B 1995 Stockton Press All rights reserved /95 $12.00 S. cells (Kelland et al., 1990). These findings indicate that 5-HT

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1 Bridsh Jouml of Phmology (I995) 116, B 1995 Stokton Press All rights reserved /95 $12. S Differentil effets of ute nd hroni fluoxetine dministrtion on the spontneous tivity of dopminergi neurones in the ventrl tegmentl re Simon Priso & 'Ennio Esposito Istituto di Rierhe Frmologihe 'Mrio Negri', Consorzio 'Mrio Negri' Sud, 663 Snt Mri Imro (Chieti), Itly 1 Eletrophysiologil tehniques were used to study the effets of fluoxetine nd itloprm on the sl tivity of dopminergi neurones in the ventrl tegmentl re (VTA) nd sustnti nigr, prs ompt (SN) of rts. 2 Aute i.v. injetion of fluoxetine (2-128,ug kg-') used dose-dependent inhiition of the firing rte of VTA dopminergi neurones, ut did not ffet the tivity of dopminergi ells in the SN. Citloprm (2-128 pg kg-', i.v.) inhiited the firing rte of dopminergi neurones in the VTA, ut its effet (mximl inhiition: 14 ± 7%) ws less pronouned thn tht of fluoxetine (mximl inhiition: 34±7%). 3 Pretretment with mesulergine (8 pg kg-', i.v.), 5-hydroxytryptmine2C/2B (5-HT2C,2B) reeptor ntgonist, loked the inhiitory effet of fluoxetine on VTA dopminergi ells. Seletive lesions of 5- hydroxytryptminergi neurones y the neurotoxin, 5,7-dihydroxytryptmine (5,7-DHT), olished the fluoxetine-indued redution of VTA dopminergi tivity. 4 In series of experiments, fluoxetine (1 mg kg-', i.p.) ws dministered one dily for 21 onseutive dys. Aute i.v. dministrtion of fluoxetine (2-128 Mg kg-', 72 h fter the lst i.p. injetion) did not use ny hnge in the sl firing rte of VTA dopminergi neurones in treted rts, wheres it indued the typil inhiitory effet in ontrol nimls. A group of rts hronilly treted with fluoxetine, reeived i.v. m-hlorophenylpiperzine (mcpp; 1-32 pg kg-'), 5-HT2C,2B reeptor gonist. This drug signifintly inhiited VTA dopminergi funtion in ontrol rts, ut did not modify the sl tivity of dopminergi ells in nimls given hroni fluoxetine. 5 It is onluded tht fluoxetine inhiits dopminergi funtion in the VTA y enhning the synpti levels of 5-HT, whih possily ts through the 5-HT2C/2B reeptor sutype. Repeted tretment with fluoxetine indues tolerne to its inhiitory effet on dopminergi tivity, possily s onsequene of down-regultion of 5-HT2C/2B reeptors. The effets of fluoxetine on VTA dopminergi ell tivity might e relevnt for its therpeuti tions nd my explin the origin of the reported ses of kthisi. Keywords: Fluoxetine; itloprm; dopminergi neurones; ventrl tegmentl re; eletrophysiology; 5-HT2C/2B reeptors. Introdution There is extensive evidene tht the tivity of midrin dopmine ontining neurones is modulted y the 5-hydroxytryptminergi system. Neurontomil studies indite tht oth the sustnti nigr, prs ompt (SN) nd the ventrl tegmentl re (VTA) reeive fferent projetions from 5-hydroxytryptmine (5-HT)-ontining xon terminls originting in the midrin rphe nulei (Azmiti & Segl, 1978; Phillipson, 1979; Steinush, 1984; Mori et l., 1987). Eletrophysiologil experiments hve shown tht seletive gonists t speifi 5-HT reeptor sutypes exert differentil effets on the sl firing tivity of midrin dopminergi neurones. For exmple, 8-hydroxy-2-(di-n-propylmino)tetrlin (8-OH- DPAT), prototypil 5-HTIA reeptor gonist, whih potently inhiits 5-HT neurones in oth dorsl nd medin rphe nulei (Sinton & Fllon, 1988; Priso et l., 1993), inresed the sl firing rte of dopminergi ells in oth the SN (Kellnd et l., 199) nd the VTA (Priso et l., 1994). Seletive lesions of 5-HT neurones y the neurotoxin, 5,7-dihydroxytryptmine (5,7-DHT), olished the exittory effets of 8-OH-DPAT on oth dopminergi nulei (Kellnd et l., 199; Priso et l., 1994). Mixed 5-HT2C/2B reeptor gonists, suh s trifluoromethylphenylpiperzine (TFMPP) nd m- hlorophenylpiperzine (mcpp) (Hoyer, 1988), signifintly redued the tivity of dopminergi neurones in the VTA (Priso et l., 1994) nd wekly inhiited SN dopminergi Author for orrespondene. ells (Kellnd et l., 199). These findings indite tht 5-HT exerts toni inhiitory influene upon the tivity of midrin dopminergi neurones. Thus, it is oneivle tht the dministrtion of seletive 5-HT reuptke inhiitors (SSRIs), whih enhne 5-HT levels in the synpti left, my ffet dopminergi funtion. Fluoxetine ws the first ompound elonging to the phrmologil lss of SSRIs whih ws found to inhiit potently 5-HT reuptke oth in vitro nd in vivo (Wong et l., 1974; Strk et l., 1985). It is now well estlished tht fluoxetine is n effetive ntidepressnt gent (Grm, 1994) whih lso hs good therpeuti tivity in the tretment of osessive-ompulsive disorders (Fontine & Chouinrd, 1986; Levine et l., 1989) nd eting disorders (Freemn & Hmpson, 1987; Kye et l., 1991). There is evidene tht the ntidepressnt