Psychopharmacology (2015) 232:583–593 DOI 10.1007/s00213-014-3696-x
ORIGINAL INVESTIGATION
Effect of selective inhibition of monoacylglycerol lipase (MAGL) on acute nausea, anticipatory nausea, and vomiting in rats and Suncus murinus Linda A. Parker & Micah J. Niphakis & Rachel Downey & Cheryl L. Limebeer & Erin M. Rock & Martin A. Sticht & Heather Morris & Rehab A. Abdullah & Aron H. Lichtman & Benjamin F. Cravatt
Received: 27 May 2014 / Accepted: 12 July 2014 / Published online: 3 August 2014 # Springer-Verlag Berlin Heidelberg 2014
Abstract Rationale To determine the role of the endocannabinoid, 2arachodonyl glycerol (2-AG), in the regulation of nausea and vomiting. Objective We evaluated the effectiveness of the potent selective monoacylglycerol lipase (MAGL) inhibitor, MJN110, which selectively elevates the endocannabinoid 2-AG, to suppress acute nausea and vomiting, as well as anticipatory nausea in rat and shrew models. Methods The rat gaping models were used to evaluate the potential of MJN110 (5, 10, and 20 mg/kg, intraperitoneally [IP]) to suppress acute nausea produced by LiCl and of MJN110 (10 and 20 mg/kg, IP) to suppress anticipatory nausea elicited by a LiCl-paired context. The potential as well of MJN110 (10 and 20 mg/kg, IP) to suppress vomiting and contextually elicited gaping in the Suncus murinus was evaluated. Results MJN110 suppressed acute nausea in rats, LiClinduced vomiting in shrews and contextually-elicited anticipatory nausea in both rats (accompanied by elevation of 2-AG in the visceral insular cortex) and shrews. These effects were L. A. Parker (*) : R. Downey : C. L. Limebeer : E. M. Rock : M. A. Sticht : H. Morris Department of Psychology and Collaborative Neuroscience Program, University of Guelph, Guelph, ON N1G2W1, Canada e-mail: [email protected] M. J. Niphakis : B. F. Cravatt The Skaggs Institute for Chemical Biology and Department of Chemical Physiology, The Scripps Research Institute, La Jolla, CA 92037, USA R. A. Abdullah : A. H. Lichtman Department of Pharmacology and Toxicology, Medical College of Virginia Campus, Virginia Commonwealth University, Richmond, VA, USA
reversed by the CB1 antagonist/inverse agonist, SR141716. The MAGL inhibitor did not modify locomotion at any dose. An activity-based protein profiling analysis of samples of tissue collected from the visceral insular cortex in rats and whole brain tissues in shrews revealed that MJN110 selectively inhibited MAGL and the alternative 2-AG hydrolase, ABHD6. Conclusions MAGL inhibition by MJN110 which selectively elevates endogenous 2-AG has therapeutic potential in the treatment of acute nausea and vomiting as well as anticipatory nausea, a distressful symptom that is resistant to currently available treatments. Keywords MAGL . Nausea . Vomiting . Rat . 2-AG . Shrew . Gaping . Conditioned response . ABPP . ABHD6 The sensation of nausea is one of the most debilitating human experiences. In chemotherapy patients, nausea frequently occurs following treatment (acute and delayed nausea), but it also happens in anticipation of treatment (anticipatory nausea). Because nausea is poorly understood, effective treatments are very limited. Current anti-emetic therapies are highly effective in reducing chemotherapy-induced vomiting, but they are only somewhat effective in treating chemotherapyinduced acute and delayed nausea (Ballatori et al. 2007; de Boer-Dennert et al. 1997; Foubert and Vaessen 2005; Hickok et al. 2003; Morrow et al. 2002; Roscoe et al. 2000). When anticipatory nausea develops, current anti-emetic agents are almost completely ineffective. Anticipatory nausea is commonly treated with non-specific anti-anxiety drugs, such as benzodiazepines (Malik et al. 1995; Razavi et al. 1993); however, benzodiazepines have sedating side effects, which severely limit their usefulness. Therefore, new treatments for both acute and anticipatory nausea are urgently required.
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Animal models of vomiting have been valuable in elucidating the neural mechanisms of the emetic reflex (Hornby 2001). However, the central neurochemical regulation of nausea is still not well understood (Andrews and Horn 2006), because, until recently, a reliable, selective animal model of nausea had not been established. We have developed such a model in the rat and have used it to explore the neural circuits (e.g., the visceral insular cortex [VIC]; Tuerke et al. 2012; Limebeer et al. 2012) involved in the regulation of nausea. Rats do not vomit (Horn et al. 2013), but they display conditioned gaping reactions (the wide opening of the mouth exposing the lower incisors) when exposed to a flavor (Grill and Norgren 1978) that has previously been paired with nausea (see Parker 2014 for review). As well, drugs that prevent vomiting in species capable of vomiting also prevent the establishment of conditioned gaping in rats by interfering with acute nausea during the conditioning trial (Parker 2014). Conditioned gaping requires similar orofacial musculature as vomiting in emetic species (Travers and Norgren 1986) and is topographically similar to the orofacial components of retching in the shrew (Parker et al 2009). We have capitalized on this behavioral response to develop a model of acute nausea in rats (Parker 2014). Conditioned gaping reactions are not only elicited by LiClpaired flavors, but they are also elicited by LiCl-paired contextual cues (albeit following several pairings) (Limebeer et al. 2013; 2006; 2008; Rock et al. 2008) revealing a model remarkably similar to that of anticipatory nausea in humans. Indeed, shrews, which vomit in response to an emetic treatment, also display conditioned gaping reactions when reintroduced to the context in which they previously vomited. Interestingly, 5-HT3 antagonists are ineffective in suppressing the expression of contextually elicited gaping reactions, as they are ineffective in suppressing anticipatory nausea in humans (Limebeer