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Diacylglycerol lipase disinhibits VTA dopamine neurons during chronic nicotine exposure Matthew W. Buczynski a,1 , Melissa A. Herman a,1 , Ku-Lung Hsu b,c,1 , Luis A. Natividad a , Cristina Irimia a , Ilham Y. Polis a , Holly Pugh c , Jae Won Chang b,c , Micah J. Niphakis b,c , Benjamin F. Cravatt b,c , Marisa Roberto a , and Loren H. Parsons a,2 a Committee on the Neurobiology of Addictive Disorders, The Scripps Research Institute, La Jolla, CA 92037; b The Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, CA 92037; and c Department of Chemical Physiology, The Scripps Research Institute, La Jolla, CA 92037 Edited by Leslie Lars Iversen, University of Oxford, Oxford, United Kingdom, and approved December 16, 2015 (received for review November 21, 2015) Chronic nicotine exposure (CNE) alters synaptic transmission in the ventral tegmental area (VTA) in a manner that enhances dopami- nergic signaling and promotes nicotine use. The present experi- ments identify a correlation between enhanced production of the endogenous cannabinoid 2-arachidonoylglycerol (2-AG) and di- minished release of the inhibitory neurotransmitter GABA in the VTA following CNE. To study the functional role of on-demand 2-AG signaling in GABAergic synapses, we used 1,2,3-triazole urea compounds to selectively inhibit 2-AG biosynthesis by diacylgly- cerol lipase (DAGL). The potency and selectivity of these inhibitors were established in rats in vitro (rat brain proteome), ex vivo (brain slices), and in vivo (intracerebroventricular administration) using activity-based protein profiling and targeted metabolomics analyses. Inhibition of DAGL (2-AG biosynthesis) rescues nicotine- induced VTA GABA signaling following CNE. Conversely, enhance- ment of 2-AG signaling in naïve rats by inhibiting 2-AG degradation recapitulates the loss of nicotine-induced GABA signaling evident following CNE. DAGL inhibition reduces nicotine self-administration without disrupting operant responding for a nondrug reinforcer or motor activity. Collectively, these findings provide a detailed char- acterization of selective inhibitors of rat brain DAGL and demon- strate that excessive 2-AG signaling contributes to a loss of inhibi- tory GABAergic constraint of VTA excitability following CNE. nicotine | 2-arachidonoylglycerol | diacylglycerol lipase | GABA | ventral tegmental area T he mesocorticolimbic dopamine (DA) system provides a critical link between the brain regions that process cognitive information and those controlling motor behavior. Precise control of these ventral tegmental area (VTA) projections facilitates seeking rewarding stimuli, retreating from aversive stimuli, con- straint of motivational state, and behavioral flexibility necessary for survival. GABAergic signaling provides robust inhibition that gates VTA DA cell excitability (1, 2), and loss of this inhibition leads to pathological dysregulation of mesocorticolimbic circuitry (3, 4). Endocannabinoids (eCBs) regulate DAergic activity through retrograde signaling from DA cell bodies onto presynaptic can- nabinoid type 1 (CB 1 ) receptors expressed on both inhibitory and excitatory inputs. Although both 2-arachidonoylglycerol (2-AG) and anandamide (AEA) function as endogenous CB 1 agonists in the brain (57), these lipids exhibit distinct pharmacological profiles in vivo (8, 9) and mediate differential behavioral effects (10, 11). Endocannabinoids are produced and degraded on- demand, and the primary enzymes responsible for eCB degra- dation have been well-characterized using selective pharmacological tools that inactivate monoacylglycerol lipase (MAGL) or fatty acid amide hydrolase (FAAH) (1113). However, a complete evaluation of the influence of eCB signaling in the brain has been hampered by the lack of appropriate corresponding tools for selectively inactivating on-demand eCB biosynthesis. Substantial evidence implicates eCB signaling in the etiology of nicotine addiction, and recent work demonstrates that chronic nicotine exposure (CNE) selectively enhances nicotine- induced increases in VTA 2-AG formation (14). The present study investigated the possible contribution of this effect to ab- errant VTA DA cell excitation present following CNE (15). We find that sensitized nicotine-induced 2-AG release (14) strongly correlates with a loss of nicotine-induced GABA release, which may contribute to impaired inhibitory constraint of VTA DA cell excitation following CNE. To test this hypothesis, we charac- terized a series of selective inhibitors of 2-AG biosynthesis by diacylglycerol lipase α and β (DAGLα and DAGLβ; hereafter referred to as DAGL) (1618) and 2-AG degradation by α/β-hydrolase domain 6 (ABHD6) and MAGL (11, 12, 19), and used these compounds to investigate the functional impact of enhanced 2-AG recruitment on GABAergic signaling at VTA synapses and nicotine self-administration. Results Chronic Nicotine Exposure Impairs Nicotine-Induced GABA Release in the Rat VTA. We previously demonstrated that nicotine-induced VTA 2-AG formation is sensitized following CNE in a manner independent of the volitional nature of drug exposure [e.g., similar sensitization induced by either nicotine self-administration (SA) or response-independent administration] (Fig. 1A) (14). To de- termine the signaling mechanisms influenced by this aberrant 2-AG response, we screened the remaining microdialysate aliquots from these same subjects for temporal changes in neurotransmitter content. Of the 11 neurotransmitters quantified, only GABA Significance Many CNS disorders result from dysregulation of the meso- corticolimbic dopamine projections arising from the ventral teg- mental area. A mechanistic understanding of these dysregulations is critical for developing treatments for these diseases. Nicotine addiction is a global health concern, and results in part from dysregulated mesocorticolimbic dopamine function. Here we present evidence that nicotine-induced 2-arachidonoylglycerol formation in the ventral tegmental area is sensitized following chronic nicotine exposure, and that this results in the loss of GABA-mediated constraint of mesocorticolimbic dopamine activ- ity. Potent and selective diacylglycerol lipase inhibitors are char- acterized and used in ex vivo electrophysiology and in vivo behavioral analyses to demonstrate that this mechanism plays a prominent role in the dysregulation of ventral tegmental dopa- mine neurons following nicotine exposure. Author contributions: M.W.B., M.A.H., K.-L.H., B.F.C., M.R., and L.H.P. designed research; M.W.B., M.A.H., K.-L.H., L.A.N., C.I., I.Y.P., and H.P. performed research; K.-L.H., J.W.C., M.J.N., and B.F.C. contributed new reagents/analytic tools; M.W.B., M.A.H., K.-L.H., L.A.N., C.I., I.Y.P., B.F.C., M.R., and L.H.P. analyzed data; and M.W.B., M.A.H., K.-L.H., B.F.C., M.R., and L.H.P. wrote the paper. Conflict of interest statement: B.F.C. is a cofounder and advisor for a biotechnology company interested in developing inhibitors of serine hydrolases as therapeutic targets. This article is a PNAS Direct Submission. 1 M.W.B., M.A.H., and K.-L.H. contributed equally to this work. 2 To whom correspondence should be addressed. Email: [email protected]. This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. 1073/pnas.1522672113/-/DCSupplemental. 10861091 | PNAS | January 26, 2016 | vol. 113 | no. 4 www.pnas.org/cgi/doi/10.1073/pnas.1522672113 Downloaded by guest on March 2, 2020

