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Mu-opioid receptor (µOR) agonists such as fentanyl have long been used for pain management, but are considered a major public health concern owing to their adverse side effects, including lethal overdose 1 . Here, in an effort to design safer therapeutic agents, we report an approach targeting a conserved sodium ion-binding site 2 found in µOR 3 and many other class A G-protein-coupled receptors with bitopic fentanyl derivatives that are functionalized via a linker with a positively charged guanidino group. Cryo-electron microscopy structures of the most potent bitopic ligands in complex with µOR highlight the key interactions between the guanidine of the ligands and the key Asp 2.50 residue in the Na + site. Two bitopics (C5 and C6 guano) maintain nanomolar potency and high efficacy at G i subtypes and show strongly reduced arrestin recruitment-one (C6 guano) also shows the lowest G z efficacy among the panel of µOR agonists, including partial and biased morphinan and fentanyl analogues. In mice, C6 guano displayed µOR-dependent antinociception with attenuated adverse effects, supporting the µOR sodium ion-binding site as a potential target for the design of safer analgesics. In general, our study suggests that bitopic ligands that engage the sodium ion-binding pocket in class A G-protein-coupled receptors can be designed to control their efficacy and functional selectivity profiles for G i , G o and G z subtypes and arrestins, thus modulating their in vivo pharmacology., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

The Locus Coeruleus (LC) is a pontine nucleus involved in many physiological processes, including the control of the sleep/wake cycle (SWC). At cellular level, the LC displays a high density of opioid receptors whose activation decreases the activity of LC noradrenergic neurons. Also, microinjections of morphine administered locally in the LC of the cat produce sleep associated with synchronized brain activity in the electroencephalogram (EEG). Even though much of the research on sleep has been done in the cat, the subcellular location of opioid receptors in the LC and their relationship with LC noradrenergic neurons is not known yet in this species. Therefore, we conducted a study to describe the ultrastructural localization of mu-opioid receptors (MOR), delta-opioid receptors (DOR) and tyrosine hydroxylase (TH) in the cat LC using high resolution electron microscopy double-immunocytochemical detection. MOR and DOR were localized mainly in dendrites (45% and 46% of the total number of profiles respectively), many of which were noradrenergic (35% and 53% for MOR and DOR, respectively). TH immunoreactivity was more frequent in dendrites (65% of the total number of profiles), which mostly also expressed opioid receptors (58% and 73% for MOR and DOR, respectively). Because the distribution of MORs and DORs are similar, it is possible that a substantial sub-population of neurons co-express both receptors, which may facilitate the formation of MOR-DOR heterodimers. Moreover, we found differences in the cat subcellular DOR distribution compared with the rat. This opens the possibility to the existence of diverse mechanisms for opioid modulation of LC activity., (Copyright © 2021 Elsevier B.V. All rights reserved.)

The nociceptin opioid receptor (NOP), the fourth member of the opioid receptor family, and its endogenous peptide ligand, nociceptin or orphanin FQ (N/OFQ), play a vital role in several central nervous system pathways regulating pain, reward, feeding, anxiety, motor control and learning/memory. Both selective NOP agonists as well as bifunctional agonists at the NOP and mu opioid receptor (MOP) have potential therapeutic applications in CNS disorders related to these processes. Using Surflex-Dock protocols, we conducted a computational structure-activity study of four scaffold classes of NOP ligands with varying NOP-MOP selectivity. By docking these compounds into the orthosteric binding sites within an active-state NOP homology model, and an active-state MOP crystal structure, the goal of this study was to use a structure-based drug design approach to modulate NOP affinity and NOP vs. MOP selectivity. We first docked four parent compounds (no side chain) to determine their binding interactions within the NOP and MOP binding pockets. Various polar sidechains were added to the heterocyclic A-pharmacophore to modulate NOP ligand affinity. The substitutions mainly contained a 1-2 carbon chain with a polar substituent such as an amine, alcohol, sulfamide, or guanidine. The SAR analysis is focused on the impact of structural changes in the sidechain, such as chain length, hydrogen bonding capability, and basic vs neutral functional groups on binding affinity and selectivity at both NOP and MOP receptors. This study highlights structural modifications that can be leveraged to rationally design both selective NOP and bifunctional NOP-MOP agonists with different ratios of functional efficacy.

