Wendy Walwyn

Ligand-Directed Trafficking of the δ-Opioid Receptor In Vivo: Two Paths Toward Analgesic Tolerance

δ-Opioid receptors are G-protein-coupled receptors that regulate nociceptive and emotional responses. It has been well established that distinct agonists acting at the same G-protein-coupled receptor can engage different signaling or regulatory responses. This concept, known as biased agonism, has important biological and therapeutic implications. Ligand-biased responses are well described in cellular models, however, demonstrating the physiological relevance of biased agonism in vivo remains a major challenge. The aim of this study was to investigate the long-term consequences of ligand-biased trafficking of the δ-opioid receptor, at both the cellular and behavioral level. We used δ agonists with similar binding and analgesic properties, but high [SNC80 ((+)-4-[(αR)-α-((2S,5R)-4-allyl-2,5-dimethyl-1-piperazinyl)-3-methoxybenzyl]-N,N-diethylbenzamide)]- or low [ARM390 (N,N-diethyl-4-(phenyl-piperidin-4-ylidenemethyl)-benzamide)]-internalization potencies. As we found previously, a single SNC80—but not ARM390—administration triggered acute desensitization of the analgesic response in mice. However, daily injections of either compound over 5 d produced full analgesic tolerance. SNC80-tolerant animals showed widespread receptor downregulation, and tolerance to analgesic, locomotor and anxiolytic effects of the agonist. Hence, internalization-dependent tolerance developed, as a result of generalized receptor degradation. In contrast, ARM390-tolerant mice showed intact receptor expression, but δ-opioid receptor coupling to Ca2+ channels was abolished in dorsal root ganglia. Concomitantly, tolerance developed for agonist-induced analgesia, but not locomotor or anxiolytic responses. Therefore, internalization-independent tolerance was produced by anatomically restricted adaptations leading to pain-specific tolerance. Hence, ligand-directed receptor trafficking of the δ-opioid receptor engages distinct adaptive responses, and this study reveals a novel aspect of biased agonism in vivo.

Elevated tonic extracellular dopamine concentration and altered dopamine modulation of synaptic activity precede dopamine loss in the striatum of mice overexpressing human α-synuclein.

Overexpression or mutation of α‐synuclein (α‐Syn), a protein associated with presynaptic vesicles, causes familial forms of Parkinsons disease in humans and is also associated with sporadic forms of the disease. We used in vivo microdialysis, tissue content analysis, behavioral assessment, and whole‐cell patch clamp recordings from striatal medium‐sized spiny neurons (MSSNs) in slices to examine dopamine transmission and dopaminergic modulation of corticostriatal synaptic function in mice overexpressing human wild‐type α‐Syn under the Thy1 promoter (α‐Syn mice). Tonic striatal extracellular dopamine and 3‐methoxytyramine levels were elevated in α‐Syn mice at 6 months of age, prior to any reduction in total striatal tissue content, and were accompanied by an increase in open‐field activity. Dopamine clearance and amphetamine‐induced dopamine efflux were unchanged. The frequency of MSSN spontaneous excitatory postsynaptic currents (sEPSCs) was lower in α‐Syn mice. Amphetamine reduced sEPSC frequency in wild types (WTs) but produced no effect in α‐Syn mice. Furthermore, whereas quinpirole reduced and sulpiride increased sEPSC frequency in WT mice, they produced the opposite effects in α‐Syn mice. These observations indicate that overexpression of α‐Syn alters dopamine efflux and D2 receptor modulation of corticostriatal glutamate release at a young age. At 14 months of age, the α‐Syn mice presented with significantly lower striatal tissue dopamine and tyrosine hydroxylase content relative to WT littermates, accompanied by an L‐DOPA‐reversible sensory motor deficit. Together, these data further validate this transgenic mouse line as a slowly progressing model of Parkinsons disease and provide evidence for early dopamine synaptic dysfunction prior to loss of striatal dopamine.