et al. 2006; Parker et al. 2006; Parker and Kemp 2001). On the other hand, cannabinoid compounds (Bolognini et al. 2013; Limebeer et al. 2013; Limebeer et al. 2006; Parker et al. 2006; Parker and Kemp 2001; Rock et al. 2008) effectively interfere with the expression of contextually-elicited conditioned gaping, suggesting that they may be successful in treating anticipatory nausea in humans. The endocannabinoid (EC) system is intimately involved in the regulation of vomiting (Kwiatkowska et al. 2004; Parker et al. 2004; 2011; Sharkey and Kroese 2001; Van Sickle et al. 2001, 2003, 2005) and both acute and anticipatory nausea (see Parker et al 2011). Cannabinoid (CB) receptors are part of a central system of lipid transmitters, the EC system, which consists of the CB1 and CB2 receptor, the ligands anandamide (AEA) (Devane et al. 1992) and 2arachidonyl glycerol (2-AG) (Mechoulam et al. 1995) and enzymes for biosynthesis and hydrolysis of the ligands (Cravatt et al. 1996; Deutsch et al. 2002; Dinh et al. 2004). Unlike most neurotransmitters, AEA and 2-AG are not stored
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in vesicles but rather are synthesized when and where they are needed. Again, unlike most neurotransmitters, their action is not postsynaptic but rather mostly presynaptic, i.e., they serve as retrograde synaptic messengers inhibiting the release of other neurotransmitters (Mechoulam and Parker 2013). Contrary to Δ9–tetrahydrocannabinol (THC), which is metabolized over several hours and excreted (or stored as one of its metabolites), endocannabinoids are rapidly removed by a membrane transport process yet to be fully characterized (Fu et al. 2012). In the postsynaptic cell, AEA is hydrolyzed to arachidonic acid and ethanolamine by fatty acid amide hydrolase (FAAH) (Cravatt et al. 1996; Deutsch et al. 2002). 2-AG i s a l s o h y d r o l y z e d e n z y m a t i c a l l y, p r i m a r i l y b y monoacylglycerol lipase (MAGL) (Blankman et al. 2007; Dinh et al. 2004; Niphakis et al. 2013; Windsor et al. 2012) found in presynaptic terminal endings. Suppression of these enzymes prolongs the activity of the respective endocannabinoid. Drugs which augment the endocannabinoid system have potential therapeutic value in the treatment of nausea (for review Parker 2014; Sharkey et al 2014). THC and other direct CB1 agonists are effective in treating nausea (Limebeer and Parker 1999; Parker and Mechoulam 2003), but their adverse effects on motor control and cognition limit their usefulness (see Mechoulam and Parker 2013). This has led to the pursuit of alternative strategies to selectively enhance endocannabinoid function. A highly attractive strategy is to inhibit FAAH or MAGL, elevating brain AEA or 2-AG, respectively, without cognitive or motor impairment. In the case of AEA, pharmacological inhibitors that selectively block intracellular hydrolysis by FAAH (e.g., URB597; PF3845) have been extensively used, leading to a better understanding of its role in the regulation of nausea and vomiting. We have shown that URB597 suppresses both acute (Cross-Mellor et al. 2007) and anticipatory nausea (Rock et al. 2008) in rats as well as LiCl-induced vomiting (Parker et al. 2009) and anticipatory retching (wide gaping; Parker et al. 2006) in musk shrews. As well, URB597 has been shown to reduce vomiting in ferrets (Sharkey et al. 2007). We have also recently shown that the dual FAAH/MAGL inhibitor, JZL195, alone and combined with AEA or 2-AG suppresses anticipatory nausea in rats in a CB1-dependent manner (Limebeer et al. 2013). The suppression of contextually elicited gaping by JZL195 was accompained by an elevation of AEA, but not 2-AG; however, when coadministered with AEA or 2-AG, both the suppression of gaping and the level of AEA and 2AG were respectively enhanced. Selective and in vivo-active MAGL inhibitors that do not cross-react with FAAH in the rat have only recently been developed (Chang et al. 2012; Niphakis et al. 2013) and have since opened new avenues for studying the role of 2-AG in mammalian physiology. One such inhibitor, MJN110, inhibits 2-AG hydrolysis in mouse and rat brain homogenates with a
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low nanomolar IC50 and has no effect on AEA hydrolysis up to 10 μM (Niphakis et al. 2013). MJN110 also displays excellent potency in vivo, inhibiting brain MAGL after administration of 1 mg/kg (intraperitoneally [IP]) in mice and 5 mg/kg (IP) in rat without affecting FAAH activity at doses up to 40 mg/kg (IP). While also targeting the alternative 2-AG hydrolase, ABHD6, MJN110 diplays high selectivity over other serine hydrolase enzymes and across the proteome (Niphakis et al. 2013; Chang et al. 2012). Owing to these factors, MJN110 is well-suited for studying the effects of MAGL blockade in rats. Indeed, this compound has been shown to alleviate mechanical allodynia in a rat model of diabetic neuropathy (Niphakis et al. 2013). Here, we evaluate the potential MJN110 to interfere with acute and anticipatory nausea in rats, as well as vomiting and anticipatory nausea in shrews. We first demostrate that MJN110 dose-dependently suppresses acute nausea produced by LiCl, by demonstrating attenuated gaping at test to a flavor previously paired with LiCl in rats pretreated with MJN110 during conditioning. This effect is reversed by the CB1 antagonist, SR141716A. Then, we show that MJN110 also interferes with the expression of previously established contextually elicited conditioned gaping in rats, as a measure of anticipatory nausea in rats. This effect is also reversed by CB1 antagonism. Finally, we demonstrate that MJN110 also dosedependently prevents LiCl-induced acute vomiting and contextually-elicited anticipatory gaping in the Suncus murinus. These findings demonstrate that drugs which prolong the action of 2-AG by blocking their rapid degradation through MAGL have promise as treatments for both acute and anticipatory nausea and vomiting.
