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Cannabis edibles are an increasingly popular form of cannabis consumption. Oral consumption of cannabis has distinct physiological and behavioural effects than injection or inhalation. An animal model is needed to understand the pharmacokinetics and physiological effects of oral cannabis consumption in rodents as a model for human cannabis edible use. Adult male and female C57BL/6 mice received a single dose of commercially available cannabis oil (5 mg/kg THC) by oral gavage. At 0.5-, 1-, 2-, 3-, and 6-hours post-exposure, plasma, brain, and adipose tissue was collected for THC, 11-OH-THC, and THC-COOH measures. We report delayed time to peak THC and 11-OH-THC concentrations in plasma, brain and adipose tissue, which is consistent with human pharmacokinetics studies. We also found sex differences in the cannabis tetrad: (1) female mice had a delayed hypothermic effect 6 hours post-consumption, which was not present in males; (2) females had stronger catalepsy than males; (3) males were less mobile following cannabis exposure, whereas female mice showed no difference in locomotion but an anxiogenic effect at 3h post exposure; and (4) male mice displayed a longer lasting antinociceptive effect of oral cannabis. Oral cannabis consumption is a translationally relevant form of administration that produces similar physiological effects as injection or vaping administration and thus should be considered as a viable approach for examining the physiological effects of cannabis moving forward. Furthermore, given the strong sex differences in metabolism of oral cannabis, these factors should be carefully considered when designing animal studies on the effects of cannabis.

Opioids are potent analgesics, but their pain-relieving effects diminish with repeated use. The reduction in analgesic potency is a hallmark of opioid analgesic tolerance, which hampers opioid pain therapy. In the central nervous system, opioid analgesia is critically modulated by adenosine, a purine nucleoside implicated in the beneficial and detrimental actions of opioid medications. Here, we focus on the A3 adenosine receptor (A3 AR) in opioid analgesic tolerance. Intrathecal administration of the A3 AR agonist MRS5698 with daily systemic morphine in male rats attenuated the reduction in morphine antinociception over 7 days. In rats with established morphine tolerance, intrathecal MRS5698 partially restored the antinociceptive effects of morphine. However, when MRS5698 was discontinued, these animals displayed a reduced antinociceptive response to morphine. Our results suggest that MRS5698 acutely and transiently potentiates morphine antinociception in tolerant rats. By contrast, in morphine-naive rats MRS5698 treatment did not impact thermal nociceptive threshold or affect antinociceptive response to a single injection of morphine. Furthermore, we found that morphine-induced adenosine release in cerebrospinal fluid was blunted in tolerant animals, but total spinal A3 AR expression was not affected. Collectively, our findings indicate that spinal A3 AR activation acutely potentiates morphine antinociception in the opioid tolerant state.

Opioids are potent analgesics used to manage pain in both young and old, but the increased use in the pregnant population has significant individual and societal implications. Infants dependent on opioids, either through maternal or iatrogenic exposure, undergo neonatal opioid withdrawal syndrome (NOWS), where they may experience withdrawal symptoms ranging from mild to severe. We present a detailed and original review of NOWS caused by maternal opioid exposure (mNOWS) and iatrogenic opioid intake (iNOWS). While these two entities have been assessed entirely separately, recognition and treatment of the clinical manifestations of NOWS overlap. Neonatal risk factors such as age, genetic predisposition, drug type, and clinical factors like type of opioid, cumulative dose of opioid exposure, and disease status affect the incidence of both mNOWS and iNOWS, as well as their severity. Recognition of withdrawal is dependent on clinical assessment of symptoms, and the use of clinical assessment tools designed to determine the need for pharmacotherapy. Treatment of NOWS relies on a combination of non-pharmacological therapies and pharmacological options. Long-term consequences of opioids and NOWS continue to generate controversy, with some evidence of anatomic brain changes, but conflicting animal and human clinical evidence of significant cognitive or behavioral impacts on school-age children. We highlight the current knowledge on clinically relevant recognition, treatment, and consequences of NOWS, and identify new advances in clinical management of the neonate. This review brings a unique clinical perspective and critically analyzes gaps between the clinical problem and our preclinical understanding of NOWS.

Stopping opioid medications can result in a debilitating withdrawal syndrome in chronic users. Opioid withdrawal can occur at all ages, but mechanistic understanding of this condition is predominantly derived from adult studies. Here, we examined whether there are age-dependent differences in the behavioural phenotype and cellular indices of opioid withdrawal. We tested this by assessing the behavioural and cFos response (a surrogate marker for neuronal activation) to morphine withdrawal in C57BL/6J mice across key developmental stages—neonatal, adolescent, and adulthood. Mice in all age groups received escalating doses of morphine (10–50 mg/kg) over 5 days and withdrawal was precipitated by a single injection of the opioid receptor antagonist naloxone (2 mg/kg) two hours after the last morphine dose. In adult and adolescent mice, withdrawal behaviours were robust, with age-related differences in autonomic and somatic signs. In both groups, cFos expression was increased in spinally projecting neurons within the Periaqueductal Grey (PAG), Rostro-ventromedial Medulla (RVM), and Locus Coeruleus. Neonatal animals displayed both a distinct behavioural withdrawal and cFos expression profile. Notably, in young animals cFos expression was increased within the PAG and LC, but decreased in the RVM. In summary, naloxone challenge precipitated robust opioid withdrawal behaviours across all developmental stages with neonatal animals displaying differences in withdrawal behaviours and unique neuronal activation patterns within key brainstem regions.

Treating chronic pain using opioids, such as morphine, is hampered by the development of opioid-induced hyperalgesia (OIH; increased pain sensitivity), antinociceptive tolerance and withdrawal which can contribute to dependence and abuse. In the central nervous system, the purine nucleoside adenosine has been implicated in beneficial and detrimental actions of morphine, but the extent of their interaction remains poorly understood. Here, we demonstrate that morphine-induced OIH and antinociceptive tolerance in rats is associated with a 2-fold increase in adenosine kinase (ADK) expression in the dorsal horn of the spinal cord (DH-SC). Blocking ADK activity in the spinal cord provided greater than 90% attenuation of OIH and antinociceptive tolerance through A3 adenosine receptor (A3AR) signaling. Supplementing adenosine signaling with selective A3AR agonists blocked OIH and antinociceptive tolerance in rodents of both sexes. Engagement of A3AR in the spinal cord with an ADK inhibitor or A3AR agonist was associated with reduced DH-SC expression of the NOD-like receptor pyrin domain-containing 3 (NLRP3; 60-75%), cleaved caspase 1 (40-60%), interleukin (IL)-1β (76-80%) and tumor necrosis factor (TNF; 50-60%). In contrast, the neuroinhibitory and anti-inflammatory cytokine IL-10 increased 2-fold. In mice, A3AR agonists prevented the development of tolerance in a model of neuropathic pain and reduced naloxone-dependent withdrawal behaviors by greater than 50%. These findings suggest A3AR-dependent adenosine signaling is compromised during sustained morphine to allow the development of morphine-induced adverse effects. These findings raise the intriguing possibility that A3AR agonists may be useful adjunct to opioids to manage their unwanted effects.

Postoperative shivering and cold hypersensitivity are major side effects of acute and chronic opioid treatments respectively. TRPM8 is a cold and menthol-sensitive channel found in a subset of dorsal root ganglion (DRG) nociceptors. Deletion or inhibition of the TRPM8 channel was found to prevent the cold hyperalgesia induced by chronic administration of morphine. Here, we examined the mechanisms by which morphine was able to promote cold hypersensitivity in DRG neurons and transfected HEK cells. Mice daily injected with morphine for 5 days developed cold hyperalgesia. Treatment with morphine did not alter the expressions of cold sensitive TREK-1, TRAAK and TRPM8 in DRGs. However, TRPM8-expressing DRG neurons isolated from morphine-treated mice exhibited hyperexcitability. Sustained morphine treatment in vitro sensitized TRPM8 responsiveness to cold or menthol and reduced activation-evoked desensitization of the channel. Blocking phospholipase C (PLC) as well as protein kinase C beta (PKCβ), but not protein kinase A (PKA) or Rho-associated protein kinase (ROCK), restored channel desensitization. Identification of two PKC phosphorylation consensus sites, S1040 and S1041, in the TRPM8 and their site-directed mutation were able to prevent the MOR-induced reduction in TRPM8 desensitization. Our results show that activation of MOR by morphine 1) promotes hyperexcitability of TRPM8-expressing neurons and 2) induces a PKCβ-mediated reduction of TRPM8 desensitization. This MOR-PKCβ dependent modulation of TRPM8 may underlie the onset of cold hyperalgesia caused by repeated administration of morphine. Our findings point to TRPM8 channel and PKCβ as important targets for opioid-induced cold hypersensitivity.