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Page 1: Diacylglycerol lipase disinhibits VTA dopamine …Diacylglycerol lipase disinhibits VTA dopamine neurons during chronic nicotine exposure Matthew W. Buczynski a,1 , Melissa A. Herman

Diacylglycerol lipase disinhibits VTA dopamine neuronsduring chronic nicotine exposureMatthew W. Buczynskia,1, Melissa A. Hermana,1, Ku-Lung Hsub,c,1, Luis A. Natividada, Cristina Irimiaa, Ilham Y. Polisa,Holly Pughc, Jae Won Changb,c, Micah J. Niphakisb,c, Benjamin F. Cravattb,c, Marisa Robertoa, and Loren H. Parsonsa,2

aCommittee on the Neurobiology of Addictive Disorders, The Scripps Research Institute, La Jolla, CA 92037; bThe Skaggs Institute for Chemical Biology, TheScripps Research Institute, La Jolla, CA 92037; and cDepartment of Chemical Physiology, The Scripps Research Institute, La Jolla, CA 92037

Edited by Leslie Lars Iversen, University of Oxford, Oxford, United Kingdom, and approved December 16, 2015 (received for review November 21, 2015)

Chronic nicotine exposure (CNE) alters synaptic transmission in theventral tegmental area (VTA) in a manner that enhances dopami-nergic signaling and promotes nicotine use. The present experi-ments identify a correlation between enhanced production of theendogenous cannabinoid 2-arachidonoylglycerol (2-AG) and di-minished release of the inhibitory neurotransmitter GABA in theVTA following CNE. To study the functional role of on-demand2-AG signaling in GABAergic synapses, we used 1,2,3-triazole ureacompounds to selectively inhibit 2-AG biosynthesis by diacylgly-cerol lipase (DAGL). The potency and selectivity of these inhibitorswere established in rats in vitro (rat brain proteome), ex vivo(brain slices), and in vivo (intracerebroventricular administration)using activity-based protein profiling and targeted metabolomicsanalyses. Inhibition of DAGL (2-AG biosynthesis) rescues nicotine-induced VTA GABA signaling following CNE. Conversely, enhance-ment of 2-AG signaling in naïve rats by inhibiting 2-AG degradationrecapitulates the loss of nicotine-induced GABA signaling evidentfollowing CNE. DAGL inhibition reduces nicotine self-administrationwithout disrupting operant responding for a nondrug reinforcer ormotor activity. Collectively, these findings provide a detailed char-acterization of selective inhibitors of rat brain DAGL and demon-strate that excessive 2-AG signaling contributes to a loss of inhibi-tory GABAergic constraint of VTA excitability following CNE.