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(lights off at 7 PM). Shrews were tested in their light cycle. They were maintained on Medi-cal/Royal Canine Feline Maintenance mixed with Harlan Ferret dry chow and water ad libitum. All shrews were weaned at 20 days of age. Drugs and materials MJN110 (2,5-dioxopyrrolidin-1-yl 4-(bis(4chlorophenyl)methyl)piperazine-1-carboxylate; Niphakis et al. 2013), PF-3845 (N-3-pyridinyl4-[[3-[[5-(trifluoromethyl)-2-pyridinyl]oxy]phenyl]methyl]1-piperidinecarboxamide; Ahn et al. 2009), KT195 (C28H28N4O2; Hsu et al. 2012), and fluorophosphosphonaterhodamine probe (FP-Rh; Patricelli et al. 2001) were synthesized as previously described. All injections were administered IP. A 0.15 M lithium chloride (LiCl; Sigma Aldrich) solution was prepared with sterile water and administered in a volume of 20 ml/kg (127.2 mg/kg). MJN110 (5, 10, or 20 mg/kg) and SR141617 (5-(4-chlorophenyl)-1-(2,4dichlorophenyl)-4-methyl-N-1-piperidinyl-1H-pyrazole-3carboxamide; 1 mg/kg; a dose that does not potentiate LiClinduced conditioned gaping or vomiting [O’Brien et al 2013]) were administered at 1 ml/kg (except 20 mg/kg MJN110 which was administered at 2 ml/kg [10 mg/ml]) and were prepared in a vehicle (VEH) consisting of a 1:1:18 mixture of ethanol, Tween 80 (Sigma), and saline (SAL). The drugs were first dissolved in ethanol then Tween 80 was added to the solution, and the ethanol was evaporated off with a nitrogen stream after which the saline was added. The final VEH consisted of 1:9 (Tween/saline) and was administered at 1 ml/kg. Tissue preparation for ABPP analysis
Methods Animals Animal procedures complied with the Canadian Council on Animal Care. The protocols were approved by the Institutional Animal Care Committee, which is accredited by the Canadian Council on Animal Care. Naive male Sprague–Dawley rats, weighing between 262 and 373 g on the first day of conditioning, obtained from Charles River Laboratories (St Constant, Quebec) were used for assessment of anti-nausealike behavior. They were pair-housed in Plexiglas cages (47× 26×20 cm) in a colony room at an ambient temperature of 21 °C with a 12/12 hour light–dark schedule (lights off at 8 AM) and maintained on food and water ad libitum. Naive male and female S. murinus (house musk shrews) ranged from 33 to 50 days old at the time of testing and were bred and raised in the University of Guelph colony. They were single-housed in opaque white mouse cages in the colony room at an ambient temperature of 21 °C on a 10/14 hour light–dark schedule
Frozen rat VIC tissue harvested from animals treated with either vehicle, MJN110 or PF-3845, was washed twice by adding cold DPBS (1.0 mL), centrifuging (3,000×g, 3 min), and removing the supernatant. Cold DPBS (300 μL) was then added to the washed VIC tissue which was then homogenized using a probe sonicator. Alternatively, hemisected shrew brains were Dounce-homogenized in cold DPBS (2.0 mL) and subsequently centrifuged (3,000×g, 3 min) to remove insoluble debris. Tissue homogenates from rat VIC and shrew brain were centrifuged at 100,000×g for 45 min and the pellets sonicated in DPBS (300 μL) to resuspend. Following the determination of their protein concentration using the DC Protein Assay (Bio-Rad), proteomes were diluted to 1.0 mg/ mL prior to activity-based protein profiling (ABPP) analysis. Ex vivo activity-based protein profiling Proteomes (1.0 mg/mL, 50 μL) were treated with FP-Rh (1.0 μL, 50 μM in dimethylsulfoxide (DMSO)), vortexed,
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and incubated at room temperature. After 30 min, each reaction was quenched with 4× sodium dodecyl sulfate (SDS) loading buffer (17 μL), and proteins were separated by SDSpolyacrylamide gel electrophoresis (PAGE). The gel was analyzed by fluorescent gel imaging on a Hitachi FMBio II flatbed scanner and activity of individual serine hydrolases assessed by comparing the fluorescent intensities of each gel band relative to samples derived from vehicle-treated animals.
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(326 > 62) and (326 > 309) for OEA; and (330 > 66) for OEAd4; in negative mode: (303>259) and (303>59) for AA and (311>267) for AA-d8. A calibration curve was constructed for each assay based on linear regression using the peak area ratios of the calibrators. The extracted standard curves ranged from 0.156 to 2.5 pmol for AEA, from 0.25 to 4 nmol for 2-AG, from 7.8 to 125 pmol for PEA and OEA, and from 1 to 16 nmol for AA. Experimental Procedures:
In vitro activity-based protein profiling of shrew brain Proteomes (1.0 mg/mL, 50 μL) derived from vehicle-treated shrew brains were treated with DMSO (1.0 μL), JZL184 (1.0 μL, 5.0 μM), KT195 (1.0 μL, 5.0 μM), or PF-3845 (1.0 μL, 5.0 μM) and incubated for 30 min at 37 °C. Each proteome was subsequently treated with FP-Rh (1.0 μL, 50 μM in DMSO), vortexed, and incubated at room temperature for 30 min. As above, after each reaction was quenched with 4× SDS loading buffer (17 μL), the proteins were separated by SDS-PAGE and analyzed by fluorescent gel imaging. Quantification of VIC endocannabinoids Lipid extractions were carried out according to previously described methods (Kinsey et al. 2013; Ramesh et al. 2011). Pre-weighed VIC samples were homogenized in 1.4 mL of buffer containing a 2:1 chloroform/methanol solution and internal standards (4 pmol AEA-d8, 1 nmol 2-AG-d8, 330 pmol PEA-d4, 300 pmol OEA-d4, and 1 nmol AA-d8), and 0.3 mL of saline (0.73 %) was added to each sample. The homogenates were subsequently vortexed for 1 min. Following centrifugation (10 min at 3,220×g; 4 °C), the aqueous phase and debris were separated and extracted another two times each with 0.8 mL of chloroform; the organic phase from each of the separations was pooled together, and the organic solvents were evaporated under a nitrogen stream (15 psi). After the samples were completely dried, they were reconstituted with 0.1 mL chloroform and, after vortexing, were mixed with 1 mL of cold acetone. Following another centrifugation (5 min at 1,811×g), the upper layer from each sample was collected and evaporated under nitrogen. The final dried constituents were reconstituted in 0.1 mL methanol and transferred to autosample vials for analysis. AEA, 2AG, OEA, PEA, and AA were quantified using liquid chromatography tandem mass spectrometry. The mobile phase consisted of methanol (90:10)/0.1 % ammonium acetate and 0.1 % formic acid. The column used was a Discovery® HS C18, 2.1×150 mm, 3 μm (Supelco, USA). Ions were analyzed in multiple-reaction monitoring mode, and the following transitions were monitored in positive mode— (348>62) and (348>91) for AEA; (356>62) for AEAd8; (379>287) and(379>269) for 2-AG; (387>96) for 2-AGd8; (300>62) and (300>283) for PEA; (304>62) for PEA-d4;
