Electron microscopic localization of photoaffinity‐labelled delta opioid receptors in the neostriatum of the rat

Abstract

The distribution of delta opioid receptors, selectively labelled in vitro with the photoaffinity probe monoiodo azido-DTLET ([D-Thr2,pN3Phe4, Leu5]enkephaly-Thr6), was analyzed by light and electron microscopic radioautography in sections from rat neostriatum. Preliminary experiments indicated that up to 65% of specific 125I-azido-DTLET binding to rat striatal sections was still detectable following prefixation of the brain with 0.5% glutaraldehyde. These experiments also showed that up to 20-30% of the specifically bound radioactivity was covalently linked following ultraviolet irradiation and was thereby retained in tissue during subsequent postfixation and dehydration steps. Accordingly, the topographic distribution of the covalently attached azido-DTLET molecules was similar to that seen in fresh frozen sections and characteristic of that previously described for delta sites. Light and electron microscopic examination of the label in prefixed, striatal sections irradiated with ultraviolet light revealed that a significant proportion of specifically bound 125I-azido-DTLET molecules was intraneuronal. Specifically, 16% of the labelled binding sites were found in dendrites, 12% in perikarya and 4% in axon terminals. These results suggest that an important proportion of delta opioid binding sites labelled in the neostriatum correspond to receptors that are undergoing synthesis, transport and/or recycling. They also imply that a major fraction of delta sites are associated with intrastriatal neurons, as opposed to afferent axons. Approximately 44% of the labelled binding sites were associated with neuronal plasma membranes. Although most of these were found at the level of axodendritic (20%) and dendrodendritic (7%) appositions, comparison of the labelling incidence of these two compartments with their frequency of occurrence in tissue suggested that delta sites are fairly widely dispersed along neuronal plasma membranes. Only a small proportion (smaller than that of mu or kappa sites labelled in the same region) was associated with synaptic specializations. These results support the concept that delta receptors correspond to molecular entities that are distinct from mu and kappa sites and suggest that delta ligands act primarily nonjunctionally on the plasma membrane of striatal neurons.

Subcellular and Subsynaptic Localization of Presynaptic and Postsynaptic Kainate Receptor Subunits in the Monkey Striatum

“…Our data demonstrate that Ͼ70% of both presynaptic and postsynaptic KAR subunit immunoreactivity is expressed intracellularly and that almost two-thirds of the plasma membrane-bound KARS are extrasynaptic. This pattern of distribution resembles that of G-protein-coupled metabotropic receptors which, for the most part, are expressed intracellularly or at nonsynapic sites along plasma membrane (Pasquini et al, 1992;Wang et al, 1997;Bernard et al, 1999;Hanson and Smith, 1999;Smith et al, 2000Smith et al, , 2001. Interestingly, both the presynaptic and postsynaptic effects of kainate receptors are consistent with those of metabotropic glutamate receptor functions (for review, see Conn and Pin, 1997;Anwyl, 1999;Cartmell and Schoepp, 2000).…”

From the Golgi–Cajal mapping to the transmitter-based characterization of the neuronal networks leading to two modes of brain communication: Wiring and volume transmission

“…The ultrastructural analysis based on transmitter and receptor immunocytochemistry has repeatedly demonstrated short distance transmitter/receptor mismatches for the peptide and classical transmitters with the receptor labeling outside synapses (Dana et al, 1989;Pasquini et al, 1992;Levey et al, 1993;Liu et al, 1994;Sesack et al, 1994;Aoki, 1992;Aoki and Pickel, 1992;Yung et al, 1995;Caillé et al, 1996;Aoki et al, 1998;Azmitia and Whitaker-Azmitia, 1991;Azmitia et al, 1996;Boudin et al, 1998;Baude and Shigemoto, 1998;Dournaud et al, 1998). Extrasynaptic location of receptors has been observed mainly for G-protein-coupled receptors, but also for ion channel linked receptors .…”

Molecular Pharmacology ofδ-Opioid Receptors

“…Ultrastructural localization using electron microscopy immunogold, photoaffinity-labeled receptors with [ 125 I]-DTLET and biochemical subcellular fractionation techniques reveal that the majority of DOPrs are localized predominantly to intracellular sites, with only a small subset of DOPrs found in association with neuronal plasma membranes throughout the central and peripheral nervous systems (Pasquini et al, 1992;Zerari et al, 1994;Arvidsson et al, 1995;Cheng et al, 1995Cheng et al, , 1997Elde et al, 1995;Zhang et al, 1998;PetajaRepo et al, 2000;Cahill et al, 2001b;Commons et al, 2001;Wang and Pickel, 2001;Petäjä-Repo et al, 2002Commons, 2003;Guan et al, 2005;Lucido et al, 2005;Gendron et al, 2006). The small number of plasma membrane-bound receptors is consistent with the observation that DOPr agonists have modest effects in modulating nociception and reward (Cahill et al, 2007;Pradhan et al, 2011); however, systemic administration of DOPr agonists such as SNC80 produces locomotor hyperactivity, anxiolytic effects, antidepressant effects, and absence seizures (Pradhan et al, 2011;Chu Sin Chung and Kieffer, 2013;Gendron et al, 2015).…”