nicotine | 2-arachidonoylglycerol | diacylglycerol lipase | GABA |ventral tegmental area

The mesocorticolimbic dopamine (DA) system provides acritical link between the brain regions that process cognitive

information and those controlling motor behavior. Precise controlof these ventral tegmental area (VTA) projections facilitatesseeking rewarding stimuli, retreating from aversive stimuli, con-straint of motivational state, and behavioral flexibility necessary forsurvival. GABAergic signaling provides robust inhibition that gatesVTA DA cell excitability (1, 2), and loss of this inhibition leads topathological dysregulation of mesocorticolimbic circuitry (3, 4).Endocannabinoids (eCBs) regulate DAergic activity through

retrograde signaling from DA cell bodies onto presynaptic can-nabinoid type 1 (CB1) receptors expressed on both inhibitory andexcitatory inputs. Although both 2-arachidonoylglycerol (2-AG)and anandamide (AEA) function as endogenous CB1 agonists inthe brain (5–7), these lipids exhibit distinct pharmacologicalprofiles in vivo (8, 9) and mediate differential behavioral effects(10, 11). Endocannabinoids are produced and degraded on-demand, and the primary enzymes responsible for eCB degra-dation have been well-characterized using selective pharmacologicaltools that inactivate monoacylglycerol lipase (MAGL) or fattyacid amide hydrolase (FAAH) (11–13). However, a completeevaluation of the influence of eCB signaling in the brain has beenhampered by the lack of appropriate corresponding tools forselectively inactivating on-demand eCB biosynthesis.Substantial evidence implicates eCB signaling in the etiology

of nicotine addiction, and recent work demonstrates thatchronic nicotine exposure (CNE) selectively enhances nicotine-induced increases in VTA 2-AG formation (14). The present

study investigated the possible contribution of this effect to ab-errant VTA DA cell excitation present following CNE (15). Wefind that sensitized nicotine-induced 2-AG release (14) stronglycorrelates with a loss of nicotine-induced GABA release, whichmay contribute to impaired inhibitory constraint of VTA DA cellexcitation following CNE. To test this hypothesis, we charac-terized a series of selective inhibitors of 2-AG biosynthesis bydiacylglycerol lipase α and β (DAGLα and DAGLβ; hereafterreferred to as DAGL) (16–18) and 2-AG degradation byα/β-hydrolase domain 6 (ABHD6) and MAGL (11, 12, 19), andused these compounds to investigate the functional impact ofenhanced 2-AG recruitment on GABAergic signaling at VTAsynapses and nicotine self-administration.

ResultsChronic Nicotine Exposure Impairs Nicotine-Induced GABA Release inthe Rat VTA. We previously demonstrated that nicotine-inducedVTA 2-AG formation is sensitized following CNE in a mannerindependent of the volitional nature of drug exposure [e.g., similarsensitization induced by either nicotine self-administration (SA)or response-independent administration] (Fig. 1A) (14). To de-termine the signaling mechanisms influenced by this aberrant 2-AGresponse, we screened the remaining microdialysate aliquots fromthese same subjects for temporal changes in neurotransmittercontent. Of the 11 neurotransmitters quantified, only GABA

Significance

Many CNS disorders result from dysregulation of the meso-corticolimbic dopamine projections arising from the ventral teg-mental area. A mechanistic understanding of these dysregulationsis critical for developing treatments for these diseases. Nicotineaddiction is a global health concern, and results in part fromdysregulated mesocorticolimbic dopamine function. Here wepresent evidence that nicotine-induced 2-arachidonoylglycerolformation in the ventral tegmental area is sensitized followingchronic nicotine exposure, and that this results in the loss ofGABA-mediated constraint of mesocorticolimbic dopamine activ-ity. Potent and selective diacylglycerol lipase inhibitors are char-acterized and used in ex vivo electrophysiology and in vivobehavioral analyses to demonstrate that this mechanism plays aprominent role in the dysregulation of ventral tegmental dopa-mine neurons following nicotine exposure.

Author contributions: M.W.B., M.A.H., K.-L.H., B.F.C., M.R., and L.H.P. designed research;M.W.B., M.A.H., K.-L.H., L.A.N., C.I., I.Y.P., and H.P. performed research; K.-L.H., J.W.C.,M.J.N., and B.F.C. contributed new reagents/analytic tools; M.W.B., M.A.H., K.-L.H., L.A.N.,C.I., I.Y.P., B.F.C., M.R., and L.H.P. analyzed data; and M.W.B., M.A.H., K.-L.H., B.F.C., M.R.,and L.H.P. wrote the paper.

Conflict of interest statement: B.F.C. is a cofounder and advisor for a biotechnologycompany interested in developing inhibitors of serine hydrolases as therapeutic targets.

This article is a PNAS Direct Submission.1M.W.B., M.A.H., and K.-L.H. contributed equally to this work.2To whom correspondence should be addressed. Email: [email protected].

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1522672113/-/DCSupplemental.

