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Reduced psychostimulant effects on dopamine dynamics in the nucleus accumbens of μ-opioid receptor knockout mice
Neuroscience 141 (2006) 1679 –1684
REDUCED PSYCHOSTIMULANT EFFECTS ON DOPAMINE DYNAMICS IN THE NUCLEUS ACCUMBENS OF -OPIOID RECEPTOR KNOCKOUT MICE tems, and in particular to -opioid receptors (MORs) located in the VTA, in modulating the reinforcing properties of drugs of abuse, including psychostimulant drugs such as cocaine (van Ree et al., 1999; Kieffer and GaveriauxRuff, 2002; Gerrits et al., 2003). Thus, infusion of the MOR antagonist naltrexone into the VTA retarded the acquisition of cocaine self-administration (Ramsey et al., 1999), and intra-VTA administration of the specific MOR agonist DAMGO enhanced cocaine self-administration in rats (Corrigall et al., 1999). Moreover, mice lacking MORs ( opioid receptor knockout (MuKO) mice) have been shown to be hyposensitive to the rewarding effects of a variety of drugs, including cocaine, as assessed using drug self-administration and place conditioning paradigms (Becker et al., 2002; Hall et al., 2004; Mathon et al., 2005a, for reviews see: Kieffer and Gaveriaux-Ruff, 2002; Gerrits et al., 2003). The most likely neural mechanism by which MORs influence drug reinforcement is by disinhibition of VTA dopamine neurons. That is, MOR stimulation inhibits local GABAergic interneurons (Gysling and Wang, 1983; Matthews and German, 1984; Johnson and North, 1992; Klitenick et al., 1992, for reviews see: Kalivas, 1993; Mathon et al., 2003), resulting in a subsequent increase in dopamine overflow in target areas (Spanagel et al., 1992). We have previously demonstrated that MuKO mice display decreased cocaine self-administration (Mathon et al., 2005a). In addition, VTA dopaminergic neurons in these mice receive an increased GABAergic input (Mathon et al., 2005a) and as a possible consequence, display decreased impulse activity (Mathon et al., 2005b). Microdialysis studies at the mesolimbic terminal area level have shown that there was no difference in the dopamine levels in the NAc between wild type and MuKO mice (Chefer et al., 2003, 2004; Robledo et al., 2004) and that the cocaineinduced increase in extracellular dopamine levels was comparable between wild type and MuKO mice (Chefer et al., 2004). As changes in neuronal activity likely result in adaptations in target areas, we here examined possible alterations in dopamine dynamics in the main terminal field of this system, the NAc, of MuKO mice. Fast scan cyclic voltammetry (FSCV) was used to measure stimulusevoked dopamine overflow at a sub-second time scale to examine detailed kinetics of dopamine overflow and reuptake. We used FSCV to 1) compare the basal overflow of stimulus-evoked dopamine in the NAc of WT and MuKO mice, and 2) examine the effects of cocaine and amphetamine on NAc dopamine overflow in these mice.
D. S. MATHON, L. J. M. J. VANDERSCHUREN AND G. M. J. RAMAKERS* Rudolf Magnus Institute of Neuroscience, Department of Pharmacology and Anatomy, University Medical Center Utrecht, Universiteitsweg 100, 3584 CG Utrecht, The Netherlands
Abstract—Dopamine neurotransmission in the nucleus accumbens plays a pivotal role in the reinforcing properties of drugs of abuse. Two interacting processes regulate nucleus accumbens dopamine overflow: release of dopamine from presynaptic terminals and the subsequent reuptake by dopamine transporters. Opioid neurotransmission, primarily through -opioid receptors has also been strongly implicated in drug reward. We have previously shown that mice lacking the -opioid receptor display decreased cocaine self-administration. In addition, we found decreased impulse activity of midbrain dopaminergic neurons and an increased GABAergic input to these neurons in -opioid receptor knockout mice. In the present study we investigated whether these changes in dopaminergic cell bodies are accompanied by altered dopamine dynamics at the terminal level. To that aim, we measured nucleus accumbens dopamine overflow using fast scan cyclic voltammetry. Our data demonstrate that in -opioid receptor knockout mice 1) the reuptake of dopamine in the nucleus accumbens is slower, and 2) the relative effect of cocaine and amphetamine on the reuptake of dopamine is smaller compared with wild type mice. These data provide a mechanism for the decreased reinforcing properties of cocaine observed in -opioid receptor knockout mice. © 2006 IBRO. Published by Elsevier Ltd. All rights reserved. Key words: dopamine reuptake, nucleus accumbens, cocaine, amphetamine, fast scan cyclic voltammetry.
The mesolimbic dopamine system, composed of dense projections from the ventral tegmental area (VTA) to the nucleus accumbens (NAc), has long been implicated in the physiology of incentive motivation and the pathophysiology of substance abuse (Koob, 1992; Berridge and Robinson, 1998; Cardinal et al., 2002; Wise, 2004; Salamone et al., 2005; Pierce and Kumaresan, 2006). Drugs of abuse, though acting on different neuronal substrates, have in common that they increase dopamine levels in the NAc (Di Chiara and Imperato, 1988; Pontieri et al., 1995). An important role has been ascribed to endogenous opioid sys-
*Corresponding author. Tel: ⫹31-30-2538413; fax: ⫹31-30-2539032. E-mail address: [email protected] (G. M. J. Ramakers). Abbreviations: aCSF, artificial cerebrospinal fluid; DAT, dopamine transporter; FSCV, fast scan cyclic voltammetry; MOR, opioid receptor; MuKO, opioid receptor knockout; NAc, nucleus accumbens; VTA, ventral tegmental area.
