© 2010 Nature America, Inc. All rights reserved. equally equally to this work. should Correspondence be addressed to A.H.L. or ([email protected]) B.F.C. ([email protected]). Wisconsin, Milwaukee, Wisconsin, USA. Department of Chemical Physiology, The Scripps Research Institute, La Jolla, California, USA. 1 brain the on effect broader a has the that suggest which lowing FAAH blockade, including hypomotility and hyperreflexia however, causes additional behavioral effects that are not observed fol behavior pain and reduces system nervous in the 2-AG levels raises MAGL, which enzyme 2-AG–degrading the pain assays. A similar outcome is observed following acute blockade of CB produces and of for enzyme anandamide degradative principal cological signaling of regulators tant involved in endocannabinoid production and degradation are impor production (on-demand be biosynthesized and released at the moment of their intended action to appear that lipids neutral hydrophobic are 2-AG and anandamide endocannabinoids the release, before vesicles membrane-delineated in stored and soluble water are which neurotransmitters, other most cells immune on mainly found instead is and brain the in marijuana as of ponent such agonists, of Δ effects neurobehavioral the most of mediates it where system, nervous the throughout expressed (2-AG) 2-arachidonoyglycerol and ligands, lipid ural CB receptors, protein–coupled G two of system (endocannabinoid) cannabinoid endogenous The functional antagonism of the brain cannabinoid system, respectively. generate endocannabinoids distinct analgesic profiles that are either sustained or transitory and associated with agonism and major anandamide, which endocannabinoid produced sustained analgesia without impairing CB brain CB blockade also caused synaptic physical plasticity dependence, and impaired endocannabinoid-dependent desensitized (CB After repeated the administration, MAGL inhibitor JZL184 lost its analgesic activity and produced to cross-tolerance cannabinoid inactivation of monoacylglycerol (MAGL), the principal degradative enzyme for the endocannabinoid 2-arachidonoylglycerol. in desensitization the nervous system. We found that a similar form of functional antagonism was produced by Prolonged exposure to drugs of abuse, such as and opioids, leads to tolerance pharmacological and receptor Dana E Selley Steven G Kinsey Joel E Schlosburg functional antagonism of the Chronic monoacylglycerol lipase blockade causes nature nature Received 6 July; accepted 19 July; published online 22 August 2010; Department Department of Pharmacology and Toxicology, Virginia Commonwealth University, Richmond, Virginia, USA. -erhdoanbnl TC, h piay scocie com psychoactive primary the (THC), 9- NEUR 12–15 1 receptors. These data contrast with blockade of amide , an enzyme that degrades the other 1 disruption of fatty acid amide hydrolase (FAAH), the the (FAAH), hydrolase amide acid fatty of disruption OSCI ) ) agonists in mice, effects that were phenocopied by genetic disruption of 1 , Laura , J Laura Sim-Selley EN 1 N 5 , , Peter T Nguyen 1 . The CB The . C , aahdnyehnlmn (anandamide) -arachidonoylethanolamine 5 E , , Jacqueline L Blankman

6 ). These features indicate that the enzymes the that indicate features These ). advance online publication online advance 7–9 . For instance, genetic instance, For . 2 receptor is only sparsely expressed expressed sparsely only is receptor 1 4 -dependent analgesia in multiple multiple in analgesia -dependent Department Department of Molecular Biology, The Scripps Research Institute, La Jolla, California, USA. 3,4 Te CB The . 1 1 1 17,18 and CB and , , Divya Ramesh , , Qing-song Liu 1 . Inhibition of . MAGL, Inhibition 6 , elevates , brain levels elevates 1 receptor is highly highly is receptor 2 10,11 , and their nat their and , 2 , 5 or pharma or , , Jonathan Z Long

1 5 d

. Unlike Unlike . consists consists o i : 1 1 3 , , Lamont Booker 0 17,19 , , Aron H Lichtman . 1 0 2 - - - - - , 3

8 / n that there are fundamental differences in the mode of signaling for for signaling of mode the in differences fundamental are there that CB intact and phenotype sic analge an maintained instead which FAAH, disrupted chronically in with mice were observed In none regions. of effects contrast, these and CB a to responses analgesic MAGL of inhibitor, to agonists, cannabinoid exogenous cross-tolerance loss a by evidenced as mice, in system endocannabinoid in brain the alterations profound of MAGL caused and 2-AG on the integrity of the endocannabinoid system? anandamide in elevations sustained of effect the be might what so, If as a morefunctioning stress-responsive restricted, endocannabinoid? anandamide with system, nervous the in 2-AG for role broader a to activity) locomotor tors appear (for functions also neurological general to example,affect component Incontrast,MAGLsubstantial containstress. of inhibi a cesses affected by FAAH inhibitors, including pain pro behavioral the of Several system. nervous the 2-AGin and mide inhibitors raise provocative questions about the respective roles of lipase- ananda diacylglycerol enzyme biosynthetic 2-AG the and synaptic that plasticity have been shown to require the CB (DSI) inhibition of pression tors, but sup not FAAH augment depolarization-induced inhibitors, cannabinoid system. In further support of this premise, MAGL inhibi n . 2 We found that prolonged pharmacological or genetic inactivation inactivation genetic or pharmacological We prolonged that found The overlapping, but distinct, behavioral effects of FAAH and MAGL 6 1 6 1 2 3 receptor downregulation and desensitization in specific brain Department Department of Pharmacology and Toxicology, Medical College of , , Daniel K Nomura 1 , , James J Burston 1 & Benjamin F Cravatt 2 17,19 The The Skaggs Institute for Chemical Biology and Mgll . Could these pharmacological profiles point profiles pharmacological these Could . (encoding MAGL). Chronic MAGL 2 1 0 receptor system. Our results suggest suggest results Our system. receptor n ectto (DSE) excitation and 1 2 receptors. Thus, individual , , Bin Pan 1 , Elizabeth A , ThomasElizabeth 5 These These authors contributed 2 3 ,

sustained sustained 12–15 t r a and anxiety 20,21 α 1 C I receptor , forms of of forms , 23,24

