Reducing cannabinoid abuse and preventing relapse by enhancing endogenous brain levels of kynurenic acid

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1 Reducing cnninoid use nd preventing relpse y enhncing endogenous rin levels of kynurenic cid Zuzn Justinov 1,2,11, Pol Msci 1,1,11, Hui-Qiu Wu 2, Mri E Secci 1, Godfrey H Redhi 1, Leigh V Pnlilio 1, Mri Scherm 3, Chnel Brnes 1, Alexndr Prshos 1, Tmr Zr 3, Wlter Frtt 3, Mrcello Solins 4,5, Mrco Pistis 3,6, Jck Bergmn 7, Brin D Kngs 7, Sergi Ferré 8, Ginluigi Tnd 9, Roert Schwrcz 2 & Steven R Golderg 1 npg 213 Nture Americ, Inc. All rights reserved. In the rewrd circuitry of the rin, -7-nicotinic cetylcholine receptors (7nAChRs) modulte effects of 9 -tetrhydrocnninol (THC), mrijun s min psychoctive ingredient. Kynurenic cid (KYNA) is n endogenous negtive llosteric modultor of 7nAChRs. Here we report tht the kynurenine 3-monooxygense (KMO) inhiitor Ro increses rin KYNA levels nd ttenutes cnninoid-induced increses in extrcellulr dopmine in rewrd-relted rin res. In the selfdministrtion model of drug use, Ro reduced the rewrding effects of THC nd the synthetic cnninoid WIN 55,212-2 in squirrel monkeys nd rts, respectively, nd it lso prevented relpse to drug-seeking induced y reexposure to cnninoids or cnninoid-ssocited cues. The effects of enhncing endogenous KYNA levels with Ro were prevented y positive llosteric modultors of 7nAChRs. Despite cler need, there re no medictions pproved for tretment of mrijun dependence. Modultion of KYNA offers phrmcologicl strtegy for chieving stinence from mrijun nd preventing relpse. The numer of people seeking tretment for mrijun use in the United Sttes per yer (1,243,) is higher thn the numer seeking tretment for cocine or heroin use (787, or 57,, respectively) 1. Like other drugs of use, mrijun s rewrding effects involve neurochemicl chnges in rin rewrd systems 2,3. Specificlly, THC, the min psychoctive ingredient in mrijun, ctivtes mesolimic dopmine circuitry y enhncing the firing of dopmine neurons in the ventrl tegmentl re (VTA) 4,5, resulting in incresed relese of dopmine from nerve terminls in the shell of the nucleus ccumens () 6,7. Developing medictions tht modulte these effects of THC on rewrd signling might provide therpeutic pproch for the tretment of mrijun dependence. α7nachrs re present in oth the VTA nd the shell, where they re locted on glutmtergic nerve terminls 8. Their ctivtion elicits the relese of glutmte, which in turn cts t ionotropic glutmte receptors on dopminergic terminls to stimulte dopmine relese 9,1. We previously found tht rewrdrelted ehviorl nd neurochemicl effects of THC or the synthetic cnninoid-receptor gonist WIN 55,212-2 could e locked y methyllycconitine (MLA), selective ntgonist t α7nachrs, pointing to modultion of α7nachr ctivity s phrmcologicl pproch for treting mrijun dependence 11,12. Unfortuntely, systemic use of cholinergic ntgonists cting directly t α7nachrs is ssocited with centrl nd peripherl side effects tht limit their therpeutic utility 13,14. Medictions tht enhnce the formtion of endogenous negtive llosteric modultors of α7nachrs might e etter tolerted thn directly cting cholinergic ntgonists Allosteric modultors chnge receptor conformtions in the presence of orthosteric lignds nd often hve no effect on their own, cting only when physiologicl receptors re ctivted Kynurenic cid (KYNA) is n endogenous neuroinhiitory metolite 18. It is produced y the irreversile trnsmintion of kynurenine, the first min ctolic product of tryptophn. Formed in strocytes 19, KYNA is present in the mmmlin rin in nnomolr concentrtions 2. Long known s competitive ntgonist of the glycine co-gonist site of the NMDA receptor 21, KYNA is lso negtive llosteric modultor of α7nachrs t endogenous concentrtions, nd somtodendritic, preterminl nd presynptic α7nachrs re eqully sensitive to KYNA Notly, fluctutions in rin KYNA hve neuromodultory consequences. Thus, reductions in rin KYNA 1 Preclinicl Phrmcology Section, Behviorl Neuroscience Reserch Brnch, Intrmurl Reserch Progrm, Ntionl Institute on Drug Ause, Ntionl Institutes of Helth, Deprtment of Helth nd Humn Services, Bltimore, Mrylnd, USA. 2 Deprtment of Psychitry, Mrylnd Psychitric Reserch Center, University of Mrylnd School of Medicine, Bltimore, Mrylnd, USA. 3 Deprtment of Biomedicl Sciences, Section of Neuroscience nd Clinicl Phrmcology, University of Cgliri, Monserrto, Itly. 4 Inserm U184, Experimentl nd Clinicl Neurosciences Lortory, Neuroiology nd Neurophrmcology Addiction Group, Poitiers, Frnce. 5 Université de Poitiers, Poitiers, Frnce. 6 Neuroscience Institute, Consiglio Nzionle delle Ricerche, Cgliri, Itly. 7 McLen Hospitl, Hrvrd Medicl School, Belmont, Msschusetts, USA. 8 Integrtive Neuroiology Section, Moleculr Trgets & Medictions Discovery Brnch, Intrmurl Reserch Progrm, Ntionl Institute on Drug Ause, Ntionl Institutes of Helth, Deprtment of Helth nd Humn Services, Bltimore, Mrylnd, USA. 9 Psychoiology Section, Moleculr Trgets & Medictions Discovery Brnch, Intrmurl Reserch Progrm, Ntionl Institute on Drug Ause, Ntionl Institutes of Helth, Deprtment of Helth nd Humn Services, Bltimore, Mrylnd, USA. 1 Present ddress: Deprtment of Psychitry, University of Msschusetts Medicl School, Worcester, Msschusetts, USA. 11 These uthors contriuted eqully to this work. Correspondence should e ddressed to S.R.G. (sgolder@mil.nih.gov). Received 27 June; ccepted 11 Septemer; pulished online 13 Octoer 213; doi:1.138/nn VOLUME 16 NUMBER 11 NOVEMBER 213 nture NEUROSCIENCE

