bs_bs_banner

Addiction Biology

PRECLINICAL STUD Y

doi:10.1111/adb.12178

Elevated dopamine in the medial prefrontal cortex suppresses cocaine seeking via D1 receptor overstimulation Paola Devoto1,2,3*, Liana Fattore3,4*, Silvia Antinori1, Pierluigi Saba1, Roberto Frau1,2, Walter Fratta1,3 & Gian Luigi Gessa1,2,4 Section of Neuroscience and Clinical Pharmacology, Department of Biomedical Sciences, University of Cagliari, Italy1, ‘Guy Everett Laboratory’, University of Cagliari, Italy2, Center of Excellence ‘Neurobiology of Addiction’, University of Cagliari, Italy3 and Institute of Neuroscience—Cagliari, National Research Council (CNR), Italy4

ABSTRACT Previous investigations indicate that the dopamine-β-hydroxylase (DBH) inhibitors disulfiram and nepicastat suppress cocaine-primed reinstatement of cocaine self-administration behaviour. Moreover, both inhibitors increase dopamine release in the rat medial prefrontal cortex (mPFC) and markedly potentiate cocaine-induced dopamine release in this region. This study was aimed to clarify if the suppressant effect of DBH inhibitors on cocaine reinstatement was mediated by the high extracellular dopamine in the rat mPFC leading to a supra-maximal stimulation of D1 receptors in the dorsal division of mPFC, an area critical for reinstatement of cocaine-seeking behaviour. In line with previous microdialysis studies in drug-naïve animals, both DBH inhibitors potentiated cocaine-induced dopamine release in the mPFC, in the same animals in which they also suppressed reinstatement of cocaine seeking. Similar to the DBH inhibitors, L-DOPA potentiated cocaine-induced dopamine release in the mPFC and suppressed cocaine-induced reinstatement of cocaine-seeking behaviour. The bilateral microinfusion of the D1 receptor antagonist SCH 23390 into the dorsal mPFC not only prevented cocaine-induced reinstatement of cocaine seeking but also reverted both disulfiram- and L-DOPA-induced suppression of reinstatement. Moreover, the bilateral microinfusion of the D1 receptor agonist chloro-APB (SKF 82958) into the dorsal mPFC markedly attenuated cocaine-induced reinstatement of cocaine seeking. These results suggest that stimulation of D1 receptors in the dorsal mPFC plays a crucial role in cocaine-induced reinstatement of cocaine seeking, whereas the suppressant effect of DBH inhibitors and L-DOPA on drug-induced reinstatement is mediated by a supra-maximal stimulation of D1 receptors leading to their inactivation. Keywords

Cocaine-seeking reinstatement, DBH inhibitor, L-DOPA, microdialysis, noradrenaline.

Correspondence to: Paola Devoto, Section of Neuroscience and Clinical Pharmacology, Department of Biomedical Sciences, University of Cagliari, Cittadella Universitaria, I-09042 Monserrato, Cagliari, Italy. E-mail: [email protected]

INTRODUCTION Cocaine dependence is a chronic disorder characterized by recurrent relapses that limit the success of therapeutic interventions after detoxification (Simpson et al. 1999). Both in humans and in experimental animals, three main factors are responsible for the relapse to cocaine seeking: cocaine-associated stimuli, cocaine primings and stress (Jaffe et al. 1989; Erb, Shaham & Stewart

1996; Robbins et al. 1997; De Vries et al. 1998; Weiss et al. 2001; Weiss 2010). In the animal model of cocaine dependence, reinstatement of cocaine seeking can be elicited even after weeks of withdrawal from cocaine self-administration, thus providing a valuable tool for investigating potential pharmacotherapies for relapse prevention (Weiss 2010). Recently, two dopamine-β-hydroxylase (DBH) inhibitors, disulfiram and nepicastat (Goldstein et al.

*These authors contributed equally to the paper. © 2014 Society for the Study of Addiction

Addiction Biology, 21, 61–71

62

Paola Devoto et al.

1964; Stanley et al. 1997), have been shown to attenuate cocaine-induced reinstatement of cocaineseeking behaviour in rats (Schroeder et al. 2010, 2013). The ability of these two DBH inhibitors to suppress reinstatement has been attributed to the inhibition of noradrenaline synthesis, resulting in a loss of α1-mediated noradrenergic tone on mesolimbic dopaminergic neurons, which is thought to play a permissive role in cocaine-induced release of dopamine in the nucleus accumbens and to be therefore essential for cocaine-induced reinstatement (Schroeder et al. 2010). At variance with this interpretation, we found that both disulfiram and nepicastat, consistent with their ability to inhibit DBH, profoundly reduced noradrenaline release in different brain regions, but also produced a selective increase of dopamine release in the medial prefrontal cortex (mPFC), and failed to modify extracellular dopamine in the nucleus accumbens (Devoto et al. 2012, 2014). Importantly, both DBH inhibitors not only reduced cocaine-induced noradrenaline release in the mPFC and nucleus accumbens but also markedly potentiated cocaine-induced dopamine release in the mPFC and failed to modify cocaine effect in the nucleus accumbens (Devoto et al. 2012, 2014). Changes in dopamine concentration in the mPFC are involved in cocaine-seeking and cocaine-induced reinstatement of drug-seeking behaviour (McFarland & Kalivas 2001; Sun & Rebec 2005). The purpose of this study was therefore to challenge the hypothesis that the suppressant effect of the DBH inhibitors on cocaineinduced reinstatement of cocaine-seeking behaviour was mediated by an excessive extracellular dopamine concentration in the mPFC, leading to the supramaximal stimulation of D1 receptors in its dorsal division and, consequently, in their functional suppression in this region (Seamans & Yang 2004), which is thought to be critical for the cocaine-seeking reinstatement (McFarland & Kalivas 2001). To this aim, we investigated if a dose of L-DOPA producing similar potentiation of cocaine effect on extracellular dopamine in the mPFC as DBH inhibitors also inhibited cocaine-induced reinstatement. Moreover, we verified whether the dopamine receptor Type 1 (D1) antagonist SCH 23390 locally infused into the dorsal mPFC would (1) antagonize cocaine-induced reinstatement of cocaine seeking and (2) reverse the suppressant effect of DBH inhibitors and L-DOPA on the reinstatement of cocaine seeking. Finally, we examined if the D1 receptor agonist chloroAPB locally perfused into the dorsal mPFC would mimic the effect of the high extracellular DA by inhibiting drug-induced reinstatement of cocaine-seeking behaviour. © 2014 Society for the Study of Addiction