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© 2010 Nature America, Inc. All rights reserved. per genotype). Data are presented as means means as presented are Data genotype). per ( sample. this in MAGL of loss complete the of treatment in protein profiling ( allele. -trapped the to corresponds band 220-bp the and allele wild-type the to corresponds band 430-bp The DNA. tail genomic ( shown). is nucleophile serine catalytic the containing sequence (GHSMG E4 exon catalytic the of upstream in vector trap ( dose) final 2 h after day, per evaluated dose one 6 d, regime: dosing chronic 2 h; intraperitoneal, kg, per mg 10 regimen: dosing (acute PF-3845 or dose) final after 2 h day, per evaluated dose one 6 d, regimen: dosing chronic 2 h; intraperitoneal, kg, per mg 40 regimen: dosing (acute JZL184 with chronically or acutely treated FAAH.or ( MAGL of disruptions 1 Figure Genomic for Institute A&M Texas the from trapping gene by ated Weobtained 2-AG. in elevations sustained for ( treatment chronic or acute ( day; per dose intraperitoneal, one kg, per mg (10 PF-3845 inhibitor FAAH selective brain in rise 15-fold the ­anand than lower much however, was, change This regimen. treatment the over treatment JZL184 to exposure cumulative of final after h 2 ( (~threefold) anandamide in elevation modest a caused repeated also dosing acute, not the but Chronic, regime. dosing throughout elevated remained 2-AG that indicating h ( 26 at for least persisted levels 2-AG brain 2-AG in the brain 2 h following final dosing ( of levels elevated highly showed chronically or dose) (single acutely levels control of brain that the tenfold in to 2-AG up of by level the increase and system nervous the in day), which has previously been shown to selectively inactivate MAGLper dose one intraperitoneal, weight, body of kg per mg (40 JZL184 inhibitor MAGL the with days model consecutive six for mice pharmacological treating by chronic a generated We system. nervous the in 2-AG in elevations sustained of consequences the examine to models and genetic pharmacological We complementary established Mouse models for chronic inactivation of MAGL RESULTS cannabinoid system modulates mammalian physiology and behavior. (CB liganddiversification animportantis endo mechanismthe by which receptor same the through occur effects these that result in either sustained agonism system or functional antagonism. Thatnervous the in pathways endocannabinoid major two these t r a  ( mice n Supplementary Fig. Supplementary 1 Fig. 1 Fig.

a = 5–6 mice per group). ( group). per mice = 5–6 2-AG (nmol g–1) We employed also 100 150 50 e a Vehicle 0 , a mide that we observed in mice treated for 1 or 6 d with the the with d 6 or 1 for treated mice in observed we that mide f

26,27 C I ), likely reflecting a partial blockade of FAAH of blockade partial a reflecting likely ), ) (Dunnett’s ) (Dunnett’s Characterization of endocannabinoid metabolism in mice with chronic chronic with mice in metabolism endocannabinoid of Characterization Mgll Acute JZL184 *** of brain membrane proteomes showing the selective loss of active MAGL MAGL active of loss selective the showing proteomes membrane brain of e l

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Mgll 4 Mgll GHSMG intron 3, intron –/– 6 5 of sustained elevations in 2-AG. in elevations sustained of behavioral and neurophysiological the examining for and establish tissues peripheral in brain and 2-AG mouse the many hydrolase MAGL is principal the not but 2-AG, ( in elevations and hydrolysis 2-AG from in tissues peripheral of panel JZL184-treated a in changes metabolic similar in in acid the brain arachidonic physiological a as 2-AG designating findings ­previous ( JZL184 with chronically or ( mice Table1 ( levels 2-AG brain in elevations tenfold ( activity hydrolytic Mgll ( proteomics spectrometry–based mass and of MAGL in absence expression including ( FAAH activities, hydrolase serine brain other in alterations ing ing ( Medicine in the expression of tolerance. Although mice treated repeatedly ( repeatedly hypoalgesic treated maintained mice PF-3845 with Although tolerance. of expression the in difference marked a in resulted enzymes these of disruption longed proIn model. contrast, (CCI) nerve of the sciatic injury constrictive ( mechanical in ( test tail-withdrawal thermal acute the in ( assays pain multiple in efficacy Acute pharmacological blockade of MAGL or FAAH produced similar assays pain in tolerance causes blockade MAGL Chronic Supplementary Fig. 4

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© 2010 Nature America, Inc. All rights reserved. The loss of analgesic responses and occurrence The of loss and of cross- cannabinoid responses occurrence analgesic CB Brain PF-3845. administered cally ­ did not precipitate paw tremors in mice that were chroni d; 6 for day per kg per mg (10 similara degree to as JZL184 mice subjectedwith totreated a mild chronically THC were chronic that dosing mice in flutters paw CB direct with treatments repeated to exposed have been that rodents in observed been has that phenotype a dependence, physical ­produces CB exogenous to cross-tolerance causes MAGLof inactivation sustained MAGL ( agonist-induced catalepsy was less affected by sustained inactivation of effects of WIN55,212-2 and PF-3845 in the CCI model ( anti-allodynic the to cross-tolerance marked elicited also treatment ( Fig. THC of effects hypothermic the and to ­antinociceptive responses reduced showed JZL184 with chronically treated CB that in ( mice ago MAGL. receptor or FAAH in disruptions cannabinoid chronic with mice of in nists effects behavioral the assessed We CB to tolerance causes blockade MAGL Chronic system. endocannabinoid the in alterations of result a was tolerance of form this whether investigated Wenext enzyme. this of inactivation sustained following lost are MAGL of blockade Mgll as latencies tail-withdrawal equivalent displayed mice ( mice as control responses pain similar ( test). (Bonferroni group treatment drug acute (Dunnett’s mice control littermate wild-type or vehicle-treated means as presented are Data ( ( administration. chronic following ( cold ( PF-3845. with treatment chronic following maintained was effect this and PF-3845 with acutely treated mice in observed was effect hypoalgesic ( JZL184 with treatment chronic following observed not was response hypoalgesic this whereas nociception, thermal for test tail-immersion the in latencies withdrawal elevated caused JZL184 2 Figure nature nature tolerance in mice of with disruptions sustained MAGL that suggested e Supplementary Fig. 5 Fig. ,

These These findings indicate that the analgesic produced effects by acute We next asked whether prolonged MAGL or FAAH blockade blockade FAAH or MAGL prolonged whether asked next We f a 1 1 ) Chronic JZL184 treatment caused cross-tolerance to the anti-allodynic effects of WIN55,212-2 and PF-3845. PF-3845. and WIN55,212-2 of effects anti-allodynic the to cross-tolerance caused treatment JZL184 ) Chronic antinociception, hypothermia and catalepsy assays, indicating indicating assays, catalepsy and hypothermia antinociception,

