Harmonized cross-species cell atlases of trigeminal and dorsal root ganglia.

Sensory neurons in the dorsal root ganglion (DRG) and trigeminal ganglion (TG) are specialized to detect and transduce diverse environmental stimuli to the central nervous system. Single-cell RNA sequencing has provided insights into the diversity of sensory ganglia cell types in rodents, nonhuman primates, and humans, but it remains difficult to compare cell types across studies and species. We thus constructed harmonized atlases of the DRG and TG that describe and facilitate comparison of 18 neuronal and 11 non-neuronal cell types across six species and 31 datasets. We then performed single-cell/nucleus RNA sequencing of DRG from both human and the highly regenerative axolotl and found that the harmonized atlas also improves cell type annotation, particularly of sparse neuronal subtypes. We observed that the transcriptomes of sensory neuron subtypes are broadly similar across vertebrates, but the expression of functionally important neuropeptides and channels can vary notably. The resources presented here can guide future studies in comparative transcriptomics, simplify cell-type nomenclature differences across studies, and help prioritize targets for future analgesic development.

Bradykinin receptor expression and bradykinin-mediated sensitization of human sensory neurons.

ABSTRACT: Bradykinin is a peptide implicated in inflammatory pain in both humans and rodents. In rodent sensory neurons, activation of B1 and B2 bradykinin receptors induces neuronal hyperexcitability. Recent evidence suggests that human and rodent dorsal root ganglia (DRG), which contain the cell bodies of sensory neurons, differ in the expression and function of key GPCRs and ion channels; whether bradykinin receptor expression and function are conserved across species has not been studied in depth. In this study, we used human DRG tissue from organ donors to provide a detailed characterization of bradykinin receptor expression and bradykinin-induced changes in the excitability of human sensory neurons. We found that B2 and, to a lesser extent, B1 receptors are expressed by human DRG neurons and satellite glial cells. B2 receptors were enriched in the nociceptor subpopulation. Using patch-clamp electrophysiology, we found that acute bradykinin increases the excitability of human sensory neurons, whereas prolonged exposure to bradykinin decreases neuronal excitability in a subpopulation of human DRG neurons. Finally, our analyses suggest that donor's history of chronic pain and age may be predictors of higher B1 receptor expression in human DRG neurons. Together, these results indicate that acute bradykinin-induced hyperexcitability, first identified in rodents, is conserved in humans and provide further evidence supporting bradykinin signaling as a potential therapeutic target for treating pain in humans.

Impact of Δ9-Tetrahydrocannabinol and oxycodone co-administration on measures of antinociception, dependence, circadian activity, and reward in mice.

Oxycodone is commonly prescribed for moderate to severe pain disorders. While efficacious, long-term use can result in tolerance, physical dependence, and the development of opioid use disorder. Cannabis and its derivatives such as Δ9-Tetrahydrocannabinol (Δ9-THC) have been reported to enhance oxycodone analgesia in animal models and in humans. However, it remains unclear if Δ9-THC may facilitate unwanted aspects of oxycodone intake, such as tolerance, dependence, and reward at analgesic doses. This study sought to evaluate the impact of co-administration of Δ9-THC and oxycodone across behavioral measures related to antinociception, dependence, circadian activity, and reward in both male and female mice. Oxycodone and Δ9-THC produced dose-dependent antinociceptive effects in the hotplate assay that were similar between sexes. Repeated treatment (twice daily for 5 days) resulted in antinociceptive tolerance. Combination treatment of oxycodone and Δ9-THC produced a greater antinociceptive effect than either administered alone, and delayed the development of antinociceptive tolerance. Repeated treatment with oxycodone produced physical dependence and alterations in circadian activity, neither of which were exacerbated by co-treatment with Δ9-THC. Combination treatment of oxycodone and Δ9-THC produced CPP when co-administered at doses that did not produce preference when administered alone. These data indicate that Δ9-THC may facilitate oxycodone-induced antinociception without augmenting certain unwanted features of opioid intake (e.g. dependence, circadian rhythm alterations). However, our findings also indicate that Δ9-THC may facilitate rewarding properties of oxycodone at therapeutically relevant doses which warrant consideration when evaluating this combination for its potential therapeutic utility.