effet of fluoxetine, ssessed in the fored swimming test in mie, is ntgonized y (±)-sulpiride dopmine D2 reeptor loker (Cesn et l., 1993). These dt re onsistent with the generl hypothesis tht n enhnement of dopminergi trnsmission in the mesolimi system is involved in the ntidepressnt effet of severl drugs (Cervo & Smnin, 1987; 1988; Cervo et l., 199). Therefore, eluidtion of the mehnisms y whih fluoxetine might influene midrin dopminergi funtion will proly e helpful in understnding the phrmodynmi sis of oth its therpeuti tions nd untowrd effets. For exmple, dministrtion of fluoxetine to mn is ssoited with severl side-effets inluding prkinsonism (Bouhrd et l., 1989; Brod, 1989; Tte, 1989) nd kthisi (Lipinski et l., 1989),

2 1924 lthough their inidene hs een very limited to dte. These extrpyrmidl symptoms might e due to redued dopminergi tone (Mrsden & Jenner, 198) whih is proly onsequent to the enhnement of 5-hydroxytryptminergi trnsmission produed y fluoxetine. Of prtiulr interest is the hypothesis out the pthophysiology of kthisi whih is thought to derive from redued dopminergi trnsmission of the mesolimi system originting in the VTA (Lipinski et l., 1989). Thus, there re severl linil hints tht fluoxetine might derese entrl dopminergi funtion. However, ler experimentl onfirmtion of this hypothesis is still lking, in tht fluoxetine ws found to redue slightly the umultion of the dopmine preursor L-dihydroxyphenyllnine (DOPA) in vrious rt rin res (Bldessrini & Mrsh, 199), ut these dt were not onfirmed in susequent study (Bldessrini et l., 1992). In the present study, single-ell reording tehniques were used to ssess the effets of ute nd hroni fluoxetine on the eletril tivity on dopminergi neurones in oth the VTA nd the SN. The effet of ute fluoxetine dministrtion ws ompred with tht of itloprm, nother potent SSRI. Methods Surgil nd reording proedures Mle Sprgue-Dwley rts (Chrles River, Clo, Itly) weighing 25 to 35 g were nesthetized with hlorl hydrte (4 mg kg-', i.p.) nd mounted on stereotxi instrument (SR-6, Nrishige, Jpn). Supplementl doses of nestheti were dministered vi lterl til vein nnul. Throughout the experiment the nimls' ody temperture ws mintined t 36-37C y thermosttilly regulted heting pd. Proedures involving nimls nd their re were onduted in onformity with the institutionl guidelines tht re in ompline with ntionl (D.L. n. 116, G.U., suppl. 4, 18 Ferury, 1992) nd interntionl lws nd poliies (EEC Counil Diretive 86/69, OJ L 358,1, De. 12, 1987; NIH Guidefor the Cre nd Use of Lortory Animls, NIH Pulition N , 1985 nd Guidelines for the Use of Animls in Biomedil Reserh, Throm. Hemost., 58, , 1987). The oordintes, reltively to the interurl line, for plement of the reording eletrode in the res studied were for the VTA: nterior 2.7 to 3.4 mm, lterl.3 to.5 mm, 7 to 8 mm ventrl to the level of exposed tissue; for the SN: nterior 2.7 to 3.4 mm, lterl 1.8 to 2.2 mm, ventrl 6.5 to 7.5 mm; nd for the dorsl rphe nuleus: nterior.7 to 1.36 mm, lterl, ventrl 5 to 6 mm (Pxinos & Wtson, 1986). Extrellulr reordings were performed with single-rrel miropipettes (4-7 MCI resistne ontining 2% pontmine sky lue dye in 2 M NCl). Dopminergi neurones were identified y their lotion, wveform, firing rte nd pttern (Bunney et l., 1973; Gre & Bunney, 198; Wng, 1981); 5-hydroxytryptminergi ells were reognized y their lotion, wide durtion (-2 ms), positive-negtive spikes, regulr rhythm nd slow firing rte ( spikes s )(Aghjnin, 1976). Eletril signls of spike tivity were pssed through high impedne mplifier the output of whih ws led into n nlog osillosope, udio monitor nd window disrimintor. Unit tivity ws then onverted to n integrted histogrm y rte-verging omputer nd displyed s spikes per 1 s intervls. After eh experiment, the reording site ws mrked y the ejetion of pontmine sky lue dye from the eletrode with -2 HA urrent for 1 min. Brins were removed nd pled in 1% uffered formlin for 2 dys efore histologil exmintion. Frozen setions were ut t 4 MM intervls nd stined with neutrl red. Mirosopi exmintion of the setions ws rried out to verify tht the eletrode tip ws in the VTA, the SN or the dorsl rphe nuleus. S. Priso & E. Esposito Fluoxetine on VTA dopminergi neurones Drug dministrtion protools In ll eletrophysiologil experiments, the drugs were dministered i.v. (vi lterl til vein) in exponentilly inresing doses every 2 min, nd the effet on the tivity of dopminergi nd 5-hydroxytryptminergi neurones ws reorded. Only one ell per niml ws studied. Fluoxetine (2-128 Mg kg-') nd itloprm (2-128 Mg kg-') were dissolved in.9% sline. The 5-HT2C/2B reeptor ntgonist mesulergine (8 Mg kg-'), dministered 1 min efore the first injetion of fluoxetine (2-128 gg kg-), ws dissolved in 1-2 M1 1% eti id, mde up to lmost required volume with.9% sline nd rought