Upregulation of dynorphin/kappa opioid receptor system in the dorsal hippocampus contributes to morphine withdrawal-induced place aversion.

Aversive emotion of opioid withdrawal generates motivational state leading to compulsive drug seeking and taking. Kappa opioid receptor (KOR) and its endogenous ligand dynorphin have been shown to participate in the regulation of aversive emotion. In the present study, we investigated the role of dynorphin/KOR system in the aversive emotion following opioid withdrawal in acute morphine-dependent mice. We found that blockade of KORs before pairing by intracerebroventricular injection of KOR antagonist norBNI (20, 40 µg) attenuated the development of morphine withdrawal-induced conditioned place aversion (CPA) behavior. We further found that morphine withdrawal increased dynorphin A expression in the dorsal hippocampus, but not in the amygdala, prefrontal cortex, nucleus accumbens, and thalamus. Microinjection of norBNI (20 µg) into the dorsal hippocampus significantly decreased morphine withdrawal-induced CPA behavior. We further found that p38 MAPK was significantly activated in the dorsal hippocampus after morphine withdrawal, and the activation of p38 MAPK was blocked by pretreatment with norBNI. Accordingly, microinjection of p38 MAPK inhibitor SB203580 (5 µg) into the dorsal hippocampus significantly decreased morphine withdrawal-produced CPA behavior. This study demonstrates that upregulation of dynorphin/KOR system in the dorsal hippocampus plays a critical role in the formation of aversive emotion associated with morphine withdrawal, suggesting that KOR antagonists may have therapeutic value for the treatment of opioid withdrawal-induced mood-related disorders.

Alteration of twinfilin1 expression underlies opioid withdrawal-induced remodeling of actin cytoskeleton at synapses and formation of aversive memory.

Exposure to drugs of abuse induces alterations of dendritic spine morphology and density that has been proposed to be a cellular basis of long-lasting addictive memory and heavily depend on remodeling of its underlying actin cytoskeleton by the actin cytoskeleton regulators. However, the actin cytoskeleton regulators involved and the specific mechanisms whereby drugs of abuse alter their expression or function are largely unknown. Twinfilin (Twf1) is a highly conserved actin-depolymerizing factor that regulates actin dynamics in organisms from yeast to mammals. Despite abundant expression of Twf1 in mammalian brain, little is known about its importance for brain functions such as experience-dependent synaptic and behavioral plasticity. Here we show that conditioned morphine withdrawal (CMW)-induced synaptic structure and behavior plasticity depends on downregulation of Twf1 in the amygdala of rats. Genetically manipulating Twf1 expression in the amygdala bidirectionally regulates CMW-induced changes in actin polymerization, spine density and behavior. We further demonstrate that downregulation of Twf1 is due to upregulation of miR101a expression via a previously unrecognized mechanism involving CMW-induced increases in miR101a nuclear processing via phosphorylation of MeCP2 at Ser421. Our findings establish the importance of Twf1 in regulating opioid-induced synaptic and behavioral plasticity and demonstrate its value as a potential therapeutic target for the treatment of opioid addiction.

Amygdala dynorphin/κ opioid receptor system modulates depressive-like behavior in mice following chronic social defeat stress.

Major depression disorder is a severe and recurrent neuropsychological disorder characterized by lowered mood and social activity and cognitive impairment. Owing to unclear molecular mechanisms of depression, limited interventions are available in clinic. In this study we investigated the role of dynorphin/κ opioid receptor system in the development of depression. Mice were subjected to chronic social defeat stress for 14 days. Chronic social defeat stress induced significant social avoidance in mice characterized by decreased time duration in the interaction zone and increased time duration in the corner zone. Pre-administration of a κ opioid receptor antagonist norBNI (10 mg/kg, i.p.) could prevent the development of social avoidance induced by chronic social defeat stress. Social avoidance was not observed in κ opioid receptor knockout mice subjected to chronic social defeat stress. We further revealed that social defeat stress activated c-fos and ERK signaling in the amygdala without affecting the NAc, hippocampus and hypothalamus, and ERK activation was blocked by systemic injection of norBNI. Finally, the expression of dynorphin A, the endogenous ligand of κ opioid receptor, was significantly increased in the amygdala following social defeat stress; microinjection of norBNI into the amygdala prevented the development of depressive-like behaviors caused by social defeat stress. The present study demonstrates that upregulated dynorphin/κ opioid receptor system in the amygdala leads to the emergence of depression following chronic social defeat stress, and sheds light on κ opioid receptor antagonists as potential therapeutic agents for the prevention and treatment of depression following chronic stress.