2 npg 213 Nture Americ, Inc. All rights reserved. cuse n increse in extrcellulr cetylcholine, dopmine nd glutmte 25 27, wheres KYNA elevtions reduce α7nachr function nd result in α7nachr-dependent, ut reltively modest, decreses in extrcellulr glutmte nd dopmine in the stritum, prefrontl cortex nd cudte nucleus 26,28,29. It hs therefore een proposed tht strocytederived KYNA, through this indirect ction, my serve s n endogenous modultor of oth physiologicl nd pthologicl glutmtergic nd dopminergic signling 3. We hypothesized tht phrmcologicl enhncement of rin KYNA levels could selectively counterct the ehviorl nd neurochemicl effects of THC responsile for mrijun use nd dependence notly, the ilities to support the development of persistent drug-tking ehvior 31, to precipitte relpse to drug-seeking ehvior in stinent sujects 32 nd to increse dopmine relese in the nucleus ccumens shell 6,7. Production of KYNA in the rin nd elsewhere cn e incresed y inhiiting kynurenine 3-monooxygense (KMO), pivotl enzyme in the kynurenine pthwy of tryptophn degrdtion 33,34. In oth rodents nd monkeys, peripherl KMO inhiition results in elevted lood levels of KYNA s precursor kynurenine 35,36, which redily penetrtes the lood-rin rrier nd ccumultes in strocytes, where it undergoes trnsmintion to KYNA 19,37. Newly formed KYNA is promptly relesed into the extrcellulr comprtment 38. Notly, no reuptke processes exist for KYNA, nd extrcellulr KYNA is not degrded enzymticlly 39 ut is slowly eliminted from the rin y non-specific cid trnsporter 2,4. In this study, we used 3,4-dimethoxy-N-[4-(3-nitrophenyl)thizol- 2-yl]enzenesulfonmide (Ro ), potent, selective, peripherlly cting KMO inhiitor 41, to indirectly increse rin KYNA. We comined neurochemicl nd ehviorl pproches to evlute effects of Ro on (i) KYNA levels in the VTA nd shell in rts; (ii) elevtions of extrcellulr dopmine in the shell nd VTA induced y THC or the synthetic cnninoid WIN 55,212-2 in rts; (iii) THC self-dministrtion in squirrel monkeys nd WIN 55,212-2 self-dministrtion in rts; (iv) drug-induced nd cue-induced relpse to cnninoid-seeking ehvior in stinent nimls; (v) cocine self-dministrtion nd food-rewrded ehvior in monkeys, to ssess specificity of the effect; nd (vi) working memory nd THC discrimintion in rts nd squirrel monkeys, to ssess potentil side effects. To further elucidte the mechnism of the oserved effects, we determined whether infusing KYNA loclly in the shell would prevent THC-induced elevtions of dopmine in the shell of rts. RESULTS Neurochemicl effects of KMO inhiition in rts We tested whether systemic dministrtion of the KMO inhiitor Ro would increse levels of KYNA in two rin regions implicted in rewrding effects of cnninoids: the shell nd VTA. In vivo microdilysis experiments in freely moving rts showed tht systemic dministrtion of 3 nd 1 mg per kilogrm ody weight intrperitonelly (i.p.) of Ro incresed extrcellulr KYNA in the shell y ~15% nd ~225%, respectively (Fig. 1; 3 mg per kg: F 11,55 = 28.59, P <.1; 1 mg per kg: F 11,55 = 15.3, P <.1). KYNA (% of sl levels) Ro mg per kg Ro mg per kg Time (min) KYNA (% of sl levels) Ro mg per kg Ro mg per kg In the VTA, the 3 nd 1 mg per kg doses of Ro elevted KYNA y ~5% nd ~2%, respectively (Fig. 1; 3 mg per kg: F 11,55 = 5.85, P <.1; 1 mg per kg: F 11,55 = 24.18, P <.1). Pek KYNA levels in oth nd VTA were oserved 8 min fter injection of 1 mg per kg Ro nd 14 min or lter fter injection of 3 mg per kg Ro We then determined whether systemic dministrtion of Ro would lock THC-induced elevtions of dopmine in the shell nd VTA in rts. In the shell, THC (3 mg per kg, i.p.) significntly incresed extrcellulr dopmine (Fig. 2,; tretment time interction, F 3,218 = 1.99, P <.3; re under the curve (AUC), F 3,22 = 6.6, P =.36), ut pretretment with 3 or 1 mg per kg of Ro dose-dependently locked this effect of THC (Fig. 2,). We sw similr effects in the VTA, where THC (3 mg per kg) lso incresed extrcellulr dopmine significntly (Fig. 2c,d; tretment time interction, F 3,188 = 4.25, P <.1; AUC, F 3,19 = 22.1, P <.1), nd pretretment with either 3 or 1 mg per kg of Ro significntly reduced this effect of THC (Fig. 2c,d). When given lone, Ro (1 mg per kg) did not significntly ffect dopmine levels in either the (Fig. 2; P =.99) or the VTA (Fig. 2c; P =.93). This finding tht systemic dministrtion of Ro locked the effects of THC on dopmine in rewrd-relted rin res, coupled with the finding tht Ro incresed KYNA in these res, led us to determine whether the effects of THC on dopmine could e locked y infusing KYNA directly into the shell. We oserved tht THC (3 mg per kg, i.p.) significntly incresed extrcellulr dopmine in the shell of freely moving rts when the locl tissue ws continuously infused with vehicle, ut not when the tissue ws continuously infused with KYNA (5 nm) (Fig. 2e,f: tretment time interction, F 14, 91 = 3.61, P <.1; AUC: F 2,13 = 13.64, P =.6). In the sence of THC, locl infusion of KYNA (5 nm) into the shell hd no effect on dopmine levels (Fig. 2e,f). To verify tht the ility of Ro to lock THC-induced dopmine elevtions in the shell ws due its ctions t α7nachrs, we reversed the effects of Ro with glntmine nd PNU12596, oth gonists t the llosteric potentiting site of α7nachrs where KYNA cts 23,42. In these two experiments (one with glntmine nd one with PNU12596), we gin found tht systemic Ro (1 mg per kg, i.p.) significntly decresed the ility of THC (3 mg per kg, i.p.) to rise dopmine in the VTA Time (min) Figure 1 Effects of tretment with Ro on the extrcellulr concentrtion of kynurenic cid (KYNA) in shell nd VTA of freely moving rts. (,) Ro (3 nd 1 mg per kg i.p.) significntly incresed extrcellulr KYNA in the shell (; sl concentrtions, 2.29 ±.18 nd 1.51 ±.1 nm, respectively) nd VTA (; sl concentrtions, 1.61 ±.1 nd 1.29 ±.1 nm, respectively). KYNA reched pek concentrtions 8 min fter the injection of 1 mg per kg Ro in oth rin res. Arrows indicte time of Ro injection. Results re expressed s men (± s.e.m.) percentge of sl vlues (n = 6 rts per group). P <.5, P <.1, P <.1, post hoc versus sl vlues. nture NEUROSCIENCE VOLUME 16 NUMBER 11 NOVEMBER