Opioid analgesics such as morphine have indispensable roles in analgesia. However, morphine use can elicit side effects such as respiratory depression and constipation. It has been reported that G protein-biased agonists as substitutes for classic opioid agonists can alleviate (or even eliminate) these side effects. The compounds PZM21 and TRV130 could be such alternatives. Nevertheless, there are controversies regarding the efficacy and G protein-biased ability of PZM21. To demonstrate a rationale for the reduced biasing agonism of PZM21 compared with that of TRV130 at the molecular level, we undertook a long-term molecular dynamics simulation of the μ-opioid receptor (MOR) upon the binding of three ligands: morphine, TRV130, and PZM21. We found that the delayed movement of the W293 6.48 (Ballesteros-Weinstein numbering) side chain was a factor determining the dose-dependent agonism of PZM21. Differences in conformational changes of W318 7.35 , Y326 7.43 , and Y336 7.53 in PZM21 and TRV130 explained the observed differences in bias between these ligands. The extent of water movements across the receptor channel was correlated with analgesic effects. Taken together, these data suggest that the observed differences in conformational changes of the studied MOR-ligand complexes point to the low-potency and lower bias effects of PZM21 compared with the other two ligands, and they lay the foundation for the development of G protein-biased agonists.

The μ-opioid receptor (μOR) is a G-protein-coupled receptor (GPCR) and the target of most clinically and recreationally used opioids. The induced positive effects of analgesia and euphoria are mediated by μOR signalling through the adenylyl cyclase-inhibiting heterotrimeric G protein G i . Here we present the 3.5 Å resolution cryo-electron microscopy structure of the μOR bound to the agonist peptide DAMGO and nucleotide-free G i . DAMGO occupies the morphinan ligand pocket, with its N terminus interacting with conserved receptor residues and its C terminus engaging regions important for opioid-ligand selectivity. Comparison of the μOR-G i complex to previously determined structures of other GPCRs bound to the stimulatory G protein G s reveals differences in the position of transmembrane receptor helix 6 and in the interactions between the G protein α-subunit and the receptor core. Together, these results shed light on the structural features that contribute to the G i protein-coupling specificity of the µOR.

A collection of crystal structures of rhodopsin, β2-adrenergic and adenosine A2A receptors in active, intermediate and inactive states were selected for structural and energetic analyses to identify the changes involved in the activation/deactivation of Class A GPCRs. A set of helix interactions exclusive to either inactive or active/intermediate states were identified. The analysis of these interactions distinguished some local conformational changes involved in receptor activation, in particular, a packing between the intracellular domains of transmembrane helices H3 and H7 and a separation between those of H2 and H6. Also, differential movements of the extracellular and intracellular domains of these helices are apparent. Moreover, a segment of residues in helix H3, including residues L/I3.40 to L3.43, is identified as a key component of the activation mechanism, acting as a conformational hinge between extracellular and intracellular regions. Remarkably, the influence on the activation process of some glutamic and aspartic acidic residues and, as a consequence, the influence of variations on local pH is highlighted. Structural hypotheses that arose from the analysis of rhodopsin, β2-adrenergic and adenosine A2A receptors were tested on the active and inactive M2 muscarinic acetylcholine receptor structures and further discussed in the context of the new mechanistic insights provided by the recently determined active and inactive crystal structures of the μ-opioid receptor. Overall, the structural and energetic analyses of the interhelical interactions present in this collection of Class A GPCRs suggests the existence of a common general activation mechanism featuring a chemical space useful for drug discovery exploration., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Receptor binding studies have shown that the density of mu opioid receptors (MORs) in the basolateral amygdala is among the highest in the brain. Activation of these receptors in the basolateral amygdala is critical for stress-induced analgesia, memory consolidation of aversive events, and stress adaptation. Despite the importance of MORs in these stress-related functions, little is known about the neural circuits that are modulated by amygdalar MORs. In the present investigation light and electron microscopy combined with immunohistochemistry was used to study the expression of MORs in the anterior basolateral nucleus (BLa). At the light microscopic level, light to moderate MOR-immunoreactivity (MOR-ir) was observed in a small number of cell bodies of nonpyramidal interneurons and in a small number of processes and puncta in the neuropil. At the electron microscopic level most MOR-ir was observed in dendritic shafts, dendritic spines, and axon terminals. MOR-ir was also observed in the Golgi apparatus of the cell bodies of pyramidal neurons (PNs) and interneurons. Some of the MOR-positive (MOR+) dendrites were spiny, suggesting that they belonged to PNs, while others received multiple asymmetrical synapses typical of interneurons. The great majority of MOR+ axon terminals (80%) that formed synapses made asymmetrical (excitatory) synapses; their main targets were spines, including some that were MOR+. The main targets of symmetrical (inhibitory and/or neuromodulatory) synapses were dendritic shafts, many of which were MOR+, but some of these terminals formed synapses with somata or spines. All of our observations were consistent with the few electrophysiological studies which have been performed on MOR activation in the basolateral amygdala. Collectively, these findings suggest that MORs may be important for filtering out weak excitatory inputs to PNs, allowing only strong inputs or synchronous inputs to influence pyramidal neuronal firing., (Copyright © 2015 IBRO. Published by Elsevier Ltd. All rights reserved.)

