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Receptor systems participating in nicotine-specific effects
NEUROCHEMISTRY International Neurochemistry International 22 "0887# 334Ð346
Receptor systems participating in nicotine!speci_c e}ects Istvan Sziraki\ Henry Sershen\ Myron Benuck\ Audrey Hashim\ Abel Lajtha Center for Neurochemistry\ The Nathan S[ Kline Institute\ 039 Old Oran`ebur` Rd\ Oran`ebur`\ NY 09851\ U[S[A[ Received 04 June 0887^ accepted 17 July 0887
Abstract It is generally accepted that self!administration of drugs is prompted primarily by a reward system driven by an increase in extracellular dopamine in the nucleus accumbens[ Recent _ndings that dopamine increase in the accumbens can be caused by many other factors\ among them stress\ suggest a more complex mechanism\ and possibly di}erences in the reward system for di}erent compounds[ In the present paper we compare the e}ects of receptor!speci_c antagonists on the increase of dopamine induced by nicotine with that induced by cocaine in the nucleus accumbens in conscious rats[ The compounds alone or together were injected intravenously\ and dopamine level changes were measured via microdialysis[ When administered together the e}ect of nicotine and cocaine on the level of dopamine in the accumbens was additive[ Apparently there is some interaction between the two compounds\ since nicotine had no e}ect after combined nicotine and cocaine administration[ Perhaps the available dopamine pool was exhausted by the prior administration[ The nicotinic antagonist mecamylamine\ the muscarinic antagonist atropine\ and the NMDA glutamate receptor antagonist MK!790 each blocked nicotine!induced dopamine release in the accumbens\ indicating the participation of more than a single receptor system in the nicotine!induced e}ect[ These three antagonists did not inhibit cocaine!induced dopamine increase in the accumbens\ indicating the lack of a role of these receptors in the cocaine e}ect under our experimental conditions[ SCH!12289\ a dopamine D0 receptor antagonist\ blocked both nicotine! and cocaine!induced e}ects\ indicating the possible role of this receptor in these reward e}ects[ The results indicate that there are di}erences in some of the receptors mediating the central e}ects of the two compounds examined\ nicotine and cocaine\ although each in~uences dopamine levels\ and that the two compounds interact[ Þ 0887 Elsevier Science Ltd[ All rights reserved[
0[ Introduction Although it has been found in numerous studies that the e}ect of nicotine constituting the major impetus for smoking is on the mesolimbic reward mechanism dopa! mine release in the nucleus accumbens "Rowell et al[\ 0876^ Corrigall et al[\ 0881^ Pontieri et al[\ 0885#\ recent _ndings suggest that the changes induced by nicotine\ and indeed by most compounds altering dopamine levels in the accumbens\ are complex and involve e}ects on several neurotransmitters[ Nicotine and cocaine have been shown to a}ect the levels of a number of other neurotransmitters and of neuropeptides in several brain structures\ in a heterogeneous manner "Naftchi et al[\ 0877^ Toth et al[\ 0881^ Reith et al[\ 0886#[ In addition to the reward system\ nicotine has in~uence on several other systems that could alter behavior\ including cognition\
Corresponding author[ Tel[] 990 803 287 4429^ Fax] 990 803 287 4420^ E!mail] lajthaÝnki[rfmh[org
learning\ memory\ arousal\ etc[ "Levin\ 0881^ Bardo\ 0887#[ It is likely that a set of the changes in the various structures and in behavior is speci_c for nicotine\ and that di}erent speci_c sets of systems are involved in various nicotine e}ects[ Although similar release of dopamine by nicotine and cocaine has been used as evidence for similar mechanisms of action "Pontieri et al[\ 0885#\ our study examines whether functional neurochemical changes similar to those caused by cocaine extend to the other receptor systems participating in nicotine e}ects[ In our long!term study of central nicotine e}ects and speci_c reward mechanisms we are attempting to identify the various changes in brain structures that are induced by nicotine administration\ and the systems mediating these changes\ and to identify those that are speci_c for nicotine\ and to understand each of the mechanisms of the various nicotine e}ects[ In the present study we compare nicotine!induced with cocaine!induced changes of the levels of dopamine in the nucleus accumbens\ using antagonists to various recep! tors to try to identify the receptor subtypes mediating the action of these two compounds in this structure[
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1[ Methods
1[2[ Nicotine assay
1[0[ Animals
In the experiments to determine the brain and blood nicotine concentrations at di}erent time points after the administration of nicotine\ the rats were killed by decapi! tation[ The brain was quickly removed and the various regions were dissected out on an ice!cold rubber sheet\ placed in plastic tubes\ and frozen on dry ice[ After the decapitation trunk blood was mixed with 9[8) saline containing EDTA "_nal concentration] 9[4 mg EDTA per ml of blood#[ The brain and blood samples were kept at −79>C until processed for extraction[ For the determination of nicotine using gas chromatography\ the procedure of Davis "0875# was followed[ Nicotine was extracted from brain homogenates or blood by adding an equal volume of 4 N NaOH\ 1 volumes of methyl chloride\ and N!ethyl!nornicotine for internal standard\ mixing for 4 min\ and separating the aqueous and organic phases by centrifugation[ The upper aqueous phase was discarded\ including the lipid interface\ by aspiration^ 2 volumes of isopropyl alcohol were added to the organic phase\ which was concentrated to 0:4 using an N!evap evaporator at 39>C and transferred to automatic injector vials\ with 1 ml injected for assay[ For gas chro! matography a Carbowax DB megabore 29!meter column was used\ the injector and detector temperature was set at 124>C\ and the column temperature was set initially at 79>C for 0 min\ increased 29>C per min to 059>C\ kept there for 4 min\ then increased again 29>C per min to 159>C\ till the end of the run[ The ND detector was run in the nitrogen mode[ In human plasma the method had an intra! and interassay RSD) of 3[6 and 4[8) respec! tively[ The extraction recovery was over 85)\ and the sensitivity of the determination was 0 ng:ml[
Male Sprague!Dawley rats bred in our animal facility\ as in our previous studies with nicotine\ were used for the present experiments[ We selected this strain because we had used it previously to measure nicotine!induced neurotransmitter changes in awake animals\ and thus our _ndings with cocaine and nicotine can be compared to our previous _ndings under similar experimental con! ditions in the same laboratory[ In experiments to determine brain and blood nicotine levels at di}erent times after intravenous nicotine injection the rats were 179Ð249 g\ and for microdialysis studies they were 179Ð 219 g[
1[1[ Administration of dru`s Nicotine\ cocaine\ and other drugs tested for their capacity to alter nicotine! or cocaine!induced changes in dopamine in the extracellular space were administered through a cannula inserted under anesthesia into the right jugular vein[ The catheterization was carried out by a standard procedure "Waynforth and Flecknell\ 0881# with some modi_cations[ A second thread was applied in the immediate proximity of the tied {anterior thread|[ The second thread had longer ends after the knot was made[ The tie was around the cannula ðTFE tubing "17 g# from Zeus Industrial ProductsŁ after it had already been inserted into the vein and secured with the {posterior| threads[ Two ligatures were applied also at the posterior position[ The cannula was _lled with heparin!saline solu! tion "39 units of heparin per ml#[ The catheter tubing was routed under the skin and exited in the back of the animal[ Cannulation of the jugular vein was followed by implan! tation of a brain guide cannula and the vein cannula was attached to a microsyringe pump "CMA# through a dual! channel liquid swivel "CMA system for freely moving rats# and heparinÐsaline was infused at a rate of 9[0 ml:min until the beginning of the microdialysis experi! ment the next day[ All drugs "from Sigma or RBI# were dissolved in 9[8) saline[ Nicotine and cocaine were always administered by bolus injection "in a volume of 099 ml# through the catheter tubing washed with an equal volume of heparinÐ saline[ All other drugs were administered _rst as bolus injections "either alone or in combination with nicotine or cocaine# and then continued via infusion at the rate of 0 ml:min using a microsyringe pump as indicated in the legends and in the _gures[ The doses given in the _gures represent the free base forms of the drugs with the excep! tion of MK!790 where the dose refers to the hydrogen maleate form of the drug[
1[3[ Brain microdialysis This was performed as described previously "Toth et al[\ 0881#[ After completion of the catheterization of the jugu! lar vein the anesthetized rats were placed in a Kopf ster! eotaxic apparatus[ A hole was drilled in the skull directly above the site selected for microdialysis[ The guide can! nula "BAS or CMA# was lowered to 1 mm above the selected site in the brain and _xed to the skull with dental cement[ The stereotaxic coordinates for nucleus accum! bens with respect to the bregma and the surface of the skull were\ as used before "Pontieri et al[\ 0885#\ AP] ¦1[9\ ML] −0[1\ DV] −5\ according to the rat brain atlas "Paxinos and Watson\ 0875#[ Experiments using the coordinates AP] ¦0[6\ ML] −9[7\ DV] −4[7 gave similar results[ After surgery the animals were placed in the bowl of the CMA system and both the vein catheter and the microdialysis probe tubing were connected to a dualÐ swivel attached to the lever arm[ The animals were given unlimited food and water during the experimental period[
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1[4[ Determination of dopamine A microdialysis probe "BAS or CMA# with a 1!mm mem! brane was inserted into the guide cannula either in the morning of the day of the experiment or in the evening of the previous day[ The probe was perfused with Ringer at 0 ml:min throughout the experiment[ At least 1 h elap! sed before the _rst injection of any of the drugs[ "If the probe was inserted on the previous day it was perfused overnight at 9[0 ml:min and the next morning the speed was set at 0 ml:min[# The dialysates of 04Ð29 min periods were collected in polyethylene microtubes[ Dialysate samples were manually injected into the injector valve of a BAS 199 A HPLC system equipped with a BAS Unijet detector cell "5 mm radial ~ow cell# set at 399 mV and a C07\ 4 mM\ 049×0 mm microbore column[ The mobile phase consisted of 099 mM NaH1PO3\ 124 mM Na1EDTA\ 09 mM NaCl\ 09 mM diethylamine\ 249 mg of sodium octyl sulfate and 44 ml of acetonitrile per liter\ adjusted to pH 4[7[ Reverse!phase liquid chro! matography with electrochemical detection was used[ After completion of the brain microdialysis the pos! ition of the catheter in the vein was checked\ then the rats were killed with an overdose of sodium pentobarbital "099 mg:kg#[ The brain was removed and kept in 09) formaldehyde[ The probe placement was veri_ed his! tologically[
2[ Results The purpose of the present study was to compare receptor subtypes participating in nicotine!induced vs cocaine! induced dopamine increase in the nucleus accumbens[ One question involved relevant doses of these com! pounds*doses for animals that are equivalent to human use of these compounds[ In the case of nicotine\ this would be a dose matching that used by smokers[ A ciga! rette yielding 0 mg of nicotine "FTC# for a 56!kg person would represent a dose of 04 mg:kg\ not in a single bolus\ but during the smoking of a cigarette[ Smokers inhale 04Ð29 mg:kg nicotine per cigarette\ and 09Ð34 mg:kg is the range for self!administration "Rose and Corrigall\ 0886#[ Using a bolus of 4 mg:kg we could not detect a measurable increase in dopamine release in the accum! bens[ With a 14 mg:kg dose\ some increase could be detected\ but it was too small and variable to examine its inhibition by receptor subtype antagonists^ therefore\ a dose of nicotine of 49 mg:kg\ 1Ð2 times the amount from smoking a cigarette\ was used in subsequent experiments[ A study of the e}ect of intravenously administered nico! tine on accumbens "shell# dopamine levels found rela! tively small changes "14Ð29)# after 14 mg:kg but signi_cant changes "59)# after 49 mg:kg "Pontieri et al[\ 0885#[ We estimate that the dose of nicotine used in the present study would result in cerebral nicotine levels 2Ð4
