Dopamine in the Medial Prefrontal Cortex Controls Genotype-Dependent Effects of Amphetamine on Mesoaccumbens Dopamine Release and Locomotion

Article (PDF Available)inNeuropsychopharmacology 29(1):72-80 · February 2004with8 Reads
DOI: 10.1038/sj.npp.1300300 · Source: PubMed
Abstract
Mice of background DBA/2J are hyporesponsive to the behavioral effects of D-amphetamine in comparison with the widely exploited murine background C57BL/6J. In view of the important role of dopamine (DA) release in the nucleus accumbens (NAc) regarding the behavioral effects of psychostimulants, we tested the hypothesis of an inverse relationship between mesocortical and mesoaccumbens DA functioning in the two backgrounds. Systemic D-amphetamine induces a sustained increase in DA release in the medial prefrontal cortex (mpFC) accompanied by a poor increase in the NAc in mice of the low-responsive DBA/2J background, as shown by intracerebral microdialysis in freely moving animals. The opposite occurs in C57BL/6J mice, which show low prefrontal cortical DA outflow accompanied by high accumbal extracellular DA. Moreover, the DBA/2J background showed lower locomotor activity than C57BL/6J mice following D-amphetamine challenge. Selective DA depletion in the mpFC of DBA/2J mice produced a clear-cut increase in D-amphetamine-induced DA outflow in the NAc as well as locomotor activity that reached levels similar to those observed in C57BL/6J mice. Finally, local infusion of D-amphetamine by reverse microdialysis produced a similar increase in extracellular DA in both the mpFC and the NAc of DBA/2J mice. This finding points to similar transporter-related mechanisms in the two brain areas and supports the hypothesis that low accumbal DA release induced by systemic D-amphetamine in the DBA/2J background is determined by the inhibitory action of prefrontal cortical DA. The present results indicate that genotype-dependent susceptibility to addictive properties of D-amphetamine involves unbalanced DA transmission in the mesocorticolimbic system.
Dopamine in the Medial Prefrontal Cortex Controls
Genotype-Dependent Effects of Amphetamine on
Mesoaccumbens Dopamine Release and Locomotion
Rossella Ventura
1,2
, Antonio Alcaro
1,2
, Simona Cabib
1,2
, Davide Conversi
1,2
, Laura Mandolesi
1,2
and Stefano
Puglisi-Allegra*
,1,2
1
Dipartimento di Psicologia, Universita
`‘La Sapienza’, Rome, Italy;
2
Fondazione Santa Lucia IRCCS, Rome, Italy
Mice of background DBA/2J are hyporesponsive to the behavioral effects of D-amphetamine in comparison with the widely exploited
murine background C57BL/6J. In view of the important role of dopamine (DA) release in the nucleus accumbens (NAc) regarding the
behavioral effects of psychostimulants, we tested the hypothesis of an inverse relationship between mesocortical and mesoaccumbens
DA functioning in the two backgrounds. Systemic D-amphetamine induces a sustained increase in DA release in the medial prefrontal
cortex (mpFC) accompanied by a poor increase in the NAc in mice of the low-responsive DBA/2J background, as shown by intracerebral
microdialysis in freely moving animals. The opposite occurs in C57BL/6J mice, which show low prefrontal cortical DA outflow
accompanied by high accumbal extracellular DA. Moreover, the DBA/2J background showed lower locomotor activity than C57BL/6J
mice following D-amphetamine challenge. Selective DA depletion in the mpFC of DBA/2J mice produced a clear-cut increase in
D-amphetamine-induced DA outflow in the NAc as well as locomotor activity that reached levels similar to those observed in C57BL/6J
mice. Finally, local infusion of D-amphetamine by reverse microdialysis produced a similar increase in extracellular DA in both the mpFC
and the NAc of DBA/2J mice. This finding points to similar transporter-related mechanisms in the two brain areas and supports the
hypothesis that low accumbal DA release induced by systemic D-amphetamine in the DBA/2J background is determined by the inhibitory
action of prefrontal cortical DA. The present results indicate that genotype-dependent susceptibility to addictive properties of
D-amphetamine involves unbalanced DA transmission in the mesocorticolimbic system.
Neuropsychopharmacology (2004) 29, 72–80, advance online publication, 10 September 2003; doi:10.1038/sj.npp.1300300
Keywords: dopamine; drugs of abuse; medial prefrontal cortex; nucleus accumbens; mesocorticolimbic system; genotype
INTRODUCTION
An increasing body of evidence indicates that psychosti-
mulants increase dopamine (DA) transmission in the
nucleus accumbens (NAc), the subcortical area that
mediates their stimulating/reinforcing effects, through
complex neural networks involving a number of brain
areas and neurotransmitters (Piazza and Le Moal, 1996;
Jackson and Moghaddam, 2001; Porrino and Lyons, 2000).