β-Arrestin2 and c-Src Regulate the Constitutive Activity and Recycling of μ Opioid Receptors in Dorsal Root Ganglion Neurons

β-Arrestins bind to agonist-activated G-protein-coupled receptors regulating signaling events and initiating endocytosis. In β-arrestin2−/− (βarr2−/−) mice, a complex phenotype is observed that includes altered sensitivity to morphine. However, little is known of how β-arrestin2 affects μ receptor signaling. We investigated the coupling of μ receptors to voltage-gated Ca2+ channels (VGCCs) in βarr2+/+ and βarr2−/− dorsal root ganglion neurons. A lack of β-arrestin2 reduced the maximum inhibition of VGCCs by morphine and DAMGO (d-Ala2-N-Me-Phe4-glycol5-enkephalin) without affecting agonist potency, the onset of receptor desensitization, or the functional contribution of N-type VGCCs. The reduction in inhibition was accompanied by increased naltrexone-sensitive constitutive inhibitory coupling of μ receptors to VGCCs. Agonist-independent μ receptor inhibitory coupling was insensitive to CTAP (Cys-Tyr-d-Trp-Arg-Thr-Pen-Thr-NH2), a neutral antagonist that inhibited the inverse agonist action of naltrexone. These functional changes were accompanied by diminished constitutive recycling and increased cell-surface μ receptor expression in βarr2−/− compared with βarr2+/+ neurons. Such changes could not be explained by the classical role of β-arrestins in agonist-induced endocytosis. The localization of the nonreceptor tyrosine kinase c-Src appeared disrupted in βarr2−/− neurons, and there was reduced activation of c-Src by DAMGO. Using the Src inhibitor PP2 [4-amino-5-(4-chlorophenyl)-(t-butyl)pyrazolo[3,4-d]pyrimidine], we demonstrated that defective Src signaling mimics the βarr2−/− cellular phenotype of reduced μ agonist efficacy, increased constitutive μ receptor activity, and reduced constitutive recycling. We propose that β-arrestin2 is required to target c-Src to constitutively active μ receptors, resulting in their internalization, providing another dimension to the complex role of β-arrestin2 and c-Src in G-protein-coupled receptor function.

Naloxone fails to produce conditioned place aversion in μ-opioid receptor knock-out mice

There is growing evidence that tonic activity of the opioid system may be important in the modulation of affective state. Naloxone produces a conditioned place aversion in rodents, an effect that is centrally mediated. Previous pharmacological data using antagonists with preferential actions at mu-, delta-, and kappa-opioid receptors indicate the importance of the mu-opioid receptor in mediating this effect. We sought to test the mu-opioid receptor selectivity of naloxone aversion using mu-opioid receptor knock-out mice. mu-Opioid receptor knock-out and wild-type mice were tested for naloxone (10 mg/kg, s.c.) aversion using a place conditioning paradigm. As a positive control for associative learning, knock-out mice were tested for conditioned place aversion to a kappa agonist, U50,488H (2 mg/kg, s.c.). Naloxone produced a significant place aversion in wild-type mice, but failed to have any effect in mu-opioid receptor knock-out mice. On the other hand, both knock-out and wild-type mice treated with U50,488H spent significantly less time in the drug-paired chamber compared to their respective vehicle controls. We conclude that the mu-opioid receptor is crucial for the acquisition of naloxone-induced conditioned place aversion. Furthermore, in a separate experiment using C57BL/6 mice, the delta-selective antagonist naltrindole (10 or 30 mg/kg, s.c.) failed to produce conditioned place aversion.Taken together, these data further support the notion that naloxone produces aversion by antagonizing tonic opioid activity at the mu-opioid receptor.

