37, 577-582, February 20, 2003, Copyright 2003 by Ce|| Press
Report Drugs of Abuse and Stress Tr|gger a Common Synapt|c Adaptat|on |n Dopam|ne Neurons is the sub[ect of debate (Berridge and Robinson, 1998}, there is no doubt that the discovery of this common action of addictive drugs, despite their very different mo|ecu|ar actions, has had a ma[or inf|uence on thinking about both the neurobio|ogica| basis of addiction as Dan|e| Saa|, 1,3 Yan Dong, 1,3 Antone||o Bonc|, 2 and Robert C. Ma|enka 1, * 1 Nancy Pritzker Laboratory Department of Psychiatry and Behaviora| Sciences Stanford University Schoo| of Medicine Pa|o A|to, Ca|ifornia 94304 we|| as the brain mechanisms mediating reinforcement and reward-based |earning (Wae|ti et a|., 2001}. 2 Ernest Ga||o C|inic and Research Center Department of Neuro|ogy and Recent|y we found that a sing|e in vivo exposure to cocaine caused an enhancement of synaptic strength Whee|er Center for the Neurobio|ogy of Addiction University of Ca|ifornia, San Francisco at excitatory synapses onmidbrain DAneurons(Ung|ess et a|., 2001} and that this shared severa| features with San Francisco, Ca|ifornia 94110 |ong-term potentiation (LTP}, the |eading mode| for the ce||u|ar changes that mediate |earning and memory (Martin et a|., 2000}. Here we examine whether in vivo Summary administration of other common|y abused drugs causes a simi|ar synaptic change. Furthermore, because in both Drug seek|ng and drug se|f-adm|n|strat|on |n both an|- ma|s and humans can be tr|ggered by drugs of abuse humans and anima|s stress faci|itates the initia| acquisi- tion and maintenance of drug se|f-administration and themse|ves or by stressfu| events. Here, we demon- strate that |n v|vo adm|n|strat|on of drugs of abuse can e|icit re|apse (i.e., reinstatement of drug taking after abstinence} (Piazza and Le Moa|, 1998, Shaham et a|., w|th d|fferent mo|ecu|ar mechan|sms of act|on as we|| as acute stress both |ncrease strength at exc|tatory 2000}, we asked whether an acute stress a|so causes a change in excitatory synaptic strength on midbrain DA synapses on m|dbra|n dopam|ne neurons. Psychoac- t|ve drugs w|th m|n|ma| abuse potent|a| do not cause neurons ana|ogous to that caused by cocaine. Our find- ings strengthen the idea that mechanisms invo|ved in th|s change. The synapt|c effects of stress, but not of coca|ne, are b|ocked by the g|ucocort|co|d receptor adaptive forms of experience-dependent p|asticity such as |earning may be patho|ogica||y usurped and contrib- antagon|st RU486. These resu|ts suggest that p|ast|c- |ty at exc|tatory synapses on dopam|ne neurons may ute to the deve|opment of addiction (Hyman and Ma- |enka, 2001, Nest|er, 2001}. be a key neura| adaptat|on contr|but|ng to add|ct|on and |ts |nteract|ons w|th stress and thus may be an attract|ve therapeut|c target for reduc|ng the r|sk of Resu|ts add|ct|on. The experimenta| approach was essentia||y identica| to Introduct|on that used previous|y (Ung|ess et a|., 2001}. Twenty-four hours after in vivo administration of the drug (or stress}, Understanding the neurobio|ogica| basis of addiction s|ices containing the VTA were prepared and who|e-ce|| has important imp|ications not on|y for the treatment recordings were obtained from DA ce||s, identified by and prevention of this devastating and common i||ness the presence of |arge / h currents (Figure 1A}. To assess but a|so as a mode| for how experiences modify neura| synaptic strength, the ratio of AMPA receptor-mediated circuitry and thereby behavior. Because humans abuse synaptic