SIRT1 Decreases Emotional Pain Vulnerability with Associated CaMKIIα Deacetylation in Central Amygdala.

Emotional disorders are common comorbid conditions that further exacerbate the severity and chronicity of chronic pain. However, individuals show considerable vulnerability to the development of chronic pain under similar pain conditions. In this study on male rat and mouse models of chronic neuropathic pain, we identify the histone deacetylase Sirtuin 1 (SIRT1) in central amygdala as a key epigenetic regulator that controls the development of comorbid emotional disorders underlying the individual vulnerability to chronic pain. We found that animals that were vulnerable to developing behaviors of anxiety and depression under the pain condition displayed reduced SIRT1 protein levels in central amygdala, but not those animals resistant to the emotional disorders. Viral overexpression of local SIRT1 reversed this vulnerability, but viral knockdown of local SIRT1 mimicked the pain effect, eliciting the pain vulnerability in pain-free animals. The SIRT1 action was associated with CaMKIIα downregulation and deacetylation of histone H3 lysine 9 at the CaMKIIα promoter. These results suggest that, by transcriptional repression of CaMKIIα in central amygdala, SIRT1 functions to guard against the emotional pain vulnerability under chronic pain conditions. This study indicates that SIRT1 may serve as a potential therapeutic molecule for individualized treatment of chronic pain with vulnerable emotional disorders.SIGNIFICANCE STATEMENT Chronic pain is a prevalent neurological disease with no effective treatment at present. Pain patients display considerably variable vulnerability to developing chronic pain, indicating individual-based molecular mechanisms underlying the pain vulnerability, which is hardly addressed in current preclinical research. In this study, we have identified the histone deacetylase Sirtuin 1 (SIRT1) as a key regulator that controls this pain vulnerability. This study reveals that the SIRT1-CaMKIIaα pathway in central amygdala acts as an epigenetic mechanism that guards against the development of comorbid emotional disorders under chronic pain, and that its dysfunction causes increased vulnerability to the development of chronic pain. These findings suggest that SIRT1 activators may be used in a novel therapeutic approach for individual-based treatment of chronic pain.

Brain Circuits Mediating Opposing Effects on Emotion and Pain.

The amygdala is important for processing emotion, including negative emotion such as anxiety and depression induced by chronic pain. Although remarkable progress has been achieved in recent years on amygdala regulation of both negative (fear) and positive (reward) behavioral responses, our current understanding is still limited regarding how the amygdala processes and integrates these negative and positive emotion responses within the amygdala circuits. In this study with optogenetic stimulation of specific brain circuits, we investigated how amygdala circuits regulate negative and positive emotion behaviors, using pain as an emotional assay in male rats. We report here that activation of the excitatory pathway from the parabrachial nucleus (PBN) that relays peripheral pain signals to the central nucleus of amygdala (CeA) is sufficient to cause behaviors of negative emotion including anxiety, depression, and aversion in normal rats. In strong contrast, activation of the excitatory pathway from basolateral amygdala (BLA) that conveys processed corticolimbic signals to CeA dramatically opposes these behaviors of negative emotion, reducing anxiety and depression, and induces behavior of reward. Surprisingly, activating the PBN-CeA pathway to simulate pain signals does not change pain sensitivity itself, but activating the BLA-CeA pathway inhibits basal and sensitized pain. These findings demonstrate that the pain signal conveyed through the PBN-CeA pathway is sufficient to drive negative emotion and that the corticolimbic signal via the BLA-CeA pathway counteracts the negative emotion, suggesting a top-down brain mechanism for cognitive control of negative emotion under stressful environmental conditions such as pain.SIGNIFICANCE STATEMENT It remains unclear how the amygdala circuits integrate both negative and positive emotional responses and the brain circuits that link peripheral pain to negative emotion are largely unknown. Using optogenetic stimulation, this study shows that the excitatory projection from the parabrachial nucleus to the central nucleus of amygdala (CeA) is sufficient to drive behaviors of negative emotion including anxiety, depression, and aversion in rats. Conversely, activation of the excitatory projection from basolateral amygdala to CeA counteracts each of these behaviors of negative emotion. Thus, this study identifies a brain pathway that mediates pain-driven negative emotion and a brain pathway that counteracts these emotion behaviors in a top-down mechanism for brain control of negative emotion.