Endomorphin-2 (EM2) demonstrates a potent antinociceptive effect via the μ-opioid receptor (MOR). To provide morphological evidence for the pain control effect of EM2, the synaptic connections between EM2-immunoreactive (IR) axonal terminals and γ-amino butyric acid (GABA)/MOR co-expressing neurons in lamina II of the spinal trigeminal caudal nucleus (Vc) were investigated in the rat. Dense EM2-, MOR- and GABA-IR fibers and terminals were mainly observed in lamina II of the Vc. Within lamina II, GABA- and MOR-neuronal cell bodies were also encountered. The results of immunofluorescent histochemical triple-staining showed that approximately 14.2 or 18.9% of GABA-IR or MOR-IR neurons also showed MOR- or GABA-immunopositive staining in lamina II; approximately 45.2 and 36.1% of the GABA-IR and MOR-IR neurons, respectively, expressed FOS protein in their nuclei induced by injecting formalin into the left lower lip of the mouth. Most of the GABA/MOR, GABA/FOS, and MOR/FOS double-labeled neurons made close contacts with EM2-IR fibers and terminals. Immuno-electron microscopy confirmed that the EM2-IR terminals formed synapses with GABA-IR or MOR-IR dendritic processes and neuronal cell bodies in lamina II of the Vc. These results suggest that EM2 might participate in pain transmission and modulation by binding to MOR-IR and GABAergic inhibitory interneuron in lamina II of the Vc to exert inhibitory effect on the excitatory interneuron in lamina II and projection neurons in laminae I and III.

Adaptive responses in glutamate and opioid receptor systems in limbic circuits are emerging as a critical component of the neural plasticity induced by chronic use of abused substances. The present commentary reviews findings from neuroanatomical studies, with superior spatial resolution, that support a cellular basis for prominent interactions of glutamate and opioid receptor systems in preclinical models of drug addiction. The review begins by highlighting the advantages of high-resolution electron microscopic immunohistochemistry for unraveling receptor interactions at the synapse. With an emphasis on a recent publication describing the anatomical relationship between the μ-opioid receptor (MOR) and the AMPA-GluR2 subunit (Beckerman, M. A., and Glass, M. J., 2011. Ultrastructural relationship between the AMPA-GluR2 receptor subunit and the mu-opioid receptor in the mouse central nucleus of the amygdala. Exp Neurol), we review the anatomical evidence for opioid-induced neural plasticity of glutamate receptors in selected brain circuits that are key integrative substrates in the brain's motivational system. The findings stress the importance of glutamate-opioid interactions as important neural mediators of adaptations to chronic use of abused drugs, particularly within the amygdaloid complex., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Activation of GluR2-expressing non-calcium-permeable AMPA-type glutamate receptors in the central nucleus of the amygdala (CeA) may play an important role in integrating emotion and memory with goal-directed behaviors involved in opioid addiction. The location of non-calcium-permeable AMPA receptors within distinct neuronal compartments (i.e., soma, dendrite, or axon) is an important functional feature of these proteins; however, their ultrastructural location and subcellular relationship with mu-opioid receptors (μOR) in the CeA are unknown. Immunocytochemical electron microscopy was used to characterize the ultrastructural distribution of GluR2 and its association with μOR in the mouse CeA. A single-labeling analysis of GluR2 distribution employing immunoperoxidase or immunogold markers revealed that this protein was frequently affiliated with intracellular vesicular organelles, as well as the plasma membrane of CeA neuronal profiles. Among all GluR2-labeled neuronal structures, over 85% were dendrites or somata. Unlabeled axon terminals frequently formed asymmetric excitatory-type synaptic junctions with GluR2-labeled dendritic profiles. Dual-labeling immunocytochemical analysis showed that GluR2 and μOR were co-localized in neuronal compartments. Among all dual-labeled structures, approximately 80% were dendritic. Synaptic inputs to these dual-labeled dendrites were frequently from unlabeled axon terminals forming asymmetric excitatory-type synapses. The presence of GluR2 in dendritic profiles receiving asymmetric synapses suggests that activation of the non-calcium-permeable AMPA receptor plays a role in the postsynaptic modulation of excitatory signaling involving CeA neuronal circuits that coordinate sensory, affective, and behavioral processes involved in drug addiction. Given the critical role of non-calcium-permeable AMPA receptor function in neural and behavioral adaptability, their dendritic association with μOR in CeA dendrites provides a neuronal substrate for opioid-mediated plasticity., (2010 Elsevier Inc. All rights reserved.)