0306-4522/06$30.00⫹0.00 © 2006 IBRO. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.neuroscience.2006.05.003
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EXPERIMENTAL PROCEDURES The MuKO mice used in these experiments have been described elsewhere (Schuller et al., 1999). Wild type and homozygous male knockout mice were obtained from heterozygous breeding. The mice (8 –9 months old) used in the present study were on a C57Bl6/Jico background after six to seven back-crossings to C57Bl6/Jico mice (Charles River, l’Arbresle, France). Animals were anesthetized with isoflurane, decapitated and the brain was rapidly removed and kept in ice-cold high-magnesium artificial cerebrospinal fluid (aCSF) containing (in mM): 124 NaCl, 3.3 KCl, 1.2 KH2PO4, 2.5 MgSO4, 2.5 CaCl2, 20 NaHCO3, 10 glucose, saturated with 95% O2–5% CO2. Slices (200 m) were cut using a vibratome (Leica VT1000S; Leica Instruments, Nussloch, Germany) and after preparation kept at room temperature in aCSF with a normal Mg2⫹ concentration (1.3 mM). Slices were then transferred to a submerged recording chamber, where they were perfused at 2–3 ml/min with aCSF at room temperature. Dopamine overflow was measured following electrical stimulation to evoke calcium-dependent vesicular dopamine release, from the NAc in 200 m thick coronal slice preparations. Dopamine release was evoked by a single pulse (0 –1 mA, 300 s) applied through a bipolar stimulation electrode (bipolar stainless steel, 100 m, insulated except from the tip) every 30 s. Dopamine was detected with 5 m carbon-fiber disk electrodes insulated with electro deposition paint (Schulte and Chow, 1996; ALA Scientific Instruments Inc., Westbury, NY, USA) using FSCV (Kawagoe et al., 1993). Cyclic voltammograms (ramps from ⫺500 mV to ⫹1000 mV and back to ⫺500 mV versus an Ag/AgCl reference, 300 V/s) were repeated every 100 ms using an EPC9 amplifier (HEKA Electronic, Lambrecht, Germany). Stimulus-evoked dopamine overflow was measured by subtracting the background current obtained before stimulation (average of 10 pre-stimulus responses) from the current measured after stimulation. The resulting voltammogram showed a typical dopamine profile, with an oxidation peak between 500 and 700 mV and a smaller reduction peak around ⫺300 mV (see inset in Fig. 1A). The concentration of the dopamine overflow was calculated after calibrating the recording electrode in known concentrations of dopamine. The resultant dopamine concentrations are plotted against time (see Fig. 1A for example). Pilot experiments showed that stimulus-induced dopamine release was constant for at least 60 min (Ramakers and Mathon, unpublished observations). The amplitude was determined at the peak of the dopamine signal (vertical arrow a in Fig. 1A). The width of the signal was determined at half-maximal amplitude (horizontal arrow b in Fig. 1A). As an indication of changes in extracellular dopamine levels, the pre-stimulus current at the dopamine oxidation peak was monitored. Dopamine uptake kinetics were determined by fitting the decay of the dopamine transient with an exponential fit. This procedure revealed values of Km and Vmax in wild type mice that are comparable with reported values for these parameters (Budygin et al., 2002, 2004). Amphetamine (d-amphetamine sulfate) and cocaine (cocaine-HCl) were obtained from OPG (Utrecht, the Netherlands). Amphetamine and cocaine were dissolved in aCSF as 1000 times concentrated stocks and applied to the slices through the perfusion medium. For statistical analysis, group averages of absolute values were compared using Student’s t-test or a two factor ANOVA followed by post hoc Student’s t-test where appropriate.
RESULTS Dopamine release was electrically evoked in the NAc shell of MuKO and wild type mice and measured with FSCV. The voltammogram constructed from the voltage ramp identifies dopamine as the measured substance, with a maximal oxidization current at approximately ⫹600 mV
Fig. 1. Dopamine removal in the NAc shell of MuKO mice is slower compared with control mice. Dopamine overflow was evoked by a single local stimulus in the NAc shell of MuKO and WT mice. Ten pre-stimulus IV-ramps were repeated at 100 ms intervals, followed by the stimulus, and 50 post-stimulus IV-ramps to measure the dopamine overflow over time. To assess the removal of the released dopamine the width of the signal at half-maximal amplitude was determined. (A) Typical responses from wild type (WT, open circles), MuKO (KO, closed circles) mice and an overlay of normalized traces from panel, to provide the same amplitude, indicating that the dopamine removal is slower in MuKO mice Left inset: overlay of a typical subtracted current response to the voltage ramp (voltammogram; solid trace) and of a voltammogram obtained in 1 M dopamine (stippled trace), demonstrating that the identified species is dopamine. Right inset: Schematic diagram of the striatum indicating the position of the stimulation electrode and carbon fiber in the NAc shell (black oval). The amplitude was determined at the peak of the dopamine signal (vertical arrow a) and the width of the signal was determined at half-maximal amplitude (horizontal arrow b). (B, C) Bar graphs summarizing the results from 12 WT and 12–13 KO recordings. The peak amplitude of dopamine overflow is not different between KO and WT mice (B), but the width at half-maximal amplitude is significantly higher (** P⬍0.01) in KO mice compared with WT mice (C).
(Fig. 1A). We found no difference between genotypes regarding the amount of dopamine released per stimulus (Fig. 1B, WT: 2.21⫾0.18 M, KO: 2.48⫾0.27 M, n⫽12 and 12 respectively, t⫽0.489, NS). The duration of the signal at half-maximal amplitude was measured to compare the removal of dopamine in MuKO and wild type mice (Fig. 1A–C). The width at half-maximal amplitude was significantly larger in MuKO compared with wild type mice (Fig. 1C, WT: 1047⫾18 ms, KO: 1290⫾25 ms, n⫽12 and 13 respectively, t⫽6.821, P⬍0.01), demonstrating a slower removal of dopamine in MuKO mice. We next examined the effects of cocaine and amphetamine on dopamine overflow. Addition of 10 M cocaine to the circulating medium increased the width in both wild type and MuKO mice (Figs. 2, 3A and 3B; genotype effect: F(1,23)⫽18.71, P⬍0.001; treatment effect F(1,23)⫽318.52, P⬍0.001; interaction: F(1,23)⫽10.07, P⫽0.05). In wild type mice the width increased from 1066⫾33 ms before cocaine to 1583⫾26 ms during cocaine, yielding a 49% increase (Fig. 3A and B; n⫽6, t⫽15.43, P⬍0.01). In MuKO the width increased from 1260⫾33 ms before cocaine to 1611⫾34 ms during cocaine (Fig. 3B; n⫽6, t⫽5.63, P⬍0.01), giving a 28% increase, which was significantly smaller than the cocaine-induced increase in width in wild
D. S. Mathon et al. / Neuroscience 141 (2006) 1679 –1684
Fig. 2. The effect of cocaine on dopamine overflow in wild type and MuKO mice. (A) Top panels: cocaine (10 M final bath concentration, gray bars) increases the width at half-maximal amplitude in both WT and KO mice. Middle panels: cocaine produces an initial rise in maximal amplitude (peak at point 2), followed by a decrease in maximal amplitude to below pre-cocaine levels (trough at point 3) in both WT and KO mice. Bottom panels: cocaine increases the pre-stimulus current at the voltage for dopamine oxidation in both WT and KO mice. (B) Example traces from points 1 (pre-cocaine baseline), 2 (peak of cocaine-induced amplitude increase), 3 (trough of cocaine-induced amplitude decrease) and 4 (washout).