MATERIALS AND METHODS Animals A total of 84 male Sprague Dawley rats (Harlan Nossan, San Pietro al Natisone, Udine, Italy) weighing 265–300 g at the beginning of the study were used. Animals were housed five per cage in a climate-controlled animal room (temperature of 21 ± 1°C, 60 ± 10 percent humidity) under a reversed 12 hours light/dark cycle (light on from 7:00 pm), with free access to food and water for at least 7 days before any treatment. After implantation surgery of intravenous (i.v.) catheter for self-administration training, rats were housed individually. Following recovery from i.v. surgery, food was restricted to 20 g/day, and given shortly after the end of each daily session, to maintain free feeding weights at ∼85 percent, with water available ad libitum. Experiments took place at the same time each day during the dark phase of the cycle. All experimental procedures were approved by the local Ethical and Animal Care Committee and performed according to the guidelines for care and use of experimental animals of the European Union (EEC Council 86/609; D.L. 27/01/1992, n. 116). All efforts were made to minimize animal suffering and reduce the number of animals used. Surgery Intravenous catheter implantation Following 7 days of acclimation from their arrival, animals under deep anaesthesia with isoflurane were surgically implanted with silastic chronic indwelling catheter (CamCaths, Ely, UK) inserted into the right jugular vein as previously described (Fattore et al. 2001). After surgery, each animal received daily intraperitoneal (i.p.) administration of antibiotic treatment (Enrofloxacin, 0.1 ml, Bayer HealthCare, Shawnee Mission, KS, USA) for 7 days before initiation of self-administration training. Bilateral guide cannulas implantation When a stable baseline for cocaine self-administration was reached, one group of rats was subjected to surgical implant of bilateral guide-cannulas aimed at the dorsal division of mPFC for local administration experiments. Rats were anaesthetized with an i.p. injection of Equithesin (0.97 g pentobarbital, 2.1 g MgSO4, 4.25 g chloral hydrate, 42.8 ml of propylene glycol, 11.5 ml of 90 percent ethanol, distilled water up to 100 ml, 5 ml/ kg) and placed in a stereotaxic apparatus (Kopf, Tujunga, CA, USA) with blunt ear bars to avoid damage of the tympanic membranes. Under aseptic conditions, rats were shaved and their scalp was retracted. Bilateral craniotomies were performed above the dorsal mPFC, and double stainless steel 22-G guide-cannulas (Plastics One, Addiction Biology, 21, 61–71

Dopamine and cocaine seeking

Roanoke, VA, USA) were lowered slowly into place and implanted using dental cement and two skull screws. The coordinates, according to Paxinos & Watson (1997) atlas, were AP = +3.0 mm, ML = ±0.5 mm; DV = −3 mm from the skull surface. The lengths of the cannulas were selected so as to end 1 mm above the targeted area with the corresponding injector projecting 1 mm beyond guide tip. Bilateral guides were used, with centre-tocentre distances between the stainless steel tubing of 1 mm. Cannulas were plugged with wire stylets, and wounds were sutured. Rats were given antibiotic therapy and allowed to recover for 24 hours, then underwent extinction training. Vertical microdialysis probe implantation The day before microdialysis experiments, rats were anaesthetized with Equithesin and placed in a Kopf stereotaxic apparatus. In-house constructed vertical microdialysis probes (AN 69-HF membrane, HospalDasco, Bologna, Italy; cut-off 40 000 Da, 3-mm dialysing membrane length) were implanted in the mPFC (AP: +3.0, L: ± 0.6, V: −6.5 from bregma) according to the coordinates of the atlas by Paxinos & Watson (1997). A microphotograph of brain section with the injection cannula track is shown in Supporting Information Fig. S1. Experimental procedures Cocaine i.v. self-administration Cocaine self-administration was performed as previously described (Fattore et al. 2009) in 12 operant chambers (Med Associates, St Albans, VT, USA), each encased in a sound- and light-attenuating box equipped with ventilating fan and a white noise. Each chamber (29.5 × 32.5 × 23.5 cm) was fitted with two retractable levers, 4 cm wide, extending 1.5 cm into the box and positioned 12 cm apart and 8 cm from the chamber floor. A white visual stimulus light (cue light, 2.5 W, 24 V) was placed between the two levers and a yellow single house light (2.5 W, 24 V), located on the opposite wall, was kept turned on during the entire session. Intravenous infusions of cocaine were delivered by a 10-ml syringe mounted on a computer-controlled infusion pump (drug delivered at a rate of 0.02 ml/second; Med Associates) placed outside each chamber. Each syringe was connected through plastic tubing with a counterbalanced single-channel swivel apparatus. Another plastic tubing, enclosed in a metal spring, was connected the swivel to the catheter fitting on the animal’s back, allowing its unrestricted movement within the operant chamber. A computer-integrated system by Med-PC interface (Med Associates) was used for programming, data collection and storage. © 2014 Society for the Study of Addiction