GPR177, the mammalian ortholog of Drosophila Wntless/Evi/Sprinter, was recently identified as a novel mu-opioid receptor (MOR) interacting protein. GPR177 is a trans-membrane protein pivotal to mediating the secretion of Wnt signaling proteins. Wnt proteins, in turn, are essential in regulating neuronal development, a phenomenon inhibited upon chronic exposure to MOR agonists such as morphine and heroin. We previously showed that GPR177 and MOR are co-localized in the mouse dorsolateral striatum; however, the nature of this interaction was not fully elucidated. Therefore, in the present study, we examined cellular substrates for interactions between GPR177 and MOR using a combined immunogold-silver and peroxidase detection approach in coronal sections in the dorsolateral segment of the striatum. Semi-quantitative analysis of the ultrastructural distribution of GPR177 and MOR in striatal somata and in dendritic processes showed that, of the somata and dendritic processes exhibiting GPR177, 32% contained MOR immunolabeling while for profiles exhibiting MOR, 37% also contained GPR177 immunoreactivity. GPR177-labeled particles were localized predominantly along both the plasma membrane and within the cytoplasm of MOR-labeled dendrites. Somata and dendritic processes that contained both GPR177 and MOR more often received symmetric (inhibitory-type) synapses from unlabeled axon terminals. To further define the phenotype of GPR177 and MOR-containing cellular profiles, triple immunofluorescence detection showed that GPR177 and MOR are localized in neurons containing the opioid peptide, enkephalin, within the dorsolateral striatum. The results provide an anatomical substrate for interactions between MOR and its interacting protein, GPR177, in striatal opioid-containing neurons that may underlie the morphological alterations produced in neurons by chronic opiate use., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Microinjection of opioid receptor agonists into the nucleus tractus solitarius (NTS) has differential effects on cardiovascular, respiratory, and gastrointestinal responses. This can be achieved either by presynaptic modulation of inputs onto neurons or by postsynaptic activation of receptors on neurons in specific regions. Therefore we sought to determine whether responses of neurons to activation of opioid receptors were dependent on their location within the NTS. Using whole cell patch-clamp recordings from neurons within the NTS, the mu opioid receptor (MOR) agonist [D-Ala(2), N-Me-Phe(4),Gly(5)-ol]-enkephalin (DAMGO, 100 nM) hyperpolarized a proportion of neurons in the medial, dorsomedial and dorsolateral NTS, whereas no postsynaptic responses were observed in remaining subdivisions. DAMGO reduced the amplitude of solitary tract-evoked excitatory postsynaptic potentials (EPSPs) in all neurons tested, regardless of subdivision. The kappa opioid receptor (KOR) agonist U69593 (10-20 microM) also hyperpolarized a small fraction of neurons (6/79) and decreased the amplitude of EPSPs in 50% of neurons. In contrast, the delta-opioid receptor agonist DPDPE (1-4 microM) had no presynaptic or postsynaptic effects on NTS neurons even after preincubation with bradykinin. Anatomical data at the light and electron microscopic level complemented electrophysiological observations with respect to MOR location and further showed that MORs were present at both presynaptic and postsynaptic sites in the dorsolateral NTS, often at the same synapse. These data demonstrate site specific responses of neurons to activation of MORs and KORs, which may underlie their ability to modulate different autonomic reflexes.