Fig. 3. Summary of the effects of cocaine on dopamine overflow in wild type and MuKO mice. Bar graphs summarizing the results from six mice/genotype. (A) The cocaine-induced percentage increase in width is significantly smaller in KO compared with WT mice (** P⬍0.01). (B) The effect of cocaine on the width at half-maximal amplitude in WT and KO mice, expressed as absolute values. Cocaine increases the width in WT (## P⬍0.01) and KO (§§ P⬍0.01). Cocaine abolishes the pre-cocaine difference (** P⬍0.01) between WT and KO mice. (C–E) Bar graphs summarizing the relative effect of cocaine on maximal current amplitude and pre-stimulus current. Cocaine initially increases the maximal current (C), that is followed by a decrease in both WT and KO mice (D). Cocaine increases the pre-stimulus current in both WT and KO mice (E).
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type mice (Fig. 3A; n⫽6 per group, t⫽6.21, P⬍0.01). The pre-cocaine difference in width between wild type and MuKO mice was completely abolished in the presence of cocaine (Fig. 3B; n⫽6 per group, t⫽0.77, NS). Cocaine induced a biphasic effect on the amplitude (Fig. 2A). Initially, cocaine induced an increase in the amplitude (⫹74⫾9% and ⫹60⫾5% in wild type and MuKO respectively, Figs. 2A and 3C), that was followed by a reduction (⫺25⫾2% and ⫺21⫾2% in wild type and MuKO respectively, Figs. 2A and 3D). The effect of cocaine on the amplitude was not different between wild type and MuKO mice (t⫽0.53, NS, n⫽6 and t⫽0.61, NS, n⫽6 comparing wild type and MuKO mice for the increase and the reduction respectively). By monitoring the pre-stimulus current at the voltage corresponding to the dopamine oxidation current, as determined by dopamine calibration of the carbon fiber, it is possible to estimate drug-induced changes in extracellular dopamine levels. Cocaine increased the prestimulus current in both wild type and MuKO mice (Fig. 2A, bottom panels). The increase tended to be smaller in MuKO compared with wild type mice (Fig. 3E; WT: 30⫾3%, KO: 22⫾2%, n⫽6 per group, t⫽2.12, P⫽0.06), suggesting that the cocaine-induced increase in extracellular dopamine is attenuated in MuKO mice. Subsequently, we examined the effect of 10 M amphetamine on dopamine overflow in the NAc (Fig. 4 and 5; effects on width: genotype effect: F(1,25)⫽20.25, P⬍0.001; treatment effect F(1,25)⫽468.13, P⬍0.001; interaction: F(1,25)⫽3.64, P⫽0.07). Amphetamine increased the width in both wild type and MuKO mice (Figs. 4, 5A and B, WT: 1041⫾25 ms before amphetamine to 2065⫾52 ms after
Fig. 4. The effect of amphetamine on dopamine overflow in wild type and MuKO mice. (A) Top panels: amphetamine (10 M final bath concentration, gray bars) increases the width at half-maximal amplitude in both WT and KO mice. Middle panels: amphetamine produces a decrease in maximal amplitude in both WT and KO mice. Bottom panels: amphetamine increases the pre-stimulus current at the voltage for dopamine oxidation in both WT and KO mice. (B) Example traces from points 1 (pre-amphetamine baseline), 2 (maximal increase in width and decrease in amplitude), and 3 (washout).
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Fig. 5. Summary of effects amphetamine on dopamine overflow in wild type and MuKO mice. Bar graph summarizing the results from six to seven mice/genotype. (A) The amphetamine-induced increase in width is significantly smaller in KO compared with WT mice (** P⬍0.01). (B) The absolute effect of amphetamine on the width at half-maximal amplitude in WT and KO mice (n⫽6 –7 mice/ group). Amphetamine increases the width in WT (## P⬍0.01) and KO (§§ P⬍0.01). Amphetamine abolishes the pre-amphetamine difference (** P⬍0.01) between WT and KO mice. (C, D) Bar graphs summarizing the relative effect of amphetamine on maximal current amplitude and pre-stimulus current. Amphetamine decreases the maximal current in both WT and KO mice (C) and increases the pre-stimulus current in both WT and KO mice (D).
amphetamine, t⫽13.51, n⫽6; KO: 1320⫾47 ms before amphetamine to 2259⫾43 ms after amphetamine, t⫽6.86, n⫽7, P⬍0.01 for both groups) and abolished the preamphetamine difference between wild type and MuKO mice (Fig. 5B, pre-amphetamine: WT versus KO t⫽4.36 P⬍0.05; post-amphetamine: WT versus KO, t⫽1.69, NS). The relative increase in width was significantly smaller in MuKO compared with wild type mice (Fig. 5A, 65⫾3% increase in MuKO vs. 99⫾8% increase in WT, n⫽6 for WT and n⫽7 for MuKO, t⫽5.02, P⬍0.001). Amphetamine decreased the amount of stimulus-evoked dopamine overflow, without causing an initial increase as seen with cocaine (Figs. 4 and 5C, WT: ⫺47⫾2%, KO: ⫺45⫾2% of baseline, WT versus KO: t⫽1.16, NS), and increased extracellular dopamine levels as seen by an augmented prestimulus current (Figs. 4 and 5D, WT: ⫹68⫾9% increase, KO: ⫹71⫾11% increase. WT versus KO: t⫽0.18, NS) to the same extent in wild type and MuKO mice. Kinetic analysis of DAT activity revealed that in control conditions, the Vmax of the DAT is reduced in MuKO mice (t⫽6.23, P⬍0.05). Application of 10 M cocaine or amphetamine strongly increased the Km in both wild type and MuKO mice (Table 1; wild type: t⫽7.21, P⬍0.05, MuKO: t⫽7.43, P⬍0.05).