63

Cocaine self-administration started 1 week after surgery and was carried out in daily 2-hour sessions, 7 days/week, at the same time every day (between 8:30 and 12:30), under a continuous (fixed ratio 1; FR-1) schedule of reinforcement and lever pressing as operandum as previously described (Fattore et al. 2009). Each session started with the extension of the two levers and the illumination of the house light. Press of one lever, defined as active, resulted in switching off the house light for 20 seconds and turning on of the white cue light for 5 seconds. Simultaneously, both levers retracted and the infusion pump was activated for 5 seconds, delivering 0.5 mg/100 μl i.v. infusion of cocaine solution. Presses of the other lever, defined as inactive, had no programmed consequences but were always recorded to provide an index of basal lever-pressing activity. The assignment of the active (drug-paired) and the inactive (no drug-paired) levers was counterbalanced and remained constant for each rat during all phases of the study. Cocaine self-administration was considered acquired when rats displayed accurate discrimination (≈70 percent) between the active and the inactive lever, with the number of active lever presses ≥ 20 per session for three consecutive days (acquisition phase) (Fattore et al. 2009). When animals developed a stable pattern of cocaine intake, i.e. less than 15 percent variation in response number over at least 5 consecutive sessions (maintenance phase), one group of rats was surgically prepared for local administration experiments by guide cannula implantation. Then, extinction condition was introduced by replacing cocaine with saline solution and leaving all the other experimental parameters unchanged (extinction phase). Drug-reinforced behaviour was considered extinguished when the number of active lever presses was ≤ 10 with no significant differences between active and inactive lever presses. Immediately after the last session of the extinction training, a second group of rats was implanted with a vertical microdialysis probe for microdialysis experiments. At the end of the extinction training, animals underwent reinstatement test sessions and received acute injection with either cocaine (10 mg/kg), test drug (disulfiram, nepicastat or L-DOPA, 50 mg/kg) or their vehicle [saline or dimethyl sulfoxide (DMSO), 1 ml/kg], alone or in combination. Specifically, each animal received 2 (cocaine and/or test drug) or 3 (saline or vehicle, cocaine and/or test drug) different priming injections, each separated by at least 4 days of extinction training to assess carryover effect of drugs. Order of drug priming presentation was counterbalanced. Throughout each phase of the study (cocaine selfadministration, extinction, reinstatement testing), locomotor activity of rats within the operant boxes was Addiction Biology, 21, 61–71

64

Paola Devoto et al.

constantly monitored by means of two series of four photocells each located at regular 3.5-cm intervals above the cage floor. The number of photocell beam breaks was recorded and served as a measure of general horizontal locomotor activity, and in particular to assess potential non-specific changes in activity induced by the experimental manipulation.

calculated as pg/20 μl dialysate and treatment-induced changes were expressed as percent of mean basal level. On completion of testing, rats were sacrificed by Equithesin overdose, the brains were removed and sectioned by a cryostat (Leica CM3050; Leica Microsystems, Milano, Italy) in 40-μm-thick coronal slices to verify locations of dialysis probes. Animals with errant location of the device (n = 2) were excluded from analysis.

Microinjection procedures During the reinstatement test sessions, a group of rats was injected with disulfiram (or its vehicle, DMSO), and after 1 hour, the injection cannulas, connected with PE 50 tubing to micro-syringes (10 μl) operated by a CMA/ 100 microinjection pump (Carnegie Medicine, Stockholm, Sweden), were inserted into the guide cannulas. SCH 23390 (0.3 or 1 μg/side), (+/−)-chloro-APB hydrobromide (SKF 82958, 3 or 5 μg/side) or an equal volume of sterile saline (0.5 μl/side) was bilaterally injected over 1 minute, and the injectors were left in place for additional 1 minute. A separate group of animals was primed with L-DOPA (50 mg/kg, i.p.) or its vehicle (saline, 1 ml/kg, i.p.), and after 20 minutes, locally injected with SCH 23390 (1 μg/side) or its vehicle (saline, 0.5 μl/side). Three minutes later, cocaine (10 mg/kg) or its vehicle (saline, 1 ml/kg) was i.p. administered, and after 10 minutes, the animals were placed into the operant box. Microdialysis experiments Microdialysis experiments took place the day after probe implantation. An artificial cerebrospinal fluid (147 mM NaCl, 4 mM KCl, 1.5 mM CaCl2, 1 mM MgCl2, pH 6–6.5) was pumped through the dialysis probes at a constant rate of 1.1 μl/minutes via a CMA/100 microinjection pump (Carnegie Medicine). Samples were collected every 20 minutes and immediately analysed for noradrenaline and dopamine content by high-performance liquid chromatography (HPLC) with electrochemical detection, as previously described (Devoto et al. 2003). When a stable baseline was obtained (three consecutive samples with a variance not exceeding 15 percent), disulfiram (50 mg/ kg) or nepicastat (50 mg/kg) or their vehicle (DMSO, 1 ml/kg) were i.p. administered, and after collection of three more samples, rats were i.p. injected with cocaine (10 mg/kg) or its solvent (saline, 1 ml/kg), and after 10 minutes, placed in the operant box. Sample collection continued during the 2-hour self-administration session for a total of 6 samples collected and measured. In a second set of experiments, aimed to evaluate L-DOPA effects on extracellular dopamine levels in the mPFC, microdialysis was performed on drug-naïve rats receiving L-DOPA alone (50 mg/kg, i.p.) or in association with cocaine (10 mg/kg, i.p.). Microdialysis data were © 2014 Society for the Study of Addiction

Drugs For self-administration training, cocaine hydrochloride (MacFarlan Smith Ltd., Edinburgh, UK) was diluted in heparinized (0.1 percent) sterile 0.9 percent saline solution. Intravenous infusions of cocaine (0.5 mg/kg per 0.1 ml/infusion) were delivered at a rate of 20 μl/second over 5 seconds. In order to ensure sterility, cocaine solution was filtered through 0.20-μm syringe filters prior to use. This dose of cocaine was previously shown to sustain robust self-administration behaviour in Sprague Dawley rats under FR-1 schedule (Fattore et al. 2009; Feltenstein, Do & See 2009). For reinstatement test (drug priming), cocaine (10 mg/kg) was dissolved in distilled water and administered 10 minutes before starting the reinstatement test session. Disulfiram (50 mg/kg, Sigma-Aldrich, Milano, Italy) and nepicastat (a gift from Biotie Therapies, Basel, Switzerland; 50 mg/kg) were dissolved in DMSO and administered 70 minutes before starting the session, i.e. 60 minutes before cocaine priming. All drugs were administered i.p. in a volume of 1 ml/kg. The dopamine D1 receptor antagonist SCH 23390 (0.3 and 1 μg/side, Sigma-Aldrich, Italy) and the D1 receptor agonist (+/−)chloro-APB hydrobromide (SKF 82958, 3 and 5 μg/side, Sigma-Aldrich) were diluted in sterile water and administered bilaterally (0.5 μl/side) into the dorsal mPFC 5 minutes prior to cocaine priming. L-DOPA (50 mg/kg, Sigma-Aldrich) was always administered concurrently with the inhibitor of peripheral DOPA-decarboxylase benserazide (6 mg/kg, Sigma-Aldrich) in a volume of 2 ml/kg (i.p.) 20 minutes before cocaine priming. Cocaine was injected 20 minutes after L-DOPA, just before its peak effect on dopamine synthesis (Spencer & Wooten 1984). Antibiotic was purchased as sterile solution from local suppliers.