Pain vulnerability and DNA methyltransferase 3a involved in the affective dimension of chronic pain.

Chronic pain with comorbid emotional disorders is a prevalent neurological disease in patients under various pathological conditions, yet patients show considerable difference in their vulnerability to developing chronic pain. Understanding the neurobiological basis underlying this pain vulnerability is essential to develop targeted therapies of higher efficiency in pain treatment of precision medicine. However, this pain vulnerability has not been addressed in preclinical pain research in animals to date. In this study, we investigated individual variance in both sensory and affective/emotional dimensions of pain behaviors in response to chronic neuropathic pain condition in a mouse model of chronic pain. We found that mice displayed considerably diverse sensitivities in the chronic pain-induced anxiety- and depression-like behaviors of affective pain. Importantly, the mouse group that was more vulnerable to developing anxiety was also more vulnerable to developing depressive behavior under the chronic pain condition. In contrast, there was relatively much less variance in individual responses in the sensory dimension of pain sensitization. Molecular analysis revealed that those mice vulnerable to developing the emotional disorders showed a significant reduction in the protein level of DNA methyltransferase 3a in the emotion-processing central nucleus of the amygdala. In addition, social stress also revealed significant individual variance in anxiety behavior in mice. These findings suggest that individual pain vulnerability may be inherent mostly in the emotional/affective component of chronic pain and remain consistent in different aspects of negative emotion, in which adaptive changes in the function of DNA methyltransferase 3a for DNA methylation in central amygdala may play an important role. This may open a new avenue of basic research into the neurobiological mechanisms underlying pain vulnerability.

Persistent pain maintains morphine-seeking behavior after morphine withdrawal through reduced MeCP2 repression of GluA1 in rat central amygdala.

As long-term opioids are increasingly used for control of chronic pain, how pain affects the rewarding effect of opioids and hence risk of prescription opioid misuse and abuse remains a healthcare concern and a challenging issue in current pain management. In this study, using a rat model of morphine self-administration, we investigated the molecular mechanisms underlying the impact of pain on operant behavior of morphine intake and morphine seeking before and after morphine withdrawal. We found that rats with persistent pain consumed a similar amount of daily morphine to that in control rats without pain, but maintained their level-pressing behavior of morphine seeking after abstinence of morphine at 0.2 mg/kg, whereas this behavior was gradually diminished in control rats. In the central nucleus of amygdala (CeA), a limbic structure critically involved in the affective dimension of pain, proteins of GluA1 subunits of glutamate AMPA receptors were upregulated during morphine withdrawal, and viral knockdown of CeA GluA1 eliminated the morphine-seeking behavior in withdrawn rats of the pain group. Chromatin immunoprecipitation analysis revealed that the methyl CpG-binding protein 2 (MeCP2) was enriched in the promoter region of Gria1 encoding GluA1 and this enrichment was significantly attenuated in withdrawn rats of the pain group. Furthermore, viral overexpression of CeA MeCP2 repressed the GluA1 level and eliminated the maintenance of morphine-seeking behavior after morphine withdrawal. These results suggest direct MeCp2 repression of GluA1 function as a likely mechanism for morphine-seeking behavior maintained by long-lasting affective pain after morphine withdrawal.

MeCP2 repression of G9a in regulation of pain and morphine reward.

Opioids are commonly used for pain relief, but their strong rewarding effects drive opioid misuse and abuse. How pain affects the liability of opioid abuse is unknown at present. In this study, we identified an epigenetic regulating cascade activated by both pain and the opioid morphine. Both persistent pain and repeated morphine upregulated the transcriptional regulator MeCP2 in mouse central nucleus of the amygdala (CeA). Chromatin immunoprecipitation analysis revealed that MeCP2 bound to and repressed the transcriptional repressor histone dimethyltransferase G9a, reducing G9a-catalyzed repressive mark H3K9me2 in CeA. Repression of G9a activity increased expression of brain-derived neurotrophic factor (BDNF). Behaviorally, persistent inflammatory pain increased the sensitivity to acquiring morphine-induced, reward-related behavior of conditioned place preference in mice. Local viral vector-mediated MeCP2 overexpression, Cre-induced G9a knockdown, and CeA application of BDNF mimicked, whereas MeCP2 knockdown inhibited, the pain effect. These results suggest that MeCP2 directly represses G9a as a shared mechanism in central amygdala for regulation of emotional responses to pain and opioid reward, and for their behavioral interaction.