Experiment 1: effect of MAGL inhibition by MJN110 on acute nausea All rats were surgically implanted with an intraoral cannula under isofluorane anesthesia, according to the procedures described by Limebeer et al. (2012). The intraoral cannula, implanted to deliver fluids to the rat’s oral cavity, consisted of a length of Intramedic plastic tubing with an inner diameter of 0.86 mm and an outer diameter of 1.27 mm with two circular elastic discs placed over the tubing at the back of the neck. Three days after the surgery, the rats received a taste reactivity (TR) adaptation trial in which they were placed in the TR chamber with their cannula attached to an infusion pump (Model KDS100, KD Scientific, Holliston, MA, USA) for fluid delivery. The TR chambers were made of clear Plexiglas (22.5×26×20 cm) that sat on a table with a clear glass top. A mirror beneath the chamber on a 45° angle facilitated viewing of the ventral surface of the rat to observe orofacial responses. Water was infused into their intraoral cannulae for 2 min at the rate of 1 mL/min. On the day following the TR adaptation trial, the rats received the TR conditioning trial in which they were administered a pretreatment injection of VEH (n=9), 5 mg/kg MJN110 (n=7), 10 mg/kg MJN110 (n=8), and 20 mg/kg MJN110 (n=8) 2 h prior to the conditioning trial. An additional group (n=8) was injected with 10 mg/kg MJN110 90 min before 1.0 mg/kg SR141716, and 30 min later, received the conditioning trial. On the conditioning trial, the rats were individually placed in the TR chamber and intraorally infused with 0.1 % saccharin solution for 2 min at the rate of 1 mL/min while the orofacial responses were video recorded from a mirror at a 45° angle beneath the chambers, with the feed from the video camera (Sony DCR-HC48, Henry’s Cameras, Waterloo, ON, Canada) fire-wired into a computer. Immediately after the saccharin infusion, all rats were injected with 20 mL kg−1 of 0.15 M LiCl and returned to their home cage. Seventy-two hours following the TR conditioning trial, the rats were given a drug-free TR test trial. Rats were intraorally infused with 0.1 % saccharin solution for 2 min at the rate of 1 mL/min while their orofacial responses were video recorded. At 1600 h on the day of the TR test trial, the rats were water-restricted (water bottles removed from cage). Eighteen hours later, on the following morning, they were given the two-bottle conditioned taste avoidance test. Each rat
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was presented with two graduated tubes side by side with one containing 0.1 % saccharin solution (always on the left side) and one containing reverse osmosis water for 6 h. The amounts consumed from each tube were measured and converted into a saccharin preference ratio: Amount of saccharin consumed divided by the amount of saccharin + water consumed. The video tapes of the TR test trial were later scored (at ½ speed) by an observer blind to the experimental conditions using ‘The Observer’ (Noldus Information Technology Inc., Leesburg, VA, USA) for the number of occurrences of gaping (large openings of the mouth and jaw, with lower incisors exposed). The behavior of gaping is extremely easy to identify and score with inter-rater reliability ratings consistently falling in the range of r=0.95 to 0.99 when the scores of highly experience raters are compared with those of raters trained for only a few hours (see Parker 2014). Experiment 2: effect of MAGL inhibition by MJN110 on anticipatory nausea The rats received four conditioning trials, during which a distinctive contextual chamber was paired with a 20-mL/kg injection of 0.15 M LiCl. On each conditioning trial, each rat was injected with LiCl and immediately placed in the distinctive context for a 30-min period. This procedure occurred on a total of four conditioning trials, with 72 h between each trial. The distinctive context utilized for conditioning used location, visual, and tactile cues different from those in the home cage environment. The room was dark with two 25-Watt lights beside the conditioning chamber. The chambers were made of black opaque Plexiglas (22.5×26× 20 cm) and sat on a table with a clear glass top. A mirror beneath the chamber on a 45° angle facilitated viewing of the ventral surface of the rat to observe orofacial responses. The test trial to assess anticipatory nausea occurred 72 h after the final conditioning trial. Two hours prior to placement in the distinctive context, the rats were injected with VEH (n= 10), 10 mg/kg MJN110 (n=8), 20 mg/kg MJN110 (n=8). An additional group (n=8) was injected with 10 mg/kg MJN110 and 90 min later with 1 mg/kg SR141716, 30 min prior to the test trial. The rats were then injected with 20 mL/kg saline solution (since the injection procedure is part of the compound conditioned stimulus) and placed in the conditioning chambers where they remained for 5 min (optimal time for measuring contextually elicited gaping; Rock et al. 2008) while their behavior was videotaped. The videotapes from the test trial were later scored by a trained observer, for the response of gaping (wide open mouth with lower incisors exposed). Immediately following the 5-min test trial, the rats were given a 15-min activity test to assess locomotor activity. The activity chamber was constructed of white Plexiglas with the dimensions of 60×25×25 cm and located in a different room than the AN chamber, illuminated with a red light. A video
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camera mounted on an extension pole captured the activity of the rat which was sent to a computer for analysis of distance (centimeters) traveled using the Ethovision software program (Noldus, Inc, NL). Immediately following the 15-min activity test trial, the VIC was bilaterally collected from the brains of four rats from group VEH and three rats from group 10 mg/kg MJN110. These rats were euthanized by rapid decapitation (restrained in a decapicone-Braintree Scientific MA), and their brains were immediately extracted; the VIC was subsequently dissected on a stainless steel platform resting atop dry ice, which resulted in rapid freezing of the tissue section. Tissue samples were stored at −80 °C until the time of processing to measure endocannabinoid levels which was performed at Virginia Commonwealth University. Experiment 3: effect of MAGL inhibition by MJN110 on LiClinduced vomiting in shrews The shrews were moved into the experimental room from the colony room and given four meal worms in an empty cage 15 min prior to receiving their pretreatment injection. Shrews were injected with VEH (n= 6), 10 mg/kg MJN110 (n=4), 20 mg/kg MJN110 (n=4) 120 min prior to an injecton of LiCl (390 mg/kg; 60 mL/kg of 0.15 M LiCl). An additional group (n=4) was injected with 20 mg/kg MJN110 and 90 min later with SR141716 (2.5 mg/kg) 30 min before being injected with LiCl. Immediately following the LiCl injection, all shrews were placed in the Plexiglas observation chamber (22.5×26×20 cm) which sat on table with a clear glass top and a mirror underneath at a 45° angle for 45 min. An observer counted the number of vomiting episodes. A vomiting episode was defined as expulsion of gastric contents from mouth. Experiment 4: effect of MAGL inhibition by MJN110 on contextually elicited conditioned gaping in shrews The shrews received three conditioning trials, separated by 72–96 h during which the contextual chamber was paired with LiCl. On each conditioning trial, the shrew as injected with LiCl (390 mg/kg; 20 mL/kg) and immediately placed in the chamber for 45 min. The chambers were thoroughly washed with lemon-scented soapy water between each animal’s trials. The test trial occurred 5 days after the final conditioning trial. On this trial, the shrews were pretreated with VEH (n=5) or 20 mg/kg MJN110 (n = 6) 120 min prior to receiving a saline injection (20 mL/kg) and being placed in the test chamber for 15 min. The shrew’s behavior in the test chamber was videotaped from a Panasonic Closed Circuit Camera (model WV-CP484, Matsushita Electric Industrial Co., Ltd) which was mounted beneath the chamber. These videotapes were later sent to a computer for analysis of distance (centimeters) traveled using Ethovision software program (Noldus Information Technology, Leesburg VA
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USA). The video tapes were scored for the number of gapes using “The Observer” Event recording software (Noldus).
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endocannabinoid levels in the VIC of a sample of rats treated with VEH or 10 mg/kg MJN110 2 h prior to the test for anticipatory nausea. As predicted, independent t tests revealed that MJN110 selectively elevated 2-AG (p=0.016) and suppressed AA (p=0.012) but had no effect on AEA, PEA, or OEA (which are each deactivated by FAAH, not MAGL).
