Acta Anaesthesiologica Taiwanica 51 (2013) 161e170

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Review Article

Targeting the cannabinoid system for pain relief? Lih-Chu Chiou 1, 2, 3, 4 *, Sherry Shu-Jung Hu 5, Yu-Cheng Ho 1 1

Graduate Institute of Pharmacology, College of Medicine, National Taiwan University, Taipei, Taiwan Department of Pharmacology, National Taiwan University, Taipei, Taiwan 3 Graduate Institute of Brain and Mind Sciences, College of Medicine, National Taiwan University, Taipei, Taiwan 4 Neurobiology and Cognitive Science Center, National Taiwan University, Taipei, Taiwan 5 Department of Psychology, National Cheng Kung University, Tainan, Taiwan 2

a r t i c l e i n f o

a b s t r a c t

Article history: Received 30 September 2013 Accepted 11 October 2013

Marijuana has been used to relieve pain for centuries, but its analgesic mechanism has only been understood during the past two decades. It is mainly mediated by its constituents, cannabinoids, through activating central cannabinoid 1 (CB1) receptors, as well as peripheral CB1 and CB2 receptors. CB2-selective agonists have the benefit of lacking CB1 receptor-mediated CNS side effects. Anandamide and 2arachidonoylglycerol (2-AG) are two intensively studied endogenous lipid ligands of cannabinoid receptors, termed endocannabinoids, which are synthesized on demand and rapidly degraded. Thus, inhibitors of their degradation enzymes, fatty acid amide hydrolase and monoacylglycerol lipase (MAGL), respectively, may be superior to direct cannabinoid receptor ligands as a promising strategy for pain relief. In addition to the antinociceptive properties of exogenous cannabinoids and endocannabinoids, involving their biosynthesis and degradation processes, we also review recent studies that revealed a novel analgesic mechanism, involving 2-AG in the periaqueductal gray (PAG), a midbrain region for initiating descending pain inhibition. It is initiated by Gq-protein-coupled receptor (GqPCR) activation of the phospholipase C (PLC)-diacylglycerol lipase (DAGL) enzymatic cascade, generating 2-AG that produces inhibition of GABAergic transmission (disinhibition) in the PAG, thereby leading to analgesia. This GqPCR-PLC-DAGL-2-AG retrograde disinhibition mechanism in the PAG can be initiated by activating type 5 metabotropic glutamate receptor (mGluR5), muscarinic acetylcholine (M1/M3), and orexin (OX1) receptors. mGluR5-mediated disinhibition can be initiated by glutamate transporter inhibitors, or indirectly by substance P, neurotensin, cholecystokinin, capsaicin, and AM404, the bioactive metabolite of acetaminophen in the brain. The putative role of 2-AG generated after activating the above neurotransmitter receptors in stress-induced analgesia is also discussed. Copyright Ó 2013, Taiwan Society of Anesthesiologists. Published by Elsevier Taiwan LLC. All rights reserved.

Key words: cannabinoids; endocannabinoids: anandamide; neuropeptides: orexin; pain; receptors: metabotropic glutamate

1. Cannabinoids Marijuana has been used for relieving pain for more than four centuries, but its analgesic mechanism was only understood during the past two decades, after cannabinoid receptors were discovered. The main active component of marijuana is D9-tetrahydrocannabinol (D9-THC),1 which exerts several pharmacological actions, including analgesia, immobilization, heightened sensory awareness, euphoria, hypothermia, impairment of short-term memory, and suppression of immune responses.2 Compounds mimicking the effects of D9-THC through activating cannabinoid receptors are termed cannabinoids with three categories. Phytocannabinoids are Conflicts of interest: The authors declare no competing financial interests. * Corresponding author. Department of Pharmacology, College of Medicine, National Taiwan University, Number 1, Jen-Ai Road, Section 1, Taipei 100, Taiwan. E-mail address: [email protected] (L.-C. Chiou).

the constituents isolated from marijuana.3 Endocannabinoids are endogenous ligands of cannabinoid receptors.4 Synthetic cannabinoids are compounds developed for potential medical uses, based on the concept that they would mimic the therapeutic effects of phytocannabinoids while having no psychoactivity. 2. CB1 and CB2 receptors The first cannabinoid receptor, CB1, was cloned by Matsuda et al5 from a rat brain cDNA library. Thereafter, Munro’s group6 cloned another cannabinoid receptor, CB2, from an HL-60 cell cDNA library. Both CB1 and CB2 receptors are seven-transmembrane G-proteincoupled receptors, mainly coupled to Gi/o proteins,7 and share 44% overall identity (68% identity for the transmembrane domains). CB1 receptors are enriched in the nervous systems and moderately expressed in peripheral tissues.5,8,9 In both rat and human

1875-4597/$ e see front matter Copyright Ó 2013, Taiwan Society of Anesthesiologists. Published by Elsevier Taiwan LLC. All rights reserved. http://dx.doi.org/10.1016/j.aat.2013.10.004

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brains, CB1 receptors are densely distributed in the frontal cerebral cortex, basal ganglia, cerebellum, hippocampus, hypothalamus, and anterior cingulate cortex, but rarely in the brainstem nuclei.10 The latter finding may account for the low toxicity of cannabinoids when given in accidental overdose. At the cellular level, CB1 receptors are mainly localized to axons and nerve terminals and are largely absent from neuronal somas or dendrites.11 This ultra-structural finding, suggesting a predominantly presynaptic localization of CB1 receptors, is consistent with the functional finding that activation of CB1 receptors inhibits calcium channels and activates potassium channels, leading to inhibition of neurotransmitter release.12,13 CB2 receptors are mainly distributed in immune cells.6,14 Although originally thought absent from the CNS, CB2 receptors were later found to be expressed in microglia, dorsal root ganglion, spinal cord, and sparsely in several brain regions, such as the cerebellum, cortex, and brainstem.15,16 Nevertheless, CB2 receptors in the CNS can be strongly induced in sensory neurons and spinal cord in neuropathic and inflammatory pain models, as well as in spinal microglia and macrophages in human postmortem spinal cord specimens of multiple sclerosis and amyotrophic lateral sclerosis.17 3. Endocannabinoids The finding of cannabinoid receptors in the brain motivated the search for their endogenous ligands to elucidate their functional roles. Indeed, several endogenous arachidonic acid derivatives have been identified to have cannabimimetic actions and are termed endocannabinoids; they include anandamide (N-arachidonoylethanolamine),18 2-arachidonoylglycerol (2-AG),19,20 noladin ether (2arachidonoylglyceryl ether),21 virodhamine (O-arachidonoylethanolamine),22 and N-arachidonoyldopamine.23 These endocannabinoids, like other lipid mediators, such as prostaglandins, are synthesized and then released locally on demand in activity-dependent and receptorregulated manners.24 Released endocannabinoids are rapidly inactivated by uptake and enzymatic degradation. Agents that interfere with the inactivation of endocannabinoids may provide a pharmacological means to modify cannabinoid-mediated functions.25,26 Anandamide and 2-AG are the two best studied endocannabinoids. Despite similar chemical structures, they have distinct pathways for biosynthesis and degradation.

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located postsynaptically, complimentary to the presynaptically located CB1 receptors, in many, but not all, brain regions.35e37 3.1.3. Anandamide uptake In addition to the degradation enzyme, a membrane transporter has been proposed to play a role in the uptake of anandamide. However, its existence remains controversial38,39 because several putative anandamide transporter inhibitors, such as AM404, VDM11, and LY218240, which enhance the effects of anandamide,40,41 also inhibit FAAH.42e44 3.1.4. Anandamide and type 1 transient receptor potential vanilloid channels Unlike 2-AG, which primarily acts at cannabinoid receptors, anandamide activates both cannabinoid receptors and type 1 transient receptor potential vanilloid (TRPV1) channels.45 The TRPV1 channel is a voltage-gated cation channel which is distributed in both the peripheral and the central nervous system. Capsaicin, the constituent of chili pepper, is a potent agonist. Anandamide and capsaicin have similar binding affinities in displacing [3H]-resiniferatoxin from TRPV1.46 Hence, activation of TRPV1 by anandamide may complicate the interpretation of the effects of experiments using FAAH or anandamide transporter inhibitors. 3.2. 2-AG 3.2.1. 2-AG synthesis 2-AG is a monoacylglycerol (MAG) and is mainly formed by the hydrolysis of diacylglycerol (DAG) through DAG lipases (DAGLs). DAG can be generated through postsynaptic depolarizationinduced Ca2þ influx and/or activation of Gq-protein-coupled receptors (GqPCRs) through phospholipase C (PLC).47,48 DAGLs are postsynaptic integral membrane proteins and exist as two isoforms, a and b.49,50 DAGLa is the major synthetic enzyme for 2-AG in the brain.51 3.2.2. 2-AG degradation 2-AG is mainly degraded by a cytosolic serine hydrolase, MAG lipase (MAGL),52,53 which is localized in presynaptic terminals, where CB1 receptors are also expressed.52 FAAH, the main degradation enzyme of anandamide, metabolizes 2-AG in vitro54,55 but not efficiently in vivo.56

3.1. Anandamide 4. Cannabinoids are antinociceptive 3.1.1. Anandamide synthesis Anandamide is an N-acylethanolamine and can be formed by a two-step biosynthesis scheme catalyzed by N-acetyltransferase and N-arachidonoyl phosphatidylethanolamine-phospholipase D (NAPE-PLD).27,28 Anandamide can also be generated from NAPE through an a/b-hydrolase-4 (ABHD4) and glycerophosphodiesterase 1 (GDE1) enzymatic pathway.29e31 However, basal levels of brain anandamide in mice lacking the putative synthetic enzymes, either NAPE-PLD32 or GDE1,33 or both GDE1 and NAPE-PLD,33 were not different from those in wild type mice. Thus, additional biosynthetic pathways are suggested. Because basal levels of anandamide were measured in these studies, it is not known if activity-dependent anandamide levels differ between the genotypes. 3.1.2. Anandamide degradation In contrast to its obscure synthetic pathway, anandamide is known to be rapidly inactivated by the degradation enzyme, fatty acid amide hydrolase (FAAH). It is a 579 amino acid serine amidohydrolase, widely distributed in the brain.34,35 FAAH is mainly

After the identification of CB1 and CB2 receptors, subtypeselective agonists and antagonists, as well as knockout mice, were actively developed. With these tools, the analgesic mechanisms of cannabinoids were explored. Systemic administration of cannabinoids can produce an analgesic effect with an efficacy comparable to opioids in acute pain animal models,57 and was even more effective than opioids in some chronic pain models.58,59 These analgesic effects are mainly mediated by CB1 receptors in the CNS,59e61 and by both CB162 and CB263,64 receptors in the periphery. 4.1. CB1 receptor-mediated central site of action In the CNS, the analgesic effect of cannabinoids is mainly mediated through CB1 receptors located in structures that mediate nociceptive neurotransmission, including the spinal dorsal horn, periaqueductal gray (PAG),65 dorsal raphe nuclei,66 and thalamic ventroposterolateral nucleus.67 At the spinal level, electrophysiological and c-fos expression studies support that cannabinoids inhibit spinal dorsal neuronal activity to produce analgesia.59,61,62,68

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Among the supraspinal areas involved in the modulation of nociception, the midbrain PAG has been extensively studied.

suggesting an analgesic tone produced by endocannabinoids via CB1 receptors, at least at the spinal level.