Intrinsic Homeostatic Plasticity in Mouse and Human Sensory Neurons.

In response to changes in activity induced by environmental cues, neurons in the central nervous system undergo homeostatic plasticity to sustain overall network function during abrupt changes in synaptic strengths. Homeostatic plasticity involves changes in synaptic scaling and regulation of intrinsic excitability. Increases in spontaneous firing and excitability of sensory neurons are evident in some forms of chronic pain in animal models and human patients. However, whether mechanisms of homeostatic plasticity are engaged in sensory neurons under normal conditions or altered after chronic pain is unknown. Here, we showed that sustained depolarization induced by 30mM KCl induces a compensatory decrease in the excitability in mouse and human sensory neurons. Moreover, voltage-gated sodium currents are robustly reduced in mouse sensory neurons contributing to the overall decrease in neuronal excitability. Decreased efficacy of these homeostatic mechanisms could potentially contribute to the development of the pathophysiology of chronic pain.

Harmonized cross-species cell atlases of trigeminal and dorsal root ganglia.

Peripheral sensory neurons in the dorsal root ganglion (DRG) and trigeminal ganglion (TG) are specialized to detect and transduce diverse environmental stimuli including touch, temperature, and pain to the central nervous system. Recent advances in single-cell RNA-sequencing (scRNA-seq) have provided new insights into the diversity of sensory ganglia cell types in rodents, non-human primates, and humans, but it remains difficult to compare transcriptomically defined cell types across studies and species. Here, we built cross-species harmonized atlases of DRG and TG cell types that describe 18 neuronal and 11 non-neuronal cell types across 6 species and 19 studies. We then demonstrate the utility of this harmonized reference atlas by using it to annotate newly profiled DRG nuclei/cells from both human and the highly regenerative axolotl. We observe that the transcriptomic profiles of sensory neuron subtypes are broadly similar across vertebrates, but the expression of functionally important neuropeptides and channels can vary notably. The new resources and data presented here can guide future studies in comparative transcriptomics, simplify cell type nomenclature differences across studies, and help prioritize targets for future pain therapy development.

Bradykinin receptor expression and bradykinin-mediated sensitization of human sensory neurons.

Bradykinin is a peptide implicated in inflammatory pain in both humans and rodents. In rodent sensory neurons, activation of B1 and B2 bradykinin receptors induces neuronal hyperexcitability. Recent evidence suggests that human and rodent dorsal root ganglia (DRG), which contain the cell bodies of sensory neurons, differ in the expression and function of key GPCRs and ion channels; whether BK receptor expression and function are conserved across species has not been studied in depth. In this study, we used human DRG tissue from organ donors to provide a detailed characterization of bradykinin receptor expression and bradykinin-induced changes in the excitability of human sensory neurons. We found that B2 and, to a lesser extent, B1 receptors are expressed by human DRG neurons and satellite glial cells. B2 receptors were enriched in the nociceptor subpopulation. Using patch-clamp electrophysiology, we found that acute bradykinin increases the excitability of human sensory neurons, while prolonged exposure to bradykinin decreases neuronal excitability in a subpopulation of human DRG neurons. Finally, our analyses suggest that donor’s history of chronic pain and age may be predictors of higher B1 receptor expression in human DRG neurons. Together, these results indicate that acute BK-induced hyperexcitability, first identified in rodents, is conserved in humans and provide further evidence supporting BK signaling as a potential therapeutic target for treating pain in humans.

Wireless, Battery-Free Implants for Electrochemical Catecholamine Sensing and Optogenetic Stimulation.

