Tritiated-6-beta-fluoro-6-desoxy-oxymorphone ([3H]FOXY): a new ligand and photoaffinity probe for the mu opioid receptors

6-Beta-fluoro-6-desoxy-oxymorphone (FOXY) is a fluorinated derivative of oxymorphone originally developed as a potential PET scanning ligand. Preliminary work (Rothman et al., Neuropeptides 4: 311-317, 1984) demonstrated that [3H]FOXY selectively labeled mu opioid binding sites with low levels of nonspecific binding. In this study the opiate receptor subtypes labeled by [3H]FOXY and [3H]D-ala2-MePhe4, Gly-ol5-enkephalin ([3H] DAGO) were compared using site directed acylating agents and binding surface analysis. Although the data indicated that both ligands selectively label mu opiate receptors, other experiments demonstrated that [3H]DAGO and [3H]FOXY labeled mu binding sites differently. Additional experiments demonstrated that [3H]FOXY can be used as a high yield photoaffinity label for the mu opiate receptor subtype.     

3H]Ethynylbicycloorthobenzoate ([3H]EBOB) binding in recombinant GABAA receptors

Ethynylbicycloorthobenzoate (EBOB) is a recently developed ligand that binds to the convulsant site of the GABAA receptor. While a few studies have examined the binding of [3H]EBOB in vertebrate brain tissue and insect preparations, none have examined [3H]EBOB binding in preparations that express known configurations of the GABAA receptor. We have thus examined [3H]EBOB binding in HEK293 cells stably expressing human alpha1beta2gamma2 and alpha2beta2gamma2 GABAA receptors, and the effects of CNS convulsants on its binding. The ability of the CNS convulsants to displace the prototypical convulsant site ligand, [35S]TBPS, was also assessed. Saturation analysis revealed [3H]EBOB binding at a single site, with a K(d) of approximately 9 nM in alpha1beta2gamma2 and alpha2beta2gamma2 receptors. Binding of both [3H]EBOB and [35S]TBPS was inhibited by dieldrin, lindane, tert-butylbicycloorthobenzoate (TBOB), PTX, TBPS, and pentylenetetrazol (PTZ) at one site in a concentration-dependent fashion. Affinities were in the high nM to low microM range for all compounds except PTZ (low mM range), and the rank order of potency for these convulsants to displace [3H]EBOB and [35S]TBPS was the same. Low [GABA] stimulated [3H]EBOB binding, while higher [GABA] (greater than 10 microM) inhibited [3H]EBOB binding. Overall, our data demonstrate that [3H]EBOB binds to a single, high affinity site in alpha1beta2gamma2 and alpha2beta2gamma2 GABAA receptors, and modulation of its binding is similar to that seen with [35S]TBPS. [3H]EBOB has a number of desirable traits that may make it preferable to [35S]TBPS for analysis of the convulsant site of the GABAA receptor.     

Developmental expression of mu and delta opioid receptors in the rat brainstem: evidence for a postnatal switch in mu isoform expression

Opioid receptors are expressed in the brain during fetal and postnatal development, and the expression patterns vary with developmental age. To investigate the role of opioids in brain development, immunoblotting and immunohistochemical techniques were used to determine mu (MOR) and delta (DOR) opioid receptor expression levels and regional distributions in fetal, early postnatal and adult rat brainstem. Two immunoreactive bands were seen on Western blots of brainstem lysates for both MOR (50 and 70 kDa) and DOR (30 and 60 kDa). The expression levels of the isoforms changed dramatically between 6 and 15 days after birth. Total MOR protein was expressed at low levels in fetal and early postnatal animals with the 50-kDa band predominating. MOR expression then increased in the older animals and the 70-kDa isoform became dominant. Total DOR protein showed the opposite pattern, being high in the fetal and neonatal brainstem and low in the juvenile and adult. A postnatal switch in isoform expression for DOR was not evident in our study. In general, regional brainstem distributions in developing and adult animals were comparable to those reported in the literature, and both receptors were localized in the same areas where opioid receptor expression was high. It was concluded that MOR and DOR are developmentally regulated in the brainstem of the rat, that the isoform ratio switches postnatally from a fetal-neonatal pattern to a juvenile-adult pattern and that both receptors are generally expressed in the same brainstem regions from E16 to adult.     