Results Experiment 1: effect of MAGL inhibition by MJN110 on acute nausea The MAGL inhibitor, MJN110, interfered with acute nausea at doses of 10 and 20 mg/kg by a CB1-dependent mechanism of action. Figure 1a shows the mean number of gapes recorded for each group on the test trial. The singlefactor ANOVA revealed a significant main effects of pretreatment, F (4, 35)=5.3; p=0.002. Subsequent Bonferroni post hoc tests revealed that rats pretreated with 10 and 20 mg/kg MJN110 displayed significantly fewer LiCl-induced conditioned gaping reactions during the test trial than Group VEH (p’s
ORIGINAL INVESTIGATION
Effect of selective inhibition of monoacylglycerol lipase (MAGL) on acute nausea, anticipatory nausea, and vomiting in rats and Suncus murinus Linda A. Parker & Micah J. Niphakis & Rachel Downey & Cheryl L. Limebeer & Erin M. Rock & Martin A. Sticht & Heather Morris & Rehab A. Abdullah & Aron H. Lichtman & Benjamin F. Cravatt
Received: 27 May 2014 / Accepted: 12 July 2014 / Published online: 3 August 2014 # Springer-Verlag Berlin Heidelberg 2014
Abstract Rationale To determine the role of the endocannabinoid, 2arachodonyl glycerol (2-AG), in the regulation of nausea and vomiting. Objective We evaluated the effectiveness of the potent selective monoacylglycerol lipase (MAGL) inhibitor, MJN110, which selectively elevates the endocannabinoid 2-AG, to suppress acute nausea and vomiting, as well as anticipatory nausea in rat and shrew models. Methods The rat gaping models were used to evaluate the potential of MJN110 (5, 10, and 20 mg/kg, intraperitoneally [IP]) to suppress acute nausea produced by LiCl and of MJN110 (10 and 20 mg/kg, IP) to suppress anticipatory nausea elicited by a LiCl-paired context. The potential as well of MJN110 (10 and 20 mg/kg, IP) to suppress vomiting and contextually elicited gaping in the Suncus murinus was evaluated. Results MJN110 suppressed acute nausea in rats, LiClinduced vomiting in shrews and contextually-elicited anticipatory nausea in both rats (accompanied by elevation of 2-AG in the visceral insular cortex) and shrews. These effects were L. A. Parker (*) : R. Downey : C. L. Limebeer : E. M. Rock : M. A. Sticht : H. Morris Department of Psychology and Collaborative Neuroscience Program, University of Guelph, Guelph, ON N1G2W1, Canada e-mail: [email protected] M. J. Niphakis : B. F. Cravatt The Skaggs Institute for Chemical Biology and Department of Chemical Physiology, The Scripps Research Institute, La Jolla, CA 92037, USA R. A. Abdullah : A. H. Lichtman Department of Pharmacology and Toxicology, Medical College of Virginia Campus, Virginia Commonwealth University, Richmond, VA, USA
reversed by the CB1 antagonist/inverse agonist, SR141716. The MAGL inhibitor did not modify locomotion at any dose. An activity-based protein profiling analysis of samples of tissue collected from the visceral insular cortex in rats and whole brain tissues in shrews revealed that MJN110 selectively inhibited MAGL and the alternative 2-AG hydrolase, ABHD6. Conclusions MAGL inhibition by MJN110 which selectively elevates endogenous 2-AG has therapeutic potential in the treatment of acute nausea and vomiting as well as anticipatory nausea, a distressful symptom that is resistant to currently available treatments. Keywords MAGL . Nausea . Vomiting . Rat . 2-AG . Shrew . Gaping . Conditioned response . ABPP . ABHD6 The sensation of nausea is one of the most debilitating human experiences. In chemotherapy patients, nausea frequently occurs following treatment (acute and delayed nausea), but it also happens in anticipation of treatment (anticipatory nausea). Because nausea is poorly understood, effective treatments are very limited. Current anti-emetic therapies are highly effective in reducing chemotherapy-induced vomiting, but they are only somewhat effective in treating chemotherapyinduced acute and delayed nausea (Ballatori et al. 2007; de Boer-Dennert et al. 1997; Foubert and Vaessen 2005; Hickok et al. 2003; Morrow et al. 2002; Roscoe et al. 2000). When anticipatory nausea develops, current anti-emetic agents are almost completely ineffective. Anticipatory nausea is commonly treated with non-specific anti-anxiety drugs, such as benzodiazepines (Malik et al. 1995; Razavi et al. 1993); however, benzodiazepines have sedating side effects, which severely limit their usefulness. Therefore, new treatments for both acute and anticipatory nausea are urgently required.
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Animal models of vomiting have been valuable in elucidating the neural mechanisms of the emetic reflex (Hornby 2001). However, the central neurochemical regulation of nausea is still not well understood (Andrews and Horn 2006), because, until recently, a reliable, selective animal model of nausea had not been established. We have developed such a model in the rat and have used it to explore the neural circuits (e.g., the visceral insular cortex [VIC]; Tuerke et al. 2012; Limebeer et al. 2012) involved in the regulation of nausea. Rats do not vomit (Horn et al. 2013), but they display conditioned gaping reactions (the wide opening of the mouth exposing the lower incisors) when exposed to a flavor (Grill and Norgren 1978) that has previously been paired with nausea (see Parker 2014 for review). As well, drugs that prevent vomiting in species capable of vomiting also prevent the establishment of conditioned gaping in rats by interfering with acute nausea during the conditioning trial (Parker 2014). Conditioned gaping requires similar orofacial musculature as vomiting in emetic species (Travers and Norgren 1986) and is topographically similar to the orofacial components of retching in the shrew (Parker et al 2009). We have capitalized on this behavioral response to develop a model of acute nausea in rats (Parker 2014). Conditioned gaping reactions are not only elicited by LiClpaired flavors, but they are also elicited by LiCl-paired contextual cues (albeit following several pairings) (Limebeer et al. 2013; 2006; 2008; Rock et al. 2008) revealing a model remarkably similar to that of anticipatory nausea in humans. Indeed, shrews, which vomit in response to an emetic treatment, also display conditioned gaping reactions when reintroduced to the context in which they previously vomited. Interestingly, 5-HT3 antagonists are ineffective in suppressing the expression of contextually elicited gaping reactions, as they are ineffective in suppressing anticipatory nausea in humans (Limebeer et al. 2006; Parker et al. 2006; Parker and Kemp 2001). On the other hand, cannabinoid compounds (Bolognini et al. 2013; Limebeer et al. 2013; Limebeer et al. 2006; Parker et al. 2006; Parker and Kemp 2001; Rock et al. 2008) effectively interfere with the expression of contextually-elicited conditioned gaping, suggesting that they may be successful in treating anticipatory nausea in humans. The endocannabinoid (EC) system is intimately involved in the regulation of vomiting (Kwiatkowska et al. 2004; Parker et al. 2004; 2011; Sharkey and Kroese 2001; Van Sickle et al. 2001, 2003, 2005) and both acute and anticipatory nausea (see Parker et al 2011). Cannabinoid (CB) receptors are part of a central system of lipid transmitters, the EC system, which consists of the CB1 and CB2 receptor, the ligands anandamide (AEA) (Devane et al. 1992) and 2arachidonyl glycerol (2-AG) (Mechoulam et al. 1995) and enzymes for biosynthesis and hydrolysis of the ligands (Cravatt et al. 1996; Deutsch et al. 2002; Dinh et al. 2004). Unlike most neurotransmitters, AEA and 2-AG are not stored