DISCUSSION Our data demonstrate that the duration of dopamine overflow in the NAc following a single electrical stimulation is longer in mice with a targeted deletion of the MOR gene. In addition, cocaine and amphetamine have a smaller relative effect on NAc dopamine reuptake in MuKO compared with wild type mice. The duration and extent of the dopamine overflow are the result of the release and subsequent reuptake of do-
pamine by the dopamine transporter (DAT) (May et al., 1988; Wightman and Zimmerman, 1990). The maximal amplitude of the signal is determined both by the initial release and by the reuptake, and the rate of reuptake is in our experiments reflected by the width of the signal at half-maximal amplitude (Suaud-Chagny et al., 1995; Benoit-Marand et al., 2000; Suaud-Chagny, 2004). The increased width at half-maximal amplitude in MuKO mice therefore demonstrates a decreased rate of DA uptake in MuKO mice. Analysis of DAT kinetics reveals that the Vmax in MuKO mice is reduced compared with wild type mice, possibly resulting from reduced expression of the DAT in MuKO mice. Altering dopamine reuptake dynamics is one way of adapting to changes in extracellular dopamine levels, as is for example observed during cocaine intake and withdrawal (Kuhar and Pilotte, 1996; Little et al., 1999) and after partial lesions of dopamine systems (Garris et al., 1997). Our findings suggest that the decreased rate of reuptake is the consequence of a decreased dopaminergic tone in the NAc of MuKO mice. Thus, we have recently demonstrated that in MuKO mice, mesolimbic dopaminergic neurons receive an increased amount of GABAergic inhibition (Mathon et al., 2005a), presumably resulting from the absence of MORs from local interneurons (Gysling and Wang, 1983; Johnson and North, 1992; Klitenick et al., 1992). In addition, we have observed in vivo that dopaminergic neurons of MuKO mice display decreased impulse activity compared with WT mice (Mathon et al., 2005b). This is in keeping with another study in MuKO mice, where unaltered basal dopamine levels were found, while dopamine uptake was decreased, providing further evidence for decreased dopamine release (Chefer et al., 2003). Thus, increased GABAergic inhibition onto VTA dopaminergic neurons in MuKO mice (Mathon et al., 2005a), results in lower impulse activity of these neurons (Mathon et al., 2005b). The resultant decreased dopaminergic impulse flow in the NAc of MuKO mice causes the rate of dopamine reuptake to be down-regulated, so that the basal dopamine levels in the NAc of MuKO mice are unaltered as compared with wild type mice (Chefer et al., 2003). Table 1. Comparison of dopamine uptake kinetics between WT and MuKO mice and the effect of cocaine and amphetamine on these parameters Km (M)
Vmax (M/s)
Cocaine Amphetamine
0.19⫾0.03 6.01⫾0.43b 8.57⫾0.15b
1.96⫾0.23 2.01⫾0.18 1.89⫾0.11
Cocaine Amphetamine
0.15⫾0.02 5.69⫾0.56b 7.84⫾0.83b
1.42⫾0.18a 1.51⫾0.21 1.47⫾0.25
Treatment group WT Basal ⫹10 M ⫹10 M MuKO Basal ⫹10 M ⫹10 M a b
Significantly different from wildtype, P⬍0.05. Significantly different from basal condition, P⬍0.05.
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The second part of this study focused on the effects of cocaine and amphetamine on NAc dopamine overflow in MuKO mice. The concentrations of cocaine and amphetamine are similar to those used by others and have maximal effects on dopamine dynamics (Jones et al., 1995a,b, 1998; Sulzer et al., 1995; Anderson et al., 1998; Schmitz et al., 2002; Mateo et al., 2005). Cocaine increases extracellular dopamine levels primarily by acting as a competitive inhibitor at the DAT, while amphetamine increases extracellular dopamine levels by i) blocking the DAT, ii) causing reverse transport of dopamine through the DAT and iii) increasing the cytoplasmic dopamine pool available for this reverse transport by depleting vesicular dopamine stores (Ritz and Kuhar, 1989; Kuhar et al., 1991; Schmitz et al., 2002). NAc dopamine reuptake was slower in MuKO mice under basal conditions (as reflected in the reduced Vmax, see Table 1) and cocaine and amphetamine abolish the difference between MuKO and wild type. Thus, the relative effect of these psychostimulant drugs on dopamine reuptake is smaller in MuKO compared with wild type mice (Richelson and Pfenning, 1984; Ritz et al., 1987; Jones et al., 1995b). The fact that the difference in dopamine overflow duration between MuKO and wild type mice is abolished after addition of both cocaine and amphetamine therefore indicates that this difference is due to decreased DAT activity. Because dopaminergic neurotransmission in the NAc plays a crucial role in the reinforcing properties of psychostimulant drugs (Koob, 1992; Wise, 2004; Pierce and Kumaresan, 2006), it is likely that the decreased relative effect of cocaine on NAc dopamine reuptake underlies the decreased self-administration of cocaine we previously observed in MuKO mice (Mathon et al., 2005a). Cocaine transiently increased the stimulus-induced overflow of dopamine (see Fig. 2A, middle panel). The initial increase in the amplitude of the stimulus-evoked dopamine signal might result from a decreased reuptake (Schmitz et al., 2002) or an increase in the release of dopamine (Lee et al., 2001). The secondary decrease in overflow amplitude is likely to be caused by enhanced D2 autoreceptor activation resulting from the enhanced dopamine overflow (Schmitz et al., 2002). Activation of D2 autoreceptors causes an up-regulation of DAT activity in a Gi/o-dependent manner (Mayfield and Zahniser, 2001). However, kinetic analysis of DAT activity in the presence of cocaine shows that the Vmax of the DAT is not changed (Table 1), indicating that in the presence of cocaine, this upregulation does not occur. In contrast to cocaine, amphetamine decreases stimulus-dependent dopamine release (Jones et al., 1998; Schmitz et al., 2002). In addition to the enhanced D2 autoreceptor activation this is caused by the depletion of vesicular dopamine stores due to a collapsing of the vesicular pH gradient (Sulzer and Rayport, 1990; Sulzer et al., 1995; Schmitz et al., 2002). We observed no clear differences in drug-induced changes in dopamine release and extracellular dopamine levels between MuKO and wild type mice, although cocaine tended to increase extracellular dopamine levels to a lesser extent in MuKO mice.
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CONCLUSION In conclusion, we have demonstrated that NAc dopamine overflow is prolonged in MuKO mice, which is likely the result of a decreased reuptake of dopamine by the DAT. In addition, cocaine and amphetamine have a decreased relative effect on dopamine reuptake in MuKO mice compared with wild type mice. The reduced reuptake, likely caused by a decreased dopaminergic impulse flow, and the decreased relative effect of these psychostimulants, provide a possible mechanism underlying the decreased reinforcing properties of cocaine in MuKO mice (Becker et al., 2002; Hall et al., 2004; Mathon et al., 2005a). Acknowledgments—The authors wish to thank A. J. A. van der Linden for her technical assistance and Dr. J. E. Pintar for the MOR KO animals.
