Estrogen binding and the actions of testosterone in the brain of the male rhesus monkey

Autoradiography and high performance liquid chromatography (HPLC) were used to determine where metabolites of testosterone interact with estrogen binding sites in the brain of the male primate. Three days after castration, animals received a subcutaneous injection of either estradiol benzoate (EB, 200 micrograms/kg, n = 4) or oil vehicle (controls, n = 4). Three hours later, 5 mCi [3H]testosterone was administered as an intravenous bolus. At 60 min, brains were rapidly removed, left halves were used for autoradiography and right halves were dissected into 14 samples for HPLC of nuclear and supernatant fractions. In control males, labeled neurons were observed in preoptic area, hypothalamus and amygdala. In EB-pretreated males, the number of labeled neurons was reduced by 35% in the anterior hypothalamus and ventromedial nucleus, and by 65% in the cortical and accessory basal amygdaloid nuclei, but was not significantly reduced in other brain regions. In hypothalamus, preoptic area and amygdala, EB-pretreatment reduced nuclear concentrations of [3H]estradiol to 37-55% of control levels, but reduced neither the nuclear concentrations of [3H]testosterone nor the supernatant concentrations of [3H]estradiol and [3H]testosterone. The data suggest that the actions of testosterone in regions such as the arcuate nucleus and lateral septal nucleus primarily involve unchanged testosterone or dihydrotestosterone, while in regions such as the amygdala, aromatization and interaction with estrogen receptors is involved also.     

Metabolism and binding of androgens in the spinal cord of the rat

The binding of [3H]androgens and estrogens, and the metabolism of [3H]androgens, were studied in the spinal cord of the adult rat. High-affinity, specific binding sites for [3H]testosterone and [3H]estradiol were detected in cytosol fractions from the spinal cords of castrate animals. Equilibrium dissociation constants for reaction of these sites with their respective ligands were similar to those of androgen and estrogen receptors from other regions of the central nervous system. Nuclear binding of [3H]estradiol was observed in the spinal cord 1 h after intravenous administration of the isotope. Likewise, exchange assay demonstrated the presence of high-affinity androgen binding sites in spinal cord nuclei from orchidectomized, testosterone propionate treated animals. 5 alpha-Reductase activity in homogenates of the spinal cord was relatively high, approximately 3 times that in the pooled hypothalamus, preoptic area, septum and amygdala. However, in contrast to the latter brain regions, estrogen formation was not detectable in spinal cord tissue. No sex differences were observed in the metabolism of [3H]testosterone by spinal cord homogenates. These results confirm the presence of androgen and estrogen receptors in the rat spinal cord. The lack of detectable aromatase activity in the spinal cord is consistent with the hypothesis that the effects of circulating testosterone on spinal reflex function are mediated primarily through the androgen receptor system.     

Nuclear retention charactristics of [H]estrogen by cells in four estrogen target regions of the rat brain

Nuclear retention of radioactivity was studied in neural estrogen target cells 15 min–7 h after i.v. injection of [³H]estradiol. Maximal uptake occured by 15 min. Cells of the medial preoptic nucleus retained the label longer than did those of the medial amygdaloid nucleus. Cells of the ventromedial nucleus and arcuate nucleus exhibited similar retention profiles which were intermediate between the medial preoptic and medial amygdaloid cells. These data are discussed in relation to observations that the duration of nuclear occupancy by estrogens is proportional to the magnitude of the cellular response.     

Sigma receptors are associated with cortical limbic areas in the primate brain.