Neurotransmitters and neuromodulators mediate communication between neurons and other cell types; knowledge of release dynamics is critical to understanding their physiological role in normal and pathological brain function. Investigation into transient neurotransmitter dynamics has largely been hindered due to electrical and material requirements for electrochemical stimulation and recording. Current systems require complex electronics for biasing and amplification and rely on materials that offer limited sensor selectivity and sensitivity. These restrictions result in bulky, tethered, or battery-powered systems impacting behavior and that require constant care of subjects. To overcome these challenges, we demonstrate a fully implantable, wireless, and battery-free platform that enables optogenetic stimulation and electrochemical recording of catecholamine dynamics in real time. The device is nearly 1/10th the size of previously reported examples and includes a probe that relies on a multilayer electrode architecture featuring a microscale light emitting diode (µ-LED) and a carbon nanotube (CNT)-based sensor with sensitivities among the highest recorded in the literature (1264.1 nA µM-1 cm-2). High sensitivity of the probe combined with a center tapped antenna design enables the realization of miniaturized, low power circuits suitable for subdermal implantation even in small animal models such as mice. A series of in vitro and in vivo experiments highlight the sensitivity and selectivity of the platform and demonstrate its capabilities in freely moving, untethered subjects. Specifically, a demonstration of changes in dopamine concentration after optogenetic stimulation of the nucleus accumbens and real-time readout of dopamine levels after opioid and naloxone exposure in freely behaving subjects highlight the experimental paradigms enabled by the platform.

Oral oxycodone self-administration leads to features of opioid misuse in male and female mice.

Use of prescription opioids, particularly oxycodone, is an initiating factor driving the current opioid epidemic. There are several challenges with modelling oxycodone abuse. First, prescription opioids including oxycodone are orally self-administered and have different pharmacokinetics and dynamics than morphine or fentanyl, which have been more commonly used in rodent research. This oral route of administration determines the pharmacokinetic profile, which then influences the establishment of drug-reinforcement associations in animals. Moreover, the pattern of intake and the environment in which addictive drugs are self-administered are critical determinants of the levels of drug intake, of behavioural sensitization and of propensity to relapse behaviour. These are all important considerations when modelling prescription opioid use, which is characterized by continuous drug access in familiar environments. Thus, to model features of prescription opioid use and the transition to abuse, we designed an oral, homecage-based oxycodone self-administration paradigm. Mice voluntarily self-administer oxycodone in this paradigm without any taste modification such as sweeteners, and the majority exhibit preference for oxycodone, escalation of intake, physical signs of dependence and reinstatement of seeking after withdrawal. In addition, a subset of animals demonstrate drug taking that is resistant to aversive consequences. This model is therefore translationally relevant and useful for studying the neurobiological substrates of prescription opioid abuse.

The cannabinoid agonist CB-13 produces peripherally mediated analgesia in mice but elicits tolerance and signs of central nervous system activity with repeated dosing.

ABSTRACT: Activation of cannabinoid receptor type 1 (CB 1 ) produces analgesia in a variety of preclinical models of pain; however, engagement of central CB 1 receptors is accompanied by unwanted side effects, such as psychoactivity, tolerance, and dependence. Therefore, some efforts to develop novel analgesics have focused on targeting peripheral CB 1 receptors to circumvent central CB 1 -related side effects. In the present study, we evaluated the effects of acute and repeated dosing with the peripherally selective CB 1 -preferring agonist CB-13 on nociception and central CB 1 -related phenotypes in a model of inflammatory pain in mice. We also evaluated cellular mechanisms underlying CB-13-induced antinociception in vitro using cultured mouse dorsal root ganglion neurons. CB-13 reduced inflammation-induced mechanical allodynia in male and female mice in a peripheral CB 1 -receptor-dependent manner and relieved inflammatory thermal hyperalgesia. In cultured mouse dorsal root ganglion neurons, CB-13 reduced TRPV1 sensitization and neuronal hyperexcitability induced by the inflammatory mediator prostaglandin E 2 , providing potential mechanistic explanations for the analgesic actions of peripheral CB 1 receptor activation. With acute dosing, phenotypes associated with central CB 1 receptor activation occurred only at a dose of CB-13 approximately 10-fold the ED 50 for reducing allodynia. Strikingly, repeated dosing resulted in both analgesic tolerance and CB 1 receptor dependence, even at a dose that did not produce central CB 1 -receptor-mediated phenotypes on acute dosing. This suggests that repeated CB-13 dosing leads to increased CNS exposure and unwanted engagement of central CB 1 receptors. Thus, caution is warranted regarding therapeutic use of CB-13 with the goal of avoiding CNS side effects. Nonetheless, the clear analgesic effect of acute peripheral CB 1 receptor activation suggests that peripherally restricted cannabinoids are a viable target for novel analgesic development.

