A Regional and Projection-Specific Role of RGSz1 in the Ventrolateral Periaqueductal Grey in the Modulation of Morphine Reward.

Opioid analgesics exert their therapeutic and adverse effects by activating µ opioid receptors (MOPR); however, functional responses to MOPR activation are modulated by distinct signal transduction complexes within the brain. The ventrolateral periaqueductal gray (vlPAG) plays a critical role in modulation of nociception and analgesia, but the exact intracellular pathways associated with opioid responses in this region are not fully understood. We previously showed that knockout of the signal transduction modulator Regulator of G protein Signaling z1 (RGSz1) enhanced analgesic responses to opioids, whereas it decreased the rewarding efficacy of morphine. Here, we applied viral mediated gene transfer methodology and delivered adeno-associated virus (AAV) expressing Cre recombinase to the vlPAG of RGSz1fl\fl mice to demonstrate that downregulation of RGSz1 in this region decreases sensitivity to morphine in the place preference paradigm, under pain-free as well as neuropathic pain states. We also used retrograde viral vectors along with flippase-dependent Cre vectors to conditionally downregulate RGSz1 in vlPAG projections to the ventral tegmental area (VTA) and show that downregulation of RGSz1 prevents the development of place conditioning to low morphine doses. Consistent with the role for RGSz1 as a negative modulator of MOPR activity, RGSz1KO enhances opioid-induced cAMP inhibition in periaqueductal gray (PAG) membranes. Furthermore, using a new generation of bioluminescence resonance energy transfer (BRET) sensors, we demonstrate that RGSz1 modulates Gαz but not other Gαi family subunits and selectively impedes MOPR-mediated Gαz signaling events invoked by morphine and other opioids. Our work highlights a regional and circuit-specific role of the G protein-signaling modulator RGSz1 in morphine reward, providing insights on midbrain intracellular pathways that control addiction-related behaviors. SIGNIFICANCE STATEMENT: This study used advanced genetic mouse models to highlight the role of the signal transduction modulator named RGSz1 in responses to clinically used opioid analgesics. We show that RGSz1 controls the rewarding efficacy of opioids by actions in ventrolateral periaqueductal gray projections to the ventral tegmental area, a key component of the midbrain dopamine pathway. These studies highlight novel mechanisms by which pain-modulating structures control the rewarding efficacy of opioids.

Suppression of RGSz1 function optimizes the actions of opioid analgesics by mechanisms that involve the Wnt/ß-catenin pathway.

Regulator of G protein signaling z1 (RGSz1), a member of the RGS family of proteins, is present in several networks expressing mu opioid receptors (MOPRs). By using genetic mouse models for global or brain region-targeted manipulations of RGSz1 expression, we demonstrated that the suppression of RGSz1 function increases the analgesic efficacy of MOPR agonists in male and female mice and delays the development of morphine tolerance while decreasing the sensitivity to rewarding and locomotor activating effects. Using biochemical assays and next-generation RNA sequencing, we identified a key role of RGSz1 in the periaqueductal gray (PAG) in morphine tolerance. Chronic morphine administration promotes RGSz1 activity in the PAG, which in turn modulates transcription mediated by the Wnt/ß-catenin signaling pathway to promote analgesic tolerance to morphine. Conversely, the suppression of RGSz1 function stabilizes Axin2-Gαz complexes near the membrane and promotes ß-catenin activation, thereby delaying the development of analgesic tolerance. These data show that the regulation of RGS complexes, particularly those involving RGSz1-Gαz, represents a promising target for optimizing the analgesic actions of opioids without increasing the risk of dependence or addiction.

RGS4 Maintains Chronic Pain Symptoms in Rodent Models.