Putative sigma receptors are a current target for antipsychotic drug development. Novel antipsychotic agents which possess selective and high affinity for sigma binding sites may serve as an alternative to the principal neuroleptic drugs currently in clinical use which mediate extrapyramidal side effects and dyskinesias through their blockade of dopamine receptors. We have used in vitro autoradiography to localize putative sigma receptors labelled with (+)-[3H]-3-(3-hydroxyphenyl)-N-(1-propyl)piperidine [(+)-[3H]-3-PPP] in the brain of the rhesus macaque. The binding characteristics of (+)-[3H]-3-PPP in the primate brain were comparable to those previously described in the rodent. Saturation analysis demonstrated a single class of sites in cerebellar and hippocampal membranes with a Kd value of 28 nM. Sigma receptors labeled with (+)-[3H]-3-PPP in the primate brain displayed the appropriate rank order of potency and stereoselectivity in competition binding assays. Haloperidol displaced (+)-[3H]-3-PPP binding in the low nanomolar range, and the (+) isomer of pentazocine was 50-fold more potent than (-) pentazocine. Computerized densitometric analysis of the autoradiograms demonstrated a striking enrichment of sigma binding sites over the paralimbic belt cortices, including the orbitofrontal, cingulate, insular, parahippocampal, and temporopolar gyri. Peak densities of sigma receptors were seen over the medial and central nuclei of the amygdala and were widely distributed within the hippocampal formation. Sigma binding sites densities were elevated over the suprachiasmatic and supraoptic nuclei of the hypothalamus. Moderate sigma receptor densities were observed over the ventromedial sectors of the caudate and the putamen. Sigma receptors were also elevated over autonomic relay nuclei of the brainstem, including the nucleus of the solitary tract and the dorsal motor nucleus of the vagus. The distribution of sigma receptors in the primate brain suggests that the paralimbic belt cortices, amygdala, hippocampus, hypothalamus, and autonomic relay nuclei of the brainstem may be interrelated by a topographic chemical linkage. The autoradiographic visualization of sigma receptor distributions in the primate brain provides further support for a role of sigma receptor mechanisms in the functions of the limbic system.     

Perinatal development of estrogen receptors in mouse brain assessed by radioautography, nuclear isolation and receptor assay

The development of estrogen receptors was investigated in vivo in the brains of fetal and neonatal mice 2 h after administering [3H]moxestrol to the pregnant mothers or neonates. Moxestrol bypasses the alpha-fetoprotein 'protective barrier' and gains access to estrogen receptors. Analysis of [3H]moxestrol uptake by radioautography and by cell nuclear isolation and counting of radioactivity revealed a marked increase in the number of estrogen receptors and estrophilic cells in the brain during late fetal and early postnatal development. Assays of cytosol estrogen receptors were conducted in parallel and revealed a comparable pattern of development. The increase in estrogen receptors and labeling was especially great from embryonic day (E) 15 to E18. Cytosol assays revealed a low level of receptors in the whole brain on E13. Radioautography revealed that clearly labeled cells in the hypothalamus and preoptic area were virtually absent on E13 but were evident on E15, with marked increases occurring between E15 and E18, both in number of labeled cells and in intensity of labeling per cell. Within the cerebral cortex the dorsal cingulate cortex was the most extensively labeled area; however, clearcut labeling was not evident on E13 or E15. Thus, the development of cortical estrogen receptors occurs somewhat later than that in the hypothalamus and preoptic area. The perinatal increase in estrogen receptors usually begins several days after the birthdates of neurons in these estrophilic regions of the brain, and corresponds to the early responsiveness of these neurons to the organizational and activational influences of estrogen.     

Biochemical and radioautographic analysis of estrogen-inducible progestin receptors in female ferret brain and pituitary: Correlations with effects of progesterone on sexual behavior and gonadotropin-releasing hormone-stimulated secretion of luteinizing hormone

Cytosolic binding molecules for the synthetic progestin [3H]R5020, were isolated in vitro from several brain regions including preoptic area/anterior hypothalamus, mediobasal hypothalamus, medial amygdala and parietal cortex as well as the pituitary gland of ovohysterectomized female ferrets. Binding of [3H]R5020 to cortical cytosols was saturable, of high affinity (apparent dissociation constant of 2.0 nM), and was steroid specific. Pretreatment of ferrets with a Silastic capsule containing estradiol caused significant increments in the concentration of cytosolic R5020 binding sites in hypothalamus and pituitary gland, but not in the other brain regions studied. Brains of additional ovohysterectomized ferrets, which had been primed with estradiol prior to receiving an i.v. injection of [3H]R5020, were processed radioautographically. Intense labeling of cells was seen in the medial and lateral preoptic area, in the lateral hypothalamus, and in the ventromedial and arcuate nuclei of the hypothalamus. Radioautograms from the brains of additional ovohysterectomized ferrets given an i.v. injection of [3H]estradiol revealed labeled cells in all of the above regions, in addition to the basolateral portion of the septum, the bed nucleus of the stria terminalis, and in the anterior amygdaloid area as well as the medial and cortical nuclei of the amygdala. This distribution of neural progestin and estrogen binding sites resembles those previously reported for these steroids in the rat, guinea pig, hamster and macaque. Functional studies showed that acute s.c. implantation of a Silastic capsule containing progesterone to ovohysterectomized ferrets, which had been primed with a low dosage of estradiol, failed to augment their sexual receptivity in limited tests with stimulus males given 4 and 8 h after progesterone treatment. This result contrasts with the well-established facilitatory effect of progesterone on sexual receptivity in rat and guinea pig. Chronic exposure to a progesterone capsule caused significant reductions in sexual receptivity in ovohysterectomized ferrets which were implanted concurrently with a second Silastic capsule containing a high dosage of estradiol. Similar effects of progesterone have been reported in rat and guinea pig, but not in the rhesus monkey. Thus species differences in the ability of progesterone to facilitate or inhibit sexual receptivity are not readily explained by species differences in the neural distribution of estrogen-induced progestin receptors.(ABSTRACT TRUNCATED AT 400 WORDS)     

