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I I I I I Elsvur Brmn Research, 4*-1989) 36-5-3G7 365 BRE ?3320 Nicotine infused into the nucleus accumbens increases synaptic dopamine as measured by in vivo microdialysis Jean-Christophe Mifsud, Luis Hernandez and Bartley G. Hoebel DtpernnenroJPr,vcholo;s, Princaon Univerriry, Prusceton, Nl0dS44-1010(U.S.A.) ( Acapted 11 October 1988) Key words: Nicotine: Dopamme: Microdialysis: Nudeus accvmbens; Addiction I 1 I I I I I I I It has been postulated that addiction to nicotine is mediated by dopanune release in the mesolimbic system. It is possible that nico- tine mtght act directly on the dopamtne terminals to releax dopamtne. This hypothesis was tested by infuutl ntconne through a nu- crodulysis probe into the nucleus accumbens of freely moving rats. Dopamine. dihydroxyphenyiaeetx sad. and homovantllic acid from the extrarellular space were collected by mtcrodulysis and measured by high pressure liquid chtornatognph,v. Nicotine tn• creased extracellular oopamine in a dose-related manner. Systemic injection of the nkotine anugottut mecamylatntne blocked the dopamtne increase induced by local nicotnx. These results suggest that nicotitx releases dopatnine by a local action in the nucleus ac- cumbens tertainal area of the mesolunbic system. Presynapttc tnducuon of dopanune release enight play a rok in oxonne addiction. Nicotine is an alkaloid naturally occurring in to- bacco leaves's. It is a primary reinforcer self-adminis- tered by humans and other animals as well7. This property seems to be centrally mediated by catechol- amines. Nicotine releases norepinephrine and dopa- mine from brain siices'-19. Rats self-inject ampheta- mine which releases dopamine and self-inject dopa- mine itself into the nucleus accttmbens°•". There• fore. a local action of nicotine to release dopamine in the atxumbens might play an important role in nico- tine self-administration. Microdialysis studies have shown that systemic administration of nicotine in- creases extncellular dopamine in the nucleus atcvm- bens of freely moving ratsu. This systemic effect might be due to a direct action of nicotine on dopa- mine terminals. To test this proposition we have stud- ied the effect of niootine on dopamine release by di- rect application of the drug to the nucleus aavm- bens. Microdialysis probes were used both for drug infusion and neurochemical sampling in freely mov- ing animalsi'. Twenty-six male Sprague-Dawley rats (250-300 g) were individually housed with food and water ad libi- tum. Under pentobarbital (23 mg/kg) and ketamine (Ketalar, 57 mg/kg) anesthesia, a 10 mm long 21- gauge guide cannula was implanted in the posterior region of the nucleus accurnbens medial to the ante- rior cornmissure. With the skull level the coordinates were 10.0 mm anterior to the interaural line, 1.2 mm lateral to the midsagittal suture, and 4.0 mm ventral to the surface of the skull. Experiments were per- formed at least 48 h after the implantation of the guide shaft. The microdialysis probes were con- stntcted of concentric 36-gauge stainless steel tubing inside of 26-gauge tubing, ending in a 3 tnm tip ot0.2 mm outside diameter cellulose tubing with a 6000 mol. wt. cut-offu. Ringer's solution was pumped through the microdialyztis probe at a flow of Iol/min. These probes have a relative recovery of 5-10% at a flow rate of 1 µl/minu. On the day of perfusion, a probe was inserted into the guide shaft and con- nected to the syringe pump and a 4001r1 polyethylene vial for collecting the output. Starting after 1 b, sam- pks were collected every 20 min in 400 µl vials corl- taining 10 NI of 0.1 N HC1 and 100 µM EDTA to re- tard oxidation of monoamines. Dopamine (DA) and the metabolites dihydroxyphenyiacstic acid (DOPAC) and homovanillic acid (HVA) were separated by re- Co.respondrnn: B.G. Hoebel. Deparunertt of hycNology, Prittatoa University, Prittcetoo, NJ 06544•1010. U.S.A. 0D06-tt993JE9/S03.50®19E9 Elsevier Science Publishers B.V. (Biomedical Division)