Highlights • Duration of joint inflammatory pain behaviour is prolonged in juvenile rats compared to adult rats. • Spinal neuron hyperexcitability mirror pain behaviours of both juvenile and adult rats. • Persistent upregulation of interleukin-6 uniquely observed in spinal cord of juvenile rats. • Inhibition of spinal interleukin-6 activity rescued pain and normalised spinal neuron activity., Pain is the most debilitating symptom in juvenile idiopathic arthritis. As pain correlates poorly to the extent of joint pathology, therapies that control joint inflammation are often inadequate as analgesics. We test the hypothesis that juvenile joint inflammation leads to sensitisation of nociceptive circuits in the central nervous system, which is maintained by cytokine expression in the spinal cord. Here, transient joint inflammation was induced in postnatal day (P)21 and P40 male Sprague-Dawley rats with a single intra-articular ankle injection of complete Freund’s adjuvant. Hindpaw mechanical pain sensitivity was assessed using von Frey hair and weight bearing tests. Spinal neuron activity was measured using in vivo extracellular recording and immunohistochemistry. Joint and spinal dorsal horn TNFα, IL1β and IL6 protein expression was quantified using western blotting. We observed greater mechanical hyperalgesia following joint inflammation in P21 compared to P40 rats, despite comparable duration of swelling and joint inflammatory cytokine levels. This is mirrored by spinal neuron hypersensitivity, which also outlasted the duration of active joint inflammation. The cytokine profile in the spinal cord differed at the two ages: prolonged upregulation of spinal IL6 was observed in P21, but not P40 rats. Finally, spinal application of anti-IL-6 antibody (30 ng) reduced the mechanical hyperalgesia and neuronal activation. Our results indicate that persistent upregulation of pro-inflammatory cytokines in the spinal dorsal horn is associated with neuronal sensitisation and mechanical hyperalgesia in juvenile rats, beyond the progress of joint pathology. In addition, we provide proof of concept that spinal IL6 is a key target for treating persistent pain in JIA.

Chronic pain is a complex sensory, cognitive, and emotional experience that imposes a great personal, psychological, and socioeconomic burden on patients. An estimated 1.5 billion people worldwide are afflicted with chronic pain, which is often difficult to treat and may be resistant to the potent pain-relieving effects of opioid analgesics. Attention has therefore focused on advancing new pain therapies directed at the cannabinoid system because of its key role in pain modulation. Endocannabinoids and exogenous cannabinoids exert their actions primarily through Gi/o-protein coupled cannabinoid CB1 and CB2 receptors expressed throughout the nervous system. CB1 receptors are found at key nodes along the pain pathway and their activity gates both the sensory and affective components of pain. CB2 receptors are typically expressed at low levels on microglia, astrocytes, and peripheral immune cells. In chronic pain states, there is a marked increase in CB2 expression which modulates the activity of these central and peripheral immune cells with important consequences for the surrounding pain circuitry. Growing evidence indicate that interventions targeting CB1 or CB2 receptors improve pain outcomes in a variety of preclinical pain models. In this mini-review, we will highlight recent advances in understanding how cannabinoids modulate microglia function and its implications for cannabinoid-mediated analgesia, focusing on microglia-neuron interactions within the spinal nociceptive circuitry.

Needle procedures are among the most common causes of pain and distress for individuals seeking health care. While needle pain is especially problematic for children needle pain and associated fear also has significant impact on adults and can lead to avoidance of appropriate medical care. Currently there is not a standard definition of needle pain. A taxonomy, or classification system, for acute needle pain would aid research efforts and enhance clinical care. To meet this need, the Analgesic, Anesthetic, and Addiction Clinical Trial Translations, Innovations, Opportunities, and Networks public-private partnership with the U.S. Food and Drug Administration, the American Pain Society, and the American Academy of Pain Medicine formed the Analgesic, Anesthetic, and Addiction Clinical Trial Translations, Innovations, Opportunities, and Networks-American Pain Society-American Academy of Pain Medicine Pain Taxonomy initiative. One of the goals of this initiative was to develop taxonomies for acute pain disorders, including needle pain. To accomplish this, a working group of experts in needle pain was convened. Based on available literature and expert opinion, the working group used a 5-dimenional structure (diagnostic criteria, common features, modulating factors, impact and/or functional consequences, and putative mechanisms) to develop an acute pain taxonomy that is specific needle pain. As part of this, a set of 4 diagnostic criteria, with 2 modifiers to account for the influence of needle associated fear, are proposed to define the types of acute needle pain. PERSPECTIVE: This article presents a taxonomy for acute needle pain. This taxonomy could help to standardize definitions of acute pain in clinical studies of patients undergoing needle procedures.

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, florescencein situhybridization, 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. Usingin vivomicrodialysis 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 KORloxPmice 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 STATEMENTWe 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).

Opioids are potent analgesics, but their pain-relieving effects diminish with repeated use. The reduction in analgesic potency is a hallmark of opioid analgesic tolerance, which hampers opioid pain therapy. In the central nervous system, opioid analgesia is critically modulated by adenosine, a purine nucleoside implicated in the beneficial and detrimental actions of opioid medications. Here, we focus on the A(3) adenosine receptor (A(3)AR) in opioid analgesic tolerance. Intrathecal administration of the A(3)AR agonist MRS5698 with daily systemic morphine in male rats attenuated the reduction in morphine antinociception over 7 days. In rats with established morphine tolerance, intrathecal MRS5698 partially restored the antinociceptive effects of morphine. However, when MRS5698 was discontinued, these animals displayed a reduced antinociceptive response to morphine. Our results suggest that MRS5698 acutely and transiently potentiates morphine antinociception in tolerant rats. By contrast, in morphine-naïve rats MRS5698 treatment did not impact thermal nociceptive threshold or affect antinociceptive response to a single injection of morphine. Furthermore, we found that morphine-induced adenosine release in cerebrospinal fluid was blunted in tolerant animals, but total spinal A(3)AR expression was not affected. Collectively, our findings indicate that spinal A(3)AR activation acutely potentiates morphine antinociception in the opioid tolerant state.

Increased afferent input resulting from painful injury augments the activity of central nociceptive circuits via both neuron-neuron and neuron-glia interactions. Microglia, resident immune cells of the central nervous system (CNS), play a crucial role in the pathogenesis of chronic pain. This study provides a framework for understanding how peripheral joint injury signals the CNS to engage spinal microglial responses. During the first week of monosodium iodoacetate (MIA)-induced knee joint injury in male rats, inflammatory and neuropathic pain were characterized by increased firing of peripheral joint afferents. This increased peripheral afferent activity was accompanied by increased Iba1 immunoreactivity within the spinal dorsal horn indicating microglial activation. Pharmacological silencing of C and A afferents with co-injections of QX-314 and bupivacaine, capsaicin, or flagellin prevented the development of mechanical allodynia and spinal microglial activity after MIA injection. Elevated levels of ATP in the cerebrospinal fluid (CSF) and increased expression of the ATP transporter vesicular nucleotide transporter (VNUT) in the ipsilateral spinal dorsal horn were also observed after MIA injections. Selective silencing of primary joint afferents subsequently inhibited ATP release into the CSF. Furthermore, increased spinal microglial reactivity, and alleviation of MIA-induced arthralgia with co-administration of QX-314 with bupivacaine were recapitulated in female rats. Our results demonstrate that early peripheral joint injury activates joint nociceptors, which triggers a central spinal microglial response. Elevation of ATP in the CSF, and spinal expression of VNUT suggest ATP signaling may modulate communication between sensory neurons and spinal microglia at 2 weeks of joint degeneration.