1086–1091 | PNAS | January 26, 2016 | vol. 113 | no. 4 www.pnas.org/cgi/doi/10.1073/pnas.1522672113

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exhibited a pattern that correlated with changes in 2-AG (Fig. 1Band Fig. S1). Specifically, dialysate 2-AG and GABA levels (Fig.1C) were inversely correlated and exhibited distinct nicotine-induced responses following chronic vs. acute nicotine exposureregardless of response contingency (14).To determine the impact of nicotine exposure on GABA

transmission, whole-cell voltage- and current-clamp recordingsof pharmacologically isolated GABAA receptor-mediated spon-taneous inhibitory postsynaptic currents (sIPSCs) were per-formed in VTA DA neurons (Fig. S2 A–C). In VTA slices fromdrug-naïve rats, bath application of nicotine produced a transientincrease in sIPSC frequency with no effect on sIPSC amplitude(Fig. 1 D and E and Fig. S3A). However, in VTA slices fromCNE rats, nicotine did not alter sIPSC frequency or amplitude(Fig. 1 D and E). There was no significant effect of prior CNE oneither baseline (Fig. S2D) or muscimol-induced (Fig. S2E)sIPSCs, indicating no CNE-induced disruption in postsynapticGABAA receptor influence. Nicotine-induced increases in sIPSCfrequency were absent in the presence of tetrodotoxin (TTX),confirming a reliance on action potential firing (Fig. 1F and Fig.S3 B and C). Collectively, these data demonstrate that CNEimpairs nicotine-induced increases in GABA signaling but notbaseline GABA transmission in VTA DA neurons.

CB1 Influence on VTA GABA Signaling Is Not Altered Following ChronicNicotine Exposure. Tests with the synthetic CB1 agonist WIN55,212-2 (WIN) and the CB1 antagonist rimonabant (RIM) wereperformed to probe for possible alterations in CB1 functionfollowing CNE. In nicotine-naïve slices, WIN reduced sIPSCfrequency and prevented subsequent nicotine-induced increasesin sIPSC frequency (Fig. 2 A, C, and D). The WIN-induced oc-clusion of nicotine-induced sIPSC increases likely reflects CB1-mediated suppression of presynaptic GABA release, as sup-ported by in vivo microdialysis data demonstrating WIN-inducedreductions in VTA GABA levels and prevention of subsequentnicotine-induced increases in VTA GABA release (Fig. 2E).RIM increased sIPSC frequency and also prevented subsequentnicotine-induced increases in sIPSC frequency (Fig. 2 B–D). Thissuggests a tonic CB1 influence on VTA GABA release, as sup-ported by a robust increase in in vivo VTA microdialysateGABA levels following RIM administration (Fig. 2F). The ro-bust per se effects of RIM on GABAA signaling may preclude

detection of additional nicotine-induced effects. WIN and RIMinduced similar effects in VTA neurons from CNE rats (Fig. 2 Cand D) and, importantly, the effects of these CB1 receptor li-gands on sIPSCs did not differ between nicotine-naïve and CNEVTA neurons.

Selective Inhibitors of Rat 2-AG Metabolic Enzymes. Determinationof the 2-AG influence on nicotine-induced alterations in VTAGABA signaling requires manipulation of 2-AG biosynthesis. Toenable this, we used an activity-based protein profiling (ABPP)assay using the broad-spectrum and DAGL-directed probes FP-Rhand HT-01 to evaluate the potency and selectivity of a series of1,2,3-triazole ureas (1,2,3-TUs) for inhibiting the 2-AG bio-synthetic enzymes DAGLα and DAGLβ (20). In the rat proteome,the 1,2,3-TU KT172 (Fig. 3 B and F) and the structurally relatedanalog KT128 (Fig. 3 C and G) displayed greater inhibitory po-tency at DAGLα than DAGLβ. Both compounds exhibited goodselectivity against other serine hydrolases including MAGL andFAAH but showed cross-reactivity with ABHD6, as expected (20–22). To identify appropriate negative control probes, we confirmedthat the ABHD6-selective inhibitors KT185 (Fig. 3 D and H) andKT195 (Fig. 3 E and I) potently inactivated rat ABHD6 (20, 21)with minimal off-target activity against other serine hydrolases in-cluding DAGLs, with the exception of FAAH cross-reactivity byKT195 (Fig. 3, Figs. S4 and S5, and Table S1).In the ex vivo brain slice conditions used for electrophysio-

logical studies, KT172 inactivated DAGLα and ABHD6 with nosignificant effect on other serine hydrolases, and the negativecontrol probe KT185 inhibited ABHD6 without disruptingDAGL activity (Fig. 3J and Fig. S6). In this same preparation,KT172 significantly and selectively reduced striatal slice 2-AGcontent (Fig. 3K), similar to the reduction in striatal 2-AGcontent evident in DAGLα-KO mice (Fig. S7A). Conversely,KT185 significantly increased striatal slice 2-AG content (Fig.3K), consistent with a role for ABHD6 as an alternative eCBhydrolase in the rat brain (11). Although KT172 also inhibitsABHD6 activity, the upstream loss of 2-AG biosynthesis byDAGLα inhibition likely precludes 2-AG accumulation thatcould result from this decrement in 2-AG hydrolysis. Indeed,DAGLα inhibition by KT172 prevents the accumulation of tissue2-AG content normally induced by the MAGL inhibitor KML29(Fig. S7B), even though coincubation with these compounds