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times higher than levels in the brain of smokers[ The time course of nicotine levels in the brain after a single injection of 4 or 14 mg:kg nicotine intravenously is shown in Fig[ 0[ Brain concentrations reach about 04 mg:kg at administration of a 4 mg:kg dose\ and about 31 mg:kg at a 14 mg:kg dose[ The half!life of nicotine in these experiments was about 29 min "Fig[ 0#[ To examine nico! tine accumulation with intermittent administration as in smoking\ we administered 1 mg:kg doses at 29 s intervals[ After 4 doses of 1 mg:kg "09 mg:kg total in 2 min# brain levels reached 29 mg:kg and after 09 doses "19 mg total# brain levels reached 49Ð59 mg\ decreasing to about half in 29 min "Fig[ 1#[ There was some regional heterogeneity in nicotine levels in the four brain areas examined "Fig[ 1#[ At each time point "2\ 5\ and 29 min#\ brain nicotine levels were 2Ð4 times higher than blood levels "Fig[ 1#[ The cocaine dose used was 149 mg:kg\ a dose su.cient for self!administration maintenance in rats "Corrigall et al[\ 0883#[ This dose is very similar to the average dose self!injected by drug addicts^ subjects have been reported to self!inject up to twice this dose "Fischman et al[\ 0865#[ An increase in dopamine levels could be measured following either 49 mg:kg nicotine or 149 mg:kg cocaine administration[ With administration at the same time\ nicotine and cocaine e}ects were additive as observed previously "Zernig et al[\ 0886#[ Cocaine administration following a nicotine plus cocaine treatment resulted in dopamine release^ however\ nicotine following nicotine plus cocaine resulted in no release of dopamine "Fig[ 2A\ 2B#[ To determine whether the additive e}ect of nicotine¦cocaine administration is due to the same or to two di}erent processes and is from similar dopamine pools\ we administered nicotine in place of cocaine in multiple administration experiments[ Whereas there was no nicotine!induced dopamine release after simultaneous administration of cocaine and nicotine "Fig[ 2B#\ dopa! mine release by nicotine could be observed when cocaine was replaced by nicotine "Fig[ 3A#[ Nicotine!induced increase in dopamine levels could be observed when nic! otine was given following an administration of cocaine "Fig[ 3B#[ This suggests that the administration of cocaine plus nicotine depletes a dopamine pool that responds to nicotine but not one responding to cocaine[ Repeated nicotine administration does not deplete this pool under our experimental conditions[ 2[0[ Effect of anta`onists on nicotine! and cocaine!induced elevation of dopamine in the accumbens We examined possible di}erences in the mechanisms of nicotine! and cocaine!induced changes by administering the three compounds\ antagonist\ nicotine\ and cocaine\ intravenously and measuring changes in extracellular dopamine levels in the accumbens via microdialysis[ The nicotinic receptor antagonist mecamylamine\ as in most systems tested before\ abolished nicotine!induced chan!
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Fig[ 0[ Half!life of nicotine in the brain[ Brain nicotine concentrations at various times after a single intravenous injection of 4 "ОÐ# or 14 "ÐEÐ# mg:kg nicotine are given[ Values are expressed as mean2SEM "N 2Ð5#[
Fig[ 1[ Nicotine concentrations in blood and various brain regions after repeated intravenous nicotine administration[ Two mg of nicotine per kg of body weight was injected in 29 s intervals 4 or 09 times[ Times given are after the _rst injection[ Values are expressed as ng per g of tissue\ mean2SEM "N 2#[
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Fig[ 2[ E}ects of nicotine "NIC# and cocaine "COC# on dopamine "DA# levels in nucleus accumbens[ Numbers in parenthesis indicate the dose "mg:kg# of nicotine or cocaine administered as a bolus intravenously either alone or in combination[ Averages of three experiments2SEM are shown[
ges^ mecamylamine did not a}ect cocaine!induced chan! ges "Fig[ 4#[ The results were similar whether the sequence of administration was nicotine followed later by cocaine "Fig[ 4A# or cocaine followed by nicotine "Fig[ 4B#[ The e}ect of atropine\ a muscarinic antagonist\ was similar to that of mecamylamine[ Nicotine!induced e}ects on accumbens dopamine levels were inhibited by atropine\ while cocaine e}ects on dopamine were not changed\ with no di}erence whether nicotine or cocaine was admin! istered _rst "Fig[ 5#[ The results shown in Figs 4 and 5 indicate that both nicotinic and muscarinic cholinergic receptors have an important role in nicotine!induced dopamine release in the accumbens\ while cholinergic receptors do not have a signi_cant role in cocaine e}ects on dopamine levels[ A similar di}erence between nicotine! and cocaine!
induced dopamine changes in the accumbens was seen after the administration of the NMDA!type glutamate receptor antagonist MK!790 "dizocilpine#[ This com! pound signi_cantly inhibited nicotine e}ects\ while cocaine e}ects were little a}ected^ as before\ the sequence of administration "nicotine _rst or cocaine _rst# did not a}ect the results "Fig[ 6#[ To see whether the reaction to other compounds is a}ected in these experiments\ or the inhibition is due to exhaustion of relevant dopamine pools\ we administered a high dose of amphetamine intra! venously at the end of these experiments[ Amphetamine was much more e}ective in increasing dopamine levels in the accumbens than either nicotine or cocaine\ and its e}ects were not altered by MK!790 infusion "Fig[ 6#[ These _ndings indicate that there was a su.ciently large dopamine pool available for release[ Our dose of
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Fig[ 3[ E}ects of repeated administration on dopamine levels in nucleus accumbens[ Numbers in parenthesis indicate the dose "mg:kg# of nicotine and amphetamine "AMPH#[ Averages of three experiments2SEM are shown[ Panel A] repeated nicotine administration^ Panel B] cocaine administration followed by nicotine[
MK!790 caused a temporary hind leg paralysis*this was not altered by amphetamine administration[ These experiments are in agreement with studies showing the interaction of dopaminergic and glutamatergic systems^ with glutamate increase releasing dopamine and increased dopamine releasing glutamate[ It seems that such interaction is more important in the action of nic! otine than in that of cocaine[ In examining dopaminergic antagonists\ the admin! istration of SCH!12289\ a D0 dopamine receptor antag! onist\ was inhibitory to both the cocaine! and the nicotine!induced e}ect on dopamine levels "Fig[ 7#[ This indicates the possible role of this receptor subtype in the e}ect of both compounds in altering dopamine levels in
the accumbens\ and shows that appropriate antagonists can block both cocaine e}ects and nicotine e}ects[ The inhibition of cocaine!induced increase in dopamine levels raises the possibility of the involvement of dopamine receptors in addition to a direct e}ect on dopamine trans! porters in cocaine!induced e}ects[
3[ Discussion Nicotine administration alters dopaminergic activity and increases dopamine levels in the nucleus accumbens "Lapin et al[\ 0876^ Mifsud et al[\ 0878^ Damsma et al[\ 0878#[ Nicotine a}ects a mechanism generally associated
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Fig[ 4[ E}ects of mecamylamine "MEC# on nicotine! and cocaine!induced dopamine increase[ Numbers in parenthesis indicate the dose "mg:kg# of nicotine\ cocaine\ and mecamylamine[ Nicotine and cocaine were administered as a bolus\ mecamylamine as a bolus followed by constant infusion[ Averages of three experiments2SEM are shown in panel A^ averages of two experiments are shown in panel B[
with reinforcement and reward] an increase in dopamine in the accumbens is proposed to operate as reinforcement for self!administration of several other compounds\ and reinforcement of other activities that may include feeding\ sexual\ and motor activity\ etc[ Eating\ among other things\ induces release of dopamine in the accumbens "Westerink et al[\ 0886#[ Because of the association of dopamine increases in the accumbens with reward\ a simi! lar e}ect of nicotine is assumed to be the one in the brain that provides the motivation for smoking[ The fact that aversive stimuli may also a}ect dopamine levels in the accumbens "for example\ foot!shock stress induces dopamine release "Takahashi et al[\ 0887# indi! cates that the mechanisms of nicotine action and reward are complex and include other structures and neuroactive
compounds[ Dopaminergic changes in the accumbens alone\ while highlighting signi_cant stimuli\ may not be su.cient to explain the feeling of pleasure "Wickelgren\ 0886#[ Increase of dopamine in the nucleus accumbens does not necessarily account for self!administration* a measure of reward as a benzodiazepine "midazolam#\ which is self!administered\ decreases extracellular con! centrations of dopamine "Finlay et al[\ 0881#\ indicating that di}erent drugs alter the function of di}erent recep! tors or transporters in various brain structures[ The com! plexity and speci_city of the e}ects of drugs on the reward system are illustrated by the _nding that in measuring neuronal activity within the mesocorticolimbic circuit during self!administration of cocaine and heroin in rats\ about half of the neurons did not respond\ and most "64Ð
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Fig[ 5[ E}ects of atropine "ATR# on nicotine! and cocaine!induced dopamine increase[ Numbers in parenthesis indicate the dose "mg:kg# of nicotine\ cocaine\ and atropine[ Averages of three experiments2SEM are shown[
73)# of those that did responded di}erently to cocaine and heroin "Chang et al[\ 0887#[ Recent results indicate that in addition to the dopaminergic system\ numerous other receptors are involved in the various reward responses\ such as serotonin\ acetylcholine\ nore! pinephrine\ glutamate\ GABA receptors\ several pepti! des\ etc[\ and many structures\ in heterogeneous fashion "Bardo\ 0887#[ A recent view of reward mechanisms examining the action of drugs on several substrates of reinforcement concluded that no single factor is likely to explain addictive behavior in general or self!admin! istration in particular "White\ 0885#[ There is het! erogeneity in the receptors mediating the drug!induced rewarding e}ects*in nicotine administration nicotine receptors have a minor role in the ventral tegmentum\ while muscarinic receptors have a major role "Yeomans and Baptista\ 0886#[ The nicotinic receptors in the som! atodendritic region play a greater role than those in the
terminal region in nicotinic e}ects on the dopaminergic system "Nisell et al[\ 0883#[ Regional heterogeneity in the mechanism of nicotine e}ects is illustrated by the blocking by tetrodotoxin of nicotine!induced dopamine release in the striatum and accumbens\ but not in the cortex "Marshall et al[\ 0886#[ Nicotine administration a}ects the release of a number of neurotransmitters\ among them norepinephrine "Toth et al[\ 0881^ Tani et al[\ 0886# and glutamic acid "Toth et al[\ 0881^ Ashworth!Preece et al[\ 0887#^ the changes are regionally heterogeneous "Marshal et al[\ 0886#[ The released neurotransmitters subsequently interact with other receptors\ resulting in a cascade of changes[ Some dopamine release may be due to prior glutamate release via glutamate heteroreceptors on dopamine!containing terminals "Garcia!Munoz et al[\ 0885#[ In addition to receptors\ neurotransmitter transport systems are a}ec! ted[ Dopamine uptake inhibition in the striatum by nic!
