Claustrum projections to the anterior cingulate modulate nociceptive and pain-associated behavior.

The anterior cingulate cortex (ACC) is critical for the perception and unpleasantness of pain.1,2,3,4,5,6 It receives nociceptive information from regions such as the thalamus and amygdala and projects to several cortical and subcortical regions of the pain neuromatrix.7,8 ACC hyperexcitability is one of many functional changes associated with chronic pain, and experimental activation of ACC pyramidal cells produces hypersensitivity to innocuous stimuli (i.e., allodynia).9,10,11,12,13,14 A less-well-studied projection to the ACC arises from a small forebrain region, the claustrum.15,16,17,18,19,20 Stimulation of excitatory claustrum projection neurons preferentially activates GABAergic interneurons, generating feed-forward inhibition onto excitatory cortical networks.21,22,23,24 Previous work has shown that claustrocingulate projections display altered activity in prolonged pain25,26,27; however, it remains unclear whether and how the claustrum participates in nociceptive processing and high-order pain behaviors. Inhibition of ACC activity reverses mechanical allodynia in animal models of persistent and neuropathic pain,1,9,28 suggesting claustrum inputs may function to attenuate pain processing. In this study, we sought to define claustrum function in acute and chronic pain. We found enhanced claustrum activity after a painful stimulus that was attenuated in chronic inflammatory pain. Selective inhibition of claustrocingulate projection neurons enhanced acute nociception but blocked pain learning. Inversely, chemogenetic activation of claustrocingulate neurons had no effect on basal nociception but rescued inflammation-induced mechanical allodynia. Together, these results suggest that claustrocingulate neurons are a critical component of the pain neuromatrix, and dysregulation of this connection may contribute to chronic pain.

Sex differences in peripheral immune cell activation: Implications for pain and pain resolution.

Decades of research into chronic pain has deepened our understanding of the cellular mechanisms behind this process. However, a failure to consider the biological variable of sex has limited the application of these breakthroughs into clinical application. In the present study, we investigate fundamental differences in chronic pain between male and female mice resulting from inflammatory activation of the innate immune system. We provide evidence that female mice are more sensitive to the effects of macrophages. Injecting small volumes of media conditioned by either unstimulated macrophages or macrophages stimulated by the inflammatory molecule TNFα lead to increased pain sensitivity only in females. Interestingly, we find that TNFα conditioned media leads to a more rapid resolution of mechanical hypersensitivity and altered immune cell recruitment to sites of injury. Furthermore, male and female macrophages exhibit differential polarization characteristics and motility after TNFα stimulation, as well as a different profile of cytokine secretions. Finally, we find that the X-linked gene Tlr7 is critical in the facilitating the adaptive resolution of pain in models of acute and chronic inflammation in both sexes. Altogether, these findings suggest that although the cellular mechanisms of pain resolution may differ between the sexes, the study of these differences may yield more targeted approaches with clinical applications.

Preclinical evidence for the use of the atypical antipsychotic, brexpiprazole, for opioid use disorder.

Opioid addiction is characterized by adaptations in the mesolimbic dopamine system that occur during chronic opioid use. Alterations in dopaminergic transmission contribute to pathological drug-seeking behavior and other symptoms associated with opioid withdrawal following drug discontinuation, making drug abstinence challenging and contributing to high rates of relapse among those suffering from substance use disorder. Recently, the use of dopamine partial agonists has been proposed as a potential strategy to restore dopaminergic signalling during drug withdrawal, while avoiding the adverse side effects associated with stronger modulators of dopaminergic transmission. We investigated the effects of the atypical antipsychotic brexpiprazole, which is a partial agonist at dopamine D2 and D3 receptors, in a mouse model of opioid dependence. The development of opioid dependence in mice is characterized by locomotor sensitization, analgesic tolerance, opioid-induced hyperalgesia, and drug-seeking behavior. We set up four paradigms to model the effects of brexpiprazole on each of these adaptations that occur during chronic opioid use in male and female C57BL/6J mice. Concomitant treatment of brexpiprazole during chronic morphine administration attenuated the development of locomotor sensitization. Brexpiprazole treatment abolished morphine place preference and blocked reinstatement of this behavior following extinction. Brexpiprazole treatment did not alter morphine analgesia, nor did it impact the development of morphine tolerance. However, brexpiprazole treatment did prevent the expression of opioid-induced hyperalgesia in a tail-withdrawal assay, while failing to improve somatic withdrawal symptoms. Altogether, these results provide preclinical evidence for the efficacy of brexpiprazole as a modulator of dopamine-dependent behaviors during opioid use and withdrawal.

