Functional and anatomical localization of mu opioid receptors in the striatum, amygdala, and extended amygdala of the nonhuman primate

Functional and Anatomical Localizationof Mu Opioid Receptors in the Striatum,

Amygdala, and Extended Amygdala ofthe Nonhuman Primate

JAMES B. DAUNAIS,1 SHARON R. LETCHWORTH,1 LAURA J. SIM-SELLEY,2

HILARY R. SMITH,1 STEVEN R. CHILDERS,1AND LINDA J. PORRINO1*

1Department of Physiology and Pharmacology, Center for the Neurobiological Investigationof Drug Abuse, Wake Forest University School of Medicine,

Winston-Salem, North Carolina 27157-10832Pharmacology and Toxicology Department and Institute for Drug and Alcohol Studies,

Virginia Commonwealth University, Medical College of Virginia,Richmond, Virginia 23298

ABSTRACTThe subregional distribution of mu opioid receptors and corresponding G-protein activation

were examined in the striatum, amygdala, and extended amygdala of cynomolgus monkeys. Thetopography of mu binding sites was defined using autoradiography with [3H]DAMGO, a selectivemu ligand. In adjacent sections, the distribution of receptor-activated G proteins was identifiedwith DAMGO-stimulated guanylyl 59(g-[35S]thio)triphosphate ([35S]GTPgS) binding. Within thestriatum, the distribution of [3H]DAMGO binding sites was characterized by a distinct dorsal–ventral gradient with a higher concentration of binding sites at more rostral levels of thestriatum. [3H]DAMGO binding was further distinguished by the presence of patch-like aggrega-tions within the caudate, as well as smaller areas of very dense receptor binding sites, previouslyidentified in human striatum as neurochemically unique domains of the accumbens and putamen(NUDAPs). The amygdala contained the highest concentration of [3H]DAMGO binding sitesmeasured in this study, with the densest levels of binding noted within the basal, accessory basal,paralaminar, and medial nuclei. In the striatum and amygdala, the distribution of DAMGO-stimulated G-protein activation largely corresponded with the distribution of mu binding sites.The central and medial nuclei of the amygdala, however, were notable exceptions. Whereas theconcentration of [3H]DAMGO binding sites in the central nucleus of the amygdala was very low,the concentration of DAMGO-stimulated G-protein activation in this nucleus, as measured with[35S]GTPgS binding, was relatively high compared to other portions of the amygdala containingmuch higher concentrations of [3H]DAMGO binding sites. The converse was true in the medialnucleus, where high concentrations of binding sites were associated with lower levels of DAMGO-stimulated G-protein activation. Finally, [3H]DAMGO and [35S]GTPgS binding within the amyg-dala, particularly the medial nucleus, formed a continuum with the substantia innominata andbed nucleus of the stria terminalis, supporting the concept of the extended amygdala in primates.J. Comp. Neurol. 433:471–485, 2001. © 2001 Wiley-Liss, Inc.

Indexing terms: [35S]GTPgS; autoradiography; limbic system; monkey

Opiates and endogenous opioid peptides induce theirbiological effects via activation of three major subtypes ofopioid receptors mu (m), delta (d), and kappa (k; Martin etal., 1976; Kosterlitz et al., 1980; Reisine and Bell, 1993),which are coupled to the pertussis toxin-sensitive Gi/Gofamily of G proteins (Kurose et al., 1983; Aghajanian andWang, 1986). Although the distribution of these receptorshas been studied extensively in rodents, far less is knownabout the anatomical distribution of opioid receptors in

primates. This is of considerable importance, because therelative levels of opiate receptors vary considerably be-

*Correspondence to: Dr. Linda J. Porrino, Department of Physiology andPharmacology, Wake Forest University School of Medicine, Medical CenterBoulevard, Winston-Salem, NC 27157-1083. E-mail: [email protected]

Received 13 October 2000; Revised 4 December 2000; Accepted 6 Febru-ary 2001

THE JOURNAL OF COMPARATIVE NEUROLOGY 433:471–485 (2001)

© 2001 WILEY-LISS, INC.

tween rats and humans. For example, the composition oftotal opioid receptors in rat brain is 41% m, 50% d, and 9%k sites, whereas the human brain contains 30% m, 34% d,and 37% k sites (Mansour et al., 1988). The patterns ofdistribution also appear to vary between species. The den-sity and distribution of m sites in the caudate and puta-men of primates (Voorn et al., 1996), for example, demon-strate clear gradients of binding that are not evident inthe rat, and the characteristic pattern of high-densitypatches in a low-density matrix readily observed in the ratis less evident in the human (Quirion et al., 1995; Voorn etal., 1996) and nonhuman primate (Porrino et al., 1991).These discrepancies suggest that findings regarding theanatomical distribution and function of the opioid systemin the rodent may not always be relevant to the primate.These distinctions further argue for the use of models thatare experimentally more similar to the human, but inwhich studies can be conducted without the confoundingelements inherent in human studies.

Previous studies reporting the localization of mu opioidreceptors in the forebrain of primates have been relativelylimited in scope. Most describe general patterns of local-ization or have been conducted in tissue homogenates.Furthermore, many of the earlier studies used nonspecificligands (Pfeiffer et al., 1982; Wamsley et al., 1982; Lewiset al., 1984; Neil et al., 1986; Cross et al., 1987; Porrino etal., 1991; Mansour and Watson, 1993). The lack of detailedinformation about the distribution of mu opioid receptorsmakes it difficult to evaluate specific alterations in thesereceptors within brain regions that are affected in variouspsychiatric and neurological disorders. The first goal ofthe present study, therefore, was to describe the topo-graphical distribution of mu opioid receptors in nonhumanprimates. Traditional in vitro receptor autoradiographywith [3H]DAMGO was used to provide a detailed descrip-tion of mu binding patterns. The present study focused onthe striatum, amygdala, and extended amygdala, loca-tions for which high concentrations of mu receptors havebeen reported (Mansour et al., 1988) and the functionalrole of opioid peptides has been extensively studied inrodents (Biggio et al., 1978; Kalivas et al., 1983; Di Chiaraand Imperato, 1988; Gosnell, 1988; Stinus et al., 1990;Hurd et al., 1992; Daunais et al., 1993).

Characterization of the density and affinities of opioidreceptor sites alone does not establish whether recep-tors are functionally coupled to an effector system.Agonist-stimulated guanylyl 59(g-[35S]thio)triphosphate([35S]GTPgS) binding was originally developed to assayreceptor activation of G proteins in purified and reconsti-tuted systems (Asano et al., 1984) as well as in isolatedmembranes (Hilf et al., 1989; Lorenzen et al., 1993). Thisassay has been successfully adapted as an in vitro auto-radiographic tool to identify anatomically receptor-activated G proteins in tissue sections, thus allowing pre-cise anatomical localization of G-protein activation inresponse to specific receptor agonists (Sim et al., 1995).This technique has been applied to numerous Gi/Go-coupled receptors in the rat and guinea pig brain, includ-ing opioid, cannabinoid, and g-aminobutyric acid (GABA)Breceptors (Sim et al., 1995, 1996a,b), and is particularlyuseful for reflecting differences in the efficiency of cou-pling of receptors to G proteins in different brain regions.We recently applied this method to an examination of muand kappa opioid receptor-activated G proteins in theprimate brain (Sim-Selley et al., 1999). That study de-

scribed the overall distribution of mu- and kappa-stimulated [35S]GTPgS binding throughout the brains ofcynomolgus monkeys and demonstrated the viability ofthis method in nonhuman primate tissue. The presentstudy provides a more detailed analysis of DAMGO-stimulated [35S]GTPgS binding in the striatum, amyg-dala, and extended amygdala and provides a direct com-parison of the distribution of [3H]DAMGO sites toDAMGO-stimulated [35S]GTPgS binding in these areas.