Fig. 1. Chronic nicotine exposure impairs nicotine-in-duced GABA release in the rat VTA. (A) Dialysate 2-AGlevels before (t −60 to 0 min) and during (t 0–120 min)nicotine exposure in naïve (n = 7), CNE (n = 7), and SA(n = 6) rats. Reprinted by permission from MacmillanPublishers Ltd. (14). (B) Summary of microdialysateneurotransmitter changes between groups during nic-otine exposure. Corresponding microdialysate profilesare shown in Fig. S1. (C) Dialysate GABA levels before(t −60 to 0 min) and during (t 0–120 min) nicotine ex-posure in naïve (n = 7), CNE (n = 7), and SA (n = 6) rats.Baseline GABA levels did not significantly differ be-tween groups. (D) Representative recordings of sIPSCsin VTA DA neurons from naïve (Left) and CNE (Right)rats during the superfusion of 1 μM nicotine (NIC). (E )Summary of sIPSC frequency during superfusion of1 μM nicotine in VTA DA neurons relative to baseline(dashed line) from naïve (n = 7) and CNE (n = 6) rats.(F) Summary of mIPSC frequency during superfusionof 1 μM nicotine in VTA DA neurons relative tobaseline (dashed line) revealed in the presence of0.5 μM TTX [naïve (n = 6), CNE (n = 8); representativetraces are in Fig. S3C]. Dashed lines reflect prenicotinebaseline levels (defined as 100%). Data are presentedas mean ± SE. *P < 0.05, **P < 0.01, ***P < 0.001.

Buczynski et al. PNAS | January 26, 2016 | vol. 113 | no. 4 | 1087

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results in the combined inhibition of ABHD6 and MAGL.Overall, KT172-induced reductions in tissue 2-AG content likelyresult from the combined reduction in 2-AG biosynthesis andunperturbed 2-AG hydrolysis by MAGL. No treatment alteredbasal AEA levels (Fig. 3K and Fig. S7C). Taken together, theseresults demonstrate that KT172 and KT185 serve as paired chemicalprobes for selectively modulating 2-AG metabolism in the rat brain.

DAGL Inactivation Restores Nicotine-Induced GABA Release in Ratswith a History of Nicotine Exposure. We tested whether inactiva-tion of DAGLs could restore nicotine-elicited GABA signalingat VTA DA synapses in slices from CNE rats. Slices weresuperfused with KT172 or KT128, which had no per se effect onbaseline sIPSC characteristics (Fig. 4 A and D). However, sub-sequent application of nicotine to drug-treated VTA slices sig-nificantly increased sIPSC frequency in DA neurons to levelssimilar to those observed in drug-naïve rats. To confirm theDAGL specificity of this effect, we treated VTA slices from CNErats with the ABHD6-selective inhibitor KT185. KT185 had noper se effect on baseline sIPSCs (Fig. 4 B and D), and did notrestore the nicotine-induced increase in GABA release. Wereplicated these experiments with the DAGL inhibitors includedin the pipette internal solution to restrict delivery to the post-synaptic cell, and showed that either KT172 or KT128 restorednicotine-induced increases in sIPSC frequency in CNE slices tolevels similar to that observed in drug-naïve slices (Fig. 4 C andD). Taken together, these data demonstrate that inhibitingpostsynaptic DAGL restores nicotine-induced GABA signalingin rats with a history of nicotine exposure.

Attenuation of 2-AG Clearance Blocks Nicotine-Induced GABA Releasein Drug-Naïve Rats. We hypothesized that enhancing 2-AG sig-naling in naïve rats would recapitulate the loss of nicotine-induced GABA release evident following CNE. Superfusion of

naïve VTA slices with the ABHD6-selective inhibitor KT185 didnot alter baseline sIPSC frequency but blocked nicotine-inducedincreases in sIPSC frequency (Fig. 4 E and G). Similarly, super-fusion of the MAGL-selective inhibitor KML29 blocked nicotine-induced sIPSCs without significantly modifying baseline sIPSC

Fig. 3. Selective inhibitors of rat 2-AG metabolic enzymes. (A) Chemicalstructures of 1,2,3-triazole urea inhibitors. (B–E) Competitive ABPP (n = 3) of(B) KT172, (C) KT128, (D) KT185, and (E) KT195 against endogenous serinehydrolases detected in rat brain proteome in vitro using either the DAGL-tailored probe (HT-01; for DAGLα, DAGLβ, and ABHD6) or broad-spectrumserine hydrolase probe (FP-Rh; for MAGL and FAAH). Proteomes were pre-incubated with the indicated dose of compound (30 min, 37 °C) followed bylabeling with activity-based probes (1 μM FP-Rh or HT-01, 30 min, 37 °C). (F–I)Representative gels for each in vitro inhibitor treatment (full gels are in Figs.S4 and S5). (J) Activity of eCB metabolic enzymes following ex vivo in-cubation of striatal tissue slices with vehicle, 1 μM KT172, or 1 μM KT185 (n =4, 10 min; full gels are in Fig. S6). (K) Levels of 2-AG and AEA following in-cubation of striatal tissue slices with vehicle, 1 μM KT172, or 1 μM KT185 (n =4–6, 4 h). Data are presented as mean ± SE. *P < 0.05, **P < 0.01.