The cannabinoid CB2 receptor agonist LY2828360 synergizes with morphine to suppress neuropathic nociception and attenuates morphine reward and physical dependence.

The opioid crisis has underscored the urgent need to identify safe and effective therapeutic strategies to overcome opioid-induced liabilities. We recently reported that LY2828360, a slowly signaling G protein-biased cannabinoid CB2 receptor agonist, suppresses neuropathic nociception and attenuates the development of tolerance to the opioid analgesic morphine in paclitaxel-treated mice. Whether beneficial effects of LY2828360 are dependent upon the presence of a pathological pain state are unknown and its impact on unwanted opioid-induced side-effects have never been investigated. Here, we asked whether LY2828360 would produce synergistic anti-allodynic effects with morphine in a paclitaxel model of chemotherapy-induced neuropathic pain and characterized its impact on opioid-induced reward and other unwanted side-effects associated with chronic opioid administration. Isobolographic analysis revealed that combinations of LY2828360 and morphine produced synergistic anti-allodynic effects in suppressing paclitaxel-induced mechanical allodynia. In wildtype (WT) mice, LY2828360 blocked morphine-induced reward in a conditioned place preference assay without producing reward or aversion when administered alone. The LY2828360-induced attenuation of morphine-induced reward was absent in CB2 knockout (CB2KO) mice. In the absence of a neuropathic pain state, LY2828360 partially attenuated naloxone-precipitated opioid withdrawal in morphine-dependent WT mice, and this withdrawal was itself markedly exacerbated in CB2KO mice. Moreover, LY2828360 did not reliably alter morphine-induced slowing of colonic transit or attenuate tolerance to morphine antinociceptive efficacy in the hot plate test of acute nociception. Our results suggest that cannabinoid CB2 receptor activation enhances the therapeutic properties of opioids while attenuating unwanted side-effects such as reward and dependence that occur with sustained opioid treatment.

Positive Allosteric Modulation of CB1 Cannabinoid Receptor Signaling Enhances Morphine Antinociception and Attenuates Morphine Tolerance Without Enhancing Morphine- Induced Dependence or Reward.

Opioid analgesics represent a critical treatment for chronic pain in the analgesic ladder of the World Health Organization. However, their use can result in a number of unwanted side-effects including incomplete efficacy, constipation, physical dependence, and overdose liability. Cannabinoids enhance the pain-relieving effects of opioids in preclinical studies and dampen unwanted side-effects resulting from excessive opioid intake. We recently reported that a CB1 positive allosteric modulator (PAM) exhibits antinociceptive efficacy in models of pathological pain and lacks the adverse side effects of direct CB1 receptor activation. In the present study, we evaluated whether a CB1 PAM would enhance morphine's therapeutic efficacy in an animal model of chemotherapy-induced neuropathic pain and characterized its impact on unwanted side-effects associated with chronic opioid administration. In paclitaxel-treated mice, both the CB1 PAM GAT211 and the opioid analgesic morphine reduced paclitaxel-induced behavioral hypersensitivities to mechanical and cold stimulation in a dose-dependent manner. Isobolographic analysis revealed that combinations of GAT211 and morphine resulted in anti-allodynic synergism. In paclitaxel-treated mice, a sub-threshold dose of GAT211 prevented the development of tolerance to the anti-allodynic effects of morphine over 20 days of once daily dosing. However, GAT211 did not reliably alter somatic withdrawal signs (i.e., jumps, paw tremors) in morphine-dependent neuropathic mice challenged with naloxone. In otherwise naïve mice, GAT211 also prolonged antinociceptive efficacy of morphine in the tail-flick test and reduced the overall right-ward shift in the ED50 for morphine to produce antinociception in the tail-flick test, consistent with attenuation of morphine tolerance. Pretreatment with GAT211 did not alter somatic signs of µ opioid receptor dependence in mice rendered dependent upon morphine via subcutaneous implantation of a morphine pellet. Moreover, GAT211 did not reliably alter µ-opioid receptor-mediated reward as measured by conditioned place preference to morphine. Our results suggest that a CB1 PAM may be beneficial in enhancing and prolonging the therapeutic properties of opioids while potentially sparing unwanted side-effects (e.g., tolerance) that occur with repeated opioid treatment.