to ph 6.5. The dose nd the time of mesulergine pretretment were hosen on the sis of previous findings showing mesulergine ntgonism of mcpp inhiitory tion on VTA dopminergi neurones (Priso et l., 1994). In hroni experiments, i.p. fluoxetine (1mg kg-') or sline ws dministered one dily for 21 onseutive dys. Therefter, eletrophysiologil reordings of dopminergi neurones in the VTA were performed to evlute the effets of ute i.v. fluoxetine (2-128 Mgkg-), given 24 or 72 h fter the lst i.p. fluoxetine dministrtion. Preliminry experiments were performed fter 24 h wsh-out period whih ws deided on the sis of previous studies (Kennett et l., 1994; Hrdin, 1987) ut, susequently, experiments were rried out 72 h fter hroni fluoxetine withdrwl, to void ny possile residul effet of the drug. In group of rts hronilly treted with i.p. fluoxetine the effet of i.v. mcpp (1-32 Mg kg-) on the tivity of VTA dopminergi neurones ws tested 72 h fter the lst injetion of fluoxetine. In nother series of experiments, the effets of fluoxetine (2-248 Mg kg-, i.v.) nd itloprm (2-128 Mg kg-', i.v.) on the tivity of 5-HT-ontining neurones in the dorsl rphe nuleus were evluted. Intrventriulr injetions of 5,7-dihydroxytryptmine All rts were nesthetized with hlorl hydrte (4 mg kg-, i.p.). The 5-HT-seletive neurotoxin 5,7-dihydroxytryptmine (5,7-DHT) ws dissolved in.9% sline solution of sori id (.5%). 5,7-DHT (15 Mg, free se) in volume of 2 M1 ws infused into the right lterl ventrile. Control rts reeived only the sori id solution. In order to protet nordrenline-ontining neurones from the tion of 5,7-DHT (Bumgrten et l., 1973), 3 min efore 5,7-DHT the rts reeived i.p. 25 mg kg-' desiprmine, n inhiitor of nordrenline uptke into the nerve endings (Smnin et l., 1975). The eletrophysiologil reordings were performed 7 dys fter tretment with 5,7-DHT or vehile. Biohemil ssys Biohemil determintions of monomines were performed in the group of rts lesioned with 5,7-DHT. After eletrophysiologil testing, vehile nd 5,7-DHT-treted rts were killed y depittion. Brins were rpidly removed, strit nd hippompi were disseted, frozen on dry ie d stored t - 8 C until ssy. Tissue smples were homogenized in 4 PM.1 N perhlori id nd entrifuged for 15 min t 12 r.p.m. An liquot of the superntnt ws filtered nd trnsferred to n Eppendorf tue. Levels of 5-HT nd 5-hydroxyindoleeti id (5-HIAA) were mesured y reversedphse high performne liquid hromtogrphy with eletrohemil detetion. The moile phse ontined 17.5% methnol, 24 mm itri id, 16 mm N2HPO4, 1.22 mm 1- heptnesulphoni id sodium slt,.19 mm EDTA, (ph 2.8). Sttistil nlysis Dt quisition nd nlysis ws omplished with n sed PC nd n integrted softwre pkge for eletophysiology (RISI, Symoli Logi, Dlls, TX, U.S.A.). Dose-response urves were onstruted y ompring the

3 S. Priso & E. Esposito Fluoxetine on VTA dopminergi neurones ) I ' C ) ) n 6 - ) O] Un Fluoxetine ~ I mmmin S* z.r ** ** ** * I* men firing rte during 2 min, strting immeditely fter the injetion of eh dose, with the sl firing rte. The dt otined with the SSIRs were sujeted to n nlysis of vrine (ANOVA) for repeted mesures. When signifint effets were found, post-ho omprisons were mde with Tukey's test. The intertions etween the following groups: fluoxetine + mesulergine, fluoxetine + 5,7-DHT; hroni fluoxetine + ute fluoxetine, hroni fluoxetine + mcpp were nlysed y two-wy ANOVA (split-plot design), followed y Tukey's test. The omputer progrmme Allfit (De Len et l., 1978) ws used to lulte the men (+ s.e.men) ED5 of fluoxetine nd itloprm on the tivity of 5-HT-ontining neurones in the dorsl rphe nuleus. Student's t test ws used to nlyse the effets of 5,7-DHT on rin levels of 5-HT nd 5-HIAA. Burst nlysis of dopminergi neurones ws performed y using the RISI progrmme running on PC omputer. A totl of 5 onseutive spikes ws reorded for eh neurone efore nd t the pek of drug effet. Burst-firing, when present, ws deteted with n lgorithm similr to tht previously desried y Gre & Bunney (1984). Comprisons etween groups were performed with Student's t test or X2 (for perentge of neurones exhiiting urst-firing) Drugs nd hemils Cumultive dose (gg kg-1 ) of fluoxetine Fluoxetine hydrohloride ws kindly provided y Ely Lilly Lortories (Indinpolis, IN, U.S.A.); itloprm ws generously donted y Dr J. Hyttel (H. Lundek A/S, Copenhgen- Vly, Denmrk); mcpp hydrohloride ws from Reserh Biohemils In. (Ntik, MA, U.S.A.); desprmine hydrohloride, 5,7-dihydroxytryptmine retinine sulphte nd pomorphine hydrohloride were from Sigm Chemil Compny (St. Louis, MO, U.S.A.); mesulergine ws kindly provided y Dr F. Frnh (Sndoz Phrm Ltd., Bsel, Switzerlnd). All dosges refer to the weight of the slt Figure 1 Effet of fluoxetine on the firing rte of VTA dopminergi neurones: () representtive rte histogrm showing the typil inhiitory