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in vesicles but rather are synthesized when and where they are needed. Again, unlike most neurotransmitters, their action is not postsynaptic but rather mostly presynaptic, i.e., they serve as retrograde synaptic messengers inhibiting the release of other neurotransmitters (Mechoulam and Parker 2013). Contrary to Δ9–tetrahydrocannabinol (THC), which is metabolized over several hours and excreted (or stored as one of its metabolites), endocannabinoids are rapidly removed by a membrane transport process yet to be fully characterized (Fu et al. 2012). In the postsynaptic cell, AEA is hydrolyzed to arachidonic acid and ethanolamine by fatty acid amide hydrolase (FAAH) (Cravatt et al. 1996; Deutsch et al. 2002). 2-AG i s a l s o h y d r o l y z e d e n z y m a t i c a l l y, p r i m a r i l y b y monoacylglycerol lipase (MAGL) (Blankman et al. 2007; Dinh et al. 2004; Niphakis et al. 2013; Windsor et al. 2012) found in presynaptic terminal endings. Suppression of these enzymes prolongs the activity of the respective endocannabinoid. Drugs which augment the endocannabinoid system have potential therapeutic value in the treatment of nausea (for review Parker 2014; Sharkey et al 2014). THC and other direct CB1 agonists are effective in treating nausea (Limebeer and Parker 1999; Parker and Mechoulam 2003), but their adverse effects on motor control and cognition limit their usefulness (see Mechoulam and Parker 2013). This has led to the pursuit of alternative strategies to selectively enhance endocannabinoid function. A highly attractive strategy is to inhibit FAAH or MAGL, elevating brain AEA or 2-AG, respectively, without cognitive or motor impairment. In the case of AEA, pharmacological inhibitors that selectively block intracellular hydrolysis by FAAH (e.g., URB597; PF3845) have been extensively used, leading to a better understanding of its role in the regulation of nausea and vomiting. We have shown that URB597 suppresses both acute (Cross-Mellor et al. 2007) and anticipatory nausea (Rock et al. 2008) in rats as well as LiCl-induced vomiting (Parker et al. 2009) and anticipatory retching (wide gaping; Parker et al. 2006) in musk shrews. As well, URB597 has been shown to reduce vomiting in ferrets (Sharkey et al. 2007). We have also recently shown that the dual FAAH/MAGL inhibitor, JZL195, alone and combined with AEA or 2-AG suppresses anticipatory nausea in rats in a CB1-dependent manner (Limebeer et al. 2013). The suppression of contextually elicited gaping by JZL195 was accompained by an elevation of AEA, but not 2-AG; however, when coadministered with AEA or 2-AG, both the suppression of gaping and the level of AEA and 2AG were respectively enhanced. Selective and in vivo-active MAGL inhibitors that do not cross-react with FAAH in the rat have only recently been developed (Chang et al. 2012; Niphakis et al. 2013) and have since opened new avenues for studying the role of 2-AG in mammalian physiology. One such inhibitor, MJN110, inhibits 2-AG hydrolysis in mouse and rat brain homogenates with a
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low nanomolar IC50 and has no effect on AEA hydrolysis up to 10 μM (Niphakis et al. 2013). MJN110 also displays excellent potency in vivo, inhibiting brain MAGL after administration of 1 mg/kg (intraperitoneally [IP]) in mice and 5 mg/kg (IP) in rat without affecting FAAH activity at doses up to 40 mg/kg (IP). While also targeting the alternative 2-AG hydrolase, ABHD6, MJN110 diplays high selectivity over other serine hydrolase enzymes and across the proteome (Niphakis et al. 2013; Chang et al. 2012). Owing to these factors, MJN110 is well-suited for studying the effects of MAGL blockade in rats. Indeed, this compound has been shown to alleviate mechanical allodynia in a rat model of diabetic neuropathy (Niphakis et al. 2013). Here, we evaluate the potential MJN110 to interfere with acute and anticipatory nausea in rats, as well as vomiting and anticipatory nausea in shrews. We first demostrate that MJN110 dose-dependently suppresses acute nausea produced by LiCl, by demonstrating attenuated gaping at test to a flavor previously paired with LiCl in rats pretreated with MJN110 during conditioning. This effect is reversed by the CB1 antagonist, SR141716A. Then, we show that MJN110 also interferes with the expression of previously established contextually elicited conditioned gaping in rats, as a measure of anticipatory nausea in rats. This effect is also reversed by CB1 antagonism. Finally, we demonstrate that MJN110 also dosedependently prevents LiCl-induced acute vomiting and contextually-elicited anticipatory gaping in the Suncus murinus. These findings demonstrate that drugs which prolong the action of 2-AG by blocking their rapid degradation through MAGL have promise as treatments for both acute and anticipatory nausea and vomiting.
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(lights off at 7 PM). Shrews were tested in their light cycle. They were maintained on Medi-cal/Royal Canine Feline Maintenance mixed with Harlan Ferret dry chow and water ad libitum. All shrews were weaned at 20 days of age. Drugs and materials MJN110 (2,5-dioxopyrrolidin-1-yl 4-(bis(4chlorophenyl)methyl)piperazine-1-carboxylate; Niphakis et al. 2013), PF-3845 (N-3-pyridinyl4-[[3-[[5-(trifluoromethyl)-2-pyridinyl]oxy]phenyl]methyl]1-piperidinecarboxamide; Ahn et al. 2009), KT195 (C28H28N4O2; Hsu et al. 2012), and fluorophosphosphonaterhodamine probe (FP-Rh; Patricelli et al. 2001) were synthesized as previously described. All injections were administered IP. A 0.15 M lithium chloride (LiCl; Sigma Aldrich) solution was prepared with sterile water and administered in a volume of 20 ml/kg (127.2 mg/kg). MJN110 (5, 10, or 20 mg/kg) and SR141617 (5-(4-chlorophenyl)-1-(2,4dichlorophenyl)-4-methyl-N-1-piperidinyl-1H-pyrazole-3carboxamide; 1 mg/kg; a dose that does not potentiate LiClinduced conditioned gaping or vomiting [O’Brien et al 2013]) were administered at 1 ml/kg (except 20 mg/kg MJN110 which was administered at 2 ml/kg [10 mg/ml]) and were prepared in a vehicle (VEH) consisting of a 1:1:18 mixture of ethanol, Tween 80 (Sigma), and saline (SAL). The drugs were first dissolved in ethanol then Tween 80 was added to the solution, and the ethanol was evaporated off with a nitrogen stream after which the saline was added. The final VEH consisted of 1:9 (Tween/saline) and was administered at 1 ml/kg. Tissue preparation for ABPP analysis