Genetic and functional analysis of a Pacific hagfish opioid system.

The actions of endogenous opioids and nociceptin/orphanin FQ are mediated by four homologous G protein-coupled receptors that constitute the opioid receptor family. However, little is known about opioid systems in cyclostomes (living jawless fish) and how opioid systems might have evolved from invertebrates. Here, we leveraged de novo transcriptome and low-coverage whole-genome assembly in the Pacific hagfish (Eptatretus stoutii) to identify and characterize the first full-length coding sequence for a functional opioid receptor in a cyclostome. Additionally, we define two novel endogenous opioid precursors in this species that predict several novel opioid peptides. Bioinformatic analysis shows no closely related opioid receptor genes in invertebrates with regard either to the genomic organization or to conserved opioid receptor-specific sequences that are common in all vertebrates. Furthermore, no proteins analogous to vertebrate opioid precursors could be identified by genomic searches despite previous claims of protein or RNA-derived sequences in several invertebrate species. The presence of an expressed orthologous receptor and opioid precursors in the Pacific hagfish confirms that a functional opioid system was likely present in the common ancestor of all extant vertebrates some 550 million years ago, earlier than all previous authenticated accounts. We discuss the premise that the cyclostome and vertebrate opioid systems evolved from invertebrate systems concerned with antimicrobial defense and speculate that the high concentrations of opioid precursors in tissues such as the testes, gut, and activated immune cells are key remnants of this evolutionary role.

Sex differences in kappa opioid receptor antinociception is influenced by the number of X chromosomes in mouse.

Kappa opioid receptor (KOR) agonists produce robust analgesia with minimal abuse liability and are considered promising pharmacological agents to manage chronic pain and itch. The KOR system is also notable for robust differences between the sexes, with females exhibiting lower analgesic response than males. Sexually dimorphic traits can be due to either the influence of gonadal hormones during development or adulthood, or due to the complement of genes expressed on the X or Y chromosome. Previous studies examining sex differences in KOR antinociception have relied on surgical or pharmacological manipulation of the gonads to determine whether sex hormones influence KOR function. While there are conflicting reports whether gonadal hormones influence KOR function, no study has examined these effects in context with sex chromosomes. Here, we use two genetic mouse models, the four core genotypes and XY*, to isolate the chromosomal and hormonal contributions to sex differences in KOR analgesia. Mice were treated with systemic KOR agonist (U50,488H) and thermal analgesia measured in the tail withdrawal assay. We found that KOR antinociception was influenced predominantly by the number of the X chromosomes. These data suggest that the dose and/or parental imprint on X gene(s) contribute significantly to the sexually dimorphism in KOR analgesia.

Central amygdala inflammation drives pain hypersensitivity and attenuates morphine analgesia in experimental autoimmune encephalomyelitis.

ABSTRACT: Chronic pain is a highly prevalent symptom associated with the autoimmune disorder multiple sclerosis (MS). The central nucleus of the amygdala plays a critical role in pain processing and modulation. Neuropathic pain alters nociceptive signaling in the central amygdala, contributing to pain chronicity and opioid tolerance. Here, we demonstrate that activated microglia within the central amygdala disrupt nociceptive sensory processing and contribute to pain hypersensitivity in experimental autoimmune encephalomyelitis (EAE), the most frequently used animal model of MS. Male and female mice with EAE exhibited differences in microglial morphology in the central amygdala, which was associated with heat hyperalgesia, impaired morphine reward, and reduced morphine antinociception in females. Animals with EAE displayed a lack of morphine-evoked activity in cells expressing somatostatin within the central amygdala, which drive antinociception. Induction of focal microglial activation in naïve mice via injection of lipopolysaccharide into the central amygdala produced a loss of morphine analgesia in females, similar to as observed in EAE animals. Our data indicate that activated microglia within the central amygdala may contribute to the sexually dimorphic effects of morphine and may drive neuronal adaptations that lead to pain hypersensitivity in EAE. Our results provide a possible mechanism underlying the decreased efficacy of opioid analgesics in the management of MS-related pain, identifying microglial activation as a potential therapeutic target for pain symptoms in this patient population.

Multiple Sclerosis and the Endogenous Opioid System.