Paraventricular nucleus-central amygdala oxytocinergic projection modulates pain-related anxiety-like behaviors in mice.

AIMS: Anxiety disorders associated with pain are a common health problem. However, the underlying mechanisms remain poorly understood. We aimed to investigate the role of paraventricular nucleus (PVN)-central nucleus of the amygdala (CeA) oxytocinergic projections in anxiety-like behaviors induced by inflammatory pain. METHODS: After inflammatory pain induction by complete Freund's adjuvant (CFA), mice underwent elevated plus maze, light-dark transition test, and marble burying test to examine the anxiety-like behaviors. Chemogenetic, optogenetic, and fiber photometry recordings were used to modulate and record the activity of the oxytocinergic projections of the PVN-CeA. RESULTS: The key results are as follows: inflammatory pain-induced anxiety-like behaviors in mice accompanied by decreased activity of PVN oxytocin neurons. Chemogenetic activation of PVN oxytocin neurons prevented pain-related anxiety-like behaviors, whereas inhibition of PVN oxytocin neurons induced anxiety-like behaviors in naïve mice. PVN oxytocin neurons projected directly to the CeA, and microinjection of oxytocin into the CeA blocked anxiety-like behaviors. Inflammatory pain also decreased the activity of CeA neurons, and optogenetic activation of PVNoxytocin -CeA circuit prevented anxiety-like behavior in response to inflammatory pain. CONCLUSION: The results of our study suggest that oxytocin has anti-anxiety effects and provide novel insights into the role of PVNoxytocin -CeA projections in the regulation of anxiety-like behaviors induced by inflammatory pain.

The claustrum-prelimbic cortex circuit through dynorphin/κ-opioid receptor signaling underlies depression-like behaviors associated with social stress etiology.

Ample evidence has suggested the stress etiology of depression, but the underlying mechanism is not fully understood yet. Here, we report that chronic social defeat stress (CSDS) attenuates the excitatory output of the claustrum (CLA) to the prelimbic cortex (PL) through the dynorphin/κ-opioid receptor (KOR) signaling, being critical for depression-related behaviors in male mice. The CSDS preferentially impairs the excitatory output from the CLA onto the parvalbumin (PV) of the PL, leading to PL micronetwork dysfunction by disinhibiting pyramidal neurons (PNs). Optogenetic activation or inhibition of this circuit suppresses or promotes depressive-like behaviors, which is reversed by chemogenetic inhibition or activation of the PV neurons. Notably, manipulating the dynorphin/KOR signaling in the CLA-PL projecting terminals controls depressive-like behaviors that is suppressed or promoted by optogenetic activation or inhibition of CLA-PL circuit. Thus, this study reveals both mechanism of the stress etiology of depression and possibly therapeutic interventions by targeting CLA-PL circuit.

Amygdalar κ-opioid receptor-dependent upregulating glutamate transporter 1 mediates depressive-like behaviors of opioid abstinence.