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© 2010 Nature America, Inc. All rights reserved. S, epciey P-85 i nt fet S i te hippocampus the ( in DSI affect not did PF-3845 respectively. DSI, and chronic ( neurons of the cingulate cortex, where acute ( treated mice ( from slices with compared when JZL184 with treated cally andtime constant( in mouse hippocampal slices acute inhibition of MAGL by bath application of JZL184 potentiatesDSIwhether affected. was also In contrast with DSI impaired in this brain region by sustained MAGL inactivation,hippocampus the in we DSI askeding Endocannabinoids regulate several forms of blockade MAGL synaptic chronic by impaired is plasticity plasticity Synaptic nature nature hippocampus ( ­hippocampus the of region CA1 the in (fEPSPs) field potentials of postsynaptic excitatory depression PF- not CP55,940-induced but attenuated JZL184, treatment 3845, Chronic hippocampus. the in transmission CB altered ment treat PF-3845 or JZL184 chronic whether examined therefore We CB of effects behavioral the of many mediate synapses CB minimal ( putamen ( significance that only did a not effect exerted modest reach statistical regions ( of in brain both IPSCs depression CP55,940-induced blocked pletely ( cortex ( hippocampus either in IPSCs of depression vivo in ( mice vehicle-treated from those in than mice JZL184–treated chronically from cortex cingulate the of the neurons pyramidal in V layer or neurons pyramidal CA1 (IPSCs) ­hippocampal currents postsynaptic inhibitory of depression less ( cuits CB of desensitization with Fig. 6 P c a > 0.05) on CP55,940-induced depression of IPSCs in the caudate caudate the in IPSCs of depression CP55,940-induced on 0.05) > Recent studies have suggested that CB have studies suggested Recent consistent is treatment JZL184 chronic by DSI of attenuation The IPSC amplitude (%) IPSC amplitude (%) 10 10 50 50 0 0 Fig. Fig. a administration of PF-3845 did not alter CP55,940-induced CP55,940-induced alter not did PF-3845 of administration ) or cingulate cortex ( V V Fig. 6 Fig. 2 0 NEUR ehicle ehicle Supplementary Fig. 11 Supplementary 1 0 2 2 1 1 Supplementary Fig. 12 Fig. Supplementary 5 1 1 4 0 ). In support of this premise, CP55,940 (3 (3 CP55,940 premise, this of support In ). 1 0 mV Fig. 6 Fig. 6 receptor adaptations ( adaptations receptor d V PF JZL1 OSCI Hippocampus Hippocampus 2 0 ehicle Supplementary Fig. 13 Fig. Supplementary ). The CB The ). 0 JZL1 JZL1 -3845 T CP 55,940 , 5 2 T 2 1 ime (min) 1 1 2 60 84 ime (s) -mediated depression of glutamatergic excitatory excitatory glutamatergic of depression -mediated b s a 84 84 τ EN ) treatment with JZL184 potentiated and disrupted ). We observed similar effects in layer V pyramidal ) of DSIin hippocampalslices from mice chroni 0 80 C PF PF E 10 2 2 2 1

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oxidase A oxidase mice and in receptors impairments in 5-HT1A CB to cross-tolerance of degree We pathways. also note that chronic treatment JZL184 produced an larger evidently endocannabinoid brain in alterations also to acid, contribute arachidonic in reductions as such inhibition, MAGL by changes caused however,that metabolic other out rule the possibility CB desensitizes eventually and activates tonically that 2-AG in elevation sustained a produces ade block MAGL chronic which in model a co-treatment supports by rimonabant with attenuated both were JZL184 by caused function DISCUSSION DSI and DSE both in defects exhibit DSI and DSE both enhances blockade FAAH, not but MAGL, acute that findings ous previ with is consistent transmission GABAergic and glutamatergic both for effects these observed we That plasticity. synaptic of forms endocannabinoid-mediated specific FAAH, impairs not but MAGL, ( controls vehicle from significantly differ not did PF-3845 with treated ( mice control vehicle-treated from those in than JZL184 with treated chronically ( hippocampus the in IPSCs of CB the ( DSI. of decay the of curves fitting exponential DSI, and magnitude both for groups vehicle-treated versus JZL184- for ( cortex cingulate the of neurons V pyramidal layer and ( neurons pyramidal CA1 hippocampal in DSI attenuated PF-3845, ( plasticity. synaptic of 6 Figure reduced activity of nicotinic receptors receptor systems following of deletion a enzyme, metabolic adaptations. receptor the in mechanisms and JZL184-treated the for 129SvEv/C57BL/6J versus (C57BL/6J strain the basis for it in this difference, differences background could reflect of disruption anandamide. and 2-AG endocannabinoids, principal two the of elevations sustained to response in adaptations CB brain Thus, function. CB in changes or tolerance of evidence out with effects analgesic persistent caused FAAH, of which disruption pharmacological sustained of that with contrasted markedly profile This plasticity. synaptic endocannabinoid-dependent in disruptions CB in reduction a agonists, CB to cross-tolerance inhibition, enzyme acute of effects to by analgesic the tolerance as manifested system, endocannabinoid of of brain the antagonism functional substantial produced MAGL also blockade pharmacological chronic or deletion genetic either by brain CB in reductions substantial by to shown be mirrored have been phenotypes behavioral and these nists leads to the development of tolerance and physical dependence ago receptor THC with and treatment Prolonged cannabinoid other P > 0.05; > 0.05; These These results, taken together, indicate that sustained inactivation of Although there are other examples of functional antagonism of of antagonism functional of examples other are there Although That the cannabinoid cross-tolerance and alterations in CB n Mgll 37,38 P = 11–15 mice per group). The lines superimposed are the single single the are superimposed lines The group). per mice = 11–15 1 < 0.05 in both brain regions). Brain regions from mice chronically chronically mice from regions Brain regions). brain both in < 0.05 agonist CP55,940 (3 (3 CP55,940 agonist

Chronic disruption of MAGL impairs CB impairs MAGL of disruption Chronic −/− . We found that sustained elevations in brain 2-AG caused caused 2-AG brain in elevations sustained Wethat . found ) mice ) n = 6–7 mice per group). Data are presented as means means as presented are Data group). per mice = 6–7 mice, respectively) or the existence of compensatory compensatory of existence the or respectively) mice, Mgll 4 0 2 , we report the first instance, to the best of our our of best the to instance, first the report we , ( 0 Mgll Fig. 3d Fig. and that - diacylglycerol that and a , b ) Chronic treatment with JZL184, but not not but JZL184, with treatment ) Chronic −/− 1 mice that counteract the observed CB observed the counteract that mice , receptors undergo markedly different different markedly undergo receptors μ 1 c e 1 receptor expression and function, and and function, and expression receptor M) induced significantly less depression depression less significantly induced M) ) and cingulate cortex ( cortex cingulate ) and ). Although we do not yet understand understand yet not do we Although ). receptor expression and activity in the the in activity and expression receptor 1 receptors in the brain. We cannot, Wecannot, brain. the in receptors 1 agonists ( agonists 3 23,24 9 in . Ache c 1 , Fig. 3a Fig. receptor expression or or expression receptor 1 d − -dependent forms forms -dependent / ) Bath application of application ) Bath Maoa −