Multiple sclerosis (MS) is an autoimmune disease characterized by chronic inflammation, neuronal degeneration and demyelinating lesions within the central nervous system. The mechanisms that underlie the pathogenesis and progression of MS are not fully known and current therapies have limited efficacy. Preclinical investigations using the murine experimental autoimmune encephalomyelitis (EAE) model of MS, as well as clinical observations in patients with MS, provide converging lines of evidence implicating the endogenous opioid system in the pathogenesis of this disease. In recent years, it has become increasingly clear that endogenous opioid peptides, binding µ- (MOR), κ- (KOR) and δ-opioid receptors (DOR), function as immunomodulatory molecules within both the immune and nervous systems. The endogenous opioid system is also well known to play a role in the development of chronic pain and negative affect, both of which are common comorbidities in MS. As such, dysregulation of the opioid system may be a mechanism that contributes to the pathogenesis of MS and associated symptoms. Here, we review the evidence for a connection between the endogenous opioid system and MS. We further explore the mechanisms by which opioidergic signaling might contribute to the pathophysiology and symptomatology of MS.

Pain, Motivation, Migraine, and the Microbiome: New Frontiers for Opioid Systems and Disease.

For decades the broad role of opioids in addiction, neuropsychiatric disorders, and pain states has been somewhat well established. However, in recent years, with the rise of technological advances, not only is the existing dogma being challenged, but we are identifying new disease areas in which opioids play a critical role. This review highlights four new areas of exploration in the opioid field. The most recent addition to the opioid family, the nociceptin receptor system, shows promise as the missing link in understanding the neurocircuitry of motivation. It is well known that activation of the kappa opioid receptor system modulates negative affect and dysphoria, but recent studies now implicate the kappa opioid system in the modulation of negative affect associated with pain. Opioids are critical in pain management; however, the often-forgotten delta opioid receptor system has been identified as a novel therapeutic target for headache disorders and migraine. Lastly, changes to the gut microbiome have been shown to directly contribute to many of the symptoms of chronic opioid use and opioid related behaviors. This review summarizes the findings from each of these areas with an emphasis on identifying new therapeutic targets. SIGNIFICANCE STATEMENT: The focus of this minireview is to highlight new disease areas or new aspects of disease in which opioids have been implicated; this includes pain, motivation, migraine, and the microbiome. In some cases, this has resulted in the pursuit of a novel therapeutic target and resultant clinical trial. We believe this is very timely and will be a refreshing take on reading about opioids and disease.

Microbes, microglia, and pain.

Globally, it is estimated that one in five people suffer from chronic pain, with prevalence increasing with age. The pathophysiology of chronic pain encompasses complex sensory, immune, and inflammatory interactions within both the central and peripheral nervous systems. Microglia, the resident macrophages of the central nervous system (CNS), are critically involved in the initiation and persistence of chronic pain. Microglia respond to local signals from the CNS but are also modulated by signals from the gastrointestinal tract. Emerging data from preclinical and clinical studies suggest that communication between the gut microbiome, the community of bacteria residing within the gut, and microglia is involved in producing chronic pain. Targeted strategies that manipulate or restore the gut microbiome have been shown to reduce microglial activation and alleviate symptoms associated with inflammation. These data indicate that manipulations of the gut microbiome in chronic pain patients might be a viable strategy in improving pain outcomes. Herein, we discuss the evidence for a connection between microglia and the gut microbiome and explore the mechanisms by which commensal bacteria might influence microglial reactivity to drive chronic pain.

Kappa Opioid Receptors Drive a Tonic Aversive Component of Chronic Pain.

Pain is a multidimensional experience and negative affect, or how much the pain is "bothersome", significantly impacts the sufferers' quality of life. It is well established that the κ opioid system contributes to depressive and dysphoric states, but whether this system contributes to the negative affect precipitated by the occurrence of chronic pain remains tenuous. Using a model of persistent pain, we show by quantitative real-time-PCR, florescence in situ hybridization, Western blotting and GTPgS autoradiography an upregulation of expression and the function of κ opioid receptors (KORs) and its endogenous ligand dynorphin in the mesolimbic circuitry in animals with chronic pain compared with surgical controls. Using in vivo microdialysis and microinjection of drugs into the mesolimbic dopamine system, we demonstrate that inhibiting KORs reinstates evoked dopamine release and reward-related behaviors in chronic pain animals. Chronic pain enhanced KOR agonist-induced place aversion in a sex-dependent manner. Using various place preference paradigms, we show that activation of KORs drives pain aversive states in male but not female mice. However, KOR antagonist treatment was effective in alleviating anxiogenic and depressive affective-like behaviors in both sexes. Finally, ablation of KORs from dopamine neurons using AAV-TH-cre in KORloxP mice prevented pain-induced aversive states as measured by place aversion assays. Our results strongly support the use of KOR antagonists as therapeutic adjuvants to alleviate the emotional, tonic-aversive component of chronic pain, which is argued to be the most significant component of the pain experience that impacts patients' quality of life.SIGNIFICANCE STATEMENT We show that KORs are sufficient to drive the tonic-aversive component of chronic pain; the emotional component of pain that is argued to significantly impact a patient's quality of life. The impact of our study is broadly relevant to affective disorders associated with disruption of reward circuitry and thus likely contributes to many of the devastating sequelae of chronic pain, including the poor response to treatment of many patients, debilitating affective disorders (other disorders including anxiety and depression that demonstrate high comorbidity with chronic pain) and substance abuse. Indeed, coexisting psychopathology increases pain intensity, pain-related disability and effectiveness of treatments (Jamison and Edwards, 2013).