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(Accepted 3 May 2006) (Available online 13 June 2006)
REDUCED PSYCHOSTIMULANT EFFECTS ON DOPAMINE DYNAMICS IN THE NUCLEUS ACCUMBENS OF -OPIOID RECEPTOR KNOCKOUT MICE tems, and in particular to -opioid receptors (MORs) located in the VTA, in modulating the reinforcing properties of drugs of abuse, including psychostimulant drugs such as cocaine (van Ree et al., 1999; Kieffer and GaveriauxRuff, 2002; Gerrits et al., 2003). Thus, infusion of the MOR antagonist naltrexone into the VTA retarded the acquisition of cocaine self-administration (Ramsey et al., 1999), and intra-VTA administration of the specific MOR agonist DAMGO enhanced cocaine self-administration in rats (Corrigall et al., 1999). Moreover, mice lacking MORs ( opioid receptor knockout (MuKO) mice) have been shown to be hyposensitive to the rewarding effects of a variety of drugs, including cocaine, as assessed using drug self-administration and place conditioning paradigms (Becker et al., 2002; Hall et al., 2004; Mathon et al., 2005a, for reviews see: Kieffer and Gaveriaux-Ruff, 2002; Gerrits et al., 2003). The most likely neural mechanism by which MORs influence drug reinforcement is by disinhibition of VTA dopamine neurons. That is, MOR stimulation inhibits local GABAergic interneurons (Gysling and Wang, 1983; Matthews and German, 1984; Johnson and North, 1992; Klitenick et al., 1992, for reviews see: Kalivas, 1993; Mathon et al., 2003), resulting in a subsequent increase in dopamine overflow in target areas (Spanagel et al., 1992). We have previously demonstrated that MuKO mice display decreased cocaine self-administration (Mathon et al., 2005a). In addition, VTA dopaminergic neurons in these mice receive an increased GABAergic input (Mathon et al., 2005a) and as a possible consequence, display decreased impulse activity (Mathon et al., 2005b). Microdialysis studies at the mesolimbic terminal area level have shown that there was no difference in the dopamine levels in the NAc between wild type and MuKO mice (Chefer et al., 2003, 2004; Robledo et al., 2004) and that the cocaineinduced increase in extracellular dopamine levels was comparable between wild type and MuKO mice (Chefer et al., 2004). As changes in neuronal activity likely result in adaptations in target areas, we here examined possible alterations in dopamine dynamics in the main terminal field of this system, the NAc, of MuKO mice. Fast scan cyclic voltammetry (FSCV) was used to measure stimulusevoked dopamine overflow at a sub-second time scale to examine detailed kinetics of dopamine overflow and reuptake. We used FSCV to 1) compare the basal overflow of stimulus-evoked dopamine in the NAc of WT and MuKO mice, and 2) examine the effects of cocaine and amphetamine on NAc dopamine overflow in these mice.
D. S. MATHON, L. J. M. J. VANDERSCHUREN AND G. M. J. RAMAKERS* Rudolf Magnus Institute of Neuroscience, Department of Pharmacology and Anatomy, University Medical Center Utrecht, Universiteitsweg 100, 3584 CG Utrecht, The Netherlands
Abstract—Dopamine neurotransmission in the nucleus accumbens plays a pivotal role in the reinforcing properties of drugs of abuse. Two interacting processes regulate nucleus accumbens dopamine overflow: release of dopamine from presynaptic terminals and the subsequent reuptake by dopamine transporters. Opioid neurotransmission, primarily through -opioid receptors has also been strongly implicated in drug reward. We have previously shown that mice lacking the -opioid receptor display decreased cocaine self-administration. In addition, we found decreased impulse activity of midbrain dopaminergic neurons and an increased GABAergic input to these neurons in -opioid receptor knockout mice. In the present study we investigated whether these changes in dopaminergic cell bodies are accompanied by altered dopamine dynamics at the terminal level. To that aim, we measured nucleus accumbens dopamine overflow using fast scan cyclic voltammetry. Our data demonstrate that in -opioid receptor knockout mice 1) the reuptake of dopamine in the nucleus accumbens is slower, and 2) the relative effect of cocaine and amphetamine on the reuptake of dopamine is smaller compared with wild type mice. These data provide a mechanism for the decreased reinforcing properties of cocaine observed in -opioid receptor knockout mice. © 2006 IBRO. Published by Elsevier Ltd. All rights reserved. Key words: dopamine reuptake, nucleus accumbens, cocaine, amphetamine, fast scan cyclic voltammetry.
The mesolimbic dopamine system, composed of dense projections from the ventral tegmental area (VTA) to the nucleus accumbens (NAc), has long been implicated in the physiology of incentive motivation and the pathophysiology of substance abuse (Koob, 1992; Berridge and Robinson, 1998; Cardinal et al., 2002; Wise, 2004; Salamone et al., 2005; Pierce and Kumaresan, 2006). Drugs of abuse, though acting on different neuronal substrates, have in common that they increase dopamine levels in the NAc (Di Chiara and Imperato, 1988; Pontieri et al., 1995). An important role has been ascribed to endogenous opioid sys-
*Corresponding author. Tel: ⫹31-30-2538413; fax: ⫹31-30-2539032. E-mail address: [email protected] (G. M. J. Ramakers). Abbreviations: aCSF, artificial cerebrospinal fluid; DAT, dopamine transporter; FSCV, fast scan cyclic voltammetry; MOR, opioid receptor; MuKO, opioid receptor knockout; NAc, nucleus accumbens; VTA, ventral tegmental area.
