Methylphenidate facilitates learning-induced amygdala plasticity

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1 Methylphenidte filittes lerning-indued mygdl plstiity Ky M Tye 1,2, Lynne D Tye 1,3, Jkson J Cone 1, Evelien F Hekkelmn 1, Ptrii H Jnk 1,2,, & Antonello Boni 1,2,, 21 Nture Ameri, In. All rights reserved. Although methylphenidte (Ritlin) hs een used therpeutilly for nerly yers, the mehnisms y whih it utely modifies ehviorl performne re poorly understood. Here we omined intr lterl mygdl in vivo phrmology nd ex vivo eletrophysiology to show tht ute dministrtion of methylphenidte, s well s seletive dopmine trnsporter inhiitor, filitted lerning-indued strengthening of ortio-mygdl synpses through postsynpti inrese in AMPA reeptor medited urrents, reltive to those in sline-treted rts. Furthermore, lol dministrtion of methylphenidte in the lterl mygdl enhned ue-rewrd lerning through dopmine D1 reeptor dependent mehnisms nd suppressed tsk-irrelevnt ehvior through D2 reeptor dependent mehnisms. These findings revel ritil nd distint roles for dopmine reeptor sutypes in mediting methylphenidte-indued enhnements of neurl trnsmission nd lerning performne. Although methylphenidte () is primrily presried for the tretment of ttention defiit hypertivity disorder (ADHD) 1, the ehviorl enhnements of re not limited to those with ADHD, s lso improves tsk performne nd dereses motor restlessness in the generl popultion 2. ADHD is hrterized y inttention, hypertivity nd impulsivity 3 nd hs een linked to impired lerning performne in sholsti settings. In reent dedes, the dignosis of ADHD nd the presription of hve mrkedly inresed 1. is highly effetive therpeuti gent for oth those with ADHD nd those without 2, improving sholsti performne in % of hildren nd dults,. Wht ellulr nd phrmologil mehnisms underlie ute -indued enhnements of ehviorl performne in the mmmlin rin? Funtionl normlities of the solterl mygdl (BLA), rin region ritil for lerning the emotionl nd motivtionl signifine of environmentl stimuli 12, hve een linked to ADHD 13. To identify neurl mehnisms underlying effets on lerning performne, we tested the effets of in the lterl mygdl, where the reltionship etween the intrinsi miroiruitry nd ute lerning performne hs een well hrterized,. The lterl mygdl is n erly site of onvergene for thlmi nd ortil fferents rrying sensory informtion out environmentl ues nd primry reinforers 1,1 nd is importnt for the quisition nd retrievl of stimulus-outome memories 1 1. Furthermore, thlmomygdl synpti strength predits the suess of ue-rewrd lerning, nd memory onsolidtion is filitted y infusions of in the mygdl 19 fter trining. Thus, the lterl mygdl rin region is well suited for studying the synpti mehnisms y whih lters quisition. Here we show tht lol dministrtion of filittes performne on surose self-dministrtion tsk nd filittes lerning-indued plstiity within single trining session. RESULTS Performne is modulted y in the mygdl To exmine the ute effets of on lerning performne, we lolly dministered into the lterl mygdl of rts (Supplementry Figs. 1 nd 2), efore trining in lterl mygdl dependent ue-rewrd lerning prdigm (Supplementry Figs. 2 nd 3). After trining, we olleted rins for ute slie preprtion nd used ex vivo eletrophysiologil reording proedures to evlute synpti funtion (Supplementry Fig. 2). Rts tht reeived intr lterl mygdl (intr-la) efore trining, reltive to those tht reeived sline, erned signifintly higher (P =.9; Fig. 1) numer of rewrds per minute ( rewrd erning ) ut did not differ in overll motor tivity (Supplementry Fig. ). We used ehviorl index, tsk effiieny, defined s the numer of rewrds erned per ue presented, to quntify the degree to whih rt hd lerned tht the presenttion of the ue indited tht surose ws ville. -treted rts performed with signifintly higher tsk effiieny thn sline-treted rts (P =.12; Fig. 1). We lso quntified the reltive distriution of ttention to goldireted ehvior using ehviorl index termed off-tsk ehvior, defined s the numer of intive port entries per rewrd port entry. Beuse surose is delivered to the rewrd port, rewrd port entries reflet gol-oriented ehvior, wheres intive port entries reflet tsk-irrelevnt ehvior. -treted rts showed ~% less off-tsk ehvior thn sline ontrols (P =.1; Fig. 1d). Thus, the enhnement in rewrd erning seen y rts reltive to sline ontrols ws 1 Ernest Gllo Clini & Reserh Center, University of Cliforni, Sn Frniso, Emeryville, Cliforni, USA. 2 Progrm in Neurosiene, University of Cliforni, Sn Frniso, Cliforni, USA. 3 Progrm in Brin nd Cognitive Sienes, Msshusetts Institute of Tehnology, Cmridge, Msshusetts, USA. Deprtment of Neurology nd Wheeler Center for the Neuroiology of Addition, University of Cliforni, Sn Frniso, Cliforni, USA. Correspondene should e ddressed to A.B. (ntonello.oni@usf.edu) or P.H.J. (pjnk@gllo.usf.edu). Reeived 2 Septemer 29; epted Jnury 21; pulished online Mrh 21; doi:1.13/nn.2 nture NEUROSCIENCE VOLUME 13 NUMBER APRIL 21