Chronic testosterone propionate treatment decreases the concentration of [3H]quipazine binding at 5-HT3 receptors in the amygdala of the castrated male rat

Chronic administration of testosterone propionate (TP) was found to decrease the concentration of [3H]quipazine binding at 5-HT3 receptors in the lateral and basal amygdaloid nuclei of the brains of castrated male rats. TP had no effect on the binding of [3H]quipazine at 5-HT3 receptors in the posterolateral or posteromedial cortical amygdaloid nuclei, or in the amygdalohippocampal areas. It is suggested that the effects of testosterone on sexual and other social behaviors in male rats may be mediated, at least in part, by decreases in the activation of 5-HT3 receptors in the amygdala.     

Distribution of cocaine recognition sites in monkey brain: I. In vitro autoradiography with [3H]CFT.

The cocaine analog [3H]CFT ([3H]WIN 35,428) was used to map and characterize cocaine recognition sites in the squirrel monkey brain by quantitative autoradiography. Coronal tissue sections were incubated with 5 nM [3H]CFT to measure total binding or with [3H]CFT in the presence of 30 microM (-)-cocaine to measure nonspecific binding. High densities of [3H]CFT binding sites were present in dopamine-rich brain regions, including the caudate nucleus, putamen, nucleus accumbens, and olfactory tubercle. In each of these regions specific binding was greater than 90% of total binding. Several additional brain regions exhibited intermediate densities of [3H]CFT binding, including the substantia nigra, the zona incerta, the amygdala, and the hypothalamus. Low, though measurable levels of binding were observed in the bed nucleus of the stria terminalis, the ventral tegmental area, the medial preoptic area, the pineal, the hippocampus, and thalamic central nuclei. Near-background levels of binding were found in white matter, cortical regions, globus pallidus, and cerebellum. The pharmacological specificity of [3H]CFT binding in various brain regions was determined in competition studies using [3H]CFT and a range of concentrations of selected monoamine uptake inhibitors. In all brain regions examined, stereoselective inhibition of [3H]CFT binding was observed for the (-) over the (+) isomer of cocaine. For other drugs tested, competition experiments indicated a rank order of potency of GBR 12909 greater than or equal to CFT greater than bupropion, suggestive of binding of [3H]CFT to elements of the dopamine transport system. The results demonstrate that although densities of [3H]CFT binding sites are highest in the caudate nucleus, putamen, and nucleus accumbens/olfactory tubercle, significant levels of binding can be detected in other brain regions that may contribute to the behavioral and physiological effects of cocaine.     

Androgen and estrogen concentrating neurons in chemosensory pathways of the male Syrian hamster brain.