Central amygdala GluA1 facilitates associative learning of opioid reward.

GluA1 subunits of AMPA glutamate receptors are implicated in the synaptic plasticity induced by drugs of abuse for behaviors of drug addiction, but GluA1 roles in emotional learning and memories of drug reward in the development of drug addiction remain unclear. In this study of the central nucleus of the amygdala (CeA), which is critical in emotional learning of drug reward, we investigated how adaptive changes in the expression of GluA1 subunits affected the learning process of opioid-induced context-reward association (associative learning) for the acquisition of reward-related behavior. In CeA neurons, we found that CeA GluA1 expression was significantly increased 2 h after conditioning treatment with morphine, but not 24 h after the conditioning when the behavior of conditioned place reference (CPP) was fully established in rats. Adenoviral overexpression of GluA1 subunits in CeA accelerated associative learning, as shown by reduced minimum time of morphine conditioning required for CPP acquisition and by facilitated CPP extinction through extinction training with no morphine involved. Adenoviral shRNA-mediated downregulation of CeA GluA1 produced opposite effects, inhibiting the processes of both CPP acquisition and CPP extinction. Adenoviral knockdown of CeA GluA2 subunits facilitated CPP acquisition, but did not alter CPP extinction. Whole-cell recording revealed enhanced electrophysiological properties of postsynaptic GluA2-lacking AMPA receptors in adenoviral GluA1-infected CeA neurons. These results suggest that increased GluA1 expression of CeA AMPA receptors facilitates the associative learning of context-drug reward, an important process in both development and relapse of drug-seeking behaviors in drug addiction.

Optogenetic activation of brainstem serotonergic neurons induces persistent pain sensitization.

BACKGROUND: The rostral ventromedial medulla (RVM) is a key brainstem structure that conveys powerful descending influence of the central pain-modulating system on spinal pain transmission and processing. Serotonergic (5-HT) neurons are a major component in the heterogeneous populations of RVM neurons and in the descending pathways from RVM. However, the descending influence of RVM 5-HT neurons on pain behaviors remains unclear. RESULTS: In this study using optogenetic stimulation in tryptophan hydroxylase 2 (TPH2)- Channelrhodopsin 2 (ChR2) transgenic mice, we determined the behavioral effects of selective activation of RVM 5-HT neurons on mechanical and thermal pain behaviors in vivo. We found that ChR2-EYFP-positive neurons strongly co-localized with TPH2-positive (5-HT) neurons in RVM. Optogenetic stimulation significantly increased c-fos expression in 5-HT cells in the RVM of TPH2-ChR2 mice, but not in wild type mice. Behaviorally, the optogenetic stimulation decreased both mechanical and thermal pain threshold in an intensity-dependent manner, with repeated stimulation producing sensitized pain behavior for up to two weeks. CONCLUSIONS: These results suggest that selective activation of RVM 5-HT neurons exerts a predominant effect of pain facilitation under control conditions.

The projection from dorsal medial prefrontal cortex to basolateral amygdala promotes behaviors of negative emotion in rats.

Brain circuits between medial prefrontal cortex (mPFC) and amygdala have been implicated in cortical control of emotion, especially anxiety. Studies in recent years focus on differential roles of subregions of mPFC and amygdala, and reciprocal pathways between mPFC and amygdala in regulation of emotional behaviors. It has been shown that, while the projection from ventral mPFC to basomedial amygdala has an anxiolytic effect, the reciprocal projections between dorsal mPFC (dmPFC) and basolateral amygdala (BLA) are generally involved in an anxiogenic effect in various conditions with increased anxiety. However, the function of the projection from dmPFC to BLA in regulation of general emotional behaviors under normal conditions remains unclear. In this study, we used optogenetic analysis to identify how this dmPFC-BLA pathway regulates various emotional behaviors in normal rats. We found that optogenetic stimulation of the dmPFC-BLA pathway promoted a behavioral state of negative emotion, increasing anxiety-like and depressive-like behaviors and producing aversive behavior of place avoidance. Conversely, optogenetic inhibition of this pathway produced opposite effects, reducing anxiety-like and depressive-like behaviors, and inducing behaviors of place preference of reward. These findings suggest that activity of the dmPFC-BLA pathway is sufficient to drive a negative emotion state and the mPFC-amygdala circuit is tonically active in cortical regulation of emotional behaviors.