Methods Animals Animal procedures complied with the Canadian Council on Animal Care. The protocols were approved by the Institutional Animal Care Committee, which is accredited by the Canadian Council on Animal Care. Naive male Sprague–Dawley rats, weighing between 262 and 373 g on the first day of conditioning, obtained from Charles River Laboratories (St Constant, Quebec) were used for assessment of anti-nausealike behavior. They were pair-housed in Plexiglas cages (47× 26×20 cm) in a colony room at an ambient temperature of 21 °C with a 12/12 hour light–dark schedule (lights off at 8 AM) and maintained on food and water ad libitum. Naive male and female S. murinus (house musk shrews) ranged from 33 to 50 days old at the time of testing and were bred and raised in the University of Guelph colony. They were single-housed in opaque white mouse cages in the colony room at an ambient temperature of 21 °C on a 10/14 hour light–dark schedule
Frozen rat VIC tissue harvested from animals treated with either vehicle, MJN110 or PF-3845, was washed twice by adding cold DPBS (1.0 mL), centrifuging (3,000×g, 3 min), and removing the supernatant. Cold DPBS (300 μL) was then added to the washed VIC tissue which was then homogenized using a probe sonicator. Alternatively, hemisected shrew brains were Dounce-homogenized in cold DPBS (2.0 mL) and subsequently centrifuged (3,000×g, 3 min) to remove insoluble debris. Tissue homogenates from rat VIC and shrew brain were centrifuged at 100,000×g for 45 min and the pellets sonicated in DPBS (300 μL) to resuspend. Following the determination of their protein concentration using the DC Protein Assay (Bio-Rad), proteomes were diluted to 1.0 mg/ mL prior to activity-based protein profiling (ABPP) analysis. Ex vivo activity-based protein profiling Proteomes (1.0 mg/mL, 50 μL) were treated with FP-Rh (1.0 μL, 50 μM in dimethylsulfoxide (DMSO)), vortexed,
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and incubated at room temperature. After 30 min, each reaction was quenched with 4× sodium dodecyl sulfate (SDS) loading buffer (17 μL), and proteins were separated by SDSpolyacrylamide gel electrophoresis (PAGE). The gel was analyzed by fluorescent gel imaging on a Hitachi FMBio II flatbed scanner and activity of individual serine hydrolases assessed by comparing the fluorescent intensities of each gel band relative to samples derived from vehicle-treated animals.
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(326 > 62) and (326 > 309) for OEA; and (330 > 66) for OEAd4; in negative mode: (303>259) and (303>59) for AA and (311>267) for AA-d8. A calibration curve was constructed for each assay based on linear regression using the peak area ratios of the calibrators. The extracted standard curves ranged from 0.156 to 2.5 pmol for AEA, from 0.25 to 4 nmol for 2-AG, from 7.8 to 125 pmol for PEA and OEA, and from 1 to 16 nmol for AA. Experimental Procedures:
In vitro activity-based protein profiling of shrew brain Proteomes (1.0 mg/mL, 50 μL) derived from vehicle-treated shrew brains were treated with DMSO (1.0 μL), JZL184 (1.0 μL, 5.0 μM), KT195 (1.0 μL, 5.0 μM), or PF-3845 (1.0 μL, 5.0 μM) and incubated for 30 min at 37 °C. Each proteome was subsequently treated with FP-Rh (1.0 μL, 50 μM in DMSO), vortexed, and incubated at room temperature for 30 min. As above, after each reaction was quenched with 4× SDS loading buffer (17 μL), the proteins were separated by SDS-PAGE and analyzed by fluorescent gel imaging. Quantification of VIC endocannabinoids Lipid extractions were carried out according to previously described methods (Kinsey et al. 2013; Ramesh et al. 2011). Pre-weighed VIC samples were homogenized in 1.4 mL of buffer containing a 2:1 chloroform/methanol solution and internal standards (4 pmol AEA-d8, 1 nmol 2-AG-d8, 330 pmol PEA-d4, 300 pmol OEA-d4, and 1 nmol AA-d8), and 0.3 mL of saline (0.73 %) was added to each sample. The homogenates were subsequently vortexed for 1 min. Following centrifugation (10 min at 3,220×g; 4 °C), the aqueous phase and debris were separated and extracted another two times each with 0.8 mL of chloroform; the organic phase from each of the separations was pooled together, and the organic solvents were evaporated under a nitrogen stream (15 psi). After the samples were completely dried, they were reconstituted with 0.1 mL chloroform and, after vortexing, were mixed with 1 mL of cold acetone. Following another centrifugation (5 min at 1,811×g), the upper layer from each sample was collected and evaporated under nitrogen. The final dried constituents were reconstituted in 0.1 mL methanol and transferred to autosample vials for analysis. AEA, 2AG, OEA, PEA, and AA were quantified using liquid chromatography tandem mass spectrometry. The mobile phase consisted of methanol (90:10)/0.1 % ammonium acetate and 0.1 % formic acid. The column used was a Discovery® HS C18, 2.1×150 mm, 3 μm (Supelco, USA). Ions were analyzed in multiple-reaction monitoring mode, and the following transitions were monitored in positive mode— (348>62) and (348>91) for AEA; (356>62) for AEAd8; (379>287) and(379>269) for 2-AG; (387>96) for 2-AGd8; (300>62) and (300>283) for PEA; (304>62) for PEA-d4;
Experiment 1: effect of MAGL inhibition by MJN110 on acute nausea All rats were surgically implanted with an intraoral cannula under isofluorane anesthesia, according to the procedures described by Limebeer et al. (2012). The intraoral cannula, implanted to deliver fluids to the rat’s oral cavity, consisted of a length of Intramedic plastic tubing with an inner diameter of 0.86 mm and an outer diameter of 1.27 mm with two circular elastic discs placed over the tubing at the back of the neck. Three days after the surgery, the rats received a taste reactivity (TR) adaptation trial in which they were placed in the TR chamber with their cannula attached to an infusion pump (Model KDS100, KD Scientific, Holliston, MA, USA) for fluid delivery. The TR chambers were made of clear Plexiglas (22.5×26×20 cm) that sat on a table with a clear glass top. A mirror beneath the chamber on a 45° angle facilitated viewing of the ventral surface of the rat to observe orofacial responses. Water was infused into their intraoral cannulae for 2 min at the rate of 1 mL/min. On the day following the TR adaptation trial, the rats received the TR conditioning trial in which they were administered a pretreatment injection of VEH (n=9), 5 mg/kg MJN110 (n=7), 10 mg/kg MJN110 (n=8), and 20 mg/kg MJN110 (n=8) 2 h prior to the conditioning trial. An additional group (n=8) was injected with 10 mg/kg MJN110 90 min before 1.0 mg/kg SR141716, and 30 min later, received the conditioning trial. On the conditioning trial, the rats were individually placed in the TR chamber and intraorally infused with 0.1 % saccharin solution for 2 min at the rate of 1 mL/min while the orofacial responses were video recorded from a mirror at a 45° angle beneath the chambers, with the feed from the video camera (Sony DCR-HC48, Henry’s Cameras, Waterloo, ON, Canada) fire-wired into a computer. Immediately after the saccharin infusion, all rats were injected with 20 mL kg−1 of 0.15 M LiCl and returned to their home cage. Seventy-two hours following the TR conditioning trial, the rats were given a drug-free TR test trial. Rats were intraorally infused with 0.1 % saccharin solution for 2 min at the rate of 1 mL/min while their orofacial responses were video recorded. At 1600 h on the day of the TR test trial, the rats were water-restricted (water bottles removed from cage). Eighteen hours later, on the following morning, they were given the two-bottle conditioned taste avoidance test. Each rat