3 npg 213 Nture Americ, Inc. All rights reserved. (% of sl levels) Vehicle + THC 3 Ro 1 Ro 3 + THC 3 Ro 1 + THC 3 THC or veh Ro or veh Time (min) shell (replicting the effect seen in Fig. 2,), reducing the AUC y out 6 to 7% (Fig. 3; F 5,27 = 8.34, P <.1; Fig. 3; F 4,17 = 9.87, P <.3). Furthermore, we oserved tht pretretment with glntmine (Fig. 3; 3 mg per kg, i.p.) or PNU12596 (Fig. 3; 1 mg per kg, i.p.) reversed this effect of Ro Neither glntmine nor PNU12596 ltered dopmine levels when given lone, nor did they lter the effects of THC (Fig. 3,). Thus, we confirmed tht α7nachrs were involved in the ility of Ro to lock THC-induced dopmine elevtions in rts. To determine whether tretment with Ro lters the effects of cnninoid CB 1 receptor gonists other thn THC, we studied dopmine elevtions induced y the synthetic gonist WIN 55, Like THC, WIN 55,212-2 (.3 mg per kg intrvenously (i.v.)) significntly incresed extrcellulr dopmine in the shell (Fig. 4; tretment time interction, F 28,98 = 3.28, P <.1). Although Ro (3 or 1 mg per kg) lone did not ffect dopmine levels (Fig. 4; oth P vlues =.99), pretretment with 3 or 1 mg per kg of Ro significntly reduced the ility of WIN 55,212-2 to increse dopmine in the shell (Fig. 4,c; AUC: F 4,14 = 3.73, P =.288). Behviorl effects of KMO inhiition in rts Hving determined tht the KMO inhiitor Ro could lock the effects of cnninoid CB 1 gonists in rewrd-relted rin res, we tested the effects of this tretment in ehviorl models of cnninoid use. We first turned to rodent model of cnninoid reinforcement in which rts intrvenously self-dminister WIN 55,212-2 (12.5 µg per kg per injection). This synthetic cnninoid hd cler reinforcing effect, cusing rts to respond significntly more on the lever tht delivered the drug thn on n inctive control lever (Fig. 4d,e; F 1,4 = 23.95, P =.8). Tretment with 3 or 1 mg per kg Ro min efore ech session significntly decresed selfdministrtion of WIN 55,212-2 (Fig. 4d, 3 mg per kg: F 3,12 = 19.5, overflow s AUC (% of THC lone) Ro 1 + veh Veh + THC # Ro 3 + THC Ro 1 + THC c (% of sl levels) e (% of sl levels) P <.1; Fig. 4e, 1 mg per kg: F 3,12 = 6.92, P =.6). The 1 mg per kg dose of Ro decresed self-dministrtion responses (on the ctive lever) over ll 3 d of testing, ut it lso significntly ffected responses on the inctive lever (Fig. 4e; F 3,12 = 18.35, P <.1). This effect on inctive-lever responses ws not seen with 3 mg per kg Ro (Fig. 4d; F 3,12 = 1.76, P =.21), yet this lower dose ws effective in locking the self-dministrtion of WIN 55, overflow s AUC (% of THC lone) Vehicle + THC 3 Ro 1 Ro 3 + THC 3 Ro 1 + THC 3 THC or veh Ro or veh Time (min) 5 Glntmine Ro THC overflow s AUC (% of THC lone) 1 5 PNU12596 Ro THC Figure 3 Prevention of the neurochemicl effects of Ro y two gonists t the llosteric potentiting site of the α7nachr, glntmine nd PNU (,) THC dministrtion (3 mg per kg i.p.;, n = 7 rts;, n = 4) elevted extrcellulr dopmine (tht is, dopmine overflow) in shell (verge sl concentrtion, ± 5.5 fmol per 1 µl) of freely moving rts. These THC-induced increses in dopmine were locked y Ro (1 mg per kg, i.p., 4 min efore THC;, n = 6;, n = 5), nd this lockde ws prevented y pretretment with glntmine (; 3 mg per kg, i.p., 6 min efore THC; n = 5) or PNU12596 (; 1 mg per kg, i.p., 6 min efore THC; n = 4). Glntmine (n = 5), PNU12596 (n = 4) or Ro (n = 5) lone did not increse dopmine, nd neither glntmine nor PNU12596 (oth n = 5) ffected THC-induced dopmine increses. levels re expressed s AUC (reltive to the men vlue in the vehicles + THC 3 mg per kg condition) over the 18 min following THC or vehicle injection. Brs represent mens ± s.e.m. P <.5, P <.1, post hoc versus vehicles + THC 3 mg per kg; Dunnett s test. Dosges re given in mg per kg; vlues of represent vehicle. VTA d overflow s AUC (% of THC lone) f overflow s AUC (% of THC lone) # # Ro 1 + veh Veh + THC Ro 3 + THC Ro 1 + THC Figure 2 Effects of elevted rin KYNA on THC-induced elevtions Vehicle + THC 3 of extrcellulr dopmine (tht is, dopmine overflow) in shell nd KYNA (5 nm in ) 15 2 VTA of freely moving rts. ( d) THC (3 mg per kg) incresed dopmine s KYNA (5 nm in ) + THC 3 compred to sl levels in oth shell (; verge sl concentrtions, 18 KYNA or veh ± 2.7 fmol per 1 µl; n = 7 rts) nd VTA (c; verge sl 16 concentrtion, ± 3.8 fmol per 1 µl; n = 7). The effects of THC 5 # 14 on dopmine in shell (,) were significntly ttenuted y 12 pretretment with 1 mg per kg Ro (Ro; n = 6), nd the 1 effects of THC on dopmine in VTA (c,d) were significntly ttenuted y 8 5 pretretment with 3 (n = 5) or 1 mg per kg Ro (n = 6). THC or veh Ro lone (oth n = 5) did not ffect extrcellulr levels of dopmine in either re. (e,f) Locl infusion of KYNA (5 nm) (n = 5) into shell significntly reduced THC-induced dopmine elevtions in shell (e,f; n = 5). Time (min) Arrows indicte time of THC, Ro or vehicle injection or locl infusion of KYNA. Dt re presented over the course of the session s percentge of sl vlues (,c,e) or s AUC during the first 12 min fter THC or vehicle injection, expressed s percentge of the men vlue in the vehicle + THC group (,d,f). Results re expressed s mens ± s.e.m. P <.5 versus seline, # P <.5 versus vehicle + THC. Dosges re given in mg per kg; Veh, vehicle KYNA + veh Veh + THC KYNA + THC 1654 VOLUME 16 NUMBER 11 NOVEMBER 213 nture neuroscience

4 npg 213 Nture Americ, Inc. All rights reserved. Figure 4 Effects of tretment with Ro on use-relted effects of the synthetic CB 1 gonist WIN 55,212-2 in rts. (,) Ro (Ro; 3 or 1 mg per kg) reduced the increses in extrcellulr dopmine levels produced y WIN 55,212-2 (WIN;.3 mg per kg) in shell (). Ro lone did not significntly ffect dopmine levels in shell (). Results expressed s percentge of sl vlues over time (n = 4 rts in ll groups). Arrows indicte time of injection. (c) levels expressed s AUC (reltive to men vlue in vehicle (Veh) + WIN condition) over the 12 min following WIN or vehicle injection. P <.5 versus seline, # P <.5 versus Veh + WIN, simultneous confidence intervls. (d,e) Presses on ctive nd inctive levers (n = 5). Ro (sessions T1 T3), ut not its vehicle (sessions B1 B3; recovery sessions R1 nd R2), dose-dependently decresed the numer of WIN injections 2 Vehicle + WIN.3 Ro 3 + WIN.3 18 Ro 1 + WIN (% of sl levels) (12.5 µg per kg; 1 injection per ctive-lever response) self-dministered over 2-h sessions. P <.5,P <.1, post hoc versus verge of sessions B1 B3, Tukey test. (f) Presses on ctive nd