2 21 Nture Ameri, In. All rights reserved. Rewrds erned per minute Corret inorret port entries Totl port entries.2.1 Rewrd erning Tsk ury Rewrds erned / ues presented due to derese in the mount of tsk-irrelevnt ehvior reltive to gol-direted ehvior, s well s n enhnement in the quisition of the ue-rewrd ssoition (Fig. 1 d). Distint effets of NET nd DAT inhiition trgets multiple phrmologil trgets, potently inhiiting oth the norepinephrine trnsporter (NET) nd the dopmine trnsporter (DAT). hs higher inding ffinity for the NET thn for the DAT 2, nd n inresingly presried lterntive tretment for ADHD, tomoxetine (Strtter), preferentilly trgets the NET. Thus, we investigted the effets of intr-la dministrtion efore trining of nisoxetine (NXT), highly seletive NET inhiitor, on lerning performne (Supplementry Fig. ). Consistent with evidene tht intr-bla enhnes memory onsolidtion 19, we found tht lthough NXT dose-dependently enhned memory retention, s mesured y the ehviorl index tsk ury on susequent test session in the sene of ny drug tretment (P =.2), there ws no ute effet on lerning performne reltive to tht of sline ontrols (Fig. 2). Tsk ury quntifies the rt s ility to reognize tht the sene of the ue indites the sene of surose nd is defined s the differene of the numer of port entries in the presene of surose nd the numer of port entries in the sene of surose, normlized to the totl port entry responses during the session. Aumulting evidene suggests tht inhiition of the DAT 2 ontriutes to its therpeuti effets 21. For exmple, sujets dignosed with ADHD show signifint inreses in DAT density 22 nd presene of ertin lleles of the DAT1 (lso known s SLCA3) gene orreltes with hypertivity nd impulsivity sores in sujets with ADHD 23. Furthermore, therpeuti doses of orlly dministrted in humns inhiit DAT funtion ( %) nd inrese d Intive / rewrd port entries Tsk effiieny Off-tsk ehvior Figure 2 Intr-LA NXT efore trining enhnes memory retention ut not ute tsk performne. Left olumn, performne on the initil trining session; right olumn, performne during 2-min memory retrievl test. No signifint differenes were oserved during trining, ut NXT dosedependently enhned tsk ury (F 3,2 =.29, P =.2) during test session on the next dy, on whih no infusions were performed, suggesting tht NET lokde my enhne memory retention in this tsk. vehile, N = ; NXT 2 µg per side, N = ; NXT µg per side, N = ; NXT µg per side, N =. P <.1. Figure 1 enhnes tsk performne y ltering different spets of ehvior through distint D1 nd D2 reeptor dependent mehnisms. () Intr-LA drug infusion lters rewrd erning (F, =.11, P <.1). Reltive to sline-treted rts, nd groups erned signifintly more rewrds per minute, wheres -treted rts erned signifintly fewer. -treted, ut not -treted, rts erned signifintly fewer thn -treted lone. () Tsk effiieny ws ltered y intr-la drug infusion (F, = 3., P =.). Reltive to sline,, nd -treted groups ll showed signifintly higher tsk effiieny. The group, ut not the, showed lower tsk effiieny thn the group treted with lone. () Reltive to sline, -treted rts showed signifintly lower tsk ury, nd -treted rts showed n ttenution of the enhnements indued y lone, ut -treted rts did not differ from those treted with lone (F, = 3., P =.19). (d) Reltive to the sline-treted group, nd -treted groups showed signifintly less off-tsk ehvior, wheres -treted rts showed signifintly more (F, =.2, P <.1). In d, numers in rs indite rts per group. All vlues re men ± s.e.m. One-wy nlysis of vrine followed y ll-pirwise multiple omprison proedure (Fisher lest signifint differene method; P <., P <.1). extrellulr dopmine 2,2. Beuse NET inhiition lone did not yield the sme enhnements of ute tsk performne s did, we hypothesized tht the effets of on lerning our y mens of DAT inhiition. If so, then intr-la infusions of the seletive DAT loker () 2 should mimi the effets of. In ontrst to NXT tretment, tretment resulted in ehviorl profile notly similr to tht of, with no differenes oserved etween Trining Rewrd erning Tsk effiieny Tsk ury. Off-tsk ehvior Test 2 VOLUME 13 NUMBER APRIL 21 nture NEUROSCIENCE

3 21 Nture Ameri, In. All rights reserved. Thlmi NMDA AMPA Cortil pa 1 ms AMPAR / NMDAR rtio Thlmi nd groups. -treted rts showed higher rewrd erning (P =.13) nd tsk effiieny (P =.2), s well s less off-tsk ehvior (P =.), thn sline-treted ontrols (Fig. 1,,d). Thus, inhiition of NET enhnes memory retention, wheres inhiition of DAT utely enhnes tsk performne. If inreses in extrellulr dopmine used these enhnements in ute tsk performne, then dopmine reeptor tivtion is likely to e required for ute -indued performne enhnement. Distint ontriutions of D1 nd D2 reeptors to performne To test this hypothesis, we performed intr-la infusions of the potent dopmine D1 reeptor (D1R) ntgonist (). treted rts showed signifintly lower rewrd erning (P =.33; Fig. 1) nd tsk ury (Fig. 1) thn did sline-treted rts. Impirments in rewrd erning nd tsk ury indite tht D1R tivtion is neessry for generl tsk performne nd ue-rewrd lerning. However, the ility to suppress tsk-irrelevnt ehvior, s mesured y off-tsk ehvior, ws spred (Fig. 1d). If enhnes tsk performne y inresing the tivtion of D1Rs, then infusion of together with () should ttenute -indued enhnements. Rts given intr-la infusions of efore trining showed lower rewrd erning (P =.13) nd tsk effiieny (P =.2) thn those treted with lone (Fig. 1,). However, there ws no hnge in off-tsk ehvior for ompred to the, or sline groups (Fig. 1d). Thus, lerning the motivtionl signifine of rewrd-preditive ue requires D1R tivtion. We next tested the role of D2 reeptors (D2Rs) in mediting lerning performne y infusing rlopride (), potent ntgonist of D2Rs, efore trining into the lterl mygdl. Wheres tretment did not hnge rewrd erning or tsk effiieny, there ws signifintly more off-tsk ehvior (P =.3) reltive to sline (Fig. 1d). This inrese in off-tsk ehvior ws due not to derese in rewrd port entries ut to >% inrese in intive port entries (Supplementry Figs. nd ). Thus, D1R funtion is ritil for ue-rewrd lerning, wheres D2R funtion is ritil for the suppression of tsk-irrelevnt ehvior. If exerts some ehviorl effets y inresing the tivtion of D2Rs, then infusion of together with () should ttenute suset of -indued ehviorl enhnements Cortil Home ge Home ge. Figure 3 Inhiition of the dopmine trnsporter gtes ortio-mygdl synpti potentition. () Representtive tres of AMPAR/NMDAR rtios evoked from thlmi nd ortil fferents for eh group. (,) AMPAR/NMDAR rtios evoked from thlmi (F,2 = 3.1, P =.3) or ortil (F,9 = 3., P =.2) fferents for eh drug-tretment group fter trining were signifintly ltered. Inset shows rts treted with sline, nd tht were returned to their home ges in lieu of trining. Numers in rs indite the numer of ells per group. () -treted rts show signifint derese in thlmomygdl AMPAR/NMDAR. () nd groups show signifint inreses in ortio-mygdl AMPAR/NMDAR. P <., P <.1. Rts treted with showed tsk effiieny tht ws higher (P =.23) thn tht of sline ontrols ut the sme s tht seen in rts treted with lone (Fig. 1). Therefore, ffets tsk effiieny independently of D2R tivtion. In ontrst to rts treted with lone, rts treted with did not show differene in off-tsk ehvior from tht seen in sline ontrols (Fig. 1d). Although these speifi ehviorl spets my e interrelted (Supplementry Figs. 1), these findings further support the hypothesis tht quisition of the ue-rewrd ssoition nd suppression of tsk-irrelevnt ehvior re medited y distint dopmine reeptor sutypes. filittes ortio-mygdl plstiity vi dopmine To test whether the -indued enhnement in lerning performne is relted to hnges in exittory synpti funtion, we performed whole-ell pth-lmp reordings within lterl mygdl slies fter intr-la infusions nd trining (Supplementry Fig. 2) in the sme sujets whose ehviorl dt re presented in Figure 1. We mesured the rtio of AMPA reeptors (AMPAR) to NMDA reeptors (NMDAR) y stimulting thlmi (internl psule) or ortil (externl psule) fferents to determine the effets of intr-la dministrtion of,, nd on lerning-indued glutmtergi synpti plstiity. Notly, rts tht reeived infusions of sline efore trining showed signifintly higher thlmomygdl AMPAR/NMDAR thn rts treted with sline in their home ge (not trined; P =.). Tretment with efore trining yielded signifintly lower thlmo-mygdl AMPAR/NMDAR reltive to sline tretment (P =.; Fig. 3,), in ddition to impiring ue-rewrd lerning (Fig. 1). Thus, D1R lokde my impir ue-rewrd lerning y ttenuting the lerning-indued inreses in thlmomygdl synpti strength 2. No other tretment signifintly ltered synpti strength in this pthwy (Fig. 3). In ontrst, rts treted efore trining with sline infusions did not show differene in ortio-mygdl synpti strength reltive to sline home-ge rts (Fig. 3), inditing tht plstiity t these synpses is not required for lerning this tsk. Only (P =.23) nd (P =.12) groups showed lerning-indued inreses in ortio-mygdl synpti strength reltive to sline ontrols (Fig. 3), suggesting tht DAT lokde hnges the inhiitory onstrints on ortio-mygdl plstiity. The enhnement in ortio-mygdl AMPAR/NMDAR y ws reversed y o-infusion with either D1R (; P =.) or D2R ntgonists (; P =.13; Fig. 3), inditing tht this hnge in synpti strength requires otivtion of these reeptor sutypes. We then onfirmed these synpti hnges were lerning indued rther thn result of ute drug exposure lone. Rts tht reeived or infusions in their home ges did not show ny inreses reltive to sline. In ontrst, rts infused with or in their home ges were signifintly lower in thlmo-mygdl (P =., P =.9, respetively) or ortio-mygdl synpti nture NEUROSCIENCE VOLUME 13 NUMBER APRIL 21

4 21 Nture Ameri, In. All rights reserved. 1 pa 1 ms strength (P =., P =.12, respetively) reltive to rts tht reeived or infusions efore trining (Fig. 3,). Beuse hnge in AMPAR/NMDAR rtio my reflet hnge in either AMPAR- or NMDAR-medited urrents, we exmined miniture exittory postsynpti urrents (mepscs), whih reflet spontneously relesed vesiles of glutmte 2. A hnge in the mplitude of mepscs typilly reflets hnge in the numer or funtion of postsynpti AMPARs, wheres hnge in the frequeny of mepscs my reflet hnge in the proility of relese t the presynpti terminl 2. We found tht intr-la (P =.12), (P <.1) or (P =.1) infusions inresed, while infusions deresed (P =.), mepsc mplitude reltive to sline infusions efore trining (Fig. ). mepsc mplitude ws not inresed y home-ge or tretment reltive to sline nd ws signifintly lower thn tht of rts tht reeived or efore trining (oth P <.1; Fig. ). Co-infusion of ttenuted (P <.1), wheres spred, -indued filittion of lerning-indued inreses in mepsc mplitude (Fig. ). We oserved no differenes in frequeny (Fig.,d,e), suggesting tht the hnge in AMPAR/NMDAR ws medited postsynptilly 2. A lk of differene mong groups for either ortio- or thlmo-mygdl fferents in pired-pulse rtio mesurements (Supplementry Fig. 11), whih reflet the proility of vesile relese 29, further supports the hypothesis tht inreses in oth thlmo- nd