MATERIALS AND METHODS

Materials

[D-Ala2,N-Me-Phe4,Gly5-ol]enkephalin (DAMGO) andlevorphanol were purchased from Sigma Chemical Co. (St.Louis, MO). [35S]GTPgS (1,250 Ci/mM) was purchasedfrom New England Nuclear Corp. (Boston, MA). GDP wasobtained from Sigma Chemical Co. [3H]Hyperfilm waspurchased from Amersham (Arlington Heights, IL) andReflections autoradiography film was purchased fromNew England Nuclear Corp. All other reagent-gradechemicals were obtained from Sigma Chemical Co. orFisher (Pittsburgh, PA).

Tissue preparation

All animal procedures were performed in accordancewith established practices as described in the NationalInstitutes of Health Guide for Care and Use of LaboratoryAnimals. In addition, all protocols were reviewed and ap-proved by the Animal Care and Use Committee of WakeForest University. Three adult male cynomolgus monkeys(Macaca fasicularis), aged 10–12 years, were killed underheavy sodium pentobarbitol anesthesia (100 mg/kg). Thebrains were immediately removed, and each was blockedin three portions, quickly frozen in isopentane at –35°C for15 minutes, and stored at –80°C until sectioning. Twentymicrometer coronal sections were cut in a cryostat main-tained at –20°C and thaw mounted onto gelatin-coatedslides. Slides were stored at –80°C until processed forautoradiography.

In vitro receptor autoradiography:[3H]DAMGO binding

Mu receptor binding sites were assessed with quantita-tive in vitro receptor autoradiography using [3H]DAMGOaccording to procedures adapted from Unterwald and col-leagues (1994). Association, washout, and saturation stud-ies were conducted to determine the optimal conditions forautoradiography in monkey tissue. Based on these stud-ies, an incubation concentration of 4 nM [3H]DAMGO waschosen (approximately 1.5–2 times the KD value). Thisconcentration is within the range used by others in humantissue (Hurd and Herkenham, 1993; Voorn et al., 1996).

For autoradiographic analysis, sections were collectedon gelatin-coated slides, dried under a vacuum at 4°C, andstored at –80°C. On the night before the assay, slides weremoved from –80°C to a –20°C environment, and they werebrought to room temperature on the morning of the assay.The sections were preincubated in four changes (5 min-utes each) of Tris buffer (50 mM Tris HCl, 120 mM NaCl,5 mM KCl), pH 7.4, at 4°C. Total binding was assessed byincubation for 60 minutes at 4°C in 4.0 nM [3H]DAMGO,followed by six 20 second rinses in Tris buffer and one 10second rinse in distilled water, all at 4°C. Nonspecific

472 J.B. DAUNAIS ET AL.

binding was determined by incubating sections underidentical conditions with the addition of 10 mM levorpha-nol (approximately 500 times the Ki value) in the incuba-tion buffer. The sections were then dried under a stream ofcool, dry air and apposed to [3H] Hyperfilm, alongwith[3H] standards (Amersham), for 12 weeks. After ex-posure, films were developed with Kodak GBX developerand stopbath and Kodak Rapid Fixer. Slides were thenNissl-stained with cresyl violet, dehydrated, and cover-slipped.

Autoradiograms were analyzed by quantitative densi-tometry using MCID (Imaging Research, St. Catharines,Ontario, Canada). Optical density measurements weremade, and tissue equivalent values (fmol/mg wet weighttissue) were determined from the optical densities of theautoradiograms and a calibration curve, obtained by den-sitometric analysis of the autoradiograms of standardscalibrated for subcortical gray matter (Orzi et al., 1983).Measurements in each region were made in a minimum oftwo (usually four) sections in each of the animals. Specificbinding was determined by subtracting nonspecific bind-ing, measured in adjacent sections, from the total binding.

In vitro [35

S]GTPgS autoradiography

The procedures for assessment of opioid-stimulated[35S]GTPgS binding in cynomolgus brain were previouslydescribed (Sim-Selley et al., 1999). Briefly, adjacent ornearly adjacent sections were processed by incubation inassay buffer (50 mM Tris-HCl, 3 mM MgCl2, 0.2 mMEGTA, 100 mM NaCl, pH 7.4) at 25°C for 10 minutes,followed by incubation with 2 mM GDP in assay buffer for15 minutes at 25°C. Slides were then incubated for 2hours at 25°C in assay buffer with [35S]GTPgS (0.04 nM),2 mM GDP, and 3 mM DAMGO. Basal [35S]GTPgS bindingwas determined in the absence of agonist and was reducedto minimal levels by the addition of high concentrations (2mM) of GDP (Sim-Selley et al., 1999). Specific agonist-stimulated binding was determined by subtracting thebasal binding levels from that stimulated by DAMGO.Following incubation, slides were rinsed twice in 50 mMTris-HCl buffer, pH 7.4, on ice and once in deionized wateron ice, dried well, and exposed to film, along with [14C]standards (Amersham). After 5 days of exposure, filmswere developed in Kodak GBX developer and stopbath andKodak Rapid Fixer.

Autoradiograms were analyzed by quantitative densi-tometry as described above. Densitometric values wereobtained by converting [14C] values to nCi/g [35S] usingbrain paste standards as previously described (Letch-worth et al., 1997). Measurements in each region weremade in a minimum of two (usually four) sections in eachof the animals. Analyses of [35S]GTPgS binding as well as[3H]DAMGO binding were conducted by the same individ-ual. Data are expressed as mean 6 SEM averaged acrossanimals.

RESULTS

Regional brain concentrations of mu opioid receptorsand the regional density of mu receptor-activated G pro-teins were measured in adjacent or closely adjacent sec-tions at multiple rostral–caudal levels through the stria-tum, amygdala, and extended amygdala of cynomolgusmonkey brains (n 5 3). In each brain region, cytoarchitec-tural criteria were used to delineate the constituent nuclei

and subregions for assessment. The patterns of distribu-tion of both [3H]DAMGO and DAMGO-stimulated[35S]GTPgS binding were highly consistent across ani-mals, although there was variability in the intensity ofsignal of both markers.

[3H]DAMGO binding sites in the dorsal and

ventral striatum

Autoradiograms and corresponding schematic represen-tations depicting the levels of the caudate, putamen, andnucleus accumbens where [3H]DAMGO and DAMGO-stimulated [35S]GTPgS binding were measured are shownin Figure 1. Mu opioid receptor binding was assessed attwo levels of the precommissural striatum that includedthe rostral precommissural striatum where the nucleusaccumbens is not readily differentiable into distinct sub-compartments (Fig. 1B) and the caudal precommissuralstriatum where the shell of the nucleus accumbens can beclearly distinguished from the core of the nucleus accum-bens (Fig. 1D). Both rostral and caudal levels of the pre-commissural striatum were further subdivided into thedorsolateral caudate, central caudate, dorsomedial cau-date, ventral caudate, nucleus accumbens (shell and coredivisions at caudal levels), dorsal putamen, central puta-men, and ventral putamen (Fig. 1A,C).

The binding of [3H]DAMGO to mu opioid receptors wasclearly heterogeneous, with appreciable differences in thedegree of binding among subregions. Considering the pre-commissural striatum as a whole, labeling was character-ized by the presence of a distinct dorsal–ventral gradient.Ventral regions expressed a higher density of mu recep-tors than more dorsal areas. Furthermore, moderatelyhigher densities of binding were present at rostral aspects(Fig. 1B) than at more caudal levels (Fig. 1D) of the stri-atum. This rostrocaudal gradient was evident within eachstriatal subdivision, including the caudate, putamen, andnucleus accumbens (Table 1).