Regulator of G-protein signaling 4 (RGS4) is a potent modulator of G-protein-coupled receptor signal transduction that is expressed throughout the pain matrix. Here, we use genetic mouse models to demonstrate a role of RGS4 in the maintenance of chronic pain states in male and female mice. Using paradigms of peripheral inflammation and nerve injury, we show that the prevention of RGS4 action leads to recovery from mechanical and cold allodynia and increases the motivation for wheel running. Similarly, RGS4KO eliminates the duration of nocifensive behavior in the second phase of the formalin assay. Using the Complete Freud's Adjuvant (CFA) model of hindpaw inflammation we also demonstrate that downregulation of RGS4 in the adult ventral posterolateral thalamic nuclei promotes recovery from mechanical and cold allodynia. RNA sequencing analysis of thalamus (THL) from RGS4WT and RGS4KO mice points to many signal transduction modulators and transcription factors that are uniquely regulated in CFA-treated RGS4WT cohorts. Ingenuity pathway analysis suggests that several components of glutamatergic signaling are differentially affected by CFA treatment between RGS4WT and RGS4KO groups. Notably, Western blot analysis shows increased expression of metabotropic glutamate receptor 2 in THL synaptosomes of RGS4KO mice at time points at which they recover from mechanical allodynia. Overall, our study provides information on a novel intracellular pathway that contributes to the maintenance of chronic pain states and points to RGS4 as a potential therapeutic target.SIGNIFICANCE STATEMENT There is an imminent need for safe and efficient chronic pain medications. Regulator of G-protein signaling 4 (RGS4) is a multifunctional signal transduction protein, widely expressed in the pain matrix. Here, we demonstrate that RGS4 plays a prominent role in the maintenance of chronic pain symptoms in male and female mice. Using genetically modified mice, we show a dynamic role of RGS4 in recovery from symptoms of sensory hypersensitivity deriving from hindpaw inflammation or hindlimb nerve injury. We also demonstrate an important role of RGS4 actions in gene expression patterns induced by chronic pain states in the mouse thalamus. Our findings provide novel insight into mechanisms associated with the maintenance of chronic pain states and demonstrate that interventions in RGS4 activity promote recovery from sensory hypersensitivity symptoms.

Regulators of G Protein Signaling in Analgesia and Addiction.

Regulator of G protein signaling (RGS) proteins are multifunctional proteins expressed in peripheral and neuronal cells, playing critical roles in development, physiologic processes, and pharmacological responses. RGS proteins primarily act as GTPase accelerators for activated Gα subunits of G-protein coupled receptors, but they may also modulate signal transduction by several other mechanisms. Over the last two decades, preclinical work identified members of the RGS family with unique and critical roles in intracellular responses to drugs of abuse. New information has emerged on the mechanisms by which RGS proteins modulate the efficacy of opioid analgesics in a brain region- and agonist-selective fashion. There has also been progress in the understanding of the protein complexes and signal transduction pathways regulated by RGS proteins in addiction and analgesia circuits. In this review, we summarize findings on the mechanisms by which RGS proteins modulate functional responses to opioids in models of analgesia and addiction. We also discuss reports on the regulation and function of RGS proteins in models of psychostimulant addiction. Using information from preclinical studies performed over the last 20 years, we highlight the diverse mechanisms by which RGS protein complexes control plasticity in response to opioid and psychostimulant drug exposure; we further discuss how the understanding of these pathways may lead to new opportunities for therapeutic interventions in G protein pathways. SIGNIFICANCE STATEMENT: Regulator of G protein signaling (RGS) proteins are signal transduction modulators, expressed widely in various tissues, including brain regions mediating addiction and analgesia. Evidence from preclinical work suggests that members of the RGS family act by unique mechanisms in specific brain regions to control drug-induced plasticity. This review highlights interesting findings on the regulation and function of RGS proteins in models of analgesia and addiction.

Abuse-Related Neurochemical Effects of Para-Substituted Methcathinone Analogs in Rats: Microdialysis Studies of Nucleus Accumbens Dopamine and Serotonin.