effet of i.v. fluoxetine (2, 2, 4, 8, 16, 32, 64, 128 jg kg'-, t rrows); () umultive dose-response urve showing men perentge hnge (± s.e.men) in firing rte of VTA dopminergi neurones fter i.v. fluoxetine. Men (± s.e.men) se-line rte=54.4±4.5 spikes los-1. *P<.5; **P<.1 ompred to sl firing rte (one-wy nlysis of vrine, followed y Tukey's test). Results Effets of 5-HT-reuptke inhiitors on the sl tivity of dopminergi neurones in the VTA Intrvenous dministrtion of fluoxetine indued dose-dependent redution in the firing rte of the VTA dopminergi ells studied. The typil effet of fluoxetine on dopminergi neurones is represented in Figure l. The inhiitory response vried mong different neurones smpled ut it did not depend on the sl firing rte of the neurones. Overll, fluoxetine (n = 18) produed mximl inhiitory effet of % t the umultive dose of 128 gg kg-' (Figure l). Administrtion of higher doses of the drug did not use dditionl effets on dopminergi ell tivity (not shown). The effet of i.v. itloprm (n = 16) on the sl firing rte of VTA dopminergi neurones ws less pronouned thn tht of fluoxetine, in tht it produed mximl inhiition of % t umultive dose of 16 jig kg-l (Figure 2). The response to itloprm ws vrile s it lerly inhiited the mjority (12 of 16) of dopminergi neurones tested (Figure 2), wheres it did not ffet the tivity of other dopminergi ells (4 of 16). The differentil effet of the drug did not depend on the sl firing rte of neurones smpled. Fluoxetine nd itloprm did not indue ny hnges in the firing pttern of VTA dopminergi neurones smpled (not shown). To understnd whether the 5-HT2C,2B reeptor sutype ws involved in the inhiitory effet of fluoxetine on dopminergi ell tivity, mesulergine, n ntgonist t this reeptor, ws given 1 min efore the first dose of the SSRI. Injetion of mesulergine (8 jg kg-', i.v.) y itself indued, in some VTA dopminergi neurones (3 of 7), slight inrese (15 + 5%) in the firing rte, whih returned to se-line levels within few minutes, i.e. efore the first dose of fluoxetine ws dminis- ) i) IUn 7 -._e. I Citloprmr-- hi 1iI, 5min I T I Cumultive dose (jg kg-1) of itloprm Figure 2 Effet of itloprm on the firing rte of VTA dopminergi neurones: () representtive rte histogrm showing the typil inhiitory effet of i.v. itloprm (2, 2, 4, 8, 16, 32, 64, 128pgkg-, t rrows); () umultive dose-response urve showing men perentge hnge (± s.e.men) in firing rte of VTA dopminergi neurones fter i.v. itloprm. Men (+ s.e.men) seline rte spikes = ± ls- 1. *P<.1 ompred to sl firing rte (one-wy nlysis of vrine, followed y Tukey's test).

4 do S. Priso & E. Esposfto Ruoxedne on VTA doprninergi mierio- neurones tered. Pretretment with mesulergine (8 jig kg', i.v.) loked ompletely the redution of VTA dopminergi tivity indued y fluoxetine, s indited y the findings tht dministrtion of fluoxetine in ontrol rts produed the typil inhiitory effet (n = 5) (Figure 3, ) whih ws prevented y pretretment with mesulergine (n = 7) (Figure 3, ). Seletive lesions of rin 5-HT-ontining neurones, produed y intrventriulr dministrtion of 5,7-DHT, olished the inhiitory effet indued y ute fluoxetine on the tivity of VTA dopminergi neurones (n = 8) (Figure 4, ). Intrvenous injetion of fluoxetine in vehile-treted rts produed, s in nive nimls, signifint redution in the firing rte of dopminergi ells (n = 7) (Figure 4, ). The sl firing rte of VTA dopminergi neurones in 5-HT-depleted rts ws higher thn in ontrol nimls, ut this differene ws not sttistilly signifint. Intrventriulr dministrtion of 5,7-DHT resulted in signifint depletions of oth 5-HT nd 5- HIAA in the orpus stritum nd hippompus. The effet of 5,7-DHT ws prtiulrly evident in the hippompus, where it produed 9% derese in 5-HT levels (Tle 1). Effet of ute fluoxetine on the sl tivity of dopminergi neurones in the SN A group of rts ws treted with fluoxetine to investigte the effet of this drug on the sl firing rte of dopminergi I3- o r._5 (D) tnq U) o5 Sl Fluoxetine _ Ir s I 1 I (Fluoxetinex',, -I 1 5 min Fluoxetine Io I I/; 6 QL n 5min =.Fluoxetine_ 2 A) O CD ) ) CU -C. U min m u... 1* -6 r Cumultive dose (jg kg_1) of fluoxetine Figure 3 Blokde y mesulergine on the inhitory tion of fluoxetine on the firing rte of VTA dopminergi neurones: () representtive rte histogrm showing the typil inhiitory effet of i.v. fluoxetine (2, 2, 4, 8, 16, 32, 64, 128 jg kg', t rrows) in ontrol rt; () typil rte histogrm showing tht i.v. mesulergine (8,ugkg-') prevents the inhiitory effet of i.v. fluoxetine (2, 2, 4, 8, 16, 32, 64pgkg-', t rrows); () umultive dose-response urves showing men perentge hnge ( s.e.men) in firing rte of VTA dopminergi neurones in ontrol () nd mesulergine () pretreted rts. Men (+s.e.men) seline rte (spikes 1s-1): sline + fluoxetine= ; mesulergine + fluoxetine = *P<.5 [F(7,14) = 1.75, two-wy