currents (AMPAR EPSCs} to NMDA receptor- and become addicted to a wide array of |ega| (e.g., mediated synaptic currents (NMDAREPSCs} was ca|cu- nicotine, ethano|} and i||icit (e.g., cocaine, heroin} drugs, |ated (Figure 1B}. This measure offers the important ad- a va|uab|e strategy has been to |ook for adaptations in vantage that it is independent of the number of synapses the brain that are common to different c|asses of ad- activated and therefore independent of such variab|es dictive substances. Such an approach |ed to the rea|iza- as the positioning of the e|ectrodes or anatomy of the tionthat onecritica||yimportant component of the neura| tissue. We first retested the effects of cocaine and com- circuitsmediatingaddictionisthemeso|imbic dopamine pared its effects to those of amphetamine, a psycho- (DA} system consisting of midbrain DA ce||s, |ocated stimu|ant that shares some mechanistic features with primari|y in the ventra| tegmenta| area (VTA} and the cocaine in that it too enhances brain DA |eve|s. Both nuc|eus accumbens (NAc}, which receives a dense pro- psychostimu|ant drugs caused a simi|ar increase in the [ection from the VTA (Everitt and Wo|f, 2002, Ke||ey and ratio of AMPAR to NMDAR EPSCs (Figure 1C} (sa|ine, Berridge, 2002, Koob et a|., 1998, Nest|er, 2001, Wise, 0.42 .09, n 14 ce||s, cocaine, 0.70 .08, n 8, 1996}. A|| drugs of abuse appear to act direct|y in the amphetamine, 0.70 .09, n 12}, indicating that |ike VTA and/or NAc to cause increases in DA |eve|s in the cocaine, in vivo administration of amphetamine causes NAc (Koob et a|., 1998, Nest|er, 2001, Wise, 1996}. A|- an increase in excitatory synaptic transmission onto DA though whether this is sufficient or even abso|ute|y nec- neurons. essary for the reinforcing properties of drugs of abuse Is this in vivo drug-induced synaptic p|asticity in mid- brain DA neurons |imited to psychostimu|ants or does it a|so occur in response to other addictive substances? *Correspondence. ma|enka@stanford.edu 3 These authors contributed equa||y to this work. Wetestedthree other common|y abuseddrugsthat have Neuron 578 Figure 1. Psychostimu|ant Drugs Enhance the AMPA/NMDA EPSC Ratio at Excitatory Synapses on Midbrain DA Ce||s (A} Examp|e of /h currents used to identify midbrain DA ce||s (ca|ibration bars. 20 pA/50 ms}. (B} Top traces show examp|e from a contro| ce|| of how AMPA/NMDA ratios were obtained. EPSCs were recorded at 40 mV (Dua| trace}, and then D-APV (50 M} was app|ied to obtain the AMPA EPSC. The NMDA EPSC was obtained by digita| subtraction of the AMPA EPSC from the dua| EPSC. Bottom traces show examp|es of AMPA and NMDA EPSCs obtained from cocaine- and amphetamine-treated anima|s (ca|ibration bars. 20 pA/15 ms}. (C} Summary of AMPA/NMDA ratios obtained from anima|s administered sa|ine, cocaine, or amphetamine (asterisk indicates p 0.02}. In this and a|| subsequent figures, numbers within the bars indicate the number of ce||s examined. very different mo|ecu|ar mechanisms of action. (1} mor- tor common|y used in the treatment of depression, nor carbamazepine, which is a common treatment for sei- phine, which acts on opioid receptors, (2} nicotine, which acts on brain nicotinic receptors, and (3} ethano|, zure disorders and bipo|ar disorder (i.e., manic- depressive i||ness} (Nest|er et a|., 2001}, had an effect which acts on a number of different neurotransmitter receptors (Koob et a|., 1998, Nest|er, 2001}. In vivo ad- on the ratio of AMPAR to NMDAR EPSCs (Figure 3} (sa|ine, 0.44 .02, n 14 ce||s, f|uoxetine, 0.44 .05, ministration of a|| three of these substances caused significant increases in the ratio of AMPAR to NMDAR n 9, carbamazepine, 0.41 .05, n 8}. These resu|ts provide further support for the hypothesis that changes EPSCs measured 24 hr |ater (Figure 2} (sa|ine, 0.46 .02, n 22 ce||s, morphine, 0.68 .04, n 9, nicotine, in strength at excitatory synapses on midbrain DA ce||s may contribute to the addictive properties of mu|tip|e 0.67 .07, n 14, ethano|, 0.64 .06, n 10}. Thus far we have demonstrated that five different c|asses of abused substances. The most difficu|t aspect of addiction treatment is the abused drugs (cocaine, amphetamine, morphine, nico- tine, and ethano|} a|| cause a simi|ar enhancement at |ong-|asting risk of re|apse (O'Brien, 1997}, individua|s can be abstinent for months or even years but sti|| are excitatory synapses on midbrain DA ce||s. These are surprising resu|ts since these substances have very dif- susceptib|e to cravings that can stimu|ate renewed drug seeking and taking. In both humans and anima|s, rein- ferent mo|ecu|ar mechanisms of action. They a|so raise the important question of whether this modification has statement of drug seeking and se|f-administration can be triggered by sing|e doses of drug, stimu|i previous|y something to do with the addictive potentia| of these substances. A|ternative|y, it might simp|y be the conse- associated with drug taking, or stressfu| events (Piazza and Le Moa|, 1998, Robinson and Berridge, 2000, Sha- quence of administering any type of psychoactive sub- stance. To address this issue, we asked whether admin- ham et a|., 2000}. Stress a|so can faci|itate the initia| acquisition of drug se|f-administration, perhaps by en- istration of common|y used therapeutic drugs, which are known to have profound effects on emotiona| states hancing the reinforcing efficacy of drugs of abuse (Pi- azza and Le Moa|, 1998}. We therefore tested whether in humans but no addictive |iabi|ity, a|so caused a change at excitatory synapses on midbrain DA ce||s. anacute stress wou|d affect excitatorysynapticstrength on midbrain DAce||s. Anima|s were sub[ect to a modified Neither f|uoxetine, a specific serotonin-reuptake inhibi- Figure 2. Common|y Abused Drugs Other than Psychostimu|ants a|so Increase the AMPA/NMDA Ratio (A} Examp|es of AMPA and NMDA EPSCs ob- tained from anima|s given the indicated sub- stance (ca|ibration bars. 20 pA/15 ms}. (B} Summary of AMPA/NMDA ratios obtained from anima|s administered sa|ine, morphine, nicotine, or ethano| (asterisk indicates p 0.03}. Drugs and Stress Cause Synaptic Changes in the VTA 579 (Figures 4A and 4B} (stress p|us RU486, 0.38 .04, n 9 ce||s}, imp|ying a critica| ro|e for GR activation. The effectiveness of RU486 a|so a||owed us to address the question of whether the synaptic effect of cocaine is due to an unrecognized drug-induced stress reaction rather than its reinforcing and/or addictive property. However, RU486 did not prevent the increase in the AMPARtoNMDAREPSCratio e|icitedby in vivo cocaine administration (Figure 4C} (sa|ine, 0.45 .03, n 14 ce||s, cocaine, 0.61 .13, n 7, cocaine p|us RU486, 0.69 .12, n 9}. This indicates that the synaptic conse- quences of in vivo cocaine exposure do not require GR activation and therefore are un|ike|y to be due to an acute stress response. Figure 3. Common|y Used Psychoactive but Nonaddictive Drugs Do Not Affect the AMPA/NMDA Ratio (A} Examp|es of AMPA and NMDA EPSCs obtained from anima|s D|scuss|on given the indicated substance (ca|ibration bars. 20 pA/15 ms}. (B} Summary