Methcathinone (MCAT) is a monoamine releaser and parent compound to a new class of designer drugs that includes the synthetic cathinones mephedrone and flephedrone. Using MCAT and a series of para-substituted (or 4-substituted) MCAT analogs, it has been previously shown that expression of abuse-related behavioral effects in rats correlates both with the volume of the para substituent and in vitro neurochemical selectivity to promote monoamine release via the dopamine (DA) versus serotonin (5-HT) transporters in rat brain synaptosomes. The present study used in vivo microdialysis to determine the relationship between these previous measures and the in vivo neurochemical selectivity of these compounds to alter nucleus accumbens (NAc) DA and 5-HT levels. Male Sprague-Dawley rats were implanted with bilateral guide cannulae targeting the NAc. MCAT and five para-substituted analogs (4-F, 4-Cl, 4-Br, 4-CH3, and 4-OCH3) produced dose- and time-dependent increases in NAc DA and/or 5-HT levels. Selectivity was determined as the dose required to increase peak 5-HT levels by 250% divided by the dose required to increase peak DA levels by 250%. This measure of in vivo neurochemical selectivity varied across compounds and correlated with 1) in vivo expression of abuse-related behavioral effects (r = 0.89, P = 0.02); 2) in vitro selectivity to promote monoamine release via DA and 5-HT transporters (r = 0.95, P < 0.01); and 3) molecular volume of the para substituent (r = -0.85, P = 0.03). These results support a relationship between these molecular, neurochemical, and behavioral measures and support a role for molecular structure as a determinant of abuse-related neurochemical and behavioral effects of MCAT analogs.

Ethylenedioxy homologs of N-methyl-(3,4-methylenedioxyphenyl)-2-aminopropane (MDMA) and its corresponding cathinone analog methylenedioxymethcathinone: Interactions with transporters for serotonin, dopamine, and norepinephrine.

N-Methyl-(3,4-methylenedioxyphenyl)-2-aminopropane (MDMA; 'Ecstasy'; 1) and its ß-keto analog methylone (MDMC; 2) are popular drugs of abuse. Little is known about their ring-expanded ethylenedioxy homologs. Here, we prepared N-methyl-(3,4-ethylenedioxyphenyl)-2-aminopropane (EDMA; 3), both of its optical isomers, and ß-keto EDMA (i.e., EDMC; 4) to examine their effects at transporters for serotonin (SERT), dopamine (DAT), and norepinephrine (NET). In general, ring-expansion of the methylenedioxy group led to a several-fold reduction in potency at all three transporters. With respect to EDMA (3), S(+)3 was 6-fold, 50-fold, and 8-fold more potent than its R(-) enantiomer at SERT, DAT, and NET, respectively. Overall, in the absence of a ß-carbonyl group, the ethylenedioxy (i.e., 1,4-dioxane) substituent seems better accommodated at SERT than at DAT and NET.

Tianeptine promotes lasting antiallodynic effects in a mouse model of neuropathic pain.

Tricyclic antidepressants (TCAs), such as desipramine (DMI), are effective at managing neuropathic pain symptoms but often take several weeks to become effective and also lead to considerable side effects. Tianeptine (TIAN) is an atypical antidepressant that activates the mu-opioid receptor but does not produce analgesic tolerance or withdrawal in mice, nor euphoria in humans, at clinically-relevant doses. Here, we evaluate the efficacy of TIAN at persistently alleviating mechanical allodynia in the spared nerve injury (SNI) model of neuropathic pain, even well after drug clearance. After finding an accelerated onset of antiallodynic action compared to DMI, we used genetically modified mice to gain insight into RGS protein-associated pathways that modulate the efficacy of TIAN relative to DMI in models of neuropathic pain. Because we observed similar behavioral responses to both TIAN and DMI treatment in RGS4, RGSz1, and RGS9 knockout mice, we performed RNA sequencing on the NAc of TIAN- and DMI-treated mice after prolonged SNI to further clarify potential mechanisms underlying TIANs faster therapeutic actions. Our bioinformatic analysis revealed distinct transcriptomic signatures between the two drugs, with TIAN more directly reversing SNI-induced differentially expressed genes, and further predicted several upstream regulators that may be implicated in onset of action. This new understanding of the molecular pathways underlying TIAN action may enable the development of novel and more efficacious pharmacological approaches for the management of neuropathic pain.

Oxycodone withdrawal induces HDAC1/HDAC2-dependent transcriptional maladaptations in the reward pathway in a mouse model of peripheral nerve injury.

The development of physical dependence and addiction disorders due to misuse of opioid analgesics is a major concern with pain therapeutics. We developed a mouse model of oxycodone exposure and subsequent withdrawal in the presence or absence of chronic neuropathic pain. Oxycodone withdrawal alone triggered robust gene expression adaptations in the nucleus accumbens, medial prefrontal cortex and ventral tegmental area, with numerous genes and pathways selectively affected by oxycodone withdrawal in mice with peripheral nerve injury. Pathway analysis predicted that histone deacetylase (HDAC) 1 is a top upstream regulator in opioid withdrawal in nucleus accumbens and medial prefrontal cortex. The novel HDAC1/HDAC2 inhibitor, Regenacy Brain Class I HDAC Inhibitor (RBC1HI), attenuated behavioral manifestations of oxycodone withdrawal, especially in mice with neuropathic pain. These findings suggest that inhibition of HDAC1/HDAC2 may provide an avenue for patients with chronic pain who are dependent on opioids to transition to non-opioid analgesics.