Autoradiographic study of irreversible binding of [3H]beta-funaltrexamine to opioid receptors in the rat forebrain: comparison with mu and delta receptor distribution.

beta-Funaltrexamine (beta-FNA) is an irreversible mu antagonist and a reversible kappa agonist in in vivo and in vitro tests. However, whether it produces irreversible delta antagonism is controversial. In binding studies, it is clear that beta-FNA does not bind irreversibly (it does reversibly) to kappa receptors. Yet there is no consensus as to whether beta-FNA binds irreversibly to mu and/or delta receptors. In this study, irreversible binding of [3H]beta-FNA to opioid receptors was examined in rat forebrain sections in the presence of 200 mM NaCl and its distribution compared with those of mu and delta opioid receptors, labeled by [3H][D-Ala2,MePhe4,Gly-ol5]enkephalin ([3H]DAMGO) and [3H][D-Pen2,D-Pen5]enkephalin ([3H]DPDPE), respectively. Irreversible binding of [3H]beta-FNA was determined as the binding that remained following 5 washes at room temp. for 1, 5, 20, 20, and 20 min each. Non-specific binding was defined by including 10 microM naloxone, beta-chlornaltrexamine (beta-CNA), or beta-FNA in the incubation mixture. At 37 degrees C, specific irreversible binding of [3H]beta-FNA to opioid receptors reached a plateau at 10 nM in 60 min, and constituted 50-70% of total irreversible binding. Series of 4 sections of similar anatomical levels were labeled with [3H]DAMGO, [3H]beta-FNA, [3H]beta-FNA + 10 microM naloxone, beta-CNA, or beta-FNA, and [3H]DPDPE, resp., and exposed to [3H]-Ultrofilm. The distribution of [3H]beta-FNA (5 nM) irreversible labeling is very similar to that of [3H]DAMGO, i.e. patches and subcallosal streaks in caudate-putamen, patches in nucleus accumbens, dense labeling in thalamus, and more binding in the rostral than caudal striatum.(ABSTRACT TRUNCATED AT 250 WORDS)     

Opiate ([3H]-naloxone) binding to hypothalamic and cerebral cortical slices of mouse brain

We have characterized the binding of [3H]-naloxone to thick (400 microns) slices of hypothalamus and cerebral cortex from mouse brain. Binding is reversible, saturable, stereospecific, thermolabile, readily displaceable by opiates and sensitive to phenoxybenzamine and phentolamine. Values for KD and Bmax are very close to published figures obtained in brain homogenates. Metabolic inhibitors (ouabain and azide) have no effect on specific binding. The assay is rapid, simple and involves minimal tissue preparation.     

Ligand selectivity of cloned human and rat opioid mu receptors

Opiate receptors play major roles in analgesic and euphoric effects of opiate drugs. Recent cloning of cDNAs encoding the rodent and human μ receptor revealed high homology between the predicted receptors but also some sequence differ- ences. To determine if these sequence differences produced significant changes in ligand-selectivity profiles, we assessed these profiles in expressing COS and CHO cell lines using the agonist ligand [¹²⁵I]IOXY-AG0 (6β-[¹²⁵Iodo]-3,14-dihydroxy-17-methyl-4,5α-epoxymorphinan). This ligand's high specific activity (2, 200 Ci/mmol) and high affinity for μ opioid receptors generated high signal-to-noise ratio binding. The resulting ligand- selectivity profiles of the human and rat mu; receptors reveal modest differences in affinities for morphine and naloxone in COS cells but not CHO cells. Ligand-selectivity profiles of the rat and human mu; receptors were otherwise similar. Interesting differences between these data and data previously obtained with the peptide agonist [³H]DAMGO suggest that the peptide and alkaloid agonists may label different domains of the μ receptor. © 1995 Wiley-Liss, Inc. 1

1 This article is a US Government work and as such, is in the public domain in the United States of America.

     

Estimation of the apparent affinity of the striatal dopamine receptors for the radioligand [3H]spiperone([3H]spiroperidol)