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© 2010 Nature America, Inc. All rights reserved. nioieto cud e trbtd o h dsniiain f CB of PAG desensitization in ( receptors the to attributed be could antinociception THC-induced and the WIN55,212-2– to and cross-tolerance JZL184 of of occurrence effects antinociceptive the to observed we that catalepsy cannabinoid-induced with ( pallidus globus and putamen caudate is catalepsy consistent with the lack of CB to cannabinoid-induced cross-tolerance The minimal of drugs. these strong to cross-tolerance the and antinociceptive effects hypothermic (ref. THC of WIN55,212-2 and effects cataleptic and hypothermic antinociceptive, the to CB of antagonism functional 2-AG cause of endogenous elevations prolonged in which model a supports regions brain multiple in DSI on effects opposing DSI ish abol to shown been has receptor this of antagonism or deletion as ( cortex cingulate and hippocampus the in function receptor are in alterations CB plasticity synaptic consistent with the observed these neuronal populations ( that repeated administration of JZL184 led to profound DSI deficits in ( cortex gulate DSI in of neurons hippocampus potentiates the inhibition DSE and DSI including plasticity, behavior. pain regulate that circuits neural the in crosstalk ( model CCI the in inhibitor MAGL FAAH a to chronic cross-tolerance produced that blockade the observation in our pathways Regardless, system. signaling nervous endocannabinoid affect differentially also might distinctions anatomical these and brain the throughout respectively) postsynaptic, and (pre- compartments subcellular and Finally, MAGL and FAAHCB are of found in different neuronaloccupancy populations greater achieve therefore may similar are endocannabinoid these of levels (see anandamide than higher much agonists exogenous of activity CB of nitude CB partial a receptor agonist) as acts which anandamide, with contrast (in agonist CB full a as 2-AG of efficacy higher the to related be may systems expression heterologous in previously in tion the and/or brain, as recycling has observed been and anandamide have differential effects on CB sic activity in acute treatment procedures. One possibility is that 2-AG Indeed, MAGL and FAAH inhibitors displayed similar relative analge correlated with the induction of superior in efficacy behavioral assays. is not necessarily effect this that unclear, but indicate would our data alterations CB1 substantial causing of capable more is 2-AG How system. endocannabinoid brain the of integrity the on anandamide than effect greater a exert 2-AG in elevations that sustained indicates FAAH, not but MAGL, inactivated chronically with and CB That behavioral enzymes. degradative of cognate their inactivation chronic following desensitization receptor and tolerance causing of capable both are versus (vesicular nonvesicular), classical neurotransmittersrelease andand lipid messengers storage of mechanisms the in differences tal fundamen despite that, indicates finding This system. nervous the in desensitization and downregulation receptor to leads transmitter lipid a degrades that enzyme an of disruption which in knowledge, t r a  antinociception cannabinoid-induced in cated

In contrast with direct CB synaptic of forms several regulates system endocannabinoid The 22,45 C I Fig. 2e Fig. . That acute and chronic inhibition of MAGL produced produced MAGL of inhibition chronic and acute That . e l 1 receptor desensitization is not related to the intrinsic intrinsic the to related not is desensitization receptor , Supplementary Fig. 4 Fig. Supplementary f ) indicates that 2-AG and anandamide pathways can pathways 2-AG that and anandamide ) indicates s 4 2 1 , , although previous research suggests that the mag Fig. Fig. receptors in the nervous system. nervous the in receptors 5 1 46), chronic MAGL disruption only caused caused only disruption MAGL chronic 46), ), a brain area that has been strongly impli strongly been has that area brain a ), receptor adaptations occurred only in only mice occurred adaptations receptor 1 Fig. agonists, which produce cross-tolerance 4 3 6 . Bulk brain levels of 2-AG are also also are 2-AG of levels brain Bulk . 4 ). ). These impairments in short-term 1 3 . This differential desensitization desensitization differential This . 4 Fig. 1 Fig. . Acute MAGL, but not FAAH, not but MAGL, Acute . ). Notably, however, we found found we however, Notably, ). 4 7 Fig. Fig. . Conversely, the tolerance tolerance the Conversely, . 1 receptor in desensitization a ; although the interstitial interstitial the although ; 5 4 ), which are ass are which ), 4 1 ), and elevated 2-AG 2-AG elevated and ), 4 receptor desensitiza 8 1 . We also note that that note Wealso . receptors receptors in vivo in 2 1 0 receptor receptor and cin remains remains in vivo in o Fig. Fig. ciated ciated 5 ), ), 1 1 1 ------.

a less suitable target for treatment of pain disorders? Perhaps, but it but Perhaps, disorders? pain of treatment for target suitable less a FAAHdisrupted Might imply chronically this systems. that MAGL is in multiple pain efficacy assays, but are effects these only in sustained for instance, that acute inhibition of FAAH and MAGL produces Consider,may implications. have discoveries translational important similar pain show in in preferentially reductions which FAAH-disrupted animals, observed phenotypes behavioral the with consistent also is idea This may reflect a more limited, stress-dependent function for anandamide. and CB system. nervous In contrast, the of preservation analgesic phenotypes the throughout 2-AG for role broad a suggest MAGL of disruption widespread behavioral and CB The system. nervous the in pathways signaling endocannabinoid of properties and functions distinct the shaping for important is tion pallidus. globus and putamen caudate the in tions CB to of effects the other cataleptic CB sustained inactivation of both MAGL and FAAH causes cross responses behavioral cataleptic promote does inhibitors these with treatment combined however, catalepsy; cause MAGL nor FAAHalone neither inhibitors reprintsandpermissions at online available is information permissions and Reprints Published online at http://www.nature.com/natureneuroscience/. The authors declare no competing interests.financial design, execution and interpretation of the experiments and wrote the manuscript. interpretation of electrophysiology experiments. A.H.L. and B.F.C. the supervised of receptor adaptation experiments. Q.-s.L. contributed to the design and experiments. D.E.S and L.J.S.-S. contributed to the design and interpretation E.A.T. contributed to the design and interpretation of experiments. P.T.N. and J.J.B. contributed to receptor adaptation experiments. L.B. contributed to behavioral experiments. B.P. performed the electrophysiology and metabolic biochemistry behavioral experiments. D.K.N., S.G.K., D.R. and experiments and contributed to behavioral experiments. J.Z.L. contributed to performed the metabolic biochemistry, proteomic and J.E.S. performed behavioral and receptor adaptation experiments. J.L.B. Institute for Biology.Chemical DA023758 to P.T.N.), the American (D.K.N.) Cancer Society and the Skaggs Fellowships (DA026261 to J.L.B., DA026279 to J.E.S., DA028333 to L.B. and and DA024741), Ruth L. Kirschstein US National Institutes of Health Predoctoral DA005274, DA015683, DA03672, DA005274, DA07027, DA014277, DA023758 the US National Institutes of Health (grants DA017259, DA009789, DA025285, laboratories for reading critical of the manuscript. This work was supported by I. Beletskaya and R. Abdullah for technical support and the Cravatt and Lichtman We thank S. Niessen and H. Hoover for assistance with proteomics studies, Note: Supplementary information is available on the online the at http://www.nature.com/natureneuroscience/. paper of the in version available are references associated any and Methods M for dependence. risk high without activity analgesic sustained producing of capable are inhibitors FAAH that suggests anandamide brain of elevations continuous to nonadaptive CB That inhibitors. about cerned the potential tolerance and of withdrawal effects MAGL In MAGL event, blockade. this one to need partial would be still con also may be possible to achieve prolonged analgesic responses through COM AUTH Acknowle et In summary, diversifica our in a which data support model 1 P h 1 O E and anxiety ods 1 TI R R receptor function in mice with sustained inactivation of FAAH CON NG advance online publication online advance 1 d receptor expression and activity in brain regions such as in brain regions and activity expression receptor FI gmen TRIBUTI N A NC 2 5 / ts . procedures with strong stress components. These 1 IA receptors, on the other hand, are surprisingly surprisingly are hand, other the on receptors, 1 9 ON L . It will be interesting to determine whether whether determine to interesting be will It . I N S T E 1 R receptor adaptations caused by chronic E STS 1 agonists and