Brain-derived neurotrophic factor-mediated downregulation of brainstem K+-Cl- cotransporter and cell-type-specific GABA impairment for activation of descending pain facilitation.

Chronic pain is thought to be partly caused by a loss of GABAergic inhibition and resultant neuronal hyperactivation in the central pain-modulating system, but the underlying mechanisms for pain-modulating neurons in the brain are unclear. In this study, we investigated the cellular mechanisms for activation of brainstem descending pain facilitation in rats under persistent pain conditions. In the nucleus raphe magnus (NRM), a critical relay in the brain's descending pain-modulating system, persistent inflammatory pain induced by complete Freund's adjuvant decreased the protein level of K(+)-Cl(-) cotransporter (KCC2) in both total and synaptosomal preparations. Persistent pain also shifted the equilibrium potential of GABAergic inhibitory postsynaptic current (EIPSC) to a more positive level and increased the firing of evoked action potentials selectively in µ-opioid receptor (MOR)-expressing NRM neurons, but not in MOR-lacking NRM neurons. Microinjection of brain-derived neurotrophic factor (BDNF) into the NRM inhibited the KCC2 protein level in the NRM, and both BDNF administration and KCC2 inhibition by furosemide mimicked the pain-induced effects on EIPSC and excitability in MOR-expressing neurons. Furthermore, inhibiting BDNF signaling by NRM infusion of tyrosine receptor kinase B-IgG or blocking KCC2 with furosemide prevented these pain effects in MOR-expressing neurons. These findings demonstrate a cellular mechanism by which the hyperactivity of NRM MOR-expressing neurons, presumably responsible for descending pain facilitation, contributes to pain sensitization through the signaling cascade of BDNF-KCC2-GABA impairment in the development of chronic pain.

Signaling cascades for δ-opioid receptor-mediated inhibition of GABA synaptic transmission and behavioral antinociception.

Membrane trafficking of the δ-opioid receptor (DOR) from intracellular compartments to plasma membrane in central neurons, induced by various pathological conditions such as long-term opioid exposure, represents unique receptor plasticity involved in the mechanisms of long-term opioid effects in opioid addiction and opioid treatment of chronic pain. However, the signaling pathways coupled to the newly emerged functional DOR in central neurons are largely unknown at present. In this study, we investigated the signaling cascades of long-term morphine-induced DOR for its cellular and behavioral effects in neurons of the rat brainstem nucleus raphe magnus (NRM), a key supraspinal site for opioid analgesia. We found that, among the three phospholipase A(2) (PLA(2))-regulated arachidonic acid (AA) metabolic pathways of lipoxygenase, cyclooxygenase, and epoxygenase, 12-lipoxygenase of the lipoxygenase pathway primarily mediated DOR inhibition of GABA synaptic transmission, because inhibitors of 12-lipoxygenase as well as lipoxygenases and PLA(2) largely blocked the DOR- or AA-induced GABA inhibition in NRM neurons in brainstem slices in vitro. Blockade of the epoxygenase pathway was ineffective, whereas blocking either 5-lipoxygenase of the lipoxygenase pathway or the cyclooxygenase pathway enhanced the DOR-mediated GABA inhibition. Behaviorally in rats in vivo, NRM infusion of 12-lipoxygenase inhibitors significantly reduced DOR-induced antinociceptive effect whereas inhibitors of 5-lipoxygenase and cyclooxygenase augmented the DOR antinociception. These findings suggest the PLA(2)-AA-12-lipoxygenase pathway as a primary signaling cascade for DOR-mediated analgesia through inhibition of GABA neurotransmission and indicate potential therapeutic benefits of combining 5-lipoxygenase and cyclooxygenase inhibitors for maximal pain inhibition.

Synaptic plasticity in two cell types of central amygdala for regulation of emotion and pain.