inctive levers. Ro (1 mg per kg) locked reinsttement of extinguished drug-seeking induced y injection of WIN (.3 mg per kg) (n = 5). P <.1, post hoc versus vehicles; P <.1, post hoc versus vehicle + WIN.3 mg per kg condition, Tukey test. All points or rs represent mens ± s.e.m. Dosges re given in mg per kg; Veh or vlues of represent vehicle. Self-dministrtion quickly recovered to seline levels when Ro tretment ws discontinued. As relpse to drug use fter long periods of stinence represents one of the gretest chllenges for the tretment of ddiction, we lso investigted whether Ro would lock reinsttement of drug seeking y stinent rts in n niml model of relpse. When WIN 55,212-2 delivery ws discontinued, rts drug-seeking ehvior decresed to low levels (Fig. 4f). A noncontingent priming injection of WIN 55,212-2 (.3 mg per kg., i.p., 1 min efore the session) reinstted drug-seeking ehvior, ut this relpse-like effect ws completely locked y pretretment with 1 mg per kg Ro (Fig. 4f; F 5,2 = , P <.1). None of these tretments significntly ffected responses on the inctive lever (Fig. 4f; oth tretment P vlues =.51). Thus, Ro prevented the relpse-like effect induced y reexposure to cnninoids in rts. KMO inhiition nd THC rewrd in squirrel monkeys Becuse THC self-dministrtion in squirrel monkeys provides the most congruent niml model of humn cnninoid use 31, we used this model to exmine the effects of Ro We lso tested the effects of Ro in monkeys trined to self-dminister food nd cocine under the sme schedule of reinforcement (fixed-rtio 1) to determine whether the effects of Ro re specific to cnninoid rewrd. At the pek of the THC self-dministrtion dose-effect curve (4 µg per kg per injection THC; Fig. 5), squirrel monkeys selfdministered n verge of 5.8 ± 1.9 injections nd lever-pressed t n verge rte of 1.2 ±.25 responses per second in the presence of green light signling THC vilility. We investigted self-dministrtion of this THC dose in monkeys cross 3 consecutive dys of tretment with Ro (1 or 2 mg per kg, 4 min efore ech session). Ro ws lwys dministered intrmusculrly (i.m.) in monkeys. Ro significntly nd dose-dependently reduced THC self-dministrtion during ll three sessions (Fig. 5; 1 mg per kg Ro : F 3,12 = 4.7, P =.33; 2 mg per kg Ro : F 3,12 = 3.93, P <.1). Selfdministrtion ehvior returned to seline levels when Ro tretment ended. d Ro WIN Time 6 8 Self-dministrtion of WIN Active Inctive Ro 3 mg per kg B1 B2 B3 T1 Session T2 T3 R1 R (% of sl levels) e Ro 3 Ro 1 Ro Veh Time 6 8 Self-dministrtion of WIN B1 B2 B3 Active Inctive T1 Ro 1 mg per kg T2 T3 R1 Session c 15 overflow s AUC (% of WIN lone) In the food self-dministrtion model (Fig. 5), monkeys selfdministered ± 1.29 food pellets on verge, with response rte of 1.97 ±.66 responses per second in the presence of green light signling food vilility. During three dily sessions with Ro pretretment, food-reinforced response in monkeys ws not ffected y either 1 or 2 mg per kg of Ro , under testing conditions tht prlleled those used to evlute THC self-dministrtion (Fig. 5; 1 mg per kg Ro : F 3,9 = 1.76, P =.22; 2 mg per kg Ro : F 3,9 = 1.77, P =.22). Thus, Ro did not produce nonspecific disruption of ehvior. Moreover, Ro (2 mg per kg, 4 min efore the session) reversed the disruptive effects of THC (.56 mg per kg i.v., immeditely efore the session) on food-mintined self-dministrtion ehvior (Supplementry Fig. 1: pellets F 3,6 = 23.29, P =.1; post hoc nlysis, THC.56 mg per kg versus Ro mg per kg + THC.56 mg per kg, P =.3; Supplementry Fig. 1: response rte F 3,6 = 15.37, P =.3; post hoc nlysis, THC.56 mg per kg versus Ro mg per kg + THC.56 mg per kg, P =.18). Tretment with.56 mg per kg THC lone significntly reduced oth the food-mintined response rte (from.9 ±.17 to.9 ±.3 responses per second) nd the numer of food pellets (from 49.5 ± 1.76 to 19. ± 4.51) s compred to vlues oserved fter vehicle tretment (post hoc nlysis, vehicle versus THC.56 mg per kg: response rte, P =.3; pellets, P =.2). Pretretment with Ro hd no effect y itself (post hoc nlysis, vehicle versus Ro mg per kg: rte. P =.28; pellets. P =.93), ut it completely reversed the effects of THC on response rte (to.62 ±.8 responses per second; post hoc nlysis, vehicle versus Ro mg per kg + THC.56 mg per kg, P =.22) nd food intke (to ± 1.45 pellets ; post hoc nlysis, P =.91). In monkeys trined to self-dminister cocine (Fig. 5c), the dose of Ro (2 mg per kg) tht effectively decresed THC selfdministrtion did not lter cocine self-dministrtion (3 µg per kg per injection). Monkeys verged ±.62 injections with men response rte of.54 ±.4 responses per second in the presence of green light signling cocine vilility. Pretretment with Ro min efore ech of three dily sessions did not R f Ro WIN 55,212-2 # Ro 3 + veh Ro 1 + veh Veh + WIN Ro 3 + WIN Ro 1 + WIN Reinsttement Active Inctive nture NEUROSCIENCE VOLUME 16 NUMBER 11 NOVEMBER

5 Injections THC 4 THC 4 µg per kg per injection + Ro THC 4 Pellets Food Food + Ro Food c Injections Cocine 3 µg per kg per injection + Ro Cocine 3 Cocine 3 npg 213 Nture Americ, Inc. All rights reserved. d Responses per second g Injections THC 4 µg per kg per injection + Ro THC 4 THC Consecutive sessions Ro mg per kg Vehicle V THC (µg per kg per injection) Vehicle Ro mg per kg Vehicle Ro mg per kg e Responses per second h Responses per second Food Food + Ro Food Consecutive sessions Ro mg per kg 1.6 Vehicle V THC (µg per kg per injection) f Responses per second i Intke (mg per kg ) Cocine 3 µg per kg per injection + Ro Cocine Consecutive sessions Ro mg per kg Vehicle Cocine THC (µg per kg per injection) Figure 5 Effects of Ro on THC, food nd cocine self-dministrtion in squirrel monkeys. ( f) Ro (1 nd 2 mg per kg, i.m.) significntly decresed the numer of THC injections self-dministered during 1-h sessions () nd decresed overll response rtes (d) y squirrel monkeys under fixed-rtio 1 schedule t THC dose of 4 µg per kg per injection (n = 5 monkeys, mens ± s.e.m.). Ro did not significntly ffect food-reinforced ehvior (,e) or