Statistical analysis Statistical significance was calculated by means of Statistica (StatSoft Inc., Tulsa, OK, USA) and Prism 6.0c (GraphPad Software Inc., San Diego, CA, USA) programs. Analyses were performed by one-way or mixed design analyses of variance (ANOVAs), with repeated measures for microdialysis data, as detailed in the Results section, followed by Tukey’s test, with Spjøtvoll–Stoline correction Addiction Biology, 21, 61–71

Dopamine and cocaine seeking

for unequal N whenever required, for post hoc comparisons of the means. Significance threshold was set at P < 0.05.

RESULTS After acquisition of cocaine self-administration criteria, the rats were subjected to extinction procedure and then tested for cocaine-induced reinstatement of drug-seeking behaviour, as described in the Methods section. Figure 1 shows the effects of treatment with disulfiram or nepicastat (50 mg/kg, i.p.) on cocaine-induced reinstatement of drug-seeking behaviour. As expected, acute

Figure 1 Reversal by disulfiram (DIS) and nepicastat (NEP) of cocaine (COC)-induced reinstatement of drug-seeking behaviour. (a) Cocaine (10 mg/kg, i.p.) resumed extinguished active lever-pressing activity up to the pre-extinction level. Disulfiram and nepicastat (50 mg/kg, i.p.) did not affect drug-seeking behaviour per se, but significantly reduced active lever pressing when administered with cocaine. **P < 0.01 versus Veh + SAL, #P < 0.01 versus Veh + COC, §P < 0.01 versus corresponding drug + SAL group. (b) Mean inactive lever presses during cocaine self-administration (BAS), extinction (Ext) and the reinstatement test sessions with different drug combinations.Values were constantly ≤ 10, which ensured the specificity of responding on the active lever © 2014 Society for the Study of Addiction

65

non-contingent cocaine priming (10 mg/kg, i.p.) reinstated active lever pressing to the basal, pre-extinction level (Fig. 1a), and marginally increased responding on the inactive lever (Fig. 1b). We first analysed cocainepriming effect on the two lever presses by one-way ANOVA. For active lever, one-way ANOVA analysis of ‘basal’, ‘extinction’, ‘vehicle + saline’ and ‘vehicle + cocaine’ data yielded significant results [F(3,64) = 622.8, P < 0.0001]. Post hoc multiple comparison Tukey’s test evidenced a significant difference between ‘basal’ versus ‘extinction’ and versus ‘vehicle + saline’ (P < 0.0001) but not versus ‘vehicle + cocaine’ group that, on the other hand, was significantly different from ‘extinction’ and ‘vehicle + saline’ groups (P < 0.0001). One-way ANOVA analysis of inactive lever data produced a significant result [F(3,64) = 5.892, P < 0.005] due to the slight increase observed in inactive lever presses of ‘vehicle + cocaine’ group, which was significantly different from ‘extinction’ but not from ‘basal’ value. Importantly, inactive lever values were constantly ≤ 10, which ensured the specificity of responding on the active lever. It is worth noting that locomotor activity during the reinstatement test sessions (mean ± SEM of photocell beam breaks) was not altered by acute primings (data not shown), thus ensuring the absence of any no-specific effect on response, since priming-induced effects were selective and not associated with motor disturbances. In line with the results from other laboratories (Schroeder et al. 2010), pre-treatment with disulfiram or nepicastat at a dose (50 mg/kg, i.p.) that per se did not affect active lever pressing efficaciously blocked cocaineprimed reinstatement of cocaine seeking (Fig. 1a). The effect of disulfiram on drug-seeking reinstatement was analysed by two-way ANOVA, which evidenced a significant effect of disulfiram pre-treatment [F(1,30) = 125, P < 0.0001], cocaine treatment [F(1,30) = 289, P < 0.0001] and their interaction [F(1,30) = 116, P < 0.0001]. A separate two-way ANOVA test yielded analogous results for nepicastat effect, with a significant effect of nepicastat pre-treatment [F(1,26) = 115, P < 0.0001], cocaine treatment [F(1,26) = 99.1, P < 0.0001] and their interaction [F(1,26) = 229, P < 0.0001]. A subgroup of these rats was tested by means of microdialysis for measuring extracellular dopamine and noradrenaline level variations in the mPFC during the 2-hour reinstatement test session. Mean ± SEM noradrenaline and dopamine basal values were 3.55 ± 0.27 and 2.13 ± 0.19 pg/20 μl dialysate, respectively. As we previously observed in drug-naïve animals (Devoto et al. 2012, 2014), pre-treatment with disulfiram or nepicastat attenuated cocaine-induced extracellular noradrenaline increase, but markedly potentiated cocaine-induced dopamine increase in the mPFC (Fig. 2). The increase in extracellular dopamine concentration Addiction Biology, 21, 61–71

66

Paola Devoto et al.

Figure 2 Effect of pre-treatment with disulfiram (DIS, 50 mg/kg, i.p.) or nepicastat (NEP, 50 mg/kg, i.p.) on the increases of extracellular noradrenaline and dopamine levels induced in the medial prefrontal cortex by cocaine priming (COC, 10 mg/kg, i.p.). Data are expressed as percent of mean basal level and are the mean ± SEM of the number of rats indicated in parentheses. The first arrow indicates the time of DIS or NEP administration, while the second arrow indicates time of COC priming

was rapid, reached the maximum in the second sample after cocaine administration, i.e. 40 minutes after the start of the session, then slowly decreased over time but remained higher than in the vehicle + cocaine group throughout the reinstatement test session. Repeated measures two-way ANOVA analysis was conducted on microdialysis data, with each DBH inhibitor or vehicle as pre-treatment, and cocaine and saline as treatment factors, and time as repeated measures. The analyses evidenced a significant effect of pre-treatment for both disulfiram [noradrenaline: F(3,23) = 23.84, P < 0.0001; dopamine: F(3,23) = 20.48, P < 0.0001] and nepicastat [noradrenaline: F(3,23) = 19.74, P < 0.0001; dopamine: F(3,23) = 19.36, P < 0.0001]. Tukey’s multiple comparison test revealed a significant difference in dopamine level increases induced by disulfiram + cocaine or nepicastat + cocaine versus all other treatment combinations (all P < 0.0001). With regard to noradrenaline level variations, significant differences were found between disulfiram + cocaine versus disulfiram + saline (P < 0.001) and vehicle + saline (P < 0.01), but not versus vehicle + cocaine, while nepicastat + cocaine effect was different from all other treatment effects (P < 0.05). To further verify the hypothesis that the marked increase in extracellular dopamine in the mPFC produced by the co-administration of DBH inhibitors with cocaine might contribute to or is responsible for the inhibition of cocaine-induced reinstatement of cocaine seeking, © 2014 Society for the Study of Addiction