Fig. 2. CB1 influence on VTA GABA signaling is not altered following CNE.(A and B) Representative recordings of sIPSCs from nicotine-naïve rats duringthe superfusion of (A) 2 μM WIN or (B) 2 μM RIM, followed by 1 μM NIC. (Cand D) Summary of sIPSC frequencies from naïve (gray) or CNE (blue) during(C) superfusion of RIM (n = 6 and n = 5) WIN (n = 5 and n = 5) and during (D)subsequent superfusion of nicotine compared with drug-treated baseline(n = 5 in all conditions). (E) Dialysate GABA levels in naïve rats treated withWIN (3 mg/kg i.p., t 0, n = 6) in the absence or presence of IV nicotine(matching the acute group in Fig. 1 A and C). (F) Dialysate GABA levels inCNE rats treated with RIM (3 mg/kg IP, t 0, n = 6) in the absence of nicotine.Data are presented as mean ± SE. *P < 0.05, **P < 0.01, ***P < 0.001.

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frequency (Fig. 4G). In contrast, superfusion with the DAGL in-hibitors KT172 or KT128 did not alter GABA signaling (Fig. 4 Fand G). Together, these data demonstrate that the loss of nico-tine-induced GABA signaling observed in the VTA of CNE ratscan be reproduced in naïve rats by inhibiting 2-AG clearance withinhibitors of ABHD6 or MAGL.

Nicotine Self-Administration Is Reduced by DAGL Inhibition. To eval-uate the influence of DAGL activity on nicotine SA behavior, weestablished that KT172 and KT185 effectively inhibit DAGL andABHD6 in the rat brain following intracerebroventricular (ICV)administration. Both DAGLα and ABHD6 activity were inhibitedfollowing ICV administration of KT172; DAGLβ activity was alsopartially inhibited in these animals (Fig. 5 A and C and Fig. S8).Treatment with KT185 resulted in comparable ABHD6 inhibition inthe absence of reduced DAGL activity (Fig. 5 B and C). Neither drugexhibited significant cross-reactivity against other serine hydrolases(including MAGL and FAAH) under these conditions. Subsequentbehavioral tests revealed that nicotine self-administration was signif-icantly reduced by KT172 but not KT185 (Fig. 5D). Neither com-pound significantly altered operant responding for water (Fig. 5E) orspontaneous locomotor activity (Fig. 5F), thereby discounting an in-fluence of nonspecific behavioral disruptions and highlighting ameaningful influence of DAGL activity on the motivation for nicotinevs. a nondrug reinforcer.

DiscussionThrough the use of selective inhibitors of DAGL, ABHD6, andMAGL, the present findings reveal that enhanced on-demand2-AG formation blunts the inhibitory control of nicotine-inducedVTA cell excitation following CNE (Fig. S9). Loss of this in-hibitory feedback mechanism likely contributes to increasedVTA sensitivity to nicotine and other stimuli (23, 24), resulting insensitized DA release in the nucleus accumbens (15) and in-creased motivation for nicotine intake.Nicotine-induced increases in VTA 2-AG formation (14)

likely contribute to the well-documented CB1 receptor influenceon behavioral and physiological response to nicotine (25). In theVTA, CB1 receptors are thought to dynamically regulate GABArelease at DAergic synapses (4), allowing the possibility that2-AG formation dampens GABAergic constraint of DA cellexcitation. A direct test of this hypothesis requires the suppressionof 2-AG formation, although studies of this type have been limitedby a lack of selective inhibitors of 2-AG biosynthesis (26) andsuitable biochemical assays to validate inhibition of endogenousDAGL activity. To address this, we evaluated the efficacy ofDAGL-selective 1,2,3-TU inhibitors for use in the rat brain usinga tailored ABPP assay (20, 22). We find that KT172 selectivelyinhibits DAGL enzymes in the exogenous brain proteome as wellas in awake animals, and that the primary off-target of this inhibitor(ABHD6) can be accounted for using the negative control probeKT185. Using targeted liquid chromatography-mass spectrometry(LC-MS) metabolomics, we show that KT172 but not KT185 sig-nificantly reduces brain 2-AG levels, supporting DAGLs as theprincipal 2-AG biosynthetic enzymes in the rat brain. Using thissuite of chemical probes, we demonstrate that DAGL inhibitionrestores nicotine-induced increases in GABA signaling in the VTA