ortio-mygdl synpti strength re medited y postsynpti inreses in AMPAR urrents. To onfirm tht D1R ntgonism, rther thn the ssoited impirment in lerning performne, ws the use of the ttenution in mepsc mplitude, we performed unilterl infusions of efore trining to provide within-sujet ontrol (Fig. ). pa d Hz mepsc mplitude 1 1 mepsc frequeny Home ge Home ge 9 Cumultive proility Cumultive proility e To proe the reltionship etween these distint spets of ehvior nd the ssoited synpti hnges, we exmined orreltions mong AMPAR/NMDAR, mepscs nd tsk performne. Both ortio-mygdl nd thlmo-mygdl AMPAR/NMDAR rtios were signifintly orrelted with rewrd erning nd tsk effiieny (Supplementry Figs. 12 nd 13). In ontrst, only the ortiomygdl AMPAR/NMDAR ws inversely orrelted with off-tsk ehvior (Supplementry Fig. 1), suggesting tht ortio-mygdl synpses my seletively modulte the ility to suppress tsk-irrelevnt ehvior, onsistent with evidene linking ADHD to norml ortiomygdl onnetivity 13. Studies in drug-nive rts show orreltions etween lerning nd AMPAR/NMDAR rtios in thlmo-mygdl, ut not ortio-mygdl synpses, result we replited in slinetreted rts (Supplementry Fig. 1). Here the nd groups were the only groups to show n inrese in ortil AMPAR/ NMDAR, nd they hevily ontriuted to the orreltion etween ortil AMPAR/NMDAR nd tsk effiieny. Additionlly, mepsc mplitude, ut not frequeny, ws orrelted with rewrd erning, tsk effiieny nd off-tsk ehvior (Supplementry Figs. 1 1), suggesting tht postsynpti AMPAR numer or funtion predits suess in ue-rewrd lerning tsk. -indued enhnements require lterl mygdl dopmine To test whether dopmine signling in the mygdl is required for mediting -indued ehviorl enhnements, we systemilly dministered, using intrperitonel (i.p.) injetion, sline or low dose of long with intr-la sline, or efore trining (Fig. nd Supplementry Fig. 19). Systemi signifintly inresed rewrd erning nd tsk effiieny, while reduing off-tsk ehvior pa 2 1, Inter-event intervl (ms) Figure Dopmine modultes lerning-indued inreses in mepsc mplitude ut not frequeny. () Smple mepscs from eh drug-tretment group. () Men mepsc mplitude for eh group vried (F,113 = 1.1, P <.1) with tretment., nd groups hd higher, wheres -treted rts hd lower, mepsc mplitude thn did sline ontrols. Inset: sline-, - nd -treted home-ge ontrols. () Cumultive proility plot of mepsc mplitude for representtive ells from eh group; 1 pa ins. (d) No signifint hnge in mepsc frequeny of ny groups reltive to sline (F,113 =.22, P =.99). (e) Cumultive proility plot of mepsc frequeny; 2-ms ins. P <., P <.1, P <.1. VOLUME 13 NUMBER APRIL 21 nture NEUROSCIENCE

5 21 Nture Ameri, In. All rights reserved. e g sline sline Rewrd erning Unilterl sline/ Tsk ury Unilterl sline/ mepsc mplitude (etween sujets) (P =.3,. nd., respetively; Fig.,,d), reltive to systemi sline. Intr-LA ttenuted systemi indued enhnements in ll ehviorl mesures tested, nd these rts were impired reltive to rts treted with systemi sline in rewrd erning, tsk effiieny nd tsk ury (Fig. ; P =.13,.1 nd.2, respetively). In ontrst, intr-la seletively ttenuted -indued redution of off-tsk ehvior (Fig. d; P =.1). Thus, D1R tivtion in the mygdl is neessry for mediting -indued enhnements in ue-rewrd lerning performne, nd D2R tivtion in the mygdl is required for mediting -indued redutions in off-tsk ehvior. Therefore, dopmine signling in the mygdl is ritil in -medited lerning performne enhnement. DISCUSSION Using omintion of in vivo phrmology nd ex vivo eletrophysiology, we show tht enhnes lterl mygdl dependent d f sline sline Tsk effiieny Unilterl sline/ Off-tsk ehvior Unilterl sline/ mepsc frequeny (etween sujets) h mepsc mplitude mepsc frequeny Unilterl (within sujets) Unilterl (within sujets) Figure Dopmine signling in the mygdl is neessry for mediting enhnements of lerning performne indued y systemi dministrtion of. Behviorl mesures of four groups of rts treted efore trining with (1) i.p. sline nd intr-la sline (N = rts), (2) i.p. nd intr-la sline (N = rts), (3) i.p. nd intr-la (N = rts) nd () i.p. nd intr-la (N = 9 rts). ( ) Intr-LA infusion of signifintly ttenuted, wheres spred, enhnements indued y systemi in rewrd erning (F 3,31 =., P <.1), tsk effiieny (F 3,31 = 2.19, P <.1) nd tsk ury (F 3,31 =., P =.3). (d) Intr-LA infusion of or signifintly ttenuted redutions indued y systemi in off-tsk ehvior (F 3,31 =., P =.11). In d, P <., P <.1,P <.1. Figure D1R ntgonism in the lterl mygdl ttenutes lerningindued synpti hnges. ( h) Rts given, efore trining, ilterl infusions of ompred with rts given unilterl infusions of nd ontrlterl infusions of sline. () Unilterl infusion of nd sline signifintly deresed rewrd erning (F 2,19 =.1, P =.1) reltive to ilterl sline infusion (P =.9) nd did not differ from ilterl. ( d) Rts treted with unilterl nd sline infusions did not show signifint differenes from rts treted with ilterl sline in tsk effiieny, tsk ury or off-tsk ehvior. (f,h) Unilterl infusions of nd sline provide within-sujets (N = rts; n = 11 ells from sline-treted side; n = 9 ells from -treted side) omprison of the effets of D1 reeptor ntgonism on lerning-indued plstiity. (e h) For oth etween-sujets nd within-sujets omprisons, tretment with signifintly ttenuted lerning-indued inreses in mepsc mplitude (P <., P <.1, P <.1, Student s t-test) reltive to sline (e,f), with no hnge in mepsc frequeny (g,h). form of ue-rewrd lerning nd provide evidene suggesting tht the lerning enhnement depends upon dopminergi modultion of exittory synpti plstiity within the lterl mygdl. These results extend previous findings tht demonstrted n importnt role for dopmine within the lterl mygdl in the formtion of oth ppetitive 3 3 nd versive 3 ssoitions y identifying potentil mehnism wherey inreses in dopmine in the mygdl modulte exittory synpti plstiity. Speifilly, we found tht, nd the dopmine uptke loker, enhned the AMPAR/ NMDAR t ortio-mygdl synpses. We lso found tht different dopmine reeptor sutypes ontriute to distint spets of lerning performne, suh tht ue-rewrd lerning depends upon dopmine D1 reeptor dependent mehnisms, nd the suppression of tskirrelevnt ehvior depends upon D2 reeptor dependent mehnisms. Together, these findings indite speifi synpti mehnism wherey my enhne ssoitive lerning through tions in the lterl mygdl. How does DAT lokde in the mygdl enhne lerning performne? At sl dopmine levels, GABAergi tivity keeps the firing rtes of pyrmidl neurons low, while D2Rs on pyrmidl neurons influene the responsiveness of the ell y modulting input resistne 1. When dopmine levels re elevted, spontneous kground inhiition from lol interneurons inreses 2,3, while feed-forwrd inhiition medited y interlted ell msses dereses owing to D1R tivtion 2,3. Thus, lterl mygdl pyrmidl neurons will eome less responsive to weker, kground inputs ut I.p. Intr-LA I.p. Intr-LA Rewrd erning Tsk ury. I.p. Intr-LA d I.p. Intr-LA Tsk effiieny Off-tsk ehvior nture NEUROSCIENCE VOLUME 13 NUMBER APRIL 21 9

6 21 Nture Ameri, In. All rights reserved. muh more responsive to stronger, oordinted exittory inputs rrying sensory informtion 39,. We hypothesize tht y elevting extrellulr dopmine 2, or relese inhiitory onstrints on ortio-mygdl plstiity nd lter the responsiveness of mygdl neurons driving different spets of ehvior through mehnisms dependent on distint D1 nd D2 reeptors. An extension of this hypothesis is tht wheres elevted dopmine is required for ortio-mygdl potentition, only sl levels of dopmine re required for thlmo-mygdl potentition. Thus, t sl nd elevted levels of dopmine, thlmo-mygdl synpses re redily potentited with lerning, ut when dopmine reeptors re ntgonized, ringing the level of dopminergi signling elow the sl level, potentition in these synpses my e ttenuted. Therefore, we suggest tht inresed tivtion of dopmine reeptors enhnes the ility to quire ue-rewrd ssoitions nd to suppress tsk-irrelevnt ehvior. Speifilly, tretment with or enhnes tsk effiieny, likely owing to the dopmineindued inrese in responsiveness to oordinted sensory inputs, nd dereses off-tsk ehvior, likely owing to the dopmine-indued derese in pyrmidl neuron responsiveness to weker kground inputs. In ontrst, D1R ntgonism y ttenutes ue-rewrd lerning nd the ssoited plstiity, whih my reflet inreses in the inhiition of lterl mygdl neurons y interlted GABA neurons, whih densely express D1Rs 2,3. In omprison, D2R inhiition seletively inreses tsk-irrelevnt ehvior, in greement with the notion tht D2R ntgonism filittes the ility of wek exittion from tsk-irrelevnt stimuli to drive neuronl exittion. In onlusion, our findings suggest tht enhnes lerning performne through dopmine-dependent mehnism y gting ortio-mygdl potentition, filitting ue-rewrd lerning through D1R-dependent mehnism nd enhning the ility to suppress tsk-irrelevnt ehvior through D2R-dependent mehnism. Although NET inhiition did not utely filitte performne of our ue-rewrd lerning tsk within the first exposure, the improvement in memory retention would likely ontriute to ehviorl enhnements oserved ross sessions. Furthermore, the NET my lso t to ler dopmine from the extrellulr spe in rin regions with low DAT expression. Although DAT inhiition enhnes tsk performne during the initil trining session, it is still unler whether DAT inhiition enhnes tsk quisition, onsolidtion, expression or omintion of these proesses. Future studies my determine whether these mehnisms of tion generlize to other rin regions involved in lerning nd ttention or to other ehviorl ssys of ognitive performne. Methods Methods nd ny ssoited referenes re ville in the online version of the pper t Note: Supplementry informtion is ville on the Nture Neurosiene wesite. Aknowledgments We thnk H.L. Fields, G.D. Stuer, J.A. Rosenkrnz nd E.E. Steinerg for helpful omments; nd A.C. Hollowell, S.L. Cho, S.J. Chng, L. Wng nd F.W. Hopf for tehnil ssistne. This reserh ws supported y the Stte of Cliforni for Medil Reserh on Alohol nd Sustne Ause through the University of Cliforni t Sn Frniso (A.B. nd P.H.J.), NIDA DA19-1 (A.B.) nd Msshusetts Institute of Tehnology Peter J. Elornt Summer Undergrdute Reserh Fellowship (L.D.T.). AUTHOR CONTRIBUTIONS K.M.T. supervised experiments nd performed ll whole-ell reordings. K.M.T., A.B. nd P.H.J. ontriuted to study design, results nlysis, interprettion nd mnusript writing. K.M.T., L.D.T., J.J.C. nd E.F.H. surgilly implnted guide nnule, performed intr-la drug infusions, onduted ehviorl experiments, setioned ute slie preprtions nd performed dt entry nd nlyses. A.B. nd P.H.J. provided mentorship nd resoures. COMPETING FINANCIAL INTERESTS The uthors delre no ompeting finnil interests. Pulished online t Reprints nd permissions informtion is ville online t reprintsndpermissions/. 