The characteristics of the receptor population labeled by the ligand [³H]spiperone were determined by several experimental procedures. Varying the assay volumes, and hence both the receptor and ligand concentrations, did not alter the specificity for the dopamine (DA) receptor. The density of binding sites estimated from saturation analyses varied little (6%) over a greater than 50-fold tissue concentration range. In contrast, variation in tissue concentration did alter the apparent affinity of [³H]spiperone for the DA receptors more than 16-fold, as determined from saturation analyses. This was most marked at large receptor or tissue concentrations. The standard correction for depletion of the free ligand by that bound to the receptors reduced the range to threefold or 200%. Two separate measurements of the DA receptor affinity, using the rates of association and dissociation and the shift in K_i for DA, gave affinity measurements in the same range as that obtained using low concentrations of receptors in saturation analyses. Therefore, three separate estimates for the affinity of [³H]spiperone binding to the striatal DA receptors agreed that it is probably in the range of 10–15 pM, and single saturation experiments reach this level when the tissue concentration is very low, ie, less than 15 μg protein per 1 ml assay volume.     

Quantitative autoradiographic analysis of [3H]carfentanil binding to mu opiate receptors in the rat brain

Fentanyl and its derivatives are considered among the most potent opiate analgesic/euphoriants. The pharmacological literature generally supports a μ opiate receptor site of action for the fentanyl derivatives, but some observations suggest that other sites of action may be involved in producing the extremely potent fentanyl effects. In order to investigate the mechanism of action of fentanyl-like drugs further, [³H]carfentanil was used as a radioligand to image high-affinity carfentanil binding sites in slidemounted sections of the rat brain (receptor autoradiography). In parallel studies the prototypical μ opiate agonist radioligand [³H]DAMGO ([D-Ala²-MePhe⁴-Glyol⁵]enkephalin) was also used. The working hypothesis was that if carfentanil was acting through another high-affinity site besides the μ opiate receptor, the distribution pattern of the autoradiographic image produced by [³H]carfentanil should be significantly different than the autoradiographic image produced by [³H]carfentanil should be significantly different than the autoradiographic pattern displayed by the well-characterized and selective μ opiate [³H]DAMGO. Thirty-five brain regions were examined for specific [³H]carfentanil and [³H]DAMGO binding. The absolute and relative densities of the sites were essentially identical. The highest levels of binding were observed in the “patch” areas of the striatum (131 ± 5 fmol/mg T. E. for [³H]carfentanil; 162 ± 13 fmol/mg T. E. for [³H]DAMGO). The lowest levels were observed in the cerebellum where no specific binding of either radioligand was observed. The overall distribution pattern of the two radioligands produced a correlation coefficient of 0.95; the distribution pattern was prototypical for the μ opiate receptor as reported previously by other groups. Despite the nearly identical distribution patterns, an intriguing difference in the interaction of DAMGO and carfentanil with the μ opiate receptor was observed. The biologically active nonhydrolyzable analog of GTP, GTPγS, was able to completely abolish or greatly diminish specific [³H]DAMGO binding depending on brain region; GTPγS had little or no effect on specific [³H]carfentanil binding. This latter difference in the molecular interaction of DAMGO and carfentanil with the μ opiate receptor may indicate that some of the observed differences in the effects of fentanyl-like opiates may be due to a difference in the intrinsic activity of the fentanyl derivatives at the μ opiate receptor. © 1993 Wiley-Liss, Inc.     

3H]thienyl-phencyclidine ([3H]TCP) binds to two different sites in rat brain. Localization by autoradiographic and biochemical techniques

A high affinity [3H]thienyl-phencyclidine ([3H]TCP) binding and its similarity to that of [3H]phencyclidine ([3H]PCP) have been demonstrated on whole rat brain homogenates. We now describe the regional distribution of the [3H]TCP binding sites in the rat brain with fixed sections and frozen slide-mounted sections visualized by autoradiography and with homogenates of 12 regions by direct binding experiments. The 3 techniques give a similar pattern for the [3H]TCP binding distribution and the biochemical study reveals that two distinct binding sites for [3H]TCP exist: one of high affinity (5-10 nM) in the forebrain, which should be responsible for the psychotropic effects and a second one of lower affinity (50-80 nM) in the hindbrain and the spinal cord, which should be involved in the extrapyramidal behavior induced by PCP and congeneers. Competition experiments have shown that muscarinic compounds interact only with the hindbrain receptor possibly in two different sites, although morphine interacts with a very low affinity with the forebrain's high affinity receptor. Results obtained with SKF-10,047 (N-allylnormetazocine) seem to indicate that TCP and sigma-receptors are different.     