The amygdala is a critical brain site for regulation of emotion-associated behaviors such as pain and anxiety. Recent studies suggest that differential cell types and synaptic circuits within the amygdala complex mediate interacting and opposing effects on emotion and pain. However, the underlying cellular and circuit mechanisms are poorly understood at present. Here we used optogenetics combined with electrophysiological analysis of synaptic inputs to investigate pain-induced synaptic plasticity within the amygdala circuits in rats. We found that 50% of the cell population in the lateral division of the central nucleus of the amygdala (CeAl) received glutamate inputs from both basolateral amygdala (BLA) and from the parabrachial nucleus (PBN), and 39% of the remaining CeAl cells received glutamate inputs only from PBN. Inflammatory pain lasting 3 days, which induced anxiety, produced sensitization in synaptic activities of the BLA-CeAl-medial division of CeA (CeAm) pathway primarily through a postsynaptic mechanism. Moreover, in CeAl cells receiving only PBN inputs, pain significantly augmented the synaptic strength of the PBN inputs. In contrast, in CeAl cells receiving both BLA and PBN inputs, pain selectively increased the synaptic strength of BLA inputs, but not the PBN inputs. Electrophysiological analysis of synaptic currents showed that the increased synaptic strength in both cases involved a postsynaptic mechanism. These findings reveal two main populations of CeAl cells that have differential profiles of synaptic inputs and show distinct plasticity in their inputs in response to anxiety-associated pain, suggesting that the specific input plasticity in the two populations of CeAl cells may encode a different role in amygdala regulation of pain and emotion.

Synaptic mechanism for functional synergism between delta- and mu-opioid receptors.

By sustained activation of mu-opioid receptors (MORs), chronic opioids cause analgesic tolerance, physical dependence, and opioid addiction, common clinical problems for which an effective treatment is still lacking. Chronic opioids recruit delta-opioid receptors (DORs) to plasma membrane through exocytotic trafficking, but the role of this new DOR and its interaction with existing MOR in brain functions and in these clinical problems remain largely unknown. In this study, we investigated the mechanisms underlying synaptic and behavioral actions of chronic morphine-induced DORs and their interaction with MORs in nucleus raphe magnus (NRM) neurons important for opioid analgesia. We found that the emerged DOR inhibited GABAergic IPSCs through both the phospholipase A(2) (PLA(2)) and cAMP/protein kinase A (PKA) signaling pathways. MOR inhibition of IPSCs, normally mediated predominantly by the PLA(2) pathway, was additionally mediated by the cAMP/PKA pathway, with MOR potency significantly increased after chronic morphine treatment. Isobologram analysis revealed a synergistic DOR-MOR interaction in their IPSC inhibition, which was dependent on upregulated activities of both the PLA(2) and cAMP/PKA pathways. Furthermore, DOR and MOR agonists microinjected into the NRM in vivo also produced a PLA(2)-dependent synergism in their antinociceptive effects. These findings suggest that the cAMP/PKA pathway, upregulated by chronic opioids, becomes more important in the mechanisms of both MOR and DOR inhibition of GABA synaptic transmission after chronic opioid exposure, and DORs and MORs are synergic both synaptically and behaviorally in producing analgesic effects in a PLA(2)-dependent fashion, supporting the potential therapeutic use of DOR agonists in pain management under chronic opioid conditions.

Nerve growth factor-regulated emergence of functional delta-opioid receptors.

Sorting of intracellular G-protein-coupled receptors (GPCRs) either to lysosomes for degradation or to plasma membrane for surface insertion and functional expression is a key process regulating signaling strength of GPCRs across the plasma membrane in adult mammalian cells. However, little is known about the molecular mechanisms governing the dynamic process of receptor sorting to the plasma membrane for functional expression under normal and pathological conditions. In this study, we demonstrate that delta-opioid receptor (DOPr), a GPCR constitutively targeted to intracellular compartments, is driven to the surface membrane of central synaptic terminals and becomes functional by the neurotrophin nerve growth factor (NGF) in native brainstem neurons. The NGF-triggered DOPr translocation is predominantly mediated by the signaling pathway involving the tyrosine receptor kinase A, Ca(2+)-mobilizing phospholipase C, and Ca(2+)/calmodulin-dependent protein kinase II. Importantly, it requires interactions with the cytoplasmic sorting protein NHERF-1 (Na(+)/H(+) exchange regulatory factor-1) and N-ethyl-maleimide-sensitive factor-regulated exocytosis. In addition, this NGF-mediated mechanism is likely responsible for the emergence of functional DOPr induced by chronic opioids. Thus, NGF may function as a key molecular switch that redirects the sorting of intracellularly targeted DOPr to plasma membrane, resulting in new functional DOPr on central synapses under chronic opioid conditions.