nlysis of vrine, split-plot design, followed y Tukey's test] Cumultive dose (gg kg-') of fluoxetine Figure 4 Effets of 5,7-dihydroxytryptmine (5,7-DHT) on the response of VTA dopminergi neurones to fluoxetine: () representtive rte histogrm showing the typil inhiitory effet of i.v. fluoxetine (2, 2, 4, 8, 16, 32ugkg', t rrows) in ontrol rt, Sl =i.v. sline injetion (.1 ml, t rrow); () typil rte histogrm showing the prevention y 5,7-DHT of the inhiitory response to i.v. fluoxetine (2, 2, 4, 8, 16, 32jgkg 1, t rrows); () umultive dose-response urves showing men perentge hnge (± s.e.men) in firing rte of VTA dopminergi neurones fter i.v. fluoxetine, in ontrol (O) nd 5,7-DHT-treted rts (A). Pretretment with 5,7-DHT olished the inhiitory effet of fluoxetine. Men (+ s.e.men) se-line rte (spikes 1 s 1); vehile + fluoxetine = ; 5,7-DHT + fluoxetine = *P<.5 [F(6,78)= 5.47, two-wy nlysis of vrine, split-plot design, followed y Tukey's test]. Tle 1 Effets of 5,7-dihydrixytryptmine (5,7-DHT) on 5-hydroxytryptmine (5-HT) nd 5-hydroxyindoleeti id (5-HIAA) levels in the hippompus nd orpus stritum Hippompus 5-HT S-HIAA (pg mg) (pg mg'1) Corpus stritum 5-HT 5-HIAA (pg mg-) (pg mg-) Control ,7-DHT 26+6* 14+3* 18+49* * Eh vlue is the men (pg mg-1 of tissue) + s.e.men of 6 nimls. *P <.1, s ompred with respetive ontrol y Student's t test.

5 neurones in the SN. Figure 5 represents typil rte histogrm showing tht fluoxetine did not modify the spontneous tivity of SN dopminergi neurones. Overll, dministr- 8 o i) - Figure 5 Sl - Fluoxetine Apo I[ fi fi ff s B y 5 min Representtive rte histogrm showing the lk of effet of i.v. fluoxetine (2, 2, 4, 8, 16, 32, 64, 128, 256, 512Ygkg'-, t rrows) on the sl tivity of dopminergi neurone in the SN. Sl =i.v. sline injetion (.1 ml, t rrow); Apo =pomorphine dministrtion (losg kg-, t rrow). S. Priso & E. Esposito Fluoxedne on VTA dopminergi neurones tion of fluoxetine (n= 1) did not use ny signifint hnge in the tivity of SN dopminergi ells. The sl firing rte of these neurones ws unffeted y doses of the drug (up to 124 fig kg-') tht were muh higher thn those whih mximlly inhiited dopminergi neurones in the VTA. Moreover, fluoxetine dministrtion did not indue ny hnge in the firing pttern of dopminergi neurones in the SN (not shown). Effets of hroni fluoxetine on the tivity of dopminergi neurones in the VTA 1927 A series of hroni experiments were rried out to test if tolerne developed to the inhiitory effet of fluoxetine on VTA dopminergi tivity. Rts reeived fluoxetine (1 mg kg-', i.p.) or sline one dily for 21 onseutive dys. At the end of hroni fluoxetine tretment, the ody weight of treted rts ws signifintly redued s ompred with ontrol nimls. Chroni fluoxetine did not use ny hnge in the T- l o 6 Fluoxetine Co 41 o I 1 mcpp \ ~I s I n Z) HIM. (1). i U) Fluoxetine mcpp o 7 o '1 I CO) ii To (A. Un 2 2-, X o. se o Cumultive dose (rg kg1) of fluoxetine Figure 6 Effets of hroni tretment with i.p. fluoxetine (lomgkg-1, for 21 dys) on the response of VTA dopminergi neurones to ute i.v. fluoxetine: () representtive rte histogrm showing the typil inhiitory effet of ute i.v. fluoxetine (2, 2, 4, 8, 16, 32.ugkg-, t rrows) in ontrol rt; () typil rte histogrm showing the prevention y hroni i.p. fluoxetine of the inhiitory response to ute i.v. fluoxetine (2, 2, 4, 8, 16, 32, 64, 128, 256ggkg-', t rrows); () umultive dose-response urves showing men perentge hnge (+ s.e.men) in firing rte of VTA dopminergi neurones fter ute i.v. fluoxetine in ontrol rts () nd in nimls treted hronilly with fluoxetine (*). Complete tolerne developed fter hroni fluoxetine dministrtion. Men (± s.e.men) se-line rte (spikes 1 s1): ontrol = 41.7 ± 6.6; hroni fluoxetine= *P<.5 [F(6,84)=6.27, two-wy nlysis of vrine, split-plot design, followed y Tukey's test] i 1 1 Cumultive dose (jg kg-1) of mcpp 1 Figure 7 Effets of hroni tretment with i.p. fluoxetine (1mgkg-1, for 21 dys) on the response of VTA dopminergi neurones to ute i.v. m-hlorophenylpiperzine (mcpp): () representtive rte histogrm showing the typil inhiitory effet of ute i.v. mcpp (1, 1, 2, 4, 8, 16, 32, 64pgkg 1, t rrows) in ontrol rt, Sl = i.v. sline injetion (.1 ml, t rrow); () typil rte histogrm showing the prevention y hroni i.p. fluoxetine of the inhiitory response to i.v. mcpp (1, 1, 2, 4, 8, 16, 32, 64, 128.ug kg-, t rrows); () umultive dose-response urves showing men perentge hnge ( + s.e.men) in firing rte of VTA dopminergi neurones fter ute i.v. mcpp in ontrol rts (*) nd in nimls treted hronilly with fluoxetine (E). Complete tolerne to mcpp developed fter hroni fluoxetine dministrtion. Men ( + s.e.men) se-line rte (spikes 1 s 1): ontrol = 23.3 ± 3.2; hroni fluoxetine=43.7±8.1. *P<.1 [F(6,72)= 1.82, two-wy nlysis of vrine, split-plot design, followed y Tukey's test].