Benzodiazepine ([3H]flunitrazepam) binding in cat visual cortex: ontogenesis of normal characteristics and the effects of dark rearing

[3H]Flunitrazepam (FNZ) binding sites were characterized in homogenates of cat visual cortex during normal postnatal development and following dark rearing from birth. In parallel experiments, the distribution and density of [3H]FNZ binding sites were examined by in vitro autoradiographic or 'scrape' methods. In homogenates, Bmax measurements showed low early values, rising to a peak in receptor density at about 60 days postnatal, followed by a decline in adulthood. At all ages, gamma-aminobutyric acid (GABA) altered the Kd, but not the Bmax of [3H]FNZ binding sites. Kd values showed a general increase with age, parallelled by an increased sensitivity to GABA. Receptor autoradiography revealed that the highest density of [3H]FNZ binding sites was in layer IV of cats of all ages. Deafferentation of extrinsic inputs to the visual cortex by surgical undercutting did not alter this pattern of laminar distribution, indicating that the receptors were associated with intrinsic cortical elements rather than subcortical inputs. Dark rearing had no effect on [3H]FNZ laminar distribution in the visual cortex. The Bmax was higher at 30 days postnatal, but did not differ significantly thereafter. Modulation by GABA was concomitantly higher at 30 days, but lower than normal in dark-reared animals at ages greater than 30 days postnatal. The results are discussed in relation to the normal and abnormal development of GABA receptors in the cat visual cortex.     

Pre- and postsynaptic distribution of cannabinoid and mu opioid receptors in rat spinal cord

In vitro receptor binding and quantitative autoradiography were used to assess the pre- and postsynaptic distribution of cannabinoid receptors in the cervical dorsal horn of the rat spinal cord. An extensive unilateral dorsal rhizotomy was performed across seven or eight successive spinal segments from C3 to T1 or T2. The densities of cannabinoid and mu opioid receptors in the central (C6) spinal segment were assessed 2, 4, 8, and 16 days post rhizotomy and compared with those of untreated rats. Rhizotomy induced approximately a 50% ipsilateral loss in the [3H]CP55,940 binding to spinal cannabinoid receptors that was maximal at 8 days post-rhizotomy. By comparison, the binding of [3H][d-Ala2-MePhe4, Gly-ol5]enkephalin (DAMGO) to mu receptors was depleted approximately 60% in near-adjacent sections. By contrast, changes in [3H]CP55,940 binding contralateral to the deafferentation were largely absent at all post-lesion delays. These data suggest that under conditions in which a spinal segment is completely deafferented, approximately 50% of cannabinoid receptors in the cervical (C6) dorsal horn reside presynaptically on central terminals of primary afferents. The present data provide anatomical evidence for presynaptic as well as postsynaptic localization of cannabinoid receptors in the spinal dorsal horn.     

Distribution of mu, delta, and kappa opioid receptors in the hypothalamus of the rat

The radioautographic distribution of mu, delta and kappa opioid binding sites was examined by in vitro radioautography in the rat hypothalamus using the highly selective ligands [125I]-FK 33-824, [125I]azidoDTLET and [125I]DPDYN, respectively. Levels of mu opioid binding sites varied considerably amongst hypothalamic nuclei. mu Opioid labeling was dense in the medial preoptic area, medial preoptic nucleus, suprachiasmatic nucleus and ventromedial nucleus, whilst the supraoptic nucleus, paraventricular nucleus, arcuate nucleus and dorsomedial nucleus were devoid of labeling. Delta opioid labeling was sparse throughout most of the hypothalamus; however, moderate binding densities were detected in the suprachiasmatic and ventromedial nucleus. kappa Opioid labeling was also scant throughout the hypothalamus with the exception of the suprachiasmatic nucleus which was very densely labeled. Our results indicate that the 3 opioid receptors types are differentially distributed within the hypothalamus, although a significant overlap exists. In general, the distribution of hypothalamic opioid receptors correlates well with that of opioid-containing terminal fibers and may represent the anatomical substrate for opioid involvement in the hypothalamic regulation of autonomic, behavioral and neuroendocrine functions.     