L-DOPA plus benserazide (50 and 6 mg/kg, respectively, i.p.) were administered prior to cocaine in order to produce an increase of extracellular dopamine of the same magnitude of that produced by the combination of DBH inhibitors with cocaine. As shown in Fig. 3, L-DOPA and cocaine given separately increased extracellular dopamine by about 200 percent, while when co-administered, they increased extracellular dopamine to about 1300 percent. The marked elevation of extracellular dopamine after co-administration of L-DOPA with cocaine is explained with cocaine-induced blockade of the uptake of dopamine formed by L-DOPA decarboxylation. Two-way ANOVA with repeated measures evidenced a significant effect of treatment [F(2,12) = 23.2, P < 0.0001], time [F(5,60) = 7.51, P < 0.0001] and time × treatment interaction [F(10,60) = 5.46, P < 0.0001]. Tukey’s multiple comparison test revealed a significant difference in dopamine level increases induced by L-DOPA + cocaine versus saline + cocaine and L-DOPA + saline (P < 0.0001), but not between L-DOPA + saline versus saline + cocaine. The effect of L-DOPA and cocaine co-administration was tested on cocaine-induced reinstatement of drugseeking behaviour. L-DOPA (50 mg/kg, i.p.) did not affect responding per se, but was very efficacious in abolishing cocaine-induced drug-seeking reinstatement (Fig. 4). Two-way ANOVA demonstrated a significant effect for L-DOPA pre-treatment [F(1,30) = 171.94, P < 0.0001], Addiction Biology, 21, 61–71

Dopamine and cocaine seeking

Figure 3 Effect of L-DOPA (50 mg/kg, i.p.) pre-treatment on cocaine (COC, 10 mg/kg, i.p.)-induced increase of extracellular dopamine levels in the medial prefrontal cortex. Results are expressed as percent of mean basal level and are the mean ± SEM of at least 4 rats. The first arrow indicates the time of L-DOPA administration; the second arrow indicates the time of COC administration. L-DOPA + COC group was significantly different both from L-DOPA + SAL and SAL + COC groups [repeated measure analysis of variance (ANOVA) followed by Tukey’s multiple comparison test, P < 0.0001]

Figure 4 Reversal by L-DOPA pre-treatment of cocaine-induced reinstatement of drug seeking. Cocaine (COC, 10 mg/kg, i.p.) increased active lever pressing to a pre-extinction level. L-DOPA (50 mg/kg, i.p.) did not affect drug-seeking behaviour per se, but significantly reduced active lever-pressing activity when administered with cocaine. *P < 0.0001 versus SAL + COC (Tukey’s multiple comparisons test)

cocaine treatment [F(1,30) = 217.75, P < 0.0001] and pre-treatment × treatment interaction [F(1,30) = 146.48, P < 0.0001]; the post hoc Tukey’s test evidenced a significant difference of cocaine treatment versus saline, L-DOPA and L-DOPA + cocaine treatments (P < 0.0001), which were not significantly different from each other. We next tested the hypothesis that the high extracellular dopamine concentration produced by the co-administration of DBH inhibitors or L-DOPA with cocaine might prevent cocaine-induced reinstatement of drug-seeking behaviour through a supra-normal stimu© 2014 Society for the Study of Addiction

67

Figure 5 Local administration of SCH 23390 into the dorsal medial prefrontal cortex dose-dependently reverted disulfiram-induced decrease of cocaine-priming effect on drug-seeking behaviour. Cocaine (10 mg/kg, i.p., first column) increased active lever-pressing activity up to basal, pre-extinction level. Both SCH 23390 (0.3 or 1 μg/side) and disulfiram (50 mg/kg, i.p.) significantly decreased cocaine-induced lever pressing. Co-administration of disulfiram and SCH 23390 (1 μg/side) reverted the decrease of cocaine effect induced by each drug when administered alone. #P < 0.001 versus all other treatment groups; oP < 0.01 versus SCH 0.3; *P < 0.001 versus SCH 1; §P < 0.05 versus disulfiram (Tukey’s multiple comparison test)

lation of D1 receptors in the dorsal mPFC. To this purpose, cocaine-induced reinstatement test was performed in animals chronically implanted with bilateral cannulas aimed at the dorsal mPFC, through which the selective D1 receptor antagonist SCH 23390 was injected 5 minutes before cocaine priming (Fig. 5). In line with previous studies (Sun & Rebec 2005), SCH 23390 infused into the dorsal mPFC dose-dependently reduced cocaineinduced reinstatement of active lever presses. Moreover, 1 μg SCH 23390 microinjection reversed disulfiraminduced inhibition of cocaine effect, whereas 0.3 μg microinjection was ineffective. Two-way ANOVA showed a significant effect for pre-treatment [disulfiram or vehicle, F(1,38) = 18.8, P = 0.0001], treatment [vehicle, SCH 0.3 and SCH 1, F(2,38) = 28.68, P < 0.0001] and their interaction [F(2,38) = 120.25, P < 0.0001]. Tukey’s test evidenced that lever-pressing activity displayed by animals pre-treated with disulfiram + 1 μg SCH 23390 was significantly higher with respect to the effect of each drug given alone (P < 0.0001), whereas disulfiram + 0.3 μg SCH 23390 effect was different from 0.3 μg SCH (P < 0.05) but not from disulfiram effect. Cocaine alone group was significantly different from all other treatment groups (P < 0.001). Similar results were obtained in rats co-administered with L-DOPA plus cocaine (Fig. 6). As already shown in Fig. 4, L-DOPA prevented cocaine-primed reinstatement of drug seeking, but 1 μg/side SCH 23390, infused prior Addiction Biology, 21, 61–71

68

Paola Devoto et al.