Within the caudate, the pattern of [3H]DAMGO bindingwas characterized by a pronounced dorsal–ventral gradi-ent (Table 1, Fig. 1) present at both rostral and caudallevels of the precommissural striatum. Thus, the ventralcaudate expressed dense concentrations of mu receptorbinding sites when contrasted with more dorsal subdivi-sions. This region of high binding density was continuousventrally with the nucleus accumbens and its subdivi-sions, in that no distinct border between the two struc-tures was apparent. Another characteristic of bindingwithin the caudate was the tendency for mu binding sitesto be concentrated medially (Fig. 1B,D, Table 1) along theedge of the caudate adjacent to the lateral ventricle. Thispattern was evident in both the ventral and the dorsome-dial regions of the caudate. Finally, binding levels withinthe caudate were somewhat denser at anterior levels thanat caudal levels (cf. Fig. 1B to D).

A similar pattern of binding was present in the puta-men. In this structure, [3H]DAMGO binding to mu recep-tors was largely confined to ventromedial aspects (Fig.1B,D). Dense aggregates of binding were detected alongthe medial edge of the ventral putamen, in an area bor-dering the internal capsule (see Fig. 2A). The dorsal andcentral regions of the putamen expressed negligible auto-radiographic signal for [3H]DAMGO (Table 1, Figs. 1, 3A).There was, therefore, a greater degree of contrast amongportions of the putamen than among portions of the cau-date.

473MU SITES IN PRIMATE STRIATUM, EXTENDED AMYGDALA

The heaviest levels of [3H]DAMGO binding within thestriatum were observed in the nucleus accumbens (Table1), where a wide range of densities was evident within thesubregions of this structure. Throughout the accumbens,the pattern of mu binding was characterized by the pres-ence of areas lacking appreciable binding density interdig-itated among areas exhibiting dense concentrations of la-bel (Figs. 1B,D, 2A, refer to bottom arrows). This patternwas most distinct at rostral levels and appeared to occupy

what eventually develops into the core-like division of theaccumbens (Fig. 1B).

At more caudal levels (Fig. 1D), the two subdivisions ofthe nucleus accumbens, core and shell, could be readilydistinguished. [3H]DAMGO binding in the core portion ofthe nucleus accumbens at this level was characterized bya more uniform distribution of moderate mu receptorbinding (Fig. 1D) than at more rostral levels. However,small areas lacking any appreciable binding were evident

Fig. 1. [3H]DAMGO binding in the precommissural striatum ofcynomolgus monkeys. Shown are coronal sections through the rostraland caudal precommissural striatum. Lateral is to the left and dorsalto the top. A and C are diagrams illustrating the striatal regionsmeasured. B and D are representative autoradiograms depicting[3H]DAMGO binding to mu receptors in the striatum. admc, Anteriordorsomedial caudate; adlc, anterior dorsolateral caudate; acc, anteriorcentral caudate; avc, anterior ventral caudate; ana, anterior nucleus

accumbens; adp, anterior dorsal putamen; acp, anterior central puta-men; avp, anterior ventral putamen; pdmc, posterior dorsomedialcaudate; pdlc, posterior dorsolateral caudate; pcc, posterior centralcaudate; pvc, posterior ventral caudate; pc, nucleus accumbens-core;ps, nucleus accumbens-shell; pdp, posterior dorsal putamen; pcp, pos-terior central putamen; pvp, posterior ventral putamen; cc, corpuscallosum; ic, internal capsule; and lv, lateral ventricle. Scale bar 5 4.5mm for B,D.


at this level as well. In contrast, binding in the shell of thenucleus accumbens was characterized by a higher averagedensity of [3H]DAMGO labeling, predominantly in thedorsal portions (Fig. 3A). Heavy concentrations were par-ticularly prominent in the area along the dorsal edge thatseparated the shell from the septal nuclei (Figs. 1D, 3A).

In its ventrolateral extent, however, the level of binding inthe shell was more diffuse and was not readily differenti-ated from the core (Fig. 1D).

Contributing to the heterogeneity of the pattern in[3H]DAMGO binding in the precommissural striatumwere patch-like aggregations containing dense levels ofmu binding sites (Figs. 1B,D, 2A,B). These aggregations ofdense binding were preferentially expressed in the cau-date, particularly within the dorsomedial and ventral por-tions of this part of the striatum. They were observed inthe rostral portions of the nucleus accumbens as well.Their distribution can be seen readily in the autoradio-grams shown in Figures 1B,D and 2A (top arrows) and B.At more rostral levels, these patch-like aggregations weresmaller, and the concentration of binding sites appearedmore intense than in more caudal levels (cf. Fig. 1B to D)where the difference in the intensity of patches, comparedto the surrounding matrix, was greatly reduced. In a fewof these aggregations (Fig. 2B), the concentration of bind-ing sites was not uniform; rather, they contained palecenters ringed with intense binding at the edges.

In addition, binding to mu receptors was assessed inclusters of particularly dense labeling located within theventral putamen and nucleus accumbens. These clustersare highly reminiscent of features recently referred to as“neurochemically unique domains of the accumbens andputamen” (NUDAPs) in human tissue by Voorn and col-leagues (1996) and, hence, have been referred to in Tables1 and 2 as “NUDAPs.” This striking feature of[3H]DAMGO binding in the ventral striatum of nonhumanprimate brain is shown in Figures 1D, 2C, and 3A. Incontrast to the patch-like aggregations described above,these clusters were predominantly expressed at more cau-dal levels of the precommissural striatum. At the levelwhere the shell of the nucleus accumbens begins toemerge, clusters were approximately 300 mm2 in size and

TABLE 1. Regional Densities of [3H]DAMGO Binding and DAMGO-Stimulated [35S]GTPgS Binding in Anterior and Posterior Levels of the

Precommissural Striatum of Cynomolgus Monkey1

[3H]DAMGO binding(fmol/mg wet weight

tissue)

DAMGO-stimulated[35S]GTPgS binding

(nCi/g)

AnteriorCaudate

Dorsolateral (adlc) 8 6 1 173 6 42Central (acc) 14 6 3 241 6 72Dorsomedial (admc) 23 6 5 291 6 71Ventral (avc) 39 6 4 427 6 71

PutamenDorsal (adp) 8 6 2 161 6 43Central (acp) 17 6 4 219 6 64Ventral (avp) 26 6 3 322 6 84

Nucleus accumbens (ana) 39 6 5 411 6 59Posterior

CaudateDorsolateral (pdlc) 6 6 1 287 6 45Central (pcc) 11 6 1 278 6 78Dorsomedial (pdmc) 19 6 2 308 6 82Ventral (pvc) 34 6 2 441 6 78

PutamenDorsal (pdp) 5 6 1 220 6 61Central (pcp) 10 6 1 222 6 77Ventral (pvp) 17 6 2 341 6 74

Nucleus accumbensShell (ps) 33 6 2 293 6 94Core (pc) 23 6 5 558 6 99

NUDAPs 54 6 2 1101 6 151

1Tissue was obtained and processed as described in Materials and Methods. Labeling ofmu receptors was performed using 4 nM [3H]DAMGO. Mu opioid-stimulated bindingwas achieved by incubation of sections with 2 mM GDP followed by 0.04 nM[35S]GTPgS, 2 mM GDP, and 3 mM DAMGO and is expressed as net stimulation(total 2 basal). Data were collected using computer-based densitometry.

