnR MC5 Mol MP MPB mtg MVe MVPO OP OPT

Region Principal oculomotor nucleus Ahducens nucleus Facial nucleus Facialnerve Dorsal motor nucleus of the vagus Hypoglossal nucleus Accessory trigeminal nucleus Nucleus ambiguus Area postrema Anterior pretectal area, dorsal part Anterior pretectal area, ventral part Brachium of the inferior colliculus,nucleus Central cervical nucleus Central (periaqueductal) gray Central gray, dorsal part Central gray, medial part Commissure of the inferior collidus Central nucleus of the inferior colliculus Caudal (central) linear nucleus of the raphe Cuneiform nucleus Cerebral peduncle, basal part Cuneate nucleus Dorsal cochlear nucleus Nucleus of Darkachewitsch Dorsal lateral geniculate nucleus Dorsal nudeus of the lateral lemniscus Deep mesencephalic nucleus Dorsal raphe nucleus Dorsal tegmental nucleus, pericentral External cortex of the inferior collidus Exkrnal cuneate nucleus Ethmoid thalamic nucleus Acceanory omdomotor nucleus (Edinger-Westphal) Flocculus Fasiculus retroflexua Genu of the f a d nerve Gigantocellular reticular nucleus, pars alpha Gigantocellular reticular nucleus Gracile nucleus Granular cell layer, cerebellar cortex Inferior colliculus Inferior cerebellarpeduncle (restifom hcdy) Intermediite nucleus of the lateral lemniscus Intermediite gray layer, superior colliculus Intermediate white layer, superior colliculus Inferior olivary nucleus Interpeduncularnucleus, apical part Interpeduncular nucleus, central part Interpeduncular nucleus, intermediate subnucleus Interpeduncular nucleus, lateral subnucleus Interpeduncular nucleus, rostral subnucleus Intermediate reticular nucleus Kolliker-Fuse nucleus Locua cceruleus Laterodorsal tegmental nucleus Linear nucleus of the medulla Lateral lemniscus Lateral mamrmllary nucleus Lateral posterior thalamic nucleus Lateral (dorsal) parabrachial nucleus Lateral paragigantocellular nucleus Lateral reticular nucleus Lateral reticular nucleus, parvocellular part Lateral superior olive Lateral terminal nucleus of the acceawry optic tract Lateral vestibular nucleus Lateroventral periolivary nucleus Dorsal reticular nucleus of the medulla Ventral reticular nucleus of the medulla Nucleus of the mesencephalic tract of the trigeminal nerve Medial geniculate nucleus, dorsal part Medial geniculate nucleus, ventral part Medial mammiUarynucleus, lateral part Medial lemniscus Medial longitudinal fasidus Medial m a m m d h y nucleus, medial part Medial mammiUary nucleus, median part Medial raphe (superior central) nucleus Motor trigeminal nucleus Molecular cell layer, cerebellar cortex Medial mamrmllary nucleus, posterior part Medial (ventral) parabrachial nucleus Mammillotegmental tract Medial vestibular nucleus Medioventral periolivary nucleus Optic nerve layer, superior colliculus Olivary pretectal nucleus

NEP

Enkephalins

C C CiF

F F F F F F CiF CiF

-

C CiF

C C/F C C C C C C C C

C C C

C -

C C C C C C C C C C C C C -

CiF C/F C C C -

C C C CiF C C C C C C C C C C -

C C C CiF CiF C CiF C CiF C C/F

C/F C C C C -

C C -

CiF C C C -

C C C C

-

SP F F F -

F

F F F F F F

-

-

CiF CiF C/F CiF CiF F C/F F CiF

CiF F CiF F F

-

F F F F

CiF CiF F F CiF CiF CiF F CiF CiF CiF F

CiF F CiF F F F CiF F F C F C

-

-

CiF F F C C/F F CiF F F F F CiF CiF CiF C/F F CiF CiF CiF F F CiF F CiF F CiF CiF CiF CiF CiF CiF F F CiF F C/F CiF F