A promising chemical series of positive allosteric modulators of the µ-opioid receptor that enhance the antinociceptive efficacy of opioids but not their adverse effects.

Positive allosteric modulators (PAMs) of the µ-opioid receptor (MOR) have been proposed to exhibit therapeutic potential by maximizing the analgesic properties of clinically used opioid drugs while limiting their adverse effects or risk of overdose as a result of using lower drug doses. We herein report in vitro and in vivo characterization of two small molecules from a chemical series of MOR PAMs that exhibit: (i) MOR PAM activity and receptor subtype selectivity in vitro, (ii) a differential potentiation of the antinociceptive effect of oxycodone, morphine, and methadone in mouse models of pain that roughly correlates with in vitro activity, and (iii) a lack of potentiation of adverse effects associated with opioid administration, such as somatic withdrawal, respiratory depression, and analgesic tolerance. This series of MOR PAMs holds promise for the development of adjuncts to opioid therapy to mitigate against overdose and opioid use disorders.

Investigation of the Optical Isomers of Methcathinone, and Two Achiral Analogs, at Monoamine Transporters and in Intracranial Self-Stimulation Studies in Rats.

Methcathinone (MCAT; 1), the progenitor of numerous and widely abused "synthetic cathinone" central stimulants, exists as a pair of optical isomers. Although S(-)MCAT is several-fold more potent than R(+)MCAT in rodent locomotor stimulation and in stimulus generalization studies in rat drug discrimination assays, the individual optical isomers of MCAT have never been directly compared for their actions at monoamine transporters that seem to underlie their actions and have never been examined for their relative abuse potential. Here, we found that the isomers of MCAT are nearly equieffective at dopamine and norepinephrine transporters (DAT and NET, respectively) as transporter substrates (i.e., as releasing agents) and are ≥63-fold less potent at the serotonin transporter (SERT). In intracranial self-stimulation (ICSS) studies to evaluate abuse-related drug effects in rats, S(-)MCAT was approximately twice as potent as its R-enantiomer. Achiral analogs, α-methyl MCAT (3) and α-des-methyl MCAT (4), also were DAT/NET substrates and also produced abuse-related ICSS effects, indicating that they retain abuse potential and that they might be useful for the further study of the stereochemistry of synthetic cathinone analogs with chiral ß- (or other) substituents.

HDAC6-selective inhibitors decrease nerve-injury and inflammation-associated mechanical hypersensitivity in mice.

BACKGROUND: HDAC6 is a class IIB histone deacetylase expressed at many levels of the nociceptive pathway. This study tested the ability of novel and selective HDAC6 inhibitors to alleviate sensory hypersensitivity behaviors in mouse models of peripheral nerve injury and peripheral inflammation. METHODS: We utilized the murine spared nerve injury (SNI) model for peripheral nerve injury and the Complete Freund's Adjuvant (CFA) model of peripheral inflammation. We applied the Von Frey assay to monitor mechanical allodynia. RESULTS: Using the SNI model, we demonstrate that daily administration of the brain-penetrant HDAC6 inhibitor, ACY-738, abolishes mechanical allodynia in male and in female mice. Importantly, there is no tolerance to the antiallodynic actions of these compounds as they produce a consistent increase in Von Frey thresholds for several weeks. We observed a similar antiallodynic effect when utilizing the HDAC6 inhibitor, ACY-257, which shows limited brain expression when administered systemically. We also demonstrate that ACY-738 and ACY-257 attenuate mechanical allodynia in the CFA model of peripheral inflammation. CONCLUSIONS: Overall, our findings suggest that inhibition of HDAC6 provides a promising therapeutic avenue for the alleviation of mechanical allodynia associated with peripheral nerve injury and peripheral inflammation.

Effects of acute and repeated treatment with serotonin 5-HT2A receptor agonist hallucinogens on intracranial self-stimulation in rats.