Fig. 2. Autoradiograms depicting the distribution of [3H]DAMGObinding sites in coronal sections of the striatum of cynomolgus mon-keys. Lateral is to the right. A: Areas lacking appreciable binding areshown (lower arrowheads) surrounded by areas of higher densitybinding. These were most evident in the rostral nucleus accumbens,as shown here. The upper arrowheads point to patch-like aggrega-

tions of higher density binding. B: Arrowheads point to patch-likeaggregations containing pale centers rimmed with intense binding atthe edges. C: Arrowheads indicate neurochemically distinct areas ofthe accumbens and putamen (NUDAPs) containing particularly denselabeling. Scale bar 5 3 mm for A,B; 2.5 mm for C.


primarily bordered the ventromedial edge of the shell and,to some extent, the transition zone between the ventralputamen and the nucleus accumbens (Fig. 1D). More pos-teriorly, they increased in size to approximately 800 mm2

and were located in more central areas of the shell asopposed to the ventromedial edge (Fig. 3A). [3H]DAMGObinding was most heavily expressed within these struc-tures in comparison to all other areas of the primatestriatum (Table 1). Clusters of this type or “NUDAPs”were not observed in any other brain region or portion ofthe striatum.

DAMGO-stimulated [35

S]GTPgS binding inthe dorsal and ventral striatum

Coronal sections, adjacent to those utilized for[3H]DAMGO receptor autoradiography, were processed toexamine basal and mu-stimulated [35S]GTPgS binding inthe striatum. Basal [35S]GTPgS binding was defined inthe absence of the agonist and was reduced to minimallevels by the addition of high concentrations (2 mM) ofGDP. Low levels of basal [35S]GTPgS binding were uni-formly distributed throughout the striatum at all levelsexamined.

Values of DAMGO-stimulated [35S]GTPgS binding inthe striatum are shown in Table 1. These values representthe difference between measurements of DAMGO-stimulated [35S]GTPgS binding and basal levels measuredas described in Materials and Methods. Figure 3 illus-trates an example of the comparison of [3H]DAMGO bind-ing (Fig. 3A) to DAMGO-stimulated [35S]GTPgS binding(Fig. 3B) in the primate striatum. There is a close corre-lation between the distribution of these markers. In gen-eral, DAMGO-stimulated [35S]GTPgS binding patterns inthe striatum exhibited the same dorsoventral and rostro-

caudal gradients that were present in the distribution ofbinding to mu receptors identified with [3H]DAMGO, al-though the relative differences between areas of highestand lowest density were not as great as those identifiedwith [3H]DAMGO binding sites (Table 1).

The pattern of DAMGO-stimulated [35S]GTPgS bindingin the caudate and putamen corresponded to the patternof [3H]DAMGO receptor binding described above. For ex-ample, high levels of mu opioid-stimulated [35S]GTPgSbinding were evident in the ventral caudate and along themedial edge of the dorsomedial caudate, closely resem-bling the distribution of receptor sites identified with[3H]DAMGO. Although it was not as readily apparent aswith [3H]DAMGO binding, there was a patch-like distri-bution of [35S]GTPgS binding in which the overlap of thetwo markers was also evident. In addition, the ventrome-dial portions of the putamen contained a higher density ofDAMGO-stimulated [35S]GTPgS binding sites than eitherthe dorsal or the lateral putamen.

The anatomical localization of agonist-stimulated[35S]GTPgS binding in the nucleus accumbens also paral-leled the distribution of these receptor sites as describedin the previous section. The overall agreement betweenDAMGO-stimulated [35S]GTPgS and [3H]DAMGO bind-ing in the ventral striatum was best exemplified by theclear overlap in binding patterns in the NUDAPs shown inFigure 3. Specifically, DAMGO-stimulated [35S]GTPgSbinding displayed the same pattern of dense localizationas was evident with mu binding sites along the dorsal edgeand in the ventral extent of the shell of the accumbens(Fig. 3B). There were, however, some discrepancies ob-served in the relative densities of the two markers. Suchdifferences were most apparent in the posterior regions ofthe ventral striatum. For example, the highest density of

Fig. 3. Autoradiograms comparing [3H]DAMGO (A) and DAMGO-stimulated [35S]GTPgS (B) binding in closely adjacent coronal sec-tions at the level of the caudal precommissural striatum of cynomol-gus monkey. Lateral is to the right. Arrowheads indicate

neurochemically distinct areas of the accumbens and putamen(NUDAPs), areas in which the high degree of anatomical overlap for[3H]DAMGO and DAMGO-stimulated [35S]GTPgS binding is clearlyillustrated. Scale bar 5 4 mm.


DAMGO-stimulated [35S]GTPgS binding sites was foundin the core of the nucleus accumbens, whereas the patternof [3H]DAMGO binding sites showed the highest concen-trations in the shell.

[3H]DAMGO binding sites in the amygdala

The distribution of mu opioid receptors in the amygda-loid complex was assessed in coronal sections located atlevels approximately 1 mm apart in the rostrocaudalplane. Schematic diagrams and representative autoradio-grams from two of these levels are shown in Figure 4. Thenomenclature used for the description of nuclei of themonkey amygdala was adapted from Amaral et al. (1992).The identification of the nuclei and their subdivisionswithin the amygdaloid complex was made using Nissl-stained sections adjacent or nearly adjacent to autoradio-grams of [3H]DAMGO binding and DAMGO-stimulated[35S]GTPgS binding. The concentration of mu receptorswas measured in the lateral, basal, accessory basal, me-dial, central, paralaminar, and cortical nuclei as well as inthe anterior amygdaloid area. In general, [3H]DAMGObinding within the majority of nuclei of the amygdala wasdenser than within most portions of the striatum. Thesedata are shown in Table 2.

For the purposes of this analysis, the lateral nucleuswas divided into dorsal and ventral subdivisions. Moder-

ate densities of [3H]DAMGO binding were evidentthroughout the rostrocaudal extent of the dorsal subdivi-sion of the lateral nucleus (Fig. 4B,E, Table 2). In contrast,a higher concentration of mu receptors was present in theventral subdivision (Fig. 4B,E, Table 2), although theheavier labeling in the ventral subdivision was most evi-dent in the posterior two-thirds of the ventral lateralnucleus than in the anterior one-third.

Within the basal nucleus, different patterns of mu re-ceptor binding were readily apparent in the magnocellularand parvicellular portions of this nucleus (Fig. 4B,E, Table2). The concentration of binding sites was greatest in theparvicellular subdivision, especially medially and at morecaudal levels. Binding was considerably less intense in theintermediate division and sparsest in the magnocellularportion of the basal nucleus.

The heaviest concentration of [3H]DAMGO bindingsites within the amygdala was found in the paralaminarnucleus (Fig. 4B,E, Table 2). The distribution of label inthis nucleus closely resembled the pattern observed in theparvicellular basal nucleus in that the entire rostrocaudalextent was densely labeled. In fact, no clear boundary wasdiscernible between these nuclei. Although the accessorybasal nucleus was divided into its parvicellular and magno-cellular divisions for the purposes of analysis, [3H]DAMGObinding appeared relatively uniform throughout these two

Fig. 4. Comparison of [3H]DAMGO and DAMGO-stimulated[35S]GTPgS binding in the amygdala of cynomolgus monkey. Shownare coronal sections at two rostrocaudal levels approximately 2 mmapart. Lateral is to the left. A and D are diagrams depicting theregions within the amygdala in which the density of [3H]DAMGObinding sites and DAMGO-stimulated [35S]GTPgS binding was quan-tified. B and E are autoradiograms depicting the constitutive bindingpatterns of mu receptors as demonstrated with [3H]DAMGO binding.

