Buprenorphine Exposure Alters the Development and Migration of Interneurons in the Cortex.

The misuse of opioids has reached epidemic proportions over the last decade, with over 2.1 million people in the United States suffering from substance use disorders related to prescription opioid pain relievers. This increase in opioid misuse affects all demographics of society, including women of child-bearing age, which has led to a rise in opioid use during pregnancy. Opioid use during pregnancy has been associated with increased risk of obstetric complications and adverse neonatal outcomes, including neonatal abstinence syndrome. Currently, opioid use disorder in pregnant women is treated with long-acting opioid agonists, including buprenorphine. Although buprenorphine reduces illicit opioid use during pregnancy and improves infant outcomes at birth, few long-term studies of the neurodevelopmental consequences have been conducted. The goal of the current experiments was to examine the effects of buprenorphine on the development of the cortex using fetal brain tissue, 3D brain cultures, and rodent models. First, we demonstrated that we can grow cortical and subpallial spheroids, which model the cellular diversity, connectivity, and activity of the developing human brain. Next, we show that cells in the developing human cortex express the nociceptin opioid (NOP) receptor and that buprenorphine can signal through this receptor in cortical spheroids. Using subpallial spheroids to grow inhibitory interneurons, we show that buprenorphine can alter interneuron development and migration into the cortex. Finally, using a rodent model of prenatal buprenorphine exposure, we demonstrate that alterations in interneuron distribution can persist into adulthood. Together, these results suggest that more research is needed into the long-lasting consequences of buprenorphine exposure on the developing human brain.

Allosterism within δ Opioid-κ Opioid Receptor Heteromers in Peripheral Sensory Neurons: Regulation of κ Opioid Agonist Efficacy.

There is abundant evidence for formation of G protein-coupled receptor heteromers in heterologous expression systems, but little is known of the function of heteromers in native systems. Heteromers of δ and κ opioid receptors (DOR-KOR heteromers) have been identified in native systems. We previously reported that activation of DOR-KOR heteromers expressed by rat pain-sensing neurons (nociceptors) produces robust, peripherally mediated antinociception. Moreover, DOR agonist potency and efficacy is regulated by KOR antagonists via allosteric interactions within the DOR-KOR heteromer in a ligand-dependent manner. Here we assessed the reciprocal regulation of KOR agonist function by DOR antagonists in adult rat nociceptors in culture and in a behavioral assay of nociception. Naltrindole enhanced the potency of the KOR agonist 2-(3,4-dichlorophenyl)-N-methyl-N-[(1S)-1-phenyl-2-pyrrolidin-1-ylethyl]acetamide (ICI-199441) 10- to 20-fold, but did not alter responses to 2-(3,4-dichlorophenyl)-N-methyl-N-[(1R,2R)-2-pyrrolidin-1-ylcyclohexyl]acetamide (U50488). By contrast, the potency of U50488 was enhanced 20-fold by 7-benzylidenenaltrexone. The efficacy of 6'-guanidinonaltrindole (6'-GNTI) to inhibit nociceptors was blocked by small interfering RNA knockdown of DOR or KOR. Replacing 6'-GNTI occupancy of DOR with either naltrindole or 7-benzylidenenaltrexone abolished 6'-GNTI efficacy. Further, peptides derived from DOR transmembrane segment 1 fused to the cell membrane-penetrating HIV transactivator of transcription peptide also blocked 6'-GNTI-mediated responses ex vivo and in vivo, suggesting that 6'-GNTI efficacy in nociceptors is due to its positive allosteric regulation of KOR via occupancy of DOR in a DOR-KOR heteromer. Together, these results provide evidence for the existence of functional DOR-KOR heteromers in rat peripheral sensory neurons and that reciprocal, ligand-dependent allosteric interactions occur between the DOR and KOR protomers.

Regulation of δ Opioid Receptor-Mediated Signaling and Antinociception in Peripheral Sensory Neurons by Arachidonic Acid-Dependent 12/15-Lipoxygenase Metabolites.