Myoblast-derived neuronal cells form glutamatergic neurons in the mouse cerebellum.

Production of neurons from non-neural cells has far-reaching clinical significance. We previously found that myoblasts can be converted to a physiologically active neuronal phenotype by transferring a single recombinant transcription factor, REST-VP16, which directly activates target genes of the transcriptional repressor, REST. However, the neuronal subtype of M-RV cells and whether they can establish synaptic communication in the brain have remained unknown. M-RV cells engineered to express green fluorescent protein (M-RV-GFP) had functional ion channels but did not establish synaptic communication in vitro. However, when transplanted into newborn mice cerebella, a site of extensive postnatal neurogenesis, these cells expressed endogenous cerebellar granule precursors and neuron proteins, such as transient axonal glycoprotein-1, neurofilament, type-III ß-tubulin, superior cervical ganglia-clone 10, glutamate receptor-2, and glutamate decarboxylase. Importantly, they exhibited action potentials and were capable of receiving glutamatergic synaptic input, similar to the native cerebellar granule neurons. These results suggest that M-RV-GFP cells differentiate into glutamatergic neurons, an important neuronal subtype, in the postnatal cerebellar milieu. Our findings suggest that although activation of REST-target genes can reprogram myoblasts to assume a general neuronal phenotype, the subtype specificity may then be directed by the brain microenvironment.

GluA1 in Central Amygdala Promotes Opioid Use and Reverses Inhibitory Effect of Pain.

Increasing evidence suggests that long-term opioids and pain induce similar adaptive changes in the brain's reward circuits, however, how pain alters the addictive properties of opioids remains poorly understood. In this study using a rat model of morphine self-administration (MSA), we found that short-term pain, induced by an intraplantar injection of complete Freund's adjuvant (CFA), acutely decreased voluntary morphine intake, but not food intake, only at a morphine dose that did not affect pain itself. Pre-treatment with indomethacin, a non-opioid inhibitor of pain, before the pain induction blocked the decrease in morphine intake. In rats with steady MSA, the protein level of GluA1 subunits of glutamate AMPA receptors (AMPARs) was significantly increased, but that of GluA2 was decreased, resulting in an increased GluA1/GluA2 ratio in central nucleus of the amygdala (CeA). In contrast, pain decreased the GluA1/GluA2 ratio in the CeA of rats with MSA. Microinjection of NASPM, a selective inhibitor of homomeric GluA1-AMPARs, into CeA inhibited morphine intake. Furthermore, viral overexpression of GluA1 protein in CeA maintained morphine intake at a higher level than controls and reversed the pain-induced reduction in morphine intake. These findings suggest that CeA GluA1 promotes opioid use and its upregulation is sufficient to increase opioid consumption, which counteracts the acute inhibitory effect of pain on opioid intake. These results demonstrate that the CeA GluA1 is a shared target of opioid and pain in regulation of opioid use, which may aid in future development of therapeutic applications in opioid abuse.

MeCP2 mediates transgenerational transmission of chronic pain.

Pain symptoms can be transmitted across generations, but the mechanisms underlying these outcomes remain poorly understood. Here, we identified an essential role for primary somatosensory cortical (S1) glutamate neuronal DNA methyl-CpG binding protein 2 (MeCP2) in the transgenerational transmission of pain. In a female mouse chronic pain model, the offspring displayed significant pain sensitization. In these mice, MeCP2 expression was increased in S1 glutamate (GluS1) neurons, correlating with increased neuronal activity. Downregulation of GluS1 neuronal MeCP2 in maternal mice with pain abolished offspring pain sensitization, whereas overexpression of MeCP2 in naïve maternal mice induced pain sensitization in offspring. Notably, single-cell sequencing and chromatin immunoprecipitation analysis showed that the expression of a wide range of genes was changed in offspring and maternal GluS1 neurons, some of which were regulated by MeCP2. These results collectively demonstrate the putative importance of MeCP2 as a key regulator in pain transgenerational transmission through actions on GluS1 neuronal maladaptation.

