Devoto Et Al. 2014 - Addiction Biology
Devoto Et Al. 2014 - Addiction Biology
Devoto Et Al. 2014 - Addiction Biology
Addiction Biology
ORIGINAL ARTICLE
doi:10.1111/adb.12178
ABSTRACT
Previous investigations indicate that the dopamine--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-nave 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
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
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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
1-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.
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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.
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 l) operated by a CMA/
100 microinjection pump (Carnegie Medicine, Stockholm, Sweden), were inserted into the guide cannulas.
SCH 23390 (0.3 or 1 g/side), (+/)-chloro-APB
hydrobromide (SKF 82958, 3 or 5 g/side) or an equal
volume of sterile saline (0.5 l/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 g/side) or its vehicle
(saline, 0.5 l/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 66.5)
was pumped through the dialysis probes at a constant
rate of 1.1 l/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-nave rats
receiving L-DOPA alone (50 mg/kg, i.p.) or in association
with cocaine (10 mg/kg, i.p.). Microdialysis data were
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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 l/second
over 5 seconds. In order to ensure sterility, cocaine solution was filtered through 0.20-m 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 g/side,
Sigma-Aldrich, Italy) and the D1 receptor agonist (+/)chloro-APB hydrobromide (SKF 82958, 3 and 5 g/side,
Sigma-Aldrich) were diluted in sterile water and administered bilaterally (0.5 l/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 Tukeys test, with SpjtvollStoline correction
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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
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 Tukeys 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 l dialysate, respectively.
As we previously observed in drug-nave 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
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DISCUSSION
In the first set of experiment, we replicated previous
studies showing that the two DBH inhibitors disulfiram
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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
1-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 1-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 1-adrenoceptros and
D1 dopamine receptors in the effect of DBH inhibitors on
reinstatement of cocaine seeking elicited by different
stimuli.
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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.
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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 245:123125.
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 38:669676.
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 5:5663.
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 10:35653571.
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 19:612622.
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) 167:7984.
Erb S, Shaham Y, Stewart J (1996) Stress reinstates cocaineseeking behavior after prolonged extinction and a drug-free
period. Psychopharmacology (Berl) 128:408412.
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) 207:401411.
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SUPPORTING INFORMATION
Additional Supporting Information may be found in the
online version of this article at the publishers 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 m thick coronal slices
were obtained by cryostat sectioning and colored with
neutral red
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