The function of δ opioid receptors (DOR) expressed by peripheral pain-sensing neurons (nociceptors) is regulated by both cyclooxygenase- and lipoxygenase (LOX)-dependent arachidonic acid (AA) metabolites. Whereas cyclooxygenase metabolites enhance responsiveness, LOX metabolites elicit a refractory, nonsignaling state of the DOR receptor system for antinociceptive signaling. In this study, using high-performance liquid chromatography-tandem mass spectrometry analyses, we have found that the 12-/15-LOX metabolites, 12-hydroxyeicosatetraenoic acid (HETE) and 15-HETE, were elevated after treatment of adult rat primary sensory neuron cultures with AA. Exogenously applied 12-HETE and 15-HETE, but not 5-HETE, completely prevented DOR and κ opioid receptor (KOR) agonist-mediated inhibition of prostaglandin E2 (PGE2)-stimulated cAMP accumulation, but not inhibition, by the 5-HT1 receptor agonist 5-carboxamidotryptamine in cultured peripheral sensory neurons and in Chinese hamster ovary (CHO) cells heterologously expressing DOR or KOR. Similarly, intraplantar injection of 12- or 15-HETE, either alone or in combination, prevented DOR agonist-mediated inhibition of PGE2-evoked thermal allodynia. Further, both AA- and carrageenan-mediated induction of the nonresponsive state of the DOR system was blocked by an intraplantar coinjection of the 12-/15-LOX inhibitors baicalein and luteolin. In contrast to the regulation of cAMP signaling, pretreatment with 12- and 15-HETE had no effect on either DOR or KOR agonist- mediated activation of extracellular signal-regulated kinase in peripheral sensory neurons or CHO cells. These results suggest that the analgesic efficacy of peripherally restricted opioids for treatment of inflammatory pain may be enhanced by adjunct inhibition of LOX activity.

Studies To Examine Potential Tolerability Differences between the 5-HT2C Receptor Selective Agonists Lorcaserin and CP-809101.

Lorcaserin (LOR) is a selective 5-HT2C receptor agonist that has been FDA approved as a treatment for obesity. The most frequently reported side-effects of LOR include nausea and headache, which can be dose limiting. We have previously reported that in the rat, while LOR produced unconditioned signs characteristic of nausea/malaise, the highly selective 5-HT2C agonist CP-809101 (CP) produced fewer equivalent signs. Because this may indicate a subclass of 5-HT2C agonists having better tolerability, the present studies were designed to further investigate this apparent difference. In a conditioned gaping model, a rodent test of nausea, LOR produced significantly higher gapes compared to CP consistent with it having higher emetogenic properties. Subsequent studies were designed to identify features of each drug that may account for such differences. In rats trained to discriminate CP-809101 from saline, both CP and LOR produced full generalization suggesting a similar interoceptive cue. In vitro tests of functional selectivity designed to examine signaling pathways activated by both drugs in CHO (Chinese hamster ovary) cells expressing h5-HT2C receptors failed to identify evidence for biased signaling differences between LOR and CP. Thus, both drugs showed similar profiles across PLC, PLA2, and ERK signaling pathways. In studies designed to examine pharmacokinetic differences between LOR and CP, while drug plasma levels correlated with increasing dose, CSF levels did not. CSF levels of LOR increased proportionally with dose; however CSF levels of CP plateaued from 6 to 12 mg/kg. Thus, the apparently improved tolerability of CP likely reflects a limit to CNS levels attained at relatively high doses.

Long-Term Reduction of Kappa Opioid Receptor Function by the Biased Ligand, Norbinaltorphimine, Requires c-Jun N-Terminal Kinase Activity and New Protein Synthesis in Peripheral Sensory Neurons.

A single administration of the κ opioid receptor (KOR) antagonist, norbinaltorphimine (norBNI), produces long-term reduction in KOR function in heterologous expression systems and brain that is mediated by activation of c-Jun N-terminal kinase (JNK). In this study, we examined the long-term effects of norBNI on adult rat peripheral sensory neurons in vivo and ex vivo. Following a single intraplantar (i.pl.) injection of norBNI into the hind paw, peripheral KOR-mediated antinociception in the ipsilateral, but not the contralateral, hindpaw was abolished for at least 9 days. By contrast, the antinociceptive response to mu and delta opioid receptor agonists was unaltered. The long-term inhibitory effect on antinociception produced by pretreatment with norBNI required occupancy of peripheral KOR and was completely blocked by i.pl. injection of the JNK inhibitor, SP600125. In cultures of peripheral sensory neurons, norBNI activated JNK for at least 30 minutes. Furthermore, norBNI blocked KOR-mediated inhibition of adenylyl cyclase activity measured 24 hours later in a JNK-dependent manner, but did not block activation of extracellular signal-regulated kinase (ERK). The long-term inhibitory effect of norBNI on KOR function in vivo and ex vivo was blocked by inhibitors of mRNA translation, cycloheximide and rapamycin. These data suggest that in peripheral sensory neurons norBNI is a KOR-biased ligand for activation of JNK signaling, resulting in long-term blockade of some (antinociception, inhibition of adenylyl cyclase activity), but not all (ERK), KOR signaling. Importantly, norBNI elicits de novo protein synthesis in sensory neuron terminals that produces selective long-term regulation of KOR.