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Fig[ 6[ E}ects of MK!790 on nicotine! and cocaine!induced dopamine increase[ Numbers in parenthesis indicate the dose "mg:kg# of nicotine\ cocaine\ MK!790\ and amphetamine[ Averages of three experiments2SEM are shown[
otine at concentrations that do not e}ect release was reported "Izenwasser et al[\ 0880#[ In the accumbens\ facilitation of dopamine transport was found "Hart and Ksir\ 0885#[ Among other central e}ects of nicotine are its in~uence on peptides\ such as substance P "Naftchi et al[\ 0877# and _broblast growth factor "Belluardo et al[\ 0887#[ Cocaine administration also results in changes in several neurotransmitters in various brain areas\ such as dopamine\ norepinephrine\ and serotonin "Reith et al[\ 0886#\ although its major e}ect is assumed to be the inhibition of dopamine uptake "Heikkila et al[\ 0864^ Woolverton and Johnson\ 0881#[ It is well established that mecamylamine blocks the central actions of nicotine\ not only dopamine release but also many other e}ects\ such as norepinephrine and ACTH release "Fu et al[\ 0886# and antinociception[ Mecamylamine is expected to inhibit nicotine!induced
dopamine release in the accumbens\ but not cocaine! induced increase\ which is mediated via uptake inhibition[ It is of interest that mecamylamine also inhibits the etha! nol!induced elevation of extracellular dopamine in the accumbens "Blomqvist et al[\ 0886#[ The fact that atropine also inhibits nicotine! but not cocaine!induced dopamine changes in the accumbens indicates that in addition to nicotinic\ muscarinic sites are also important[ There are a number of di}erences in alterations between nicotine! induced and cocaine!induced central e}ects[ The nic! otinic antagonist dihydroerythroidine infused in the ven! tral tegmental area inhibited nicotine self!administration but did not a}ect cocaine self!administration\ and atro! pine did not a}ect nicotine self!administration in these experiments "Corrigall et al[\ 0883#[ We previously found\ as did other laboratories\ that mecamylamine and atro! pine inhibit nicotine!induced dopamine release "Toth et
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Fig[ 7[ E}ects of SCH!12289 on nicotine! and cocaine!induced dopamine increase[ Numbers in parenthesis indicate the dose "mg:kg# of nicotine\ cocaine\ and SCH!12289[ Averages of three experiments2SEM are shown[
al[\ 0881#[ The relationship between dopamine and glu! tamate receptors has already been discussed*dopa! minergic stimulation a}ects glutamatergic activity and glutamatergic stimulation a}ects dopaminergic activity[ We reported previously that nicotine releases glutamate and that AMPA\ kainate\ and NMDA receptor antag! onists inhibit nicotine!induced dopamine release\ indi! cating that nicotine e}ects are partially mediated via glutamate release "Toth et al[\ 0881#[ Our present results with MK!790 con_rm this and indicate in addition that glutamate release does not have a major role in cocaine e}ects[ In a previous study intraperitoneally administered MK!790 inhibited behavioral sensitization to cocaine\ amphetamine\ and morphine but did not a}ect cocaine! or amphetamine!induced increase in dopamine levels in
the accumbens "Wolf et al[\ 0883#[ When MK!790 was administered in the nucleus accumbens via microdialysis probe before cocaine injection\ it signi_cantly inhibited the cocaine!induced increase in dopamine "Pap and Brad! berry\ 0884#[ At levels where nicotine e}ects are inhibited\ MK!790 does not a}ect cocaine!induced dopamine increase*at higher levels it may have an inhibitory e}ect on cocaine!induced changes as well[ It is not possible to unequivocally de_ne the mechanism of the inhibitory e}ect of MK!790[ Recent _ndings show that MK!790 is also an inhibitor of nicotinic receptors "Briggs and McKenna\ 0885^ Buisson and Bertrand\ 0887#^ therefore\ at lower levels it may act via nicotinic receptors[ We are now testing the e}ect of additional NMDA receptor antagonists with no reported a.nity to nicotinic recep!
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tors to de_ne the role of the glutamatergic system in nicotine! vs cocaine!induced dopamine release[ The e}ect of SCH!12289\ a dopamine D0 receptor antagonist\ indicates an important role for D0 receptors for both nicotine! and cocaine!induced changes[ It is of interest that in rodent tests SCH!12289 decreased nicotine self!administration\ while it increased cocaine self!administration "Corrigall and Coen\ 0880#\ since attenuation of elevation of dopamine levels tends to increase self!administration[ SCH!12289 injected in the amygdala or striatum also increased cocaine self!admin! istration "Caine et al[\ 0884#[ It did not inhibit nicotine! produced EEG alterations "Ferger and Kuschinsky\ 0883#[ SCH!12289 injected into the ventral tegmental area blocked cocaine!induced accumbens dopamine release\ but not cocaine!induced behavioral "motor stimulation# sensitization "Steketee\ 0887#[ The fact that cocaine!induced behavioral stimulation is not inhibited under these conditions indicates that the processes involved in the two e}ects of cocaine "dopamine increase and behavioral stimulation# are at least in part di}erent\ and not all processes depend on dopamine D0 receptor activity[ Although SCH!12289 was also reported to inhibit the dopamine transporter "Tomiyama et al[\ 0884# we feel it is more likely that it a}ected D0 receptors in our experiments[ When SCH!12289 was administered\ dopamine levels in the accumbens were not a}ected\ and when amphetamine was given after nicotine and cocaine administration\ SCH!12289 did not a}ect the amphet! amine!induced large increase in dopamine levels[ The _nding that dopamine!transporter knockout mice self! administer cocaine "Rocha et al[\ 0887# was interpreted to mean that serotonergic activity can sustain cocaine self!administration or other transporters can assume the role of dopamine transporter[ The above _ndings show that both nicotine and cocaine a}ect numerous compounds*neurotransmitters and other neuroactive compounds\ receptor and trans! port systems*to a di}erent degree in various areas of the brain via heterogeneous speci_c mechanisms[ Some of the changes are caused directly\ such as the action of nicotine on nicotinic receptors located on dopaminergic\ glutamatergic\ etc[\ cells^ some changes are indirect\ such as the released dopamine or glutamate in~uencing activity at additional sites[ The regional heterogeneity of the changes can be explained by the regional het! erogeneity of the distribution of the receptor systems involved[ It is now well known that the nicotinic ace! tylcholine receptor has multiple forms composed of di}erent subunits[ The di}erent pairing of the subunits generates di}erent types of receptors\ which have di}er! ent patterns of sensitivity to nicotine\ thus di}ering phar! macological character "Luetje and Patrick\ 0880#[ For example\ presynaptic nicotinic receptors associated with striatal dopamine and hippocampal norepinephrine ter! minals di}er pharmacologically "Clarke and Reuben\
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0885#\ and di}erent nicotinic receptor agonists di}er in their e}ects on dopamine release in the accumbens "Mirza et al[\ 0885#[ The subunit composition of receptors media! ting nicotine action is of importance*the b1 subunit of the acetylcholine receptor plays an important role\ since b1 subunit!de_cient mutant mice do not respond to nic! otine "Picciotto et al[\ 0887#[ Anabaseine and its deriva! tive "GTS!10#\ two nicotinic agonists\ showed di}erent e}ects in the cortex] anabaseine increased acetylcholine and had no e}ect on dopamine^ its derivative increased dopamine and had no e}ect on acetylcholine "Summers et al[\ 0886#[ Norepinephrine release seems to depend on a nicotinic receptor subunit di}erent from that for serotonin release "Xiangyang et al[\ 0887#[ Our results in this study are consistent with the multiple pharmacological character of both nicotine! and cocaine! induced changes in the brain\ involving alteration in a number of compounds and participation of a number of receptor systems\ with the pattern of changes in com! pounds and the pattern of participation of receptor sys! tems showing di}erences between nicotine! and cocaine! induced e}ects[ The heterogeneous distribution of phar! macologically di}erent receptors results in a drug!speci_c pattern of changes in compounds\ areas\ and receptors involved[ Our study was limited in that it focused on one brain area and measured the dopamine released[ It is likely that there are many other di}erences between the changes induced by nicotine and those induced by cocaine or other drugs in other brain areas*in neurotransmitter changes or in receptor subtypes involved[ It is important to realize that changes in the nucleus accumbens are in~uenced by those occurring in other areas[ The relation! ship of nicotine!induced release in the accumbens to that in the ventral tegmental area is well established\ with the nicotine receptors in the ventral tegmental area being more active in mediating nicotine!induced dopamine release in the accumbens than the nicotinic receptors in the accumbens itself "Nisell et al[\ 0884#[ It is likely that other receptors are also involved] metabotropic glu! tamate receptors stimulate dopamine release "Verma and Moghaddam\ 0887#\ and GABA receptors on ventral tegmental dopamine neurons stimulate accumbens dopa! mine release "Westerink et al[\ 0885#[ Recent studies of functional MRI imaging the brains of cocaine!dependent subjects given a dose of cocaine showed changes in activity in numerous brain areas in addition to the nucleus accumbens "among others in caudate\ putamen\ thalamus\ hippocampus\ pons\ several cortical regions\ etc[#\ with some di}erences between cocaine!induced euphoria and cocaine!induced craving "Breiter et al[\ 0886#\ showing the involvement of numerous areas and circuits in the brain[ Regional di}erences between cocaine and nicotine e}ects remain to be demonstrated[ It also has to be emphasized that both nicotine and cocaine may have several central e}ects not related to reward