Dopamine is well known to regulate movement through the differential control of direct and indirect pathways in the striatum that express D1 and D2 receptors respectively. The spinal cord also expresses all dopamine receptors however; how the specific receptors regulate spinal network output in mammals is poorly understood. We explore the receptor-specific mechanisms that underlie dopaminergic control of spinal network output of neonatal mice during changes in spinal network excitability. During spontaneous activity, which is a characteristic of developing spinal networks operating in a low excitability state, we found that dopamine is primarily inhibitory. We uncover an excitatory D1-mediated effect of dopamine on motoneurons and network output that also involves co-activation with D2 receptors. Critically, these excitatory actions require higher concentrations of dopamine; however, analysis of dopamine concentrations of neonates indicates that endogenous levels of spinal dopamine are low. Because endogenous levels of spinal dopamine are low, this excitatory dopaminergic pathway is likely physiologically-silent at this stage in development. In contrast, the inhibitory effect of dopamine, at low physiological concentrations is mediated by parallel activation of D2, D3, D4 and α2 receptors which is reproduced when endogenous dopamine levels are increased by blocking dopamine reuptake and metabolism. We provide evidence in support of dedicated spinal network components that are controlled by excitatory D1 and inhibitory D2 receptors that is reminiscent of the classic dopaminergic indirect and direct pathway within the striatum. These results indicate that network state is an important factor that dictates receptor-specific and therefore dose-dependent control of neuromodulators on spinal network output and advances our understanding of how neuromodulators regulate neural networks under dynamically changing excitability.Significance statementMonoaminergic neuromodulation of neural networks is dependent not only on target receptors but also on network state. We studied the concentration-dependent control of spinal networks of the neonatal mouse, in vitro, during a low excitability state characterized by spontaneous network activity. Spontaneous activity is an essential element for the development of networks. Under these conditions, we defined converging receptor and cellular mechanisms that contribute to the diverse, concentration-dependent control of spinal motor networks by dopamine, in vitro. These experiments advance understanding of how monoamines modulate neuronal networks under dynamically changing excitability conditions and provide evidence of dedicated D1 and D2 regulated network components in the spinal cord that are consistent with those reported in the striatum.

Pannexin 1 (Panx1) channels are transmembrane proteins that release adenosine triphosphate and play an important role in intercellular communication. They are widely expressed in somatic and nervous system tissues, and their activity has been associated with many pathologies such as stroke, epilepsy, inflammation, and chronic pain. While there are a variety of small molecules known to inhibit Panx1, currently little is known about the mechanism of channel inhibition, and there is a dearth of sufficiently potent and selective drugs targeting Panx1. Herein we provide a review of the current literature on Panx1 structural biology and known pharmacological agents that will help provide a basis for rational development of Panx1 chemical modulators.

Stopping opioid medications can result in a debilitating withdrawal syndrome in chronic users. Opioid withdrawal can occur at all ages, but mechanistic understanding of this condition is predominantly derived from adult studies. Here, we examined whether there are age-dependent differences in the behavioural phenotype and cellular indices of opioid withdrawal. We tested this by assessing the behavioural and cFos response (a surrogate marker for neuronal activation) to morphine withdrawal in C57BL/6J mice across key developmental stages-neonatal, adolescent, and adulthood. Mice in all age groups received escalating doses of morphine (10-50 mg/kg) over 5 days and withdrawal was precipitated by a single injection of the opioid receptor antagonist naloxone (2 mg/kg) two hours after the last morphine dose. In adult and adolescent mice, withdrawal behaviours were robust, with age-related differences in autonomic and somatic signs. In both groups, cFos expression was increased in spinally projecting neurons within the Periaqueductal Grey (PAG), Rostro-ventromedial Medulla (RVM), and Locus Coeruleus. Neonatal animals displayed both a distinct behavioural withdrawal and cFos expression profile. Notably, in young animals cFos expression was increased within the PAG and LC, but decreased in the RVM. In summary, naloxone challenge precipitated robust opioid withdrawal behaviours across all developmental stages with neonatal animals displaying differences in withdrawal behaviours and unique neuronal activation patterns within key brainstem regions.

Treating chronic pain by using opioids, such as morphine, is hampered by the development of opioid-induced hyperalgesia (OIH; increased pain sensitivity), antinociceptive tolerance, and withdrawal, which can contribute to dependence and abuse. In the central nervous system, the purine nucleoside adenosine has been implicated in beneficial and detrimental actions of morphine, but the extent of their interaction remains poorly understood. Here, we demonstrate that morphine-induced OIH and antinociceptive tolerance in rats is associated with a twofold increase in adenosine kinase (ADK) expression in the dorsal horn of the spinal cord. Blocking ADK activity in the spinal cord provided greater than 90% attenuation of OIH and antinociceptive tolerance through A(3) adenosine receptor (A(3)AR) signaling. Supplementing adenosine signaling with selective A(3)AR agonists blocked OIH and antinociceptive tolerance in rodents of both sexes. Engagement of A(3)AR in the spinal cord with an ADK inhibitor or A(3)AR agonist was associated with reduced dorsal horn of the spinal cord expression of the NOD-like receptor pyrin domain-containing 3 (60%–75%), cleaved caspase 1 (40%–60%), interleukin (IL)-1β (76%–80%), and tumor necrosis factor (50%–60%). In contrast, the neuroinhibitory and anti-inflammatory cytokine IL-10 increased twofold. In mice, A(3)AR agonists prevented the development of tolerance in a model of neuropathic pain and reduced naloxone-dependent withdrawal behaviors by greater than 50%. These findings suggest A(3)AR-dependent adenosine signaling is compromised during sustained morphine to allow the development of morphine-induced adverse effects. These findings raise the intriguing possibility that A(3)AR agonists may be useful adjunct to opioids to manage their unwanted effects. SIGNIFICANCE STATEMENT: The development of hyperalgesia and antinociceptive tolerance during prolonged opioid use are noteworthy opioid-induced adverse effects that reduce opioid efficacy for treating chronic pain and increase the risk of dependence and abuse. We report that in rodents, these adverse effects are due to reduced adenosine signaling at the A(3)AR, resulting in NOD-like receptor pyrin domain-containing 3–interleukin-1β neuroinflammation in spinal cord. These effects are attenuated by A(3)AR agonists, suggesting that A(3)AR may be a target for therapeutic intervention with selective A(3)AR agonist as opioid adjuncts.

Postoperative shivering and cold hypersensitivity are major side effects of acute and chronic opioid treatments respectively. TRPM8 is a cold and menthol-sensitive channel found in a subset of dorsal root ganglion (DRG) nociceptors. Deletion or inhibition of the TRPM8 channel was found to prevent the cold hyperalgesia induced by chronic administration of morphine. Here, we examined the mechanisms by which morphine was able to promote cold hypersensitivity in DRG neurons and transfected HEK cells. Mice daily injected with morphine for five days developed cold hyperalgesia. Treatment with morphine did not alter the expressions of cold sensitive TREK-1, TRAAK and TRPM8 in DRGs. However, TRPM8-expressing DRG neurons isolated from morphine-treated mice exhibited hyperexcitability. Sustained morphine treatment in vitro sensitized TRPM8 responsiveness to cold or menthol and reduced activation-evoked desensitization of the channel. Blocking the phospholipase C (PLC) as well as protein kinase C beta (PKCβ), but not protein kinase A (PKA) or Rho-associated protein kinase (ROCK), restored channel desensitization. Identification of two PKC phosphorylation consensus sites, S1040 and S1041, in the TRPM8 and their site-directed mutation were able to prevent the MOR-induced reduction in TRPM8 desensitization. Our results show that activation of MOR by morphine 1) promotes hyperexcitability of TRPM8-expressing neurons and 2) induces a PKCβ-mediated reduction of TRPM8 desensitization. This MOR-PKCβ dependent modulation of TRPM8 may underlie the onset of cold hyperalgesia caused by repeated administration of morphine. Our findings point to TRPM8 channel and PKCβ as important targets for opioid-induced cold hypersensitivity.