The gut microbiota mediates reward and sensory responses associated with regimen-selective morphine dependence.

Opioid use for long-term pain management is limited by adverse side effects, such as hyperalgesia and negative affect. Neuroinflammation in the brain and spinal cord is a contributing factor to the development of symptoms associated with chronic opioid use. Recent studies have described a link between neuroinflammation and behavior that is mediated by a gut-brain signaling axis, where alterations in indigenous gut bacteria contribute to several inflammation-related psychopathologies. As opioid receptors are highly expressed within the digestive tract and opioids influence gut motility, we hypothesized that systemic opioid treatment will impact the composition of the gut microbiota. Here, we explored how opioid treatments, and cessation, impacts the mouse gut microbiome and whether opioid-induced changes in the gut microbiota influences inflammation-driven hyperalgesia and impaired reward behavior. Male C57Bl6/J mice were treated with either intermittent or sustained morphine. Using 16S rDNA sequencing, we describe changes in gut microbiota composition following different morphine regimens. Manipulation of the gut microbiome was used to assess the causal relationship between the gut microbiome and opioid-dependent behaviors. Intermittent, but not sustained, morphine treatment was associated with microglial activation, hyperalgesia, and impaired reward response. Depletion of the gut microbiota via antibiotic treatment surprisingly recapitulated neuroinflammation and sequelae, including reduced opioid analgesic potency and impaired cocaine reward following intermittent morphine treatment. Colonization of antibiotic-treated mice with a control microbiota restored microglial activation state and behaviors. Our findings suggest that differing opioid regimens uniquely influence the gut microbiome that is causally related to behaviors associated with opioid dependence.

Reducing Astrocyte Calcium Signaling In Vivo Alters Striatal Microcircuits and Causes Repetitive Behavior.

Astrocytes tile the central nervous system, but their functions in neural microcircuits in vivo and their roles in mammalian behavior remain incompletely defined. We used two-photon laser scanning microscopy, electrophysiology, MINIscopes, RNA-seq, and a genetic approach to explore the effects of reduced striatal astrocyte Ca2+ signaling in vivo. In wild-type mice, reducing striatal astrocyte Ca2+-dependent signaling increased repetitive self-grooming behaviors by altering medium spiny neuron (MSN) activity. The mechanism involved astrocyte-mediated neuromodulation facilitated by ambient GABA and was corrected by blocking astrocyte GABA transporter 3 (GAT-3). Furthermore, in a mouse model of Huntington's disease, dysregulation of GABA and astrocyte Ca2+ signaling accompanied excessive self-grooming, which was relieved by blocking GAT-3. Assessments with RNA-seq revealed astrocyte genes and pathways regulated by Ca2+ signaling in a cell-autonomous and non-cell-autonomous manner, including Rab11a, a regulator of GAT-3 functional expression. Thus, striatal astrocytes contribute to neuromodulation controlling mouse obsessive-compulsive-like behavior.

Corticolimbic circuitry in the modulation of chronic pain and substance abuse.

The transition from acute to chronic pain is accompanied by increased engagement of emotional and motivational circuits. Adaptations within this corticolimbic circuitry contribute to the cellular and behavioral maladaptations associated with chronic pain. Central regions within the corticolimbic brain include the mesolimbic dopamine system, the amygdala, and the medial prefrontal cortex. The evidence reviewed herein supports the notion that chronic pain induces significant changes within these corticolimbic regions that contribute to the chronicity and intractability of pain. In addition, pain-induced changes in corticolimbic circuitry are poised to impact motivated behavior and reward responsiveness to environmental stimuli, and may modulate the addiction liability of drugs of abuse, such as opioids.

Topography of microglial activation in sensory- and affect-related brain regions in chronic pain.