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mechanisms[ For any therapeutic approach to cocaine or nicotine use\ and better understanding of reward systems\ knowledge of the speci_c mechanisms involved in various reward systems is of essential importance[
Acknowledgements The work was supported in part by a grant from Philip Morris\ USA[ The advice and help of Thomas Cooper and David Allen are gratefully acknowledged[
References Ashworth!Preece\ M[\ Jarrott\ B[\ Lawrence\ A[J[\ 0887[ Nicotinic ace! tylcholine receptors in the rat and primate nucleus tractus solitarius and on rat and human inferior vagal "nodose# ganglia] evidence from in vivo microdialysis and ð014IŁa!bungarotoxin auto! radiography[ Neuroscience 72\ 0002Ð0011[ Bardo\ M[T[\ 0887[ Neuropharmacological mechanisms of drug reward] beyond dopamine in the nucleus accumbens[ Critical Reviews in Neurobiology 01\ 26Ð56[ Belluardo\ N[\ Blum\ M[\ Mudo\ G[\ Andbjer\ B[\ Fuxe\ K[\ 0887[ Acute intermittent nicotine treatment produces regional increases of basic _broblast growth factor messenger RNA and protein in the tel! and diencephalon of the rat[ Neuroscience 72\ 612Ð639[ Blomqvist\ O[\ Ericson\ M[\ Engel\ J[A[\ Soderpalm\ B[\ 0886[ Accumbal dopamine over~ow after ethanol] localization of the antagonizing e}ect of mecamylamine[ European Journal of Pharmacology 223\ 038Ð045[ Breiter\ H[C[\ Gollub\ R[L[\ Weissko}\ R[M[\ Kennedy\ D[N[\ Makris\ N[\ Berke\ J[D[\ Goodman\ J[M[\ Kantor\ H[L[\ Gastfriend\ D[R[\ Riorden\ J[P[\ Mathew\ R[T[\ Rosen\ B[R[\ Hyman\ S[E[\ 0886[ Acute e}ects of cocaine on human brain activity and emotion[ Neuron 08\ 480Ð500[ Briggs\ C[A[\ McKenna\ D[G[\ 0885[ E}ect of MK!790 at the human alpha 6 nicotinic acetylcholine receptor[ Neuropharmacology 24\ 396Ð303[ Buisson\ B[\ Bertrand\ D[\ 0887[ Open!channel blockers at the human alpha3beta1 neuronal nicotinic acetylcholine receptor[ Molecular Pharmacology 42\ 444Ð452[ Caine\ S[B[\ Heinrichs\ S[C[\ Co.n\ V[L[\ Koob\ G[F[\ 0884[ E}ects of the dopamine D!0 antagonist SCH!12289 microinjected into the accumbens\ amygdala or striatum on cocaine self!administration in the rat[ Brain Research 581\ 36Ð45[ Chang\ J[!Y[\ Janak\ P[H[\ Woodward\ D[J[\ 0887[ Comparison of mesocorticolimbic neuronal responses during cocaine and heroin self!administration in freely moving rats[ The Journal of Neu! roscience 07\ 2987Ð2004[ Clarke\ P[B[\ Reuben\ M[\ 0885[ Release of ð2HŁnoradrenaline from rat hippocampal synaptosomes by nicotine] mediation by di}erent nicotinic receptor subtypes from striatal ð2HŁdopamine release[ Brit! ish Journal of Pharmacology 006\ 484Ð595[ Corrigall\ W[A[\ Coen\ K[M[\ 0880[ Selective dopamine antagonists reduce nicotine self!administration[ Psychopharmacology 093\ 060Ð 065[ Corrigall\ W[A[\ Cohen\ K[M[\ Adamson\ K[L[\ 0883[ Self!administered nicotine activates the mesolimbic dopamine system through the ventral tegmental area[ Brain Research 542\ 167Ð173[ Corrigall\ W[A[\ Franklin\ K[B[\ Coen\ K[M[\ Clarke\ P[B[\ 0881[ The mesolimbic dopaminergic system is implicated in the reinforcing e}ects of nicotine[ Psychopharmacology 096\ 174Ð178[ Damsma\ G[\ Day\ J[\ Fibiger\ 0878[ Lack of tolerance to nicotine!
induced dopamine release in the nucleus accumbens[ European Journal of Pharmacology 057\ 252Ð257[ Davis\ R[A[\ 0875[ The determination of nicotine and cotinine in plasma[ Journal of Chromatographic Science 13\ 023Ð030[ Ferger\ B[\ Kuschinsky\ K[\ 0883[ Activation of dopamine D0 receptors of alpha 0 adrenoceptors is not involved in the EEG e}ect of nicotine in rats[ Naunyn!Schmiedebergs Archives of Pharmacology 249\ 235Ð240[ Finlay\ J[M[\ Damsma\ G[\ Fibiger\ H[C[\ 0881[ Benzodiazepine! induced decreases in extracellular concentrations of dopamine in the nucleus accumbens after acute and repeated administration[ Psychopharmacology 095\ 191Ð197[ Fischman\ M[W[\ Schuster\ C[R[\ Resnekov\ L[\ Shick\ J[F[E[\ Kras! negor\ N[A[\ Fennell\ W[\ Freedman\ D[X[\ 0865[ Cardiovascular and subjective e}ects of intravenous cocaine administration in humans[ Archives of General Psychiatry 22\ 872Ð878[ Fu\ Y[\ Matta\ S[G[\ Valentine\ J[D[\ Sharp\ B[M[\ 0886[ Adre! nocorticotropin response and nicotine!induced norepinephrine secretion in the rat paraventricular nucleus are mediated through brainstem receptors[ Endocrinology 027\ 0824Ð0832[ Garcia!Munoz\ M[\ Patino\ P[\ Young\ S[J[\ Groves\ P[M[\ 0885[ E}ects of nicotine on dopaminergic nigrostratal axons requires stimulation of presynaptic glutamatergic receptors[ Journal of Pharmacology + Experimental Therapeutics 166\ 0574Ð0582[ Hart\ C[\ Ksir\ C[\ 0885[ Nicotine e}ects on dopamine clearance in rat nucleus accumbens[ Journal of Neurochemistry 55\ 105Ð110[ Heikkila\ R[E[\ Orlansky\ H[\ Cohen\ G[\ 0864[ Studies on the dis! tinction between uptake inhibition and release of ð2HŁdopamine in rat brain tissue slices[ Biochemical Pharmacology 13\ 736Ð741[ Izenwasser\ S[\ Jacocks\ H[M[\ Rosenberger\ J[G[\ Cox\ B[M[\ 0880[ Nicotine indirectly inhibits ð2HŁdopamine uptake at concentrations that do not directly promote ð2HŁdopamine release in rat striatum[ Journal of Neurochemistry 45\ 592Ð509[ Lapin\ E[P[\ Maker\ H[S[\ Sershen\ H[\ Hurd\ Y[\ Lajtha\ A[\ 0876[ Dopamine!like action of nicotine] lack of tolerance and reverse tolerance[ Brain Research 396\ 240Ð252[ Levin\ E[D[\ 0881[ Nicotinic systems and cognitive function[ Psy! chopharmacology 097\ 306Ð320[ Luetje\ C[W[\ Patrick\ J[\ 0880[ Both a! and b!subunits contribute to the agonist sensitivity of neuronal nicotinic acetylcholine receptors[ The Journal of Neuroscience 00\ 726Ð734[ Marshall\ D[L[\ Redfern\ P[H[\ Wonnacott\ S[\ 0886[ Presynaptic nic! otinic modulation of dopamine release in the three ascending path! ways studied by in vivo microdialysis] comparison of na(ve and chronic nicotine!treated rats[ Journal of Neurochemistry 57\ 0400Ð 0408[ Mifsud\ J[!C[\ Hernandez\ L[\ Hoebel\ B[G[\ 0878[ Nicotine infused into the nucleus accumbens increases synaptic dopamine as measured by in vivo microdialysis[ Brain Research 367\ 254Ð256[ Mirza\ N[R[\ Pei\ Q[\ Stolerman\ I[P[\ Zetterstrom\ T[S[\ 0885[ The nicotinic receptor agonists "−#!nicotine and isoarecolone di}er in their e}ects on dopamine release in the nucleus accumbens[ Eur! opean Journal of Pharmacology 184\ 196Ð109[ Naftchi\ N[E[\ Maker\ H[\ Lapin\ E[\ Sleis\ J[\ Lajtha\ A[\ Leeman\ S[\ 0877[ Acute reduction of brain substance P induced by nicotine[ Neurochemical Research 02\ 294Ð298[ Nisell\ M[\ Nomikos\ G[G[\ Svensson\ T[H[\ 0883[ Systemic nicotine! induced dopamine release in the rat nucleus accumbens is regulated by nicotinic receptors in the ventral tegmental area[ Synapse 05\ 25Ð 33[ Nisell\ M[\ Nomikos\ G[G[\ Svensson\ T[H[\ 0884[ Nicotine dependence\ midbrain dopamine systems and psychiatric disorders[ Phar! macology + Toxicology 65\ 046Ð051[ Pap\ A[\ Bradberry\ C[W[\ 0884[ Excitatory amino acid antagonists attenuate the e}ects of cocaine on extracellular dopamine in the nucleus accumbens[ The Journal of Pharmacology and Exper! imental Therapeutics 163\ 016Ð022[
I[ Sziraki et al[:Neurochem[ Int[ 22 "0887# 334Ð346 Paxinos\ G[\ Watson\ C[\ 0875[ The rat brain in stereotaxic coordinates[ Academic Press\ New York[ Picciotto\ M[R[\ Zoli\ M[\ Rimondini\ R[\ Lena\ C[\ Marubio\ L[M[\ Pich\ E[M[\ Fuxe\ K[\ Changeux\ J[!P[\ 0887[ Acetylcholine recep! tors containing the b1 subunit are involved in the reinforcing proper! ties of nicotine[ Nature 280\ 062Ð066[ Pontieri\ F[E[\ Tanda\ G[\ Orzi\ F[\ Di Chiara\ G[\ 0885[ E}ects of nicotine on the nucleus accumbens and similarity to those of addict! ive drugs[ Nature 271\ 144Ð146[ Reith\ M[E[A[\ Li\ M[!Y[\ Yan\ Q[!S[\ 0886[ Extracellular dopamine\ norepinephrine\ and serotonin in the ventral tegmental area and nucleus accumbens of freely moving rats during intracerebral dialy! sis following systemic administration of cocaine and other uptake blockers[ Psychopharmacology 023\ 298Ð206[ Rocha\ B[A[\ Fumagalli\ F[\ Gainetdinov\ R[R[\ Jones\ S[R[\ Ator\ R[\ Giros\ B[\ Miller\ G[W[\ Caron\ M[C[\ 0887[ Cocaine self!admin! istration in dopamine!transporter knockout mice[ Nature Neu! roscience 0\ 021Ð026[ Rose\ J[E[\ Corrigall\ W[A[\ 0886[ Nicotine self!administration in ani! mals and humans] similarities and di}erences[ Psychopharmacology 029\ 17Ð39[ Rowell\ P[P[\ Carr\ L[A[\ Garner\ A[C[\ 0876[ Stimulation of ð2HŁdo! pamine release by nicotine in rat nucleus accumbens[ Journal of Neurochemistry 38\ 0338Ð0343[ Steketee\ J[D[\ 0887[ Injection of SCH!12289 into the ventral tegmental area blocks the development of neurochemical but not behavioral sensitization to cocaine[ Behavioural Pharmacology 8\ 58Ð65[ Summers\ K[L[\ Kem\ W[R[\ Giacobini\ E[\ 0886[ Nicotinic agonist modulation of neurotransmitter levels in the rat frontoparietal cortex[ Japanese Journal of Pharmacology 63\ 028Ð035[ Takahashi\ H[\ Takada\ Y[\ Nagai\ N[\ Urano\ T[\ Takada\ A[\ 0887[ E}ects of nicotine and footshock stress on dopamine release in the striatum and nucleus accumbens[ Brain Research Bulletin 34\ 046Ð 051[ Tani\ Y[\ Saito\ K[\ Tsuneyoshi\ A[\ Imoto\ M[\ Ohno\ T[\ 0886[ Nic! otinic acetylcholine receptor "nACh!R# agonist!induced changes in brain monoamine turnover in mice[ Psychopharmacology 018\ 114Ð 121[ Tomiyama\ K[\ Koshikawa\ N[\ Funada\ K[\ Oka\ K[\ and Kobayashi\