Tolerance to the analgesic effects of opioids is a major problem in chronic pain management. Microglia are implicated in opioid tolerance, but the core mechanisms regulating their response to opioids remain obscure. By selectively ablating microglia in the spinal cord using a saporin-conjugated antibody to Mac1, we demonstrate a causal role for microglia in the development, but not maintenance, of morphine tolerance in male rats. Increased P2X7 receptor (P2X7R) activity is a cardinal feature of microglial activation, and in this study we found that morphine potentiates P2X7R-mediated Ca2+responses in resident spinal microglia acutely isolated from morphine tolerant rats. The increased P2X7R function was blocked in cultured microglia by PP2, a Src family protein tyrosine kinase inhibitor. We identified Src family kinase activation mediated by μ-receptors as a key mechanistic step required for morphine potentiation of P2X7R function. Furthermore, we show by site-directed mutagenesis that tyrosine (Y382–384) within the P2X7R C-terminus is differentially modulated by repeated morphine treatment and has no bearing on normal P2X7R function. Intrathecal administration of a palmitoylated peptide corresponding to the Y382–384site suppressed morphine-induced microglial reactivity and preserved the antinociceptive effects of morphine in male rats. Thus, site-specific regulation of P2X7R function mediated by Y382–384is a novel cellular determinant of the microglial response to morphine that critically underlies the development of morphine analgesic tolerance.SIGNIFICANCE STATEMENTControlling pain is one of the most difficult challenges in medicine and its management is a requirement of a large diversity of illnesses. Although morphine and other opioids offer dramatic and impressive relief of pain, their impact is truncated by loss of efficacy (analgesic tolerance). Understanding why this occurs and how to prevent it are of critical importance in improving pain therapies. We uncovered a novel site (Y382–384) within the P2X7 receptor that can be targeted to blunt the development of morphine analgesic tolerance, without affecting normal P2X7 receptor function. Our findings provide a critical missing mechanistic piece, site-specific modulation by Y382–384, that unifies P2X7R function to the activation of spinal microglia and the development of morphine tolerance.

Opioids are among the most powerful analgesics for managing pain, yet their repeated use can lead to the development of severe adverse effects. In a recent study, we identified the microglial pannexin-1 channel (Panx1) as a critical substrate for opioid withdrawal. Here, we investigated whether microglial Panx1 contributes to opioid-induced hyperalgesia (OIH) and opioid analgesic tolerance using mice with a tamoxifen-inducible deletion of microglial Panx1. We determined that escalating doses of morphine resulted in thermal pain hypersensitivity in both Panx1-expressing and microglial Panx1-deficient mice. In microglial Panx1-deficient mice, we also found that acute morphine antinociception remained intact, and repeated morphine treatment at a constant dose resulted in a progressive decline in morphine antinociception and a reduction in morphine potency. This reduction in morphine antinociceptive potency was indistinguishable from that observed in Panx1-expressing mice. Notably, morphine tolerant animals displayed increased spinal microglial reactivity, but no change of microglial Panx1 expression. Collectively, our findings indicate microglial Panx1 differentially contributes to opioid withdrawal, but not the development of opioid-induced hyperalgesia or tolerance.

Opiates are essential for treating pain, but termination of opiate therapy can cause a debilitating withdrawal syndrome in chronic users. To alleviate or avoid the aversive symptoms of withdrawal, many of these individuals continue to use opiates. Withdrawal is therefore a key determinant of opiate use in dependent individuals, yet its underlying mechanisms are poorly understood and effective therapies are lacking. Here, we identify the pannexin-1 (Panx1) channel as a therapeutic target in opiate withdrawal. We show that withdrawal from morphine induces long-term synaptic facilitation in lamina I and II neurons within the rodent spinal dorsal horn, a principal site of action for opiate analgesia. Genetic ablation of Panx1 in microglia abolished the spinal synaptic facilitation and ameliorated the sequelae of morphine withdrawal. Panx1 is unique in its permeability to molecules up to 1 kDa in size and its release of ATP. We show that Panx1 activation drives ATP release from microglia during morphine withdrawal and that degrading endogenous spinal ATP by administering apyrase produces a reduction in withdrawal behaviors. Conversely, we found that pharmacological inhibition of ATP breakdown exacerbates withdrawal. Treatment with a Panx1-blocking peptide (10panx) or the clinically used broad-spectrum Panx1 blockers, mefloquine or probenecid, suppressed ATP release and reduced withdrawal severity. Our results demonstrate that Panx1-mediated ATP release from microglia is required for morphine withdrawal in rodents and that blocking Panx1 alleviates the severity of withdrawal without affecting opiate analgesia.

Pain and inflammation are inherently linked responses to injury, infection, or chronic diseases. Given that acute inflammation in humans or mice enhances the analgesic properties of opioids, there is much interest in determining the inflammatory transducers that prime opioid receptor signaling in primary afferent nociceptors. Here, we found that activation of the transient receptor potential vanilloid type 1 (TRPV1) channel stimulated a mitogen-activated protein kinase (MAPK) signaling pathway that was accompanied by the shuttling of the scaffold protein β-arrestin2 to the nucleus. The nuclear translocation of β-arrestin2 in turn prevented its recruitment to the μ-opioid receptor (MOR), the subsequent internalization of agonist-bound MOR, and the suppression of MOR activity that occurs upon receptor desensitization. Using the complete Freund's adjuvant (CFA) inflammatory pain model to examine the role of TRPV1 in regulating endogenous opioid analgesia in mice, we found that naloxone methiodide (Nal-M), a peripherally restricted, nonselective, and competitive opioid receptor antagonist, slowed the recovery from CFA-induced hypersensitivity in wild-type, but not TRPV1-deficient, mice. Furthermore, we showed that inflammation prolonged morphine-induced antinociception in a mouse model of opioid receptor desensitization, a process that depended on TRPV1. Together, our data reveal a TRPV1-mediated signaling pathway that serves as an endogenous pain-resolution mechanism by promoting the nuclear translocation of β-arrestin2 to minimize MOR desensitization. This previously uncharacterized mechanism may underlie the peripheral opioid control of inflammatory pain. Dysregulation of the TRPV1-β-arrestin2 axis may thus contribute to the transition from acute to chronic pain.

New Findings What is the topic of this review? This review discusses the origins and development of microglia, and how stress, pain or inflammation in early life disturbs microglial function during critical developmental periods, leading to altered pain sensitivity and/or increased risk of chronic pain in later life. What advances does it highlight? We highlight recent advances in understanding how disrupted microglial function impacts the developing nervous system and the consequences for pain processing and susceptibility for development of chronic pain in later life. The discovery of microglia is accredited to Pio del Rio-Hortega, who recognized this ‘third element’ of CNS cells as being morphologically distinct from neurons and astrocytes. For decades after this finding, microglia were altogether ignored or relegated as simply being support cells. Emerging from virtual obscurity, microglia have now gained notoriety as immune cells that assume a leading role in the development, maintenance and protection of a healthy CNS. Pioneering studies have recently shed light on the origins of microglia, their role in the developing nervous system and the complex roles they play beyond the immune response. These studies reveal that altered microglial function can have a profoundly negative impact on the developing brain and may be a determinant in a range of neurodevelopmental disorders and neurodegenerative diseases. The realization that aberrant microglial function also critically underlies chronic pain, a debilitating disorder that afflicts over 1.5 billion people worldwide, was a major conceptual leap forward in the pain field. Adding to this advance is emerging evidence that early life noxious experiences can have a long-lasting impact on central pain processing and adult pain sensitivity. With microglia now coming of age, in this review we examine the association between adverse early life events, such as stress, injury or inflammation, and the influence of sex differences, on the role of microglia in pain physiology in adulthood.