0306-4522/06$30.00⫹0.00 © 2006 IBRO. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.neuroscience.2006.05.003
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EXPERIMENTAL PROCEDURES The MuKO mice used in these experiments have been described elsewhere (Schuller et al., 1999). Wild type and homozygous male knockout mice were obtained from heterozygous breeding. The mice (8 –9 months old) used in the present study were on a C57Bl6/Jico background after six to seven back-crossings to C57Bl6/Jico mice (Charles River, l’Arbresle, France). Animals were anesthetized with isoflurane, decapitated and the brain was rapidly removed and kept in ice-cold high-magnesium artificial cerebrospinal fluid (aCSF) containing (in mM): 124 NaCl, 3.3 KCl, 1.2 KH2PO4, 2.5 MgSO4, 2.5 CaCl2, 20 NaHCO3, 10 glucose, saturated with 95% O2–5% CO2. Slices (200 m) were cut using a vibratome (Leica VT1000S; Leica Instruments, Nussloch, Germany) and after preparation kept at room temperature in aCSF with a normal Mg2⫹ concentration (1.3 mM). Slices were then transferred to a submerged recording chamber, where they were perfused at 2–3 ml/min with aCSF at room temperature. Dopamine overflow was measured following electrical stimulation to evoke calcium-dependent vesicular dopamine release, from the NAc in 200 m thick coronal slice preparations. Dopamine release was evoked by a single pulse (0 –1 mA, 300 s) applied through a bipolar stimulation electrode (bipolar stainless steel, 100 m, insulated except from the tip) every 30 s. Dopamine was detected with 5 m carbon-fiber disk electrodes insulated with electro deposition paint (Schulte and Chow, 1996; ALA Scientific Instruments Inc., Westbury, NY, USA) using FSCV (Kawagoe et al., 1993). Cyclic voltammograms (ramps from ⫺500 mV to ⫹1000 mV and back to ⫺500 mV versus an Ag/AgCl reference, 300 V/s) were repeated every 100 ms using an EPC9 amplifier (HEKA Electronic, Lambrecht, Germany). Stimulus-evoked dopamine overflow was measured by subtracting the background current obtained before stimulation (average of 10 pre-stimulus responses) from the current measured after stimulation. The resulting voltammogram showed a typical dopamine profile, with an oxidation peak between 500 and 700 mV and a smaller reduction peak around ⫺300 mV (see inset in Fig. 1A). The concentration of the dopamine overflow was calculated after calibrating the recording electrode in known concentrations of dopamine. The resultant dopamine concentrations are plotted against time (see Fig. 1A for example). Pilot experiments showed that stimulus-induced dopamine release was constant for at least 60 min (Ramakers and Mathon, unpublished observations). The amplitude was determined at the peak of the dopamine signal (vertical arrow a in Fig. 1A). The width of the signal was determined at half-maximal amplitude (horizontal arrow b in Fig. 1A). As an indication of changes in extracellular dopamine levels, the pre-stimulus current at the dopamine oxidation peak was monitored. Dopamine uptake kinetics were determined by fitting the decay of the dopamine transient with an exponential fit. This procedure revealed values of Km and Vmax in wild type mice that are comparable with reported values for these parameters (Budygin et al., 2002, 2004). Amphetamine (d-amphetamine sulfate) and cocaine (cocaine-HCl) were obtained from OPG (Utrecht, the Netherlands). Amphetamine and cocaine were dissolved in aCSF as 1000 times concentrated stocks and applied to the slices through the perfusion medium. For statistical analysis, group averages of absolute values were compared using Student’s t-test or a two factor ANOVA followed by post hoc Student’s t-test where appropriate.
RESULTS Dopamine release was electrically evoked in the NAc shell of MuKO and wild type mice and measured with FSCV. The voltammogram constructed from the voltage ramp identifies dopamine as the measured substance, with a maximal oxidization current at approximately ⫹600 mV
Fig. 1. Dopamine removal in the NAc shell of MuKO mice is slower compared with control mice. Dopamine overflow was evoked by a single local stimulus in the NAc shell of MuKO and WT mice. Ten pre-stimulus IV-ramps were repeated at 100 ms intervals, followed by the stimulus, and 50 post-stimulus IV-ramps to measure the dopamine overflow over time. To assess the removal of the released dopamine the width of the signal at half-maximal amplitude was determined. (A) Typical responses from wild type (WT, open circles), MuKO (KO, closed circles) mice and an overlay of normalized traces from panel, to provide the same amplitude, indicating that the dopamine removal is slower in MuKO mice Left inset: overlay of a typical subtracted current response to the voltage ramp (voltammogram; solid trace) and of a voltammogram obtained in 1 M dopamine (stippled trace), demonstrating that the identified species is dopamine. Right inset: Schematic diagram of the striatum indicating the position of the stimulation electrode and carbon fiber in the NAc shell (black oval). The amplitude was determined at the peak of the dopamine signal (vertical arrow a) and the width of the signal was determined at half-maximal amplitude (horizontal arrow b). (B, C) Bar graphs summarizing the results from 12 WT and 12–13 KO recordings. The peak amplitude of dopamine overflow is not different between KO and WT mice (B), but the width at half-maximal amplitude is significantly higher (** P⬍0.01) in KO mice compared with WT mice (C).
(Fig. 1A). We found no difference between genotypes regarding the amount of dopamine released per stimulus (Fig. 1B, WT: 2.21⫾0.18 M, KO: 2.48⫾0.27 M, n⫽12 and 12 respectively, t⫽0.489, NS). The duration of the signal at half-maximal amplitude was measured to compare the removal of dopamine in MuKO and wild type mice (Fig. 1A–C). The width at half-maximal amplitude was significantly larger in MuKO compared with wild type mice (Fig. 1C, WT: 1047⫾18 ms, KO: 1290⫾25 ms, n⫽12 and 13 respectively, t⫽6.821, P⬍0.01), demonstrating a slower removal of dopamine in MuKO mice. We next examined the effects of cocaine and amphetamine on dopamine overflow. Addition of 10 M cocaine to the circulating medium increased the width in both wild type and MuKO mice (Figs. 2, 3A and 3B; genotype effect: F(1,23)⫽18.71, P⬍0.001; treatment effect F(1,23)⫽318.52, P⬍0.001; interaction: F(1,23)⫽10.07, P⫽0.05). In wild type mice the width increased from 1066⫾33 ms before cocaine to 1583⫾26 ms during cocaine, yielding a 49% increase (Fig. 3A and B; n⫽6, t⫽15.43, P⬍0.01). In MuKO the width increased from 1260⫾33 ms before cocaine to 1611⫾34 ms during cocaine (Fig. 3B; n⫽6, t⫽5.63, P⬍0.01), giving a 28% increase, which was significantly smaller than the cocaine-induced increase in width in wild
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Fig. 2. The effect of cocaine on dopamine overflow in wild type and MuKO mice. (A) Top panels: cocaine (10 M final bath concentration, gray bars) increases the width at half-maximal amplitude in both WT and KO mice. Middle panels: cocaine produces an initial rise in maximal amplitude (peak at point 2), followed by a decrease in maximal amplitude to below pre-cocaine levels (trough at point 3) in both WT and KO mice. Bottom panels: cocaine increases the pre-stimulus current at the voltage for dopamine oxidation in both WT and KO mice. (B) Example traces from points 1 (pre-cocaine baseline), 2 (peak of cocaine-induced amplitude increase), 3 (trough of cocaine-induced amplitude decrease) and 4 (washout).