of AMPA/NMDAratios obtained fromanima|s adminis- We have demonstrated that in vivo administration of five tered sa|ine, f|uoxetine, or carbamazepine. different drugs of abuse with very different mo|ecu|ar mechanisms of action a|| e|icit an enhancement of strength at excitatory synapses on midbrain DA neu- Porso|t forced swimtask, a common|y used acute stres- sor (Huber et a|., 2001}, and s|ices were prepared 1 day rons. Two psychoactive but therapeutic and nonad- dictive medications did not cause such a change. This |ater. This resu|ted in an increase in the ratio of AMPAR to NMDAREPSCs even |arger than that e|icited by drugs degree of specificity suggests that this in vivo, drug- induced synaptic p|asticity in DA neurons may be one of abuse (Figures 4A and 4B} (sa|ine, 0.46 .03, n 11 ce||s, stress p|us sa|ine, 0.82 .04, n 24}. To begin important component of the neura| circuit adaptations that contribute to core features of addiction. Because to exp|ore whether this stress-induced synaptic modifi- cation shares mechanisms with the cocaine-e|icited syn- of theprofoundeffect of stressin faci|itatingthe initiation and reinstatement of drug se|f-administration, we a|so aptic enhancement, we administered the NMDAR antago- nist MK-801 prior to the co|d water swim. This stress examined whether an acute stress might have an effect simi|ar to that of drugs of abuse and found that it too protoco| b|ocked the increase in the ratio of AMPAR to NMDAR EPSCs (Figures 4A and 4B} (stress p|us MK801, causeda |arge increase in synaptic strength at this same popu|ation of excitatory synapses. Based on these ob- 0.51 .06, n 15}, indicating that, |ike the effects of cocaine (Ung|ess et a|., 2001}, the synaptic changes due servations, we suggest that this enhancement of strength at excitatory synapses on midbrain DA neurons a|so is a to the acute stress required activation of NMDARs. One of the ma[or consequences of acute stress is key neura| adaptation that contributes to the effects of stress on drug seeking and re|apse. increased secretion of corticosteroids and activation of g|ucocorticoid receptors (GRs}. To test whether GR The conc|usion that the increases in the AMPAR to NMDAREPSCratio e|icitedby drugs of abuse andstress activation p|ayed a ro|e in mediating the effects of stress on synaptic strength in midbrain DAneurons, we admin- ref|ect an enhancement of AMPAR-mediated excitatory synaptic transmission is based on previous work that ex- istered the GRantagonist RU486 (Cadepondet a|., 1997, Marine||i et a|., 1998} prior to p|acing anima|s in the co|d amined the mechanisms under|ying the cocaine-induced increase in this ratio (Ung|ess et a|., 2001}. We cannot water. This comp|ete|y b|ocked the synaptic change Figure 4. Acute Stress Increases the AMPA/NMDA Ratio (A} Examp|es of AMPA and NMDA EPSCs obtained from a contro| anima| and anima|s experiencing stress p|us the indicated substance (ca|ibration bars. 20 pA/15 ms}. (B} Summary of AMPA/NMDA ratios obtained from anima|s administered sa|ine or experiencing stress a|ong with administration of sa|ine, the NMDA receptor antagonist MK801, or the g|ucocorticoid receptor antagonist RU486 (asterisk indicates p 0.01}. (C} Summary of AMPA/NMDA ratios obtained from anima|s administered sa|ine, cocaine, or cocaine p|us RU486 (asterisk indicates p 0.05}. Neuron 580 ru|e out, however, additiona| mechanisms that cou|d with DA ce|| firing are granted high appetitive or motiva- contribute to this increase in the AMPAR to NMDAR tiona| significance. We suggest that by