C and F are representative autoradiograms illustrating DAMGO-stimulated [35S]GTPgS binding. AAA, anterior amygdaloid area;ABm, accessory basal nucleus, magnocellular division; ABp, accessorybasal nucleus, parvicellular division; Bm, basal nucleus, magnocellu-lar division; Bp, basal nucleus, parvicellular division; CE, centralnucleus; COA, cortical nucleus; LATd, lateral nucleus, dorsal division;LATv, lateral nucleus, ventral division; ME, medial nucleus; PAL,paralaminar nucleus. Scale bar 5 2.2 mm for B,C,E,F.


subdivisions (Fig. 4B,E, Table 2). A higher density of bind-ing in the ventromedial subdivision located along the me-dial border of the accessory basal nucleus, however, wasclearly evident (Fig. 4B).

Low to moderate concentrations of [3H]DAMGO bindingsites were measured in the anterior amygdaloid area andin the cortical nucleus (Fig. 4B, Table 2). In contrast, avery high concentration of binding sites was observed inthe medial nucleus (Fig. 4E, Table 2). Dense labeling wasprominent throughout its entire rostrocaudal extent fromjust posterior to the anterior cortical nucleus caudally tothe caudal pole of amygdaloid complex. Furthermore, lit-tle differentiation among its subdivisions was apparent.By far the lowest concentrations of [3H]DAMGO bindingsites were found in the central nucleus of the amygdala(Fig. 4E, Table 2). There was uniformly low binding withinthe lateral and medial divisions of the central amygdalaregardless of the anterior–posterior level considered.

[3H]DAMGO binding sites in the

extended amygdala

Figure 5A–C shows a series of schematic drawings thatdepict [3H]DAMGO binding in the bed nucleus of the striaterminalis (BST), the substantia innominata (SI), and thecentral and medial amygdala. Nomenclature for the de-scription of regions within the bed nucleus and its contin-uum with the amygdala was adapted from the work ofMartin et al. (1991), in which the cytoarchitecture andneuropeptide distribution within the BST, SI, and amyg-dala of both human and monkey were compared. Theidentification of the nuclei and their subdivisions withinthe extended amygdala was made using Nissl-stained sec-tions adjacent or nearly adjacent to autoradiograms of[3H]DAMGO binding and DAMGO-stimulated [35S]GTPgSbinding. For the purposes of this analysis, the BST was

subdivided into lateral (BSTL) and medial (BSTM) divi-sions. The SI was divided into a dorsal and ventral aspect.Although Martin and colleagues (1991) parcellate theseregions further, only two subdivisions were utilized in thepresent analysis because of the limited number of sectionsavailable at each rostrocaudal level.

The concentrations and distribution of [3H]DAMGObinding in these areas is shown in Table 2, as well asschematically in Figure 5A–C. The bed nucleus of the striaterminalis emerges rostrally at the level of the posterioraspects of the nucleus accumbens, and it extends caudally,where it is bounded by the columns of the fornix mediallyand anterior hypothalamic regions ventrally. [3H]DAMGObinding in the medial division of the bed nucleus (Fig.5A–C, Table 2) was characterized by dense labeling inregions both dorsal and ventral to the anterior commis-sure (Fig. 6). This pattern of dense labeling extendedthroughout the rostral caudal extent of the medial subdi-vision of the BST, although labeling was considerablydenser at more rostral levels, especially adjacent to theanterior commissure. In contrast, the lateral component ofthe BST expressed lower levels of [3H]DAMGO labelingcompared to the medial components (Figs. 5A–C, 6, Table2). This pattern of [3H]DAMGO binding in the lateraldivision of the BST was consistent throughout the rostro-caudal extent of all portions of the BSTL, including thosedorsal and ventral to the anterior commissure, althoughthe distinction between medial and lateral componentswas most evident rostrally.

In general, labeling within the substantia innominata(Figs. 5B,C, 6, Table 2) was less intense than in the stri-atum, amygdala, and BST. Regardless of the low levels of[3H]DAMGO binding in this area, differences in bindingdensities were evident between the dorsal and the ventraldivisions of the SI. The ventral portion of the SI, whichappeared to form a continuum between the medial BSTand the medial nucleus of the amygdala, expressed light tomoderate levels of [3H]DAMGO binding. In contrast, thedorsal SI, which in turn appeared to form a continuumbetween the lateral BST and the central nucleus of theamygdala, contained very low levels of [3H]DAMGO bind-ing (Figs. 5B,C, 6).

DAMGO-stimulated [35

S]GTPgS binding inthe amygdala and extended amygdala

Basal [35S]GTPgS binding in the amygdala and ex-tended amygdala was defined in the absence of agonist bythe addition of high concentrations (2 mM) of GDP. Lowlevels of basal [35S]GTPgS binding were generally ob-served in the amygdala and extended amygdala, althoughhigher basal binding levels were detected in some regions.In the central amygdala, for example, levels of basal bind-ing were higher than in other nuclei within the amygdala.It is important to note that, although higher basal levelsare present in some areas, the values of net stimulated[35S]GTPgS binding shown in Table 2 represent the dif-ference between measurements of DAMGO-stimulated[35S]GTPgS binding and basal levels measured as de-scribed above.

DAMGO-stimulated [35S]GTPgS binding in the amyg-dala is illustrated in Figure 4C,F. In the amygdala, as inother areas of the brain, the degree of DAMGO-stimulated[35S]GTPgS binding tended to be considerably more vari-able among animals than was the [3H]DAMGO binding.Despite the variability in the general levels of signal

TABLE 2. Regional Densities of [3H]DAMGO Binding and DAMGO-Stimulated [35S]GTPgS Binding in the Amygdala and Extended Amygdala

of Cynomolgus Monkey1

[3H]DAMGO binding(fmol/mg wet weight

tissue)

DAMGO-stimulated[35S]GTPgS binding

(nCi/g)

AmygdalaLateral nucleus (LAT)

Dorsal 29 6 3 428 6 94Ventral 55 6 4 653 6 98

Basal nucleus (B)Magnocellular 56 6 10 540 6 96Parvicellular 85 6 10 921 6 23

Accessory basal nucleus (AB)Magnocellular 64 6 9 686 6 117Parvicellular 76 6 6 920 6 123

Central nucleus (CE) 10 6 5 623 6 113Medial nucleus (ME) 84 6 3 761 6 112Paralaminar nucleus (PAL) 109 6 7 1077 6 108Cortical nucleus (COA) 62 6 8 890 6 38Anterior amygdaloid area

(AAA)44 6 8 576 6 69

Extended amygdalaBed nucleus of the stria

terminalisLateral (BSTL) 23 6 6 448 6 37Medial (BSTM) 46 6 9 536 6 11

Substantia innominataDorsal (SId) 9 6 3 221 6 30Ventral (SIv) 29 6 3 460 6 5

1Tissue was obtained and processed as described in Materials and Methods. Labeling ofmu receptors was performed using 4 nM [3H]DAMGO. Mu opioid-stimulated bindingwas achieved by incubation of sections with 2 mM GDP followed by 0.04 nM[35S]GTPgS, 2 mM GDP, and 3 mM DAMGO and is expressed as net stimulation(total 2 basal). Data were collected using computer-based densitometry.


among animals, the overall patterns of DAMGO-stimulated [35S]GTPgS binding in the amygdala werehighly consistent. Furthermore, these patterns were verysimilar to the patterns of distribution of receptor bindingsites identified with [3H]DAMGO.

The distribution of mu receptor-stimulated [35S]GTPgSbinding paralleled the anatomical distribution of[3H]DAMGO binding sites in the lateral and basal amyg-daloid nuclei. In the caudal two-thirds of the lateral nu-cleus of the amygdala, the highest levels of DAMGO-stimulated [35S]GTPgS binding were confined to theventral subdivision, whereas considerably lower levelswere evident in the more dorsal subdivision. A similarpattern was present in the basal nucleus, where the ven-tral parvicellular portion of this nucleus contained agreater concentration of DAMGO-stimulated [35S]GTPgSbinding sites than did the more dorsal magnocellular andintermediate subdivisions. The distribution of mu-

stimulated [35S]GTPgS binding also corresponded withthe anatomical distribution of [3H]DAMGO sites in theaccessory basal nucleus, paralaminar, and cortical nucleias well as in the anterior amygdaloid area.