Chronic pain is a global problem that has reached epidemic proportions. An estimated 20% of adults suffer from pain, and another 10% are diagnosed with chronic pain each year (Goldberg and McGee, ). Despite the high prevalence of chronic pain (an estimated 1.5 billion people are afflicted worldwide), much remains to be understood about the underlying causes of this condition, and there is an urgent requirement for better pain therapies. The discovery of novel targets and the development of better analgesics rely on an assortment of preclinical animal models; however, there are major challenges to translating discoveries made in animal models to realized pain therapies in humans. This review discusses common animal models used to recapitulate clinical chronic pain conditions (such as neuropathic, inflammatory, and visceral pain) and the methods for assessing the sensory and affective components of pain in animals. We also discuss the advantages and limitations of modeling chronic pain in animals as well as highlighting strategies for improving the predictive validity of preclinical pain studies. © 2016 Wiley Periodicals, Inc.

Key points Inflammatory kinins are released following spinal cord injury or neurotrauma. The effects of these kinins on ongoing locomotor activity of central pattern generator networks are unknown. In the present study, kinins were shown to have short- and long-term effects on motor networks. The short-term effects included direct depolarization of interneurons and motoneurons in the ventral horn accompanied by modulation of transient receptor potential vanilloid 1-sensitive nociceptors in the dorsal horn. Over the long-term, we observed a bradykinin-mediated effect on promoting plasticity in the spinal cord. In a model of spinal cord injury, we observed an increase in microglia numbers in both the dorsal and ventral horn and, in a microglia cell culture model, we observed bradykinin-induced expression of glial-derived neurotrophic factor. Abstract The expression and function of inflammatory mediators in the developing spinal cord remain poorly characterized. We discovered novel, short and long-term roles for the inflammatory nonapeptide bradykinin (BK) and its receptor bradykinin receptor B2 (B2R) in the neuromodulation of developing sensorimotor networks following a spinal cord injury (SCI), suggesting that BK participates in an excitotoxic cascade. Functional expression of B2R was confirmed by a transient disruptive action of BK on fictive locomotion generated by a combination of NMDA, 5-HT and dopamine. The role of BK in the dorsal horn nociceptive afferents was tested using spinal cord attached to one-hind-limb (HL) preparations. In the HL preparations, BK at a subthreshold concentration induced transient disruption of fictive locomotion only in the presence of: (1) noxious heat applied to the hind paw and (2) the heat sensing ion channel transient receptor potential vanilloid 1 (TRPV1), known to be restricted to nociceptors in the superficial dorsal horn. BK directly depolarized motoneurons and ascending interneurons in the ventrolateral funiculus. We found a key mechanism for BK in promoting long-term plasticity within the spinal cord. Using a model of neonatal SCI and a microglial cell culture model, we examined the role of BK in inducing activation of microglia and expression of glial-derived neurotrophic factor (GDNF). In the neonatal SCI model, we observed an increase in microglia numbers and increased GDNF expression restricted to microglia. In the microglia cell culture model, we observed a BK-induced increased expression of GDNF via B2R, suggesting a novel mechanism for BK spinal-mediated plasticity.

Pain hypersensitivity resulting from peripheral nerve injury depends on pathological microglial activation in the dorsal horn of the spinal cord. This microglial activity is critically modulated by P2X7 receptors (P2X7R) and ATP stimulation of these receptors produces mechanical allodynia, a defining feature of neuropathic pain. Peripheral nerve injury increases P2X7R expression and potentiates its cation channel function in spinal microglia. Here, we report a means to preferentially block the potentiation of P2X7R function by delivering a membrane permeant small interfering peptide that targets Y382-384, a putative tyrosine phosphorylation site within the P2X7R intracellular C-terminal domain. Intrathecal administration of this palmitoylated peptide (P2X7R379-389) transiently reversed mechanical allodynia caused by peripheral nerve injury in both male and female rats. Furthermore, targeting Y382-384 suppressed P2X7R-mediated release of cytokine tumor necrosis factor alpha and blocked the adoptive transfer of mechanical allodynia caused by intrathecal injection of P2X7R-stimulated microglia. Thus, Y382-384 site-specific modulation of P2X7R is an important microglial mechanism in neuropathic pain.

A new therapeutic option for treating arthritis pain., Chronic joint pain such as mechanical allodynia is the most debilitating symptom of arthritis, yet effective therapies are lacking. We identify the pannexin-1 (Panx1) channel as a therapeutic target for alleviating mechanical allodynia, a cardinal sign of arthritis. In rats, joint pain caused by intra-articular injection of monosodium iodoacetate (MIA) was associated with spinal adenosine 5′-triphosphate (ATP) release and a microglia-specific up-regulation of P2X7 receptors (P2X7Rs). Blockade of P2X7R or ablation of spinal microglia prevented and reversed mechanical allodynia. P2X7Rs drive Panx1 channel activation, and in rats with mechanical allodynia, Panx1 function was increased in spinal microglia. Specifically, microglial Panx1-mediated release of the proinflammatory cytokine interleukin-1β (IL-1β) induced mechanical allodynia in the MIA-injected hindlimb. Intrathecal administration of the Panx1-blocking peptide 10panx suppressed the aberrant discharge of spinal laminae I-II neurons evoked by innocuous mechanical hindpaw stimulation in arthritic rats. Furthermore, mice with a microglia-specific genetic deletion of Panx1 were protected from developing mechanical allodynia. Treatment with probenecid, a clinically used broad-spectrum Panx1 blocker, resulted in a striking attenuation of MIA-induced mechanical allodynia and normalized responses in the dynamic weight-bearing test, without affecting acute nociception. Probenecid reversal of mechanical allodynia was also observed in rats 13 weeks after anterior cruciate ligament transection, a model of posttraumatic osteoarthritis. Thus, Panx1-targeted therapy is a new mechanistic approach for alleviating joint pain.

Microglia-neuron signalling in the spinal cord is a key mediator of mechanical allodynia caused by peripheral nerve injury. We recently reported sex differences in microglia in pain signalling in mice: spinal mechanisms underlying nerve injury-induced allodynia are microglial dependent in male but not female mice. Whether this sex difference in pain hypersensitivity mechanisms is conserved in other species is unknown. Here, we show that in rats, the spinal mechanisms of nerve injury-induced hypersensitivity in males differ from those in females, with microglial P2X4 receptors (P2X4Rs) being a key point of divergence. In rats, nerve injury produced comparable allodynia and reactive microgliosis in both sexes. However, inhibiting microglia in the spinal cord reversed allodynia in male rats but not female rats. In addition, pharmacological blockade of P2X4Rs, by an intrathecally administered antagonist, attenuated pain hypersensitivity in male rats only. Consistent with the behavioural findings, nerve injury increased cell surface expression and function of P2X4Rs in acutely isolated spinal microglia from male rats but not from female rats. Moreover, in microglia cultured from male rats, but not in those from female rats, stimulating P2X4Rs drove intracellular signalling through p38 mitogen-activated protein kinase. Furthermore, chromatin immunoprecipitation-qPCR revealed that the transcription factor IRF5 differentially binds to the P2rx4 promoter region in female rats vs male rats. Finally, mechanical allodynia was produced in otherwise naive rats by intrathecally administering P2X4R-stimulated microglia from male rats but not those from female rats. Together, our findings demonstrate the existence of sexually dimorphic pain signalling in rats, suggesting that this sex difference is evolutionarily conserved, at least across rodent species.