CiF F F

CiF

-

CiF F F C/F F CiF CiF -

CiF CiF F CiF

__

-

F

C F F F CiF F F CiF F F F CiF F F F F F F F C/F F F F F F F F F F F ~

F F -

F -

F -

F F (continued)

BRAINSTEM ENKEPHALINASE LOCALIZATION

155

TABLE 1. (Continued) ~~

Abbreviation P Pa5 PBP POT PCGS PCRt PDTg PFI PGi PMV PMn PN Pn PnC PnO PP PPTg PR PI5 PrH RLi RMC RMg Rob RPa RPC RPO RPn RRF RtTg RtTgP RVL Sag SCP SG SMT SNC SNL SNR Sol Spb SPX Sp5I Sp50 SPO

s m

SPVe SUI SubCD SuG SUM

SuVe TZ VCA VLGMC VLGPC VLL VPO VTA VTg ZI

zo

Region

NEP

Purkinjecell layer, cerebellarcortex Paratrigeminal nucleus Parabrachial pigmented nucleus Posterior thalamic nucleus gr., triangular P a r a d e a r glial substance Parvocellularreticular nucleus Pcatercdorsal tegmental nucleus (Gudden) P~aflOLXUllU Paragignnhellular reticular nucleus PremammiUarynucleus, ventral part Paramdan reticular nucleus Paranigral nucleus Pontine nuclei Pontine reticular nucleus, caudal part Pontine reticular nucleus, oral part Peripeduncular nucleus Pedunculopontine tegmental nucleus Preruhral field Principalsensory trigeminal nucleus Prepaitus hypglwal nucleus Rastral linear nucleus of the raphe Red nucleus, magnocellular part Raphe magnus nucleus Raphe obseurus nucleus Raphe pallidus nucleus Red nucleus,parvocellular part Rcatral periolivary region Raphe pontia nucleus Retroruhralfield Reticulotegmentalnucleusof the pons Reticulotegmentalnucleus ofthe pons, pericentral part Rmtral ventrolateral reticular nucleus Saguhun nucleus Superior cerebellar peduncle Suprageniculatethalamic nucleus S u h d o t h a l a m i c nucleus Substantianigr4 compact part Substantianigra, lateral part Substantianiga, reticular part Nucleus of the solitary tract Sphenoid nucleus Nucleus of the spinal tract of the trigeminal nerve, caudal part Nucleus of the spinal tract of the trigeminal nerve, interpositus part Nucleus of the spinal tract of the trigeminal nerve, oral part Superior paraolivarynucleus Subpeduncular tegmental nucleus Spinal vestibular nucleus Suprafacialnucleus Subcoeruleusnucleus, dorsal part Superficialgray layer, superior colliculus SupramammiUary nucleus Superior vestibular nucleus Nucleus of the trapezoid body Ventral cochleararea Ventral lateral geniculate nucleus, magnocellularpart Ventral lateralgeniculatenucleus, parvocellular part Ventral nucleus of the lateral lemniscua Ventral periolivary nucleus Ventral tegmental area Ventral m e n t a l nucleus zonaincerta zonal layer, superior colliculus

Enkephalins

SP

-

-

-

C C C C CiF C C CiF C CiF C C C C C CiF C C CiF C C C C/F CiF C C C C C C C C

CiF F CiF

-

F F F -

CiF C/F

F F

-

C C CiF CiF CiF C C CiF C/F CiF C C CiF C C C C C C C C C C C C C

C C

-

-

CiF CiF

C/F F F

F F F F CiF

-

F

CiF

F F F F F

-

-

F CiF

F F F F CiF CIF CiF

F

F CiF CiF CiF F F CiF F F

F F CiF F C F CiF F F CiF CfF C/F CIF F CiF F CiF

F F CiF F F F F F -

F F F F F C/F F CiF F F F -

F

CiF F

CiF

-

CiF

C/F F F F F F F F

F F CiF CiF CiF

CW CiF CiF CiF F F F

F F F C/F

'TheregionaldiatrihutionoftheenkephalinsandsubstancePshowninthetableis summarizedfromstudiescitedinthetext(seeDiscuasion).Abhreviationsinthetahleand thefigureaareaccordu to those used in the atlas of Paxinos and Watson ('8s).