Moreover, DA transmission in subcortical structures, such
as the NAc, appears to be modulated by the DA
mesocortical system in an inhibitory way (Herve et al,
1981; Deutch et al, 1990; Le Moal and Simon, 1991; Piazza
et al, 1991; Thompson and Moss, 1995; Doherty and
Gratton, 1996; Karreman and Moghaddam, 1996; Harden
et al, 1998; Jentsch et al, 1998, Beyer and Steketee, 1999),
thus strongly suggesting that the DA response in the
accumbens is inversely related to that elicited in the
prefrontal cortex.
Mice of the DBA/2J background have been shown to be
poorly responsive to the stimulating and the reinforcing
effects of D-amphetamine (Cabib et al, 2000) as well as to its
enhancing effects on dopamine outflow in the NAc (Zocchi
et al, 1998) compared with the widely exploited C57BL/6J
background. The C57BL/6J background, commonly used
in molecular approaches, has been chosen by the NIH
as one of its two standard strains to be evaluated in depth
and compared with contrasting inbred strains (The Trans-
NIH Mouse Initiative, http://www.nih.gov/science/Models/
mouse/).
In the light of the aforementioned studies indicat-
ing that DA response in the accumbens is inversely
related to that elicited in the prefrontal cortex, we
hypothesize that this neurobehavioral phenotype
depends on high responsiveness of DA transmission
in the medial prefrontal cortex (mpFC) of the DBA/2J
background.
Online publication: 4 August 2003 at http://www.acnp.org/citations/
NPP08040303109/default.pdf
Received 15 March 2003; revised 26 July 2003; accepted 29 July 2003
*Correspondence: Dr S Puglisi-Allegra, Dipartimento di Psicologia,
Universita
`‘La Sapienza’, via dei Marsi n. 78, 00185 Rome, Italy, Tel:
+39 06 49917523, Fax: +39 06 49917712,
E-mail: stefano.puglisi-allegra@uniroma1.it
Neuropsychopharmacology (2004) 29, 7280
&
2004 Nature Publishing Group All rights reserved 0893-133X/04
$
25.00
www.neuropsychopharmacology.org
In particular, a sustained increase in extracellular DA in
the mpFC of DBA/2J mice is expected, which would have a
strong inhibitory effect on accumbal DA outflow, thus
leading to low extracellular DA levels being induced by
amphetamine. The opposite is expected in the C57BL/6J
background, known to be highly responsive to ampheta-
mine (Cabib et al, 2000), in which low DA outflow in the
mpFC should lead to permissive action on accumbal DA,
resulting in high accumbal release.
We also predict that in the DBA/2J background, in the
absence of dopaminergic prefrontal cortical influence,
amphetamine induces higher DA outflow in the NAc and
higher locomotor activity. This can be assessed by
evaluating the effects of selective prefrontal dopamine
depletion on those produced by systemic amphetamine on
accumbal DA and behavior.
Lastly, if low DA outflow in the NAc of DBA/2J mice is
due to the inhibitory action of prefrontal DA on accumbal
DA, it is conceivable that locally infused amphetamine does
produce a similar increase in extracellular DA in both the
NAc and the mpFC, unless dopamine transporter (DAT)-
related mechanisms lead to differences in DA release in the
two brain areas (Moron et al, 2002).
Here, we report the results of experiments aimed at
assessing (1) if in DBA/2J mice, systemic amphetamine
induces a sustained increase of DA release in the mpFC
accompanied by a low increase in the NAc, and if the
opposite occurs in mice of the C57/BL/6J background,
which is highly responsive to amphetamine; (2) if selective
prefrontal DA depletion in the DBA/2J background
potentiates the effects of the psychostimulant on accumbal
DA and on locomotion; (3) if, unlike systemic administra-
tion, intracortical or intra-accumbens infusion of amphe-
tamine in the DBA/2J background produces a similar
increase in dopamine release.
MATERIALS AND METHODS
Animals
Male mice of the inbred C57BL/6JIco (C57) and DBA/2JIco
(DBA) strains (Charles River Italy) were used for these
experiments. All mice were purchased at 6 weeks of age.