Figure 6 Cocaine (10 mg/kg, i.p.) increased active lever pressing to basal, pre-extinction level. L-DOPA (50 mg/kg, i.p.) and SCH 23390 (1 μg/side into the dorsal medial prefrontal cortex) reverted cocaine priming-induced drug-seeking behaviour, but their co-administration abolished this effect. *P < 0.0001 versus cocaine; §P < 0.0001 versus L-DOPA + SCH + cocaine [Tukey’s HSD (honest significant difference) test]

to cocaine challenge, totally reversed L-DOPA inhibition, so that responding activity reached the same level as after cocaine alone (Fig. 6). Three-way ANOVA evidenced a main effect of treatment [F(1,47) = 166.25, P < 0.0001], and Tukey’s HSD (honest significant difference) test showed that cocaine group was not significantly different from L-DOPA + SCH 23390 + cocaine, whereas all other groups were significantly different both versus cocaine alone and versus L-DOPA + SCH 23390 + cocaine group (P < 0.0001). To further verify the hypothesis that the suppressant effect of DBH inhibitors on the reinstatement of cocaine seeking is mediated by supra-normal stimulation of D1 receptors, the selective D1 receptor agonist chloro-APB was locally injected into the dorsal mPFC. As shown in Fig. 7, chloro-APB, at doses of 3 and 5 μg/side, drastically reduced cocaine-primed reinstatement of cocaine seeking. Interestingly, chloro-APB given alone failed to reinstate cocaine seeking. One-way ANOVA evidenced a significant effect of treatment [F(4,40) = 17.36, P < 0.0001]; Tukey’s post hoc revealed that cocaine priming yielded a lever pressing activity significantly different from extinction (P < 0.0001) and from both doses of chloro-APB + cocaine (P < 0.05 and P < 0.01 for 3 and 5 μg, respectively). Most importantly, both chloroAPB + cocaine groups were not significantly different from extinction value.

DISCUSSION In the first set of experiment, we replicated previous studies showing that the two DBH inhibitors disulfiram © 2014 Society for the Study of Addiction

Figure 7 Reversal by chloro-APB local injection into the dorsal medial prefrontal cortex of cocaine-induced reinstatement of drug seeking. Cocaine (COC, 10 mg/kg, i.p.) increased active lever pressing to a pre-extinction level. Chloro-APB (3 and 5 μg/side) significantly reduced active lever pressing when administered with cocaine. *P < 0.05; **P < 0.01; ***P < 0.0001 versus SAL + COC. ++P < 0.0001; +P < 0.001 versus Basal (Tukey’s multiple comparison test)

and nepicastat attenuate cocaine-induced reinstatement of cocaine-seeking behaviour (Schroeder et al. 2010). While confirming our previous results in drug-naïve animals, we also found that both DBH inhibitors markedly potentiated cocaine-induced dopamine release in the mPFC in the same rats in which they concomitantly suppressed cocaine-induced reinstatement. In addition, we demonstrated that the administration of L-DOPA suppressed cocaine-induced reinstatement of cocaine seeking at a dose that, similar to DBH inhibitors, markedly potentiated cocaine-induced dopamine release in the mPFC. These results support the hypothesis that an excessive extracellular dopamine accumulation in the mPFC is causally related to the inhibition of cocaineseeking reinstatement. Accordingly, the finding that blockade of D1 receptors with SCH 23390 reversed the reinstatement suppressant effect of L-DOPA and disulfiram, while supra-normal stimulation of D1 receptors with chloro-APB suppressed the ability of cocaine to reinstate cocaine seeking, suggests that D1 receptor stimulation in the dorsal mPFC plays a crucial role in the inhibitory effect of L-DOPA and DBH inhibitors on cocaine-induced reinstatement of cocaine seeking. This interpretation is in apparent contrast to the notion that activation of dopamine receptors in the dorsal mPFC facilitates the activity of glutamatergic neurons projecting to the nucleus accumbens core and, thereby, reinstates cocaine-seeking activity (McFarland & Kalivas 2001; Kalivas & McFarland 2003; McFarland, Lapish & Kalivas 2003). A likely explanation for these Addiction Biology, 21, 61–71

Dopamine and cocaine seeking

apparently contradictory findings might be that cocaineinduced reinstatement requires an optimal activation of D1 receptor signalling in the dorsal mPFC, so that neither supra- nor sub-normal D1 activation would permit cocaine-induced reinstatement. Accordingly, SCH 23390 would reverse the reinstatement inhibition induced by L-DOPA or disulfiram by reducing a supra-normal D1 receptor stimulation produced by an excess of dopamine; vice versa, it would suppress cocaine-induced reinstatement by reducing D1 receptor signalling below the level needed for eliciting reinstatement. The hypothesis that DBH inhibitors and L-DOPA suppress cocaine-induced reinstatement via a supra-normal stimulation of D1 receptors in the dorsal mPFC is consistent with the recent results by Lauzon et al. (2013) showing that supra-normal stimulation of dopamine D1 receptors in the dorsal mPFC, obtained with local microinfusion of a D1 receptor agonist, blocked the behavioural expression of both aversive and rewarding associative memories through a cAMP-dependent signalling pathway. They postulate that an optimal level of D1 receptor signalling is required for spontaneous expression of previously acquired associative memories, which can trigger drug-seeking behaviour or relapse. Our results are also consistent with previous research that demonstrated an inverted ‘U’ shaped function for the effect of D1 receptor stimulation mPFC neuronal activity and correlated cognitive performances in primates (Vijayraghavan et al. 2007). While the foregoing results indicate that suppression of cocaine-induced reinstatement by DBH inhibitors, is likely mediated by a marked elevation of extracellular dopamine in the mPFC, leading to a supra-normal stimulation of D1 receptor signalling in the dorsal mPFC, they do not exclude the possibility that disulfiram and nepicastat might inhibit reinstatement primed by drug-associated cues or by stress via reduction of α1-adrenoceptor signalling required for promoting dopaminergic transmission, as suggested by several studies (Schroeder et al. 2010, 2013). These authors suggested that disulfiram and nepicastat block cocaine-, cueand foot shock-induced reinstatement of cocaine seeking by reducing α1-receptor-dependent signalling, which would play a permissive role in cocaine-induced dopamine release (Schank et al. 2006; Mitrano et al. 2012). However, while both DBH inhibitors do profoundly reduce noradrenaline release, either in the mPFC or in the nucleus accumbens, they fail to modify dopamine release and cocaine-induced dopamine release in the nucleus accumbens (Devoto et al. 2012, 2014). Future research should clarify the relative role of α1-adrenoceptros and D1 dopamine receptors in the effect of DBH inhibitors on reinstatement of cocaine seeking elicited by different stimuli. © 2014 Society for the Study of Addiction