Microglial activation in the spinal cord plays a central role in the development and maintenance of chronic pain after a peripheral nerve injury (PNI). There has not yet been a thorough assessment of microglial activation in brain regions associated with pain and reward. To this end, this study uses a mouse model of neuropathic pain in which the left sciatic nerve of male C57Bl/6J mice is loosely constricted (chronic constriction injury) to assess microglial activation in several brain regions 2 weeks after injury, a time point at which pain hypersensitivity is well established. We found significant microglial activation in brain regions associated with sensory pain transmission and affect, including the thalamus, sensory cortex, and amygdala. Activation was consistently most robust in brain regions contralateral to the side of injury. Brain regions not directly involved in either sensory or affective dimensions of pain, such as the motor cortex, did not display microglial activation. This study confirms that PNI induces microglial activation in regions involved with both sensory and affective components of pain. © 2016 Wiley Periodicals, Inc.

Allostatic Mechanisms of Opioid Tolerance Beyond Desensitization and Downregulation.

Mechanisms of opioid tolerance have focused on adaptive modifications within cells containing opioid receptors, defined here as cellular allostasis, emphasizing regulation of the opioid receptor signalosome. We review additional regulatory and opponent processes involved in behavioral tolerance, and include mechanistic differences both between agonists (agonist bias), and between µ- and δ-opioid receptors. In a process we will refer to as pass-forward allostasis, cells modified directly by opioid drugs impute allostatic changes to downstream circuitry. Because of the broad distribution of opioid systems, every brain cell may be touched by pass-forward allostasis in the opioid-dependent/tolerant state. We will implicate neurons and microglia as interactive contributors to the cumulative allostatic processes creating analgesic and hedonic tolerance to opioid drugs.

Neuroimmune Regulation of GABAergic Neurons Within the Ventral Tegmental Area During Withdrawal from Chronic Morphine.

Opioid dependence is accompanied by neuroplastic changes in reward circuitry leading to a negative affective state contributing to addictive behaviors and risk of relapse. The current study presents a neuroimmune mechanism through which chronic opioids disrupt the ventral tegmental area (VTA) dopaminergic circuitry that contributes to impaired reward behavior. Opioid dependence was induced in rodents by treatment with escalating doses of morphine. Microglial activation was observed in the VTA following spontaneous withdrawal from chronic morphine treatment. Opioid-induced microglial activation resulted in an increase in brain-derived neurotrophic factor (BDNF) expression and a reduction in the expression and function of the K(+)Cl(-) co-transporter KCC2 within VTA GABAergic neurons. Inhibition of microglial activation or interfering with BDNF signaling prevented the loss of Cl(-) extrusion capacity and restored the rewarding effects of cocaine in opioid-dependent animals. Consistent with a microglial-derived BDNF-induced disruption of reward, intra-VTA injection of BDNF or a KCC2 inhibitor resulted in a loss of cocaine-induced place preference in opioid-naïve animals. The loss of the extracellular Cl(-) gradient undermines GABAA-mediated inhibition, and represents a mechanism by which chronic opioid treatments can result in blunted reward circuitry. This study directly implicates microglial-derived BDNF as a negative regulator of reward in opioid-dependent states, identifying new therapeutic targets for opiate addictive behaviors.

Microglia disrupt mesolimbic reward circuitry in chronic pain.

Chronic pain attenuates midbrain dopamine (DA) transmission, as evidenced by a decrease in opioid-evoked DA release in the ventral striatum, suggesting that the occurrence of chronic pain impairs reward-related behaviors. However, mechanisms by which pain modifies DA transmission remain elusive. Using in vivo microdialysis and microinjection of drugs into the mesolimbic DA system, we demonstrate in mice and rats that microglial activation in the VTA compromises not only opioid-evoked release of DA, but also other DA-stimulating drugs, such as cocaine. Our data show that loss of stimulated extracellular DA is due to impaired chloride homeostasis in midbrain GABAergic interneurons. Treatment with minocycline or interfering with BDNF signaling restored chloride transport within these neurons and recovered DA-dependent reward behavior. Our findings demonstrate that a peripheral nerve injury causes activated microglia within reward circuitry that result in disruption of dopaminergic signaling and reward behavior. These results have broad implications that are not restricted to the problem of pain, but are also relevant to affective disorders associated with disruption of reward circuitry. Because chronic pain causes glial activation in areas of the CNS important for mood and affect, our findings may translate to other disorders, including anxiety and depression, that demonstrate high comorbidity with chronic pain.