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© 2010 Nature America, Inc. All rights reserved. 25. 24. 23. 22. 21. 20. 19. 18. 17. 16. 15. 14. 13. 12. 11. 10. 9. 8. 7. 6. 5. 4. 3. 2. 1. nature nature

Haller,J. Y. Gao, A. Tanimura, brain. the in signaling Endocannabinoid R.A. Nicoll, & R.I. Wilson, A. Straiker, B. Pan, J.Z. Long, S.G. Kinsey, J.Z. Long, B.F. Cravatt, K. Ahn, Jhaveri, M.D., Richardson, D., Kendall, D.A., Barrett, D.A. & Chapman, V. Analgesic Lichtman, A.H. Kathuria, S. acid fatty lacking Mice B.F. Cravatt, & T. Advani, C.C., Shelton, A.H., Lichtman, B.F. Cravatt, anandamide, of degradation and synthesis Enzymatic S.A. Chin, & regulate D.G. Deutsch, that pathways Enzymatic B.F. Cravatt, & M.K. McKinney, K., Ahn, Fowler, C.J. The cannabinoid system and its pharmacological manipulation–a review, G. Marsicano, targets. therapeutic as receptors Cannabinoid K. Mackie, Sugiura, T. R. Mechoulam, W.A.Devane, emerging an as system endocannabinoid The G. Kunos, & S. Bátkai, P.,Pacher, fet o ehne cnaiod inln b FA ihbto i rats. in inhibition FAAH by signaling cannabinoid enhanced (Berl.) Psychopharmacology of effects (2010). mice. knock-out lipase diacylglycerol in neurogenesis Neuron diacylglycerol lipase alpha mediates retrograde suppression of synaptic transmission. (2002). 678–682 neurons. hippocampal autaptic in excitation of suppression depolarization-induced mediated Ther.Pharmacol.Exp. J. signaling. endocannabinoid retrograde enhancespiperidine-1-carboxylate (JZL184) ylglycerollipaseinhibitor 4-nitrophenyl 4-(dibenzo[d][1,3]dioxol-5-yl(hydroxy)methyl) (2009). 20270–20275 crosstalk endocannabinoid by regulated pain. neuropathic effects. behavioral cannabinoid amides. fatty-acid neuromodulatory pain. inflammatory reduces that Neurosci. J. pain. neuropathic of model rat a in inhibition hydrolase amide acid fatty of effects Ther. Exp. Pharmacol. J. selectivity. and potency of combination unprecedented an for evidence analgesia: Med. Nat. Pain hypoalgesia. phenotypic receptor–mediated cannabinoid a exhibit hydrolase amide USA Sci. hydrolase. amide acid fatty lacking mice in signaling cannabinoid agonist. receptor cannabinoid a (2008). system. nervous the in signaling endocannabinoid anandamide. of hydrolysis and Pharmacol. uptake the upon emphasis with excitotoxicity. Toxicol. brain. in ligand (1995). receptors. cannabinoid binds that gut, canine receptor. cannabinoid pharmacotherapy. of target

109 NEUR

65 46 t al. et et al. et t al. et

Mol. Pharmacol. Mol. , 319–327 (2004). 319–327 , et al. et 98

et al. et , 320–327 (2010). 320–327 , 9 , 101–122 (2006). 101–122 , t al. et

et al.

20 et al. et , 76–81 (2003). 76–81 , et al. , 9371–9376 (2001). 9371–9376 , 26 t al. et et al. et OSCI Discovery and characterization of a highly selective FAAH inhibitor FAAH selective highly a of characterization and Discovery lcae f -rcioollcrl yrlss y eetv monoac selective by 2-arachidonoylglycerol hydrolysis of Blockade t al. et os f ergae noanbni sgaig n rdcd adult reduced and signaling endocannabinoid retrograde of Loss Science , 549–562 (2006). 549–562 , t al. et Interactions between environmental aversiveness and the anxiolytic the and aversiveness environmental between Interactions t al. et t al. et et al. , 13318–13327 (2006). 13318–13327 , Dual blockade of FAAH and MAGL identifies behavioral processes behavioral identifies MAGL FAAHand of blockade Dual et al. et 2-Arachidonylglycerol: a possible endogenous cannabinoid receptor Biochem. Biophys. Res. Commun. Res. Biophys. Biochem. eetv bokd o 2aahdnygyeo hdoyi produces hydrolysis 2-arachidonoylglycerol of blockade Selective Modulation of anxiety through blockade of anandamide hydrolysis. Monoacylglycerol lipase limits the duration of endocannabinoid- of duration the limits lipase Monoacylglycerol J. Pharmacol. Exp. Ther. Exp. Pharmacol. J. Isolation and structure of a brain constituent that binds to the to binds that constituent brain a of structure and Isolation lcae f noanbni-erdn ezms attenuates enzymes endocannabinoid-degrading of Blockade uesniiiy o nnaie n ehne endogenous enhanced and anandamide to Supersensitivity Reversible inhibitors of fatty acid amide hydrolase that promote B cnaiod eetr ad ndmn dfne against defense on-demand and receptors cannabinoid CB1 EN h edcnaiod -rcioollcrl rdcd by produced 2-arachidonoylglycerol endocannabinoid The oeua caatrzto o a ezm ta degrades that enzyme an of characterization Molecular Identification of an endogenous 2-, present in present 2-monoglyceride, endogenous an of Identification

Science 302 C

331 311

E 76 , 84–88 (2003). 84–88 ,

Pharmacol. Rev. Pharmacol. 204 , 591–597 (2009).591–597 , , 1220–1227 (2009). 1220–1227 , advance online publication online advance , 441–448 (2004). 441–448 ,

258 Nat. Chem. Biol. Chem. Nat. Biochem. Pharmacol. Biochem. , 607–616 (2009). 607–616 , Chem. Biol. Chem. , 1946–1949 (1992). 1946–1949 , Nature in vivo in

384

330

58 16 . , 389–462 (2006). 389–462 , , 83–87 (1996). 83–87 , , 902–910 (2009). 902–910 , , 411–420 (2009). 411–420 , ice. Pharmacol. Biochem.

Proc. Natl. Acad. Sci. USA Sci. Acad. Natl. Proc. 5

, 37–44 (2009). 37–44 , 215 hm Rev. Chem. J. Neurosci. J.

46 , 89–97 (1995). 89–97 , , 791–796 (1993). 791–796 , nu Rv Pharmacol. Rev. Annu.