Only moderate levels of mu-stimulated [35S]GTPgSbinding were evident in the medial nucleus of the amyg-dala, despite the very heavy concentrations of mu receptorbinding in this region (Fig. 4E,F, Table 2). Furthermore,in striking contrast to very low levels of [3H]DAMGObinding in the central nucleus, moderate levels ofDAMGO-stimulated [35S]GTPgS binding were found inthis area (cf. Fig. 4E to F).

The distribution of mu-stimulated [35S]GTPgS bindingsites in the bed nucleus of the stria terminalis and sub-stantia innominata corresponded closely to the distribu-tion of mu receptor sites (cf. Fig. 5A–C to D–F). Specifi-cally, a higher concentration of DAMGO-stimulated[35S]GTPgS binding sites was evident in the medial por-

Fig. 5. Line drawings through three rostrocaudal levels approxi-mately 1 mm apart of the extended amygdala continuum showing thedensity and distribution of [3H]DAMGO binding sites (A–C) andDAMGO-stimulated [35S]GTPgS binding (D–F). [3H]DAMGO bindingdensity (A–C) is shown in increasingly dark shades of gray rangingfrom very low density (,10 fmol/mg tissue) shown in the lightestshade of gray to very dense binding (.80 fmol/mg tissue) shown in thedarkest shade of gray. DAMGO-stimulated [35S]GTPgS binding (D–F)is also shown in increasingly dark shades of gray ranging from verylow density (,250 nCi/g) shown in the lightest shade of gray to very

dense binding (.600 nCi/g) shown in the darkest shade of gray. ac,Anterior commissure; BSTL, lateral division of the bed nucleus of thestria terminalis; BSTM, medial division of the bed nucleus of the striaterminalis; cc, corpus callosum; Cd, caudate; Ce, central nucleus of theamygdala; GP, globus pallidus; ic, internal capsule; LS, lateral sep-tum; lv, lateral ventricle; Me, medial nucleus of the amygdala; ot,optic tract; Pu, putamen; SFi, septofimbrial nucleus; SId, dorsal divi-sion of the substantia innominata; SIv, ventral division of the sub-stantia innominata; st, stria terminalis; 3v, third ventricle.


tions of the BST than in the lateral subdivisions and in theventral substantia innominata compared to the dorsalsubstantia innominata (Fig. 5D–F, Table 2).

DISCUSSION

This study of the topographical distribution of mu opioidreceptors and mu agonist-activated sites in the striatum,amygdala, and extended amygdala of cynomolgus mon-keys demonstrates a highly heterogeneous pattern ofbinding in each of these regions. Although there have beenprevious reports of the distribution of mu receptors inprimate brain, these have been limited in that some of thestudies employed ligands relatively less selective for themu receptor (Kuhar et al., 1973; Lewis et al., 1984; Porrinoet al., 1991), or their assessments of the topographythroughout the brain were of a more general nature(Wamsley et al., 1982; Maurer et al., 1983; Pilapil et al.,1987). The present study provides a more detailed descrip-tion of the distribution of [3H]DAMGO binding sites, aligand more selective for mu receptors than either the[3H]naloxone (Porrino et al., 1991) or [3H]dihydromor-phine (Kuhar et al., 1973) utilized in some previous stud-ies.

Several findings emerge from the current observations.First, the distribution of mu receptors in the striatum ofnonhuman primates is concentrated in the more ventralportions and is characterized by the presence of patch-likeaggregations, low-density areas, and “NUDAPs.” Second,the highest concentrations of receptors are localizedwithin the amygdaloid complex. Third, the receptorswithin the amygdala form part of a continuum extendingthrough the substantia innominata to the bed nucleus ofthe stria terminalis, providing further support for theconcept of the extended amygdala in primates. Fourth,functional mu opioid receptor activity is demonstratedwithin these brain regions using DAMGO-stimulated

[35S]GTPgS binding, with close correlations between thesebinding sites and the anatomical distribution of mu recep-tors. There were, however, several instances of mis-matches between these distributions, most notably in thecentral and medial nuclei of the amygdala.

Striatum

The distribution of mu opioid receptors in monkey stri-atum, as defined by [3H]DAMGO binding, was character-ized by a distinct dorsoventral low-to-high gradient. Asimilar dorsoventral topography of the distribution of mureceptors was reported in rhesus monkeys when [3H]nal-oxone was used to label mu sites (Lewis et al., 1984;Porrino et al., 1991). Thus, the highest levels of mu recep-tors in the nonhuman primate were expressed within thenucleus accumbens and surrounding territories of themost ventral and most medial parts of the caudate andputamen. The topography of the areas containing thedensest concentrations of mu receptor binding sites coin-cides closely with the area designated as the ventral stri-atum as defined in nonhuman primate by Haber and hercolleagues (Haber et al., 1995; Haber and Fudge, 1997;Haber and McFarland, 1999).

[3H]DAMGO binding within the striatum was furthercharacterized by aggregations of binding in the caudateand ventral striatum. In the caudate, both solid and ring-shaped patches were observed. The solid clusters wereseen along the medial edge of the caudate and resembledpatches of mu binding sites, which are readily visible inthe rat (Herkenham and Pert, 1981; Mansour et al., 1987;Jongen-Relo et al., 1993). In contrast, [3H]DAMGO bind-ing in the human caudate was more homogeneous(Quirion et al., 1995; Voorn et al., 1996). It appears, then,that patches of heavier [3H]DAMGO binding are presentin the three species, with a rank order of rat . monkey .human. In addition, ring-shaped patches were observed inthe ventral caudate, similar to those previously reported

Fig. 6. Autoradiograms depicting the distribution of [3H]DAMGObinding sites in coronal sections of the bed nucleus of the stria termi-nalis of cynomolgus monkeys. Lateral is to the left. A: Coronal sectionthrough the level of the bed nucleus of the stria terminalis depictingthe constitutive binding patterns of mu receptors as demonstratedwith [3H]DAMGO binding. The boxed area in A is magnified in C.B: Diagram depicting the regions (corresponding to C) within the bednucleus and extended amygdala in which the density of [3H]DAMGObinding sites was quantified. C: Magnified view of the bed nucleus ofthe stria terminalis and the extended amygdala. ac, Anterior commis-

sure; BSTL, lateral division of the bed nucleus of the stria terminalis;BSTLd, the dorsal portion of the lateral division of the bed nucleus ofthe stria terminalis; BSTM, medial division of the bed nucleus of thestria terminalis; cc, corpus callosum; Cd, caudate; Ce, central nucleusof the amygdala; GP, globus pallidus; ic, internal capsule; Me, medialnucleus of the amygdala; ot, optic tract; SFi, septofimbrial nucleus;SId, dorsal division of the substantia innominata; and SIv, ventraldivision of the substantia innominata. Scale bar 5 14 mm for A; 2.2mm for C.


for D1 dopamine receptors in cynomolgus monkeys (Bes-son et al., 1988) and substance P (Graybiel, 1986) andenkephalin immunoreactivity (Graybiel and Ragsdale,1983) in rhesus macaques.