1. Swnson, J.M., Lerner, M. & Willims, L. More frequent dignosis of ttention defiit-hypertivity disorder. N. Engl. J. Med. 333, 9 (199). 2. Amn, M.G., Vmos, M. & Werry, J.S. Effets of methylphenidte in norml dults with referene to drug tion in hypertivity. Aust. N. Z. J. Psyhitry 1, (19). 3. Amerin Psyhitri Assoition. ed. Dignosti nd Sttistil Mnul of Mentl Disorders (Amerin Psyhitri Pulishing, Arlington, Virgini, USA, 199).. Rodriguez, A. et l. Do inttention nd hypertivity symptoms equl sholsti impirment? Evidene from three Europen ohorts. BMC Puli Helth, 32 (2).. Greenhill, L.L. et l. Prtie prmeter for the use of stimulnt meditions in the tretment of hildren, dolesents, nd dults. J. Am. Ad. Child Adoles. Psyhitry 1, 2S 9S (22).. Yng, P., Chung, L.C., Chen, C.S. & Chen, C.C. Rpid improvement in demi grdes following methylphenidte tretment in ttention-defiit hypertivity disorder. Psyhitry Clin. Neurosi., 3 1 (2).. LeDoux, J. The emotionl rin, fer, nd the mygdl. Cell. Mol. Neuroiol. 23, 2 3 (23).. Tye, K.M., Stuer, G.D., de Ridder, B., Boni, A. & Jnk, P.H. Rpid strengthening of thlmo-mygdl synpses medites ue-rewrd lerning. Nture 3, (2). 9. Crdinl, R.N., Prkinson, J.A., Hll, J. & Everitt, B.J. Emotion nd motivtion: the role of the mygdl, ventrl stritum, nd prefrontl ortex. Neurosi. Bioehv. Rev. 2, (22). 1. Cdor, M., Roins, T.W. & Everitt, B.J. Involvement of the mygdl in stimulusrewrd ssoitions: intertion with the ventrl stritum. Neurosiene 3, (199). 11. Dvis, M. The role of the mygdl in emotionl lerning. Int. Rev. Neuroiol. 3, 22 2 (199). 12. Shoenum, G., Chi, A.A. & Gllgher, M. Oritofrontl ortex nd solterl mygdl enode expeted outomes during lerning. Nt. Neurosi. 1, 1 19 (199). 13. Plessen, K.J. et l. Hippompus nd mygdl morphology in ttention-defiit/ hypertivity disorder. Arh. Gen. Psyhitry 3, 9 (2). 1. Doron, N.N. & Ledoux, J.E. Orgniztion of projetions to the lterl mygdl from uditory nd visul res of the thlmus in the rt. J. Comp. Neurol. 12, 33 9 (1999). 1. Nkshim, M. et l. An nterogrde nd retrogrde trt-tring study on the projetions from the thlmi gusttory re in the rt: distriution of neurons projeting to the insulr ortex nd mygdloid omplex. Neurosi. Res. 3, (2). 1. Reijmers, L.G., Perkins, B.L., Mtsuo, N. & Myford, M. Loliztion of stle neurl orrelte of ssoitive memory. Siene 31, (2). 1. Hn, J.H. et l. Seletive ersure of fer memory. Siene 323, (29). 1. Tye, K.M. & Jnk, P.H. Amygdl neurons differentilly enode motivtion nd reinforement. J. Neurosi. 2, (2). 19. Zheng, X., Liu, F., Wu, X. & Li, B. Infusion of methylphenidte into the solterl nuleus of mygdl or nterior ingulte ortex enhnes fer memory onsolidtion in rts. Si. Chin C Life Si. 1, 13 (2). 2. Mrkowitz, J.S., DeVne, C.L., Pestreih, L.K., Ptrik, K.S. & Muniz, R. A omprehensive in vitro sreening of d-, l-, nd dl-threo-methylphenidte: n explortory study. J. Child Adoles. Psyhophrmol. 1, 9 (2). 21. Solnto, M.V. Neuropsyhophrmologil mehnisms of stimulnt drug tion in ttention-defiit hypertivity disorder: review nd integrtion. Behv. Brin Res. 9, (199). 22. Dougherty, D.D. et l. Dopmine trnsporter density in ptients with ttention defiit hypertivity disorder. Lnet 3, (1999). 23. Wldmn, I.D. et l. Assoition nd linkge of the dopmine trnsporter gene nd ttention-defiit hypertivity disorder in hildren: heterogeneity owing to dignosti sutype nd severity. Am. J. Hum. Genet. 3, 1 1 (199). 2. Volkow, N.D. et l. Therpeuti doses of orl methylphenidte signifintly inrese extrellulr dopmine in the humn rin. J. Neurosi. 21, RC121 (21). 2. Volkow, N.D. et l. Dopmine trnsporter oupnies in the humn rin indued y therpeuti doses of orl methylphenidte. Am. J. Psyhitry 1, (199). 2. Andersen, P.H. The dopmine inhiitor 1299: seletivity nd moleulr mehnism of tion. Eur. J. Phrmol. 1, 93 (199). 2. Bissiere, S., Humeu, Y. & Luthi, A. Dopmine gtes LTP indution in lterl mygdl y suppressing feedforwrd inhiition. Nt. Neurosi., 92 (23). VOLUME 13 NUMBER APRIL 21 nture NEUROSCIENCE

7 2. Mlenk, R.C. & Nioll, R.A. Long-term potentition dede of progress? Siene 2, 1 1 (1999). 29. Hess, G., Kuhnt, U. & Voronin, L.L. Quntl nlysis of pired-pulse filittion in guine pig hippompl slies. Neurosi. Lett., (19). 3. Hithott, P.K., Hrmer, C.J. & Phillips, G.D. Enhned quisition of disrimintive pproh following intr-mygdl d-mphetmine. Psyhophrmology (Berl.) 132, 23 2 (199). 31. Bernl, S. et l. Role of mygdl dopmine D1 nd D2 reeptors in the quisition nd expression of frutose-onditioned flvor preferenes in rts. Behv. Brin Res. 2, (29). 32. Hithott, P.K., Bonrdi, C.M. & Phillips, G.D. Enhned stimulus-rewrd lerning y intr-mygdl dministrtion of D3 dopmine reeptor gonist. Psyhophrmology (Berl.) 133, 2 2 (199). 33. Touzni, K., Bodnr, R.J. & Slfni, A. Dopmine D1-like reeptor ntgonism in mygdl impirs the quisition of gluose-onditioned flvor preferene in rts. Eur. J. Neurosi. 3, (29). 3. Andrzejewski, M.E., Spener, R.C. & Kelley, A.E. Instrumentl lerning, ut not performne, requires dopmine D1-reeptor tivtion in the mygdl. Neurosiene 13, 33 3 (2). 3. Lmont, E.W. & Kokkinidis, L. Infusion of the dopmine D1 reeptor ntgonist 2339 into the mygdl loks fer expression in potentited strtle prdigm. Brin Res. 9, (199). 