Opioid receptors: from binding sites to visible molecules in vivo

Abstract

Opioid drugs such as heroin interact directly with opioid receptors whilst other addictive drugs, including marijuana, alcohol and nicotine indirectly activate endogenous opioid systems to contribute to their rewarding properties. The opioid system therefore plays a key role in addiction neurobiology and continues to be a primary focus for NIDA-supported research. Opioid receptors and their peptide ligands, the endorphins and enkephalins, form an extensive heterogeneous network throughout the central and peripheral nervous system. In addition to reward, opioid drugs regulate many functions such that opioid receptors are targets of choice in several physiological, neurological and psychiatric disorders. Because of the multiplicity and diversity of ligands and receptors, opioid receptors have served as an optimal model for G protein coupled receptor (GPCR) research. The isolation of opioid receptor genes opened the way to molecular manipulations of the receptors, both in artificial systems and in vivo, contributing to our current understanding of the diversity of opioid receptor biology at the behavioral, cellular and molecular levels. This review will briefly summarize some aspects of current knowledge that has accumulated since the very early characterization of opioid receptor genes. Importantly, we will identify a number of research directions that are likely to develop during the next decade.

Fig. 1. Receptor structure and signaling

(A) Lateral view of a 3D model of the human delta opioid receptor (from Décaillot and Kieffer, 2004). Helices are indicated as ribbons, side chains of aminoacids implicated in binding (dark gray) or both binding and activation (light grey are shown as sticks. The opioid binding site forms a pocket penetrating half-way into the helical bundle, and is similar across mu, delta and kappa receptors. (B) Opioid receptors are coupled to inhibitory G proteins and form signaling complexes with many protein partners. Opioid receptor activation modifies ion channel activities (decreased neuronal excitability or neurotransmitter release), decreases cAMP levels via inhibition of adenylate (Ad.) cyclase and activates phosphorylation pathways that lead to transcriptional regulations. As for all GPCRs, signaling is highly regulated by receptor phosphorylation and trafficking via scaffolding proteins. Signaling and regulatory proteins that have been identified are indicated in the figure. A current hypothesis is that different agonist ligands confer different patterns of receptor signaling and trafficking in vivo.

Fig. 2. Receptor imaging in delta-eGFP knock-in mice

Images are adapted from Scherrer et al. (Scherrer et al., 2006) (A) Epifluorescence macroscopy shows the general anatomical distribution of delta receptors. Top left, whole brain from wild-type (+/+) and knock-in (eGFP/eGFP) mice; top midde, coronal section at the level of the caudate putamen (Cpu); top right, sagittal section at the level of the hippocampus (Hip). Confocal microscopy of regions delimited by insets reveals receptor distribution with a cellular resolution (middle panels). Confocal microscopy of primary neurons from caudate putamen and hippocampus highlights the subcellular distribution of delta receptors (bottom panels). (B) Immunostaining with GAD antibodies (red) identifies GABAergic neurons expressing fluorescent delta receptors in neurons from the Cpu (Cpu-GABA) and Hip (Hip-GABA) in either brain sections (top) or primary cultures (bottom). Nuclei are stained in blue. (C) Delta receptor internalization in vivo, upon exposure to delta agonists. Left panels show prominent surface labeling in cortical (Ctx) and hippocampal (Hip) neurons in brain sections of vehicle-treated animals (left) and the typical punctate pattern of internalized receptors in brain sections of SNC80-treated animals (right); Scale bars 8 µm. Right panels shows delta-eGFP labeling in striatal primary neurons before (0 min) and 20 minutes after exposure to both a non-peptidic (SNC80) and a peptidic (deltorphin II) delta agonist; scale bars 12 µm. In those preparations, the receptor internalization process can be observed in real time, using time-lapse confocal microscopy (see Scherrer et al., 2006).

Fig. 2. Receptor imaging in delta-eGFP knock-in mice

Images are adapted from Scherrer et al. (Scherrer et al., 2006) (A) Epifluorescence macroscopy shows the general anatomical distribution of delta receptors. Top left, whole brain from wild-type (+/+) and knock-in (eGFP/eGFP) mice; top midde, coronal section at the level of the caudate putamen (Cpu); top right, sagittal section at the level of the hippocampus (Hip). Confocal microscopy of regions delimited by insets reveals receptor distribution with a cellular resolution (middle panels). Confocal microscopy of primary neurons from caudate putamen and hippocampus highlights the subcellular distribution of delta receptors (bottom panels). (B) Immunostaining with GAD antibodies (red) identifies GABAergic neurons expressing fluorescent delta receptors in neurons from the Cpu (Cpu-GABA) and Hip (Hip-GABA) in either brain sections (top) or primary cultures (bottom). Nuclei are stained in blue. (C) Delta receptor internalization in vivo, upon exposure to delta agonists. Left panels show prominent surface labeling in cortical (Ctx) and hippocampal (Hip) neurons in brain sections of vehicle-treated animals (left) and the typical punctate pattern of internalized receptors in brain sections of SNC80-treated animals (right); Scale bars 8 µm. Right panels shows delta-eGFP labeling in striatal primary neurons before (0 min) and 20 minutes after exposure to both a non-peptidic (SNC80) and a peptidic (deltorphin II) delta agonist; scale bars 12 µm. In those preparations, the receptor internalization process can be observed in real time, using time-lapse confocal microscopy (see Scherrer et al., 2006).

Fig. 3. Delta opioid receptors are localized both pre- and post-synaptically in the hippocampus (D. Massotte and B. Kieffer, unpublished)

Confocal imaging of brain sections from delta-eGFP mice, labeled with a MAP2 antibody that labels somatodendritic, but not axonal, compartments. MAP immunostaining is shown in red, fluorescently labeled delta receptors are shown in green, and cell nuclei in blue (DAPI). (A) A general view at the level of the hippocampus. Dentate gyrus (left panel), CA1 region (central panel) and CA3 region (right panel). Scale bars 100 µm. (B) Top panels: the delta receptor is expressed in dendrites. In this neuron MAP2 and delta-eGFP are co-localized (merged image on the right), suggesting a postsynaptic localization of the receptor. Scale bar 10 µm. Bottom panels: the delta receptor is also expressed in axons. In this neuron, lack of MAP2 and delta-eGFP co-staining (merged image on the right) indicates a presynaptic receptor localization. Scale bar 10µm.