Within the rat hippocampal formation, cholinergic afferents and mu-opioid receptors (MORs) are involved in many crucial learning processes, including those associated with drug reward. Pharmacological data, and the overlapping distributions of cholinergic and mu-opioid systems, particularly in the dentate gyrus, suggest that MOR activation is a potential mechanism for endogenous opioid modulation of cholinergic activity. To date, anatomical evidence supporting this has not been reported. To delineate the relationship between cholinergic afferents and MOR-containing processes in the dentate gyrus, hippocampal sections were dually immunolabeled for vesicular acetylcholine transporter (VAChT) and MOR-1 and examined by electron microscopy. VAChT immunoreactivity was in unmyelinated axons and axon terminals, and was most often associated with small synaptic vesicles. MOR immunoreactivity was found in axons, axon terminals and, to a lesser extent, perikarya, which resembled GABAergic basket cells. Semi-quantitative ultrastructural analysis revealed that from 5% to 13% (depending on laminar location) of VAChT-immunoreactive (ir) presynaptic profiles contained MOR immunoreactivity. Additionally, 7% of VAChT-ir presynaptic profiles directly apposed MOR-ir axons and terminals, and there were almost no appositions to MOR-ir dendrites. These data suggest that opioids may directly and indirectly modulate acetylcholine release and/or reuptake. In the hilus and molecular layer, 4% of VAChT-ir terminals contacted dendritic shafts that were also contacted by MOR-ir terminals. This suggests that cholinergic afferents and MOR-containing afferents can converge on granule cell dendrites (which are restricted to the molecular layer) and on interneuron dendrites in the hilus. The results of this study provide ultrastructural evidence for direct and indirect modulation of cholinergic systems by mu-opioids in the hippocampal formation.

Morphine stimulates the internalization of mu-opioid receptors (MORs) in transfected cell models to a lesser degree than opioid peptides and other analgesic drugs, such as methadone, and previous studies have reported that morphine does not produce a detectable redistribution of MORs in neural tissue after either acute or chronic administration. Nevertheless, morphine produces profound physiological effects, raising the question of whether receptor trafficking plays any role in the in vivo actions of morphine. We investigated the effects of opiate drugs on recombinant and native opioid receptors in the nucleus accumbens, which plays an important role in mediating the behavioral effects of opiate drugs. Morphine and methadone differed in their effects on the internalization of epitope-tagged MORs in cell bodies, introduced by viral gene transfer and imaged by fluorescence microscopy. A mutation of the cytoplasmic tail that confers morphine-induced internalization in cultured cells had a similar effect on receptor trafficking in nucleus accumbens cell bodies. Surprisingly, in contrast to its failure to affect MOR distribution detectably in cell bodies, acute morphine administration produced a pronounced change in MOR distribution visualized in the processes of the same neurons. A similar effect of acute morphine administration was observed for endogenously expressed MORs by immunoelectron microscopy; the acute administration of morphine increased the density of MORs associated with internal membrane structures specifically in dendrites. These results provide the first evidence that morphine regulates the distribution of MORs in neuronal processes, suggesting that "compartment-selective" membrane trafficking represents a previously unanticipated type of opioid receptor regulation contributing to the in vivo effects of opiate drugs on a physiologically relevant population of CNS neurons.

Endomorphin 2 is a newly discovered peptide that has high affinity and specificity for the mu-opioid receptor. One criterion for establishing that endomorphin serves as an endogenous agonist for the mu receptor is that it be anatomically distributed in close proximity to that receptor. We tested this idea with a preembedding double immunostaining technique to study synaptic relationships between them. The distributions of both endomorphin 2 and the mu-opioid receptor were similar in the dorsal horn of the cervical spinal cord at the light microscopic level. At the electron microscopic level, axon terminals with dense-cored vesicles containing endomorphin 2-like immunoreactivity were observed making mostly asymmetrical synapses on profiles immunostained for the mu-opioid receptor. The immunostaining for the mu-opioid receptor was found mostly in postsynaptic membranes in profiles having dendrite-like appearance. The results support the idea that endomorphin 2 is an endogenous ligand for the mu-opioid receptor. Furthermore, the results indicate that such a role is mediated at least in part through synaptic relationships.

Single particle tracking is a powerful tool for probing the organization and dynamics of the plasma membrane constituents. We used this technique to study the micro -opioid receptor belonging to the large family of the G-protein-coupled receptors involved with other partners in a signal transduction pathway. The specific labeling of the receptor coupled to a T7-tag at its N-terminus, stably expressed in fibroblastic cells, was achieved by colloidal gold coupled to a monoclonal anti T7-tag antibody. The lateral movements of the particles were followed by nanovideomicroscopy at 40 ms time resolution during 2 min with a spatial precision of 15 nm. The receptors were found to have either a slow or directed diffusion mode (10%) or a walking confined diffusion mode (90%) composed of a long-term random diffusion and a short-term confined diffusion, and corresponding to a diffusion confined within a domain that itself diffuses. The results indicate that the confinement is due to an effective harmonic potential generated by long-range attraction between the membrane proteins. A simple model for interacting membrane proteins diffusion is proposed that explains the variations with the domain size of the short-term and long-term diffusion coefficients.