Acute inflammation in humans or mice enhances the analgesic properties of opioids. However, the inflammatory transducers that prime opioid receptor signaling in nociceptors are unknown. We found that TRPV1−/− mice are insensitive to peripheral opioid analgesia in an inflammatory pain model. We report that TRPV1 channel activation drives a MAPK signaling pathway accompanied by the shuttling of β-arrestin2 to the nucleus. This shuttling in turn prevents: β-arrestin2-receptor recruitment, subsequent internalization of agonist-bound mu opioid receptor (MOR), and suppression of DAMGO-induced inhibition of N-type calcium current observed upon desensitization. Consequently, inflammation-induced activation of TRPV1 preserves opioid analgesic potency in a mouse model of opioid receptor desensitization. Overall, our work reveals a TRPV1-mediated signaling mechanism, involving β-arrestin2 nuclear translocation, that underlies the peripheral opioid control of inflammatory pain. Our data single out TRPV1 channels as modulators of opioid analgesia.

Neonatal injury is associated with persistent changes in sensory function and altered nociceptive thresholds that give rise to aberrant pain sensitivity in later life. Although these changes are well documented in adult rodents, little is known about the consequences of neonatal injury during adolescence. Because adolescence is a critical developmental period during which persistent pain conditions can arise, we examined the effect of neonatal injury on nociception, social behavior, and response to morphine in adolescent Sprague Dawley rats. Male and female rats exposed to plantar incision injury at postnatal day 3 displayed mechanical hypersensitivity that resolved by 24 hours after incision. When these animals reached adolescence (postnatal day 28–40), neonatally-injured male rats showed ipsilaterally restricted mechanical, heat, and cold hypersensitivity, as well as social behavioral deficits. In contrast, these effects were not seen in female rats. Neonatal injury did not alter acute morphine antinociception or the development of analgesic tolerance in either sex. Morphine-induced conditioned place preference, behavioral sensitization, and physical withdrawal were also not affected by neonatal incision. Thus, early-life injury results in sex-dependent pain-related hypersensitivity and social behavior deficits during adolescence, without altering the response to opioids. Perspective Neonatal surgery has greater effects on adolescent male than female rats, resulting in pain-related hypersensitivity and social behavioral deficits. Neonatal surgery does not alter the antinociceptive effects of morphine or abuse liability.

The detection and processing of painful stimuli in afferent sensory neurons is critically dependent on a wide range of different types of voltage- and ligand-gated ion channels, including sodium, calcium, and TRP channels, to name a few. The functions of these channels include the detection of mechanical and chemical insults, the generation of action potentials and regulation of neuronal firing patterns, the initiation of neurotransmitter release at dorsal horn synapses, and the ensuing activation of spinal cord neurons that project to pain centers in the brain. Long-term changes in ion channel expression and function are thought to contribute to chronic pain states. Many of the channels involved in the afferent pain pathway are permeable to calcium ions, suggesting a role in cell signaling beyond the mere generation of electrical activity. In this article, we provide a broad overview of different calcium-permeable ion channels in the afferent pain pathway and their role in pain pathophysiology.

What is the topic of this review? This review discusses the origins and development of microglia, and how stress, pain or inflammation in early life disturbs microglial function during critical developmental periods, leading to altered pain sensitivity and/or increased risk of chronic pain in later life. What advances does it highlight? We highlight recent advances in understanding how disrupted microglial function impacts the developing nervous system and the consequences for pain processing and susceptibility for development of chronic pain in later life. The discovery of microglia is accredited to Pío del Río-Hortega, who recognized this 'third element' of CNS cells as being morphologically distinct from neurons and astrocytes. For decades after this finding, microglia were altogether ignored or relegated as simply being support cells. Emerging from virtual obscurity, microglia have now gained notoriety as immune cells that assume a leading role in the development, maintenance and protection of a healthy CNS. Pioneering studies have recently shed light on the origins of microglia, their role in the developing nervous system and the complex roles they play beyond the immune response. These studies reveal that altered microglial function can have a profoundly negative impact on the developing brain and may be a determinant in a range of neurodevelopmental disorders and neurodegenerative diseases. The realization that aberrant microglial function also critically underlies chronic pain, a debilitating disorder that afflicts over 1.5 billion people worldwide, was a major conceptual leap forward in the pain field. Adding to this advance is emerging evidence that early life noxious experiences can have a long-lasting impact on central pain processing and adult pain sensitivity. With microglia now coming of age, in this review we examine the association between adverse early life events, such as stress, injury or inflammation, and the influence of sex differences, on the role of microglia in pain physiology in adulthood.

Chronic pain is a global problem that has reached epidemic proportions. An estimated 20% of adults suffer from pain, and another 10% are diagnosed with chronic pain each year (Goldberg and McGee, ). Despite the high prevalence of chronic pain (an estimated 1.5 billion people are afflicted worldwide), much remains to be understood about the underlying causes of this condition, and there is an urgent requirement for better pain therapies. The discovery of novel targets and the development of better analgesics rely on an assortment of preclinical animal models; however, there are major challenges to translating discoveries made in animal models to realized pain therapies in humans. This review discusses common animal models used to recapitulate clinical chronic pain conditions (such as neuropathic, inflammatory, and visceral pain) and the methods for assessing the sensory and affective components of pain in animals. We also discuss the advantages and limitations of modeling chronic pain in animals as well as highlighting strategies for improving the predictive validity of preclinical pain studies. © 2016 Wiley Periodicals, Inc.

Neuropathic pain, the most debilitating of all clinical pain syndromes, may be a consequence of trauma, infection or pathology from diseases that affect peripheral nerves. Here we provide a framework for understanding the spinal mechanisms of neuropathic pain as distinct from those of acute pain or inflammatory pain. Recent work suggests that a specific microglia response phenotype characterized by de novo expression of the purinergic receptor P2X4 is critical for the pathogenesis of pain hypersensitivity caused by injury to peripheral nerves. Stimulating P2X4 receptors initiates a core pain signaling pathway mediated by release of brain-derived neurotrophic factor, which produces a disinhibitory increase in intracellular chloride in nociceptive (pain-transmitting) neurons in the spinal dorsal horn. The changes caused by signaling from P2X4R(+) microglia to nociceptive transmission neurons may account for the main symptoms of neuropathic pain in humans, and they point to specific interventions to alleviate this debilitating condition.

ATP, acting via P2 purinergic receptors, is a known mediator of inflammatory and neuropathic pain. There is increasing evidence that the ATP-gated P2X4 receptor (P2X4R) subtype is a locus through which activity of spinal microglia and peripheral macrophages instigate pain hypersensitivity caused by inflammation or by injury to a peripheral nerve. The present article highlights the recent advances in our understanding of microglia–neuron interactions in neuropathic pain by focusing on the signaling and regulation of the P2X4R. We will also develop a framework for understanding converging lines of evidence for involvement of P2X4Rs expressed on macrophages in peripheral inflammatory pain.

One of the most significant advances in pain research is the realization that neurons are not the only cell type involved in the etiology of chronic pain. This realization has caused a radical shift from the previous dogma that neuronal dysfunction alone accounts for pain pathologies to the current framework of thinking that takes into account all cell types within the central nervous system (CNS). This shift in thinking stems from growing evidence that glia can modulate the function and directly shape the cellular architecture of nociceptive networks in the CNS. Microglia, in particular, are increasingly recognized as active principal players that respond to changes in physiological homeostasis by extending their processes toward the site of neural damage, and by releasing specific factors that have profound consequences on neuronal function and that contribute to CNS pathologies caused by disease or injury. A key molecule that modulates microglia activity is ATP, an endogenous ligand of the P2 receptor family. Microglia expresses several P2 receptor subtypes, and of these the P2X4 receptor subtype has emerged as a core microglia–neuron signaling pathway: activation of this receptor drives the release of brain-derived neurotrophic factor (BDNF), a cellular substrate that causes disinhibition of pain-transmitting spinal lamina I neurons. Converging evidence points to BDNF from spinal microglia as being a critical microglia–neuron signaling molecule that gates aberrant nociceptive processing in the spinal cord. The present review highlights recent advances in our understanding of P2X4 receptor-mediated signaling and regulation of BDNF in microglia, as well as the implications for microglia–neuron interactions in the pathobiology of neuropathic pain.