Fig. 3. Summary of the effects of cocaine on dopamine overflow in wild type and MuKO mice. Bar graphs summarizing the results from six mice/genotype. (A) The cocaine-induced percentage increase in width is significantly smaller in KO compared with WT mice (** P⬍0.01). (B) The effect of cocaine on the width at half-maximal amplitude in WT and KO mice, expressed as absolute values. Cocaine increases the width in WT (## P⬍0.01) and KO (§§ P⬍0.01). Cocaine abolishes the pre-cocaine difference (** P⬍0.01) between WT and KO mice. (C–E) Bar graphs summarizing the relative effect of cocaine on maximal current amplitude and pre-stimulus current. Cocaine initially increases the maximal current (C), that is followed by a decrease in both WT and KO mice (D). Cocaine increases the pre-stimulus current in both WT and KO mice (E).
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type mice (Fig. 3A; n⫽6 per group, t⫽6.21, P⬍0.01). The pre-cocaine difference in width between wild type and MuKO mice was completely abolished in the presence of cocaine (Fig. 3B; n⫽6 per group, t⫽0.77, NS). Cocaine induced a biphasic effect on the amplitude (Fig. 2A). Initially, cocaine induced an increase in the amplitude (⫹74⫾9% and ⫹60⫾5% in wild type and MuKO respectively, Figs. 2A and 3C), that was followed by a reduction (⫺25⫾2% and ⫺21⫾2% in wild type and MuKO respectively, Figs. 2A and 3D). The effect of cocaine on the amplitude was not different between wild type and MuKO mice (t⫽0.53, NS, n⫽6 and t⫽0.61, NS, n⫽6 comparing wild type and MuKO mice for the increase and the reduction respectively). By monitoring the pre-stimulus current at the voltage corresponding to the dopamine oxidation current, as determined by dopamine calibration of the carbon fiber, it is possible to estimate drug-induced changes in extracellular dopamine levels. Cocaine increased the prestimulus current in both wild type and MuKO mice (Fig. 2A, bottom panels). The increase tended to be smaller in MuKO compared with wild type mice (Fig. 3E; WT: 30⫾3%, KO: 22⫾2%, n⫽6 per group, t⫽2.12, P⫽0.06), suggesting that the cocaine-induced increase in extracellular dopamine is attenuated in MuKO mice. Subsequently, we examined the effect of 10 M amphetamine on dopamine overflow in the NAc (Fig. 4 and 5; effects on width: genotype effect: F(1,25)⫽20.25, P⬍0.001; treatment effect F(1,25)⫽468.13, P⬍0.001; interaction: F(1,25)⫽3.64, P⫽0.07). Amphetamine increased the width in both wild type and MuKO mice (Figs. 4, 5A and B, WT: 1041⫾25 ms before amphetamine to 2065⫾52 ms after
Fig. 4. The effect of amphetamine on dopamine overflow in wild type and MuKO mice. (A) Top panels: amphetamine (10 M final bath concentration, gray bars) increases the width at half-maximal amplitude in both WT and KO mice. Middle panels: amphetamine produces a decrease in maximal amplitude in both WT and KO mice. Bottom panels: amphetamine increases the pre-stimulus current at the voltage for dopamine oxidation in both WT and KO mice. (B) Example traces from points 1 (pre-amphetamine baseline), 2 (maximal increase in width and decrease in amplitude), and 3 (washout).
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Fig. 5. Summary of effects amphetamine on dopamine overflow in wild type and MuKO mice. Bar graph summarizing the results from six to seven mice/genotype. (A) The amphetamine-induced increase in width is significantly smaller in KO compared with WT mice (** P⬍0.01). (B) The absolute effect of amphetamine on the width at half-maximal amplitude in WT and KO mice (n⫽6 –7 mice/ group). Amphetamine increases the width in WT (## P⬍0.01) and KO (§§ P⬍0.01). Amphetamine abolishes the pre-amphetamine difference (** P⬍0.01) between WT and KO mice. (C, D) Bar graphs summarizing the relative effect of amphetamine on maximal current amplitude and pre-stimulus current. Amphetamine decreases the maximal current in both WT and KO mice (C) and increases the pre-stimulus current in both WT and KO mice (D).
amphetamine, t⫽13.51, n⫽6; KO: 1320⫾47 ms before amphetamine to 2259⫾43 ms after amphetamine, t⫽6.86, n⫽7, P⬍0.01 for both groups) and abolished the preamphetamine difference between wild type and MuKO mice (Fig. 5B, pre-amphetamine: WT versus KO t⫽4.36 P⬍0.05; post-amphetamine: WT versus KO, t⫽1.69, NS). The relative increase in width was significantly smaller in MuKO compared with wild type mice (Fig. 5A, 65⫾3% increase in MuKO vs. 99⫾8% increase in WT, n⫽6 for WT and n⫽7 for MuKO, t⫽5.02, P⬍0.001). Amphetamine decreased the amount of stimulus-evoked dopamine overflow, without causing an initial increase as seen with cocaine (Figs. 4 and 5C, WT: ⫺47⫾2%, KO: ⫺45⫾2% of baseline, WT versus KO: t⫽1.16, NS), and increased extracellular dopamine levels as seen by an augmented prestimulus current (Figs. 4 and 5D, WT: ⫹68⫾9% increase, KO: ⫹71⫾11% increase. WT versus KO: t⫽0.18, NS) to the same extent in wild type and MuKO mice. Kinetic analysis of DAT activity revealed that in control conditions, the Vmax of the DAT is reduced in MuKO mice (t⫽6.23, P⬍0.05). Application of 10 M cocaine or amphetamine strongly increased the Km in both wild type and MuKO mice (Table 1; wild type: t⫽7.21, P⬍0.05, MuKO: t⫽7.43, P⬍0.05).