6 1928 gross ehviour of rts. Aute i.v. hllenge with fluoxetine ws performed in two seprte series of experiments either 24 h or 72 h fter the lst i.p. fluoxetine injetion. Sine results otined fter 24 or 72 h were lmost superimposle, only dt regrding the 72 h wsh-out period re presented. Aute i.v. dministrtion of fluoxetine (2-128 jg kg-') did not use ny hnge in the sl firing rte of VTA dopminergi neurones in the group of rts treted hronilly with i.p. fluoxetine (n = 9) (Figure 6, ), wheres it indued the typil inhiitory effet in ontrol nimls (n = 7) (Figure 6, ). In seprte experiment, rts hronilly treted with i.p. fluoxetine reeived i.v. mcpp, 5-HT2C/2B reeptor gonist, to hek if down-regultion of these reeptors ould e involved in the mehnism of tolerne to fluoxetine. Aute i.v. dministrtion of mcpp (1-32 jig kg-') 72 h fter the lst i.p. fluoxetine tretment used signifint inhiition of VTA dopminergi ell tivity in ontrol rts (mximl inhiition: %; n = 7) (Figure 7, ), ut it did not modify the sl firing rte of dopminergi ells in nimls given hroni fluoxetine (n=7) (Figure 7, ). Sine the typil inhiitory tion of ute fluoxetine nd mcpp disppered in rts treted hronilly with fluoxetine, it is likely tht prolonged dministrtion of this drug indued down-regultion of 5- HT reeptor, possily the 5-HT2C sutype. S. Priso & E. Esposito Fluoxetne on VTA dopminergi neurones Effets of S-HT reuptke inhiitors on the sl firing rte of 5-hydroxytryptminergi neurones in the dorsl rphe nuleus The evidene tht seletive lesions of 5-hydroxytryptminergi neurones y 5,7-DHT olished the inhiitory effet of fluoxetine on VTA dopminergi ells suggested n indiret tion of fluoxetine on dopminergi neurones of the VTA. Thus, series of experiments ws rried out to investigte the effet of 5-HT reuptke lokers on the sl tivity of 5-HTontining ells in the dorsl rphe nuleus. Intrvenous injetions of fluoxetine (2-248 jig kg-'; n = 7) nd itloprm (2-128 jig kg-'; n = 7) used dose-dependent derese in the sl firing rte of 5-hydroxytryptminergi neurones; ll the ells studied were ompletely inhiited, lthough t different doses (Figure 8). Thus, itloprm stopped the spontneous firing of 5-hydroxytryptminergi neurones in the dorsl rphe nuleus t the umultive dose of 128 pg kg-' (Figure 8, ), wheres fluoxetine produed the sme effet only t the umultive dose of 248 jig kg-' (Figure 8, ). These differenes in poteny etween the two SSRIs were refleted y the lulted ED5 whih ws lower for itloprm ( jg kg-'; men+s.e.men) thn for fluoxetine ( jig kg-'; men + s.e.men). 4- (- Fluoxetine Ad C ) -4- C Figure 8 X - & C,) I' 12 Citloprm 1 i.. 5 min Cumultive dose (jg kg-1) Effets of fluoxetine nd itloprm on the sl tivity of 5-hydroxytryptminergi neurones in the dorsl rphe nuleus: representtive rte histogrms showing the inhiitory effets of () i.v. fluoxetine (2, 2, 4, 8, 16, 32, 64, 128jg kg', t rrows) nd () i.v. itloprm (2, 2, 4, 8, 161gkg-1, t rrows); () umultive dose-response urves showing men perentge hnge (± s.e.men) in firing rte of 5-hydroxytryptminergi neurones fter i.v. fluoxetine (El) or i.v. itloprm (). Men (±s.e.men) seline rte (spikes 1 s'): fluoxetine = 12.7 ± 1.4; itloprm = 12.4±2.8. I 5 min Disussion The present study shows tht ute dministrtion of fluoxetine inhiits the sl firing rte of dopminergi neurones in the VTA, whih is the nuleus of origin of the mesolimi system (Moore & Bloom, 1978). Interestingly, the effet of ute fluoxetine ppers to e seletive in tht it did not ffet the tivity of nigrostritl dopminergi neurones. The inhiitory tion of fluoxetine upon dopminergi neurones in the VTA ws prevented ompletely y mesulergine, drug whih loks 5-HT2C,2B nd 5-HT2A reeptors (Hoyer, 1988). The dose of mesulergine used in the present study ws similr to tht employed y Priso et l. (1994), who found tht 8 jig kg' i.v. mesulergine ffeted dopminergi funtion in the VTA y ting on 5-HT2C,2B reeptors. As reported y Priso et l. (1994), 8 jig kg-' i.v. mesulergine used slight exittion of VTA dopminergi neurones, ut it seems unlikely tht mesulergine ould e ting y loking dopminergi reeptors euse t the dose used in the present study, it did not prevent the inhiitory tion of pomorphine (Priso et l., 1994). Therefore, it is possile to rgue tht ute fluoxetine redues mesolimi dopminergi funtion y ting on 5-HT2C,2B reeptors. This finding is onsistent with previous report showing tht the nti-immoility effet of fluoxetine in the fored swimming test ws loked y mesulergine, ut not y the mixed 5-HT2A/5-HT2C/2 ntgonist ritnserin (Cesn et l., 1993) thus suggesting tht this phrmologil effet ws medited y the 5-HT2C,2B reeptor sutype. Therefore, the possiility exists tht fluoxetine ould exert its effet on the mesolimi dopminergi system y ting diretly on the 5-HT2C/2B reeptors insmuh s there is evidene tht it n