The medial preoptic area (MPOA), bed nucleus of the stria terminalis (BNST), and medial amygdaloid nucleus (Me) are essential for male sexual behavior in the Syrian hamster. These nuclei received chemosensory stimuli and gonadal steroid signals, both of which are required for mating behavior. The objective of this study was to compare the distribution of androgen- and estrogen-concentrating neurons in MPOA, BNST, and Me in the adult male hamster using steroid autoradiography for estradiol (E2), testosterone (T) and dihydrotestosterone (DHT). Adult males (n = 4 per group) received two i.p. injections of tritiated steroid 4-7 days after castration. Six-microns frozen sections through the brain were mounted onto emulsion-coated slides, and exposed for 11-16 months. In MPOA, BNST, and Me, neurons were more abundant and heavily labelled after [3H]E2 treatment than after either [3H]T or [3H]DHT. Tritiated estradiol- and DHT-labeled cells were found throughout the rostrocaudal extent of Me, with a high concentration in posterodorsal Me. Tritiated testosterone treatment labelled cells largely within posterodorsal Me. In MPOA, the majority of E2-, T-, and DHT-labelled neurons were in the medial preoptic nucleus (MPN) and the preoptic continuation of the posteromedial bed nucleus of the stria terminalis (BNSTpm). Few T-labelled cells were present outside these subdivisions. In the BNST, E2- and DHT-labelled neurons were present in all subdivisions, whereas T labelling was confined to the antero- and posteromedial subdivisions of BNST. These results suggest that the distribution of androgen- and estrogen receptor-containing neurons overlap considerably in nuclei which transmit chemosensory signals in the control of mating behavior.     

A topographic quantitative method for measuring brain tissue pH under physiological and pathophysiological conditions

The autoradiographic regional localization of [3H]beta-endorphin binding in rat brain differed from that of either [3H]dihydromorphine or [3H]D-Ala2-D-Leu5-enkephalin. Comparisons were made from sequential sections through 3 regions of rat brain: striatum, hypothalamus/thalamus, and brainstem. [3H]beta-endorphin labeled some clusters as well as the subcallosal streak in the striatum, the nucleus accumbens, lamina IV of the cortex, medial regions of the thalamus, hippocampus, inferior colliculus, dorsal raphe, median raphe and pontine nuclei. White matter regions had little binding. Although many of these structures were also labeled with either [3H]dihydromorphine or [3H]D-Ala2-D-Leu5-enkephalin, the overall pattern of [3H]beta-endorphin labeling appeared unique, consistent with the proposal of central epsilon receptors.     

Simultaneous presence of estrogen receptors and gonadotropin-releasing hormone in the hypothalamus and of estrogen receptors and follicle-stimulating hormone in the pituitary of the fetal guinea pig

Early studies in our laboratory have shown the presence of estrogen receptors (ER) in the fetal brain of guinea pig. Ontogenetic evaluation indicated maximal values of these specific binding sites between 50 and 62 days of gestation. We have now characterized ER in both the hypothalamus and the pituitary at the end of gestation. Autoradiographic studies after subcutaneous injection of [3H]estradiol to the fetus showed the nuclear localization of radioactivity in the hypothalamus to be in the neurons of the preoptic area and the nucleus arcuatus where gonadotropin-releasing hormone was also present. Nuclear localization of radioactivity also occurred in the anterior pituitary where the number of labeled cells increased from the border of the intermediate part to the periphery of the anterior lobe. These labeled cells were predominantly gonadotropes containing follicle-stimulating hormone. Since the fetal uterus responds very actively to estrogens, it is suggested that reciprocal influences of the hypothalamus-hypophyseal axis and gonadal steroid hormones are present in the fetal guinea pig.     

The distribution and plasticity of [3H]vasopressin-labelled specific binding sites in the canary brain

[3H]Vasopressin was used to detect and to quantitate specific binding sites with in vitro receptor autoradiography in the canary brain. A discrete regional distribution of [3H]vasopressin-labelled binding sites was observed. A high density of specific binding sites was present in the medial posterior hypothalamic nucleus, the superficial layer of the optic tectum, the area ventralis of Tsai (AVT), the nucleus pretectalis, the habenula, the nucleus of Darkschewitch and the nucleus interstitialis. A low density occurred in the nucleus robustus archistriatalis of female birds. However, after testosterone treatment the density of the sites in this area as well as in the AVT increased 2-fold. [3H]Vasopressin binding was displaced to the same extent by excess vasopressin as well as by vasotocin. Moreover, the [3H]vasopressin-labelled brain regions are innervated by immunoreactive vasotocin fibers and terminals. It seems likely therefore that the labelled sites represent putative vasotocin receptors.     