DISCUSSION Our data demonstrate that the duration of dopamine overflow in the NAc following a single electrical stimulation is longer in mice with a targeted deletion of the MOR gene. In addition, cocaine and amphetamine have a smaller relative effect on NAc dopamine reuptake in MuKO compared with wild type mice. The duration and extent of the dopamine overflow are the result of the release and subsequent reuptake of do-
pamine by the dopamine transporter (DAT) (May et al., 1988; Wightman and Zimmerman, 1990). The maximal amplitude of the signal is determined both by the initial release and by the reuptake, and the rate of reuptake is in our experiments reflected by the width of the signal at half-maximal amplitude (Suaud-Chagny et al., 1995; Benoit-Marand et al., 2000; Suaud-Chagny, 2004). The increased width at half-maximal amplitude in MuKO mice therefore demonstrates a decreased rate of DA uptake in MuKO mice. Analysis of DAT kinetics reveals that the Vmax in MuKO mice is reduced compared with wild type mice, possibly resulting from reduced expression of the DAT in MuKO mice. Altering dopamine reuptake dynamics is one way of adapting to changes in extracellular dopamine levels, as is for example observed during cocaine intake and withdrawal (Kuhar and Pilotte, 1996; Little et al., 1999) and after partial lesions of dopamine systems (Garris et al., 1997). Our findings suggest that the decreased rate of reuptake is the consequence of a decreased dopaminergic tone in the NAc of MuKO mice. Thus, we have recently demonstrated that in MuKO mice, mesolimbic dopaminergic neurons receive an increased amount of GABAergic inhibition (Mathon et al., 2005a), presumably resulting from the absence of MORs from local interneurons (Gysling and Wang, 1983; Johnson and North, 1992; Klitenick et al., 1992). In addition, we have observed in vivo that dopaminergic neurons of MuKO mice display decreased impulse activity compared with WT mice (Mathon et al., 2005b). This is in keeping with another study in MuKO mice, where unaltered basal dopamine levels were found, while dopamine uptake was decreased, providing further evidence for decreased dopamine release (Chefer et al., 2003). Thus, increased GABAergic inhibition onto VTA dopaminergic neurons in MuKO mice (Mathon et al., 2005a), results in lower impulse activity of these neurons (Mathon et al., 2005b). The resultant decreased dopaminergic impulse flow in the NAc of MuKO mice causes the rate of dopamine reuptake to be down-regulated, so that the basal dopamine levels in the NAc of MuKO mice are unaltered as compared with wild type mice (Chefer et al., 2003). Table 1. Comparison of dopamine uptake kinetics between WT and MuKO mice and the effect of cocaine and amphetamine on these parameters Km (M)
Vmax (M/s)
Cocaine Amphetamine
0.19⫾0.03 6.01⫾0.43b 8.57⫾0.15b
1.96⫾0.23 2.01⫾0.18 1.89⫾0.11
Cocaine Amphetamine
0.15⫾0.02 5.69⫾0.56b 7.84⫾0.83b
1.42⫾0.18a 1.51⫾0.21 1.47⫾0.25
Treatment group WT Basal ⫹10 M ⫹10 M MuKO Basal ⫹10 M ⫹10 M a b
Significantly different from wildtype, P⬍0.05. Significantly different from basal condition, P⬍0.05.
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The second part of this study focused on the effects of cocaine and amphetamine on NAc dopamine overflow in MuKO mice. The concentrations of cocaine and amphetamine are similar to those used by others and have maximal effects on dopamine dynamics (Jones et al., 1995a,b, 1998; Sulzer et al., 1995; Anderson et al., 1998; Schmitz et al., 2002; Mateo et al., 2005). Cocaine increases extracellular dopamine levels primarily by acting as a competitive inhibitor at the DAT, while amphetamine increases extracellular dopamine levels by i) blocking the DAT, ii) causing reverse transport of dopamine through the DAT and iii) increasing the cytoplasmic dopamine pool available for this reverse transport by depleting vesicular dopamine stores (Ritz and Kuhar, 1989; Kuhar et al., 1991; Schmitz et al., 2002). NAc dopamine reuptake was slower in MuKO mice under basal conditions (as reflected in the reduced Vmax, see Table 1) and cocaine and amphetamine abolish the difference between MuKO and wild type. Thus, the relative effect of these psychostimulant drugs on dopamine reuptake is smaller in MuKO compared with wild type mice (Richelson and Pfenning, 1984; Ritz et al., 1987; Jones et al., 1995b). The fact that the difference in dopamine overflow duration between MuKO and wild type mice is abolished after addition of both cocaine and amphetamine therefore indicates that this difference is due to decreased DAT activity. Because dopaminergic neurotransmission in the NAc plays a crucial role in the reinforcing properties of psychostimulant drugs (Koob, 1992; Wise, 2004; Pierce and Kumaresan, 2006), it is likely that the decreased relative effect of cocaine on NAc dopamine reuptake underlies the decreased self-administration of cocaine we previously observed in MuKO mice (Mathon et al., 2005a). Cocaine transiently increased the stimulus-induced overflow of dopamine (see Fig. 2A, middle panel). The initial increase in the amplitude of the stimulus-evoked dopamine signal might result from a decreased reuptake (Schmitz et al., 2002) or an increase in the release of dopamine (Lee et al., 2001). The secondary decrease in overflow amplitude is likely to be caused by enhanced D2 autoreceptor activation resulting from the enhanced dopamine overflow (Schmitz et al., 2002). Activation of D2 autoreceptors causes an up-regulation of DAT activity in a Gi/o-dependent manner (Mayfield and Zahniser, 2001). However, kinetic analysis of DAT activity in the presence of cocaine shows that the Vmax of the DAT is not changed (Table 1), indicating that in the presence of cocaine, this upregulation does not occur. In contrast to cocaine, amphetamine decreases stimulus-dependent dopamine release (Jones et al., 1998; Schmitz et al., 2002). In addition to the enhanced D2 autoreceptor activation this is caused by the depletion of vesicular dopamine stores due to a collapsing of the vesicular pH gradient (Sulzer and Rayport, 1990; Sulzer et al., 1995; Schmitz et al., 2002). We observed no clear differences in drug-induced changes in dopamine release and extracellular dopamine levels between MuKO and wild type mice, although cocaine tended to increase extracellular dopamine levels to a lesser extent in MuKO mice.
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CONCLUSION In conclusion, we have demonstrated that NAc dopamine overflow is prolonged in MuKO mice, which is likely the result of a decreased reuptake of dopamine by the DAT. In addition, cocaine and amphetamine have a decreased relative effect on dopamine reuptake in MuKO mice compared with wild type mice. The reduced reuptake, likely caused by a decreased dopaminergic impulse flow, and the decreased relative effect of these psychostimulants, provide a possible mechanism underlying the decreased reinforcing properties of cocaine in MuKO mice (Becker et al., 2002; Hall et al., 2004; Mathon et al., 2005a). Acknowledgments—The authors wish to thank A. J. A. van der Linden for her technical assistance and Dr. J. E. Pintar for the MOR KO animals.
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(Accepted 3 May 2006) (Available online 13 June 2006)