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Addiction Biology

ORIGINAL ARTICLE

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 NeuroscienceCagliari, National Research Council
(CNR), Italy4

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

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--hydroxylase (DBH)
inhibitors, disulfiram and nepicastat (Goldstein et al.

*These authors contributed equally to the paper.

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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
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.
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 265300 g
at the beginning of the study were used. Animals were
housed five per cage in a climate-controlled animal room
(temperature of 21 1C, 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,
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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 animals 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.
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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 l 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
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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 l 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-m-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 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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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
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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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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]. Tukeys 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,
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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]. Tukeys 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],
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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 Tukeys 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 (Tukeys 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 Tukeys 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

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 g/side) and disulfiram (50 mg/kg, i.p.) significantly decreased
cocaine-induced lever pressing. Co-administration of disulfiram and
SCH 23390 (1 g/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 (Tukeys 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 g SCH 23390 microinjection reversed disulfiraminduced inhibition of cocaine effect, whereas 0.3 g
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]. Tukeys
test evidenced that lever-pressing activity displayed by
animals pre-treated with disulfiram + 1 g SCH 23390
was significantly higher with respect to the effect of
each drug given alone (P < 0.0001), whereas disulfiram + 0.3 g SCH 23390 effect was different from 0.3 g
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 g/side SCH 23390, infused prior
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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 g/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 [Tukeys 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 Tukeys 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 g/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]; Tukeys 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 g, 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 g/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 (Tukeys multiple comparison
test)

and nepicastat attenuate cocaine-induced reinstatement

of cocaine-seeking behaviour (Schroeder et al. 2010).
While confirming our previous results in drug-nave
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
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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
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.
2014 Society for the Study of Addiction

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-Lpez 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.
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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.
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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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