Acute ethanol administration differentially modulates mu opioid receptors in the rat meso-accumbens and mesocortical pathways

Biochemical and pharmacological evidence suggest that the dopaminergic mesolimbic system plays a key role in mediating the reinforcing properties of alcohol and other drugs of abuse. Alcohol reinforcement and high alcohol drinking behavior have been postulated to be partially mediated by a neurobiological mechanism involving the alcohol-induced activation of the endogenous opioid system. The aim of this work was to study the effect of the in vivo acute administration of ethanol on mu (mu) opioid receptors in the rat dopaminergic meso-accumbens and mesocortical pathways by quantitative receptor autoradiography. [(3)H]DAMGO binding was significantly decreased in the ventral tegmental area (VTA) 30 min after ethanol administration. A small ethanol-induced reduction was observed in the shell region of the nucleus accumbens 1 h after exposure. In contrast, 2 h after ethanol administration, [(3)H]DAMGO binding was significantly increased in the frontal and prefrontal cortices. The observed changes correlated well with high ethanol plasma levels. Our results suggest that the reinforcing properties of ethanol may be partially mediated by mechanisms involving the ethanol-induced down- and up-regulation of mu receptors in the dopaminergic mesolimbic system. Mu receptors in the VTA and the frontal and prefrontal cortices may be involved in the in vivo acute responses to ethanol and could play a key role in modulating the dopaminergic activity of the mesocortical pathway in response to the drug. In contrast, the contribution of both mu and delta receptors in the nucleus accumbens might be relevant in these processes.     

Morphine can produce analgesia via spinal kappa opioid receptors in the absence of mu opioid receptors

Previous studies have demonstrated the virtual lack of analgesia in mu opioid receptor knockout mice after systemic administration of morphine. Thus, it has been suggested that analgesic actions of morphine are produced via the mu opioid receptor, despite its ability to bind to kappa and delta receptors in vitro. However, it is not clear whether the results of these studies reflect the effect of morphine in the spinal cord. In the present study, we report study of the analgesic actions of spinally-administered morphine and other opioid receptor agonists in mu opioid receptor knockout and wild type mice. Morphine produced a dose-dependent antinociceptive effect in the tail flick test in the knockout mice, although higher doses were needed to produce antinociception than in wild type mice. The antinociceptive effect of morphine was completely blocked by naloxone (a non-selective opioid antagonist) and nor-binaltorphimine (nor-BNI, a selective kappa-opioid receptor antagonist), but not by naltrindole (a selective delta-opioid receptor antagonist). U-50,488H (a selective kappa-opioid receptor agonist) also produced a dose-dependent antinociceptive effect in knockout mice but presented lower analgesic potency in knockout mice than in wild type mice. Analgesic effects of [d-Pen2,d-Pen5]enkephalin (DPDPE, a selective delta-opioid receptor agonist) were observed in wild type mice but abolished in knockout mice. SNC80 (a selective delta-opioid receptor agonist) was not antinociceptive even in wild type mice. The present study demonstrated that morphine can produce thermal antinociception via the kappa opioid receptor in the spinal cord in the absence of the mu opioid receptor. Lower potency of U50,488H in mu opioid receptor knockout mice suggests interaction between kappa and mu opioid receptors at the spinal level.     

A monoclonal anti-idiotypic antibody to mu and delta opioid receptors.