4.2. The midbrain PAG is a supraspinal site of action

5.1.2. CB1 knockout mice Pain sensitivity has been examined in three lines of CB1 knockout mice of distinct genetic backgrounds, C57BL/6J,87 CD1,88 and 129/SvJ.89 Knockout of CB1 in C57BL/6J mice resulted in lower pain sensitivity in the hot-plate and formalin tests, but not the tail-flick test. However, the antinociceptive responses in these mutants may be confounded by their impaired locomotor activity.87 The other two lines of CB1 knockout mice displayed normal pain sensitivity in several nociceptive tests (Table 1). These results also suggest that tonic levels of endocannabinoids play a minor role in setting pain thresholds.

Activation of the PAG produces analgesia,69 through activating the downstream rostroventral medulla (RVM), which sends inhibitory projections to the dorsal horn of the spinal cord.70 This PAGRVM-spinal dorsal horn circuit constitutes a key endogenous descending pain inhibitory pathway. Studies using microinjection techniques in pain models suggest that the PAG is the site for cannabinoids producing CB1-mediated analgesic effects.66,71 Immunohistochemical72 and electrophysiological73 studies support that cannabinoids produce analgesic effects in the PAG via a disinhibition (inhibition of GABAergic transmission) mechanism mediated by CB1 receptors. 4.3. CB2 receptor-mediated peripheral site of action In the periphery, the antinociceptive effect of cannabinoids can be mediated both by CB162 and CB2 receptors. Peripherally mediated CB2 antinociception has been an especially attractive therapeutic target, as compounds that selectively activate CB2 receptors have the merit of avoiding CB1-mediated CNS side effects, such as hypomotility, catalepsy, hypothermia, and cognitive impairment. Intraplantar (i.pl.) administration of the CB2-preferring agonists, AM1241 or GW405833, effectively reduced nociceptive responses in several inflammatory pain models.74e76 These antinociceptive effects were blocked by i.pl. injection of a CB2, but not a CB1 antagonist74 and were absent in CB2 receptor-knockout mice.76,77 However, activation of CB2 receptors may also have a proinflammatory effect.78 In addition, CB2 receptors were strongly induced in sensory neurons and the spinal cord in neuropathic and inflamed rats.79,80 This high inducibility of CB2 receptors renders them an attractive potential therapeutic target for pain treatment. A small scale, proof-of-concept clinical trial (32 patients, single site, double-blind, two-way crossover) using a CB2 receptor agonist, LY2828360 (80 mg/kg), for the treatment of osteoarthritis was conducted by Eli Lilly (Indianapolis, IN, USA). However, it failed to meet the primary endpoint.81

5.2. CB2 receptor-mediated 5.2.1. CB2 antagonists SR144528, the first CB2 receptor antagonist, was used to investigate the role of CB2 receptor activation in the effect of endocannabinoids on pain regulation, mainly in inflammatory pain models (Table 1). In the formalin test, it was hyperalgesic when given by Intravenous (i.v.) injection63 but was ineffective by i.p. administration.90,91 This CB2 antagonist induced hyperalgesia and enhanced edema when given by i.p. injection in the carrageenan model,92 but displayed anti-inflammatory effects by oral administration in the same model.78 Again, the inverse agonist property of SR144528 might also explain its hyperalgesic effects in these studies. 5.2.2. CB2 knockout mice CB2 knockout mice, as compared with wild type mice, had increased thermal nociceptive responses in the plantar test, while having normal pain sensitivity in the hot-plate and tail-immersion assays.77 In a neuropathic pain model, CB2 knockout mice displayed enhanced nociceptive responses in the contralateral paw compared to control mice, which was also associated with enhanced interferon-g and microglial expression in the contralateral spinal dorsal horn.93,94 These results suggest that endocannabinoids play a CB2 receptor-mediated tonic analgesic role, but also exert a proinflammatory tone during neuropathic pain state.

5. The role of endocannabinoids in pain regulation 5.3. Role of anandamide in pain regulation Our understanding of the role of endocannabinoids in pain regulation comes from studies using both pharmacological and genetic approaches by blocking or silencing CB1 and CB2 receptors, or by inhibiting or silencing degradation or synthetic enzymes of anandamide and 2-AG. Table 1 summarizes the effects of inactivating the endocannabinoid system on nociceptive responses in various pain models. 5.1. CB1 receptor-mediated 5.1.1. CB1 antagonists Systemic administration of SR141716A, the first CB1 antagonist,82 was initially found to have no effect in several pain models (Table 1). Later, it was reported to increase nociceptive responses when given by intrathecal (i.t.) or intraperitoneal (i.p.), but not i.pl., routes of administration, although replication of these findings has been variable (Table 1). Because SR141716A also acts as an inverse agonist of CB1 receptors,83 this might lead to pronociceptive effects in the above studies. The finding that systemic administration of AM4113,84 a neutral CB1 antagonist, produced no changes in nociception suggests there is little tonic endocannabinoid modulation of pain thresholds. However, i.t. injection of CB1 receptor antisense nucleotide caused hyperalgesia in the hot-plate test in mice,85,86

5.3.1. FAAH inhibitors Several lines of evidence suggest that inhibition of FAAH, the degradation enzyme of anandamide, effectively reduced nociceptive responses in various pain models (Table 1). A series of irreversible carbamate FAAH inhibitors, such as URB532 and URB597, has been developed and patented95,96 for pain treatment. Later, an a-ketooxazole reversible FAAH inhibitor, OL-135, was demonstrated to have high in vivo efficacy for reducing nociceptive responses in tail-immersion, hot-plate, formalin tests97 and neuropathic pain models.98 Thereafter, a highly selective ureabased irreversible FAAH inhibitor, PF-3845, was developed by Pfizer (NYC, NY, USA) and shown to be an efficacious analgesic in inflammatory99,100 and neuropathic101 pain models. These FAAH inhibitors significantly increased anandamide levels in the brain and mainly induced a CB1 receptor-mediated antinociception, suggesting that endogenous anandamide, when protected from degradation, can produce antinociception through CB1 receptors. Chronic systemic treatment with PF-3845 produced a persistent analgesic effect without tolerance in mice.101 Interestingly, this compound was also effective when given by i.pl. administration.100 Recently, a peripherally acting FAAH inhibitor, URB937, was shown to be effective in inflammatory pain, neuropathic pain, and arthritis

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Table 1 Changes in nociceptive responses after inactivating cannabinoid 1 (CB1) or CB2 receptors or inhibiting endocannabinoid degradation by pharmacological or genetic approach.

CB1 antagonist

Compound

Species

Route

Effects on nociceptive responses

Refs

SR141716A SR141716A SR141716A SR141716A SR141716A SR141716A SR141716A

Rat Mice Mice Rat Rat Rat Mice/Rat/Mice

i.p. i.p. i.t. i.pl. i.p. i.t. i.p.

82 34,97 85 64 166 167 63,90,168

AM4113

Rat Mice Mice Mice Mice Mice Mice/Rat Mice Rat Mice Mice Mice Mice Mice Rat Rat Rat Mice Rat Rat Mice Mice Mice Mice Mice Rat Mice Mice Rat Rat Mice Mice Mice Rat Rat Mice Mice Rat

i.p.

4 tail-flick 4 tail-immersion, hot-plate [ hot-plate 4 paw withdraw [ tail-flick [ non-potentiated C-fiber evoked neuronal response [ formalin test Y formalin test (low dose), 4 formalin test (high dose) 4 tail-flick Y hot-plate, Y formalin test, 4 tail-flick 4 hot-plate, tail-pressure, writhing and Hargreaves tests [ hot-plate [ paw withdraw, hot-plate, tail-flick [ formalin test 4 formalin test (low and high dose) Y carrageenan-induced edema [ carrageenan-mechanical paw withdrawal [ Hargreaves test, 4 hot-plate, 4 tail-immersion [ nerve ligation contralateral hyperalgesia Y hot-plate Y tail-immersion, hot-plate, Y formalin test Y tail-flick Y cholestasis-induced tail-flick Hargreaves test (Y TRPV1-mediated; [ CB1-mediated) Y mechanical and thermal allodynia (PNL) Y mechanical and thermal allodynia (CCI) Y mechanical and cold allodynia (cisplatin) Y mechanical allodynia (CFA), Y joint compression (arthritis pain) Y mechanical (CCI) and cold allodynia (acetone) Y mechanical and cold allodynia (CCI) Y hot-plate, tail-immersion and formalin test 4 noxious heat latency (CCI model) 4 mechanical (CCI) and cold allodynia (acetone) Y tail-flick Y tail-immersion Y tail-immersion, Y mechanical and cord allodynia (CCI) Y mechanical and cold allodynia (cisplatin) Y receptive field expansion (carrageenan) Y mechanical allodynia (carrageenan) Y mechanical allodynia (bone cancer pain) Y mechanical and cold allodynia (CCI) Y formalin test Y mechanical and thermal allodynia (PNL) Y mechanical (CCI) and cold allodynia (acetone) 4 hot-plate (SNL, CFA), mechanical (SNL, CFA) Y tail-immersion

CB1 KO CB1 KD CB2 antagonist

CB2 KO CB2 KO FAAH inhibitor

SR144528 SR144528 SR144528/JTE-907 SR144528

URB532, URB597 OL-135 URB597 URB597 URB597 URB597 URB597 URB597, URB937 PF-04457845 URB597/OL-135 PF-3845

FAAH KO

MGL inhibitor

MGL KO Dual antagonist of FAAH-MGL

URB 602 JZL184 JZL184 JZL184 JZL184 JZL184 JZL184 JZL184 JZL184/URB602 URB 602 JZL184 JZL195

i.t. i.t. i.v. i.p. p.o. i.p.

i.p. i.p. i.p. i.p. i.vlPAG s.c. p.o. i.p. p.o. i.p. i.p.

i.dlPAG i.p. i.p. i.p. i.t. i.p. i.pl i.p. i.pl s.c. i.p. i.p.