Upon their arrival, animals were housed in groups of four
per standard breeding cage (27 21 13.5 cm
3
) with food
and water ad libitum on a 12/12 h dark/light cycle (lights on
between 0700 and 1900). Experiments started when animals
reached 8 weeks of age and were carried out in a room
separated from the colony room between 1400 and 1800. All
the mice used were handled and accustomed to the
environment where the experiment was to be performed
and then randomly assigned to different treatments. Each
experimental group comprised six to 11 animals. All mice
were housed individually 24 h before surgery for micro-
dialysis. Naive animals were used for each experiment.
The procedures used in this study were in strict
accordance with European legislation (EEC no. 86/609),
with Italian national legislation (DL no. 116/92) governing
the use of animals for research, and with the guidelines of
the National Institutes of Health on the use and care of
laboratory animals.
Drugs
D-Amphetamine sulfate (amphetamine), chloral hydrate,
6-hydroxydopamine (6-OHDA), and desipramine hydro-
chloride (DMI) were purchased from Sigma (Sigma Aldrich,
MI). Amphetamine (2.5 mg/kg), chloral hydrate (450 mg/
kg), and DMI (35 mg/kg) were dissolved in saline (0.9%
NaCl) and injected intraperitoneally (i.p.) in a volume of
10 ml/kg. Amphetamine (1, 5, 10, 100, 1000 mM) was
dissolved in artificial CSF for reverse microdialysis experi-
ments. 6-OHDA was dissolved in saline containing
Na-metabisulfite (0.1 M).
Microdialysis
Animals were anesthetized with chloral hydrate, mounted in
a stereotaxic frame (David Kopf Instruments, Tujunga, CA)
equipped with a mouse adapter and implanted unilaterally
with a guide cannula (stainless steel, shaft OD 0.38 mm,
Metalant AB, Stockholm, Sweden,) in the mpFC or in the
NAc. The length of the guide cannula was 1 mm for mpFC
and 4.5 mm (C57) or 4.0 mm (DBA) for NAc. The guide
cannula was fixed with epoxy glue, and dental cement was
added for further stabilization. The coordinates from
bregma (measured according to the atlas of Franklin and
Paxinos (1998) and Mouse Brain Atlases, The Mouse Brain
Library, www.nervenet.org/mbl/) were as follows: mpFC:
C57 ¼+2.5 AP, 0.6 L; DBA ¼+2.0 AP, 0.6 L; NAc:
C57 ¼+1.6 AP, 0.6 l; DBA:+1.1 AP, 0.6 L. The probe
(dialysis membrane length 2 mm for mpFC and 1 mm for
NAc; OD 0.24 mm, MAB 4 cuprophane microdialysis probe,
Metalant AB) was introduced 24 h after implantation of the
guide cannula. The probe was then fastened with the locking
system provided by Metalant AB. The animals were lightly
anesthetized to facilitate manual insertion of the micro-
dialysis probe into the guide cannula. The membranes were
tested for in vitro recovery of DA (relative recovery
(%) ¼10.9+0.86; n¼21) on the day before use to verify
recovery.
The microdialysis probe was connected to a CMA/100
pump (Carnegie Medicine Stockholm, Sweden) through a
PE-20 tubing and an ultralow torque dual channel liquid
swivel (Model 375/D/22QM, Instech Laboratories, Inc.,
Plymouth Meeting, PA) to allow free movement. Artificial
CSF (147 mM NaCl, 2.2 mM CaCl
2
and 4 mM KCl) was
pumped through the dialysis probe at a constant flow rate of
2ml/min. Experiments were carried out 22–24 h after probe
placement. Each animal was placed in a circular cage
provided with microdialysis equipment (Instech Labora-
tories, Inc.) and with home cage bedding on the floor.
Dialysis perfusion was started 1 h later. Following the start
of the dialysis perfusion, mice were left undisturbed for
approximately 2 h before the collection of baseline samples.
The dialysate was collected every 20 min for 180 min.
Placements were judged by methylene blue staining. Only
data from mice with correctly placed cannula have been
reported. Within the mpFC, the probe position mainly
included the prelimbic cortex in both strains (Figure 1).
Within the NAc, the probe position mainly included the
shell subdivision (Figure 2). In total, 20 mm of the dialysate
samples was analyzed by high-performance liquid chroma-
tography (HPLC). The remaining 20 ml were kept for
Prefrontal cortex, genotype, and amphetamine
R Ventura et al
73
Neuropsychopharmacology
possible subsequent analysis. Concentrations (pg/20 ml)
were not corrected for probe recovery. The mean concen-
tration of the three samples collected immediately before
treatment (less than 10% variation) was taken as the basal
concentration. The average basal values of dopamine for
each group did not differ significantly. Therefore, they have
been grouped together here. Basal values (pg/20 ml) were as
follows: mpFC: DA: C57 ¼0.286 70.013; DBA ¼0.356 7
0.006; NAc: DA: C57 ¼1.27 70.14; DBA ¼1.38 70.23.