In both accumbens core and shell, regions of variable[3H]DAMGO binding were observed. [3H]DAMGO-poorregions were seen in the accumbens core and may corre-late with regions of low mu binding reported for humantissue (Hurd and Herkenham, 1993; Voorn et al., 1996). Inthe monkey, these [3H]DAMGO-poor regions were limitedin size and were primarily located in the rostral ventralstriatum. In the human striatum, the [3H]DAMGO-poorregions encompassed a much larger area and extendedfrom the rostral ventral striatum through the accumbenscore and into the putamen (Hurd and Herkenham, 1993;Voorn et al., 1996; but see Quirion et al., 1995). Theseregions in the human striatum give the appearance of adorsoventral high-to-low gradient of binding site density,in contrast to the low-to-high gradient reported in thepresent study. Furthermore, the low [3H]DAMGO bindinglevels in the human accumbens core form a clear demar-cation with the adjacent accumbens shell, which containsintermediate [3H]DAMGO levels (Voorn et al., 1996). Be-cause the levels of [3H]DAMGO binding sites, especially inventromedial aspects, are more similar between the shelland the core in the monkey, the distinction between thesetwo subregions of the nucleus accumbens is not as clear asin the human.

In contrast to the regions of low [3H]DAMGO binding,clusters of dense binding were found in the shell of thenucleus accumbens, as well as in the transition zone be-tween the accumbens and putamen. These clusters con-tained the highest levels of [3H]DAMGO sites in the stri-atum. Such regions have previously been described asNUDAPs in human tissue by Voorn and colleagues (1994,1996). They differ from the patches described above basedon their greater density of [3H]DAMGO binding and theirlocalization to the accumbens shell rather than the ven-tral caudate. In human tissue, mu opioid NUDAPs overlapwith clusters for kappa opioid receptors, dopamine D1receptors, and acetylcholinesterase activity (Voorn et al.,1994, 1996). Moreover, they can be distinguished fromislands of Calleja based on cell size (Voorn et al., 1996) andlack of dopamine D3 receptor binding (Voorn et al., 1994),which is a marker for the islands of Calleja (Landwehrm-eyer et al., 1993; Murray et al., 1994). Although moreresearch is needed to characterize these cell groupings,they may represent, as suggested by Voorn et al. (1996), aunique feature of the organization of the striatum of pri-mates, both human and nonhuman. Moreover, theNUDAPs appear to be a part of a more extensive networkof cell clusters or “interface islands” within the basal fore-brain, first described by Sanides (1957), that has receivedincreasing attention of late (see Heimer, 2000). This sys-tem of interface islands includes the islands of Calleja(Meyer et al., 1989), the parvicellular and granular is-lands within the ventral pallidum, and the intercalatedislands of the amygdala (De Olmos, 1990). Heimer (2000)has suggested that abnormalities in these “interface is-lands,” because of their unique histochemistry and imma-ture morphology, may in fact be implicated in the etiologyof schizophrenia.

In addition to [3H]DAMGO binding sites, we examinedlevels of DAMGO-stimulated G-protein activation in mon-key striatum. As expected, the anatomical distribution of

DAMGO-stimulated [35S]GTPgS binding in the striatumcorresponds closely to that of [3H]DAMGO sites, exhibit-ing the same dorsoventral and mediolateral gradients.Similar dorsoventral low-to-high gradients were observedfor DAMGO-stimulated [35S]GTPgS binding in postmor-tem human tissue (Rodrıguez-Puertas et al., 2000), de-spite reports of an opposite, high-to-low gradient for[3H]DAMGO sites in the human striatum (Hurd andHerkenham, 1993; Quirion et al., 1995; Voorn et al., 1996).

In this study, we observed an anatomical overlap for[3H]DAMGO and DAMGO-stimulated [35S]GTPgS bind-ing in the NUDAPs of the ventral striatum. Furthermore,there was a close correspondence between the high den-sity of [3H]DAMGO binding sites and the intensity of theDAMGO-stimulated [35S]GTPgS signal. Not all regions inthe striatum had relatively similar levels of binding sitesand stimulated [35S]GTPgS binding. The accumbens shell,for example, exhibited lower levels of DAMGO-stimulated[35S]GTPgS than would be expected from the density of[3H]DAMGO sites in that area, whereas the accumbenscore exhibited higher than expected levels of DAMGO-stimulated [35S]GTPgS. These regions may represent ar-eas of less and more signal amplification, respectively.

Patches of DAMGO-stimulated [35S]GTPgS binding inthe primate caudate were not as readily visible as with[3H]DAMGO binding. It is possible that the lower resolu-tion of the DAMGO-stimulated [35S]GTPgS assay, com-pared to the [3H]DAMGO binding assay, made it difficultto detect the patches. One limiting factor determining theresolution of the signal is the maximum energy (Emax) ofthe two radioisotopes. The Emax for [35S] is approximately10 times that of [3H]. Therefore, the [35S] signal travelsfarther from the point of origin to expose silver grains inthe film, decreasing clarity. For this reason, the[3H]DAMGO signal would be expected to be much crisperthan that of the [35S]GTPgS-labeled ligand, which was thecase in this study.

Amygdala

In the present study, a high concentration of mu recep-tors was observed throughout the amygdala. This concen-tration was among the highest observed in any portion ofthe brain considered in this report. These findings areconsistent with those of previous studies in nonhumanprimates (Kuhar et al., 1973; LaMotte et al., 1978; Wams-ley et al., 1982; Mansour and Watson, 1993), as well ashumans (Pilapil et al., 1987; Quirion et al., 1995), in whichvery dense concentrations of mu receptors were also ob-served in this area. In humans, mu receptors have alsobeen characterized with positron emission tomography,and, consistent with in vitro studies, high levels of ligandbinding were found in the amygdala (Frost et al., 1989).These levels in the amygdala have been shown to fluctuatewith changes in circulating estradiol levels (Smith et al.,1998), as a function of age and gender (Zubieta et al.,1999), and in psychiatric (Mayberg et al., 1991; Zubieta etal., 1996) and neurological (Madar et al., 1997) diseasestates. Few studies, however, have described the distribu-tion of receptors in individual nuclei of nonhuman primateor human amygdala.

In the rodent brain, dense levels of mu receptors havebeen shown within various components of the amygdala(Herkenham and Pert, 1981; Mansour et al., 1988), whichcontains among the highest concentrations of bindingsites of all brain regions. Dense mu binding (Mansour et


al., 1987) and high levels of mu opioid receptor immuno-reactivity (Ding et al., 1996) have been shown in themedial nucleus of the amygdala, as well as in the basalcomplex and the lateral amygdala, whereas lower levelshave been observed in the central nucleus. The amygdalardistribution in rodents, therefore, is quite analogous to thepresent findings in the monkey, suggesting a high degreeof homology across species.

There was considerable heterogeneity in the patterns of[3H]DAMGO binding within nuclei of the amygdala. Forexample, within the lateral nucleus, mu receptors wereheavily concentrated within the ventral subdivision,whereas only sparse labeling was observed in the dorsalsubdivision. Within the basal nucleus, a particularly highlevel of binding was observed in the parvicellular portion,and the more dorsal magnocellular and intermediate sub-divisions displayed only light binding. It is interesting tonote that in many cases the subdivisions and nuclei withintense levels of binding appear to have strong intrinsicinterconnections. The ventral portions of the lateral nu-cleus have extensive connections with the parvicellularportion of the basal nucleus and with the medial andaccessory basal nuclei (Pitkanen and Amaral, 1998), eachof which contains dense concentrations of mu bindingsites. In contrast, the ventral lateral nucleus has fewconnections to the magnocellular basal and the centralnuclei (Pitkanen and Amaral, 1998), each of which con-tains low levels of mu receptor binding sites. The centralnucleus, containing only sparse mu receptor binding sites,projects to the anterior amygdaloid area and anterior cor-tical nucleus (Price and Amaral, 1981; Aggleton, 1985),areas with only moderate levels of mu binding sites.