cocine (3 µg per kg per injection) self-dministrtion ehvior (c,f) in monkeys under conditions tht prlleled the THC self-dministrtion procedure (n = 4 for food, n = 3 for cocine, mens ± s.e.m.). The Ro vehicle ws given 4 min efore ech seline session. (g i) Numer of THC injections (g), overll response rtes in the presence of the green light signling THC vilility (h) nd totl THC intke (i) s function of the THC dose. Ech dt point represents the men ± s.e.m. of the lst three sessions under ech THC condition nd under vehicle conditions (n = 3 5 monkeys). Pretretment with Ro (2 mg per kg) cused rightwrd shift of the THC dose-response curves compred to vehicle pretretment. P <.5, P <.1, post hoc versus the lst session with vehicle pretretment (session 3) ( f) or vehicle conditions (g,h), Bonferroni test. V represents THC vehicle significntly ffect cocine self-dministrtion during those sessions (Fig. 5c; F 3,6 = 1.33, P =.35). Lever response rtes were significntly ffected during Ro tretment only in the group self-dministering THC (Fig. 5d; 1 mg per kg Ro : F 3,12 = 9.82, P =.1; 2 mg per kg Ro : F 3,12 = 15.92, P <.1). In monkeys self-dministering food or cocine, response rtes were not ffected y tretment with Ro (Fig. 5e, food selfdministrtion: 1 mg per kg Ro , F 3,9 = 1.1, P =.4; 2 mg per kg Ro , F 3,9 = 1.61, P =.26; Fig. 5f, cocine selfdministrtion: F 3,6 =.69, P =.59). To further chrcterize the nture of the effects of Ro on THC self-dministrtion, we vried the dose of THC nd otined clssic inverted U-shped dose-effect curves (Fig. 5g,h). THC mintined significntly more self-injections (Fig. 5g; F 6,22 = 29.34, P <.1) thn vehicle t.5, 1, 2, 4 nd 8 µg per kg per injection nd significntly higher response rtes (Fig. 5h; F 6,22 = 36.76, P <.1) thn vehicle t 4 µg per kg per injection. We found tht pretretment with 2 mg per kg of Ro significntly shifted the THC dose-response curve for injections down nd to the right (Fig. 5g; interction of THC nd Ro , F 5,17 = 35.45, P <.1), consistent with decrese in THC s rewrding effects. This Ro dose lso produced significnt downwrd nd rightwrd shift for response rtes (Fig. 5h; interction of THC nd Ro , F 5,17 = 16.1, P <.1). Post hoc pirwise comprisons reveled significnt differences in the effects of 1, 2, 4 nd 16 µg THC per kg per injection fter Ro pretretment on the numer of selfdministered injections (ll P <.1) nd significnt differences in effects of 2, 4 nd 16 µg per kg THC per injection fter Ro pretretment on response rtes (2 µg per kg, P =.4; 4 µg per kg, P <.1; 16 µg per kg, P =.12). The totl mount of THC received during the session ws significntly decresed y Ro cross most of the dose-effect function (Fig. 5i; F 5,17 = 59.4, P <.1), ut it incresed t the highest dose per injection. We then sked whether positive llosteric modultors of α7nachrs (glntmine nd PNU12596) would prevent the effects of Ro on THC self-dministrtion in monkeys. Glntmine 1656 VOLUME 16 NUMBER 11 NOVEMBER 213 nture neuroscience

6 npg 213 Nture Americ, Inc. All rights reserved. Figure 6 Reversl of ehviorl effects of Ro y positive llosteric modultors of α7nachrs. ( d) Glntmine () or PNU12596 (c) dose-dependently reversed the lockde of THC (4 µg per kg per injection) self-dministrtion cused y pretretment with Ro (2 mg per kg). Glntmine () nd PNU12596 (d) lone hd no significnt effect. P <.1, post hoc versus vehicles + THC 4 µg per kg; # P <.5, P <.1, post hoc versus vehicle + Ro mg per kg + THC 4 µg per kg, Tukey test. (e,f) Tretment with Ro locked the reinsttement of extinguished THC-seeking responses produced y priming injection of THC (4 µg per kg, i.v.), nd this effect ws prevented y pretretment with glntmine (e) or PNU12596 (f). P <.5, P <.1, post hoc versus vehicles; P <.1, post hoc versus vehicles + THC 4 µg per kg, $$ P <.1, post hoc versus vehicle + Ro mg per kg + THC 4 µg per kg, Tukey test. (g,h) Tretment with Ro lso locked the reinsttement of extinguished THC-seeking responses induced y reintroduction of cues previously ssocited with THC, nd this effect of Ro ws reversed y pretretment with glntmine (g) or PNU12596 (h). P <.1, post hoc versus vehicles + no cues; P <.1, post hoc versus vehicles + cues; $ P <.5, $$ P <.1, post hoc versus vehicle + Ro mg per kg + cues, Tukey test. n = 4 monkeys for ll conditions, except n = 3 in h. Brs represent mens ± s.e.m. represents vehicle in ll pnels. THC dosges expressed in µg per kg per injection; ll other dosges expressed in mg per kg. (.3 3 mg per kg, i.m.) dose-dependently prevented the effects of Ro (2 mg per kg, i.m.) on THC self-dministrtion in monkeys (Fig. 6; F 4,12 = 36.48, P <.1), ut glntmine lone (.3 3 mg per kg, i.m.) hd no significnt effect (Fig. 6; F 3,9 = 3.41, P =.67). Like glntmine, PNU12596 (.3 3 mg per kg, i.m.) lso dose-dependently prevented the effects of Ro (2 mg per kg, i.m.) on THC self-dministrtion in monkeys (Fig. 6c; F 4,12 = 35.71, P <.1) ut hd no significnt effect when given lone (Fig. 6d: F 3,9 = 1.98, P =.19). Thus, we confirmed tht α7nachrs were involved in the ility of Ro to lock the reinforcing effects of THC in nonhumn primtes. KMO inhiition nd relpse in squirrel monkeys To further study the effects of KMO inhiition in niml models of relpse to THC seeking, we determined whether Ro locked reinsttement induced y reexposure to THC or THC-ssocited cues in squirrel monkeys nd whether α7nachrs were involved in this lockde. When lever-press responses for THC hd een extinguished y discontinuing THC delivery, dministrtion of noncontingent priming injection of THC (4 µg per kg, i.v.) efore the session reinstted drug-seeking (Fig. 6e, F 5,14 = 34.37, P <.1; Fig. 6f, F 5,13 = 77.81, P <.1). Tretment with Ro (2 mg per kg) locked this THC-induced reinsttement (Fig. 6e,f; oth P <.1 versus THC), nd pretretment with either glntmine (3 mg per kg) or PNU12596 (1 mg per kg) prevented this lockde (Fig. 6e,f). Ro lone did not reinstte drug-seeking ehvior (Fig. 6e,f). Both glntmine nd PNU12596 produced low level of reinsttement of drug-seeking ehvior (Fig. 6e, P =.33 versus vehicles; Fig. 6g, P =.12 versus vehicles), ut this effect ws significntly smller thn the reinsttement produced y priming injection of THC (Fig. 6e, P <.1 versus THC; Fig. 6f, P <.1 versus THC). Becuse relpse cn e triggered y reexposure to drug-relted environmentl cues, we looked t cue-induced reinsttement of THC seeking. When oth THC delivery nd presenttion of cues signling delivery of THC were discontinued, THC seeking y the monkeys decresed to very low levels (Fig. 6g,h). When visul cue presenttion ws restored nd i.v. vehicle ws delivered contingent on lever response, THC-seeking ehvior ws reinstted (Fig. 6g, F 5,13 = 21.16, THC self-dministrtion Glntmine Ro THC THC injections c THC self-dministrtion d THC self-dministrtion # PNU PNU Ro THC THC e THC-induced reinsttement f THC-induced reinsttement 5 5 $$ 4 $$ Glntmine 3 3 PNU Ro Ro THC THC g Cue-induced reinsttement h Cue-induced reinsttement $$ 5 5 $ Glntmine 3 3 PNU Ro Ro Cues Cues THC injections THC injections P <.1; Fig. 6h, F 5,1 = 57.87, P <.1). This cue-induced reinsttement ws significntly decresed y Ro (2 mg per kg) (Fig. 6g,h). Pretretment with either glntmine (3 mg per kg) or PNU12596 (1 mg per kg) prevented these effects of Ro (Fig. 6g,h). When Ro , glntmine or PNU12596 were given with the THC vehicle without presenttion of cues, THCseeking ehvior ws not reinstted (Fig. 6g, post hoc nlysis versus vehicle + no cues: Ro , P =.95; glntmine, P = 1.; Fig. 6h: Ro , P =.79; PNU12596, P =.69). These results suggest tht tretment with KMO inhiitor could prevent relpse cused y reexposure to THC or to THC-ssocited cues, nd tht this effect of KMO inhiition occurs through n α7nachrmedited mechnism. Effects of KMO inhiition nd THC on working memory Becuse excessive levels of KYNA my e ssocited with cognitive impirment 43,44 nd ecuse THC is well known to impir memory, we tested the effects of Ro on working memory in rts trined with delyed nonmtching-to-position procedure nd in squirrel monkeys trined with delyed mtching-to-smple procedure. In rts, THC (3 or 5.6 mg per kg) nd Ro (1 mg per kg) were dministered lone nd in comintion. Ro hd no effect on memory when given lone (Fig. 7), ut THC decresed ccurcy in dely-dependent mnner, consistent with selective impirment of working memory (Fig. 7). Ro did THC injections Glntmine THC THC self-dministrtion nture NEUROSCIENCE VOLUME 16 NUMBER 11 NOVEMBER

7 npg 213 Nture Americ, Inc. All rights reserved. Figure 7 Effects of Ro nd THC on working memory in rts nd squirrel monkeys. ( d) The 1 mg per kg dose of Ro , which ws effective in locking the effects of THC in rewrd-relted rin res in rts, did not hve deleterious effects on short-term memory in rts when given lone () or in comintion with THC (3 or 5.6 mg per kg, i.p.; c,d) in delyed nonmtching-to-position model of working memory. Both doses of THC significntly decresed ccurcy (; P <.7), ut this ws not excerted y Ro (c,d). (e,f) The 2 mg per kg dose of Ro , which ws effective in locking the effects of THC in rewrd-relted rin res in monkeys, did not hve deleterious effects on short-term memory in monkeys when given lone (e) or in comintion with THC (f) in delyed mtching-to-smple model of working memory. THC (.1 mg per kg, i.m.) significntly decresed ccurcy (f), nd this ws reversed y Ro (f). Accurcy (percentge of trils with correct response) is shown (mens ± s.e.m.; n = 8 rts, n = 3 monkeys) s function of dely nd of drug tretment. Dosges re given in mg per kg. not lter the effects of THC (Fig. 7c,d). The min effects of THC (F 2,13 = 13.56, P <.1) nd dely (F 4,28 = 47.34, P <.1) were oth significnt, ut the effects of Ro were not (P =.85). Pired comprisons indicted tht oth doses of THC significntly impired working memory (P <.35). In squirrel monkeys, working memory ws lso impired y THC lone (.1 mg per kg) ut not y Ro (2 mg per kg) lone (Fig. 7e,f). THC t short dely vlues lso decresed ccurcy slightly in monkeys, suggesting tht impirments might hve een due in prt to nonselective disruption y THC, similr to the disruptions in food-mintined ehvior descried ove. THC-induced impirments in monkeys were reversed y Ro (Fig. 7f; min effect of Ro , F 1,2 = 2.46, P <.5; min effect of THC, F 1,2 = 42.42, P <.5; min effect of dely, F 4,8 = 59.65, P <.1). KMO inhiition nd discrimintive-stimulus effects of THC To determine whether Ro could ffect not only the reinforcing effects of THC ut lso its sujective effects, we studied effects of Ro in rodent nd primte models of cnninoid discrimintion. In rts trined to detect whether they hd een injected with Percentge CB 1 lever selection c Percentge CB 1 lever selection Rts Rts THC.5 THC + Ro mg per kg THC + Ro mg per kg V V THC dose (mg per kg) THC dose (mg per kg) Squirrel monkeys d Squirrel monkeys Vehicle 2 AM THC.5 THC + Ro mg per kg THC + Ro mg per kg Cumultive THC dose (mg per kg) Cumultive THC dose (mg per kg) Responses per second Responses per second Percentge correct c Percentge correct e Percentge correct Rts Vehicle Ro Dely (s) Rts Vehicle + THC 3 Ro THC Dely (s) Squirrel monkeys Vehicle Ro Dely (s) 16 Percentge correct d Percentge correct f Percentge correct Dely (s) Squirrel monkeys Vehicle Vehicle + THC.1 Ro THC.1 THC or its vehicle, lever selection ws dose dependent, with mximl selection of the drug lever (99.66%) t the 3 mg per kg trining dose of THC (Fig. 8; F 5,4 = 28.3, P <.1). Notly, the THC dose-effect curve ws not significntly shifted y tretment with either 3 or 1 mg per kg of Ro (Fig. 8; F 2,45 = 2.66, P =.1), indicting tht Ro does not lock ll of the sujective effects of cnninoid CB 1 gonists in rts. Also in this procedure, Ro did Rts Vehicle THC 3 THC Dely (s) Rts Vehicle + THC 5.6 Ro THC Dely (s) Figure 8 Effects of Ro on discrimintive-stimulus effects of THC in rts nd squirrel monkeys. (,) Rts trined to discriminte THC (3 mg per kg, i.p.) from vehicle (V) under fixed-rtio 1 schedule of food delivery were tested with vrious doses of THC. The percentge of responses on the CB 1 gonist pproprite lever ws monotoniclly