ind to this reeptor sutype with sumiromolr ffinity (Wong et l., 1991) ting s n ntgonist (Lightowler et l., 1994). However, this possiility seems unlikely sine the inhiitory effet of fluoxetine on the tivity of VTA dopminergi neurones ws olished y pretretment with the neurotoxin 5,7-DHT, whih used mrked nd seletive redution of 5-HT levels in the rin. This finding indites tht ute fluoxetine ts presynptilly y enhning 5-hydroxytryptminergi trnsmission whih, in turn, redues the dopminergi tone in the VTA. Tht ute dministrtion of fluoxetine n inrese the synpti vilility of 5-HT is demonstrted y severl studies showing tht this drug inreses the extrellulr onentrtion of 5-HT, s mesured y in vivo mirodilysis, in different rin res (Perry & Fuller, 1992; 1993; Ruttler & Auerh, 1993). Thus, fluoxetine inreses the synpti levels of 5-HT, whih inhiits the

7 tivity of the mesolimi dopminergi system y stimulting the 5-HT2C,2B reeptor sutypes. Like fluoxetine, itloprm, potent SSRI, deresed the tivity of mesolimi dopminergi neurones leit to lesser extent. It is tempting to speulte tht the redued inhiition of VTA dopminergi neurones y itloprm, s ompred to fluoxetine, might depend on its greter pility of inhiiting 5-hydroxytryptminergi neurones in the dorsl rphe nuleus. Thus, itloprm ws seven fold more potent tht fluoxetine in inhiiting the tivity of 5-HT-ontining neurones in the dorsl rphe nuleus, s lulted from the ED5. This is onsistent with previous findings inditing tht the EDM for itloprm (.23 mg kg-') (Chput et l., 1986) is muh lower thn the ED5 for fluoxetine (1.8 mg kg-') (Cunninghm & Lkoski, 199). Proly, the different potenies of these two SSRIs s inhiitors of 5-hydroxytryptminergi neurones reflet in vitro dt inditing tht itloprm is more effetive thn fluoxetine in loking the reuptke of 5-HT from rt rin synptosomes (Hyttel, 1982; Thoms et l., 1987). However, ute dministrtion of itloprm only slightly inreses extrellulr 5-HT onentrtions in the rt frontl ortex, proly s onsequene of onomitnt redution in the impulse flow of 5-hydroxytryptminergi neurones in the dorsl rphe nuleus (Invernizzi et l., 1992). On the sis of these findings, it possile to rgue tht the slight inhiitory effet of itloprm on the tivity of VTA dopminergi neurones might depend on its low pility of inresing the synpti levels of 5-HT in terminl regions of the 5-hydroxytryptminergi system. The typil inhiitory effet of ute fluoxetine upon the sl tivity of VTA dopminergi neurones disppered fter repeted tretment with this SSRI for 21 onseutive dys. This finding indites tht omplete tolerne to fluoxetine's tion develops fter hroni tretment. The tolerne to the ute hllenge with fluoxetine ws lerly evident 24 h fter the esstion of hroni tretment with this drug. However, 72 h wsh-out period ws preferred to void possile residul effets of fluoxetine whih hs een shown to umulte in the rt rin following repeted tretment (Ci et l., 1992). Nevertheless, rin levels of fluoxetine fll elow detetle limits 72 h fter withdrwl from hroni i.p. fluoxetine dministrtion (1 mg kg-' for 21 onseutive dys) (Grdier et l., 1993). Interestingly, the results otined t 24 h nd 72 h were very similr, inditing tht the puttive phrmodynmi hnges indued y hroni fluoxetine persisted for severl dys. This sttement is strengthened y the evidene tht hroni fluoxetine dministrtion indued omplete tolerne to the inhiitory tion of mcpp, 5-HT2C/2B reeptor gonist (Curzon & Kennett, 199). Thus, mcpp given to ontrol rts used mrked inhiition of the sl firing rte of dopminergi neurones in the VTA, wheres it ws ineffetive when injeted 72 h fter the withdrwl of hroni fluoxetine tretment. These findings re onsistent with previous dt showing tht repeted orl dministrtion of fluoxetine nd proxetine for 21 dys indues tolerne to the hypoloomotor effet of mcpp in rts (Kennett et l., 1994). It is unlikely tht redued disposition of mcpp might hve een responsile for its redued effet, insmuh s mrked inrese in rin levels of mcpp hs een found following repeted dministrtion of fluoxetine to rts (Kennett et l., 1994). Moreover, hroni tretments with itloprm nd sertline, two potent SSRIs, were found to ttenute the hypoloomotor effet of mcpp (Mj & Moryl, 1992; Kennedy et l., 1993). Sine it is thought tht mcpp redues loomotor tivity in rodents y stimulting the 5- HT2C/2B reeptors (Kennett & Curzon, 1988; Kennett et l., 1994), it hs een rgued tht down-regultion of 5-HT2,2B repetors might e responsile for the ehviourl tolerne to mcpp ourring fter hroni dministrtion of SSRIs (Kennedy et l., 1993; Kennett et l., 1994). This is reonille with the evidene tht mcpp inhiits the tivity of dopminergi neurones in the VTA y ting S. Priso & E. Esposito Fluoxetine on VTA dopminergi neurones 1929 through the 5-HT2C/2B reeptors (Priso et l., 1994). Thus, it