84 87 88,89 169 86 63 90,91 78 92 77 94 170 97 171 172 148 109 173 103 104,105 98 101,174 34,91 91 98 150 175 101 103 176 117 177 174 178 109 98 111 175

[ ¼ increase; Y ¼ decrease; 4 ¼ no change; CCI ¼ chronic constriction injury; CFA ¼ Freund’s complete adjuvant; FAAH ¼ fatty acid amide hydrolase; i.dlPAG ¼ intradorsolateral PAG; i.p. ¼ intraperitoneal; i.pl. ¼ intraplantar; i.t. ¼ intrathecal; i.v. ¼ intravenous; i.vlPAG ¼ intra-ventrolateral PAG; KD ¼ knock down; KO ¼ knockout; MGL ¼ monoacylglycerol lipase; PNL ¼ partial sciatic nerve ligation; p.o. ¼ oral; s.c. ¼ subcutaneous; SNL ¼ spinal nerve ligation.

pain models.102 URB597 and URB937 were also found to be effective in the cisplatin (a chemotherapeutic agent) neuropathic pain model.103 An orally bioavailable urea-based irreversible carbamate FAAH inhibitor, PF-04457845, was developed by Pfizer and displayed significant CB1 receptor-dependent reductions in inflammatory and arthritis pain models.104,105 The promising findings with FAAH inhibitors in reducing nociceptive responses in various animal pain models (Table 1) motivated advancing an FAAH inhibitor to a clinical pain trial. However, in a randomized, placebo-controlled clinical trial, chronic administration of PF-04457845 did not have significant analgesic effects in patients with osteoarthritis, although their plasma levels of endocannabinoids were significantly elevated and no significant side effects were reported.106 Although this Phase II clinical trial was terminated early due to ineffectiveness in an interim analysis, it remains to be further elucidated if FAAH inhibitors would be clinically effective for other pain indications,107 such as migraine, cancer pain or neuropathic pain induced by chemotherapy,103 diabetes, or herpes virus. The

possible species difference in the FAAH between human and rodents should also be investigated.106 Interestingly, a noncovalent, reversible and noncompetitive FAAH inhibitor, AZ513, was published by AstraZenica,108 with a higher potency in inhibiting human FAAH than rat FAAH. It is not known if this compound will be more promising if there is any clinical trial in the future. 5.3.2. FAAH knockout mice FAAH knockout mice exhibited reduced CB1-mediated pain sensitivity, but locomotor activity and body temperature were similar to those of wild type mice.34 This suggests that FAAH plays an important role in regulating endogenous anandamide-mediated analgesic tone but not in its motor- or thermo-regulation. CB1mediated nociceptive responses in acute and inflammatory pain models were significantly reduced in FAAH knockout mice, as seen in animals treated with FAAH inhibitors. However, FAAH knockout mice had unchanged sensitivity in neuropathic pain models, as compared with wild type mice (Table 1).

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5.4. Role of 2-AG in pain regulation

6.2. Inhibiting glutamate transport

5.4.1. MAGL inhibitors URB602, the first MAGL inhibitor, given by i.pl. injection, significantly reduced mechanical and thermal allodynia in a neuropathic pain model.109 JZL184, a much more potent and selective MAGL inhibitor,110 was also effective in reducing nociceptive responses in several inflammatory and neuropathic pain models by either i.p., i.t., or i.pl. injection (Table 1). This suggests that acute administration of MAGL inhibitors has an analgesic effect.

As postsynaptic mGluR5s are mainly located perisynaptically,132 they are primarily activated by spillover glutamate, such as when glutamate transporters are inhibited or overwhelmed, subsequently leading to disinhibition of the PAG through the mGluR5PLC-DAGL-2-AG signaling.123

5.4.2. MAGL knockout mice Brain 2-AG levels were elevated more than 10-fold in MAGL knockout mice101,111 close to the 8-fold elevation induced by acute administration of JZL184.110 However, because of this elevation of 2-AG desensitized CB1 receptors in MAGL knockout mice, fewer and less active CB1 receptors existed in the brains of these mutants.101,111 Similarly, treatment with a high dose of JZL184 for 6 consecutive days also led to desensitization of CB1 receptors, loss of analgesic activity, and cross-tolerance to the antiallodynic effects of CB1 agonists and FAAH inhibitors.101,111 This is significantly different from the finding that CB1 receptor function remained normal in mice lacking FAAH or which were chronically treated with FAAH inhibitors.34,101,112 The difference between FAAH and MAGL inhibition may be because 2-AG has a higher efficacy than anandamide at CB1 receptors.113e115 Thus, after long-term inhibition of its degradation enzyme, the enhanced and prolonged action of 2-AG, but not anandamide, leads to CB1 receptor desensitization. Interestingly, lower doses of MAGL inhibitors produce significant therapeutic benefits in preclinical models, while avoiding desensitization and tolerance of CB1 signaling.116e118 6. GqPCR activation-initiated 2-AG retrograde disinhibition in the PAG: A novel analgesic mechanism A major pathway to generate 2-AG is by GqPCR activation. Activation of postsynaptic GqPCRs results in phospholipid hydrolysis by PLCb to yield DAG. DAG is then hydrolyzed by DAGLa located at postsynaptic membranes to generate 2-AG, which then diffuses retrogradely across the synapse to activate presynaptic CB1 receptors and inhibit transmitter release.119 This neurotransmission inhibitory mechanism mediated by a GqPCR-PLC-DAGL-2-AG retrograde signaling cascade has been reported in several brain regions,119 including the PAG. Stimulation of the type 5 metabotropic glutamate receptor (mGluR5),120e125 M1/M3 muscarinic acetylcholine receptor (M1/M3 mAChR),126 and orexin 1 receptor (OX1R),127 has been reported to initiate the GqPCR-PLC-DAGL-2-AG signaling-mediated retrograde inhibition of GABAergic transmission (disinhibition) in PAG slices. This 2-AG-mediated disinhibition mechanism may contribute to the analgesic effects induced by activation of these GqPCRs. 6.1. mGluR5 activation There are eight subtypes of mGluRs classified into three groups according to their coupling signaling pathways: Group I (mGluR1/ 5), Group II (mGluR2/3), and Group III (mGluR4/6-8).128,129 Only Group I mGluRs belong to the GqPCR family.130 Drew and Vaughan131 found that activation of all three groups of mGluRs inhibits PAG GABAergic transmission, but only the effect of activating Group I mGluRs, specifically mGluR5, is mediated through endocannabinoids.123

6.3. mGluR5 agonists Recently, Gregg and colleagues,120 found that microinjection of an mGluR5 agonist into the dorsolateral PAG (dlPAG) triggered the release of 2-AG, but not anandamide. This effect was reversed by intra-dorsolateral PAG (intra-dlPAG) injection of a CB1 antagonist and DAGL inhibitor, or by expressing siRNA to silence DAGLa in the dlPAG. Immunohistochemical staining showed that mGluR5s were colocalized with DAGLa in the postsynaptic dendritic site, which is juxtaposed to the presynaptic localization of CB1 receptors. These data support the fact that postsynaptic mGluR5-DAGLa cascade triggers retrograde 2-AG signaling in the PAG in vivo. 6.3.1. Activating NK1, NTS1/2, and CCK1 receptors In addition to inhibiting glutamate transporter or directly applying mGluR agonists, there are several ways to release glutamate in sufficient amounts to activate perisynaptic mGluR5s, leading to disinhibition of the PAG through the mGluR5-PLC-DAGL-2-AG signaling. Indeed, Vaughan and colleagues proved that this 2-AG-mediated disinhibition signaling triggered by mGluR5 activation in ventrolateral PAG (vlPAG) slices can be induced by several analgesic neuropeptides.122,124,125 Substance P,122 neurotensin,125 and cholecystokinin (CCK),124 activated neurokinin 1, neurotensin 1/2, cholecystokinin 1 receptors, respectively, on glutamatergic somas to release glutamate, which then activated perisynaptic mGluR5s, and produced disinhibition in the PAG through 2-AG. It remains to be elucidated by in vivo studies if this PAG disinhibition mechanism mediated by the mGluR5-PLC-DAGL-2-AG signaling contributes to the antinociceptive effects of intra-PAG injection of substance P,133 neurotensin,134 and CCK.135 6.3.2. TRPV1 activation Capsaicin, when injected into the PAG, induced antinociception, in contrast to its pronociceptive effect in the periphery.136,137 In a study examining the analgesic mechanism of capsaicin at the supraspinal level, we recently found that capsaicin can also induce substantial glutamate release in the vlPAG to activate the mGluR5PLC-DAGL-2-AG disinhibition mechanism, leading to analgesia.121 We found that capsaicin markedly increased the frequency of miniature excitatory postsynaptic currents (mEPSCs), but decreased evoked inhibitory postsynaptic currents (eIPSCs) in PAG slices. This IPSC inhibitory effect of capsaicin was antagonized by TRPV1, mGluR5, and CB1 antagonists and by a DAGL inhibitor. These results suggest that capsaicin activates the TRPV1 channels on glutamatergic terminals to release massive amounts of glutamate to activate postsynaptic mGluR5s, leading to disinhibition in the PAG through the mGluR5-PLC-DAGL-2-AG signaling. Finally, we proved that this mechanism contributes to the analgesic effect induced by intra-ventrolateral PAG (intra-vlPAG) injection of capsaicin,121 because this analgesic effect was reversed by intravlPAG injection of AM251 and 2-methyl-6-(phenylethynyl)pyridine, an mGluR5 antagonist. In agreement with our findings, Starowicz et al137 also found that intra-vlPAG capsaicin induced analgesia by releasing glutamate from the PAG to activate the OFF neurons in the RVM.