I ~ I I I I I I i I I I I ~ I I , 366 verse phase liquid chromatography according to the procedures described elsewhere (Hernandez et ai., 1986). The sample was directly injected into an HPLC equipped with a 20,u1 injection loop and 10 cm long, 3.2 mm bore, 3,um particle, C-1S column. The chemicals were electrochemically detected in an am- perometric detector (BAS Co., Model LC-4B) by oxidation on a glassy carbon electrode at 0.71 V with respect to an Ag-AgCI reference electrode. When stable levels of chemicals were observed a 30 cm piece of PE-10 tubing was fully loaded with 20 µl of 2.4 µM nicotine solution and intercalated into the in- put line. This allowed gentle application of nicotine into the nucleus accumbens for 20 min without me- chanical disturbance of the tissue. Nicotine diffused out of the probe at the same time DA, DOPAC, and HVA diffused along their concentration gradients. For a dose-response curve the monoamine data were plotted directly in terms of picdtrams per 100 min (5 samples following the injection). For the rest of the experiments the data were normalized as per- cent of the first baseline sample and then analyzed by ANOVA followed by Newman-Keuts test when war- ranted. In the first experiment 4 rats were used to establish the phenomenon. As shown in Fig. 1, the 2.4µM nic- otine dose stimulated DA release by 490% (Fr,7t - 20.5, P < 0.001). The dopamine level reached its maximum 40 min after the beginning of the 20-min perfusion period and returned to the initial baseline 60 min later. No dartge in DOPAC or HVA oc• ctured. In the second experiment with 16 rats, 4 more doses of nicotine, two higher aad two {ower, were each infused in 4 naive rats to aeate a dose-response •A • +•• :e• MINUTES ® . Fig. 1. Aa infusiott M 2.4MM ttioonne in the period iadiaatsd by  tbe arto.vs maeised doQantine in the earsoeUular ooaapart. ' taent of the audetn aocutntxo: (meae t S.E.M.. 01 a 4, •Pc0.001)ia4rats. • . J.i. . NICOTINE 4+IA) •. Fig. 2. Dose-response reiationship between nicotitte cottan• tration put into the infu:ate for 20 nun and DA eoneentrauon recovered dunng the sutuquent 100 min. The Unear eorrela- twn of 0.79 was stgnificant ( P< 0.05). curve. Fig. 2 suggests a sigmoidal relationship be- tween nicotine concentration and extracelhilar DA. The regression analysis showed a significantly linear correlation between nicotine dose and extracellular dopamine (F., - 11.26, P < 0.05). Specificity of the stimulatory effect of nicotine on DA release was tested with a nicotinic receptor blocker. In 6 rats nicotine solution (2.4,uM) was in- fused twice 160 min apart. Half the animals were in- jeaed s.c. with 1 mg/kg of inecatnylamine hydrochlo- ride 40 tnin before the second infusion. Results of this experiment show that the first infusion of nicotine in- creased dopamine in all 6 rats. In the control condi- tion without meamyiamine, the second infusion of ~ $ rMMIANR.~ • tM aM aM M• •M FiS. 3. Sqstetaic meamytamine b{oets local ttiaodae effea oa enraaUutar DA ia tbe arodetat aowaobea•. Six rats wm Rvea a 20 ttsia lod 'tnfnt+ioe of tncooae (open yuats•. aeae t S.E.M. ). T3ea 3 of tbem teoeived an s.e. iajaetioa of tbe arta tiaie t,iodter meeaaaytamine fotio.•ed 40 =a Wer by a ncoed loal inftaioo of t3icodne (blaek t4rslM). The txber 3 rats (opes tarda) did not reoeive meamylamia. Poiats with anwislts rere sipti5caady peater than b.aeiiae. Meet+myiamiee, i.p.. r+pti6caatty aataaoeixd the doQamioe-releatiaS etIaa ot loo! amootiae (P < 0.01).

I I I I ~ i I I I I I I 1 I I I I I nicotine had somewhat less effect than the first infu- sion (first nicotine produced 476% DA, n= 6, vs sec• ond nicotine 390% DA, n - 3, not significant). In the group of 3 that received systemic mecamylamine, the second infusion of nicotine increased extracellular dopamine only 10% (i.e. DA 110% of baseline with nicotine plus blocker vs 390% without the blocker, F1,4 - 25.89, P< 0.01). Histological examination showed tracks of the probes in the intended posterior region of the nucleus aocumbens medial to the ante- rior commissure. These experiments extend previous microdialysis results which showed that systemic nicotine increases exaracellular doparnine in the nucleus accumbensls Tte present results suggest that nicotine relaases dopamine by acting locally via the dopamine tettni- tlals in the nucleus accumbens. This effect was sup- pressed by pretreatment with meanzylamine, a nic- otinic cholinergic receptor blocker. Therefore it ap• pears that the release of DA induced by nicotine can have its origin in centrally located nicotinic choliner- gic recepton172Q. These receptors have besn de• scribed as specific binding sites for nicotine. Our re- I Arguero. L., Naguira, D. and Z.unino, E., NKocine•in- duoed release of catecAolatnines from rat hippocampus and strtattun, siothent. Pbenn.col., 2r (1978) 2667-2674. 