Targeted expression of μ-opioid receptors in a subset of striatal direct-pathway neurons restores opiate reward

μ-opioid receptors (MORs) are necessary for the analgesic and addictive effects of opioids such as morphine, but the MOR-expressing neuronal populations that mediate the distinct opiate effects remain elusive. Here we devised a new conditional bacterial artificial chromosome rescue strategy to show, in mice, that targeted MOR expression in a subpopulation of striatal direct-pathway neurons enriched in the striosome and nucleus accumbens, in an otherwise MOR-null background, restores opiate reward and opiate-induced striatal dopamine release and partially restores motivation to self administer an opiate. However, these mice lack opiate analgesia or withdrawal. We used Cre-mediated deletion of the rescued MOR transgene to establish that expression of the MOR transgene in the striatum, rather than in extrastriatal sites, is needed for the restoration of opiate reward. Our study demonstrates that a subpopulation of striatal direct-pathway neurons is sufficient to support opiate reward-driven behaviors and provides a new intersectional genetic approach to dissecting neurocircuit-specific gene function in vivo.

p38 MAPK and β-Arrestin 2 Mediate Functional Interactions between Endogenous μ-Opioid and α2A-Adrenergic Receptors in Neurons

Formation of receptor complexes between μ-opioid and α2A-adrenergic receptors has been demonstrated in transfected cells. The functional significance and underlying mechanisms of such receptor interactions remain to be determined in neuronal systems. We examined functional interactions between endogenous μ and α2A receptors in mouse dorsal root ganglion neurons. Acute application of the μ agonist [d-Ala2,N-MePhe4, Gly-ol5]enkephalin (DAMGO) or the α2 agonist clonidine inhibited voltage-gated Ca2+ currents in these neurons. Prolonged treatment with either DAMGO or clonidine induced a mutual cross-desensitization between μ and α2A receptor-mediated current inhibition. The cross-desensitization was closely associated with simultaneous internalization of μ and α2A receptors. Morphine, a μ agonist triggering little μ receptor endocytosis, induced neither cross-desensitization nor internalization of α2A receptors. Furthermore, inhibition of p38 MAPK prevented the cross-desensitization as well as cointernalization of μ and α2A receptors. Changes in receptor trafficking profiles suggested that p38 MAPK activity was required for initiating μ receptor internalization and maintaining possible μ-α2A association during their cointernalization. Finally, the μ-α2A cross-desensitization was absent in dorsal root ganglion neurons lacking β-arrestin 2. These findings demonstrated p38 MAPK- and β-arrestin 2-dependent cross-regulation between neuronal μ and α2A receptors. By promoting receptor cross-desensitization and cointernalization, such functional interactions may serve as negative feedback mechanisms triggered by prolonged agonist exposure to modulate the signaling of functionally related G protein-coupled receptors.

Pentobarbital enhances GABAergic neurotransmission to cardiac parasympathetic neurons, which is prevented by expression of GABA(A) epsilon subunit.

BACKGROUND Pentobarbital decreases the gain of the baroreceptor reflex on the order of 50%, and this blunting is caused nearly entirely by decreasing cardioinhibitory parasympathetic activity. The most likely site of action of pentobarbital is the gamma-aminobutyric acid type A (GABA(A)) receptor. The authors tested whether pentobarbital augments the inhibitory GABAergic neurotransmission to cardiac parasympathetic neurons, and whether expression of the GABA(A) epsilon subunit prevents this facilitation. METHODS The authors used a novel approach to study the effect of pentobarbital on identified cardiac parasympathetic preganglionic neurons in rat brainstem slices. The cardiac parasympathetic neurons in the nucleus ambiguus were retrogradely prelabeled with a fluorescent tracer and were visually identified for patch clamp recording. The effects of pentobarbital on spontaneous GABAergic synaptic events were tested. An adenovirus was used to express the epsilon subunit of the GABA(A) receptor in cardiac parasympathetic neurons to examine whether this transfection alters pentobarbital-mediated changes in GABAergic neurotransmission. RESULTS Pentobarbital increased the duration but not the frequency or amplitude of spontaneous GABAergic currents in cardiac parasympathetic neurons. Transfection of cardiac parasympathetic neurons with the epsilon subunit of the GABA(A) receptor prevented the pentobarbital-evoked facilitation of GABAergic currents. CONCLUSIONS Pentobarbital, at clinically relevant concentrations, prolongs the duration of spontaneous inhibitory postsynaptic currents that impinge on cardiac parasympathetic neurons. This action would augment the inhibition of cardiac parasympathetic neurons, reduce parasympathetic cardioinhibitory activity, and increase heart rate. Expression of the GABA(A) receptor epsilon subunit in cardiac parasympathetic neurons renders the GABA receptors insensitive to pentobarbital.