Voluntary exercise reduces both chemotherapy-induced neuropathic nociception and deficits in hippocampal cellular proliferation in a mouse model of paclitaxel-induced peripheral neuropathy.

Chemotherapy-induced peripheral neuropathy (CIPN) is a common dose-limiting side-effect of all major chemotherapeutic agents. Here, we explored efficacy of voluntary exercise as a nonpharmacological strategy for suppressing two distinct adverse side effects of chemotherapy treatment. We evaluated whether voluntary running would suppress both neuropathic pain and deficits in hippocampal cell proliferation in a mouse model of CIPN induced by the taxane chemotherapeutic agent paclitaxel. Mice were given free access to running wheels or were housed without running wheels during one of three different intervention phases: 1) during the onset (i.e. development phase) of paclitaxel-induced neuropathy, 2) prior to dosing with paclitaxel or its vehicle, or 3) following the establishment (i.e. maintenance phase) of paclitaxel-induced neuropathy. Paclitaxel treatment did not alter running wheel behavior relative to vehicle-treated animals in any study. Animals that engaged in voluntary running during the development phase of paclitaxel-induced neuropathy failed to display mechanical or cold hypersensitivities relative to sedentary control animals that did not have access to running wheels. A prior history of voluntary running delayed the onset of, but did not fully prevent, development of paclitaxel-induced neuropathic pain behavior. Voluntary running reduced already established mechanical and cold allodynia induced by paclitaxel. Importantly, voluntary running did not alter mechanical or cold responsivity in vehicle-treated animals, suggesting that the observed antinociceptive effect of exercise was dependent upon the presence of the pathological pain state. In the same animals evaluated for nociceptive responding, paclitaxel also reduced cellular proliferation but not cellular survival in the dentate gyrus of the hippocampus, as measured by immunohistochemistry for Ki67 and BrdU expression, respectively. Voluntary running abrogated paclitaxel-induced reductions in cellular proliferation to levels observed in vehicle-treated mice and also increased BrdU expression levels irrespective of chemotherapy treatment. Our studies support the hypothesis that voluntary exercise may be beneficial in suppressing both neuropathic pain and markers of hippocampal cellular function that are impacted by toxic challenge with chemotherapeutic agents.

Brain permeant and impermeant inhibitors of fatty-acid amide hydrolase suppress the development and maintenance of paclitaxel-induced neuropathic pain without producing tolerance or physical dependence in vivo and synergize with paclitaxel to reduce tumor cell line viability in vitro.