3. Gurri, F.A., Frohrdt, R.J., Young, S.L. & Kpp, B.S. A funtionl role for dopmine trnsmission in the mygdl during onditioned fer. Ann. NY Ad. Si., 32 3 (1999). 3. Gurri, F.A., Frohrdt, R.J., Flls, W.A. & Kpp, B.S. The effets of intrmygdloid infusions of D2 dopmine reeptor ntgonist on Pvlovin fer onditioning. Behv. Neurosi. 11, 1 (2). 3. Gre, Q., Gifkins, A. & Kokkinidis, L. Inhiition of mygdloid dopmine D2 reeptors impirs emotionl lerning mesured with fer-potentited strtle. Brin Res. 99, (21). 39. Rosenkrnz, J.A. & Gre, A.A. Dopmine-medited modultion of odour-evoked mygdl potentils during pvlovin onditioning. Nture 1, 22 2 (22).. Kienst, T. et l. Dopmine in mygdl gtes limi proessing of versive stimuli in humns. Nt. Neurosi. 11, (2). 1. Kroner, S., Rosenkrnz, J.A., Gre, A.A. & Brrionuevo, G. Dopmine modultes exitility of solterl mygdl neurons in vitro. J. Neurophysiol. 93, (2). 2. Loretn, K., Bissiere, S. & Luthi, A. Dopminergi modultion of spontneous inhiitory network tivity in the lterl mygdl. Neurophrmology, (2). 3. Mrowsky, A., Yngw, Y., Ot, K. & Vogt, K.E. A speilized sulss of interneurons medites dopminergi filittion of mygdl funtion. Neuron, (2).. Rosenkrnz, J.A. & Gre, A.A. Dopmine ttenutes prefrontl ortil suppression of sensory inputs to the solterl mygdl of rts. J. Neurosi. 21, 9 13 (21).. Moron, J.A., Brokington, A., Wise, R.A., Roh, B.A. & Hope, B.T. Dopmine uptke through the norepinephrine trnsporter in rin regions with low levels of the dopmine trnsporter: evidene from knok-out mouse lines. J. Neurosi. 22, (22). 21 Nture Ameri, In. All rights reserved. nture NEUROSCIENCE VOLUME 13 NUMBER APRIL 21 1

8 21 Nture Ameri, In. All rights reserved. ONLINE METHODS Experimentl sujets. Adult mle Sprgue-Dwley rts (29 3 g) were food restrited to 9% of free-feeding ody weight nd mintined on 12 h:12 h light: drk yle. Eh rt ws only used for single drug tretment, nd ll rts used for ex vivo experimenttion were only trined on single session. In ll ses, nose-poke responses were reinfored on pseudo-rndom-rtio 2 shedule with -s ompound light-tone stimulus, nd.1 ml of 1% surose solution delivered 1 s fter ue onset. All rts, inluding home ge treted rts, reeived 2 ml of surose solution efore deth. After intr-la infusions, home-ge rts were returned to their home ges for the sme durtion s the trining session efore ute slie preprtion. For ll ex vivo experiments, ehviorl performne ws nlyzed fter whole-ell reordings were ompleted. All experimentl proedures were onduted in ordne with the Guide for the Cre nd Use of Lortory Animls, s dopted y the US Ntionl Institutes of Helth nd with pprovl of Gllo Institutionl Animl Cre nd Use Committee. Intr lterl mygdl infusions. Adult mle Sprgue-Dwley rts (29 3 g) were surgilly implnted with guide nnule imed just ove the ventrolterl mygdl (nteroposterior, 2. to 3.3; mediolterl, ±.9; dorsoventrl,.3 mm, reltive to regm). One week fter surgery, rts were food restrited to pproximtely 9% of their free-feeding weight. Ten to 1 minutes efore the trining session, restrited rts were ilterlly infused with. µl of drug or vehile (sline) t rte of.1 µl min 1 to minimize tissue dmge. Infusion needles extended pproximtely 1. mm eyond the tip of the guide nnule to minimize dmge t the trget sites. On the dy efore trining, we piered the tissues of ll rts to lessen the response to tissue piering on the dy of trining. Drug dosges were seleted to ensure tht side effets (suh s hnges in loomotor tivity) did not onfound our study of tsk quisition. For methylphenidte infusions (Sigm), we used µg in. µl per side, dose tht hs een shown to improve fer memory onsolidtion when infused in the BLA in rts 19. For dihydrohloride (Toris), we used dose of 3.1 µg in. µl per side, moderte dose within rnges tht hve een ehviorlly tested with intrrnil injetion in rts 2,,. For hydrohloride infusions (Toris), we used dose of ng in. µl per side, t onentrtion of.11 mm. Infusions of t even higher onentrtions of 1.22 nd 3. mm (1, or 2, ng in.2 µl per side) in the BLA of rts tested in n open-field test does not produe hnges in gross loomotor tivity. For rlopride (Toris), we used dose of µg in. µl per side, moderte dose tht flls within the rnge of doses ehviorlly tested with intr-la or intr-bla infusions in rts 3,9,. This hs een found to e the lowest effetive dosge for effets on fer quisition, nd even higher dosge of µg per side does not hnge movement levels during shok dministrtion 33. For, we infused the sme onentrtions of eh drug in the sme volume: µg nd ng in. µl of sline per side. For, we infused µg nd µg in. µl sline per side. Guide nnule plements were visulized with n upright mirosope using infrred illumintion (Supplementry Fig. 1). Behviorl proedures. Before trining session, ll rts were wter deprived for ~12 h. All rts were trined on the sme ehviorl proedure in sessions lsting pproximtely h, with the sme ue nd 1% surose solution. In our ue-rewrd lerning prdigm, rts were enourged to nose-poke t the nose-poke operndum with pltle odor ue. Cues nd surose were presented ontingently fter nose-poke response on prtil reinforement shedule to ensure tht the rt ssoited the ue nd the surose rewrd rther thn the opernt response nd the surose rewrd. Speifilly, fter ~% of nose-poke responses, ue would e presented immeditely ( ms fter em rek t nose-poke operndum) nd surose would e