108

Proc. Natl. Acad. Natl. Proc. 30 udm Clin. Fundam. 1687–1707 , , 2017–2024 , Science

50 83–90 ,

296 106 ­ , ,

37. 31. 38. 36. 35. 34. 33. 32. 30. 29. 28. 27. 26. 48. 47. 46. 45. 44. 43. 42. 41. 40. 39.

i, .. Hmsn RE, edye, .. Cidr, .. fet o chronic of Effects S.R. Childers, & S.A. Deadwyler, R.E., Hampson, L.J., Sim, K.W. Falenski, oeo J. Romero, dependence. and tolerance Cannabinoid B.R. Martin, & A.H. Lichtman, K. Monory, Kreitzer, A.C. & Regehr, W.G. Retrograde signaling by endocannabinoids. endocannabinoid of specificity Presynaptic R.A. Nicoll, & G. Kunos, R.I., Wilson, activity B.F. Pharmacological Cravatt, & G. Griffin, E.G., Hawkins, A.H., Lichtman, precipitated Cannabinoid B.R. Martin, & J.A. Lowe, S.M., Scates, M.D., Aceto, Long, J.Z., Nomura, D.K. & Cravatt, B.F. Characterization of monoacylglycerol lipase Nomura, D.K. brain of profile comprehensive A B.F. Cravatt, & G.M. Simon, J.L., Blankman, serine the profiling: protein Activity-based B.F. Cravatt, & M.P. Patricelli, Y., Liu, ihmn AH, ok SA & atn BR Ivsiain f ri sts mediating sites brain of Investigation B.R. Martin, & S.A. Cook, A.H., Lichtman, catalepsy of chlordiazepoxide by Enhancement A.P. Wickens, & R.G. Pertwee, cross- of Development B.R. Martin, & L. Melvin, S., Ward,F., D.R., Fan, Compton, mediate cannabinoids Endogenous M. Kano, & T. Maejima, T., Ohno-Shosaku, Caillé, S., Alvarez-Jaimes, L., Polis, I., Stouffer, R-(+)- D.G. & Parsons, of L.H. Specific alterations administration chronic of endocannabinoids Effect B.R. the Martin, & of L.J. Sim-Selley, pharmacology and Biochemistry C.J. Hillard, Luk, T. in receptors 5-HT1A functional of density Reduced I. Seif, & G. Hilaire, Lanoir,J., sympathetic in function receptor nicotinic Reduced H.S. Shin, & C.J. Lee, M., Sun, anbni rcpo bnig n mN lvl i svrl a ban regions. brain rat several in levels mRNA and binding receptor cannabinoid brain. rat in autoradiography with treatment Pharmacol. Exp. Δ Neurobiol. hippocampus. the in signaling vivo in hydrolase amide acid fatty by mediated, not but regulated, is amides acid fatty of administration. cannabinoid receptor adaptation following Pharmacol. 141716A. SR antagonist, receptor cannabinoid selective the by withdrawal Biol. Chem. inhibition reveals differences in central and peripheral endocannabinoid metabolism. agents. nerve 14 2-arachidonoylglycerol. endocannabinoid the hydrolyze that enzymes nue i rt b itaeos r nrplia ijcin o enantiomeric involvement. gray periaqueductal supporting of evidence rats: in injections antinociception cannabinoid-induced intrapallidal or intravenous by cannabinoids. rats in induced Ther. Exp. Pharmacol. J. between tolerance Neuron terminals. presynaptic to neurons postsynaptic depolarized from signals retrograde cocaineandself-administration. extracellularof endocannabinoid nucleusaccumbenstheethanol,levels inheroinby (2002). mice. in adaptation receptor cannabinoid on or (WIN55,212–2) mesylate (1-naphthalenyl)methanone enzoxazinyl]- [2,3-dihydro-5-methyl-3-[(morpholinyl)methyl]pyrrolo[1,2,3-de]-1,4-b Mediat. 2-arachidonylglycerol. and arachidonylethanolamide agonist. receptor cannabinoid CB1 mouse knockout oxidase-A monoamine of cord spinal neonates. and medulla brain, the knockout acetylcholinesterase the in mouse. hypothermia the for responsible is ganglia Res. Brain Mol. Res. Brain . 9-tetrahydrocannabinol in mice. in 9-tetrahydrocannabinol , 1347–1356 (2007). 1347–1356 , .

et al.

J. Physiol. (Lond.) Physiol. J. 29 J. Pharmacol. Exp. Ther. Exp. Pharmacol. J. 61

J. Comp. Neurol. Comp. J. , 729–738 (2001). 729–738 ,

, 3–18 (2000). 3–18 , 12

Proc. Natl. Acad. Sci. USA Sci. Acad. Natl. Proc. 282 Identification of a potent and highly efficacious, yet slowly desensitizing t al. et 16 t al. et , 324–330 (2002). 324–330 , Nat. Chem. Biol. Chem. Nat. et al. Neuropharmacology et al. et , 744–753 (2009). 744–753 , Neuropsychopharmacology , R1–R2 (1995). R1–R2 ,

Δ 168 -erhdoanbnl n anbni-tmltd [35S]GTP cannabinoid-stimulated on 9-tetrahydrocannabinol J. Pharmacol. Exp. Ther. Exp. Pharmacol. J. eei dseto o bhvorl n atnmc fet of effects autonomic and behavioural of dissection Genetic fet o crnc xoue to exposure chronic of Effects Activation of the endocannabinoid system by organophosphorus

Δ , 691–717 (2005). 691–717 , Faah -erhdoanbnl C 5,4 ad I 55,212. WIN and 55,940 CP 9-tetrahydrocannabinol,

271

−/−

578

46 495 mice display differential tolerance, dependence and dependence tolerance, differential display mice J. Neurosci. J. , 1383–1390 (1994). 1383–1390 , , 100–108 (1997). 100–108 ,

Neuron , 751–764 (2007). 751–764 , J. Neurosci.J. 4 , 607–623 (2006). 607–623 ,

, 373–378 (2008). 373–378 , 302

PLoS Biol. PLoS 30 Br. J. Pharmacol. J. Br. , 237–244 (1991). 237–244 , , 73–79 (2002). 73–79 ,

31 96

Δ

, 453–462 (2001). 453–462 , 16 , 14694–14699 (1999). 14694–14699 , 9-tetrahydrocannabinol and anandamide 35

27 276

J. Pharmacol. Exp. Ther.Exp. Pharmacol. J. , 8057–8066 (1996). 8057–8066 , , 1775–1787 (2010). 1775–1787 , 5 , 3695–3702, (2007). , e269 (2007). e269 , , 585–593 (1996). 585–593 ,

142 Δ rsalnis te Lipid Other Prostaglandins -erhdoanbnl on 9-tetrahydrocannabinol , 495–500 (2004). 495–500 , Δ t r a 9-tetrahydrocannabinol C I