A mouse monoclonal, anti-idiotypic, anti-opioid receptor antibody (Ab2-AOR) has been generated from monoclonal anti-morphine antibodies (Ab1). Hybridoma culture supernatants were screened by a solid phase radioimmunoassay (RIA), based on their competition with radiolabelled morphine for Ab1. One of the Ab2s that gave a positive RIA also competed at rat brain opioid receptors with tritiated opioid ligands dihydromorphine (DHM), naloxone, etorphine, Tyr-D-Ala-Gly-Phe-D-Leu (DADLE), Tyr-D-Ala-Gly-NMe-Phe-Gly-ol (DAMGE) and Tyr-D-Pen-Gly-Phe-D-Pen (DPDPE). SDS-PAGE revealed Ab2-AOR to be highly purified after successive affinity and protein A-Sepharose chromatography. Ab2-AOR at concentrations of 10-100 nM competed with both mu- and delta-selective specific ligands for brain opioid receptors. Less than 13 micrograms/ml Ab2-AOR completely inhibited specific opioid radioligand binding to both soluble and membrane-bound opioid receptors. To demonstrate its anti-delta receptor activity further, a double-antibody ELISA procedure was developed that is based on the binding of Ab2-AOR to immobilized NG 108-15 cells (which contain only delta opioid receptors). Dose-dependent, opioid peptide- and opiate alkaloid-competitive binding of Ab2-AOR-containing ascites fluid to NG 108-15 cells was observed. A mu opioid agonist effect was demonstrated for Ab2-AOR, in that it decreased by 70% [3H]thymidine incorporation into DNA of fetal brain cell aggregates. This agonist-like action of Ab2-AOR was blocked by naltrexone. The antibody bound specifically to brain tissue sections and the presence of diprenorphine blocked this interaction. Hence, an Ab2 with mu and delta specificity has been characterized.     

Developmental profile of kappa, mu and delta opioid receptors in the rat and guinea pig cerebellum

The phenotypic expression of the three opioid receptors kappa, mu and delta was determined in the rat and guinea pig cerebellum during ontogeny. In both species, the increase in cerebellum weight was accompanied with a gradual increase in the total number of opioid receptors. At the same time, however, the receptor concentration (femtomoles per milligram protein) fluctuated, and finally declined by 3.3- and 2.0-fold in the adult rat and guinea pig cerebella, respectively. In the guinea pig, kappa receptors constituted about 80% of the receptors at the earliest developmental stage tested, day 20 of the embryo, but then there was a sharp decrease in this value, resulting from an increase in both mu and delta receptors. This decrease was, however, transient, and the percentage of the kappa receptors increased again to reach 77% of the receptors in the adult cerebella. A similar transient increase in the percentage of mu and delta receptors was observed during the development of the rat cerebellum, although the receptor density in this species was 1/4-1/8 that of the guinea pig. Cultures of aggregating guinea pig cerebellar cells were used to further study the factors regulating the expression of the three opioid receptors in this brain region. The possibility that the development in vivo of cells expressing different opioid receptors is associated with the ontogeny of the endogenous opioid peptides is discussed.     

Autoradiographic differentiation of mu, delta, and kappa opioid receptors in the rat forebrain and midbrain

While there is an abundance of pharmacological and biochemical evidence to suggest the existence of multiple opioid receptors, their precise localization within the brain is unclear. To help clarify this issue, the present study examined the distributions of the mu, delta, and kappa opioid receptor subtypes in the rat forebrain and midbrain using in vitro autoradiography. Mu and delta receptors were labeled with the selective ligands 3H-DAGO (Tyr- D-Ala-Gly-MePhe-Gly-ol), and 3H-DPDPE (D-Pen2, D-Pen5-enkephalin), respectively, while the kappa receptors were labeled with 3H-(-)bremazocine in the presence of unlabeled DAGO and DPDPE. Based on previous findings in our laboratory, the labeling conditions were such that each ligand selectively occupied approximately 75% of each of the opioid sites. The results demonstrated that all 3 opioid receptor subtypes were differentially distributed in the rat brain. Mu binding was dense in anterior cingulate cortex, neocortex, amygdala, hippocampus, ventral dentate gyrus, presubiculum, nucleus accumbens, caudate putamen, thalamus, habenula, interpeduncular nucleus, pars compacta of the substantia nigra, superior and inferior colliculi, and raphe nuclei. In contrast, delta binding was restricted to only a few brain areas, including anterior cingulate cortex, neocortex, amygdala, olfactory tubercle, nucleus accumbens, and caudate putamen. Kappa binding, while not as widespread as observed with mu binding, was densely distributed in the amygdala, olfactory tubercle, nucleus accumbens, caudate putamen, medial preoptic area, hypothalamus, median eminence, periventricular thalamus, and interpeduncular nucleus. While all 3 opioid receptor subtypes could sometimes be localized within the same brain area, their precise distribution within the region often varied widely. For example, in the caudate putamen, mu binding had a patchy distribution, while delta and kappa sites were diffusely distributed, with delta sites being particularly dense ventrolaterally and kappa sites being concentrated ventromedially. These results support the existence of at least 3 distinct opioid receptors with possibly separate functional roles.     