incresing function of dose. Tretment with Ro (3 or 1 mg per kg, i.p.) did not significntly lter this curve (), nd Ro did not significntly lter the food-reinforced response rte fter THC or vehicle dministrtion (). Ascisse, dose, log scle; ordinte, percentge of responses on the THC-ssocited lever () or response rte (). (c,d) When monkeys, trined under stimulusshock termintion schedule to discriminte injection of the selective cnninoid CB 1 gonist AM454 (1 µg per kg, i.m.) from vehicle, were injected i.m. with vrious doses of THC, the percentge of responses on the CB 1 -pproprite lever ws monotoniclly incresing function of cumultive dose (c). Ro (2 mg per kg, i.m.) reduced the monkeys ility to detect interoceptive effects of THC in the cnninoid CB 1 discrimintion procedure (c). Ro did not significntly ffect response rtes fter THC dministrtion in this procedure (d). Ascisse, cumultive THC dose, log scle; ordinte, percentge of responses on the AM454-ssocited lever (c) or response rte (d). Symols left of the sciss reks indicte performnce during vehicle nd AM454 control sessions. All dt re presented s mens ± s.e.m. (n = 9 rts, n = 3 monkeys) VOLUME 16 NUMBER 11 NOVEMBER 213 nture neuroscience

8 npg 213 Nture Americ, Inc. All rights reserved. not disrupt food-reinforced ehvior when given lone (Fig. 8; F 2,16 =.53, P =.59) or in comintion with different doses of THC (Fig. 8; F 2,45 =.24, P =.79), indicting tht decreses in WIN 55,212-2 self-dministrtion produced y Ro in rts (Fig. 4d,e) were not due to nonspecific ehviorl disruption. In squirrel monkeys trined to discriminte the cnninoid CB 1 gonist AM454 (1 µg per kg) from vehicle, THC generlized to the cnninoid CB 1 trining stimulus in dose-dependent mnner (Fig. 8c; F 3,6 = 864., P <.1). This dose-effect curve ws significntly shifted to the right y tretment with 2 mg per kg of Ro (Fig. 8c; F 2,15 = , P <.1) ut not y 1 mg per kg of Ro (Fig. 8c; P =.58). Ro did not ffect response rtes in this tsk (Fig. 8d; F 2,11 = 4.54, P =.9). DISCUSSION The present results indicte tht phrmcologicl modultion of rin KYNA levels y KMO inhiitors could provide n effective pproch for the tretment of mrijun dependence. It is well estlished tht THC, like other drugs of use, elevtes extrcellulr dopmine in the shell 2,7,11, n effect tht is medited y cnninoid CB 1 receptors nd presumly underlies the rewrding nd dependenceinducing effects of mrijun. Systemic dministrtion of the KMO inhiitor Ro in rts incresed extrcellulr KYNA levels in the VTA nd shell nd sustntilly reduced the ility of THC or the synthetic cnninoid WIN 55,212-2 to stimulte dopmine relese in these res. This lockde of THC s effects ppers to e due to ctions of KYNA in the shell, s, like systemic dministrtion, locl infusion of KYNA into the shell lso prevented THC from elevting extrcellulr dopmine levels. Ro lso countercted the effects of THC nd WIN 55,212-2 in ehviorl models of drug use. In rts, it mrkedly reduced self-dministrtion of the synthetic cnninoid WIN 55, In monkeys, Ro decresed the rewrding effects of THC, s demonstrted y shift of the self-dministrtion dose-response curves of THC down nd to the right. After pretretment with Ro , THC intke ws reduced over wide rnge of THC doses nd incresed only t the highest THC dose. This increse is consistent with reduced rewrding effect of THC nd my lso e due to the reversl y Ro of rte-depressing effects of THC such s those we oserved in monkeys self-dministering food. Relpse to drug use (s opposed to initil chievement of stinence) is typiclly the min ostcle to successful cesstion of drug use. In stinent monkeys with extensive histories of THC selfdministrtion, Ro prevented relpse-like THC-seeking ehvior induced y reexposure to THC. Prllel effects occurred in rts, where Ro prevented drug-induced seeking of WIN 55, Moreover, Ro ws le to lock the relpseinducing effects of THC-ssocited cues in monkeys, suggesting tht it might reduce drug crving in humns. The ility of the KMO inhiitor Ro to reduce neurochemicl nd ehviorl effects of THC in rts nd monkeys ws prevented y glntmine, n gonist t the llosteric potentiting site of α7nachrs tht overlps with the site where KYNA cts s n ntgonist 24. As glntmine is lso wek cholinesterse inhiitor, we confirmed prevention of the effects of Ro using PNU12596, selective positive llosteric modultor of α7nachrs tht does not inhiit cholinesterse 42. These results indicte tht the ntiuse ctions of KMO inhiition re due to KYNA-induced negtive llosteric modultion of α7nachrs. Although further experimenttion will e required to fully elucidte the circuitry nd mechnisms involved in KYNA s ility to lock cnninoid rewrd, the ville evidence supports the following hypothesis. THC nd WIN 55,212-2 fcilitte dopmine relese in the shell 5,6,45, nd this is elieved to e due t lest in prt to ctivtion of excittory glutmtergic pyrmidl neurons tht project from the prefrontl cortex to the VTA nd shell 46,47. Becuse α7nachrs re loclized on the terminls of these glutmtergic cells 8, negtive llosteric modultion of α7nachrs y KYNA could reduce the relese of glutmte y these cells nd therey reduce glutmte-induced dopmine relese in the VTA nd shell 9,1,45. As elevted levels of dopmine in the shell re considered centrl to the rewrding effects of cnninoid drugs 2 nd s locl infusion of KYNA directly into the shell ws sufficient to completely lock THC-induced dopmine elevtions, it is likely tht effects of KYNA in the shell of the re min fctor underlying the ility of Ro to reduce the rewrding effects of cnninoids. The sfety of KMO inhiitors in humns will hve to e considered in trnsltionl studies. Although high levels of KYNA hve een ssocited with cognitive deficits 43,44, Ro hs nticonvulsnt effects nd neuroprotective effects in niml models of focl or glol ischemi nd l-dopa induced dyskinesi 3. In our experiments, the effects of Ro were specific to cnninoid rewrd nd were not ssocited with dverse effects such s reduced ppetite or nonspecific suppression of ehvior. Of specil relevnce, the modest increse in