Rewarding morphine-induced synaptic function of delta-opioid receptors on central glutamate synapses.

The rewarding effect of opioids, the driving force for compulsive behaviors of opioid abuse and addiction, is primarily mediated by the mu-opioid receptor. However, the role of the delta-opioid receptor (DOR) in opioid reward and addiction is still poorly understood. The recently discovered adaptive DOR property of exocytotic translocation in sensory neurons after chronic opioid exposure provides a new avenue of conceptual thoughts to exploring the DOR function in this psychoneurological disease. In this study, we investigated potential adaptive function of DOR in neurons of the central nucleus of the amygdala (CeA), a forebrain structure increasingly recognized for mediating stimulus reward learning in drug addiction. Using whole-cell recordings in CeA slices, we found that in rats displaying morphine-induced behavior of conditioned place preference, a behavioral measure of drug reward, the overall synaptic strength of glutamate synapses in CeA neurons was significantly enhanced. The selective DOR agonist [D-Pen(2),D-Pen(5)]-enkephalin, having no apparent effect on glutamatergic excitatory postsynaptic current (EPSC) in neurons from control rats, produced a significant, dose-dependent inhibition of the synaptic current in neurons from those morphine-treated rats. Detailed analyses of EPSC properties revealed that DOR activation inhibited the EPSC by reducing presynaptic release of glutamate, indicating functional DOR emerging on presynaptic glutamate terminals. The morphine treatment also significantly increased DOR proteins in CeA preparations of synaptosomes. These findings provide functional evidence for an adaptive modulation by presynaptic DOR of a key synaptic activity altered by morphine, thus implying likely important involvement of DOR in opioid reward and addiction.

A Central Amygdala Input to the Parafascicular Nucleus Controls Comorbid Pain in Depression.

While comorbid pain in depression (CP) occurs at a high rate worldwide, the neural connections underlying the core symptoms of CP have yet to be elucidated. Here, we define a pathway whereby GABAergic neurons from the central nucleus of the amygdala (GABACeA) project to glutamatergic neurons in the parafascicular nucleus (GluPF). These GluPF neurons relay directly to neurons in the second somatosensory cortex (S2), a well-known area involved in pain signal processing. Enhanced inhibition of the GABACeA→GluPF→S2 pathway is found in mice exhibiting CP symptoms. Reversing this pathway using chemogenetic or optogenetic approaches alleviates CP symptoms. Together, the current study demonstrates the putative importance of the GABACeA→GluPF→S2 pathway in controlling at least some aspects of CP.

Involvement of non-NMDA glutamate receptors in central amygdala in synaptic actions of ethanol and ethanol-induced reward behavior.

The central nucleus of the amygdala (CeA) plays a critical role in positive emotional responses that involve stimulus-reward learning and are induced by the reinforcing effects of many drugs of abuse, including alcohol. Behavioral studies have implicated CeA as a key brain structure in alcohol reward, but the underlying mechanisms are still poorly understood. Recent studies have demonstrated that both NMDA and non-NMDA receptors in CeA neurons are targets of acute and chronic alcohol in naive and alcohol-dependent animals. However, little is known about the role of CeA non-NMDA receptors in synaptic actions of alcohol and, particularly, in the behavior of alcohol reward. In the present study with both whole-cell voltage-clamp recordings in CeA slices in vitro and analysis of an animal model of conditioned place preference (CPP) in vivo, we investigated the synaptic mechanisms for actions of acute and chronic ethanol on CeA non-NMDA receptor functions and their contribution to ethanol-induced reward behavior. Acute ethanol significantly inhibited evoked and miniature synaptic currents mediated by non-NMDA receptors through inhibitions of both postsynaptic non-NMDA receptors and presynaptic glutamate release involving N-type Ca2+ channels. CeA neurons from rats exhibiting the ethanol-induced CPP behavior showed a significant increase in non-NMDA synaptic transmission. Blockade of this increased synaptic transmission through CeA microinjection abolished the CPP behavior. These results suggest that acute alcohol inhibits CeA non-NMDA synaptic transmission through both presynaptic and postsynaptic mechanisms, and chronic alcohol upregulates this synaptic activity, which is required for the alcohol-induced reward behavior.