Local injection of mu-opioid receptor specific neurotoxin, dermorphin-saporin, into the striatum resulted in selective degeneration of striatal neurons in the patch compartment. We analyzed subsequent anterograde degeneration of axons and terminals at light and electron microscopic level. Light microscopic examination after silver impregnation method revealed that degenerating axons and terminals arising from the striatal patch compartment were distributed in the globus pallidus, entopeduncular nucleus, and substantia nigra. They were found in both pars reticulata and compacta of the substantia nigra. Electron microscopic examination revealed that the degenerating axon terminals contained large pleomorphic vesicles and formed symmetric synapses on dendrites. The present results suggest that patch neurons expressing mu-opioid receptor send projection fibers to multiple nuclei of the basal ganglia.

A direct action of mu-opioid agonists on neurons in the spinal dorsal horn is thought to contribute to opiate-induced analgesia. In this study we have investigated neurons that express the mu-opioid receptor MOR-1 in rat spinal cord to provide further evidence about their role in nociceptive processing. MOR-1-immunoreactive cells were largely restricted to lamina II, where they comprised approximately 10% of the neuronal population. The cells received few contacts from nonpeptidergic unmyelinated afferents, but many from substance P-containing afferents. However, electron microscopy revealed that most of these contacts were not associated with synapses. None of the MOR-1 cells in lamina II expressed the neurokinin 1 receptor; however, the mu-selective opioid peptide endomorphin-2 was present in the majority (62-82%) of substance P axons that contacted them. Noxious thermal stimulation of the foot induced c-Fos expression in approximately 15% of MOR-1 cells in the medial third of the ipsilateral dorsal horn at mid-lumbar level. However, following pinching of the foot or intraplantar injection of formalin very few MOR-1 cells expressed c-Fos, and for intraplantar formalin injection this result was not altered significantly by pretreatment with systemic naloxone. Although these findings indicate that at least some of the neurons in lamina II with MOR-1 are activated by noxious thermal stimulation, the results do not support the hypothesis that the cells have a role in transmitting nociceptive information following acute mechanical or chemical noxious stimuli.

Agonists of the alpha-2A-adrenergic- (alpha(2A)-AR) and the mu-opioid-receptor (muOR) jointly affect autonomic functions that are also disregulated in animals undergoing withdrawal from chronic administration of the muOR agonist morphine. Cardiovascular and gastrointestinal reflexes are mediated, in part, by the medial nucleus of the solitary tract (mNTS) at caudal (cNTS) and intermediate (iNTS) subregions. Together, this evidence suggests that alpha(2A)-AR and muOR may be colocalized within many of the same neuronal profiles in both the intermediate and caudal mNTS. In order to examine whether alpha(2A)-AR and muOR are present within common somata, dendrites, or axon terminals in the mNTS, we used electron microscopic immunocytochemistry for the detection of antisera against each receptor at intermediate and caudal levels of this brain region. Most of the dually labeled profiles were somata and dendrites. Of all dual-labeled profiles in the iNTS 49% were somata and were 47% dendrites, whereas in the cNTS 61% were somata and 32% were dendrites. Within dual-labeled profiles, the intracellular distribution of alpha(2A)-AR and muOR differed. MuOR was more frequently associated with the plasmalemma, whereas alpha(2A)-AR was often affiliated with vesicular organelles. Few axon terminals, and even fewer glia, contained both markers. We also frequently observed single-labeled alpha(2A)-AR glia that apposed exclusively muOR-containing dendrites or axon terminals. These findings indicate that somata and dendrites contain functional sites for convergent muOR and alpha(2A)-AR activation. In addition, each receptor is positioned for involvement in intercellular signaling between apposed neurons and glia. Activation of alpha(2A)-AR on muOR-containing somata or dendrites, or on glia apposed to muOR-containing neurons, may help to account for the efficacy of alpha(2A)-AR agonists in relieving some of the autonomic symptoms of opiate withdrawal., (Copyright 2002 Wiley-Liss, Inc.)