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6.4. M1/M3 mAChR activation The PAG receives dense cholinergic projections from the pontine tegmentum.138 Microinjection of cholinergic agonists into the PAG produces analgesia and related behaviors.139,140 In PAG slices, Lau and Vaughan126 found that carbachol suppressed IPSCs in PAG slices. This effect was mimicked by inhibiting acetylcholinesterase, occluded by a CB1 agonist, and reduced by blocking M1/M3 mAChRs and CB1 receptors, and inhibiting DAGL. These results suggest that activation of postsynaptic M1/M3 mAChR, like other GqPCRs, initiates the GqPCRPLC-DAGL-2-AG disinhibition mechanism in the PAG. 6.5. OX1 receptor activation Orexin A and orexin B, also named hypocretin 1 and 2, respectively, are a pair of hypothalamic neuropeptides and exert their biological functions through two GqPCRs, OX1 and OX2 receptors.141,142 Orexin-expressing neurons are localized in the perifornical area and the lateral hypothalamus (LH),141,142 but project widely throughout the brain, including the PAG.143e145 Orexins have been implicated in a myriad of physiological functions, such as sleep, reward, energy homeostasis, autonomic central control, and pain.146,147 However, the mechanism(s) of how orexins regulate pain, especially at the supraspinal level, remained unclear until our findings in 2011.127 We found that orexin A depressed IPSCs in PAG slices and that this effect was inhibited by OX1 and CB1, but not OX2 antagonists, as well as by PLC and DAGL inhibitors. Moreover, the effect of orexin A was mimicked by a cannabinoid agonist and enhanced by an MAGL inhibitor. These results suggest that activation of postsynaptic OX1 receptors, like mGluR5s and M1/M3 mAChRs, initiates the GqPCR-PLC-DAGL-2-AG disinhibition mechanism in the PAG. We also proved that this mechanism contributes to the antinociceptive effect induced by intra-vlPAG injection of orexin A in the rat hot-plate test.127 Taken together, in the PAG, mGluR5 can be activated directly by an mGluR5 agonist,120 or by endogenous spillover glutamate: (1) when glutamate transport is inhibited;123 (2) when glutamatergic terminals are depolarized by TRPV1 activation by capsaicin121 or anandamide;148,149 and (3) when glutamatergic cell bodies were excited by substance P via NK1 receptors,122 neurotensin via NTS1/2 receptors,125 or CCK via CCK1 receptors.124 After mGluR5 activation, 2-AG was generated through the PLC-DAGL enzymatic cascade to produce retrograde inhibition of GABAergic transmission in the PAG, leading to analgesia. 7. Functional role of endocannabinoid analgesia in the PAG 7.1. Stress-induced analgesia In 2005, Hohmann et al150 demonstrated that stress-induced analgesia (SIA), a phenomenon believed to represent the evolutionary impetus for the development of central pain inhibition mechanisms in humans and animals,151 is associated with the rapid formation of 2-AG and anandamide in the dlPAG. SIA induced by foot shock stress was blocked by CB1 antagonists and enhanced by the MAGL or FAAH inhibitor given by intra-dlPAG injection, but not affected by the CB2, opioid, or TRPV1 antagonist. Valverde et al152 also reported that forced swim stress-induced analgesia was absent in CB1 knockout mice. Olango et al153 and Butler et al154 also demonstrated that endocannabinoids are involved in fear-conditioned analgesia (FCA) in rats re-exposed to the context previously associated with foot shock. They found that intra-dlPAG injection of the CB1 antagonist attenuated, and the FAAH inhibitor enhanced, FCA in rats injected with i.pl. formalin. Elevated anandamide in the dlPAG was specifically associated with FCA.153

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Together, these results suggest that endocannabinoids, either anandamide or 2-AG, can be induced to release by various stressors, either electrical foot shock, swimming or fear, or during a pain state to activate the CB1 receptors in the PAG, serving as an endogenous analgesic protector. However, how a stress triggers endocannabinoid release to induce SIA remains unclear. The disinhibition mechanism mediated by 2-AG in the PAG through the GqPCR-PLCDAGL signaling might be such a mechanism. 7.2. mGluR5 involvement Recently, Gregg and colleagues120 found that intra-dlPAG injection of an mGluR5 agonist increased PAG 2-AG and enhanced SIA. This effect was reversed by intra-dlPAG injection of the CB1 receptor antagonist and DAGL inhibitors, and by silencing DAGLa in the dlPAG.120 These data suggest that the mGluR5-PLC-DAGLa-2AG disinhibition mechanism in the PAG contributes to SIA. 7.3. Orexin involvement Two studies have suggested that endogenous orexins play a role in SIA. Watanabe et al155 reported that SIA induced by electrical foot shock was absent in prepro-orexin knockout mice. Xie et al156 found that in mice with hypothalamic orexin neurons degenerated by ataxin-3 expression, restraint stress-induced antinociception was significantly reduced, as compared to wild type controls. The reduction of SIA in orexin/ataxin-3 mice can be reversed by intracerebroventricular (i.c.v.) injection of orexin A. We recently found that a 30-min restraint stress in mice induced analgesia in the hot-plate test accompanied with increased c-fos expression in LH orexin neurons. This SIA was blocked by intravlPAG injection of OX1 and CB1 antagonists, respectively. These results suggest that restraint stress activates hypothalamic orexin neurons, releasing orexins to activate OX1 receptors in the vlPAG, initiating the GqPCR-PLC-DAGL-2-AG disinhibition mechanism in the PAG, leading to analgesia.157 7.4. Substance P/neurotensin involvements Substance P is antinociceptive at the supraspinal level, in contrast to a pronociceptive action in the periphery. The PAG is one of the sites of action because intra-PAG injection of substance P induced antinociception.133 Chemical (carbachol) stimulation of LH triggered substance P release in the PAG, leading to analgesia.158 Interestingly, activation of LH by restraint stress also released orexins, which in turn acted in the PAG to induce analgesia through 2-AG mediated disinhibition.157 It is interesting to know if substance P also plays a role in endocannabinoid-mediated SIA. Moreover, both orexin and substance P were released after LH activation, so it will be interesting to examine if there are interactions between these two neuropeptide systems during SIA. Similarly, neurotensin was also suggested to be involved in SIA.159 It will also be interesting to see if neurotensin plays a role in endocannabinoid-mediated SIA. 7.5. Analgesic mechanism of acetaminophen Acetaminophen, one of the most popular analgesic drugs, has been used for more than a century. Its mode of analgesic action is still a matter of debate. In 2005e2006, a new look for the analgesic effect of acetaminophen, involving the CB1 receptor, was proposed by two groups simultaneously.160e162 Hogestatt et al162 demonstrated that N-arachidonoylphenolamine, known as AM404, can be formed in the brain after systemic administration of acetaminophen in rats. They suggested that after systemic administration,

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acetaminophen is deacetylated in the liver into p-aminophenol, which is then conjugated with arachidonic acid to form AM404 in the brain by FAAH, the degradation enzyme of anandamide. Further studies showed that the analgesic effects of acetaminophen were reduced by CB1 antagonists,160,161 and absent in CB1 knockout mice.161 AM404 has complicated pharmacological properties. Like anandamide, it is a C20 unsaturated fatty acid amide, originally developed as a potent inhibitor for anandamide uptake,40 but it is also an FAAH inhibitor. Furthermore, AM404 is also a TRPV1 agonist, as potent as capsaicin.163 Recently, Mallet et al164 found that the TRPV1 channel in the brain is involved in acetaminopheninduced antinociception. They found that the oral antinociceptive effects in several pain models induced by acetaminophen at a clinically effective dose range observed in wild type mice, were absent in TRPV1 or FAAH knockout mice. The analgesic effect of AM404 (i.c.v.) was also absent in TRPV1 knockout mice. Together, these studies suggest that the analgesic effects of oral acetaminophen, or its central active metabolite, AM404, are mediated by TRPV1 channels and CB1 receptors. However, TRPV1 activation usually causes neuroexcitation, whereas CB1R activation decreases neurotransmitter release. We recently proved that the mechanism we reported for the analgesic mechanism of capsaicin,121 can explain how acetaminophen can produce a CB1Rmediated analgesic effect via TRPV1 activation.165 We found that oral acetaminophen or intra-PAG microinjection of AM404 produced comparable antinociceptive effects in the rat hot-plate test. Both antinociceptive effects were antagonized by intra-PAG microinjection of antagonists of TRPV1, mGluR5, or CB1R. It is suggested that after oral administration, acetaminophen is deacetylated in the liver into p-aminophenol, which is then converted into AM404 in the brain. AM404 then activates TRPV1, like capsaicin, in the vlPAG, to release a great amount of glutamate, which activates the mGluR5-PLC-DAGLa-2-AG disinhibition mechanism in the PAG, leading to analgesia. 8. Conclusions and perspectives After the identification of cannabinoid receptors and endocannabinoids, accumulating studies have emerged during the past two decades that have aimed to elucidate the regulatory mechanisms of the cannabinoid system in pain control, with a goal of developing new pain therapies. CB2 selective agonists have the merit of avoiding CB1 receptor-mediated CNS side effects. Inhibitors of anandamide and 2-AG degradation enzymes, FAAH and MAGL, respectively, have the merit of focusing action at generating sites. Despite promising preclinical results, the first clinical trial with an FAAH inhibitor failed. The efficacy of MAGL inhibitors remains to be determined. Because of its synthesis on demand, endogenous 2-AG levels can be elevated in the PAG upon GqPCR activation and lead to analgesia via PAG disinhibition. Several endogenous analgesic neuropeptides may serve as endogenous analgesics during SIA via this GqPCR-PLC-DAGL-2-AG disinhibition mechanism in the PAG. As an effective endogenous pain relief mechanism during evolution, it offers a promising strategy for analgesic drug development. Acknowledgments This study was supported by the grants to LCC from the National Science Council, Taipei, Taiwan (NSC 101-2325-B002-048 and NSC 102-2321-B002-066) and National Health Research Institutes, Miaoli, Taiwan (NHRI-EX102-10251NI).