Fig. 4. DAGL inhibition restores nicotine-induced GABA release in rats witha history of nicotine exposure. (A and B) Representative recordings of sIPSCsin VTA DA neurons from CNE rats during superfusion of (A) 1 μM KT172followed by 1 μM NIC and (B) 1 μM KT185 followed by 1 μM NIC. (C) Rep-resentative recordings of sIPSCs in a VTA DA neuron from a CNE rat duringadministration of KT172 (1 μM in the pipette solution) before (Left) andduring (Right) superfusion of 1 μM NIC. (D) Summary of sIPSC frequencies inVTA DA neurons from CNE rats during superfusion of either 1 μM KT172 (n =5), 1 μM KT128 (n = 6), or 1 μM KT185 (n = 6) and during subsequent nicotinesuperfusion. Summary of sIPSC frequencies in VTA DA neurons before andduring nicotine superfusion with either 1 μM KT172 (n = 6) or 1 μM KT128(n = 6) in the pipette solution. Attenuation of 2-AG clearance in nicotine-

naïve subjects recapitulates the effects of CNE. (E and F) Representativerecordings of sIPSCs in VTA DA neurons from naïve rats during superfusionof (E) 1 μM KT185 followed by 1 μM NIC and (F) 1 μM KT172 followed by1 μM NIC. (G) Summary of sIPSC frequencies in VTA DA neurons from naïverats during superfusion of either 1 μM KT172 (n = 5), 1 μM KT128 (n = 5),1 μM KT185 (n = 6), or 1 μM KML29 (n = 8) and during subsequent nicotinesuperfusion. The response to NIC in naïve and CNE rats is shown for com-parison [open bars (Left) from Fig. 1]. Data are presented as mean ± SE. *P <0.05, **P < 0.01.

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of CNE rats. Conversely, attenuation of 2-AG hydrolysis by eitherABHD6 or MAGL in nicotine-naïve rats recapitulates the loss ofnicotine-induced GABA signaling observed following CNE. Theselatter effects are consistent with previous studies on hippocampalGABAergic signaling using MAGL inhibitors (27) or MAGL-knockout mice (28). Collectively, these findings support the hy-pothesis that nicotine-induced increases in 2-AG dampen theGABAergic constraint of DA cell excitation following CNE.We find that the functional influence of CB1 receptors on

VTA GABA release is unaltered by CNE, and demonstrate thatCB1 receptor antagonism robustly increases VTA GABA levelsand GABAA receptor signaling. The resultant decrease in DAcell excitability likely underlies the ability of CB1 antagonists toblock nicotine-induced increases in nucleus accumbens DA re-lease and to suppress nicotine self-administration (25, 29–34).Nicotine and other drugs of abuse alter synaptic transmission

in the VTA through additional mechanisms that may be facili-tated by the loss of local inhibitory signaling. For example, nic-otine enhances glutamatergic transmission in the VTA andstrengthens excitatory synapses onto VTA DA neurons via theinduction of long-term potentiation (35–37). A significant body of

work highlights the importance of glutamatergic synaptic plasticityin long-term changes that result from a history of drug exposure(37, 38), although in the context of the present studies it is notablethat the functional influence of these excitatory adaptations re-quires a concomitant loss of inhibitory signaling (39).In summary, on-demand 2-AG biosynthesis represents a mo-

lecular switch that regulates inhibitory signaling in the VTA.Following CNE, enhanced 2-AG production following cholin-ergic receptor activation imbalances this system in a manner thatfacilitates DAergic cell excitability. The present results providenovel, direct evidence that pharmacological modulation of 2-AGbiosynthesis is a viable approach for evaluating the influence ofthe eCB system on substance abuse disorders.

Experimental ProceduresAnimals. Studies involving male Wistar rats (300–450 g; Charles RiverLaboratories) were conducted in accordance with NIH guidelines and asapproved by the Scripps Research Institute Institutional Animal Care andUse Committee. Intravenous catheterization (14), microdialysis probeimplantation (14), and intracerebroventricular administration of drugsusing homemade cannulae and infusers (40) were performed usingpublished methodology.

Nicotine Administration Procedures. For volitional nicotine exposure, SA ratswere trained to operantly respond for IV nicotine (75 μg/kg per infusion;0.1 mL per infusion over 4 s) under a continuous (fixed ratio 1; FR-1)schedule of reinforcement in daily 2-h sessions using previously publishedprocedures (14). Rats from the SA group were individually paired withanimals receiving noncontingent forced nicotine exposure (CNE) thatreceived nicotine infusions based on the partner SA rats’ patterns of in-take during the training and collection sessions. Rats receiving acutenoncontingent forced nicotine exposure (acute) were also individuallypaired with SA rats, but received saline infusions in all training sessions,and nicotine infusions during the collection session (infusion patternsbased on partner SA rats’ volitional intake).

Neurotransmitter Measurements. Quantification of neurotransmitters in di-alysate samples was performed by LC-MS/MS using established methodology(41, 42). Briefly, 1 μL dialysate was added to 6.5 μL borate (100 mM),derivatized with benzoyl chloride [2% (vol/vol), acetonitrile (ACN)], andsupplemented with an internal standard.