Opiates produce a strong rewarding effect, but abstinence from opiate use emerges with severe negative emotions. Depression is one of the most frequent emotion disorders associated with opiate abstinence, which is thought to be a main cause for relapse. However, neurobiological bases of such an aversive emotion processing are poorly understood. Here, we find that morphine abstinence activates κ-opioid receptors (KORs) by increasing endogenous KOR ligand dynorphin expression in the amygdala, which in turn facilitates glutamate transporter 1 (GLT1) expression by activation of p38 mitogen-activated protein kinase (MAPK). Upregulation of GLT1 expression contributes to opiate-abstinence-elicited depressive-like behaviors through modulating amygdalar glutamatergic inputs to the nucleus accumbens (NAc). Intra-amygdala injection of GLT1 inhibitor DHK or knockdown of GLT1 expression in the amygdala significantly suppresses morphine-abstinence-induced depressive-like behaviors. Pharmacological and pharmacogenetic activation of amygdala-NAc projections prevents morphine-abstinence-induced behaviors. Overall, our study provides key molecular and circuit insights into the mechanisms of depression associated with opiate abstinence.

Anteromedial thalamic nucleus to anterior cingulate cortex inputs modulate histaminergic itch sensation.

Itch is an unpleasant feeling that triggers scratching behavior. Much progress has been made in identifying the mechanism of itch at the peripheral and spinal levels, however, itch circuits in the brain remain largely unexplored. We previously found that anterior cingulate cortex (ACC) to dorsal medial striatum (DMS) inputs modulated histamine-induced itch sensation, but how itch information was transmitted to ACC remained unclear. Here, we demonstrated that the anteromedial thalamic nucleus (AM) was activated during histaminergic itch, and there existed reciprocal neuronal projections between AM and ACC. Disconnection between AM and ACC resulted in a significant reduction of histaminergic, but not nonhistaminergic, itch-related scratching behavior. Optogenetic activation of AM-ACC, but not ACC-AM, projections evoked histaminergic itch sensation. Thus, our studies firstly reveal that AM is critical for histaminergic itch sensation and AM-ACC projections modulate histaminergic itch-induced scratching behavior.

Low-frequency repetitive transcranial magnetic stimulation inhibits the development of methamphetamine-induced conditioned place preference.

The abuse of amphetamine-type stimulants (ATS) has become a global public health issue in recent years, these new-type drugs can cause addiction and serious cognitive impairment. However, there are no effective methods for the prevention and treatment of ATS addiction at present. Repetitive transcranial magnetic stimulation (rTMS) is a painless and non-invasive new therapeutic approach that has been used for the treatment of depression and other neuropsychiatric disorders, but whether it can be used to treat drug addiction is unclear. In the present study, we investigated the possible effects of rTMS on methamphetamine(METH)-induced conditioned place preference (CPP). High-frequency (10 Hz) and low-frequency stimulation patterns (1 Hz) were applied to test the effect of rTMS on METH-induced CPP. The results showed that low-frequency but not high-frequency rTMS could block METH-CPP, accompanied with a downregulation of gamma-aminobutyric acid type B receptor subunit 1 (GABABR1) expression in rat dorsolateral striatum. These results suggested that low-frequency rTMS could effectively inhibit the development of METH addiction and shed light on the rTMS as a potential approach for the prevention of drug addiction.

Pharmacological Characterization of Dezocine, a Potent Analgesic Acting as a κ Partial Agonist and µ Partial Agonist.

Dezocine is becoming dominated in China market for relieving moderate to severe pain. It is believed that Dezocine's clinical efficacy and little chance to provoke adverse events during the therapeutic process are mainly attributed to its partial agonist activity at the µ opioid receptor. In the present work, we comprehensively studied the pharmacological characterization of Dezocine and identified that the analgesic effect of Dezocine was a result of action at both the κ and µ opioid receptors. We firstly found that Dezocine displayed preferential binding to µ opioid receptor over κ and δ opioid receptors. Dezocine, on its own, weakly stimulated G protein activation in cells expressing κ and µ receptors, but in the presence of full κ agonist U50,488 H and µ agonist DAMGO, Dezocine inhibited U50,488H- and DAMGO-mediated G protein activation, indicating that Dezocine was a κ partial agonist and µ partial agonist. Then the in intro results were verified by in vivo studies in mice. We observed that Dezocine-produced antinociception was significantly inhibited by κ antagonist nor-BNI and µ antagonist ß-FNA pretreatment, indicating that Dezocine-mediated antinociception was via both the κ and µ opioid receptors. When co-administrating of Dezocine with U50,488 H or morphine, Dezocine was capable of inhibiting U50,488H- or morphine-induced antinociception. Finally, κ receptor activation-associated side effect sedation was investigated. We found that Dezocine displayed limited sedative effect with a ceiling effecting at a moderate dose. Thus, our work led to a better understanding of the analgesic mechanism of action of Dezocine in vivo.