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M[\ 0884[ In vivo microdialysis evidence for transient dopamine release by benzazepines in rat striatum[ Journal of Neurochemistry 54\ 1689Ð1684[ Toth\ E[\ Sershen\ H[\ Hashim\ A[\ Vizi\ E[S[\ Lajtha\ A[\ 0881[ E}ect of nicotine on extracellular levels of neurotransmitters assessed by microdialysis in various brain regions] role of glutamic acid[ Neu! rochemical Research 06\ 154Ð160[ Verma\ A[\ Moghaddam\ B[\ 0887[ Regulation of striatal dopamine release by metabotropic glutamate receptors[ Synapse 17\ 119Ð115[ Wayneforth\ H[B[\ Flecknell\ P[A[\ 0881[ Catheterisation of the jugular vein[ In] Waynforth\ H[B[\ Flecknell\ P[A[ "Eds[# Experimental and Surgical Technique in the Rat[ 1nd ed[\ Academic Press\ pp[ 104Ð 111[ Westerink\ B[H[\ Kwint\ H[F[\ de Vries\ J[B[\ 0885[ The pharmacology of mesolimbic dopamine neurons] a dual!probe microdialysis study in the ventral tegmental area and nucleus accumbens of the rat brain[ Journal of Neuroscience 05\ 1594Ð1500[ Westerink\ B[H[C[\ Kwint\ H[!F[\ de Vries\ J[B[\ 0886[ Eating!induced dopamine release from mesolimbic neurons is mediated by NMDA receptors in the ventral tegmental area] A dual!probe microdialysis study[ Journal of Neurochemistry 58\ 551Ð557[ White\ N[M[\ 0885[ Addictive drugs are reinforcers] multiple partial actions on memory systems[ Addiction 80\ 8101Ð838[ Wickelgren\ I[\ 0886[ Getting the brain|s attention[ Science 167\ 24Ð26[ Wolf\ M[E[\ Xue\ C[!J[\ White\ F[J[\ Dahlin\ S[L[\ 0883[ MK!790 does not prevent acute stimulatory e}ects of amphetamine or cocaine on locomotor activity or extracellular dopamine levels in rat nucleus accumbens[ Brain Research 555\ 112Ð120[ Woolverton\ W[L[\ Johnson\ K[M[\ 0881[ Neurobiology of cocaine abuse[ Trends in Pharmacological Sciences 02\ 082Ð199[ Xiangyang\ L[\ Rainnie\ D[G[\ McCarley\ R[W[\ Greene\ R[W[\ 0887[ Presynaptic nicotinic receptors facilitate monoaminergic trans! mission[ The Journal of Neuroscience 07\ 0893Ð0801[ Yeomans\ J[\ Baptista\ M[\ 0886[ Both nicotinic and muscarinic recep! tors in ventral tegmental area contribute to brain!stimulation reward[ Pharmacology\ Biochemistry + Behavior 46\ 804Ð810[ Zernig\ G[\ O|Laughlin\ I[A[\ Fibiger\ H[C[\ 0886[ Nicotine and heroin augment cocaine!induced dopamine over~ow in nucleus accumbens[ European Journal of Pharmacology 226\ 0Ð09[
Receptor systems participating in nicotine!speci_c e}ects Istvan Sziraki\ Henry Sershen\ Myron Benuck\ Audrey Hashim\ Abel Lajtha Center for Neurochemistry\ The Nathan S[ Kline Institute\ 039 Old Oran`ebur` Rd\ Oran`ebur`\ NY 09851\ U[S[A[ Received 04 June 0887^ accepted 17 July 0887
Abstract It is generally accepted that self!administration of drugs is prompted primarily by a reward system driven by an increase in extracellular dopamine in the nucleus accumbens[ Recent _ndings that dopamine increase in the accumbens can be caused by many other factors\ among them stress\ suggest a more complex mechanism\ and possibly di}erences in the reward system for di}erent compounds[ In the present paper we compare the e}ects of receptor!speci_c antagonists on the increase of dopamine induced by nicotine with that induced by cocaine in the nucleus accumbens in conscious rats[ The compounds alone or together were injected intravenously\ and dopamine level changes were measured via microdialysis[ When administered together the e}ect of nicotine and cocaine on the level of dopamine in the accumbens was additive[ Apparently there is some interaction between the two compounds\ since nicotine had no e}ect after combined nicotine and cocaine administration[ Perhaps the available dopamine pool was exhausted by the prior administration[ The nicotinic antagonist mecamylamine\ the muscarinic antagonist atropine\ and the NMDA glutamate receptor antagonist MK!790 each blocked nicotine!induced dopamine release in the accumbens\ indicating the participation of more than a single receptor system in the nicotine!induced e}ect[ These three antagonists did not inhibit cocaine!induced dopamine increase in the accumbens\ indicating the lack of a role of these receptors in the cocaine e}ect under our experimental conditions[ SCH!12289\ a dopamine D0 receptor antagonist\ blocked both nicotine! and cocaine!induced e}ects\ indicating the possible role of this receptor in these reward e}ects[ The results indicate that there are di}erences in some of the receptors mediating the central e}ects of the two compounds examined\ nicotine and cocaine\ although each in~uences dopamine levels\ and that the two compounds interact[ Þ 0887 Elsevier Science Ltd[ All rights reserved[
0[ Introduction Although it has been found in numerous studies that the e}ect of nicotine constituting the major impetus for smoking is on the mesolimbic reward mechanism dopa! mine release in the nucleus accumbens "Rowell et al[\ 0876^ Corrigall et al[\ 0881^ Pontieri et al[\ 0885#\ recent _ndings suggest that the changes induced by nicotine\ and indeed by most compounds altering dopamine levels in the accumbens\ are complex and involve e}ects on several neurotransmitters[ Nicotine and cocaine have been shown to a}ect the levels of a number of other neurotransmitters and of neuropeptides in several brain structures\ in a heterogeneous manner "Naftchi et al[\ 0877^ Toth et al[\ 0881^ Reith et al[\ 0886#[ In addition to the reward system\ nicotine has in~uence on several other systems that could alter behavior\ including cognition\
Corresponding author[ Tel[] 990 803 287 4429^ Fax] 990 803 287 4420^ E!mail] lajthaÝnki[rfmh[org
learning\ memory\ arousal\ etc[ "Levin\ 0881^ Bardo\ 0887#[ It is likely that a set of the changes in the various structures and in behavior is speci_c for nicotine\ and that di}erent speci_c sets of systems are involved in various nicotine e}ects[ Although similar release of dopamine by nicotine and cocaine has been used as evidence for similar mechanisms of action "Pontieri et al[\ 0885#\ our study examines whether functional neurochemical changes similar to those caused by cocaine extend to the other receptor systems participating in nicotine e}ects[ In our long!term study of central nicotine e}ects and speci_c reward mechanisms we are attempting to identify the various changes in brain structures that are induced by nicotine administration\ and the systems mediating these changes\ and to identify those that are speci_c for nicotine\ and to understand each of the mechanisms of the various nicotine e}ects[ In the present study we compare nicotine!induced with cocaine!induced changes of the levels of dopamine in the nucleus accumbens\ using antagonists to various recep! tors to try to identify the receptor subtypes mediating the action of these two compounds in this structure[
9086Ð9075:87:, ! see front matter Þ 0887 Elsevier Science Ltd[ All rights reserved PII] S 9 0 8 6 Ð 9 0 7 5 " 8 7 # 9 9 9 3 8 Ð 6
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1[ Methods
1[2[ Nicotine assay
1[0[ Animals
In the experiments to determine the brain and blood nicotine concentrations at di}erent time points after the administration of nicotine\ the rats were killed by decapi! tation[ The brain was quickly removed and the various regions were dissected out on an ice!cold rubber sheet\ placed in plastic tubes\ and frozen on dry ice[ After the decapitation trunk blood was mixed with 9[8) saline containing EDTA "_nal concentration] 9[4 mg EDTA per ml of blood#[ The brain and blood samples were kept at −79>C until processed for extraction[ For the determination of nicotine using gas chromatography\ the procedure of Davis "0875# was followed[ Nicotine was extracted from brain homogenates or blood by adding an equal volume of 4 N NaOH\ 1 volumes of methyl chloride\ and N!ethyl!nornicotine for internal standard\ mixing for 4 min\ and separating the aqueous and organic phases by centrifugation[ The upper aqueous phase was discarded\ including the lipid interface\ by aspiration^ 2 volumes of isopropyl alcohol were added to the organic phase\ which was concentrated to 0:4 using an N!evap evaporator at 39>C and transferred to automatic injector vials\ with 1 ml injected for assay[ For gas chro! matography a Carbowax DB megabore 29!meter column was used\ the injector and detector temperature was set at 124>C\ and the column temperature was set initially at 79>C for 0 min\ increased 29>C per min to 059>C\ kept there for 4 min\ then increased again 29>C per min to 159>C\ till the end of the run[ The ND detector was run in the nitrogen mode[ In human plasma the method had an intra! and interassay RSD) of 3[6 and 4[8) respec! tively[ The extraction recovery was over 85)\ and the sensitivity of the determination was 0 ng:ml[