increasing syn- EPSC ratio. A decrease in the magnitude of the NMDAR aptic strength at excitatory synapses on midbrain DA EPSC due to changes in NMDAR function and/or num- ce||s, drugs of abuse or stress enhance the motivationa| ber c|ear|y wou|d increase the ratio, as wou|d decreases significance of drugs themse|ves as we|| as stimu|i in the NMDAR EPSC due to the extent of g|utamate c|ose|y associated with drug seeking and se|f-adminis- spi||over (Ku||mann, 2000} shou|d spi||over occur nor- tration. ma||y in this brain region. In previous work an attempt Consistent with this hypothesis, there is considerab|e was made to address the first of these possibi|ities by evidence that stress can enhance the rewarding efficacy examining the inward currents generated by app|ication of drugs, |ike|y via inf|uences on the meso|imbic DA of NMDA (Ung|ess et a|., 2001}. However, this manipu|a- system (Piazza and Le Moa|, 1998}. Indeed, stress, |ike tion activates extrasynaptic NMDARs and thus negative drugs of abuse, causes an increase in DA |eve|s in the resu|ts are not conc|usive. Another possibi|ity is that NAc and prefronta| cortex (Horger and Roth, 1996, Pi- if norma||y synaptic AMPARs on midbrain DA neurons azza and Le Moa|, 1998}. Stress has a|so been reported exhibit inward rectification (i.e., pass more current in to affect synaptic p|asticity in the hippocampus. Specifi- the inward than in the outward direction}, a drug- or ca||y, acute stress appears to rapid|y inhibit the genera- stress-induced decrease in the degree of inward rectifi- tion of LTP and faci|itate the generation of LTD (Shors cation wou|d resu|t in an increase in the AMPAR to et a|., 1989, Kim et a|., 1996, Xu et a|., 1997, 1998}. NMDAR EPSC ratio when measured at 40 mV (since Whether this invo|ves an increase in basa| synaptic the modifiedAMPARs wi|| nowpass more current at 40 strength or rather some modification of the triggering mV a|though the same current at hyperpo|arized mem- mechanisms for synaptic p|asticity is debated (Kim et brane potentia|s}. However, such a change in the bio- a|., 1996, Xu et a|., 1997}. physica| properties of AMPARs cannot account for our One important unanswered question is whether a|| or resu|ts since a c|ear increase in the AMPAR to NMDAR critica| subsets of excitatory afferent inputs to midbrain EPSC ratio in response to cocaine administration was DA ce||s are strengthened by in vivo drugs or stress. detected when AMPAR EPSCs were measured at 70 Strengthening of prefronta| cortica| inputs may be par- mV rather than 40 mV (unpub|ished observations}. ticu|ar|y important, as this has been suggested to p|ay There are severa| corre|ative findings that can be a critica| ro|e in the deve|opment of behaviora| sensitiza- viewed as being consistent with the hypothesis that tion, a prominent mode| for core features of addiction drugs of abuse enhance AMPAR-mediated synaptic inc|uding craving (Everitt and Wo|f, 2002, Robinson and transmission in midbrain DAce||s. First, repeatedadmin- Berridge, 2000}. On the other hand, modifications of the istration of cocaine or amphetamine caused an increase recent|y described input from the bed nuc|eus of the in the sing|e unit responses of VTA DA ce||s to AMPA stria termina|is (Georges and Aston-Jones, 2002} may but not to NMDA, an effect that was observed 3 but not be particu|ar|y important for mediating the effects of 14 days after cessation of drug administration (Zhang stress (Shahamet a|., 2000}. Whatever sets of synapses et a|., 1997}. Second, both repeated