The neuroprotective effect of memantine on methamphetamine-induced cognitive deficits.

Repeated exposure to methamphetamine (METH) can cause severe neurotoxicity to the cortical neurons. In the present study, we investigated the effect of METH on cognitive function deficits, and determined the neuroprotective effects of memantine (MEM) on memory impairment induced by METH. The protein levels of Bcl-2 and cleaved caspase-3 in prefrontal cortex (PFC) were further examined to exploring the underlying mechanism. We found that repeated METH administration impaired long term (24h) memory retention without affecting short term (5min) memory retention. Co-administration of MEM with METH before training session significantly improved METH-induced cognitive function. METH significantly decreased expression level of Bcl-2 and increased expression level of cleaved caspase-3 in the PFC. The changes can be prevented by MEM pretreatment. Thus, these results demonstrated that MEM pretreatment reversed METH-induced changes of protein levels of apoptotic-related gene, and produced protective effects against METH-induced cognitive deficits, suggesting the effectiveness of MEM may be due to its anti-apoptotic activity.

The anxiolytic- and antidepressant-like effects of ATPM-ET, a novel κ agonist and µ partial agonist, in mice.

RATIONALE: Opioid receptors are implicated in the regulation of motivation and emotion. However, animal studies show that activation of κ opioid receptor produces contrasting mood-altering effects in models of anxiety-like and depressive-like behaviors, and consequently, the role of κ receptor in mood control remains unsettled. The effect of κ/µ opioid combination in emotion regulation was unexplored. OBJECTIVES: The aim of the study was to investigate the effects of (-)-3-N-ethylaminothiazolo [5,4-b]-N-cyclopropylmethylmorphinan hydrochloride (ATPM-ET), a novel κ agonist and µ partial agonist, in regulating emotional responses. METHODS: The emotional responses of ATPM-ET were detected in the elevated plus maze (EPM), open field test (OFT), forced swim test (FST), and tail suspension test (TST). Selective κ antagonist nor-binaltorphimine (nor-BNI) and µ antagonist ß-funaltrexamine (ß-FNA) were applied to determine the type of receptor involved. The conditioned place aversion model was used to evaluate the effects on aversive emotion. RESULTS: In the EPM and OFT, ATPM-ET (1 and 2 mg/kg, s.c.) significantly increased the time spent in the open arm and in the central area, respectively. In the FST and TST, ATPM-ET (0.5 and 1 mg/kg, s.c.) significantly reduced the duration of immobility. These effects were prevented by nor-BNI (10 mg/kg, i.p., -24 h), but not by ß-FNA (10 and20 mg/kg, i.p., -24 h) pretreatment. At the dose of 2 mg/kg, ATPM-ET did not induce conditioned place aversion. CONCLUSIONS: ATPM-ET, at doses from 0.5 to 2 mg/kg, produced anxiolytic- and antidepressant-like effects without inducing aversive emotion. These effects were more closely mediated by activation of κ receptor than µ receptor.

κ Opioid receptor activation in different brain regions differentially modulates anxiety-related behaviors in mice.