303 hm Biol. Chem. Curr. Opin. , 36–44 , e l Handb. Eur. J. Eur. s γ S 

© 2010 Nature America, Inc. All rights reserved. type) were precipitated with 1:4 chloroform:methanol and denatured with with denatured 25 mM ammonium and bicarbonate chloroform:methanol in 6 M 1:4 urea. Samples with were reduced precipitated with 10 were mM type) fromprotein) total mg (0.75 M cRNA probes to described hybridization25-performedonwas mice were postfixed, cryoprotected and frozen as previously described In situ to a palmitic acid calibration curve. brains were measured on an Agilent 1100 series LC-MS and quantifiedously compareddescribed Brainmetabolite measurements. liquid chromatography–mass spectrometry assay determinedpreviouslywasusing°C)DMSO a vehicleat 25 min (30 described brain homogenates ( 2-A described previously treated with 5 analysis. profiling protein Activity-based genotype) were prepared as previously described homogenatesfrom C57BL6/Jand homogenates. brain mouse of Preparation receptor adaptation) or vehicle for 6 d. per kg), rimonabant (3 mg per kg for behavioral analysis, 10 mg per kg for CB daily injection of JZL184 (40 mg per kg), PF-3845 (10 mg per kg), THC (10 mg 10 of volume a in administration of route intraperitoneal the via administered were drugs All 1:1:18. of ratio a in saline and (Sanofi-aventis) Alkamuls-620 were filters fiber obtained through glass Fisher Scientific. GF/B Whatman and Scientific Fisher Thermo from purchased was 1) Econo (ScinitSafe fluid Scintillation Pharmacia.Amersham and Analytical Sciences. [ [ Aldrich. sine deaminase and bovine serum albumin (BSA) were purchased from Sigma- Institute on Drug Abuse. AM251 was obtained from Tocris. GDP,THC andGTP CP55,940 were obtained from the Drug Supply Programviously of the National Drugs and chemicals. the Scripps Research Institute and Virginia Commonwealth University. ance with the guidelines of the Institutional Animal Care and Use Committees Mgll of expected Mendelian frequency, and display normal cage behavior comparedallele. Mice homozygouswith for the gene-trap ( 430-bp product for the wild-type allele and a 220-bp producttrap reversefor 5 the gene-trapped C-3 Genomic Medicine: of genomic tail DNA using the following primers designed by Texas Institute of of the ACT GGA T(gene trap insertion)CT TGG GCC TTC TGT TC,The gene whichtrap vectoris insertion upstreamsite was mapped to the sequence GCC TTG TGG ofthe cell line OST113734) containing a gene trap vector inserted into the third intron 6J background. as male and female Animals. ONLINE MET nature nature urea M 2 to diluted and iodoacetamide mM 40 with alkylated dithiothreitol, ultidimensional ultidimensional rg wr dsovd i snfcto i a eil cnitn o ethanol, of consisting vehicle a in sonification via dissolved were Drugs μ ′ G , l per g of body mass.For body of chronicadministration,g drug per l subjectsreceived a +/+ Mgll hydrolysis assays. Mgll hybridization. Mgll 15,17 and Subjects consisted of male C57BL/6J mice (Jackson Laboratories) as well 4 reverse 5 NEUR 35 catalytic exon 4. 9 genewere obtained fromTexasthe Institute ofGenomic Medicine. . WIN55,212 was purchasedWIN55,212was . fromCayman Chemical.Rimonabant, with [ S]GTP Mgll ′ μ -ATA AAC CCT GCA CTT GCAGTT TC-3 2 M JZL184 or DMSO vehicle (30 min at 25 °C) was performed as Mgll OSCI 9 Mgll − H except that free fatty acid levels in / 35 − Mgll γ ODS ′ littermates. Animal experiments were conducted in accord -GGG AGT CAA GAC ACT GGG GAA TCC T-3 S]UTP-labeled, single-stranded antisense and sense control S (1250 Ci mmol Ci (1250 S Mgll Gt1 n 2 LC cDNA (bases 285–600). Perfused brains from 12-week-old male 7 = 4 per genotype) pretreated with either 1 EN . JZL184 and PF-3845 were synthesized as described pre +/+ (neo) - 2-AG hydrolytic activity of forward 5 M 3 C , H]SR141716A (44.0 Ci mmol Mgll Mgll poemc analysis. proteomic S E mutant mice (TG0078; derived from OmniBank ES Mgll +/− genotype was determined by PCR amplification and +/+ Mgll Brainlipid levelswereprevi determined as ′ -TTG -TTG CCT GCT TGC TCT TAA CTC TTG μ and − m-thick free-floatingm-thickcoronal as sections Mgll 1 +/+ ) was obtained from PerkinElmer Life PerkinElmer from obtained was ) , Mgll −/− Mgll Mgll Analysis of brain proteomes pre- proteomes brain of Analysis mice on a mixed 129SvEv/C57BL/ ebae n slbe brain soluble and Membrane −/− 2 +/− −/− 7 2 on an Agilent 6520 QTOF MS. 9 mice ( mice and . mice) are viable, born at the Mgll Mgll Mouse brain proteomes proteomes brain Mouse Mgll − +/+ +/+ 1 n ) was purchased from = 3 mice per geno per mice 3 = , , ′ Mgll Mgll , which amplified a −/− Mgll mice ( mice +/− +/− +/+ μ M JZL184 or and and and ′ γ , and gene n S, adeno 4 9 = 4 per per 4 = Mgll Mgll . Mgll In situ −/− −/− −/− 1 - - - - -

the final chronic drug injection. Cross-tolerance studies in the CCI model were performed starting 26 h followingin the acetone-induced paw lifting model, with a maximum cut-off time of 20 s. Coast Medical) and approximately 30 min later were evaluated for cold allodynia Subjects were assessed for mechanical allodynia using von Frey filaments (North sciatic nerve and allodynia assessment were performed as previously described tail-immersion test at 56.0 °C using a 10-s cut-off or the final chronic injection. Acute thermal antinociception was assessed in the occurring on day 6. Subjects were evaluated 2 h after acute drug administration mentgroups received ofd 5 daily vehicle injections, with acute treatment drug assays. Behavioral greater than 0.08 were included in the spectral counting analysis. scoresCNdeltaand correlation(+3) 3.5 (+2), 2.5scores (+1),greater 1.8 than DTASELECT.with groupedand filteredwere results andcross- Peptideswith searched against the mouseIPI database usingthe SEQUEST search algorithm type, 45 ( HPLC series 1200 Agilent an to coupled(ThermoFinnigan) 30 (ThermoFinnigan) coupled to an Agilent 1100 series HPLC ( analysis was performed as previously described −80 °C until use. Multidimensional protein identification technology (MudPIT) samples were acidified with 5% formic acid (wt/vol) and aliquots were frozen at performed overnight at 37 °C in the presence of 1 mM CaCl ammoniumwithmM25 bicarbonate. Digestion (0.5 withtrypsin 590siuae [ 55,940–stimulated described previously as conducted were brains whole in binding Binding assays. observation period 1-h a for recorded were paws, front the of shaking or tremors any including afterchronich final 2kg) the per injection and incidentsthe of paw fluttering, 4 h than less in completed being assessment dose-response entire the with injection, each after min 30 measure each for evaluated were subjects and min 40 every given were injections assessed, were endpoints behavioral baseline which in regimen dosing cumulative a using relationships dose-response evaluated we To study, for rectum. into this cm the required 2.0 of mice number the reduce immersion test at 52.0 °C test bar the in catalepsy for mice evaluating by assessed was activity Cannabimimetic injection. chronic final the after h 48 10 mM HEPES, 0.2 mM EGTA, 2 mM MgCl tion containing 80 mM cesium methanesulfonate, 60 mM CsCl, 5 mM QX-314, we clamped the neurons at −60 mV and filled the pipettes with an internal solu 1440A) and analysis software pClamp 10 (Molecular Devices). To record IPSCs, scopy. micro Data acquisition contrast and analysis interference were performed differential using a digitizer infrared (DigiData under 700B) (Multiclamp (1 previously slices (300 putamen caudate and cortical Hippocampal, injection. final the after h decapitated24–26 and inhalation isoflurane by anaesthetized were subjects E landmarks and measured using ImageJ (US National Institutes of Health). anatomicalusing selected interestwereof Regions camera. video XC-77Sony with [ ized water. The rinsed slides were dried and exposed to Kodak BioMax MR film were rinsed twice in 50 mM Tris buffer (pH 7.4) at 4 °C of and concentrationsthen (3 CP55,940 rinsed effective in deion maximally of (basal) absence or presence the in ­incubated in assay buffer and 0.5% BSA (wt/vol) containing 0.04 nM [ stored were cut on a cryostat at −20 °C, thaw-mounted onto gelatin-subbed slides and lectrophysiology slice preparationlectrophysiologyslice testing.and μ Subjects were evaluated for cross-tolerance to WIN55,212-2 or THC 26 or 26 THC or WIN55,212-2 to cross-tolerance for evaluated were Subjects Forprecipitated withdrawal, weremice challenged rimonabantwith mg(10 Whole-cell voltage-clamp recordings were made using patch clamp amplifier μ M) or PF-3845 (10 g protein, 5-step MudPIT) or an LTQ Orbitrap Velos mass spectrometer VelosLTQOrbitrapmass an or MudPIT) 5-step protein, g μ 14