Summary: Pro-regenerative macrophages are well known for their role in promoting tissue repair; however, their specific roles in promoting regeneration of the injured nerve are not well defined. Specifically, how macrophages interact with Schwann cells following injury during remyelination has been largely unexplored. We demonstrate that after injury, including in humans, macrophages function to clear debris and persist within the nerve microenvironment. Macrophage ablation immediately preceding remyelination results in an increase in immature Schwann cell density, a reduction in remyelination, and long-term deficits in conduction velocity. Targeted RNA-seq of macrophages from injured nerve identified Gas6 as one of several candidate factors involved in regulating Schwann cell dynamics. Functional studies show that the absence of Gas6 within monocyte lineage cells impairs Schwann cell remyelination within the injured nerve. These results demonstrate a role for macrophages in regulating Schwann cell function during nerve regeneration and highlight a molecular mechanism by which this occurs. : Stratton et al. demonstrate that macrophages persist in the injured rodent and human nerve and regulate Schwann cells. Macrophages have a unique transcriptional profile, including the expression of Gas6, that functions to regulate Schwann cell remyelination. Keywords: nerve injury, macrophage, Schwann cell, regeneration, remyelination, population-based RNA-seq

Repeated exposure to opioid drugs can lead to the development of tolerance, which manifests as a reduction in analgesic potency, and physical dependence, a response indicated by a withdrawal syndrome. Accumulating evidence suggests that the nerve growth factor (NGF) family of neurotrophins may have an important modulatory role in the induction of opioid analgesia and opioid addiction. Because neurotrophins universally bind the p75 neurotrophin receptor (p75NTR), we investigated whether the activity of this receptor is involved in the development of opioid analgesic tolerance and physical dependence. We found that in both the wild-type and p75NTR-/- mice an acute systemic (i.p.) injection of morphine produced a maximal analgesic response as measured by the thermal tail-immersion test. Repeated injection of morphine over 5 days in wild-type mice resulted in a progressive decline of the analgesic effect and a concomitant loss of the agonist potency, reflecting development of morphine tolerance. However, the loss of morphine analgesia was not observed in p75NTR-/- mice. In the second part of this study, mice were given escalating doses of systemic (i.p.) morphine over 5 days and subsequently challenged with the opioid receptor antagonist naloxone. This challenge precipitated a robust withdrawal syndrome that was comparable in wild-type mice and p75NTR-/- mice. The findings suggest that p75NTR activity plays a critical role in the development of opioid analgesic tolerance but not in the induction or the expression of opioid physical dependence.

Sustained exposure to opioid agonists such as morphine increases levels of calcitonin gene-related peptide (CGRP) in the spinal dorsal horn, a response implicated in the development of opioid tolerance and physical dependence. Recent evidence suggests that both the opioid-induced increase in CGRP and the development of opioid physical dependence are suppressed by blockade of spinal cannabinoid (CB 1 )-receptors. The present study examined whether CB 1 -receptor activity also has a role in the development of opioid tolerance. In rats implanted with spinal catheters, repeated acute injections of morphine (15 μg) delivered over 4 h resulted in a rapid decline of thermal and mechanical antinociception and a significant loss of analgesic potency, reflecting development of acute opioid tolerance. In another set of experiments, chronic administration of spinal morphine (15 μg) once daily for 5 days produced a similar loss of analgesic effect and a marked increase in CGRP-immunoreactivity in the superficial laminae of the dorsal horn. Consistent with the in vivo findings, primary cultures of adult dorsal root ganglion (DRG) neurons exposed to morphine for 5 days showed a significant increase in the number of CGRP-immunoreactive neurons. Co-administration of acute or chronic morphine with a CB 1 -receptor antagonist/inverse agonist, 1-(2,4-dichlorophenyl)-5-(4-iodophenyl)-4-methyl-N-1-piperidinyl-1H-pyrazole-3-carboxamide (AM-251), inhibited the development of both acute and chronic analgesic tolerance. In animals already exhibiting tolerance to morphine, intervention with AM-251 restored morphine analgesic potency. Co-administration with AM-251 attenuated the morphine-induced increase in CGRP-immunoreactivity in the spinal cord and in DRG cultured neurons. Collectively, the results of this study suggest that activity of endocannabinoids, mediated via CB 1 -receptors, contributes to both the development and maintenance of opioid tolerance by influencing the opioid-induced increase in spinal CGRP.

Treating pain is one of the most difficult challenges in medicine and a key facet of disease management. The isolation of morphine by Friedrich Sertürner in 1804 added an essential pharmacological tool in the treatment of pain and spawned the discovery of a new class of drugs known collectively as opioid analgesics. Revered for their potent pain-relieving effects, even Morpheus the god of dreams could not have dreamt that his opium tincture would be both a gift and a burden to humankind. To date, morphine and other opioids remain essential analgesics for alleviating pain. However, their use is plagued by major side effects, such as analgesic tolerance (diminished pain-relieving effects), hyperalgesia (increased pain sensitivity), and drug dependence. This review highlights recent advances in understanding the key causes of these adverse effects and explores the effect of chronic pain on opioid reward.SIGNIFICANCE STATEMENTChronic pain is pervasive and afflicts >100 million Americans. Treating pain in these individuals is notoriously difficult and often requires opioids, one of the most powerful and effective classes of drugs used for controlling pain. However, their use is plagued by major side effects, such as a loss of pain-relieving effects (analgesic tolerance), paradoxical pain (hyperalgesia), and addiction. Despite the potential side effects, opioids remain the pharmacological cornerstone of modern pain therapy. This review highlights recent breakthroughs in understanding the key causes of these adverse effects and explores the cellular control of opioid systems in reward and aversion. The findings will challenge traditional views of the good, the bad, and the ugly of opioids.

Emerging evidence indicates that microglia play a critical role in the pathogenesis of neuropathic pain, a debilitating chronic pain condition that can occur after peripheral nerve damage caused by disease, infection, or physical injury. Microglia are immunocompetent cells of the central nervous system and express various ionotropic P2X and metabotropic P2Y purinoceptors. After injury to a peripheral nerve, microglia in the spinal cord become activated and upregulate expression of the P2X4 receptor. Recent findings suggest that activation of P2X4 receptors evokes release of brain-derived neurotrophic factor from microglia and that this mediates microglia-neuron signaling leading to pain hypersensitivity. Thus, P2X4 receptors and the intracellular signaling mediators in microglia are promising therapeutic targets for the development of novel pharmacological agents in the management of neuropathic pain.

Chronic opioid use in the management of pain is limited by development of analgesic tolerance and physical dependence. The mechanisms underlying tolerance-dependence are not entirely clear, however, recent evidence suggests that spinal adaptations leading to increased activity of sensory neuropeptides (calcitonin gene-related peptide (CGRP), substance P) and their downstream signaling messengers derived from metabolism of arachidonic acid: prostaglandins (PG), lipoxygenase (LOX) metabolites, and endocannabinoids, plays an important role in this phenomenon. In this communication we review the evidence implicating these factors in the induction and expression of opioid tolerance and physical dependence at the spinal level.

1. This study investigated the role of spinal lipoxygenase (LOX) products in the induction and expression of opioid physical dependence using behavioural assessment of withdrawal and immunostaining for CGRP and Fos protein expression in the spinal cord. 2. Administration of escalating doses (5-50 mg kg-1; i.p.) of morphine for 5 days markedly elevated CGRP-like immunoreactivity in the dorsal horn of the rat spinal cord. Naloxone (2 mg kg-1; i.p.) challenge precipitated a robust withdrawal syndrome that depleted CGRP-like immunoreactivity and increased the number of Fos-like immunoreactive neurons in the dorsal horn. 3. Intrathecal administration of NDGA (10, 20 microg), a nonselective LOX inhibitor, AA-861 (1.5, 3 microg), a 5-LOX selective inhibitor, or baicalein (1.4, 2.8 microg), a 12-LOX selective inhibitor, concurrently with systemic morphine for 5 days or as a single injection immediately preceding naloxone challenge, blocked the depletion of CGRP-like immunoreactivity, prevented increase in the number of Fos-like immunoreactive neurons in the dorsal horn, and significantly attenuated the morphine withdrawal syndrome. 4. The results of this study suggest that activity of LOX products, at the spinal level, contributes to the expression of opioid physical dependence, and that this activity may be expressed through increased sensory neuropeptide release.