Activation of cannabinoid CB1 receptors suppresses pathological pain but also produces unwanted side effects, including tolerance and physical dependence. Inhibition of fatty-acid amide hydrolase (FAAH), the major enzyme catalyzing the degradation of anandamide (AEA), an endocannabinoid, and other fatty-acid amides, suppresses pain without unwanted side effects typical of direct CB1 agonists. However, FAAH inhibitors have failed to show efficacy in several clinical trials suggesting that the right partnership of FAAH inhibition and pathology has yet to be identified. We compared efficacy of chronic treatments with a centrally penetrant FAAH inhibitor (URB597), a peripherally restricted FAAH inhibitor (URB937) and an orthosteric pan-cannabinoid agonist (WIN55,212-2) in suppressing neuropathic pain induced by the chemotherapeutic agent paclitaxel. Each FAAH inhibitor suppressed the development of paclitaxel-induced neuropathic pain and reduced the maintenance of already established allodynia with sustained efficacy. Tolerance developed to the anti-allodynic efficacy of WIN55,212-2, but not to that of URB597 or URB937, in each dosing paradigm. Challenge with the CB1 antagonist rimonabant precipitated CB1-dependent withdrawal in paclitaxel-treated mice receiving WIN55,212-2 but not URB597 or URB937. When dosing with either URB597 or URB937 was restricted to the development of neuropathy, paclitaxel-induced allodynia emerged following termination of drug delivery. These observations suggest that both FAAH inhibitors were anti-allodynic rather than curative. Moreover, neither URB597 nor URB937 impeded the ability of paclitaxel to reduce breast (4T1) or ovarian (HeyA8) tumor cell line viability. In fact, URB597 and URB937 alone reduced 4T1 tumor cell line viability, albeit with low potency, and the dose matrix of each combination with paclitaxel was synergistic in reducing 4T1 and HeyA8 tumor cell line viability according to Bliss, Highest Single Agent (HSA) and Loewe additivity models. Both FAAH inhibitors synergized with paclitaxel to reduce 4T1 and HeyA8 tumor cell line viability without reducing viability of non-tumor HEK293 cells. Neither FAAH inhibitor reduced viability of non-tumor HEK293 cells in either the presence or absence of paclitaxel, suggesting that nonspecific cytotoxic effects were not produced by the same treatments. Our results suggest that FAAH inhibitors reduce paclitaxel-induced allodynia without the occurrence of CB1-dependence in vivo and may, in fact, enhance the anti-tumor actions of paclitaxel in vitro.

Brain-Permeant and -Impermeant Inhibitors of Fatty Acid Amide Hydrolase Synergize with the Opioid Analgesic Morphine to Suppress Chemotherapy-Induced Neuropathic Nociception Without Enhancing Effects of Morphine on Gastrointestinal Transit.

Opioid-based therapies remain a mainstay for chronic pain management, but unwanted side effects limit therapeutic use. We compared efficacies of brain-permeant and -impermeant inhibitors of fatty acid amide hydrolase (FAAH) in suppressing neuropathic pain induced by the chemotherapeutic agent paclitaxel. Paclitaxel produced mechanical and cold allodynia without altering nestlet shredding or marble burying behaviors. We compared FAAH inhibitors that differ in their ability to penetrate the central nervous system for antiallodynic efficacy, pharmacological specificity, and synergism with the opioid analgesic morphine. (3'-(aminocarbonyl)[1,1'-biphenyl]- 3-yl)-cyclohexylcarbamate (URB597), a brain-permeant FAAH inhibitor, attenuated paclitaxel-induced allodynia via cannabinoid receptor 1 (CB1) and cannabinoid receptor 2 (CB2) mechanisms. URB937, a brain-impermeant FAAH inhibitor, suppressed paclitaxel-induced allodynia through a CB1 mechanism only. 5-[4-(4-cyano-1-butyn-1-yl)phenyl]-1-(2,4-dichlorophenyl)-N-(1,1-dioxido-4-thiomorpholinyl)-4-methyl-1H-pyrazole-3-carboxamide (AM6545), a peripherally restricted CB1 antagonist, fully reversed the antiallodynic efficacy of N-cyclohexyl-carbamic acid, 3'-(aminocarbonyl)-6-hydroxy[1,1'- biphenyl]-3-yl ester (URB937) but only partially reversed that of URB597. Thus, URB937 suppressed paclitaxel-induced allodynia through a mechanism that was dependent upon peripheral CB1 receptor activation only. Antiallodynic effects of both FAAH inhibitors were reversed by N-(piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (AM251). Antiallodynic effects of URB597, but not URB937, were reversed by 6-iodo-2-methyl-1-[2-(4-morpholinyl)ethyl]-1H-indol-3-yl](4-methoxyphenyl)methanone (AM630). Isobolographic analysis revealed synergistic interactions between morphine and either URB597 or URB937 in reducing paclitaxel-induced allodynia. A leftward shift in the dose-response curve of morphine antinociception was observed when morphine was coadministered with either URB597 or URB937, consistent with morphine sparing. However, neither URB937 nor URB597 enhanced morphine-induced deficits in colonic transit. Thus, our findings suggest that FAAH inhibition may represent a therapeutic avenue to reduce the overall amount of opioid needed for treating neuropathic pain with potential to reduce unwanted side effects that accompany opioid administration.