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References 1. Gaoni Y, Mechoulam R. Isolation, structure and partial synthesis of an active constituent of hashish. J Am Chem Soc 1964;86:1646e7. 2. Mechoulam R. Cannabinoids as therapeutic agents. Florida: Chapman and Hall/ CRC; 1986. 3. Russo EB, McPartland JM. Cannabis is more than simply delta(9)-tetrahydrocannabinol. Psychopharmacology (Berl) 2003;165:431e2. author reply 433e434. 4. Pertwee RG. Inverse agonism and neutral antagonism at cannabinoid CB1 receptors. Life Sci 2005;76:1307e24. 5. Matsuda LA, Lolait SJ, Brownstein MJ, Young AC, Bonner TI. Structure of a cannabinoid receptor and functional expression of the cloned cDNA. Nature 1990;346:561e4. 6. Munro S, Thomas KL, Abu-Shaar M. Molecular characterization of a peripheral receptor for cannabinoids. Nature 1993;365:61e5. 7. Rhee MH, Bayewitch M, Avidor-Reiss T, Levy R, Vogel Z. Cannabinoid receptor activation differentially regulates the various adenylyl cyclase isozymes. J Neurochem 1998;71:1525e34. 8. Gerard CM, Mollereau C, Vassart G, Parmentier M. Molecular cloning of a human cannabinoid receptor which is also expressed in testis. Biochem J 1991;279(Pt 1):129e34. 9. Howlett AC, Breivogel CS, Childers SR, Deadwyler SA, Hampson RE, Porrino LJ. Cannabinoid physiology and pharmacology: 30 years of progress. Neuropharmacology 2004;47(Suppl. 1):345e58. 10. Herkenham M, Lynn AB, Little MD, Johnson MR, Melvin LS, de Costa BR, et al. Cannabinoid receptor localization in brain. Proc Natl Acad Sci U S A 1990;87:1932e6. 11. Katona I, Sperlagh B, Sik A, Käfalvi A, Vizi ES, Mackie K, et al. Presynaptically located CB1 cannabinoid receptors regulate GABA release from axon terminals of specific hippocampal interneurons. J Neurosci 1999;19:4544e58. 12. Mackie K, Lai Y, Westenbroek R, Mitchell R. Cannabinoids activate an inwardly rectifying potassium conductance and inhibit Q-type calcium currents in AtT20 cells transfected with rat brain cannabinoid receptor. J Neurosci 1995;15:6552e61. 13. Shen M, Piser TM, Seybold VS, Thayer SA. Cannabinoid receptor agonists inhibit glutamatergic synaptic transmission in rat hippocampal cultures. J Neurosci 1996;16:4322e34. 14. Galiegue S, Mary S, Marchand J, Dussossoy D, Carrière D, Carayon P, et al. Expression of central and peripheral cannabinoid receptors in human immune tissues and leukocyte subpopulations. Eur J Biochem 1995;232:54e61. 15. Van Sickle MD, Duncan M, Kingsley PJ, Mouihate A, Urbani P, Mackie K, et al. Identification and functional characterization of brainstem cannabinoid CB2 receptors. Science 2005;310:329e32. 16. Walczak JS, Pichette V, Leblond F, Desbiens K, Beaulieu P. Characterization of chronic constriction of the saphenous nerve, a model of neuropathic pain in mice showing rapid molecular and electrophysiological changes. J Neurosci Res 2006;83:1310e22. 17. Atwood BK, Mackie K. CB2: a cannabinoid receptor with an identity crisis. Br J Pharmacol 2010;160:467e79. 18. Devane WA, Hanus L, Breuer A, Pertwee RG, Stevenson LA, Griffin G, et al. Isolation and structure of a brain constituent that binds to the cannabinoid receptor. Science 1992;258:1946e9. 19. Mechoulam R, Ben-Shabat S, Hanus L, Ligumsky M, Kaminski NE, Schatz AR, et al. Identification of an endogenous 2-monoglyceride, present in canine gut, that binds to cannabinoid receptors. Biochem Pharmacol 1995;50:83e90. 20. Sugiura T, Kondo S, Sukagawa A, Nakane S, Shinoda A, Itoh K, et al. 2Arachidonoylglycerol: a possible endogenous cannabinoid receptor ligand in brain. Biochem Biophys Res Commun 1995;215:89e97. 21. Hanus L, Abu-Lafi S, Fride E, Breuer A, Vogel Z, Shalev DE, et al. 2-Arachidonyl glyceryl ether, an endogenous agonist of the cannabinoid CB1 receptor. Proc Natl Acad Sci U S A 2001;98:3662e5. 22. Porter AC, Sauer JM, Knierman MD, Becker GW, Berna MJ, Bao J, et al. Characterization of a novel endocannabinoid, virodhamine, with antagonist activity at the CB1 receptor. J Pharmacol Exp Ther 2002;301:1020e4. 23. Huang SM, Bisogno T, Trevisani M, Al-Hayani A, De Petrocellis L, Fezza F, et al. An endogenous capsaicin-like substance with high potency at recombinant and native vanilloid VR1 receptors. Proc Natl Acad Sci U S A 2002;99:8400e5. 24. Di Marzo V, Fontana A, Cadas H, Schinelli S, Cimino G, Schwartz JC, et al. Formation and inactivation of endogenous cannabinoid anandamide in central neurons. Nature 1994;372:686e91. 25. Piomelli D, Giuffrida A, Calignano A, Rodriguez de Fonseca F. The endocannabinoid system as a target for therapeutic drugs. Trends Pharmacol Sci 2000;21:218e24. 26. Di Marzo V. Endocannabinoids: synthesis and degradation. Rev Physiol Biochem Pharmacol 2008;160:1e24. 27. Schmid PC, Reddy PV, Natarajan V, Schmid HH. Metabolism of N-acylethanolamine phospholipids by a mammalian phosphodiesterase of the phospholipase D type. J Biol Chem 1983;258:9302e6. 28. Okamoto Y, Wang J, Morishita J, Ueda N. Biosynthetic pathways of the endocannabinoid anandamide. Chem Biodivers 2007;4:1842e57. 29. Sun YX, Tsuboi K, Okamoto Y, Tonai T, Murakami M, Kudo I, et al. Biosynthesis of anandamide and N-palmitoylethanolamine by sequential actions of phospholipase A2 and lysophospholipase D. Biochem J 2004;380(Pt 3):749e56.

168 30. Simon GM, Cravatt BF. Endocannabinoid biosynthesis proceeding through glycerophospho-N-acyl ethanolamine and a role for alpha/beta hydrolase 4 in this pathway. J Biol Chem 2006;281:26465e72. 31. Liu J, Wang L, Harvey-White J, Huang BX, Kim HY, Luquet S, et al. Multiple pathways involved in the biosynthesis of anandamide. Neuropharmacology 2008;54:1e7. 32. Leung D, Saghatelian A, Simon GM, Cravatt BF. Inactivation of N-acyl phosphatidylethanolamine phospholipase D reveals multiple mechanisms for the biosynthesis of endocannabinoids. Biochemistry 2006;45:4720e6. 33. Simon GM, Cravatt BF. Characterization of mice lacking candidate N-acyl ethanolamine biosynthetic enzymes provides evidence for multiple pathways that contribute to endocannabinoid production in vivo. Mol Biosyst 2010;6: 1411e8. 34. Cravatt BF, Demarest K, Patricelli MP, Bracey MH, Giang DK, Martin BR, et al. Supersensitivity to anandamide and enhanced endogenous cannabinoid signaling in mice lacking fatty acid amide hydrolase. Proc Natl Acad Sci U S A 2001;98:9371e6. 35. Egertova M, Cravatt BF, Elphick MR. Comparative analysis of fatty acid amide hydrolase and cb(1) cannabinoid receptor expression in the mouse brain: evidence of a widespread role for fatty acid amide hydrolase in regulation of endocannabinoid signaling. Neuroscience 2003;119:481e96. 36. Egertova M, Giang DK, Cravatt BF, Elphick MR. A new perspective on cannabinoid signalling: complementary localization of fatty acid amide hydrolase and the CB1 receptor in rat brain. Proc R Soc Lond B Biol Sci 1998;265:2081e5. 37. Tsou K, Nogueron MI, Muthian S, Sañudo-Pena MC, Hillard CJ, Deutsch DG, et al. Fatty acid amide hydrolase is located preferentially in large neurons in the rat central nervous system as revealed by immunohistochemistry. Neurosci Lett 1998;254:137e40. 38. Glaser ST, Kaczocha M, Deutsch DG. Anandamide transport: a critical review. Life Sci 2005;77:1584e604. 39. Fowler CJ. Transport of endocannabinoids across the plasma membrane and within the cell. Febs J 2013;280:1895e904. 40. Beltramo M, Stella N, Calignano A, Lin SY, Makriyannis A, Piomelli D. Functional role of high-affinity anandamide transport, as revealed by selective inhibition. Science 1997;277:1094e7. 41. Di Marzo V, Bifulco M, De Petrocellis L. The endocannabinoid system and its therapeutic exploitation. Nat Rev Drug Discov 2004;3:771e84. 42. Jarrahian A, Manna S, Edgemond WS, Campbell WB, Hillard CJ. Structure-activity relationships among N-arachidonylethanolamine (Anandamide) head group analogues for the anandamide transporter. J Neurochem 2000;74:2597e606. 43. Vandevoorde S, Fowler CJ. Inhibition of fatty acid amide hydrolase and monoacylglycerol lipase by the anandamide uptake inhibitor VDM11: evidence that VDM11 acts as an FAAH substrate. Br J Pharmacol 2005;145:885e93. 44. Alexander JP, Cravatt BF. The putative endocannabinoid transport blocker LY2183240 is a potent inhibitor of FAAH and several other brain serine hydrolases. J Am Chem Soc 2006;128:9699e704. 45. Zygmunt PM, Petersson J, Andersson DA, Chuang H, Sørgård M, Di Marzo V, et al. Vanilloid receptors on sensory nerves mediate the vasodilator action of anandamide. Nature 1999;400:452e7. 46. De Petrocellis L, Bisogno T, Maccarrone M, Davis JB, Finazzi-Agro A, Di Marzo V. The activity of anandamide at vanilloid VR1 receptors requires facilitated transport across the cell membrane and is limited by intracellular metabolism. J Biol Chem 2001;276:12856e63. 47. Hashimotodani Y, Ohno-Shosaku T, Tsubokawa H, Ogata H, Emoto K, Maejima T, et al. Phospholipase Cbeta serves as a coincidence detector through its Ca(2þ) dependency for triggering retrograde endocannabinoid signal. Neuron 2005;45:257e68. 48. Hashimotodani Y, Ohno-Shosaku T, Kano M. Ca(2þ)-assisted receptor-driven endocannabinoid release: mechanisms that associate presynaptic and postsynaptic activities. Curr Opin Neurobiol 2007;17:360e5. 49. Bisogno T, Howell F, Williams G, Minassi A, Cascio MG, Ligresti A, et al. Cloning of the first sn1-DAG lipases points to the spatial and temporal regulation of endocannabinoid signaling in the brain. J Cell Biol 2003;163:463e8. 50. Matyas F, Urban GM, Watanabe M, Mackie K, Zimmer A, Freund TF, et al. Identification of the sites of 2-arachidonoylglycerol synthesis and action imply retrograde endocannabinoid signaling at both GABAergic and glutamatergic synapses in the ventral tegmental area. Neuropharmacology 2008;54:95e107. 51. Gao Y, Vasilyev DV, Goncalves MB, Howell FV, Hobbs C, Reisenberg M, et al. Loss of retrograde endocannabinoid signaling and reduced adult neurogenesis in diacylglycerol lipase knock-out mice. J Neurosci 2010;30:2017e24. 52. Dinh TP, Freund TF, Piomelli D. A role for monoglyceride lipase in 2arachidonoylglycerol inactivation. Chem Phys Lipids 2002;121:149e58. 53. Dinh TP, Kathuria S, Piomelli D. RNA interference suggests a primary role for monoacylglycerol lipase in the degradation of the endocannabinoid 2arachidonoylglycerol. Mol Pharmacol 2004;66:1260e4. 54. Di Marzo V, Bisogno T, Sugiura T, Melck D, De Petrocellis L. The novel endogenous cannabinoid 2-arachidonoylglycerol is inactivated by neuronal- and basophil-like cells: connections with anandamide. Biochem J 1998;331(Pt 1):15e9. 55. Goparaju SK, Ueda N, Taniguchi K, Yamamoto S. Enzymes of porcine brain hydrolyzing 2-arachidonoylglycerol, an endogenous ligand of cannabinoid receptors. Biochem Pharmacol 1999;57:417e23. 56. Blankman JL, Simon GM, Cravatt BF. A comprehensive profile of brain enzymes that hydrolyze the endocannabinoid 2-arachidonoylglycerol. Chem Biol 2007;14:1347e56.