This pattern of intrinsic interconnectivity between re-gions of high receptor density within the amygdala alsoappears to characterize the relationship between amyg-dala and striatal binding sites. In the primate, the amyg-dala projects heavily to the striatum, particularly the ven-tral striatum (Parent et al., 1983; Russchen et al., 1985).These projections arise predominantly from the parvicel-lular portion of the basal nucleus, which has the densestpopulation of mu receptors within the basal nucleus andprojects preferentially to the nucleus accumbens, anotherregion expressing a high density of mu receptors. Themagnocellular subdivision of the basal nucleus, in con-trast, sends its efferents to more dorsal striatal areascontaining lower mu receptor concentrations. Further-more, although the studies were carried out in cats, Rags-dale and Graybiel (1988) demonstrated that amygdalo-striatal efferents terminate preferentially in patch-likeportions of the feline striatum. In the present investiga-tion, high densities of mu receptors were observed inpatch-like aggregations within the striatum. Althoughthere may be significant species differences and the patch-like organization is not as prominent in the monkey, asimilar topographical arrangement is likely to exist in theprimate as in the cat.

The central nucleus of the amygdala was almost devoidof [3H]DAMGO binding sites in the present study. This isin contrast to a previous study by Mansour and Watson(1993) from nonhuman primate tissue in which a highconcentration of mu receptors was reported. The reasonsfor this discrepancy are not readily apparent. It is possiblethat they result from a species difference. The presentstudy was conducted in cynomolgus monkeys, but similarlow levels of mu receptors within the central nucleus of the

amygdala were also observed in rhesus monkeys (Daunaisand Porrino, unpublished observations). The species ofmonkey was not specified by Mansour and Watson in theirdescription, complicating direct comparisons.

In the present study, the low concentration of receptorsin the central nucleus, however, was accompanied by arelatively high level of mu-stimulated [35S]GTPgS bind-ing. This apparent mismatch suggests that the centralnucleus may be an area where there is a high efficiency ofagonist-stimulated G-protein activation such that one re-ceptor could activate a large number of G proteins. Such ahigh level of amplification of the coupling of mu receptorshas been shown in tissue homogenates of the amygdala ofthe rat (Maher et al., 2000). In sharp contrast to thecentral nucleus, the medial nucleus, where dense levels of[3H]DAMGO binding were expressed, displayed relativelylower levels of DAMGO-stimulated [35S]GTPgS binding,indicating less efficiency in G-protein activation. Differ-ences in the degree of amplification have been demon-strated in a number of systems (Sim et al., 1996a; Breivo-gel et al., 1997). Another factor contributing to thedifferences between receptor binding and G-protein acti-vation is the existence of high- and low-affinity agonistbinding sites. [3H]DAMGO binding is performed underconditions that favor high-affinity states, whereas the so-dium and GDP in the [35S]GTPgS assay favor low-affinitystates. Differences in proportions of these states couldcontribute to regional differences in these two assays.

Extended Amygdala

The bed nucleus of the stria terminalis has been impli-cated recently in a number of motivational behavioralprocesses (Davis and Shi, 1999; Aston-Jones et al., 1999;Koob, 1999) and has been the subject of intense anatom-ical investigation (Alheid and Heimer, 1988; Alheid et al.,1995; Heimer et al., 1997). Despite this increasing atten-tion, few studies have characterized the bed nucleus of thestria terminalis in primates, human or nonhuman. In fact,previous studies of the distribution of opioid receptorshave not even included this brain region in the analyses.The present study, therefore, represents the first descrip-tion of mu opioid receptors in the bed nucleus of the striaterminalis in nonhuman primates. Both [3H]DAMGObinding and DAMGO-stimulated G proteins were ex-pressed in high densities in the medial portions of the bednucleus, but more moderately in the lateral cytoarchitec-turally defined subdivisions.

This distinction between medial and lateral subdivi-sions has been evident in studies of other markers. Thelateral subdivisions of the bed nucleus are densely inner-vated by neurotensin-, somatostatin-, and leu-enkephalin-immunoreactive neurons and processes in the human andnonhuman primate, particularly compared to more medialportions of the bed nucleus (Lesur et al., 1989; Martin etal., 1991). In humans, tyrosine hydroxylase-immuno-reactive fibers and neurons are also more concentrated inthe more lateral portions of the bed nucleus. In general,then, the lateral portions appear to be rich in neuropep-tide and aminergic innervation, whereas the more medialsubdivisions are devoid of such innervations (Haber andElde, 1982; Martin et al., 1991). This is in contrast to thepresent findings, in which mu receptors are far moredense medially than laterally within the BST. Character-ization of the BST is of great importance, because this


nucleus appears to expand in size as the phylogeneticscale is ascended (Andy and Stephan, 1976).

The bed nucleus has come to be considered a part of acontinuous structure, termed the extended amygdala,stretching from the central and medial nuclei of the amyg-dala through the substantia innominata to the bed nu-cleus of the stria terminalis. This notion of a commonanatomical entity, first proposed by Johnston in 1923, hasgained increasing acceptance anatomically and function-ally in recent years. Columns of cells have been shown toform bridges that connect these two brain regions in bothrodent brain (De Olmos et al., 1985; Alheid and Heimer,1988; Heimer and Alheid, 1991) and primate brain (Mar-tin et al., 1991). Although the characteristics of the ex-tended amygdala in primates has not received nearly asmuch attention as in rodents, reports by De Olmos et al.(1985), Heimer (2000), and Martin et al. (1991) includeconvincing evidence that this continuum is present in bothhuman and nonhuman primates. The extended amygdala,as delineated in rodents and primates (Martin et al., 1991;Alheid et al., 1995), has various branches. Although theexact trajectory and the number of branches is the subjectof some debate, generally it is considered that one branchextends from the medial nucleus of the amygdala throughthe ventral substantia innominata to the most medialportions of the BST, whereas a second extends from thecentral amygdala though the dorsal substantia innomi-nata to the more lateral subdivisions of the bed nucleus.Our present observations support these distinctions, inthat mu receptor binding was far more concentrated ineach of the elements of the medial branch than in theconstituent regions that comprise the more lateral branchof the extended amygdala. In addition, these data providea potentially useful marker for the more medial subdivi-sion of the extended amygdala in primate brain.

CONCLUSIONS

We have described here the topography of the distribu-tion of mu opioid receptors and mu receptor-activated Gproteins in the striatum, amygdala, and extended amyg-dala of the nonhuman primate. High concentrations of mureceptors and receptor-activated G proteins suggest thatfunctional receptors are present in the ventral striatum,amygdala, and extended amygdala encompassing the areafrom the amygdala to the BST. These brain regions haveclose anatomical connections but are also related function-ally. Each has been shown to play crucial roles in theprocessing of motivational and emotional information andreward-related stimuli. In fact, the ventral striatum isconsidered the part of the striatum receiving afferent pro-jections from brain regions (cortical, thalamic, amygdalar,and midbrain) associated with the development and ex-pression of motivational and emotional processes (Haberet al., 1995; Haber and Fudge, 1997; Haber and McFar-land, 1999). The presence of high densities of mu opioidreceptors in these limbic areas further reinforces the im-portance of these systems for the processing of emotionaland reward-related information in the brain as well as thedisruption of these systems in psychiatric and neurode-generative disorders.

ACKNOWLEDGMENTS

Drs. David Friedman and Pieter Voorn provided helpfuldiscussions regarding the manuscript. The authors thankMack Miller for his valued assistance in the preparation ofthe figures and Stephanie Hart for her assistance in thepreparation of the manuscript. These studies were sup-ported by PHS grants DA-09085 (L.J.P.), DA-00287(L.J.S.-S.), DA-07246 (J.B.D.), and DA-02904 (S.R.C.)from the National Institute on Drug Abuse.

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Functional and anatomical localization of mu opioid receptors in the striatum, amygdala, and extended amygdala of the nonhuman primate