For reverse microdialysis experiments, increasing doses
of amphetamine (1, 5, 10, 100, 1000 mM) were perfused for
60 min each (three blocks of 20 min summed over each
60 min point) (Hedou et al, 2000).
The HPLC system consisted of an Alliance (Waters
Corporation, Milford, MA) system and a coulometric
detector (ESA Model 5200A Coulochem II) provided with
a guard cell (M 5021) and an analytical cell (M 5011). The
guard cell was set at 400 mV, electrode 1 at 200 mV, and
electrode 2 at 250 mV. A Nova-Pack C18 column
(3.9 150 mm
2
, Waters) maintained at 331C was used.
The flow rate was 1.1 ml/min. The mobile phase was as
previously described (Westerink et al, 1998). The detection
limit of the assay was 0.1 pg.
Visualization of Probe Placement
Two types of visualization of probe placement were carried
out. Visualization in the mpFC was evidentiated by Nissl’s
staining. In addition, we used DAT immunostaining in
order to identify core–shell borders in the NAc of each
strain. Indeed, a number of studies have shown differential
expression of DAT in the NAc shell and core at the regional
as well as subcellular level (Jones et al, 1996; Nirenberg et al,
1997).
Animals were deeply anesthetized with chloral hydrate
and transcardially perfused with saline followed by ice-cold
probe
membrane
probe
membrane
0.5mm 0.5mm
C57 DBA
Figure 1 Location of microdialysis probe in the mpFC of C57BL/6 (C57) and DBA/2J (DBA) mice. Nissl’s stained coronal sections of mouse brain
hemispheres show the segment of the probe membrane in the two backgrounds.
Sh
Co
Sh
Sh
Co
Sh
0.5mm
0.5mm
C57
DBA
Figure 2 Anatomical location of microdialysis probes in the NAc of
C57BL/6J (C57) and DBA/2J (DBA) mice. The inset represents the areas
containing the tracks of the microdialysis probes and the range of
implantation sites. DAT immunostaining was used to reveal core–shell
borders. Sh ¼NAc shell; Co ¼NAc core.
Prefrontal cortex, genotype, and amphetamine
R Ventura et al
74
Neuropsychopharmacology
10% neutral buffered formalin. Brains were dissected,
further fixed, and cryoprotected in 30% sucrose.
The frontal lobes were dissected, and the extent of the
cortical lesion was determined from Nissl-stained 40 mm
frozen sections (Figure 1).
Sections including the NAc (30 mm sections) were serially
collected in 10 mM pH 7.4 phosphate-buffered saline (PBS)
and processed for DAT immunostaining (Figure 2). Sections
were processed free-floating at room temperature in 24-well
culture plates placed on gentle orbital agitation. After a
15 min endogenous peroxidase inactivation step with 0.3%
hydrogen peroxide in PBS, sections were rinsed in PBS, then
incubated overnight in a primary antibody (monoclonal rat
anti-DAT, Sigma) diluted 1/10 000 in PBS with 1% bovine
serum albumin (BSA) and 0.25% Triton X-100. After rinsing
in PBS, sections were incubated for 1.30 h in secondary
antiserum (polyclonal mouse anti-rat, Jackson immuno-
research) diluted 1/500 in PBS with 1% BSA and 0.25%
Triton X-100. Sections were then thoroughly rinsed and
incubated for 1 h in an avidin–biotin complex (Vector
Laboratories) diluted 1/500 in PBS with 0.25% Triton X-100.
After further rinsing, tissue-bound horseradish peroxidase
was revealed by incubating sections in cobalt-enhanced
diaminobenzidine substrate solution prepared according to
the manufacturer’s instructions (Sigma). After rinsing in
10 mM pH 8.0 Tris-buffered saline, sections were mounted
on superfrost slides, dehydrated with ascending grades of
EtOH, cleared in xylene, and coverslipped with Entellan
(Merck).Visual examination and digital imaging were
performed with a light-transmission microscope equipped
with a CCD camera.
Dopamine Depletion
Anesthesia and surgical set have been described in the
previous section.