L.-C. Chiou et al. 57. Walker JM, Strangman NM, Huang SM. Cannabinoids and pain. Pain Res Manag Summer 2001;6:74e9. 58. Bloom AS, Dewey WL, Harris LS, Brosius KK. 9-nor-9beta-hydroxyhexahydrocannabinol, a cannabinoid with potent antinociceptive activity: comparisons with morphine. J Pharmacol Exp Ther 1977;200:263e70. 59. Tsou K, Lowitz KA, Hohmann AG, Martin WJ, Hathaway CB, Bereiter DA, et al. Suppression of noxious stimulus-evoked expression of Fos protein-like immunoreactivity in rat spinal cord by a selective cannabinoid agonist. Neuroscience 1996;70:791e8. 60. Hohmann AG, Martin WJ, Tsou K, Walker JM. Inhibition of noxious stimulusevoked activity of spinal cord dorsal horn neurons by the cannabinoid WIN 55,212-2. Life Sci 1995;56:2111e8. 61. Hohmann AG, Tsou K, Walker JM. Intrathecal cannabinoid administration suppresses noxious stimulus-evoked Fos protein-like immunoreactivity in rat spinal cord: comparison with morphine. Zhongguo Yao Li Xue Bao 1999;20: 1132e6. 62. Agarwal N, Pacher P, Tegeder I, Amaya F, Constantin CE, Brenner GJ, et al. Cannabinoids mediate analgesia largely via peripheral type 1 cannabinoid receptors in nociceptors. Nat Neurosci 2007;10:870e9. 63. Calignano A, La Rana G, Giuffrida A, Piomelli D. Control of pain initiation by endogenous cannabinoids. Nature 1998;394:277e81. 64. Richardson JD, Kilo S, Hargreaves KM. Cannabinoids reduce hyperalgesia and inflammation via interaction with peripheral CB1 receptors. Pain 1998;75: 111e9. 65. Herkenham M, Lynn AB, Johnson MR, Melvin LS, de Costa BR, Rice KC. Characterization and localization of cannabinoid receptors in rat brain: a quantitative in vitro autoradiographic study. J Neurosci 1991;11:563e83. 66. Martin WJ, Patrick SL, Coffin PO, Tsou K, Walker JM. An examination of the central sites of action of cannabinoid-induced antinociception in the rat. Life Sci 1995;56:2103e9. 67. Martin WJ, Hohmann AG, Walker JM. Suppression of noxious stimulus-evoked activity in the ventral posterolateral nucleus of the thalamus by a cannabinoid agonist: correlation between electrophysiological and antinociceptive effects. J Neurosci 1996;16:6601e11. 68. Nackley AG, Suplita 2nd RL, Hohmann AG. A peripheral cannabinoid mechanism suppresses spinal fos protein expression and pain behavior in a rat model of inflammation. Neuroscience 2003;117:659e70. 69. Reynolds DV. Surgery in the rat during electrical analgesia induced by focal brain stimulation. Science 1969;164:444e5. 70. Millan MJ. Descending control of pain. Prog Neurobiol 2002;66:355e474. 71. Lichtman AH, Cook SA, Martin BR. Investigation of brain sites mediating cannabinoid-induced antinociception in rats: evidence supporting periaqueductal gray involvement. J Pharmacol Exp Ther 1996;276:585e93. 72. Tsou K, Brown S, Sanudo-Pena MC, Mackie K, Walker JM. Immunohistochemical distribution of cannabinoid CB1 receptors in the rat central nervous system. Neuroscience 1998;83:393e411. 73. Vaughan CW, Connor M, Bagley EE, Christie MJ. Actions of cannabinoids on membrane properties and synaptic transmission in rat periaqueductal gray neurons in vitro. Mol Pharmacol 2000;57:288e95. 74. Malan Jr TP, Ibrahim MM, Deng H, Liu Q, Mata HP, Vanderah T, et al. CB2 cannabinoid receptor-mediated peripheral antinociception. Pain 2001;93: 239e45. 75. Nackley AG, Makriyannis A, Hohmann AG. Selective activation of cannabinoid CB(2) receptors suppresses spinal fos protein expression and pain behavior in a rat model of inflammation. Neuroscience 2003;119:747e57. 76. Whiteside GT, Gottshall SL, Boulet JM, Chaffer SM, Harrison JE, Pearson MS, et al. A role for cannabinoid receptors, but not endogenous opioids, in the antinociceptive activity of the CB2-selective agonist, GW405833. Eur J Pharmacol 2005;528:65e72. 77. Ibrahim MM, Rude ML, Stagg NJ, Mata HP, Lai J, Vanderah TW, et al. CB(2) cannabinoid receptor mediation of antinociception. Pain 2006;122:36e42. 78. Iwamura H, Suzuki H, Ueda Y, Kaya T, Inaba T. In vitro and in vivo pharmacological characterization of JTE-907, a novel selective ligand for cannabinoid CB2 receptor. J Pharmacol Exp Ther 2001;296:420e5. 79. Zhang J, Hoffert C, Vu HK, Groblewski T, Ahmad S, O’Donnell D. Induction of CB2 receptor expression in the rat spinal cord of neuropathic but not inflammatory chronic pain models. Eur J Neurosci 2003;17:2750e4. 80. Wotherspoon G, Fox A, McIntyre P, Colley S, Bevan S, Winter J. Peripheral nerve injury induces cannabinoid receptor 2 protein expression in rat sensory neurons. Neuroscience 2005;135:235e45. 81. Pereira A, Chappell A, Dethy J, Hoeck H, Arendt-Nielsen L, Verfaille S, et al. A proof-of-concept (POC) study including experimental pain models (EPMs) to assess the effects of a CB2 agonist (LY2828360) in the treatment of patients with osteoarthritic (OA) knee pain. Clin Pharmacol Thera 2013;93(S1):S56e7. No. PII-11. 82. Rinaldi-Carmona M, Barth F, Heaulme M, Shire D, Calandra B, Congy C, et al. SR141716A, a potent and selective antagonist of the brain cannabinoid receptor. FEBS Lett 1994;350:240e4. 83. Landsman RS, Burkey TH, Consroe P, Roeske WR, Yamamura HI. SR141716A is an inverse agonist at the human cannabinoid CB1 receptor. Eur J Pharmacol 1997;334:R1e2. 84. Sink KS, McLaughlin PJ, Wood JA, Brown C, Fan P, Vemuri VK, et al. The novel cannabinoid CB1 receptor neutral antagonist AM4113 suppresses food intake and food-reinforced behavior but does not induce signs of nausea in rats. Neuropsychopharmacology 2008;33:946e55.