administration of are affected, by demonstrating a synaptic modification cocaine or morphine as we|| as repeated restraint stress common|y e|icited by addictive drugs and stress, we increased |eve|s of the AMPAR subunit G|uR1 in the VTA provide a neura| mechanism that may contribute to the (as we|| as the NMDAR subunit NR1} (Fitzgera|d et a|., c|inica||y important interactions between addiction and 1996, but see Lu et a|., 2002}. Third, vira|-mediated over- stress as we|| as one potentia||y important target for expression of G|uR1 in the VTA enhanced the |ocomotor therapeutic interventions in treatment of addictive dis- stimu|ating and rewarding properties of morphine (Car- orders. |ezon et a|., 1997}. These sorts of findings have |ed to a hypothesis that e|evated |eve|s of G|uR1 in the VTA are one primary trigger |eading to the comp|ex cascade Exper|menta| Procedures of mo|ecu|ar and circuit adaptations that under|ie sensi- tization to drugs of abuse (Car|ezon and Nest|er, 2002}. An|ma|s and In V|vo Man|pu|at|ons Accepting that drugs of abuse and stress do enhance C57/B|6 mice (21- to 30-days-o|d} were used for a|| experiments. For drug administration, anima|s were in[ected i.p. at the same time the strength of excitatory afferent inputs to midbrain DA each day with sa|ine (vo|ume matched for experimenta| in[ections}, ce||s, how might this contribute to the deve|opment of cocaine (15 mg/kg}, d-amphetamine (10 mg/kg}, morphine (10 mg/kg}, addiction and/or re|apse? There are three genera| hypoth- nicotine (0.5 mg/kg}, ethano| (20 mg/kg}, f|uoxetine (10 mg/kg}, or eses concerning the functions of midbrain DA ce||s and carbamazepine (15 mg/kg}. Drugs were obtained from Sigma (ex- their pro[ections to the NAc. DAce|| firing and the conse- cept for ethano|, which was obtained from the Stanford Hospita| quent re|ease of DA in the NAc may. (1} signify reward pharmacy}. Acute stress was e|icited with a modified Porso|t forced (Wise, 1996} or changes in the positive incentive va|ue swim task. Anima|s were p|aced in a 1 | graduated cy|inder con- of stimu|i (Koob, 1996}, (2} serve as a reward prediction taining 400 m| of co|d (6 C} water for 4-6 min. MK801 (1 mg/kg in sa|ine} or RU486 (40 mg/kg in DMSO} was administered 15-30 min signa| and thereby p|ay an important ro|e in reward- prior to the co|d water swim. RU486 was administered 30 min prior based |earning (Wae|ti et a|., 2001}, or (3} be invo|ved in to cocaine administration for the experiments i||ustrated in Figure signa|ing "incentive sa|ience," which in the context of 4C. The dose of RU486 was chosen based on pub|ished reports addiction suggests that adaptations in the meso|imbic showing variab|e efficacy of 20-25 mg/kg (Pieretti et a|., 1991, DA system |ead to "wanting" (i.e., craving} drugs a|- Douma et a|., 1998, Xu et a|., 1998} and the concern that because though not necessari|y "|iking" them(Robinson and Ber- rodents |ack a specific binding protein for RU486, brain concentra- ridge, 2000}. These non-mutua||y exc|usive hypotheses tions of RU486 fo||owing periphera| administration are |ow (Heikin- heimo and Kekkonen, 1993}. share the featurethat externa| stimu|i that are associated Drugs and Stress Cause Synaptic Changes in the VTA 581 E|ectrophys|o|ogy pristone}. mechanisms of action and c|inica| uses. Annu. Rev. Med. 48, 129-156. Anima|s were anesthetized with ha|othane and sacrificed 24-30 hr after the in vivo manipu|ation. A|| remaining procedures were essen- Cameron, D.L., Wessendorf, M.W., and Wi||iams, J.T. (1997}. A sub- tia||y as described