m thick) were cut using a vibrating slicer (Leica) and prepared as described desiccated at −80 °C until use. For assay, slides were brought to 20–25 °C, C] standards for 24–36 h. Films were digitized at 8-bits per pixel with a μ 3 1 g protein, 10-step MudPIT). The tandem mass spectrometry data were . 2 0 . In the acute experiments, the slices were perfused with JZL184 with perfused were slices the experiments, acute the In . μ M) and/or vehicle for 2 h at 25 °C. After final incubation,slides final After °C. at25 and/orh vehicleM) for2 CP-55,940–stimulated [ 5 To control for stress of repeated injection, all acute treat acute all injection, repeated of stress forTo control 0 . 35 S]GTP μ M) for 40–80 min. 3 1 and hypothermia by inserting a thermocouple probe γ atrdorps crnl etos (20 sections coronal autoradiographs, S 35 S]GTP 2 , 4 mM MgATP, 0.3 mM Na 2 γ 1 7 S binding and [ 9 on an LTQ mass spectrometer 1 . Surgery for CCI model of the 9 In the chronicInthe experiments, , antinociception in the tail tail the in antinociception , 2 doi:10.1038/nn.2616 . The tryptic peptide n = 2 per genotype, n 3 H]-SR141716A = 1 per geno per 1 = μ 3 g 35 1 . For CP- For . μ S]GTP l −1 2 ) was) GTP

μ m) m) 1 γ 8 S ­ - - - - .

© 2010 Nature America, Inc. All rights reserved. basal. All receptor binding experiments wereabovestimulation percentage asperformed reported are values and bindingin basal duplicateminus and reported binding data. Stimulated binding was determined as agonist-stimulated binding ± experiments were performed in triplicate and all data points are usingreported a Bonferronias testmean to correct used, for when multiplecaptions figure comparisons.in noted[ are treatments within-drug specific and formed for comparisons to treatment or genotypic dose control.ANOVA, significantDunnett’s (cumulative Planneda Following ANOVA comparisonsresponses). repeated-measures or genotype) or (treatment ANOVAby evaluated initially were endpoints behavioral and quantification noted. otherwise Results were considered significantto be at statistics.and analysis Data 1 °C by using an automatic temperature controller. cingulate cortex or the caudate putamen. All recordings were performed at 32 in the in the stratum radiatum of the CA1 region of hippocampus, in layer V of Toevoke IPSCs or fEPSPs, bipolara tungsten stimulation electrode was placed 1 M NaCl, and picrotoxin (50 ance changed by more than 20%. To record fEPSPs, the pipettes were filled with was monitored throughout the recordings, and data were discarded if the resist cerebrospinalthroughoutfluid resistanceexperiments. the Series M (15–30 amino-5-phosphonovaleric( acid 20 (CNQX,6-cyano-7-nitroquinoxaline-2,3-dione antagonists and 10 mM sodium phosphocreatine (pH 7.2 with CsOH). Glutamate receptor doi:10.1038/nn.2616 s.e.m.offour experiments. Nonspecific binding subtractedfirstwas from all μ All results are expressed as mean as expressedareresults All M) was present in the artificial cerebrospinal fluid. d -AP5, 25 -AP5, μ M) were present in the artificial artificial the presentwerein M) post hoc post 35 P S]GTP < 0.05. All lipid0.05.< All ± μ test was per was test s.e.m. unless s.e.m. M) and M) γ S binding d -2- Ω ± - - )

50. 49. compared with Student’s treatment divided by mean amplitude of baseline IPSCs/fEPSPs). Data sets were was calculated as 100 × (mean amplitude of IPSCs/fEPSPs during the last 5-min were averaged for each neuron. The depression (%) of IPSCs/fEPSPsmeanamplitude beforeIPSCsoffive depolarization)]. Values by DSItrials of2–3 CP55,940 [1 − (mean amplitude of two IPSCs immediately after depolarization divided by y the baseline. The to normalized was amplitude EPSP IPSC/f analysis, electrophysiological For Student’s [ [ mean as reported are autoradiographydata stimulation protein G Regional results. Software). Values from regressions are reported as mean ­sigmoidal dose-response model or specific a bindingusing 5.0 Prism with performed wereof curves concentrationagonist effect singleof site model (GraphPad from total binding, yielding specific binding data. Nonlinear regression analyses asmean 35 35 = S]GTP S]GTP

Schlosburg, J.E. Schlosburg, Thomas, E.A. (2009). precipitated withdrawal responses in THC-dependent mice. THC-dependent in responses withdrawal precipitated pharmacotherapy. neuroleptic for (2001). 789–796 mechanism a towards y 0 + k × e × k + ± s.e.m. of triplicate sections from seven to eight brains per group.Net per brains eight to seven from sections triplicate of s.e.m. ± γ γ t s.e.m. of four experiments. Nonspecific binding wasfirst subtracted test between the two treatments for each individual region S binding. Analysis was performed in GraphPad Prism Version 5 using S binding is defined as agonist-stimulated [ − x et al. / τ τ . The magnitude of DSI was calculated as DSI (%) = 100 × 100 = (%) DSI as calculated was DSI of magnitude The . of DSI was measured using a single exponential function of et al. et Clozapine increases apolipoprotein D expression in rodent brain: Inhibitors of endocannabinoid-metabolizing enzymes reduce enzymes endocannabinoid-metabolizing of Inhibitors t test. nature nature 35 S]GTP ± s.e.m. for interpolated AAPS J. AAPS . Neurochem. J. NEUR γ S binding − basal

11 OSCI , 342–352 ,

analyzed. EN

76 C E ,