the extensive localization of Enk and SP to virtually every region of the cord (Ljungdahl et al., '78; Bresnahan, '84; c.f., Fig. 24, Petrusz et al., '85). Similarly, a continuous network of EnkIR and SPIR fibers have been observed to extend throughout the middle level of the medulla oblongata (Ljungdahl et al., '78; c.f., Fig. 19, Petrusz et al., '85). This observation is compatible with our finding that NEPpositive cell bodies are extensively localized throughout this level in the medulla (e.g., Fig. 10A). Thus, NEP is associated with extensive populations of cell bodies which appear to receive converging peptidergic inputs from multiple sources. Hence, in regions of the brainstem where the endogenous Enk or SP levels are sufficient to observe immunoreactive fibers or terminals, there does not appear to be a mismatch between the distribution of NEP and its putative sub-

strates. Using combined fluorescent visualization of the enkephalins and NEP in the same tissue section, we have also observed EnkIR varicosities to be extensively localized in the neuropil surrounding NEP stained motor neurons in the hypoglossal nucleus (Back et al., '89b). In areas where the level or distribution of NEP appears to differ from one of its putative peptide substrates (e.g., in the cerebellum, inferior colliculus, or locus coeruleus), the mismatch may reflect 1)a level of the peptide which is too low to detect in postmortem tissue; 2) a level of NEP in terminal fields which may be too low to detect; 3) the existence of multiple peptide substrates for NEP in a given region; 4) the fact that NEP may degrade a peptide released globally, in a neuroendocrine fashion, which acts on sites distal to the site of its release; 5 ) continued expression of NEP in cells which,

S.A. BACK AND C. GORENSTEIN

156 during development, displayed transient peptidergic expression which is undetectable in the adult animal; 6) the lower affinity and rate of cleavage of peptide neuromodulators compared to classical neurotransmitters (the IC50 of N E P for most peptides is in the micromolar rather than the nanomolar range; Gorenstein and Snyder, ’80). NEP is an enzyme that is broadly associated with multiple functional systems involving sensory, special sensory, motor and proprioceptive processing. I t localizes to cholinergic, catecholaminergic, and serotonergic nuclei, where it may regulate peptide neuromodulation of the action of these primary neurotransmitters in the postsynaptic cell. In many regions, NEP may be colocalized in the cells which express the biosynthetic enzymes that synthesize primary neurotransmitters. We have found support for this in the hypoglossal n., for example, where N E P and choline acetyltransferase are each found to localize to all the motor neurons of the nucleus (Back et al., ’89). A postsynaptic site of expression of NEP relative to Enk or SP terminal inputs could account for the strong correlation in many brainstem regions between the distribution of EnkIR or SPIR fibers or varicosities and NEP-positive cells. It appears likely that N E P inhibitors could potentially affect multiple levels of information processing in the brainstem. In animals studies, no pronounced side effects or behavioral changes (e.g., respiratory depression; Chipkin et al., ’88) have been reported from N E P inhibitors. This suggests that more sensitive behavioral tests may be needed or that N E P inhibitors may be well tolerated despite their extensive sites of action in the CNS. The present data on the distribution of sites of action of NEP inhibitors should, therefore, be useful in designing future physiological studies to assess the role of NEP-inactivated peptides in the whole animal or in isolated functional systems.

ACKNOWLEDGMENTS This research was supported by NIDA grant DA03131 and predoctoral fellowship DA05255. S.A.B. is a recipient of a Pharmaceutical Manufacturers Association Foundation Medical Student Research Fellowship. We thank Drs. James Fallon, Stewart Hendry, Sandra Loughlin, Charles Ribak, and John Swett for many helpful discussions and suggestions. We are grateful to Dr. Bernard Roques for his generous gift of JHF-26.

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