69

Another issue to be clarified is why L-DOPA and DBH inhibitors increase extracellular dopamine in the mPFC to the same extent as cocaine but, unlike the latter, they fail to elicit reinstatement of cocaine seeking. A possible explanation for this divergence is that cocaine, in nonanaesthetized rats, increases the firing rate and bursting of midbrain DA neurons (Koulchitsky et al. 2012), while L-DOPA, via the dopamine formed, inhibits the firing of dopaminergic neurons (Bunney, Aghajanian & Roth 1973). This explanation would imply that phasic dopamine release by nerve activity is required for cocaineinduced reinstatement. Moreover, it should be clarified if L-DOPA, like DBH inhibitors (Devoto et al. 2012, 2014), fails to modify the cocaine-induced dopamine release in the nucleus accumbens. Alternatively, it might be suggested that dopamine signalling on D1 receptors in the mPFC is permissive for cocaine-induced reinstatement of cocaine seeking, but is not sufficient to elicit this behaviour. The results of interaction between L-DOPA and cocaine require a separate comment. While L-DOPA has been clinically used in the pharmacotherapy of cocaine dependence, although with uncertain results (Wolfsohn, Sanfilipo & Angrist 1993; Mooney 2007; Schmitz et al. 2008), our study is, to the best of our knowledge, the first one to examine the effect of L-DOPA in an animal model of cocaine dependence. While our results suggest that L-DOPA may be useful in the suppression of relapse to cocaine seeking, additional experiments are needed in order to provide translational value to animal research and render the pre-clinical data a useful background to guide the clinical use. Thus, it should be clarified if L-DOPA, like nepicastat (Schroeder et al. 2013), also prevents cocaine-seeking reinstatement triggered by other stimuli besides cocaine priming (i.e. cues, stress), and if it may attenuate other aspects of cocaine-seeking behaviour. An important caveat to be considered is whether the marked elevation of extracellular dopamine in the mPFC produced by the combination of L-DOPA or disulfiram with cocaine might impair the acquisition and expression of different types of associative memories (Lauzon et al. 2013) and may constitute a risk factor for L-DOPA and disulfiram-induced psychosis (Carey et al. 1995; Cubells et al. 2000; Kaiser et al. 2003; Mutschler, Diehl & Kiefer 2009; Grau-López et al. 2012).

Acknowledgements The authors wish to thank Dr. B. Tuveri for her excellent technical assistance and animal care, and Dr. V. Bini for her precious help with statistic analysis. Nepicastat was a generous gift from Biotie Therapies. This research was supported by the ‘Guy Everett Laboratory’ Foundation. Addiction Biology, 21, 61–71

70

Paola Devoto et al.

Conflict of Interest The authors declare no competing financial interests. Authors Contribution PD and LF cooperated for the study concept and design, supervised experiments and analysed the data. PD, LF, WF and GLG discussed and drafted the manuscript; LF and SA were responsible for and executed the selfadministration experiments; PD, PS and RF performed microdialysis and HPLC analysis. All authors reviewed content and approved final version for publication. References Bunney BS, Aghajanian GK, Roth RH (1973) Comparison of effects of L-DOPA, amphetamine and apomorphine on firing rate of rat dopaminergic neurons. Nat New Biol 245:123–125. Carey RJ, Pinheiro-Carrera M, Dai H, Tomaz C, Huston JP (1995) L-DOPA and psychosis: evidence for L-DOPA-induced increases in prefrontal cortex dopamine and in serum corticosterone. Biol Psychiatry 38:669–676. Cubells JF, Kranzler HR, McCance-Katz E, Anderson GM, Malison RT, Price LH, Gelernter J (2000) A haplotype at the DBH locus, associated with low plasma dopamine betahydroxylase activity, also associates with cocaine-induced paranoia. Mol Psychiatry 5:56–63. De Vries TJ, Schoffelmeer AN, Binnekade R, Mulder AH, Vanderschuren LJ (1998) Drug-induced reinstatement of heroin- and cocaine-seeking behaviour following long-term extinction is associated with expression of behavioural sensitization. Eur J Neurosci 10:3565–3571. Devoto P, Flore G, Saba P, Cadeddu R, Gessa GL (2012) Disulfiram stimulates dopamine release from noradrenergic terminals and potentiates cocaine-induced dopamine release in the prefrontal cortex. Psychopharmacology (Berl) 219:1153– 1164. Devoto P, Flore G, Saba P, Bini V, Gessa GL (2014) The dopamine beta-hydroxylase inhibitor nepicastat increases dopamine release and potentiates psychostimulant-induced dopamine release in the prefrontal cortex. Addict Biol 19:612–622. Devoto P, Flore G, Vacca G, Pira L, Arca A, Casu MA, Pani L, Gessa GL (2003) Co-release of noradrenaline and dopamine from noradrenergic neurons in the occipital cortex induced by clozapine, the prototype atypical antipsychotic. Psychopharmacology (Berl) 167:79–84. Erb S, Shaham Y, Stewart J (1996) Stress reinstates cocaineseeking behavior after prolonged extinction and a drug-free period. Psychopharmacology (Berl) 128:408–412. Fattore L, Cossu G, Martellotta CM, Fratta W (2001) Intravenous self-administration of the cannabinoid CB1 receptor agonist WIN55,212-2 in rats. Psychopharmacology (Berl) 156:410– 416. Fattore L, Piras G, Corda MG, Giorgi O (2009) The Roman highand low-avoidance rat lines differ in the acquisition, maintenance, extinction, and reinstatement of intravenous cocaine self-administration. Neuropsychopharmacology 34:1091– 1101. Feltenstein MW, Do PH, See RE (2009) Repeated aripiprazole administration attenuates cocaine seeking in a rat model of relapse. Psychopharmacology (Berl) 207:401–411. © 2014 Society for the Study of Addiction