Positive Allosteric Modulation of Cannabinoid Receptor Type 1 Suppresses Pathological Pain Without Producing Tolerance or Dependence.

BACKGROUND: Activation of cannabinoid CB1 receptors suppresses pathological pain but also produces unwanted central side effects. We hypothesized that a positive allosteric modulator of CB1 signaling would suppress inflammatory and neuropathic pain without producing cannabimimetic effects or physical dependence. We also asked whether a CB1 positive allosteric modulator would synergize with inhibitors of endocannabinoid deactivation and/or an orthosteric cannabinoid agonist. METHODS: GAT211, a novel CB1 positive allosteric modulator, was evaluated for antinociceptive efficacy and tolerance in models of neuropathic and/or inflammatory pain. Cardinal signs of direct CB1-receptor activation were evaluated together with the propensity to induce reward or aversion and physical dependence. Comparisons were made with inhibitors of endocannabinoid deactivation (JZL184, URB597) or an orthosteric cannabinoid agonist (WIN55,212-2). All studies used 4 to 11 subjects per group. RESULTS: GAT211 suppressed allodynia induced by complete Freund's adjuvant and the chemotherapeutic agent paclitaxel in wild-type but not CB1 knockout mice. GAT211 did not impede paclitaxel-induced tumor cell line toxicity. GAT211 did not produce cardinal signs of direct CB1-receptor activation in the presence or absence of pathological pain. GAT211 produced synergistic antiallodynic effects with fatty acid amide hydrolase and monoacylglycerol lipase inhibitors in paclitaxel-treated mice. Therapeutic efficacy was preserved over 19 days of chronic dosing with GAT211, but it was not preserved with the monoacylglycerol lipase inhibitor JZL184. The CB1 antagonist rimonabant precipitated withdrawal in mice treated chronically with WIN55,212-2 but not in mice treated with GAT211. GAT211 did not induce conditioned place preference or aversion. CONCLUSIONS: Positive allosteric modulation of CB1-receptor signaling shows promise as a safe and effective analgesic strategy that lacks tolerance, dependence, and abuse liability.

The chemotherapeutic agent paclitaxel selectively impairs learning while sparing source memory and spatial memory.

Chemotherapeutic agents are widely used to treat patients with systemic cancer. The efficacy of these therapies is undermined by their adverse side-effect profiles such as cognitive deficits that have a negative impact on the quality of life of cancer survivors. Cognitive side effects occur across a variety of domains, including memory, executive function, and processing speed. Such impairments are exacerbated under cognitive challenges and a subgroup of patients experience long-term impairments. Episodic memory in rats can be examined using a source memory task. In the current study, rats received paclitaxel, a taxane-derived chemotherapeutic agent, and learning and memory functioning was examined using the source memory task. Treatment with paclitaxel did not impair spatial and episodic memory, and paclitaxel treated rats were not more susceptible to cognitive challenges. Under conditions in which memory was not impaired, paclitaxel treatment impaired learning of new rules, documenting a decreased sensitivity to changes in experimental contingencies. These findings provide new information on the nature of cancer chemotherapy-induced cognitive impairments, particularly regarding the incongruent vulnerability of episodic memory and new learning following treatment with paclitaxel.

A pro-nociceptive phenotype unmasked in mice lacking fatty-acid amide hydrolase.