Cannabinoids and pain regulation 85. Richardson JD, Aanonsen L, Hargreaves KM. SR 141716A, a cannabinoid receptor antagonist, produces hyperalgesia in untreated mice. Eur J Pharmacol 1997;319:R3e4. 86. Dogrul A, Gardell LR, Ma S, Ossipov MH, Porreca F, Lai J. ‘Knock-down’ of spinal CB1 receptors produces abnormal pain and elevates spinal dynorphin content in mice. Pain 2002;100:203e9. 87. Zimmer A, Zimmer AM, Hohmann AG, Herkenham M, Bonner TI. Increased mortality, hypoactivity, and hypoalgesia in cannabinoid CB1 receptor knockout mice. Proc Natl Acad Sci U S A 1999;96:5780e5. 88. Ledent C, Valverde O, Cossu G, Petitet F, Aubert JF, Beslot F, et al. Unresponsiveness to cannabinoids and reduced addictive effects of opiates in CB1 receptor knockout mice. Science 1999;283:401e4. 89. Ibrahim MM, Deng H, Zvonok A, Cockayne DA, Kwan J, Mata HP, et al. Activation of CB2 cannabinoid receptors by AM1241 inhibits experimental neuropathic pain: pain inhibition by receptors not present in the CNS. Proc Natl Acad Sci U S A 2003;100:10529e33. 90. Beaulieu P, Bisogno T, Punwar S, Farquhar-Smith WP, Ambrosino G, Di Marzo V, et al. Role of the endogenous cannabinoid system in the formalin test of persistent pain in the rat. Eur J Pharmacol 2000;396:85e92. 91. Lichtman AH, Shelton CC, Advani T, Cravatt BF. Mice lacking fatty acid amide hydrolase exhibit a cannabinoid receptor-mediated phenotypic hypoalgesia. Pain 2004;109:319e27. 92. Clayton N, Marshall FH, Bountra C, O’Shaughnessy CT. CB1 and CB2 cannabinoid receptors are implicated in inflammatory pain. Pain 2002;96:253e60. 93. Racz I, Nadal X, Alferink J, Baños JE, Rehnelt J, Martín M, et al. Interferongamma is a critical modulator of CB(2) cannabinoid receptor signaling during neuropathic pain. J Neurosci 2008;28:12136e45. 94. Racz I, Nadal X, Alferink J, Baños JE, Rehnelt J, Martín M, et al. Crucial role of CB(2) cannabinoid receptor in the regulation of central immune responses during neuropathic pain. J Neurosci 2008;28:12125e35. 95. Sit S, Xie K. Inventors. Bisarylimidazolyl fatty acid amide hydrolase inhibitors. Princeton, NJ: Bristol-Myers Squibb Company, US20020188009-A1; 2002. 96. Sit S, Xie K. Inventors. (Oxime) carbamoyl fatty acid amide hydrolase inhibitors. Princeton, NJ: Bristol-Myers Squibb Company, US2003003222; 2003. 97. Lichtman AH, Leung D, Shelton CC, Saghatelian A, Hardouin C, Boger DL, et al. Reversible inhibitors of fatty acid amide hydrolase that promote analgesia: evidence for an unprecedented combination of potency and selectivity. J Pharmacol Exp Ther 2004;311:441e8. 98. Kinsey SG, Long JZ, O’Neal ST, Abdullah RA, Poklis JL, Boger DL, et al. Blockade of endocannabinoid-degrading enzymes attenuates neuropathic pain. J Pharmacol Exp Ther 2009;330:902e10. 99. Ahn K, Johnson DS, Mileni M, Beidler D, Long JZ, McKinney MK, et al. Discovery and characterization of a highly selective FAAH inhibitor that reduces inflammatory pain. Chem Biol 2009;16:411e20. 100. Booker L, Kinsey SG, Abdullah RA, Blankman JL, Long JZ, Ezzili C, et al. The fatty acid amide hydrolase (FAAH) inhibitor PF-3845 acts in the nervous system to reverse LPS-induced tactile allodynia in mice. Br J Pharmacol 2012;165:2485e96. 101. Schlosburg JE, Blankman JL, Long JZ, Nomura DK, Pan B, Kinsey SG, et al. Chronic monoacylglycerol lipase blockade causes functional antagonism of the endocannabinoid system. Nat Neurosci 2010;13:1113e9. 102. Sasso O, Bertorelli R, Bandiera T, Scarpelli R, Colombano G, Armirotti A, et al. Peripheral FAAH inhibition causes profound antinociception and protects against indomethacin-induced gastric lesions. Pharmacol Res 2012;65:553e63. 103. Guindon J, Lai Y, Takacs SM, Bradshaw HB, Hohmann AG. Alterations in endocannabinoid tone following chemotherapy-induced peripheral neuropathy: effects of endocannabinoid deactivation inhibitors targeting fatty-acid amide hydrolase and monoacylglycerol lipase in comparison to reference analgesics following cisplatin treatment. Pharmacol Res 2013;67:94e109. 104. Ahn K, Smith SE, Liimatta MB, Beidler D, Sadagopan N, Dudley DT, et al. Mechanistic and pharmacological characterization of PF-04457845: a highly potent and selective fatty acid amide hydrolase inhibitor that reduces inflammatory and noninflammatory pain. J Pharmacol Exp Ther 2011;338:114e 24. 105. Johnson DS, Stiff C, Lazerwith SE, Kesten SR, Fay LK, Morris M, et al. Discovery of pf-04457845: a highly potent, orally bioavailable, and selective urea FAAH inhibitor. ACS Med Chem Lett 2011;2:91e6. 106. Huggins JP, Smart TS, Langman S, Taylor L, Young T. An efficient randomised, placebo-controlled clinical trial with the irreversible fatty acid amide hydrolase-1 inhibitor PF-04457845, which modulates endocannabinoids but fails to induce effective analgesia in patients with pain due to osteoarthritis of the knee. Pain 2012;153:1837e46. 107. Di Marzo V. Inhibitors of endocannabinoid breakdown for pain: not so FA(AH) cile, after all. Pain 2012;153:1785e6. 108. Scott CW, Tian G, Yu XH, Paschetto KA, Wilkins DE, Meury L, et al. Biochemical characterization and in vitro activity of AZ513, a noncovalent, reversible, and noncompetitive inhibitor of fatty acid amide hydrolase. Eur J Pharmacol 2011;667:74e9. 109. Desroches J, Guindon J, Lambert C, Beaulieu P. Modulation of the antinociceptive effects of 2-arachidonoyl glycerol by peripherally administered FAAH and MGL inhibitors in a neuropathic pain model. Br J Pharmacol 2008;155:913e24. 110. Long JZ, Li W, Booker L, Burston JJ, Kinsey SG, Schlosburg JE, et al. Selective blockade of 2-arachidonoylglycerol hydrolysis produces cannabinoid behavioral effects. Nat Chem Biol 2009;5:37e44.

169 111. Chanda PK, Gao Y, Mark L, Btesh J, Strassle BW, Lu P, et al. Monoacylglycerol lipase activity is a critical modulator of the tone and integrity of the endocannabinoid system. Mol Pharmacol 2010;78:996e1003. 112. Lichtman AH, Hawkins EG, Griffin G, Cravatt BF. Pharmacological activity of fatty acid amides is regulated, but not mediated, by fatty acid amide hydrolase in vivo. J Pharmacol Exp Ther 2002;302:73e9. 113. Hillard CJ. Biochemistry and pharmacology of the endocannabinoids arachidonylethanolamide and 2-arachidonylglycerol. Prostaglandins Other Lipid Mediat 2000;61:3e18. 114. Sugiura T, Kondo S, Kishimoto S, Miyashita T, Nakane S, Kodaka T, et al. Evidence that 2-arachidonoylglycerol but not N-palmitoylethanolamine or anandamide is the physiological ligand for the cannabinoid CB2 receptor. Comparison of the agonistic activities of various cannabinoid receptor ligands in HL-60 cells. J Biol Chem 2000;275:605e12. 115. Mackie K, Devane WA, Hille B. Anandamide, an endogenous cannabinoid, inhibits calcium currents as a partial agonist in N18 neuroblastoma cells. Mol Pharmacol. 1993;44:498e503. 116. Busquets-Garcia A, Puighermanal E, Pastor A, de la Torre R, Maldonado R, Ozaita A. Differential role of anandamide and 2-arachidonoylglycerol in memory and anxiety-like responses. Biol Psychiatry 2011;70:479e86. 117. Ghosh S, Wise LE, Chen Y, Gujjar R, Mahadevan A, Cravatt BF, et al. The monoacylglycerol lipase inhibitor JZL184 suppresses inflammatory pain in the mouse carrageenan model. Life Sci 2013;92:498e505. 118. Kinsey SG, Wise LE, Ramesh D, Abdullah R, Selley DE, Cravatt BF, et al. Repeated low-dose administration of the monoacylglycerol lipase inhibitor JZL184 retains cannabinoid receptor type 1-mediated antinociceptive and gastroprotective effects. J Pharmacol Exp Ther 2013;345:492e501. 119. Kano M, Ohno-Shosaku T, Hashimotodani Y, Uchigashima M, Watanabe M. Endocannabinoid-mediated control of synaptic transmission. Physiol Rev 2009;89:309e80. 120. Gregg LC, Jung KM, Spradley JM, Nyilas R, Suplita 2nd RL, Zimmer A, et al. Activation of type 5 metabotropic glutamate receptors and diacylglycerol lipase-alpha initiates 2-arachidonoylglycerol formation and endocannabinoid-mediated analgesia. J Neurosci 2012;32:9457e68. 121. Liao HT, Lee HJ, Ho YC, Chiou LC. Capsaicin in the periaqueductal gray induces analgesia via metabotropic glutamate receptor-mediated endocannabinoid retrograde disinhibition. Br J Pharmacol 2011;163:330e45. 122. Drew GM, Lau BK, Vaughan CW. Substance P drives endocannabinoidmediated disinhibition in a midbrain descending analgesic pathway. J Neurosci 2009;29:7220e9. 123. Drew GM, Mitchell VA, Vaughan CW. Glutamate spillover modulates GABAergic synaptic transmission in the rat midbrain periaqueductal grey via metabotropic glutamate receptors and endocannabinoid signaling. J Neurosci 2008;28:808e15. 124. Mitchell VA, Jeong HJ, Drew GM, Vaughan CW. Cholecystokinin exerts an effect via the endocannabinoid system to inhibit GABAergic transmission in midbrain periaqueductal gray. Neuropsychopharmacology 2011;36:1801e10. 125. Mitchell VA, Kawahara H, Vaughan CW. Neurotensin inhibition of GABAergic transmission via mGluR-induced endocannabinoid signalling in rat periaqueductal grey. J Physiol 2009;587(Pt 11):2511e20. 126. Lau BK, Vaughan CW. Muscarinic modulation of synaptic transmission via endocannabinoid signalling in the rat midbrain periaqueductal gray. Mol Pharmacol 2008;74:1392e8. 127. Ho YC, Lee HJ, Tung LW, Liao YY, Fu SY, Teng SF, et al. Activation of orexin 1 receptors in the periaqueductal gray of male rats leads to antinociception via retrograde endocannabinoid (2-arachidonoylglycerol)-induced disinhibition. J Neurosci 2011;31:14600e10. 128. Riedel G, Wetzel W, Reymann KG. Comparing the role of metabotropic glutamate receptors in long-term potentiation and in learning and memory. Prog Neuropsychopharmacol Biol Psychiatry 1996;20:761e89. 129. Conn PJ, Pin JP. Pharmacology and functions of metabotropic glutamate receptors. Annu Rev Pharmacol Toxicol 1997;37:205e37. 130. Pin JP, Duvoisin R. The metabotropic glutamate receptors: structure and functions. Neuropharmacology 1995;34:1e26. 131. Drew GM, Vaughan CW. Multiple metabotropic glutamate receptor subtypes modulate GABAergic neurotransmission in rat periaqueductal grey neurons in vitro. Neuropharmacology 2004;46:927e34. 132. Nusser Z, Mulvihill E, Streit P, Somogyi P. Subsynaptic segregation of metabotropic and ionotropic glutamate receptors as revealed by immunogold localization. Neuroscience 1994;61:421e7. 133. Rosen A, Zhang YX, Lund I, Lundeberg T, Yu LC. Substance P microinjected into the periaqueductal gray matter induces antinociception and is released following morphine administration. Brain Res 2004;1001:87e94. 134. Kalivas PW, Jennes L, Nemeroff CB, Prange Jr AJ. Neurotensin: topographical distribution of brain sites involved in hypothermia and antinociception. J Comp Neurol 1982;210:225e38. 135. Chen XH, Geller EB, Adler MW. CCK(B) receptors in the periaqueductal grey are involved in electroacupuncture antinociception in the rat cold water tailflick test. Neuropharmacology 1998;37:751e7. 136. Palazzo E, de Novellis V, Marabese I, Cuomo D, Rossi F, Berrino L, et al. Interaction between vanilloid and glutamate receptors in the central modulation of nociception. Eur J Pharmacol 2002;439:69e75. 137. Starowicz K, Maione S, Cristino L, Palazzo E, Marabese I, Rossi F, et al. Tonic endovanilloid facilitation of glutamate release in brainstem descending antinociceptive pathways. J Neurosci 2007;27:13739e49.