delivered 1 s fter nose-poke. The durtion of the ompound light-nd-tone ue presenttion ws s, nd it ompletely overlpped with the surose delivery, whih ourred over 3 s. The ue lwys predited surose delivery, nd surose ws never delivered in the sene of the ue. Furthermore, if surose ws delivered ut the rt did not onsume it, ll susequent nose-pokes were pired with the ue to mintin the ue-rewrd ontingeny. Tsk effiieny nd tsk ury mesure distint spets of the quisition of the ue-rewrd ssoition. Tsk ury is defined s the totl numer of orret port entries minus inorret port entries, normlized to the totl numer of rewrd port entries. A orret port entry ws defined s nose-poke response yielding ue presenttion nd susequent port entry (within 1 s or efore performing different ehviorl event (nose-poke, port entry or intive port entry)). Inorret port entries were defined s entering the port fter nose-poke without the ue. Finlly, for ll rts, ny unerned surose ws delivered in dish in the home ge during the interim efore ute slie preprtion to ensure tht the volume of surose onsumed did not onfound ny lerning-indued hnges in plstiity. For home-ge nd groups, infusions were performed nd the sme volume of surose ws delivered in the home ge, where they remined for the durtion of the session. Eh rt ws only used for single tretment, nd ll rts were trined for single session only. After the trining session, rts were depitted, guide nnule hed ps were removed nd rins were prepred for ute slies for whole-ell reordings. Ex vivo eletrophysiology. Adult mle Sprgue-Dwley rts (29 3 g) were put to deth ~3 min fter session end. Rts were nesthetized with mg kg 1 pentoritl (i.p.) nd trnsrdilly perfused with ~3 ml of ie-old modified rtifiil ererospinl fluid (ACSF) t rte of ~2 ml min 1. The modified ACSF for perfusion ontined (in mm) 22 surose, 119 NCl, 2. KCl, 1. NH 2 PO,.9 MgCl 2,.1 CCl 2, 2.2 NHCO 3, 1.2 gluose; 3 kynureni id. After perfusion, the rin ws quikly removed nd pled into ie-old ACSF for 1 2 min. Coronl setions (32 µm) ontining the lterl mygdl were prepred with virtome (Lei). Slies were pled in holding hmer (ontining ACSF with 1 mm sori id) nd llowed to reover for t lest 1 h efore eing pled in the reording hmer nd superfused with ironte-uffered solution sturted with 9% O 2 nd % CO 2 nd ontining (in mm) 119 NCl, 2. KCl, 1. NH 2 PO, 1.3 MgCl 2, 2. CCl 2, 2.2 NHCO 3,.1 pirotoxin nd 11 gluose t 32 3 C. Exittory postsynpti urrents (EPSCs) were filtered t 2 khz nd stored using IgorPro softwre (Wvemetris). AMPAR/NMDAR rtio ws lulted y verging 2 3 EPSCs t mv efore nd fter pplition of the NMDAR loker d-( )-2-mino--phosphonopentnoi id (AP-) ( µm) for min. NMDAR responses were lulted y sutrting the verge response in the presene of AP- (AMPAR only) from tht seen in its sene. mepsc tres were filtered t 1 khz, olleted using Clmpex (Axon Instruments) nd nlyzed using Mini Anlysis Progrm (Synptosoft). AMPAR mepscs were reorded in ells voltge-lmped t mv nd in the ontinul presene of lidoine ( µm) nd AP- ( µm). The detetion riterion ws set t > pa. All vlues re expressed s men ± s.e.m. In ll experiments, n individul rt s ehviorl performne ws not nlyzed until fter whole-ell reordings were ompleted. Nisoxetine study. For the nisoxetine hydrohloride (Toris) infusions, we used doses of 2, nd µg per side in. µl per side. We then trined these rts in the sme mnner s ove, exept tht fter trining, these rts were returned to their home ges overnight nd then tested for memory retention of the tsk on n revited version of the sme prdigm (2 min) in the sene of drug tretment. Systemi dministrtion. We dministered sline or (.2 mg kg 1 ) dissolved in sline y i.p. injetion through 2-guge needle immeditely fter intr-la infusions of sline, or (sme onentrtions nd volumes s ove). Dt nlysis. All vlues were expressed s men ± s.e.m. Sttistil signifine for multigroup dt ws ssessed using one-wy nlysis of vrine followed y Fisher lest signifint differene method post ho when pplile, unless stted otherwise. Sttistil signifine for two-group dt ws ssessed using two-tiled Student s t-tests, exept where stted otherwise. In the se of orreltions, Person s orreltion test ws used to determine the orreltion oeffiient. For ll orreltions, unless otherwise indited, sttistis were performed on the rw individul dt.. Cornish, J.L. & Klivs, P.W. Repeted oine dministrtion into the rt ventrl tegmentl re produes ehviorl sensitiztion to systemi oine hllenge. Behv. Brin Res. 12, 2 29 (21).. Steketee, J.D. Repeted injetion of 1299, ut not oine or WIN 3,-2, into the ventrl tegmentl re indues ehviorl sensitiztion. Behv. Brin Res. 9, 39 (199).. Medo, C.E., Mrtinez, R.C., Alrehet-Souz, L., Molin, V.A. & Brndo, M.L. -HT2- nd D1-mehnisms of the solterl nuleus of the mygdl enhne onditioned fer nd impir unonditioned fer. Behv. Brin Res. 1, 1 1 (2). 9. Berglind, W.J., Cse, J.M., Prker, M.P., Fuhs, R.A. & See, R.E. Dopmine D1 or D2 reeptor ntgonism within the solterl mygdl differentilly lters the quisition of oine-ue ssoitions neessry for ue-indued reinsttement of oine-seeking. Neurosiene 13, 99 (2).. See, R.E., Kruzih, P.J. & Grimm, J.W. Dopmine, ut not glutmte, reeptor lokde in the solterl mygdl ttenutes onditioned rewrd in rt model of relpse to oine-seeking ehvior. Psyhophrmology (Berl.) 1, (21). nture NEUROSCIENCE doi:1.13/nn.2