Male Sprague!Dawley rats bred in our animal facility\ as in our previous studies with nicotine\ were used for the present experiments[ We selected this strain because we had used it previously to measure nicotine!induced neurotransmitter changes in awake animals\ and thus our _ndings with cocaine and nicotine can be compared to our previous _ndings under similar experimental con! ditions in the same laboratory[ In experiments to determine brain and blood nicotine levels at di}erent times after intravenous nicotine injection the rats were 179Ð249 g\ and for microdialysis studies they were 179Ð 219 g[
1[1[ Administration of dru`s Nicotine\ cocaine\ and other drugs tested for their capacity to alter nicotine! or cocaine!induced changes in dopamine in the extracellular space were administered through a cannula inserted under anesthesia into the right jugular vein[ The catheterization was carried out by a standard procedure "Waynforth and Flecknell\ 0881# with some modi_cations[ A second thread was applied in the immediate proximity of the tied {anterior thread|[ The second thread had longer ends after the knot was made[ The tie was around the cannula ðTFE tubing "17 g# from Zeus Industrial ProductsŁ after it had already been inserted into the vein and secured with the {posterior| threads[ Two ligatures were applied also at the posterior position[ The cannula was _lled with heparin!saline solu! tion "39 units of heparin per ml#[ The catheter tubing was routed under the skin and exited in the back of the animal[ Cannulation of the jugular vein was followed by implan! tation of a brain guide cannula and the vein cannula was attached to a microsyringe pump "CMA# through a dual! channel liquid swivel "CMA system for freely moving rats# and heparinÐsaline was infused at a rate of 9[0 ml:min until the beginning of the microdialysis experi! ment the next day[ All drugs "from Sigma or RBI# were dissolved in 9[8) saline[ Nicotine and cocaine were always administered by bolus injection "in a volume of 099 ml# through the catheter tubing washed with an equal volume of heparinÐ saline[ All other drugs were administered _rst as bolus injections "either alone or in combination with nicotine or cocaine# and then continued via infusion at the rate of 0 ml:min using a microsyringe pump as indicated in the legends and in the _gures[ The doses given in the _gures represent the free base forms of the drugs with the excep! tion of MK!790 where the dose refers to the hydrogen maleate form of the drug[
1[3[ Brain microdialysis This was performed as described previously "Toth et al[\ 0881#[ After completion of the catheterization of the jugu! lar vein the anesthetized rats were placed in a Kopf ster! eotaxic apparatus[ A hole was drilled in the skull directly above the site selected for microdialysis[ The guide can! nula "BAS or CMA# was lowered to 1 mm above the selected site in the brain and _xed to the skull with dental cement[ The stereotaxic coordinates for nucleus accum! bens with respect to the bregma and the surface of the skull were\ as used before "Pontieri et al[\ 0885#\ AP] ¦1[9\ ML] −0[1\ DV] −5\ according to the rat brain atlas "Paxinos and Watson\ 0875#[ Experiments using the coordinates AP] ¦0[6\ ML] −9[7\ DV] −4[7 gave similar results[ After surgery the animals were placed in the bowl of the CMA system and both the vein catheter and the microdialysis probe tubing were connected to a dualÐ swivel attached to the lever arm[ The animals were given unlimited food and water during the experimental period[
I[ Sziraki et al[:Neurochem[ Int[ 22 "0887# 334Ð346
1[4[ Determination of dopamine A microdialysis probe "BAS or CMA# with a 1!mm mem! brane was inserted into the guide cannula either in the morning of the day of the experiment or in the evening of the previous day[ The probe was perfused with Ringer at 0 ml:min throughout the experiment[ At least 1 h elap! sed before the _rst injection of any of the drugs[ "If the probe was inserted on the previous day it was perfused overnight at 9[0 ml:min and the next morning the speed was set at 0 ml:min[# The dialysates of 04Ð29 min periods were collected in polyethylene microtubes[ Dialysate samples were manually injected into the injector valve of a BAS 199 A HPLC system equipped with a BAS Unijet detector cell "5 mm radial ~ow cell# set at 399 mV and a C07\ 4 mM\ 049×0 mm microbore column[ The mobile phase consisted of 099 mM NaH1PO3\ 124 mM Na1EDTA\ 09 mM NaCl\ 09 mM diethylamine\ 249 mg of sodium octyl sulfate and 44 ml of acetonitrile per liter\ adjusted to pH 4[7[ Reverse!phase liquid chro! matography with electrochemical detection was used[ After completion of the brain microdialysis the pos! ition of the catheter in the vein was checked\ then the rats were killed with an overdose of sodium pentobarbital "099 mg:kg#[ The brain was removed and kept in 09) formaldehyde[ The probe placement was veri_ed his! tologically[
2[ Results The purpose of the present study was to compare receptor subtypes participating in nicotine!induced vs cocaine! induced dopamine increase in the nucleus accumbens[ One question involved relevant doses of these com! pounds*doses for animals that are equivalent to human use of these compounds[ In the case of nicotine\ this would be a dose matching that used by smokers[ A ciga! rette yielding 0 mg of nicotine "FTC# for a 56!kg person would represent a dose of 04 mg:kg\ not in a single bolus\ but during the smoking of a cigarette[ Smokers inhale 04Ð29 mg:kg nicotine per cigarette\ and 09Ð34 mg:kg is the range for self!administration "Rose and Corrigall\ 0886#[ Using a bolus of 4 mg:kg we could not detect a measurable increase in dopamine release in the accum! bens[ With a 14 mg:kg dose\ some increase could be detected\ but it was too small and variable to examine its inhibition by receptor subtype antagonists^ therefore\ a dose of nicotine of 49 mg:kg\ 1Ð2 times the amount from smoking a cigarette\ was used in subsequent experiments[ A study of the e}ect of intravenously administered nico! tine on accumbens "shell# dopamine levels found rela! tively small changes "14Ð29)# after 14 mg:kg but signi_cant changes "59)# after 49 mg:kg "Pontieri et al[\ 0885#[ We estimate that the dose of nicotine used in the present study would result in cerebral nicotine levels 2Ð4
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times higher than levels in the brain of smokers[ The time course of nicotine levels in the brain after a single injection of 4 or 14 mg:kg nicotine intravenously is shown in Fig[ 0[ Brain concentrations reach about 04 mg:kg at administration of a 4 mg:kg dose\ and about 31 mg:kg at a 14 mg:kg dose[ The half!life of nicotine in these experiments was about 29 min "Fig[ 0#[ To examine nico! tine accumulation with intermittent administration as in smoking\ we administered 1 mg:kg doses at 29 s intervals[ After 4 doses of 1 mg:kg "09 mg:kg total in 2 min# brain levels reached 29 mg:kg and after 09 doses "19 mg total# brain levels reached 49Ð59 mg\ decreasing to about half in 29 min "Fig[ 1#[ There was some regional heterogeneity in nicotine levels in the four brain areas examined "Fig[ 1#[ At each time point "2\ 5\ and 29 min#\ brain nicotine levels were 2Ð4 times higher than blood levels "Fig[ 1#[ The cocaine dose used was 149 mg:kg\ a dose su.cient for self!administration maintenance in rats "Corrigall et al[\ 0883#[ This dose is very similar to the average dose self!injected by drug addicts^ subjects have been reported to self!inject up to twice this dose "Fischman et al[\ 0865#[ An increase in dopamine levels could be measured following either 49 mg:kg nicotine or 149 mg:kg cocaine administration[ With administration at the same time\ nicotine and cocaine e}ects were additive as observed previously "Zernig et al[\ 0886#[ Cocaine administration following a nicotine plus cocaine treatment resulted in dopamine release^ however\ nicotine following nicotine plus cocaine resulted in no release of dopamine "Fig[ 2A\ 2B#[ To determine whether the additive e}ect of nicotine¦cocaine administration is due to the same or to two di}erent processes and is from similar dopamine pools\ we administered nicotine in place of cocaine in multiple administration experiments[ Whereas there was no nicotine!induced dopamine release after simultaneous administration of cocaine and nicotine "Fig[ 2B#\ dopa! mine release by nicotine could be observed when cocaine was replaced by nicotine "Fig[ 3A#[ Nicotine!induced increase in dopamine levels could be observed when nic! otine was given following an administration of cocaine "Fig[ 3B#[ This suggests that the administration of cocaine plus nicotine depletes a dopamine pool that responds to nicotine but not one responding to cocaine[ Repeated nicotine administration does not deplete this pool under our experimental conditions[ 2[0[ Effect of anta`onists on nicotine! and cocaine!induced elevation of dopamine in the accumbens We examined possible di}erences in the mechanisms of nicotine! and cocaine!induced changes by administering the three compounds\ antagonist\ nicotine\ and cocaine\ intravenously and measuring changes in extracellular dopamine levels in the accumbens via microdialysis[ The nicotinic receptor antagonist mecamylamine\ as in most systems tested before\ abolished nicotine!induced chan!