previous|y (Ung|ess et a|., 2001}. Brief|y, a b|ock set of ventra| tegmenta| area neurons is inhibited by dopamine, of tissue containing midbrain was s|iced in the horizonta| p|ane (250 5-hydroxytryptamine and opioids. Neuroscience 77, 155-166. m} in ice-co|d |ow Ca 2 so|ution (containing in mM. 126 NaC|, Car|ezon, W.A., and Nest|er, E.J. (2002}. E|evated |eve|s of G|uR1 in 1.6 KC|, 1.2 NaH 2 PO 4 , 1.2 MgC| 2 , 0.625 CaC| 2 , 18 NaHCO 3 , and 11 the midbrain. a trigger for sensitization to drugs of abuse? Trends g|ucose}. S|ices were transferred to a ho|ding chamber containing Neurosci. 25, 610-615. artificia| cerebrospina| f|uid (ACSF, in mM. 126 NaC|, 1.6 KC|, 1.2 Car|ezon, W.A., Boundy, V.A., Hai|e, C.N., Lane, S.B., Ka|b, R.G., NaH2PO4, 1.2 MgC|2, 2.5 CaC|2, 18 NaHCO3, and 11 g|ucose} and Neve, R.L., and Nest|er, E.J. (1997}. Sensitization to morphine in- equi|ibriated at 31 C -34 C for at |east 1 hr. Picrotoxin (100 M} was duced by vira|-mediated gene transfer. Science 277, 812-814. added to the ACSF for recording, to b|ock GABA A receptor-mediated inhibitory postsynaptic potentia|s. A|| so|utions were bubb|ed with Douma, B.R.K., Korte, S.M., Buwa|da, B., |a F|eur, S.E., Bohus, B., 95¾ O2/5¾ CO2 and perfused over the s|ice at a rate of 2.5 m|/min. and Luiten, P.G.M. (1998}. Repeated b|ockade of minera|ocorticoid Ce||s were visua|ized with an upright microscope using infrared receptors, but not of g|ucocorticoid receptors impairs food re- i||umination, and who|e-ce|| vo|tage-c|amp recordings were made warded spatia| |earning. Psychoneuroendocrino|ogy 23, 33-44. with an Axopatch 1D amp|ifier (Axon Instruments}. E|ectrodes (2-6 Everitt, B.J., and Wo|f, M.E. (2002}. Psychomotor stimu|ant addic- M } contained in mM. 117 cesium methansu|fonic acid, 20 HEPES, tion. a neura| systems perspective. J. Neurosci. 22, 3312-3320. 0.4 EGTA, 2.8 NaC|, 5 TEA-C|, 2.5 MgATP, 0.25 MgGTP (pH 7.2-7.4}, Fitzgera|d, L.W., Ortiz, J., Hamedani, A.G., and Nest|er, E.J. (1996}. 275-285 mOsm. Dopamine ce||s were identified by the presence of Drugs of abuse and stress increase the expression of G|uR1 and a |arge / h current, which was evoked by ho|ding ce||s at 70 mV NMDAR1 g|utamate receptor subunits in the rat ventra| tegmenta| and stepping to 120 mV in 10 mV increments. Whi|e / h is present area. common adaptations among cross-sensitizing agents. J. Neu- in 90¾ of dopamine neurons (Cameron et a|., 1997, Neuhoff et rosci. 16, 274-282. a|., 2002}, we recognize that its presence does not unequivoca||y Georges, F., and Aston-Jones, G. (2002}. Activation of ventra| teg- identify dopamine ce||s in midbrain s|ices. However, both in previous menta| area ce||s by the bed nuc|eus of the stria termina|is. a nove| work (Ung|ess et a|., 2001} and in the present experiments, this excitatory amino acid input to midbrain dopamine neurons. J. Neu- criterion was sufficient to obtain c|ear differences between contro| rosci. 22, 5173-5187. and experimenta| ce||s. A bipo|ar stimu|ating e|ectrode was p|aced 100-300 m rostra| to Heikinheimo, O., and Kekkonen, R. (1993}. Dose-response re|ation- the recording e|ectrode and was used to stimu|ate excitatory affer- ships of RU 486. Ann. Med. 25, 71-76. ents at 0.1 Hz. Ce||s were initia||y he|d at 70 mV for 5-15 min to Horger, B.A., and Roth, R.H. (1996}. The ro|e of mesoprefronta| dopa- ensure the stabi|ity of EPSCs and then were depo|arized to 40 mine neurons in stress. Crit. Rev. Neurobio|. 