κ Opioid receptor system is widely implicated in the regulation of emotion. However, the findings about the role on anxiety in rodents are highly controversial, since both anxiogenic- and anxiolytic-like effects have been reported with κ opioid receptor activation. The mechanism and the underlying neuroanatomical substrates are unexplored. In the present study, we first investigated the effects of κ agonist U50,488H on anxiety-related behaviors over a wide range of doses, and we found that U50,488H produced dual effects in anxiety, with low dose being anxiogenic and high dose being anxiolytic. To assess the potential neuroanatomical substrates, we used phosphorylation of extracellular signal-related kinase1/2 (pERK1/2) to map the underlying neural circuits. We found that the anxiogenic effect of U50,488H was paralleled by an increase of pERK1/2 in the nucleus accumbens, whereas the anxiolytic effect was paralleled by an increase of pERK1/2 in the lateral septal nucleus. We then examined the behavioral consequences with locally microinjection of U50,488H, and we found that microinjection of U50,488H into the nucleus accumbens exerted anxiogenic-like effects, whereas microinjection of U50,488H into the lateral septal nucleus. Both effects can be abolished by κ antagonist nor-BNI pretreatment. To the best of our knowledge, the present work firstly provides the neuroanatomical sites that mediating the dual anxiogenic- and anxiolytic-like effects of U50,488H in mice. This study may help to explain current controversial role of κ receptor activation in anxiety-related behaviors in rodents, and may open new perspectives in the areas of anxiety disorders and κ receptor function.

Role for engagement of ß-arrestin2 by the transactivated EGFR in agonist-specific regulation of δ receptor activation of ERK1/2.

BACKGROUND AND PURPOSE: ß-Arrestins function as signal transducers linking GPCRs to ERK1/2 signalling either by scaffolding members of ERK1/2s cascades or by transactivating receptor tyrosine kinases through Src-mediated release of transactivating factor. Recruitment of ß-arrestins to the activated GPCRs is required for ERK1/2 activation. Our previous studies showed that δ receptors activate ERK1/2 through a ß-arrestin-dependent mechanism without inducing ß-arrestin binding to the δ receptors. However, the precise mechanisms involved remain to be established. EXPERIMENTAL APPROACH: ERK1/2 activation by δ receptor ligands was assessed using HEK293 cells in vitro and male Sprague Dawley rats in vivo. Immunoprecipitation, immunoblotting, siRNA transfection, intracerebroventricular injection and immunohistochemistry were used to elucidate the underlying mechanism. KEY RESULTS: We identified a new signalling pathway in which recruitment of ß-arrestin2 to the EGFR rather than δ receptor was required for its role in δ receptor-mediated ERK1/2 activation in response to H-Tyr-Tic-Phe-Phe-OH (TIPP) or morphine stimulation. Stimulation of the δ receptor with ligands leads to the phosphorylation of PKCδ, which acts upstream of EGFR transactivation and is needed for the release of the EGFR-activating factor, whereas ß-arrestin2 was found to act downstream of the EGFR transactivation. Moreover, we demonstrated that coupling of the PKCδ/EGFR/ß-arrestin2 transactivation pathway to δ receptor-mediated ERK1/2 activation was ligand-specific and the Ser(363) of δ receptors was crucial for ligand-specific implementation of this ERK1/2 activation pathway. CONCLUSIONS AND IMPLICATIONS: The δ receptor-mediated activation of ERK1/2 is via ligand-specific transactivation of EGFR. This study adds new insights into the mechanism by which δ receptors activate ERK1/2.

Antagonism of κ opioid receptor in the nucleus accumbens prevents the depressive-like behaviors following prolonged morphine abstinence.

The association between morphine withdrawal and depressive-like symptoms is well documented, however, the role of dynorphin/κ opioid receptor system and the underlying neural substrates have not been fully understood. In the present study, we found that four weeks morphine abstinence after a chronic escalating morphine regimen significantly induced depressive-like behaviors in mice. Prodynorphin mRNA and protein levels were increased in the nucleus accumbens (NAc) after four weeks of morphine withdrawal. Local injection of κ opioid receptor antagonist nor-Binaltorphimine (norBNI) in the NAc significantly blocked the expression of depressive-like behaviors without influencing general locomotor activity. Thus, the present study extends previous findings by showing that prolonged morphine withdrawal-induced depressive-like behaviors are regulated by dynorphin/κ opioid receptor system, and shed light on the κ opioid receptor antagonists as potential therapeutic agents for the treatment of depressive-like behaviors induced by opiate withdrawal.