Physiological studies have demonstrated that long-term potentiation (LTP) induction in N-methyl-D-aspartate (NMDA) receptor containing dentate granule cells following lateral perforant path stimulation is opioid dependent, involving mu-opioid receptors (MORs) on gamma-aminobutyric acid (GABA)-ergic neurons. To determine the cellular relationships of MORs to postsynaptic NMDA receptor-containing dendrites, immunoreactivity (-I) against MOR and the NMDA receptor subunit 1 (NMDAR1) was examined in the outer molecular layer of the dentate gyrus using electron microscopy. MOR-I was predominantly in axons and axon terminals. NMDAR1-I was almost exclusively in spiny dendrites, but was also in a few terminals. Using immunogold particles to localize precisely NMDAR1, one-third of the NMDAR1-I was detected on the dendritic plasmalemma; in dendritic spines plasmalemmal immunogold particles were near synaptic densities. Many MOR-labeled axons and terminals contacted NMDAR1-labeled dendrites. MOR-labeled terminals formed symmetric (inhibitory-type) synapses on NMDAR1-labeled dendritic shafts or nonsynaptically contacted NMDAR1-labeled shafts and spines. MOR-labeled axons often abutted NMDAR1-containing dendritic spines which received asymmetric (excitatory-type) synapses from unlabeled terminals. Occasionally, MOR-labeled terminals and dendrites were apposed to NMDAR1-containing terminals. These results provide anatomical evidence that endogenous enkephalins or exogenous opioid agonists could inhibit GABAergic terminals that modulate granule cell dendrites, thus boosting depolarizing events in granule cells and facilitating the activation of NMDA receptors located on their dendrites.

Substance P (SP) is a peptide that is present in unmyelinated primary afferents to the dorsal horn and is released in response to painful or noxious stimuli. Opiates active at the mu-opiate receptor (MOR) produce antinociception, in part, through modulation of responses to SP. MOR ligands may either inhibit the release of SP or reduce the excitatory responses of second-order neurons to SP. We examined potential functional sites for interactions between SP and MOR with dual electron microscopic immmunocytochemical localization of the SP receptor (NK1) and MOR in rat trigeminal dorsal horn. We also examined the relationship between SP-containing profiles and NK1-bearing profiles. We found that 56% of SP-immunoreactive terminals contact NK1 dendrites, whereas 34% of NK1-immunoreactive dendrites receive SP afferents. This result indicates that there is not a significant mismatch between sites of SP release and available NK1 receptors, although receptive neurons may contain receptors at sites distant from the peptide release site. With regard to opioid receptors, we found that many MOR-immunoreactive dendrites also contain NK1 (32%), whereas a smaller proportion of NK1-immunoreactive dendrites contain MOR (17%). Few NK1 dendrites (2%) were contacted by MOR-immunoreactive afferents. These results provide the first direct evidence that MORs are on the same neurons as NK1 receptors, suggesting that MOR ligands directly modulate SP-induced nociceptive responses primarily at postsynaptic sites, rather than through inhibition of SP release from primary afferents. This colocalization of NK1 and MORs has significant implications for the development of pain therapies targeted at these nociceptive neurons.

Opiates active at the mu-opiate receptor (MOR) produce antinociception, in part, through actions involving substance P (SP), a peptide present in both unmyelinated primary afferents and interneurons within the dorsal horn. We examined potential functional sites for interactions between SP and MOR by using dual electron microscopic immunocytochemical localization of antisera against SP and a sequence-specific antipeptide antibody against MOR in rat cervical spinal dorsal horn. The distribution was compared with that of the functionally analogous dorsal horn of the trigeminal nucleus caudalis. Many of the SP-immunoreactive terminals in the dorsal horn contacted dendrites that contain MOR (53% in trigeminal; 70% in cervical spinal cord). Conversely, within the cervical spinal dorsal horn 79% of the MOR-labeled dendrites that received any afferent input were contacted by at least one SP-containing axon or terminal. Although SP-immunoreactive dendrites were rare, many of these (48%) contained MOR, suggesting that the activity of SP-containing spinal interneurons may be regulated by MOR ligands. A few SP-labeled terminals also contained MOR (12% in trigeminal; 6% in cervical spinal cord). These data support the idea that MOR ligands produce antinociception primarily through modulation of postsynaptic second-order nociceptive neurons in the dorsal horns of spinal cord and spinal trigeminal nuclei, some of which contain SP. They also suggest, however, that in each region, MOR agonists can act presynaptically to control the release of SP and/or glutamate from afferent terminals. The post- and presynaptic MOR sites are likely to account for the potency of MOR agonists as analgesics., (Copyright 2000 Wiley-Liss, Inc.)