Goldstein M, Anagnoste B, Lauber E, McKeregham MR (1964) Inhibition of dopamine- beta-hydroxylase by disulfiram. Life Sci 3:763–767. Grau-López L, Roncero C, Navarro MC, Casas M (2012) Psychosis induced by the interaction between disulfiram and methylphenidate may be dose dependent. Subst Abus 33:186–188. Jaffe JH, Cascella NG, Kumor KM, Sherer MA (1989) Cocaineinduced cocaine craving. Psychopharmacology (Berl) 97:59– 64. Kaiser R, Hofer A, Grapengiesser A, Gasser T, Kupsch A, Roots I, Brockmöller J (2003) L-dopa-induced adverse effects in PD and dopamine transporter gene polymorphism. Neurology 60:1750–1755. Kalivas PW, McFarland K (2003) Brain circuitry and the reinstatement of cocaine-seeking behavior. Psychopharmacology (Berl) 168:44–56. Koulchitsky S, De Backer B, Quertemont E, Charlier C, Seutin V (2012) Differential effects of cocaine on dopamine neuron firing in awake and anesthetized rats. Neuropsychopharmacology 37:1559–1571. Lauzon NM, Bechard M, Ahmad T, Laviolette S (2013) Supranornal stimulation of dopamine D1 receptors in the prelimbic cortex blocks behavioral expression of both aversive and rewarding associative memories through a cyclic-AMPdependent signaling pathway. Neuropharmacology 67:104– 114. McFarland K, Kalivas PW (2001) The circuitry mediating cocaine-induced reinstatement of drug-seeking behavior. J Neurosci 21:8655–8663. McFarland K, Lapish CC, Kalivas PW (2003) Prefrontal glutamate release into the core of the nucleus accumbens mediates cocaine-induced reinstatement of drug-seeking behavior. J Neurosci 23:3531–3537. Mitrano DA, Schroeder JP, Smith Y, Cortright JJ, Bubula N, Vezina P, Weinshenker D (2012) Alpha-1 adrenergic receptors are localized on presynaptic elements in the nucleus accumbens and regulate mesolimbic dopamine transmission. Neuropsychopharmacology 37:2161–2172. Mooney ME (2007) Safety, tolerability and efficacy of levodopacarbidopa treatment for cocaine dependence: two doubleblind randomized clinical trials. Drug Alcohol Depend 88:214–223. Mutschler J, Diehl A, Kiefer F (2009) Pronounced paranoia as a result of cocaine-disulfiram interaction: case report and mode of action. J Clin Psychopharmacol 29:99–101. Paxinos G, Watson C (1997) The Rat Brain in Stereotaxic Coordinates, 3rd edn. San Diego, CA: Academic Press. Robbins SJ, Ehrman RN, Childress AR, O’Brien CP (1997) Relationships among physiological and self-report responses produced by cocaine-related cues. Addict Behav 22:157– 167. Schank JR, Ventura R, Puglisi-Allegra S, Alcaro A, Cole CD, Liles LC, Seeman P, Weinshenker D (2006) Dopamine betahydroxylase knockout mice have alterations in dopamine signaling and are hypersensitive to cocaine. Neuropsychopharmacology 31:2221–2230. Schmitz JM, Mooney ME, Moeller FG, Stotts AL, Green C, Grabowski J (2008) Levodopa pharmacotherapy for cocaine dependence: choosing the optimal behavioral therapy platform. Drug Alcohol Depend 94:142–150. Schroeder JP, Alisha Epps S, Grice TW, Weinshenker D (2013) The selective dopamine β-hydroxylase inhibitor nepicastat attenuates multiple aspects of cocaine-seeking behavior. Neuropsychopharmacology 38:1032–1038. Addiction Biology, 21, 61–71

Dopamine and cocaine seeking

Schroeder JP, Cooper DA, Schank JR et al. (2010) Disulfiram attenuates drug-primed reinstatement of cocaine seeking via inhibition of dopamine β-hydroxylase. Neuropsychopharmacology 35:2440–2449. Seamans JK, Yang CR (2004) The principal features and mechanism of dopamine modulation in the prefrontal cortex. Prog Neurobiol 74:1–57. Simpson DD, Joe GW, Fletcher BW, Hubbard RL, Anglin MD (1999) A national evaluation of treatment outcomes for cocaine dependence. Arch Gen Psychiatry 56:507–514. Spencer SE, Wooten GF (1984) Pharmacologic effects of L-dopa are not closely linked temporally to striatal dopamine concentration. Neurology 34:1609–1611. Stanley WC, Li B, Bonhaus DW, Johnson LG, Lee K, Porter S, Walker K, Martinez G, Eglen RM, Whiting RL, Hegde SS (1997) Catecholamine modulatory effects of nepicastat (RS-25560197), a novel, potent and selective inhibitor of dopamine-betahydroxylase. Br J Pharmacol 121:1803–1809. Sun WL, Rebec GV (2005) The role of prefrontal cortex D1-like receptors in cocaine-seeking in rats. Psychopharmacology (Berl) 177:315–323. Vijayraghavan S, Wang M, Birnbaum SG, Williams GV, Arnsten AF (2007) Inverted-U dopamine D1 receptor actions on prefrontal neurons engaged in working memory. Nat Neurosci 10:376–384.

© 2014 Society for the Study of Addiction

71

Weiss F (2010) Advances in animal models of relapse for addiction research. In: Kuhn CM, Koob GF, eds. Advances in the Neuroscience of Addiction, 2nd edn. Boca Raton, FL: CRC Press. Available at: http://www.ncbi.nlm.nih.gov/books/ NBK53361/. Accessed 13 August 2014. Weiss F, Martin-Fardon R, Ciccocioppo R, Kerr TM, Smith DL, Ben-Shahar O (2001) Enduring resistance to extinction of cocaine-seeking behavior induced by drug-related cues. Neuropsychopharmacology 25:361–372. Wolfsohn R, Sanfilipo M, Angrist B (1993) A placebo-controlled trial of L-DOPA/carbidopa in early cocaine abstinence. Neuropsychopharmacology 9:49–53.

SUPPORTING INFORMATION Additional Supporting Information may be found in the online version of this article at the publisher’s web-site: Figure S1 Microphotograph showing injection cannulae positioning into the dorsal division of the medial prefrontal cortex. Rats were trans-cardially perfused with 4 percent formaldehyde; 40 μm thick coronal slices were obtained by cryostat sectioning and colored with neutral red

Addiction Biology, 21, 61–71