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Fig[ 0[ Half!life of nicotine in the brain[ Brain nicotine concentrations at various times after a single intravenous injection of 4 "ОÐ# or 14 "ÐEÐ# mg:kg nicotine are given[ Values are expressed as mean2SEM "N 2Ð5#[
Fig[ 1[ Nicotine concentrations in blood and various brain regions after repeated intravenous nicotine administration[ Two mg of nicotine per kg of body weight was injected in 29 s intervals 4 or 09 times[ Times given are after the _rst injection[ Values are expressed as ng per g of tissue\ mean2SEM "N 2#[
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Fig[ 2[ E}ects of nicotine "NIC# and cocaine "COC# on dopamine "DA# levels in nucleus accumbens[ Numbers in parenthesis indicate the dose "mg:kg# of nicotine or cocaine administered as a bolus intravenously either alone or in combination[ Averages of three experiments2SEM are shown[
ges^ mecamylamine did not a}ect cocaine!induced chan! ges "Fig[ 4#[ The results were similar whether the sequence of administration was nicotine followed later by cocaine "Fig[ 4A# or cocaine followed by nicotine "Fig[ 4B#[ The e}ect of atropine\ a muscarinic antagonist\ was similar to that of mecamylamine[ Nicotine!induced e}ects on accumbens dopamine levels were inhibited by atropine\ while cocaine e}ects on dopamine were not changed\ with no di}erence whether nicotine or cocaine was admin! istered _rst "Fig[ 5#[ The results shown in Figs 4 and 5 indicate that both nicotinic and muscarinic cholinergic receptors have an important role in nicotine!induced dopamine release in the accumbens\ while cholinergic receptors do not have a signi_cant role in cocaine e}ects on dopamine levels[ A similar di}erence between nicotine! and cocaine!
induced dopamine changes in the accumbens was seen after the administration of the NMDA!type glutamate receptor antagonist MK!790 "dizocilpine#[ This com! pound signi_cantly inhibited nicotine e}ects\ while cocaine e}ects were little a}ected^ as before\ the sequence of administration "nicotine _rst or cocaine _rst# did not a}ect the results "Fig[ 6#[ To see whether the reaction to other compounds is a}ected in these experiments\ or the inhibition is due to exhaustion of relevant dopamine pools\ we administered a high dose of amphetamine intra! venously at the end of these experiments[ Amphetamine was much more e}ective in increasing dopamine levels in the accumbens than either nicotine or cocaine\ and its e}ects were not altered by MK!790 infusion "Fig[ 6#[ These _ndings indicate that there was a su.ciently large dopamine pool available for release[ Our dose of
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Fig[ 3[ E}ects of repeated administration on dopamine levels in nucleus accumbens[ Numbers in parenthesis indicate the dose "mg:kg# of nicotine and amphetamine "AMPH#[ Averages of three experiments2SEM are shown[ Panel A] repeated nicotine administration^ Panel B] cocaine administration followed by nicotine[
MK!790 caused a temporary hind leg paralysis*this was not altered by amphetamine administration[ These experiments are in agreement with studies showing the interaction of dopaminergic and glutamatergic systems^ with glutamate increase releasing dopamine and increased dopamine releasing glutamate[ It seems that such interaction is more important in the action of nic! otine than in that of cocaine[ In examining dopaminergic antagonists\ the admin! istration of SCH!12289\ a D0 dopamine receptor antag! onist\ was inhibitory to both the cocaine! and the nicotine!induced e}ect on dopamine levels "Fig[ 7#[ This indicates the possible role of this receptor subtype in the e}ect of both compounds in altering dopamine levels in
the accumbens\ and shows that appropriate antagonists can block both cocaine e}ects and nicotine e}ects[ The inhibition of cocaine!induced increase in dopamine levels raises the possibility of the involvement of dopamine receptors in addition to a direct e}ect on dopamine trans! porters in cocaine!induced e}ects[
3[ Discussion Nicotine administration alters dopaminergic activity and increases dopamine levels in the nucleus accumbens "Lapin et al[\ 0876^ Mifsud et al[\ 0878^ Damsma et al[\ 0878#[ Nicotine a}ects a mechanism generally associated
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Fig[ 4[ E}ects of mecamylamine "MEC# on nicotine! and cocaine!induced dopamine increase[ Numbers in parenthesis indicate the dose "mg:kg# of nicotine\ cocaine\ and mecamylamine[ Nicotine and cocaine were administered as a bolus\ mecamylamine as a bolus followed by constant infusion[ Averages of three experiments2SEM are shown in panel A^ averages of two experiments are shown in panel B[
with reinforcement and reward] an increase in dopamine in the accumbens is proposed to operate as reinforcement for self!administration of several other compounds\ and reinforcement of other activities that may include feeding\ sexual\ and motor activity\ etc[ Eating\ among other things\ induces release of dopamine in the accumbens "Westerink et al[\ 0886#[ Because of the association of dopamine increases in the accumbens with reward\ a simi! lar e}ect of nicotine is assumed to be the one in the brain that provides the motivation for smoking[ The fact that aversive stimuli may also a}ect dopamine levels in the accumbens "for example\ foot!shock stress induces dopamine release "Takahashi et al[\ 0887# indi! cates that the mechanisms of nicotine action and reward are complex and include other structures and neuroactive
compounds[ Dopaminergic changes in the accumbens alone\ while highlighting signi_cant stimuli\ may not be su.cient to explain the feeling of pleasure "Wickelgren\ 0886#[ Increase of dopamine in the nucleus accumbens does not necessarily account for self!administration* a measure of reward as a benzodiazepine "midazolam#\ which is self!administered\ decreases extracellular con! centrations of dopamine "Finlay et al[\ 0881#\ indicating that di}erent drugs alter the function of di}erent recep! tors or transporters in various brain structures[ The com! plexity and speci_city of the e}ects of drugs on the reward system are illustrated by the _nding that in measuring neuronal activity within the mesocorticolimbic circuit during self!administration of cocaine and heroin in rats\ about half of the neurons did not respond\ and most "64Ð
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Fig[ 5[ E}ects of atropine "ATR# on nicotine! and cocaine!induced dopamine increase[ Numbers in parenthesis indicate the dose "mg:kg# of nicotine\ cocaine\ and atropine[ Averages of three experiments2SEM are shown[
73)# of those that did responded di}erently to cocaine and heroin "Chang et al[\ 0887#[ Recent results indicate that in addition to the dopaminergic system\ numerous other receptors are involved in the various reward responses\ such as serotonin\ acetylcholine\ nore! pinephrine\ glutamate\ GABA receptors\ several pepti! des\ etc[\ and many structures\ in heterogeneous fashion "Bardo\ 0887#[ A recent view of reward mechanisms examining the action of drugs on several substrates of reinforcement concluded that no single factor is likely to explain addictive behavior in general or self!admin! istration in particular "White\ 0885#[ There is het! erogeneity in the receptors mediating the drug!induced rewarding e}ects*in nicotine administration nicotine receptors have a minor role in the ventral tegmentum\ while muscarinic receptors have a major role "Yeomans and Baptista\ 0886#[ The nicotinic receptors in the som! atodendritic region play a greater role than those in the
terminal region in nicotinic e}ects on the dopaminergic system "Nisell et al[\ 0883#[ Regional heterogeneity in the mechanism of nicotine e}ects is illustrated by the blocking by tetrodotoxin of nicotine!induced dopamine release in the striatum and accumbens\ but not in the cortex "Marshall et al[\ 0886#[ Nicotine administration a}ects the release of a number of neurotransmitters\ among them norepinephrine "Toth et al[\ 0881^ Tani et al[\ 0886# and glutamic acid "Toth et al[\ 0881^ Ashworth!Preece et al[\ 0887#^ the changes are regionally heterogeneous "Marshal et al[\ 0886#[ The released neurotransmitters subsequently interact with other receptors\ resulting in a cascade of changes[ Some dopamine release may be due to prior glutamate release via glutamate heteroreceptors on dopamine!containing terminals "Garcia!Munoz et al[\ 0885#[ In addition to receptors\ neurotransmitter transport systems are a}ec! ted[ Dopamine uptake inhibition in the striatum by nic!