2 Blander, D.S., Mark. G.P., HerAandez, L. and Hoebel, B.G., Anpotensin and drinking induce doptmine relesse in the nucleus aaumbees, Soc. Netuoacr. Abstr., 14 (1968) 3527. 3 Chan;, V.C., Mark. G.P.. Hernandez, L. and Hoebel. B.G.. EslraceUular dopamiee incrt:ase to tise nudevs ae• cuatbers following rehYdruioa or sodium repietioa in rats, Soc. Nesvoui. Abra., 14 (1968) 3527. 4 Di Chiara, G. and Imperato, A., Ethanol prefsrsatially stimulates dopaaune rokase in the nudeus Bocumbem o( fmly worn2 nts, Ew. J. PN.nnwol., 113 (19aS) 131-132. 3 Di Chiara. G. and Imperato, A., Drugs abired by bmsm prtferentiaUy iaaeaee sraaptie dopaeeins ooooeatrarioas in the mesolimbic sritem of freely atorinS tatt, rroc. Naet. AaI. Sef. U.S.A., tS (19N)3274-s27a. 6 Dworkin, S.L, Go.dertc, N.E. n.d Smith,l.E.. Is lWdo~.t turimu on Dnq Abwt Ra.rtb MawjrqA Se+ie:, 67, trobkrv of Dnej DePwAlwcr, U.S. Publie HeahS Ssrvirs. 1963, pp. l6-144. 7 Haaaa, H.M., Iwaeer, C.A. a.d h/orto., B.R., Ia Nrion - .f lwrdnue oe Drrq AMue Rae.rdt Moraojr.pb Series, 2J. U.S. Pubtic Health Serriee.1979. E Heraaada, L., Atierb.cit. S. and Hoebel, B.G., Pbeery- didine (PCt) iajeeted in the tsudeus Bavmtxsis iaaaaes aaraaellular dopamioe and aetosonin ac meawrad by mi- ctodialysit. [iJt Scr., 42 (19li)1713-1723. 9 Heraandez. L. aard Hotbel, B.G., Food re.nrd and co- aine increase artrsodlular dop.mine in the nudeua ae- cumbees as eeamred by asnsodiatysis, Life Sci.. 42 (19l8) 1705-1712. 10 Hernandez. L. and Hoebel. B.G.. Feeding and hypotha- lantic uimulation utereaae dopantine tutt+over ia the aca+sr beut, Phr:iol. ddi.v., in ptess. 367 suits show both disparities and similarities between nicotine and other drugs of abuse. Intra-accutnbens nicotine did not affect DOPAC or HVA; wbereas io- cal amphetamine, cocaine, and phencydidine de• creased DOPACr.'.tt. 'Tbis suggests that nicotine does not act as a reuptake blocker. The overriding similarity between nicotine and other drugs of abuse such as amphetaminelt, phencyclidinei, alcohol', aAd ooairte' is that they all increase extracellular dopafnine in the a=umbettss. Food, too, has this ef- fect'.lo.l', as do some other natural rewards such as water and Salt", sugjesting that nicotine could sub- stitute for food and some other natural reinforcets in this regard. Conversely, animals or people under- going nicotine withdrawal may substitute food for nicotine and thereby overeat. In any case, it is clear that nicotine can have a local action on DA neuro- transmission in the mesolintbic system. We thank Kenneth Kellar for advice and Dawn Davidson for technical assistance. Tbis research was supported by USPHS Grant DA-03597 to B.G.H. and Rhone-Poulenc Inc. support for 1.-C.M. 1I Hernandez. L.. Les, F. and Hoebel, B.G., Simultatmosn miarodialyus and ataphetamine tnfusioe ia the nudeus ae- cvmbens and uriatum of freely enovinS rats: inaease in a- traeeliulu dopamine and serotonia, Br.irt Ra. OWL, 19 (19a?)623-62d. 12 Hernandez, L.. Rada, P. and Hoebel, B.G., Mierodia)nis tatM'sque for sitnultaneous ssses>mant of dopatniae fuae• tio4 ia three ditterent tetmiaal 5eida (unpublisltsd). 13 Herttandez. L.. Sunley, B.G. and Hosbel, B.G., A amall, reawvable mmodiaryrds peobe, 1,iJe Sd., 39 (1966) 2629-2637. 14 Hoebel, B.G.. Moasco, A.P., Hernanda, L.. Auiisai, E., Stanley. B.G. and Lenud, L. Sett•iajeoawa of ampbeta- miee direNy into the brai., Pzrdropien.eodoV , 11 (19113)156-163. 15 Imperato, A.. MuW, A. sad Di Cliara, G., Nioooae pW- ereatiaUy stimulates dopamias eslwe in the limbic trysttm of freely aartng rata, Eiv. !. Plrmw.cW., 132 (1966) 337-33a. 16 RadAakishsa. F.S., ra. Rse.1.M. asd weatstiek, B.H.C., Scheduled eating inereaies dop.mirie teJelae in the atsdeua aocumbens of food depnved tas rs arrwd Mritb ot.ai.e .uaodiatvsis. Nervosci. Ler., tS (19q) 331-356. 17 Romano. 0. and Goldpsia, A., Stetraaped6e aiootine ro- ceptors on rat btai. ae.br~. Satase:, 210 (1910) N7-6S0. 18 Taylor. P., Ganglionie sasttdatiaS and ttiodciaS aBsats. Ia A.G. Giltman. L. Goadmaa, T.W. RaY atsd F. Murad (Eda. ).17re PAsr+wafotiaiJ d.eir oJ?itier.pwier, MaMil- Iau. New York,19RS. 19 Westfall. T.C., EHeet of lieoti=e aid other drugs oa the rr- karc of 1H•noreptkpArme aa0 JH-dopramiee froea rat t>fai.n tlices. NewopM.rnuoeJoV,13 (1974) 693-700. 20 Yos?uda. K. aad Imura. H.. Niootinie, dsobnerpc ttap- ton in bnia syaal+toaoiaes. 3min Roevc>r, 172 (1979) 4S3-iS9. I