Functional selectivity of kappa opioid receptor agonists in peripheral sensory neurons.

Activation of kappa opioid receptors (KORs) expressed by peripheral sensory neurons that respond to noxious stimuli (nociceptors) can reduce neurotransmission of pain stimuli from the periphery to the central nervous system. We have previously shown that the antinociception dose-response curve for peripherally restricted doses of the KOR agonist (-)-(trans)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)cyclohexyl]benzeneacetamide (U50488) has an inverted U shape. Here, we found that the downward phase of the U50488 dose-response curve was blocked by an inhibitor of extracellular signal-regulated kinase (ERK) activation U0126. Local administration of the selective KOR agonist salvinorin A (Sal-A), also resulted in an inverted U-shaped curve; however, the downward phase was insensitive to U0126. By contrast, inhibition of c-Jun N-terminal kinase (JNK) partially blocked the downward phase of the dose-response curve to Sal-A, suggesting a role for JNK. In cultures of peripheral sensory neurons, U50488 and Sal-A inhibited adenylyl cyclase activity with similar efficacies; however, their ability to activate ERK and JNK differed. Whereas U50488 activated ERK but not JNK, Sal-A activated JNK but not ERK. Moreover, although both U50488 and Sal-A produced homologous desensitization, desensitization to U50488 was blocked by inhibition of ERK activation, whereas desensitization to Sal-A was blocked by inhibition of JNK. Substitution of an ethoxymethyl ether for the C2 position acetyl group of Sal-A reduced stimulation of JNK, prevented desensitization by ethoxymethyl ether for the C2 position acetyl group of Sal-A, and resulted in a monotonic antinociception dose-response curve. Collectively, these data demonstrate the functional selectivity of KOR ligands for signaling in peripheral sensory neurons, which results in differential effects on behavioral responses in vivo.

G protein-coupled receptor 30 (GPR30) forms a plasma membrane complex with membrane-associated guanylate kinases (MAGUKs) and protein kinase A-anchoring protein 5 (AKAP5) that constitutively inhibits cAMP production.

GPR30, or G protein-coupled estrogen receptor, is a G protein-coupled receptor reported to bind 17ß-estradiol (E2), couple to the G proteins Gs and Gi/o, and mediate non-genomic estrogenic responses. However, controversies exist regarding the receptor pharmacological profile, effector coupling, and subcellular localization. We addressed the role of the type I PDZ motif at the receptor C terminus in receptor trafficking and coupling to cAMP production in HEK293 cells and CHO cells ectopically expressing the receptor and in Madin-Darby canine kidney cells expressing the native receptor. GPR30 was localized both intracellularly and in the plasma membrane and subject to limited basal endocytosis. E2 and G-1, reported GPR30 agonists, neither stimulated nor inhibited cAMP production through GPR30, nor did they influence receptor localization. Instead, GPR30 constitutively inhibited cAMP production stimulated by a heterologous agonist independently of Gi/o. Moreover, siRNA knockdown of native GPR30 increased cAMP production. Deletion of the receptor PDZ motif interfered with inhibition of cAMP production and increased basal receptor endocytosis. GPR30 interacted with membrane-associated guanylate kinases, including SAP97 and PSD-95, and protein kinase A-anchoring protein (AKAP) 5 in the plasma membrane in a PDZ-dependent manner. Knockdown of AKAP5 or St-Ht31 treatment, to disrupt AKAP interaction with the PKA RIIß regulatory subunit, decreased inhibition of cAMP production, and St-Ht31 increased basal receptor endocytosis. Therefore, GPR30 forms a plasma membrane complex with a membrane-associated guanylate kinase and AKAP5, which constitutively attenuates cAMP production in response to heterologous agonists independently of Gi/o and retains receptors in the plasma membrane.