Fatty-acid amide hydrolase (FAAH) is the major enzyme responsible for degradation of anandamide, an endocannabinoid. Pharmacological inhibition or genetic deletion of FAAH (FAAH KO) produces antinociception in preclinical pain models that is largely attributed to anandamide-induced activation of cannabinoid receptors. However, FAAH metabolizes a wide range of structurally related, biologically active lipid signaling molecules whose functions remain largely unknown. Some of these endogenous lipids, including anandamide itself, may exert pro-nociceptive effects under certain conditions. In our study, FAAH KO mice exhibited a characteristic analgesic phenotype in the tail flick test and in both formalin and carrageenan models of inflammatory nociception. Nonetheless, intradermal injection of the transient receptor potential channel V1 (TRPV1) agonist capsaicin increased nocifensive behavior as well as mechanical and heat hypersensitivity in FAAH KO relative to wild-type mice. This pro-nociceptive phenotype was accompanied by increases in capsaicin-evoked Fos-like immunoreactive (FLI) cells in spinal dorsal horn regions implicated in nociceptive processing and was attenuated by CB1 (AM251) and TRPV1 (AMG9810) antagonists. When central sensitization was established, FAAH KO mice displayed elevated levels of anandamide, other fatty-acid amides, and endogenous TRPV1 agonists in both paw skin and lumbar spinal cord relative to wild-type mice. Capsaicin decreased spinal cord 2-AG levels and increased arachidonic acid and prostaglandin E2 levels in both spinal cord and paw skin irrespective of genotype. Our studies identify a previously unrecognized pro-nociceptive phenotype in FAAH KO mice that was unmasked by capsaicin challenge. The heightened nociceptive response was mediated by CB1 and TRPV1 receptors and accompanied by enhanced spinal neuronal activation. Moreover, genetic deletion of FAAH has a profound impact on the peripheral and central lipidome. Thus, genetic deletion of FAAH may predispose animals to increased sensitivity to certain types of pain. More work is necessary to determine whether such changes could explain the lack of efficacy of FAAH inhibitors in clinical trials.

Impact of Genetic Reduction of NMNAT2 on Chemotherapy-Induced Losses in Cell Viability In Vitro and Peripheral Neuropathy In Vivo.

Nicotinamide mononucleotide adenylyl transferases (NMNATs) are essential neuronal maintenance factors postulated to preserve neuronal function and protect against axonal degeneration in various neurodegenerative disease states. We used in vitro and in vivo approaches to assess the impact of NMNAT2 reduction on cellular and physiological functions induced by treatment with a vinca alkaloid (vincristine) and a taxane-based (paclitaxel) chemotherapeutic agent. NMNAT2 null (NMNAT2-/-) mutant mice die at birth and cannot be used to probe functions of NMNAT2 in adult animals. Nonetheless, primary cortical cultures derived from NMNAT2-/- embryos showed reduced cell viability in response to either vincristine or paclitaxel treatment whereas those derived from NMNAT2 heterozygous (NMNAT2+/-) mice were preferentially sensitive to vincristine-induced degeneration. Adult NMNAT2+/- mice, which survive to adulthood, exhibited a 50% reduction of NMNAT2 protein levels in dorsal root ganglia relative to wildtype (WT) mice with no change in levels of other NMNAT isoforms (NMNAT1 or NMNAT3), NMNAT enzyme activity (i.e. NAD/NADH levels) or microtubule associated protein-2 (MAP2) or neurofilament protein levels. We therefore compared the impact of NMNAT2 knockdown on the development and maintenance of chemotherapy-induced peripheral neuropathy induced by vincristine and paclitaxel treatment using NMNAT2+/- and WT mice. NMNAT2+/- did not differ from WT mice in either the development or maintenance of either mechanical or cold allodynia induced by either vincristine or paclitaxel treatment. Intradermal injection of capsaicin, the pungent ingredient in hot chili peppers, produced equivalent hypersensitivity in NMNAT2+/- and WT mice receiving vehicle in lieu of paclitaxel. Capsaicin-evoked hypersensitivity was enhanced by prior paclitaxel treatment but did not differ in either NMNAT2+/- or WT mice. Thus, capsaicin failed to unmask differences in nociceptive behaviors in either paclitaxel-treated or paclitaxel-untreated NMNAT2+/- and WT mice. Moreover, no differences in motor behavior were detected between genotypes in the rotarod test. Our studies do not preclude the possibility that complete knockout of NMNAT2 in a conditional knockout animal could unmask a role for NMNAT2 in protection against detrimental effects of chemotherapeutic treatment.