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Fig[ 6[ E}ects of MK!790 on nicotine! and cocaine!induced dopamine increase[ Numbers in parenthesis indicate the dose "mg:kg# of nicotine\ cocaine\ MK!790\ and amphetamine[ Averages of three experiments2SEM are shown[
otine at concentrations that do not e}ect release was reported "Izenwasser et al[\ 0880#[ In the accumbens\ facilitation of dopamine transport was found "Hart and Ksir\ 0885#[ Among other central e}ects of nicotine are its in~uence on peptides\ such as substance P "Naftchi et al[\ 0877# and _broblast growth factor "Belluardo et al[\ 0887#[ Cocaine administration also results in changes in several neurotransmitters in various brain areas\ such as dopamine\ norepinephrine\ and serotonin "Reith et al[\ 0886#\ although its major e}ect is assumed to be the inhibition of dopamine uptake "Heikkila et al[\ 0864^ Woolverton and Johnson\ 0881#[ It is well established that mecamylamine blocks the central actions of nicotine\ not only dopamine release but also many other e}ects\ such as norepinephrine and ACTH release "Fu et al[\ 0886# and antinociception[ Mecamylamine is expected to inhibit nicotine!induced
dopamine release in the accumbens\ but not cocaine! induced increase\ which is mediated via uptake inhibition[ It is of interest that mecamylamine also inhibits the etha! nol!induced elevation of extracellular dopamine in the accumbens "Blomqvist et al[\ 0886#[ The fact that atropine also inhibits nicotine! but not cocaine!induced dopamine changes in the accumbens indicates that in addition to nicotinic\ muscarinic sites are also important[ There are a number of di}erences in alterations between nicotine! induced and cocaine!induced central e}ects[ The nic! otinic antagonist dihydroerythroidine infused in the ven! tral tegmental area inhibited nicotine self!administration but did not a}ect cocaine self!administration\ and atro! pine did not a}ect nicotine self!administration in these experiments "Corrigall et al[\ 0883#[ We previously found\ as did other laboratories\ that mecamylamine and atro! pine inhibit nicotine!induced dopamine release "Toth et
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Fig[ 7[ E}ects of SCH!12289 on nicotine! and cocaine!induced dopamine increase[ Numbers in parenthesis indicate the dose "mg:kg# of nicotine\ cocaine\ and SCH!12289[ Averages of three experiments2SEM are shown[
al[\ 0881#[ The relationship between dopamine and glu! tamate receptors has already been discussed*dopa! minergic stimulation a}ects glutamatergic activity and glutamatergic stimulation a}ects dopaminergic activity[ We reported previously that nicotine releases glutamate and that AMPA\ kainate\ and NMDA receptor antag! onists inhibit nicotine!induced dopamine release\ indi! cating that nicotine e}ects are partially mediated via glutamate release "Toth et al[\ 0881#[ Our present results with MK!790 con_rm this and indicate in addition that glutamate release does not have a major role in cocaine e}ects[ In a previous study intraperitoneally administered MK!790 inhibited behavioral sensitization to cocaine\ amphetamine\ and morphine but did not a}ect cocaine! or amphetamine!induced increase in dopamine levels in
the accumbens "Wolf et al[\ 0883#[ When MK!790 was administered in the nucleus accumbens via microdialysis probe before cocaine injection\ it signi_cantly inhibited the cocaine!induced increase in dopamine "Pap and Brad! berry\ 0884#[ At levels where nicotine e}ects are inhibited\ MK!790 does not a}ect cocaine!induced dopamine increase*at higher levels it may have an inhibitory e}ect on cocaine!induced changes as well[ It is not possible to unequivocally de_ne the mechanism of the inhibitory e}ect of MK!790[ Recent _ndings show that MK!790 is also an inhibitor of nicotinic receptors "Briggs and McKenna\ 0885^ Buisson and Bertrand\ 0887#^ therefore\ at lower levels it may act via nicotinic receptors[ We are now testing the e}ect of additional NMDA receptor antagonists with no reported a.nity to nicotinic recep!
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tors to de_ne the role of the glutamatergic system in nicotine! vs cocaine!induced dopamine release[ The e}ect of SCH!12289\ a dopamine D0 receptor antagonist\ indicates an important role for D0 receptors for both nicotine! and cocaine!induced changes[ It is of interest that in rodent tests SCH!12289 decreased nicotine self!administration\ while it increased cocaine self!administration "Corrigall and Coen\ 0880#\ since attenuation of elevation of dopamine levels tends to increase self!administration[ SCH!12289 injected in the amygdala or striatum also increased cocaine self!admin! istration "Caine et al[\ 0884#[ It did not inhibit nicotine! produced EEG alterations "Ferger and Kuschinsky\ 0883#[ SCH!12289 injected into the ventral tegmental area blocked cocaine!induced accumbens dopamine release\ but not cocaine!induced behavioral "motor stimulation# sensitization "Steketee\ 0887#[ The fact that cocaine!induced behavioral stimulation is not inhibited under these conditions indicates that the processes involved in the two e}ects of cocaine "dopamine increase and behavioral stimulation# are at least in part di}erent\ and not all processes depend on dopamine D0 receptor activity[ Although SCH!12289 was also reported to inhibit the dopamine transporter "Tomiyama et al[\ 0884# we feel it is more likely that it a}ected D0 receptors in our experiments[ When SCH!12289 was administered\ dopamine levels in the accumbens were not a}ected\ and when amphetamine was given after nicotine and cocaine administration\ SCH!12289 did not a}ect the amphet! amine!induced large increase in dopamine levels[ The _nding that dopamine!transporter knockout mice self! administer cocaine "Rocha et al[\ 0887# was interpreted to mean that serotonergic activity can sustain cocaine self!administration or other transporters can assume the role of dopamine transporter[ The above _ndings show that both nicotine and cocaine a}ect numerous compounds*neurotransmitters and other neuroactive compounds\ receptor and trans! port systems*to a di}erent degree in various areas of the brain via heterogeneous speci_c mechanisms[ Some of the changes are caused directly\ such as the action of nicotine on nicotinic receptors located on dopaminergic\ glutamatergic\ etc[\ cells^ some changes are indirect\ such as the released dopamine or glutamate in~uencing activity at additional sites[ The regional heterogeneity of the changes can be explained by the regional het! erogeneity of the distribution of the receptor systems involved[ It is now well known that the nicotinic ace! tylcholine receptor has multiple forms composed of di}erent subunits[ The di}erent pairing of the subunits generates di}erent types of receptors\ which have di}er! ent patterns of sensitivity to nicotine\ thus di}ering phar! macological character "Luetje and Patrick\ 0880#[ For example\ presynaptic nicotinic receptors associated with striatal dopamine and hippocampal norepinephrine ter! minals di}er pharmacologically "Clarke and Reuben\
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0885#\ and di}erent nicotinic receptor agonists di}er in their e}ects on dopamine release in the accumbens "Mirza et al[\ 0885#[ The subunit composition of receptors media! ting nicotine action is of importance*the b1 subunit of the acetylcholine receptor plays an important role\ since b1 subunit!de_cient mutant mice do not respond to nic! otine "Picciotto et al[\ 0887#[ Anabaseine and its deriva! tive "GTS!10#\ two nicotinic agonists\ showed di}erent e}ects in the cortex] anabaseine increased acetylcholine and had no e}ect on dopamine^ its derivative increased dopamine and had no e}ect on acetylcholine "Summers et al[\ 0886#[ Norepinephrine release seems to depend on a nicotinic receptor subunit di}erent from that for serotonin release "Xiangyang et al[\ 0887#[ Our results in this study are consistent with the multiple pharmacological character of both nicotine! and cocaine! induced changes in the brain\ involving alteration in a number of compounds and participation of a number of receptor systems\ with the pattern of changes in com! pounds and the pattern of participation of receptor sys! tems showing di}erences between nicotine! and cocaine! induced e}ects[ The heterogeneous distribution of phar! macologically di}erent receptors results in a drug!speci_c pattern of changes in compounds\ areas\ and receptors involved[ Our study was limited in that it focused on one brain area and measured the dopamine released[ It is likely that there are many other di}erences between the changes induced by nicotine and those induced by cocaine or other drugs in other brain areas*in neurotransmitter changes or in receptor subtypes involved[ It is important to realize that changes in the nucleus accumbens are in~uenced by those occurring in other areas[ The relation! ship of nicotine!induced release in the accumbens to that in the ventral tegmental area is well established\ with the nicotine receptors in the ventral tegmental area being more active in mediating nicotine!induced dopamine release in the accumbens than the nicotinic receptors in the accumbens itself "Nisell et al[\ 0884#[ It is likely that other receptors are also involved] metabotropic glu! tamate receptors stimulate dopamine release "Verma and Moghaddam\ 0887#\ and GABA receptors on ventral tegmental dopamine neurons stimulate accumbens dopa! mine release "Westerink et al[\ 0885#[ Recent studies of functional MRI imaging the brains of cocaine!dependent subjects given a dose of cocaine showed changes in activity in numerous brain areas in addition to the nucleus accumbens "among others in caudate\ putamen\ thalamus\ hippocampus\ pons\ several cortical regions\ etc[#\ with some di}erences between cocaine!induced euphoria and cocaine!induced craving "Breiter et al[\ 0886#\ showing the involvement of numerous areas and circuits in the brain[ Regional di}erences between cocaine and nicotine e}ects remain to be demonstrated[ It also has to be emphasized that both nicotine and cocaine may have several central e}ects not related to reward
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mechanisms[ For any therapeutic approach to cocaine or nicotine use\ and better understanding of reward systems\ knowledge of the speci_c mechanisms involved in various reward systems is of essential importance[
Acknowledgements The work was supported in part by a grant from Philip Morris\ USA[ The advice and help of Thomas Cooper and David Allen are gratefully acknowledged[
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