Nuclear localization of testosterone, dihydrotestosterone and estradiol-17beta in basal rat brain

(1) One-half hour after the intravenous injection of [³H]testosterone, basal brain cell nuclei isolated from castrated male rats contained more radioactivity with the thin layer chromatography mobility of estradiol-17β and dihydrotestosterone (DHT) than nuclei from female rats. (2) The sex differences in the subcellular localization of [³H]DHT and [³H]estradiol-17β were not reflected in the ratio of the nuclear to whole homogenate radioactivity. (3) In the basal brain, estradiol-17β was the only metabolite examined that showed more DPM/mg protein in the nuclear fraction than in the whole homogenate. (4) In the basal brain and pituitary, unlabeled DHT was more effective than testosterone in blocking the nuclear localization of [³H]testosterone and [³H]DHT. (5) Unlabeled estradiol-17β only blocked the nuclear concentration of [³H]estradiol-17β and not [³H]testosterone or [³H]DHT.     

Direct autoradiographic determination of M1 and M2 muscarinic acetylcholine receptor distribution in the rat brain: Relation to cholinergic nuclei and projections

The autoradiographic distributions of receptors with high affinity for [3H]oxotremorine-M (the M2 receptor) and [3H]pirenzepine (the M1 receptor) were studied in the rat brain. M1 receptors were seen in highest density only in telencephalic structures: cerebral cortex (layers I-II), hippocampus, dentate gyrus, medial and basolateral amygdala, nucleus accumbens and caudate/putamen. M2 receptors were detected throughout the brain, with highest levels observed in cerebral cortical layers III and V, forebrain cholinergic nuclei, caudate/putamen, various thalamic areas, inferior and superior colliculus, interpeduncular and pontine nuclei, brainstem cholinergic nuclei and cervical spinal cord regions. M2 receptors were found to be good markers for cholinergic cell groups and the majority of cholinergic projection areas, whereas M1 receptors were only found in a large sub-group of telencephalic cholinergic projection areas, and the pattern of distribution of receptors in these areas differed from that of M2 receptors. Scatchard analysis of [3H]oxotremorine-M binding to inferior collicular slices revealed one site with a dissociation constant (Kd) of 1.9 nM and a receptor density (Bmax) of 1.4 pmol/mg protein. Our data support the hypothesis that M1 and M2 receptors are physically distinct sub-types of the muscarinic acetylcholine receptor.     

Postnatal development and sex difference in neurons containing estrogen receptor-α immunoreactivity in the preoptic brain,the diencephalon,and the amygdala in the rat

Estrogen has been considered as a key substance that induces sexual differentiation of the brain during fetal and neonatal life in the rat. Thus, to define the brain regions involved in the brain sexual differentiation, we examined the regions where the estrogen receptor (ER) is located in the developing rat brain. We examined immunohistochemical distribution of the cells containing estrogen receptor-α (ER-α) in the preoptic region, the diencephalon, and the amygdala in male and female rats on postnatal days 1–35 (PD1–PD35). The antibody used recognizes ER-α equally well for both occupied and unoccupied forms. ER-α immunostaining was restricted to the cell nuclei of specific cell groups. In PD1 rats, ER-α-immunoreactive (ER-IR) signals were detected in the lateral septum, the organum vasculosum lamina terminalis, the medial preoptic nucleus (MPN), the median preoptic nucleus, the bed nucleus of the stria terminalis, the hypothalamic periventricular nucleus, the lateral habenula, the posterodorsal part of the medial amygdala nucleus, the posterior part of the cortical amygdala nucleus, the hypothalamic ventromedial nucleus (VMH), the hypothalamic arcuate nucleus, and the posterior hypothalamic periventricular nucleus. The distribution pattern of ER-IR cells in the newborn rat was much the same as that in the adult in the preoptic-hypothalamic and amygdala regions. Moreover, the signals in the MPN and the VMH were stronger in the female than in the male, perhaps reflecting the ability of estrogen generated by aromatization of testosterone in the male to down-regulate the ER signal. Thus, the brain regions showing sex differences may be sites of sexual differentiation of the brain by aromatizable androgen during the neonatal period. J. Comp. Neurol. 389:81–93, 1997. © 1997 Wiley-Liss, Inc.     

3H]Rauwolscine binding sites in the brains of male tree shrews are related to social status.