10, 395-418. mV. Ce||s were again monitored for 5-15 min, at which point D-APV Huber, J.D., Dar|ing, S.F., Park, K.K., and So|iman, K.F. (2001}. The (50 M} was app|iedfor 10-20 min. To ca|cu|ate the AMPAR/NMDAR ro|e of NMDA receptors in neonata| cocaine-induced neurotoxicity. ratio, a contro| dua| component EPSC was obtained by averaging Pharmaco|. Biochem. Behav. 69, 451-459. 20-30 consecutive responses immediate|y before app|ication of Hyman, S.E., and Ma|enka, R.C. (2001}. Addiction and the brain. the D-APV. An average AMPAR EPSC was then obtained by averaging neurobio|ogy of compu|sion and its persistence. Nat. Rev. Neurosci. 20-30 consecutive responses beginning 5 min after app|ication of 2, 695-703. D-APV. The average response in the presence of D-APV was sub- tracted from that seen in its absence to obtain an average NMDAR Ke||ey, A.E., and Berridge, K.C. (2002}. The neuroscience of natura| EPSC. The peak of the AMPAR EPSC was divided by the peak of rewards. re|evance to addictive drugs. J. Neurosci. 22, 3306-3311. the NMDAR EPSCto give an AMPAR/NMDAR ratio. Input and series Kim, J.J., Foy, M.R., and Thompson, R.F. (1996}. Behaviora| stress resistances were monitored on|ine throughout each experiment. modifies hippocampa| p|asticity through N-methy|-D-aspartate re- Over 65¾ of the data were co||ected and ana|yzed without know|- ceptor activation. Proc. Nat|. Acad. Sci. USA 93, 4750-4753. edge of the treatment that the anima|s had received. There were no Koob, G.F. (1996}. Hedonic va|ence, dopamine and motivation. Mo|. differences in the resu|ts from b|inded and nonb|inded experiments Psychiatry 1, 186-189. and therefore resu|ts were combined. Contro|, sa|ine-in[ected ani- Koob, G.F., Sanna, P.P., and B|oom, F.E. (1998}. Neuroscience of ma|s were inter|eaved throughout the course of a|| experiments, and addiction. Neuron 21, 467-476. the sa|ine contro|s presented in each bar graph represent the ce||s obtained during the time period when the experimenta| manipu|a- Ku||mann, D.M. (2000}. Spi||over and synaptic cross ta|k mediated tions shown in that graph were performed. On|y two s|ices were by g|utamate and GABA in the mamma|ian brain. Prog. Brain Res. obtained from each anima| and a sing|e ce|| was examined from 125, 339-351. each s|ice. A|| va|ues are expressed as mean and SEM. Statistica| Lu, W., Monteggia, L.M., and Wo|f, M.E. (2002}. Repeated adminis- significance was assessed using two-tai|ed Student's t tests. tration of amphetamine or cocaine does not a|ter AMPA receptor subunit expression in the rat midbrain. Neuropsychopharmaco|ogy. Acknow|edgments 26, 1-13. Marine||i, M., Aouizerate, B., Barrot, M., Le Moa|, M., and Piazza, This work was supported by grants from NIDA (R.C.M. and A.B.}, P.V. (1998}. Dopamine-dependent responses to morphine depend the Zaffaroni Innovation Fund for Addiction Research (R.C.M.}, and on g|ucocorticoid receptors. Proc. Nat|. Acad. Sci. USA 95, 7742- Department of the Army (Award No. DAMD17-01-10736}, U.S. Army 7747. Medica| Research Acquisition Activity (Fort Detrick, MD} (A.B.}. D.S. Martin, S.J., Grimwood, P.D., and Morris, R.G. (2000}. Synaptic p|as- was supported by a Pfizer Postdoctora| Fe||owship. We thank Mark ticity and memory. an eva|uation of the hypothesis. Annu. Rev. Neu- Ung|ess and David Lyons for technica| assistance and discussions. rosci. 23, 649-711. Nest|er, E.J. (2001}. Mo|ecu|ar basis of |ong-term p|asticity under|y- Received. October 22, 2002 ing addiction. Nat. Rev. Neurosci. 2, 119-128. Revised. December 17, 2002 Nest|er, E.J., Hyman, S.E., and Ma|enka, R.C. (2001}. 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