is oneivle tht down-regultion of 5-HT2C,2B reeptors might underlie the tolerne to the inhiitory effet of mcpp eliited y hroni fluoxetine. It is tempting to speulte tht the differentil effets of ute nd hroni fluoxetine on the tivity of the mesolimi dopminergi system might explin, t lest in prt, the phrmodynmi sis of its therpeuti tions. For exmple, it is known tht the ntidepressnt effet of fluoxetine eomes evident three weeks fter the eginning of therpy (Strk & Hrdison, 1985; Chouinrd, 1985). Thus, in view of the hypothesis tht disinhiition of the mesolimi dopminergi system underlies the mehnism of tion of severl ntidepressnt gents (Cervo & Smnin, 1987; 1988; Cervo et l., 199), it is oneivle tht inhiition y fluoxetine of VTA dopminergi funtion my msk its linil effiy during the first weeks of tretment. Bsed on this ssumption, it is possile to rgue tht the ntidepressnt tivity of fluoxetine eomes mnifest when tolerne develops to the inhiition of the VTA dopminergi system. There is evidene tht fluoxetine is n effetive gent in the tretment of osessive-ompulsive disorders (OCD) (Turner et l., 1985; Fontine & Chouinrd, 1986; Levine et l., 1989; Jenike et l., 199). However, there is lteny of out four weeks etween the eginning of tretment nd the pperne of signifint linil effet (Levine et l., 1989). It hs een suggested tht repeted dministrtion of fluoxetine in humn sujets indues down-regultion of 5- HT2C/2B reeptors, whih is ultimtely responsile for its therpeuti effet in OCD (Hollnder et l., 1991). This hypothesis is sed on the evidene tht dministrtion of the 5-HT2C,2B reeptor gonist, mcpp, in ptients with OCD worsens osessive-ompulsive (OC) symptoms in drug-free sujets, wheres it does not exerte OC symptoms in ptients treted hronilly with fluoxetine (Hollnder et l., 1991). These linil dt re onsistent with the findings of the present study showing tht hroni tretment with fluoxetine indues tolerne to the inhiitory effet of mcpp on the tivity of mesolimi dopminergi neurones. Although the role of entrl dopminergi systems in the pthophysiology of OCD is still unler (Goodmn et l., 199; 1992), the possiility tht the effet of fluoxetine on VTA dopminergi neurones might ply role in its ntiosessive tivity nnot e ruled out. The therpeuti use of fluoxetine lone or ssoited with hloperidol n use dysfuntion of the extrpyrmidl system whih my result in prkinsonism (Meltzer et l., 1979; Bouhrd et l., 1989; Brod, 1989; Tte, 1989) or kthisi (Lipinski et l., 1989; Bldwin et l., 1991). It hs een speulted tht the extrpyrmidl side-effets of fluoxetine might hve een used y redued dopminergi tone in the sl gngli (Meltzer et l., 1979; Bldessrini & Mrsh, 199; Bldessrini et l., 1992). However, fluoxetine ws found to redue only slightly dopmine synthesis in the rt stritum nd frontl ortex (Bldessrini & Mrsh, 199) nd these dt were not replited in susequent study (Bldessrini et l., 1992). Moreover, fluoxetine does not hnge in vivo dopmine relese either in the stritum (Perry & Fuller, 1992) or in the nuleus umens (Tnd et l., 1994). Tht dopmine relese in the nuleus umens is unffeted y fluoxetine (Tnd et l., 1994) is pprently in ontrst with the findings of this study. The resons for this disrepny re presently unknown ut, proly, eletrophysiologil tehniques re more sensitive thn in vivo mirodilysis in deteting hnges in neuronl funtion. Thus, the present study provides the first ler evidene of n inhiitory tion of fluoxetine on the dopminergi system originting in the VTA, whih might e of relevne in the pthophysiology of kthisi (Mrsden & Jenner, 198). In this regrd, it is interesting to note tht Lipinski et l. (1989) rgued, on the sis of linil dt, tht fluoxetine might hve inhiited dopminergi neurones in the VTA ut not in the SN, whih is

8 193 S. Priso & E. Esposito Fluoxetine on VTA dopminergi neurones extly wht ws found in the present study. However, evidene from this study does not support linil oservtions tht fluoxetine indues prkinson-like effets sine it did not ffet the tivity of dopminergi nigro-stritl neurones, whose redued funtion is generlly thought to e the use of drug indued prkinson-like syndrome (Mrsden & Jenner, 198). In onlusion, the present study provides evidene tht ute tretment with fluoxetine redues the tivity of dopminergi neurones in the VTA ut not in the SN. This ute effet of fluoxetine, whih is proly medited y the 5-HT2C/2B reeptor sutype, disppers following hroni fluoxetine dministrtion. However, more seletive gents re requried to onfirm the involvement of the 5-HT2C or 5-HT2B in these responses. It is hypothesized tht the differentil effets of ute nd hroni fluoxetine on the tivity of mesolimi dopminergi funtion might e relevnt mehnism underlying the lg in its ntidepressnt tion. 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