This study examined the role of spinal calcitonin gene-related peptide (CGRP), substance P, and prostaglandins in the development and expression of opioid physical dependence. Administration of escalating doses (5 – 100 mg kg−1, i.p.) of morphine for 7 days markedly elevated CGRP and substance P− immunoreactivity in the dorsal horn of the rat spinal cord. Naloxone (2 mg kg−1, i.p.) challenge decreased both CGRP and substance P immunoreactivity and precipitated a robust withdrawal syndrome. Acute intrathecal pre-treatment with a CGRP receptor antagonist, CGRP8 – 37 (4, 8 μg), a substance P receptor antagonist, SR 140333 (1.4, 2.8 μg), a cyclo-oxygenase (COX) inhibitor, ketorolac (30, 45 μg), and COX-2 selective inhibitors, DuP 697 (10, 30 μg) and nimesulide (30 μg), 30 min before naloxone challenge, partially attenuated the symptoms of morphine withdrawal. CGRP8 – 37 (8 μg), but no other agents, inhibited the decrease in CGRP immunoreactivity. Chronic intrathecal treatment with CGRP8 – 37 (4, 8 μg), SR 140333 (1.4 μg), ketorolac (15, 30 μg), DuP 697 (10, 30 μg), and nimesulide (30 μg), delivered with daily morphine injection significantly attenuated both the symptoms of withdrawal and the decrease in CGRP but not substance P immunoreactivity. The results of this study suggest that activation of CGRP and substance P receptors, at the spinal level, contributes to the induction and expression of opioid physical dependence and that this activity may be partially expressed through the intermediary actions of prostaglandins. Keywords: Opioid physical dependence, opioid withdrawal, prostaglandins, neuropeptides Introduction Morphine and related opioid drugs are widely used as analgesics in the management of severe pain. However, repeated administration of these agents leads to the development of opioid tolerance and physical dependence, factors that limit their therapeutic usefulness. Opioid tolerance manifests as a loss in analgesic potency, whereas physical dependence is indicated by the onset of a characteristic withdrawal syndrome precipitated by cessation of opioid drug treatment or a challenge with an opioid receptor antagonist such as naloxone. The mechanisms underlying the development and expression of opioid tolerance-dependence are not completely understood, however recent evidence suggests that increased activity of spinal excitatory amino acid (L-glutamate/L-aspartate) and neuropeptide transmitters (calcitonin gene-related peptide (CGRP), substance P) may play an important role in these phenomena (see Jhamandas et al., 2000). Considerable attention has been focused on the role of excitatory amino acids and the activity of the NMDA receptor (an excitatory amino acid receptor sub-type) in the development of opioid tolerance and physical dependence. Several studies have shown that blockade of spinal NMDA receptors effectively inhibits development of tolerance to the antinociceptive actions of morphine (Trujillo & Akil, 1991; Mao et al., 1994; Dunbar & Yaksh, 1996; Shimoyama et al., 1996). Additionally, blockade of this receptor also inhibits the expression of naloxone-precipitated morphine withdrawal (Trujillo & Akil, 1991; Dunbar & Yaksh, 1996; Dambisya & Lee, 1996). These and other findings have led to the proposal that chronic exposure to opioid drugs induces a latent increase in NMDA receptor activity that physiologically antagonizes the inhibitory effects of these agents and compromises the analgesic response (Mao et al., 1994; Mao, 1999). Cessation of drug treatment unmasks this increased NMDA receptor activity and gives rise to the autonomic and behavioural hyperactivity that constitutes the opioid withdrawal syndrome. In addition to the excitatory amino acid activity, the activity of sensory neuropeptide transmitters also contributes to the genesis of the opioid tolerant-dependent state (Menard et al., 1995; 1996; Powell et al., 1999; 2000). In nociceptive primary afferents that terminate in the superficial laminae of the spinal dorsal horn, L-glutamate is co-localized with CGRP and substance P (Merighi et al., 1991). In recent studies, we reported that repeated daily intrathecal administration of morphine significantly increased CGRP and substance P immunoreactivity in the rat spinal cord (Menard et al., 1996; Powell et al., 2000) and in dorsal root ganglion neurons which gives rise to neuropeptide expressing primary afferent fibres (Ma et al., 2000). Co-treatment with a CGRP receptor antagonist consistently blocked this effect and prevented the development of morphine tolerance (Powell et al., 2000). Preliminary findings indicated that treatment with an NK-1 receptor antagonist can also inhibit and reverse spinal morphine tolerance (Powell et al., 2000). These findings suggest that the activity of spinal CGRP and substance P contribute to the induction and expression of opioid analgesic tolerance, however their role in the development of opioid physical dependence is relatively unknown. A recent study demonstrating that morphine withdrawal is attenuated in CGRP deficient transgenic mice supports the involvement of this neuropeptide in the genesis of opioid physical dependence (Salmon et al., 2001). Previous studies have also shown that blockade of the NK-1 receptor reduces the magnitude of opioid withdrawal associated contractions in isolated guinea-pig ileum (Johnston & Chahl, 1991) and inhibits some signs of morphine withdrawal in the rat (Buccafusco & Shuster, 1997; Maldonado et al., 1993). Thus, these studies suggest the involvement of CGRP and substance P in the development of opioid physical dependence. The present study examined this possibility by determining the changes in CGRP and substance P immunoreactivity that accompany precipitated morphine withdrawal, and by assessing the effects of spinally administered CGRP and NK-1 receptor antagonists on physiological and behavioural manifestations of the withdrawal syndrome. The mechanisms by which increased neuropeptide activity contributes to the development of opioid withdrawal are not known, but evidence from tolerance studies suggests an intermediary role of prostaglandins (Powell et al., 1999). Activation of neuropeptide and amino acid receptor activity in the spinal cord results in prostaglandin release (Malmberg & Yaksh, 1992; Hua et al., 1999; Marriott et al., 1991a, 1991b). Prostaglandins in turn act on terminals of primary afferents to further release CGRP, substance P, and glutamate, initiating a positive feedback loop (Vasko et al., 1994; Malmberg & Yaksh, 1992). Thus, to investigate the potential role of spinal prostaglandins in opioid withdrawal, we examined the effects of inhibitors of cyclo-oxygenase (COX), the enzyme catalysing prostaglandin synthesis, on the morphine withdrawal associated responses.

A major unresolved issue in treating pain is the paradoxical hyperalgesia produced by the gold-standard analgesic morphine and other opiates. We found that hyperalgesia-inducing treatment with morphine resulted in downregulation of the K(+)-Cl(-) co-transporter KCC2, impairing Cl(-) homeostasis in rat spinal lamina l neurons. Restoring the anion equilibrium potential reversed the morphine-induced hyperalgesia without affecting tolerance. The hyperalgesia was also reversed by ablating spinal microglia. Morphine hyperalgesia, but not tolerance, required μ opioid receptor-dependent expression of P2X4 receptors (P2X4Rs) in microglia and μ-independent gating of the release of brain-derived neurotrophic factor (BDNF) by P2X4Rs. Blocking BDNF-TrkB signaling preserved Cl(-) homeostasis and reversed the hyperalgesia. Gene-targeted mice in which Bdnf was deleted from microglia did not develop hyperalgesia to morphine. However, neither morphine antinociception nor tolerance was affected in these mice. Our findings dissociate morphine-induced hyperalgesia from tolerance and suggest the microglia-to-neuron P2X4-BDNF-KCC2 pathway as a therapeutic target for preventing hyperalgesia without affecting morphine analgesia.