rin KYNA produced y Ro did not dversely ffect working memory in rts or squirrel monkeys in tests highly sensitive to impirments induced y THC nd other mnesic gents 48. Moreover, in rts, KMO inhiition y itself neither produced THC-like sujective effects nor ltered the effects of THC itself, which most likely comprise oth rewrd-relted nd non-rewrd components. However, in squirrel monkeys, KMO inhiition ttenuted the discrimintive-stimulus effects of THC. The reson(s) for this species-specific effect my e relted to differences in cnninoid mechnisms etween rodents nd primtes 31. Notly, moderte KMO inhiition did not ffect seline levels of dopmine in the shell or VTA in the present study, nd is known not to ffect rin levels of the neurotoxic kynurenine pthwy metolites 3-hydroxykynurenine nd quinolinic cid 36. The decreses in cnninoid self-dministrtion oserved here were not due to nonspecific suppression of opernt ehvior. Although responses on n inctive lever were decresed long with those on the ctive lever in the WIN 55,212-2 self-dministrtion experiment when rts received 1 mg per kg Ro , the 3 mg per kg dose decresed WIN 55,212-2 self-dministrtion without significntly ffecting inctive-lever responses. Moreover, the higher dose of Ro did not lter food-mintined ehvior in rts. In monkeys, Ro did not ffect food or cocine self-dministrtion ehvior, nd, in fct, it reversed rtedepressnt effects of THC on food self-dministrtion. Tken together, our results suggest tht KMO inhiitors could e sfe nd effective, decresing cnninoid rewrd nd relpse t doses devoid of dverse ehviorl or neurotoxic effects. Becuse enhncing endogenous KYNA levels countercts the use-relted effects of THC through negtive llosteric modultion of α7nachrs, rther thn y direct interference with CB 1 receptor function, drugs such s Ro might e etter tolerted thn orthosteric inverse gonists or ntgonists of CB 1 receptors, which cn hve dverse side effects due to ctions t CB 1 receptors not directly relted to THC use 49. A mediction tht would sfely nd effectively ssist in the tretment of mrijun dependence would e n importnt step forwrd in deling with cnnis-use disorders. In the present study, KMO nture NEUROSCIENCE VOLUME 16 NUMBER 11 NOVEMBER

9 npg 213 Nture Americ, Inc. All rights reserved. inhiition selectively locked cnninoid rewrd nd lso countercted the ility of drugs nd drug-relted cues to trigger relpse to cnninoid seeking. As in rodent or nonhumn primte models of neurologicl diseses, where KMO inhiition provides mrked enefits rnging from ehviorl remedition to neuroprotection 35,36,5, phrmcologicl elevtion of rin KYNA offers n ttrctive strtegy for treting humn mrijun dependence. Methods Methods nd ny ssocited references re ville in the online version of the pper. Note: Any Supplementry Informtion nd Source Dt files re ville in the online version of the pper. Acknowledgments We thnk E. Thorndike for excellent technicl ssistnce during the rodent studies. We thnk I. Bum, S. Stevens nd P. White for excellent veterinry ssistnce during the primte studies. This study ws supported in prt y the Intrmurl Reserch Progrm of the Ntionl Institute on Drug Ause (NIDA), Ntionl Institutes of Helth, Deprtment of Helth nd Humn Services, NIDA Residentil Reserch Support Services Contrct N1 DA5999 (principl investigtor D. Kelly), y the Itlin Ministry of University nd Scientific Reserch, y DA23142 (J.B.) nd DA35974 (B.D.K.) nd y the Mrylnd Psychitric Reserch Center, Deprtment of Psychitry, University of Mrylnd School of Medicine. T.Z. ws supported y Grnt from Regione Autonom dell Srdegn nd y the Europen Socil Fund, LR7 27. AUTHOR CONTRIBUTIONS Z.J. ws involved in the design of the study, supervised nd nlyzed the primte self-dministrtion nd reinsttement experiments nd rodent drug discrimintion experiments, nd wrote the first drft of the mnuscript. P.M. ws involved in design of the study, conducted microdilysis experiments including collection of ll smples, performed dopmine ssy, nlyzed microdilysis dt nd helped prepre the finl drft of the mnuscript. H.-Q.W. nlyzed KYNA levels in microdilysis smples. M.E.S. conducted nd nlyzed microdilysis experiments with locl KYNA infusion nd PNU G.H.R. conducted the primte selfdministrtion nd reinsttement experiments. L.V.P designed nd nlyzed the experiments with delyed nonmtching to position nd ws involved in discussions of the dt. C.B. conducted rodent drug discrimintion experiments nd ws involved in discussions of the dt. T.Z. conducted the rt self-dministrtion nd microdilysis experiments with WIN 55, M. Scherm supervised the rt self-dministrtion nd microdilysis experiments with WIN 55,212-2, nlyzed the dt nd ws involved in the discussions of the dt. W.F. ws involved in the discussions of the dt. A.P. ssisted in conducting the microdilysis experiments with THC. J.B. nd B.D.K. designed, conducted nd nlyzed the dt from the primte discrimintion nd memory experiments. S.F., M. Solins, M.P. nd G.T. were involved in the design of the study nd discussions of the dt. R.S. nd S.R.G. conceived, designed nd supervised the study nd edited the mnuscript. COMPETING FINANCIAL INTERESTS The uthors declre no competing finncil interests. Reprints nd permissions informtion is ville online t reprints/index.html. 1. Sustnce Ause nd Mentl Helth Services Administrtion. Results from the 29 Ntionl Survey on Drug Use nd Helth: Volume I. Summry of Ntionl Findings (Rockville, Mrylnd, USA, 21). 2. Grdner, E.L. Endocnninoid signling system nd rin rewrd: emphsis on dopmine. Phrmcol. Biochem. Behv. 81, (25). 3. Solins, M., Golderg, S.R. & Piomelli, D. The endocnninoid system in rin rewrd processes. Br. J. Phrmcol. 154, (28). 4. Din, M., Melis, M. & Gess, G.L. Increse in meso-prefrontl dopminergic ctivity fter stimultion of CB1 receptors y cnninoids. Eur. J. Neurosci. 1, (1998). 5. French, E.D., Dillon, K. & Wu, X. Cnninoids excite dopmine neurons in the ventrl tegmentum nd sustnti nigr. Neuroreport 8, (1997). 6. Chen, J.P. et l. 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