170 138. Woolf NJ, Harrison JB, Buchwald JS. Cholinergic neurons of the feline pontomesencephalon. II. Ascending anatomical projections. Brain Res 1990;520: 55e72. 139. Monassi CR, Hoffmann A, Menescal-de-Oliveira L. Involvement of the cholinergic system and periaqueductal gray matter in the modulation of tonic immobility in the guinea pig. Physiol Behav 1997;62:53e9. 140. Guimaraes AP, Guimaraes FS, Prado WA. Modulation of carbachol-induced antinociception from the rat periaqueductal gray. Brain Res Bull 2000;51: 471e8. 141. Sakurai T, Amemiya A, Ishii M, Matsuzaki I, Chemelli RM, Tanaka H, et al. Orexins and orexin receptors: a family of hypothalamic neuropeptides and G protein-coupled receptors that regulate feeding behavior. Cell 1998;92:573e85. 142. de Lecea L, Kilduff TS, Peyron C, Gao X, Foye PE, Danielson PE, et al. The hypocretins: hypothalamus-specific peptides with neuroexcitatory activity. Proc Natl Acad Sci U S A 1998;95:322e7. 143. Peyron C, Tighe DK, van den Pol AN, de Lecea L, Heller HC, Sutcliffe JG, et al. Neurons containing hypocretin (orexin) project to multiple neuronal systems. J Neurosci 1998;18:9996e10015. 144. Horvath TL, Peyron C, Diano S, Ivanov A, Aston-Jones G, Kilduff TS, et al. Hypocretin (orexin) activation and synaptic innervation of the locus coeruleus noradrenergic system. J Comp Neurol 1999;415:145e59. 145. van den Pol AN. Hypothalamic hypocretin (orexin): robust innervation of the spinal cord. J Neurosci 1999;19:3171e82. 146. Tsujino N, Sakurai T. Orexin/hypocretin: a neuropeptide at the interface of sleep, energy homeostasis, and reward system. Pharmacol Rev 2009;61:162e76. 147. Chiou LC, Lee HJ, Ho YC, Chen SP, Liao YY, Ma CH, et al. Orexins/hypocretins: pain regulation and cellular actions. Curr Pharm Des 2010;16:3089e100. 148. Maione S, Bisogno T, de Novellis V, Palazzo E, Cristino L, Valenti M, et al. Elevation of endocannabinoid levels in the ventrolateral periaqueductal grey through inhibition of fatty acid amide hydrolase affects descending nociceptive pathways via both cannabinoid receptor type 1 and transient receptor potential vanilloid type-1 receptors. J Pharmacol Exp Ther 2006;316:969e82. 149. Kawahara H, Drew GM, Christie MJ, Vaughan CW. Inhibition of fatty acid amide hydrolase unmasks CB1 receptor and TRPV1 channel-mediated modulation of glutamatergic synaptic transmission in midbrain periaqueductal grey. Br J Pharmacol 2011;163:1214e22. 150. Hohmann AG, Suplita RL, Bolton NM, Neely MH, Fegley D, Mangieri R, et al. An endocannabinoid mechanism for stress-induced analgesia. Nature 2005;435: 1108e12. 151. Flor H, Birbaumer N, Schulz R, Grusser SM, Mucha RF. Pavlovian conditioning of opioid and nonopioid pain inhibitory mechanisms in humans. Eur J Pain 2002;6:395e402. 152. Valverde O, Ledent C, Beslot F, Parmentier M, Roques BP. Reduction of stressinduced analgesia but not of exogenous opioid effects in mice lacking CB1 receptors. Eur J Neurosci 2000;12:533e9. 153. Olango WM, Roche M, Ford GK, Harhen B, Finn DP. The endocannabinoid system in the rat dorsolateral periaqueductal grey mediates fear-conditioned analgesia and controls fear expression in the presence of nociceptive tone. Br J Pharmacol 2012;165:2549e60. 154. Butler RK, Ford GK, Hogan M, Roche M, Doyle KM, Kelly JP, et al. Fear-induced suppression of nociceptive behaviour and activation of Akt signalling in the rat periaqueductal grey: role of fatty acid amide hydrolase. J Psychopharmacol 2012;26:83e91. 155. Watanabe S, Kuwaki T, Yanagisawa M, Fukuda Y, Shimoyama M. Persistent pain and stress activate pain-inhibitory orexin pathways. Neuroreport 2005;16:5e8. 156. Xie X, Wisor JP, Hara J, Crowder TL, LeWinter R, Khroyan TV, et al. Hypocretin/ orexin and nociceptin/orphanin FQ coordinately regulate analgesia in a mouse model of stress-induced analgesia. J Clin Invest 2008;118:2471e81. 157. Chiou LC, Teng SF, Chang LY. Restraint stress induces analgesia through endogenous orexins via OX1 receptor-mediated endocannabinoid retrograde disinhibition in the ventrolateral periaqueductal gray. San Diego, CA: Annual meeting of neuroscience; November, 2010. Prog. No. 844.1. 158. Holden JE, Pizzi JA, Jeong Y. An NK1 receptor antagonist microinjected into the periaqueductal gray blocks lateral hypothalamic-induced antinociception in rats. Neurosci Lett 2009;453:115e9.

L.-C. Chiou et al. 159. Gui X, Carraway RE, Dobner PR. Endogenous neurotensin facilitates visceral nociception and is required for stress-induced antinociception in mice and rats. Neuroscience 2004;126:1023e32. 160. Ottani A, Leone S, Sandrini M, Ferrari A, Bertolini A. The analgesic activity of paracetamol is prevented by the blockade of cannabinoid CB1 receptors. Eur J Pharmacol 2006;531:280e1. 161. Mallet C, Daulhac L, Bonnefont J, Ledent C, Etienne M, Chapuy E, et al. Endocannabinoid and serotonergic systems are needed for acetaminopheninduced analgesia. Pain 2008;139:190e200. 162. Hogestatt ED, Jonsson BA, Ermund A, Andersson DA, Björk H, Alexander JP, et al. Conversion of acetaminophen to the bioactive N-acylphenolamine AM404 via fatty acid amide hydrolase-dependent arachidonic acid conjugation in the nervous system. J Biol Chem 2005;280:31405e12. 163. Zygmunt PM, Chuang H, Movahed P, Julius D, Hogestatt ED. The anandamide transport inhibitor AM404 activates vanilloid receptors. Eur J Pharmacol 2000;396:39e42. 164. Mallet C, Barriere DA, Ermund A, Jönsson BAG, Eschalier A, Zygmunt PM, et al. TRPV1 in brain is involved in acetaminophen-induced antinociception. PLoS One 2010;5:e12748. 165. Chiou LC, Lee HJ, Tung LW. Acetaminophen induces analgesia via the mGluR5mediated endocannabinoid retrograde disinhibition mechanism initiated by TRPV1 activation in the periaqueductal gray 639.29/C30. 42nd Annual Meeting of Neuroscience; 2012. New Orleans. 166. Costa B, Colleoni M. SR141716A induces in rats a behavioral pattern opposite to that of CB1 receptor agonists. Zhongguo Yao Li Xue Bao. 1999;20:1103e8. 167. Chapman V. The cannabinoid CB1 receptor antagonist, SR141716A, selectively facilitates nociceptive responses of dorsal horn neurones in the rat. Br J Pharmacol 1999;127:1765e7. 168. Strangman NM, Patrick SL, Hohmann AG, Tsou K, Walker JM. Evidence for a role of endogenous cannabinoids in the modulation of acute and tonic pain sensitivity. Brain Res 1998;813:323e8. 169. Richardson JD, Aanonsen L, Hargreaves KM. Hypoactivity of the spinal cannabinoid system results in NMDA-dependent hyperalgesia. J Neurosci 1998;18:451e7. 170. Kathuria S, Gaetani S, Fegley D, Valiño F, Duranti A, Tontini A, et al. Modulation of anxiety through blockade of anandamide hydrolysis. Nat Med 2003;9:76e81. 171. Haller VL, Stevens DL, Welch SP. Modulation of opioids via protection of anandamide degradation by fatty acid amide hydrolase. Eur J Pharmacol 2008;600:50e8. 172. Hasanein P, Shahidi S, Komaki A, Mirazi N. Effects of URB597 as an inhibitor of fatty acid amide hydrolase on modulation of nociception in a rat model of cholestasis. Eur J Pharmacol 2008;591:132e5. 173. Russo R, Loverme J, La Rana G, Compton TR, Parrott J, Duranti A, et al. The fatty acid amide hydrolase inhibitor URB597 (cyclohexylcarbamic acid 3’-carbamoylbiphenyl-3-yl ester) reduces neuropathic pain after oral administration in mice. J Pharmacol Exp Ther 2007;322:236e42. 174. Kinsey SG, Long JZ, Cravatt BF, Lichtman AH. Fatty acid amide hydrolase and monoacylglycerol lipase inhibitors produce anti-allodynic effects in mice through distinct cannabinoid receptor mechanisms. J Pain 2010;11:1420e8. 175. Long JZ, Nomura DK, Vann RE, Walentiny DM, Booker L, Jin X, et al. Dual blockade of FAAH and MAGL identifies behavioral processes regulated by endocannabinoid crosstalk in vivo. Proc Natl Acad Sci U S A 2009;106: 20270e5. 176. Woodhams SG, Wong A, Barrett DA, Bennett AJ, Chapman V, Alexander SP. Spinal administration of the monoacylglycerol lipase inhibitor JZL184 produces robust inhibitory effects on nociceptive processing and the development of central sensitization in the rat. Br J Pharmacol 2012;167:1609e19. 177. Khasabova IA, Chandiramani A, Harding-Rose C, Simone DA, Seybold VS. Increasing 2-arachidonoyl glycerol signaling in the periphery attenuates mechanical hyperalgesia in a model of bone cancer pain. Pharmacol Res 2011;64:60e7. 178. Guindon J, Guijarro A, Piomelli D, Hohmann AG. Peripheral antinociceptive effects of inhibitors of monoacylglycerol lipase in a rat model of inflammatory pain. Br J Pharmacol 2011;163:1464e78.