Gel-Based Competitive ABPP.Gel-based competitive ABPP experiments wereperformed as previously described (20, 43). For experiments using the FP-rhodamine probe, proteomes (1 mg/mL) were treated with FP-rhodamine(1 μM) in a 50-μL total reaction volume for 30 min at 37 °C. For experi-ments using the HT-01 probe, proteomes (2 mg/mL) were treated with HT-01 (1 μM) in a 50-μL total reaction volume for 30 min at 37 °C. Reactionswere quenched with SDS/PAGE loading buffer. Proteins were separatedby SDS/PAGE (10% acrylamide) and visualized using a fluorescence scan-ner (Hitachi; FMBIO IIe).

Electrophysiological Recording. Electrophysiology procedures were as re-cently reported (44, 45) and are described briefly. Coronal sections(300 μm) through the midbrain were cut on a Leica VT1000S (Leica Micro-systems) and placed in oxygenated (95% O2/5% CO2) artificial cerebro-spinal fluid (aCSF) solution composed of 130 mM NaCl, 3.5 mM KCl, 2 mMCaCl2, 1.5 mM MgClSO4, 1.25 mM NaH2PO4, 24 mM NaHCO3, 10 mMglucose. Whole-cell recordings were made using glass pipettes (3–5 MΩ)filled with an intracellular solution containing 145 mM KCl, 5 mM EGTA,5 mM MgCl2, 10 mM Hepes, 2 mM Na-ATP, 0.2 mM Na-GTP coupled to aMultiClamp 700B amplifier (Molecular Devices), low-pass-filtered at 2–5kHz, digitized (Digidata 1440A; Axon Instruments), and stored on acomputer using pCLAMP 10 software (Axon Instruments). To isolateinhibitory currents mediated by GABAA receptors, recordings (Vhold −60mV) were performed in the presence of dinitroquinoxaline-2,3-dione(DNQX) (20 μM), DL-2-amino-5-phosphonovalerate (AP-5) (50 μM), andCGP55845A (1 μM). Miniature IPSCs (mIPSCs) were recorded in the addi-tional presence of TTX (0.5 μM).

Statistics. For microdialysis analyses, significance was determined byrepeated measures ANOVA for effects of group, nicotine, and group xnicotine interaction. Significance for microdialysis baseline levels and

Fig. 5. Nicotine self-administration is reduced by DAGL inhibition. (A andB) Gel-based ABPP using HT-01 and FP-Rh assessed the efficacy and selec-tivity of KT172 and KT185 in vivo by ICV injection. KT172 (A) (100 μg, 4 h, n= 4) reduces DAGLα, DAGLβ, and ABHD6 activity, whereas treatment withKT185 (B) (100 μg, 4 h, n = 4) reduces ABHD6 activity with negligible ac-tivity against DAGLs. (C ) Representative HT-01 (Left) and FP-Rh (Right) gelsfor each inhibitor. (D) Pretreatment with KT172 (100 μg, 4 h, n = 16), butnot KT185 (100 μg, 4 h, n = 10), significantly reduced nicotine self-ad-ministration (75 μg/kg per infusion; FR-1 reinforcement schedule). (E) Pre-treatment with either KT172 (n = 9) or KT185 (n = 9) has no effect on oralwater self-administration. (F) Pretreatment with either KT172 (n = 9) orKT185 (n = 9) has no effect on motor activity. Data are presented as mean ±SE. **P < 0.01, ***P < 0.001.

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area-under-the-curve (AUC) values was determined by ANOVA followed byDunnett’s post hoc. For the ABPP studies, a Student’s t test (unpaired, two-tailed) was used to determine the effect of each drug. For electrophysiologyexperiments, sIPSC frequency was examined for a 2-min period followingdrug or nicotine application (36), and events were analyzed for independentsignificance using a one-sample t test and compared using a two-tailed t testfor independent samples, a paired two-tailed t test for comparisons madewithin the same recording, and a one-way ANOVA with a Bonferroni posthoc analysis for comparisons made between three or more groups. All sta-tistical analysis was performed using SPSS 18 (IBM) or Prism 5.02 (GraphPad).

Data are presented as mean ± SE. In all cases, the criterion for significancewas as follows: *P < 0.05, **P < 0.01, ***P < 0.001.

Supplemental Procedures. For more details, see SI Experimental Procedures.

ACKNOWLEDGMENTS. This work was supported by NIH Grants R01AA020404 (to L.H.P.), P60 AA006420 (to L.H.P. and M.R.), R01 AA013498(to M.R.), R01 DA009789 (to B.F.C.), R01 DA033760 (to B.F.C.), R01 MH084512(to B.F.C.), K99 DA035864 (to K.-L.H.), F32 AA020430 (to M.A.H.), F32DA029994 (to M.W.B.), and K99 DA035865 (to M.W.B.). This is manuscriptno. 28034 from The Scripps Research Institute.

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