The aim of the present study was to investigate the influence of social status on central nervous alpha 2-adrenoceptors. Using the specific alpha 2-adrenoceptor antagonist [3H]rauwolscine, binding sites in the brains of dominant and subordinate male tree shrews were quantified by in vitro autoradiography. In 5 of the 14 brain structures investigated, subordinates had significantly lower numbers of binding sites than dominants. These structures were the solitary tract nucleus, the dorsal motor nucleus of the vagus, the periaqueductal gray, the perifornical region of the hypothalamus and the medial nucleus of the amygdala. These brain areas are all intimately involved in the regulation of autonomic functions and of emotional behavior. Also the affinities for [3H]rauwolscine differed between the groups. In 3 nuclei, the solitary tract nucleus, the periaqueductal gray and the medial nucleus of the amygdala, dominants had significantly higher Kd-values than subordinates. This demonstrates the presence of low affinity binding sites in dominants which do not exist in subordinates. It is suggested that the low number of [3H]rauwolscine binding sites in subordinates results from down-regulation of alpha 2-adrenoceptors by high levels of noradrenaline and/or adrenaline. The disappearance of low affinity [3H]rauwolscine binding sites may play an important role in the etiology of psychosocial stress.     

Autoradiographic localization and quantification of dopamine D2 receptors in normal human brain with [3H]N-n-propylnorapomorphine

The precise distribution of dopamine receptors has been studied autoradiographically in the normal human brain using [3H]N-n-propylnorapomorphine ([3H]NPA) as a ligand. Preliminary experiments aimed at optimizing the binding assay conditions revealed that preincubation washing of caudate nucleus sections was a prerequisite to obtain a good ratio of specific to non-specific binding. The binding of [3H]NPA to caudate-putamen sections was saturable, stereospecific, reversible, of high affinity (Kd = 0.27-0.35 nM) and occurred at a single population of sites. Competition experiments with various drugs indicated that in the caudate-putamen the specific [3H]NPA binding sites possess the pharmacological features of the dopamine D2 receptor. The highest levels of [3H]NPA binding sites were found in the caudate nucleus, putamen, globus pallidus and nucleus accumbens. There were also intermediate to low concentrations of the 3H-ligand in the hippocampus, the insular and cingular cortices and in the occipito-temporal gyrus, while almost undetectable levels of binding were found in the anterior frontal cortex. Thorough examination of the subregional distribution of [3H]NPA binding sites in the caudate-putamen-pallidum complex revealed heterogeneous patterns of radioactivity. In these brain regions, the distribution of autoradiographic grains was punctate and islands of high and low densities were observed. Moreover, in the caudate nucleus, there was a subtle high lateral to low medial gradient in the topography of the [3H]NPA binding sites and a more pronounced gradient along the rostrocaudal axis; the highest levels of binding being located at the midbody of the nucleus. No gradients of [3H]NPA binding were observed in the putamen. The present data indicate that [3H]NPA is a suitable ligand for accurate autoradiographic labeling of dopamine D2 receptors in human postmortem brain tissue and that dopamine receptors are heterogeneously distributed and topographically organized in patches and gradients in the basal ganglia regions.     

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

The subregional distribution of mu opioid receptors and corresponding G-protein activation were examined in the striatum, amygdala, and extended amygdala of cynomolgus monkeys. The topography of mu binding sites was defined using autoradiography with [(3)H]DAMGO, a selective mu ligand. In adjacent sections, the distribution of receptor-activated G proteins was identified with DAMGO-stimulated guanylyl 5'(gamma-[(35)S]thio)triphosphate ([(35)S]GTPgammaS) binding. Within the striatum, the distribution of [(3)H]DAMGO binding sites was characterized by a distinct dorsal-ventral gradient with a higher concentration of binding sites at more rostral levels of the striatum. [(3)H]DAMGO binding was further distinguished by the presence of patch-like aggregations within the caudate, as well as smaller areas of very dense receptor binding sites, previously identified in human striatum as neurochemically unique domains of the accumbens and putamen (NUDAPs). The amygdala contained the highest concentration of [(3)H]DAMGO binding sites measured 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 the concentration of [(3)H]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 [(35)S]GTPgammaS binding, was relatively high compared to other portions of the amygdala containing much higher concentrations of [(3)H]DAMGO binding sites. The converse was true in the medial nucleus, where high concentrations of binding sites were associated with lower levels of DAMGO-stimulated G-protein activation. Finally, [(3)H]DAMGO and [(35)S]GTPgammaS binding within the amygdala, particularly the medial nucleus, formed a continuum with the substantia innominata and bed nucleus of the stria terminalis, supporting the concept of the extended amygdala in primates.