Neurons with mu opioid receptors interact indirectly with enkephalin-containing neurons in the rat dentate gyrus

In the dentate gyrus, mu opioid receptors (MORs) and their enkephalin agonists have overlapping distributions and influence excitability and plasticity. Released endogenous enkephalins can activate at least some of these MORs; however, whether these interactions involve synaptically associated profiles or more distant associations and whether some subcellular compartments (e.g., terminals or dendrites) are more likely to be targeted than others are not known. To elucidate the relationships between potential sites of enkephalin release and MORs, MOR1 and leucine-enkephalin (LE) immunoreactivities were localized in the hilus by electron microscopy, using immunoperoxidase and immunogold markers. Of the 573 MOR-immunoreactive (ir) profiles analyzed, most were axons and terminals (51 and 30%, respectively), and fewer were dendrites (12%), glia (3%), or unclassifiable (4%). Most MOR-ir profiles resembled interneuron processes, while most LE-ir terminals resembled mossy fibers. One third of MOR-ir profiles were within 3 microm and approximately half were within 4 microm of the nearest LE-ir profile. In contrast, few (3%) MOR-ir profiles contacted LE-ir profiles; only 16% of these contacts included observable synapses, and very few profiles (0.5%) colocalized MOR and LE immunoreactivity. MOR-ir axons, terminals, and dendrites were not distributed differently relative to LE-ir profiles. These results suggest that activation of hilar MORs by LE usually involves short-range volume transmission and that dendritic MORs are as likely as axonal and terminal MORs to be activated by released LE. However, the greater abundance of MOR-ir axons and terminals compared to dendrites indicates that presynaptic profiles are a more prominent target for enkephalins and exogenous MOR agonists such as morphine.     

Differential postnatal development of mu and delta opiate receptors

We found a differential postnatal development of mu and delta opiate receptors. Mu receptors labelled with low concentrations of [3H]naloxone appeared to develop earlier than did delta receptors labelled with [3H]D-Ala2-D-Leu5-enkephalin (0.5 nM). Competition binding studies also revealed a delayed appearance of delta receptors (day 12 postnatal).     

Ex vivo determination of opiate antagonist binding at mu-opioid ([3H]-DAGO) receptors in hypothalamic micropunches from maturing female rats: comparison between SDZ 210-096 and nalmefene.

We have previously shown that the duration of opioid receptor blockade is critical in determining the degree of opioid antagonist effect following peripheral injection of naloxone and naltrexone. In the present work, we have used this ex vivo technique to compare receptor occupancy of a new opiate antagonist, SDZ 210-096 (SDZ), to that of nalmefene (NLM) in maturing female rats. Two doses (SDZ, 5.6 and 50 mg/kg; NLM, 2.5 and 50 mg/kg) were injected subcutaneously into 3 groups of rats (infantile, juvenile and peripubertal). Micropunches from hypothalamic coronal slices (300 microns) were removed at various times post-injection for quantification of mu-opioid receptors with [3H]-DAGO. Acute administration of the lower dose of SDZ inhibited ligand binding almost completely by 3 h but 50% recovery was observed in all age groups by 12 h. In contrast, SDZ 50 mg/kg provided 80-100% antagonism for at least 24 h. Age-related differences in the ability of SDZ to inhibit [3H]-DAGO binding were observed in that hypothalamic mu-opioid receptors were blocked for longer periods in younger rats. Determination of receptor occupation following NLM injection confirmed that it too has prolonged duration of action but a 24 h blockade is not achieved with either dose of this antagonist. Age-related and dose-related changes in receptor occupancy were minimal compared to SDZ. These studies clarify the interaction of these antagonists at hypothalamic mu-opioid receptors and provide information which allows a clearer interpretation of results in experiments involving opioid blockade.