The prototype 5-HT2A receptor agonist hallucinogens LSD, mescaline, and psilocybin are classified as Schedule 1 drugs of abuse by the U.S. Drug Enforcement Administration. Accumulating clinical evidence has also suggested that acute or repeated "microdosing" with these drugs may have utility for treatment of some mental health disorders, including drug abuse and depression. The goal of the present study was to evaluate LSD, mescaline, and psilocybin effects on intracranial self-stimulation (ICSS), a procedure that has been used to evaluate abuse-related effects of other classes of abused drugs. Effects of repeated LSD were also examined to evaluate potential changes in its own effects on ICSS or changes in effects produced by the abused psychostimulant methamphetamine or the prodepressant kappa opioid receptor (KOR) agonist U69,593. Male Sprague-Dawley rats were implanted with microelectrodes targeting the medial forebrain bundle and trained to respond under a "frequency-rate" ICSS procedure, in which many drugs of abuse increase (or "facilitate") ICSS. In acute dose-effect and time-course studies, evidence for abuse-related ICSS facilitation was weak and inconsistent; the predominant effect of all 3 drugs was dose- and time-dependent ICSS depression. Repeated LSD treatment failed to alter either its own ICSS depressant effects or the abuse-related effects of methamphetamine; however, repeated LSD did attenuate ICSS depression by U69,593. These results extend those of previous preclinical studies to suggest weak expression of abuse-related effects by 5-HT2A agonist hallucinogens and provide supportive evidence for therapeutic effects of repeated LSD dosing to attenuate KOR-mediated depressant effects but not abuse potential of psychostimulants. (PsycINFO Database Record (c) 2019 APA, all rights reserved).

Naltrexone maintenance fails to alter amphetamine effects on intracranial self-stimulation in rats.

Pharmacotherapy to treat stimulant use disorders continues to be an unmet medical need. Some evidence supports both the role of opioids in mediating abuse-related amphetamine effects and the potential utility of opioid antagonists as therapeutic candidates for treating amphetamine abuse. This study used intracranial self-stimulation (ICSS) to evaluate effects of exposure to and termination of naltrexone maintenance on rewarding amphetamine effects in an ICSS procedure in rats. Morphine and cocaine were included as positive and negative controls, respectively. Male Sprague-Dawley rats (N = 40) were trained to lever press for electrical brain stimulation to the medial forebrain bundle via an implanted electrode. Rats were then implanted with osmotic pumps delivering naltrexone (0.001 mg/kg/h, SC, 0.01 mg/kg/h, SC, or 0.1 mg/kg/h, SC) or saline for 14 days. Cumulative dose-effect curves were determined for amphetamine (0.032 mg/kg to 0.32 mg/kg), cocaine (1 mg/kg to 10 mg/kg), and morphine (1 mg/kg to 10 mg/kg) during the 2nd week of naltrexone maintenance. Additionally, dose-effect curves for morphine and amphetamine were determined again 24 hr after pump removal. Our results suggest that (a) exposure to and termination of naltrexone maintenance do not affect baseline ICSS responding, (b) naltrexone doses sufficient to antagonize morphine did not alter amphetamine or cocaine effects, and (c) termination of naltrexone treatment produced weak evidence for increased morphine sensitivity but no change in amphetamine effects. Our results do not support naltrexone as a pharmacotherapy for amphetamine and cocaine abuse and also suggest that termination from chronic naltrexone does not increase sensitivity to abuse-related morphine or amphetamine effects in ICSS. (PsycINFO Database Record

Effects of N-Alkyl-4-Methylamphetamine Optical Isomers on Plasma Membrane Monoamine Transporters and Abuse-Related Behavior.