The delta opioid receptor (DOR) and mu opioid receptor (MOR) are abundantly distributed in the dorsal horn of the spinal cord. Simultaneous activation of each receptor by selective opiate agonists has been shown to result in synergistic analgesic effects. To determine the cellular basis for these functional associations, we examined the electron microscopic immunocytochemical localization of DOR and MOR in single sections through the superficial layers of the dorsal horn in the adult rat spinal cord (C2-C4). From a total of 270 DOR-labeled profiles, 49% were soma and dendrites, 46% were axon terminals and small unmyelinated axons, and 5% were glial processes. 6% of the DOR-labeled soma and dendrites, and < 1% of the glial processes also showed MOR-like immunoreactivity (MOR-LI). Of 339 MOR-labeled profiles, 87% were axon terminals and small unmyelinated axons, 12% were soma and dendrites, and 2% were glial processes. 21% of the MOR-labeled soma and dendrites, but none of the axon terminals also contain DOR-LI. The subcellular distributions of MOR and DOR were distinct in axon terminals. In axon terminals, both DOR-LI and MOR-LI were detected along the plasmalemma, but only DOR-LI was associated with large dense core vesicles. DOR-labeled terminals formed synapses with dendrites containing MOR and conversely, MOR-labeled terminals formed synapses with DOR-labeled dendrites. These results suggest that the synergistic actions of selective MOR- and DOR-agonists may be attributed to dual modulation of the same or synaptically linked neurons in the superficial layers of the spinal cord.

The distribution of mu opioid receptors was examined by light and electron microscopic autoradiography in the locus coeruleus of the rat following in vitro labelling with the iodinated agonist [125I]FK-33824. At the light microscopic level, specific mu opioid binding sites were concentrated over the perikarya and dendrites of neurons that were tyrosine hydroxylase-immunopositive in adjacent sections. Accordingly, both the number of tyrosine hydroxylase-immunoreactive neurons and the density of labelled mu receptors decreased markedly throughout the rostrocaudal extent of the nucleus following treatment with the catecholaminergic neurotoxin 6-hydroxydopamine. By electron microscopy, specifically labelled receptors were detected both inside and on the surface of locus coeruleus neurons. Intracellular sites were found by resolution circle analysis to be highly concentrated within the endoplasmic reticulum and Golgi apparatus, suggesting that the ligand recognizes both glycosylated and preglycosylated forms of receptor. The remainder were found mainly over the cytoplasmic matrix or intracytoplasmic vesicles, and were attributed to newly synthesized or recycled receptors in transit. Cell surface receptors were present over both dendritic and perikaryal membranes of noradrenergic cells. These were most highly concentrated opposite abutting axon terminals, suggesting the existence of receptor 'hot spots' at sites of putative endogenous ligand release. However, only a small proportion of these sites was associated with synaptic specializations. Furthermore, an important contingent was detected opposite non-axonal elements, such as dendrites and glial cells, suggesting that mu opioid ligands act mainly parasynaptically on locus coeruleus neurons. Finally, approximately 5% of labelled receptors were associated with axoglial interfaces, indicating that a minor action of mu opioids in the locus may be presynaptic and/or glial.

To delineate the cellular sites for the motor effects of opiates acting at the mu opioid receptor (MOR) in the rat caudate-putamen nucleus, we examined the ultrastructural immunogold and immunoperoxidase labeling of an antipeptide antiserum specific for the MOR. We also combined these labeling methods to examine the subcellular relationship between the MOR and the endogenous opioid peptide, Leu5-enkephalin (LE). By light microscopy, MOR-labeling was seen in a heterogeneous patchy distribution. Electron microscopic analysis of these patches showed that more than 80% of the total neuronal profiles (n = 1,586) containing MOR-like immunoreactivity (MOR-IR) were dendrites and dendritic spines. The remaining labeled profiles included a few perikarya and many axon terminals. MOR-IR was predominantly localized to extrasynaptic plasma membranes of dendrites, and to both synaptic vesicles and plasma membranes in terminals. Ten percent of the total MOR-labeled terminals (n = 272) formed asymmetric synapses with unlabeled or MOR-labeled dendritic spines. Terminals containing LE-IR formed synapses, in almost equal proportions, on MOR-labeled dendrites and dendritic spines, while over 80% of the unlabeled terminals formed synapses on MOR-labeled dendritic spines. Moreover, colocalization of MOR- and LE-IR was often seen in both dendrites and terminals. These results indicate that in patch compartments of the caudate-putamen nucleus, the MOR is mainly involved in extrasynaptic modulation of spiny neurons, including those that contain LE. In addition, the findings provide a cellular basis for presynaptic opioid modulation of neurotransmitter release through MOR located on axon terminals.

Neuropil within the terminal nuclei of the accessory optic tract of the rat showed intense to moderate mu-opioid receptor-like immunoreactivity (MOR-LI). After unilateral enucleation, MOR-LI within the terminal nuclei almost disappeared or was markedly reduced on the side contralateral to the operation. Electron microscopy revealed that MOR-LI axon terminals within the terminal nuclei were filled with round synaptic vesicles and in asymmetric synaptic contact mainly with dendritic profiles, and occasionally with somatic profiles.