Recent advances on the δ opioid receptor: from trafficking to function

Within the opioid family of receptors, δ (DOPrs) and μ opioid receptors (MOPrs) are typical GPCRs that activate canonical second‐messenger signalling cascades to influence diverse cellular functions in neuronal and non‐neuronal cell types. These receptors activate well‐known pathways to influence ion channel function and pathways such as the map kinase cascade, AC and PI3K. In addition new information regarding opioid receptor‐interacting proteins, downstream signalling pathways and resultant functional effects has recently come to light. In this review, we will examine these novel findings focusing on the DOPr and, in doing so, will contrast and compare DOPrs with MOPrs in terms of differences and similarities in function, signalling pathways, distribution and interactions. We will also discuss and clarify issues that have recently surfaced regarding the expression and function of DOPrs in different cell types and analgesia.

Opioid pharmaceuticals and addiction: The issues, and research directions seeking solutions

There are few pharmaceuticals superior to opiates for the treatment of pain. However, with concerns of addiction, withdrawal and questionable efficacy for all types of pain, these compounds are far from a magical panacea for pain-relief. As it is unlikely that other classes of compounds will supersede the opioids in the very near future, it is important to both optimize current opioid therapies and curb the astounding diversion of opioids from their intended analgesic use to non-medical abuse. In optimizing opioid therapeutics it is necessary to enhance the clinical awareness of the benefits of treating pain and combine this with aggressive strategies to reduce diversion for non-medical use. At the heart of the issue of opioid misuse is the role of opioid systems in the reward circuitry, and the adaptive processes associated with repetitive opioid use that manifest during withdrawal. Emerging pharmacological insights of opioid receptors will be reviewed that provide future hope for developing opioid-based analgesics with reduced addictive properties and perhaps, reduced opponent processes. In addition, with the increased understanding of nociceptive circuitry and the molecules involved in transmitting pain, new therapeutic targets have become evident that may result in effective analgesics either alone or in combination with current opioid therapies.

BDNF released during neuropathic pain potentiates NMDA receptors in primary afferent terminals

NMDA receptors in primary afferent terminals can contribute to hyperalgesia by increasing neurotransmitter release. In rats and mice, we found that the ability of intrathecal NMDA to induce neurokinin 1 receptor (NK1R) internalization (a measure of substance P release) required a previous injection of BDNF. Selective knock‐down of NMDA receptors in primary afferents decreased NMDA‐induced NK1R internalization, confirming the presynaptic location of these receptors. The effect of BDNF was mediated by tropomyosin‐related kinase B (trkB) receptors and not p75 neurotrophin receptors (p75NTR), because it was not produced by proBDNF and was inhibited by the trkB antagonist ANA‐12 but not by the p75NTR inhibitor TAT‐Pep5. These effects are probably mediated through the truncated form of the trkB receptor as there is little expression of full‐length trkB in dorsal root ganglion (DRG) neurons. Src family kinase inhibitors blocked the effect of BDNF, suggesting that trkB receptors promote the activation of these NMDA receptors by Src family kinase phosphorylation. Western blots of cultured DRG neurons revealed that BDNF increased Tyr1472 phosphorylation of the NR2B subunit of the NMDA receptor, known to have a potentiating effect. Patch‐clamp recordings showed that BDNF, but not proBDNF, increased NMDA receptor currents in cultured DRG neurons. NMDA‐induced NK1R internalization was also enabled in a neuropathic pain model or by activating dorsal horn microglia with lipopolysaccharide. These effects were decreased by a BDNF scavenger, a trkB receptor antagonist and a Src family kinase inhibitor, indicating that BDNF released by microglia potentiates NMDA receptors in primary afferents during neuropathic pain.