4-Methylamphetamine (4-MA) is an emerging drug of abuse that acts as a substrate at plasma membrane transporters for dopamine (DAT), norepinephrine (NET), and serotonin (SERT), thereby causing nonexocytotic release of monoamine transmitters via reverse transport. Prior studies by us showed that increasing the N-alkyl chain length of N-substituted 4-MA analogues converts 4-MA from a transportable substrate (i.e., releaser) at DAT and NET to a nontransported blocker at these sites. Here, we studied the effects of the individual optical isomers of N-methyl-, N-ethyl-, and N- n-propyl 4-MA on monoamine transporters and abuse-related behavior in rats because action/function might be related to stereochemistry. Uptake inhibition and release assays were conducted in rat brain synaptosomes whereas electrophysiological assessments of drug-transporter interactions were examined using cell-based biosensors. Intracranial-self-stimulation in rats was employed to assess abuse potential in vivo. The experimental evidence demonstrates that S(+) N-methyl 4-MA is a potent and efficacious releaser at DAT, NET, and SERT with the highest abuse potential among the test drugs, whereas R(-) N-methyl 4-MA is a less potent releaser with reduced abuse potential. The S(+)ethyl analogue has decreased efficacy as a releaser at DAT but retains full release activity at NET and SERT with a reduction in abuse-related effects; the R(-)ethyl analogue has a similar profile but is less potent. S(+) N-Propyl 4-MA is a nontransported blocker at DAT and NET but an efficacious releaser at SERT, whereas the R enantiomer is almost inactive. In conclusion, the S enantiomers of the N-alkyl 4-MA analogues are most potent. Lengthening the N-alkyl chain converts compounds from potent nonselective releasers showing abuse-related effects to more selective SERT releasers with no apparent abuse potential.

N-Alkylated Analogs of 4-Methylamphetamine (4-MA) Differentially Affect Monoamine Transporters and Abuse Liability.

Clandestine chemists synthesize novel stimulant drugs by exploiting structural templates known to target monoamine transporters for dopamine, norepinephrine, and serotonin (DAT, NET, and SERT, respectively). 4-Methylamphetamine (4-MA) is an emerging drug of abuse that interacts with transporters, but limited structure-activity data are available for its analogs. Here we employed uptake and release assays in rat brain synaptosomes, voltage-clamp current measurements in cells expressing transporters, and calcium flux assays in cells coexpressing transporters and calcium channels to study the effects of increasing N-alkyl chain length of 4-MA on interactions at DAT, NET, and SERT. In addition, we performed intracranial self-stimulation in rats to understand how the chemical modifications affect abuse liability. All 4-MA analogs inhibited uptake at DAT, NET, and SERT, but lengthening the amine substituent from methyl to ethyl, propyl, and butyl produced a stepwise decrease in potency. N-methyl 4-MA was an efficacious substrate-type releaser at DAT that evoked an inward depolarizing current and calcium influx, whereas other analogs did not exhibit these effects. N-methyl and N-ethyl 4-MA were substrates at NET, whereas N-propyl and N-butyl 4-MA were not. All analogs acted as SERT substrates, though N-butyl 4-MA had very weak effects. Intracranial self-stimulation in rats showed that elongating the N-alkyl chain decreased abuse-related effects in vivo that appeared to parallel reductions in DAT activity. Overall, converging lines of evidence show that lengthening the N-alkyl substituent of 4-MA reduces potency to inhibit transporters, eliminates substrate activity at DAT and NET, and decreases abuse liability of the compounds.

"Deconstruction" of the abused synthetic cathinone methylenedioxypyrovalerone (MDPV) and an examination of effects at the human dopamine transporter.

Synthetic cathinones, ß-keto analogues of amphetamine (or, more correctly, of phenylalkylamines), represent a new and growing class of abused substances. Several such analogues have been demonstrated to act as dopamine (DA) releasing agents. Methylenedioxypyrovalerone (MDPV) was the first synthetic cathinone shown to act as a cocaine-like DA reuptake inhibitor. MDPV and seven deconstructed analogues were examined to determine which of MDPV's structural features account(s) for uptake inhibition. In voltage-clamped (-60 mV) Xenopus oocytes transfected with the human DA transporter (hDAT), all analogues elicited inhibitor-like behavior shown as hDAT-mediated outward currents. Using hDAT-expressing mammalian cells we determined the affinities of MDPV and its analogues to inhibit uptake of [3H]DA by hDAT that varied over a broad range (IC50 values ca. 135 to >25,000 nM). The methylenedioxy group of MDPV made a minimal contribution to affinity, the carbonyl group and a tertiary amine are more important, and the extended α-alkyl group seems most important. Either a tertiary amine, or the extended α-alkyl group (but not both), are required for the potent nature of MDPV as an hDAT inhibitor.