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was presented with two graduated tubes side by side with one containing 0.1 % saccharin solution (always on the left side) and one containing reverse osmosis water for 6 h. The amounts consumed from each tube were measured and converted into a saccharin preference ratio: Amount of saccharin consumed divided by the amount of saccharin + water consumed. The video tapes of the TR test trial were later scored (at ½ speed) by an observer blind to the experimental conditions using ‘The Observer’ (Noldus Information Technology Inc., Leesburg, VA, USA) for the number of occurrences of gaping (large openings of the mouth and jaw, with lower incisors exposed). The behavior of gaping is extremely easy to identify and score with inter-rater reliability ratings consistently falling in the range of r=0.95 to 0.99 when the scores of highly experience raters are compared with those of raters trained for only a few hours (see Parker 2014). Experiment 2: effect of MAGL inhibition by MJN110 on anticipatory nausea The rats received four conditioning trials, during which a distinctive contextual chamber was paired with a 20-mL/kg injection of 0.15 M LiCl. On each conditioning trial, each rat was injected with LiCl and immediately placed in the distinctive context for a 30-min period. This procedure occurred on a total of four conditioning trials, with 72 h between each trial. The distinctive context utilized for conditioning used location, visual, and tactile cues different from those in the home cage environment. The room was dark with two 25-Watt lights beside the conditioning chamber. The chambers were made of black opaque Plexiglas (22.5×26× 20 cm) and sat on a table with a clear glass top. A mirror beneath the chamber on a 45° angle facilitated viewing of the ventral surface of the rat to observe orofacial responses. The test trial to assess anticipatory nausea occurred 72 h after the final conditioning trial. Two hours prior to placement in the distinctive context, the rats were injected with VEH (n= 10), 10 mg/kg MJN110 (n=8), 20 mg/kg MJN110 (n=8). An additional group (n=8) was injected with 10 mg/kg MJN110 and 90 min later with 1 mg/kg SR141716, 30 min prior to the test trial. The rats were then injected with 20 mL/kg saline solution (since the injection procedure is part of the compound conditioned stimulus) and placed in the conditioning chambers where they remained for 5 min (optimal time for measuring contextually elicited gaping; Rock et al. 2008) while their behavior was videotaped. The videotapes from the test trial were later scored by a trained observer, for the response of gaping (wide open mouth with lower incisors exposed). Immediately following the 5-min test trial, the rats were given a 15-min activity test to assess locomotor activity. The activity chamber was constructed of white Plexiglas with the dimensions of 60×25×25 cm and located in a different room than the AN chamber, illuminated with a red light. A video
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camera mounted on an extension pole captured the activity of the rat which was sent to a computer for analysis of distance (centimeters) traveled using the Ethovision software program (Noldus, Inc, NL). Immediately following the 15-min activity test trial, the VIC was bilaterally collected from the brains of four rats from group VEH and three rats from group 10 mg/kg MJN110. These rats were euthanized by rapid decapitation (restrained in a decapicone-Braintree Scientific MA), and their brains were immediately extracted; the VIC was subsequently dissected on a stainless steel platform resting atop dry ice, which resulted in rapid freezing of the tissue section. Tissue samples were stored at −80 °C until the time of processing to measure endocannabinoid levels which was performed at Virginia Commonwealth University. Experiment 3: effect of MAGL inhibition by MJN110 on LiClinduced vomiting in shrews The shrews were moved into the experimental room from the colony room and given four meal worms in an empty cage 15 min prior to receiving their pretreatment injection. Shrews were injected with VEH (n= 6), 10 mg/kg MJN110 (n=4), 20 mg/kg MJN110 (n=4) 120 min prior to an injecton of LiCl (390 mg/kg; 60 mL/kg of 0.15 M LiCl). An additional group (n=4) was injected with 20 mg/kg MJN110 and 90 min later with SR141716 (2.5 mg/kg) 30 min before being injected with LiCl. Immediately following the LiCl injection, all shrews were placed in the Plexiglas observation chamber (22.5×26×20 cm) which sat on table with a clear glass top and a mirror underneath at a 45° angle for 45 min. An observer counted the number of vomiting episodes. A vomiting episode was defined as expulsion of gastric contents from mouth. Experiment 4: effect of MAGL inhibition by MJN110 on contextually elicited conditioned gaping in shrews The shrews received three conditioning trials, separated by 72–96 h during which the contextual chamber was paired with LiCl. On each conditioning trial, the shrew as injected with LiCl (390 mg/kg; 20 mL/kg) and immediately placed in the chamber for 45 min. The chambers were thoroughly washed with lemon-scented soapy water between each animal’s trials. The test trial occurred 5 days after the final conditioning trial. On this trial, the shrews were pretreated with VEH (n=5) or 20 mg/kg MJN110 (n = 6) 120 min prior to receiving a saline injection (20 mL/kg) and being placed in the test chamber for 15 min. The shrew’s behavior in the test chamber was videotaped from a Panasonic Closed Circuit Camera (model WV-CP484, Matsushita Electric Industrial Co., Ltd) which was mounted beneath the chamber. These videotapes were later sent to a computer for analysis of distance (centimeters) traveled using Ethovision software program (Noldus Information Technology, Leesburg VA
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USA). The video tapes were scored for the number of gapes using “The Observer” Event recording software (Noldus).
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endocannabinoid levels in the VIC of a sample of rats treated with VEH or 10 mg/kg MJN110 2 h prior to the test for anticipatory nausea. As predicted, independent t tests revealed that MJN110 selectively elevated 2-AG (p=0.016) and suppressed AA (p=0.012) but had no effect on AEA, PEA, or OEA (which are each deactivated by FAAH, not MAGL).
Results Experiment 1: effect of MAGL inhibition by MJN110 on acute nausea The MAGL inhibitor, MJN110, interfered with acute nausea at doses of 10 and 20 mg/kg by a CB1-dependent mechanism of action. Figure 1a shows the mean number of gapes recorded for each group on the test trial. The singlefactor ANOVA revealed a significant main effects of pretreatment, F (4, 35)=5.3; p=0.002. Subsequent Bonferroni post hoc tests revealed that rats pretreated with 10 and 20 mg/kg MJN110 displayed significantly fewer LiCl-induced conditioned gaping reactions during the test trial than Group VEH (p’s