DBA mice were injected with DMI (35 mg/kg) 30 min
before 6-OHDA microinjection in order to protect nor-
adrenergic neurons. Bilateral injections of 6-OHDA (1.5 mg/
0.1 ml/2 min for each side) were made into the mpFC
(coordinates: +2.0 AP; 70.6 L; 2.0 V with respect to
bregma) through a stainless-steel cannula (0.15 mm OD,
UNIMED, Switzerland) connected to a 1 ml syringe by a
polyethylene tube and driven by a CMA/100 pump. The
cannula was left in place for an additional 2 min after the
end of the infusion. Sham animals were subjected to the
same treatment, but received intracerebral vehicle.
Norepinephrine (NE) and DA tissue levels in the mpFC
were assessed as previously described (Ventura et al, 2003)
to evaluate the amount and the extent of depletion. The
brain was fixed vertically on the freeze plate of a freezing
microtome. Punches of both hemispheres were obtained
from brain slices (coronal sections) no thicker than 300 mm.
Stainless-steel tubing of 2.3 mm internal diameter was
used. The coordinates were measured according to the atlas
of Franklin and Paxinos (1998) and adapted according to
the Mouse Brain Atlases (The Mouse Brain Library,
www.nervenet.org/mbl/) and to the previous study (Ventura
et al, 2001). The punches were stored in liquid nitrogen
until the day of analysis.
DA and NE were determined simultaneously, utilizing a
reverse phase HPLC procedure coupled with coulochem
electrochemical detection. On the day of the analysis, frozen
samples were weighed and homogenized in HC1O
4
0.1 N
containing Na-metabisulfite 6 mM and EDTA 1 mM. The
homogenates were centrifuged at 10 000 rpm for 20 min at
41C. Aliquots of the supernatant were then transferred to
the HPLC system.
The HPLC system is described above, the potentials being
set at +450 and +100 mV at the analytical and the
conditioning cell, respectively. The columns, a Nova-Pack
Phenyl column (3.9 150 mm
2
), and a Sentry Guard Nova-
Pack precolumn (3.9 20 mm
2
), were purchased from
Waters Assoc. The flow rate was 1 ml/min. The mobile
phase consisted of 3% methanol in 0.1 M Na-phosphate
buffer pH 3, Na
2
EDTA 0.1 mM, and 1-octane sulfonic acid
Na salt (Aldrich) 0.5 mM.
Animals were used for microdialysis or behavioral
experiments 7 days after surgery.
Locomotor Activity
Locomotor activity was assessed in animals other than those
tested in microdialysis experiments. They were either naive
(C57 and DBA) or had been subjected to DA depletion as
described in the previous section (DBA: Sham or DA
depleted). Mice from each background (C57 or DBA) or
pretreatment (Sham or DA depleted) were challenged with
amphetamine or vehicle before testing. The apparatus
comprised eight gray opaque Plexiglas chambers divided
into two compartments (20 10), with removable floors,
placed inside a sound-attenuated room. Individual mice
were introduced into each chamber and accustomed to the
apparatus for 60 min. Then, all mice were removed and left
undisturbed inside their home cages for the following
60 min. Immediately before testing, subjects were weighed
and injected (i.p.) with 2.5 mg/kg of amphetamine or with
10 ml/kg of vehicle. Then, mice were placed individually in
the same cages they had experienced and tested for the
following 60 min.
Behavioral data were collected and analyzed by the
‘EthoVision’ (Noldus, The Netherlands) fully automated
video tracking system (Spink et al, 2001). Briefly, a CCD
video camera was used to record the experimental system.
The signal was then digitized (using a hardware device
called a frame grabber) and passed on to the computer’s
memory. Later on, digital data were analyzed using
EthoVision software to measure the number of crossings
between compartments.
Statistics
Microdialysis data were analyzed for each brain area by
repeated-measures ANOVA with two between factors
(treatment, two levels: saline, amphetamine; strain, two
levels: C57, DBA, or pretreatment, two levels: Sham, DA
depleted), and one within factor (time, seven levels: 0, 20,
40, 60, 80, 100, 120 min from drug injection).
Reverse microdialysis was analyzed by repeated-measures
ANOVA with one within factor (doses, six levels: basal, 1, 5,
10, 100, 1000 mM).
The locomotor activity was analyzed by two-way ANOVA,
the factors being: strain (two levels: C57, DBA) or
pretreatment (two levels: Sham, DA depleted) and treatment
Prefrontal cortex, genotype, and amphetamine
R Ventura et al
75
Neuropsychopharmacology
(two levels: saline, amphetamine). In the case of significant
interactions, post hoc comparisons were performed by a
Duncan test. The Student’s t-test (two-tailed) was also used.
RESULTS
Effects of Systemic Amphetamine on Extracellular DA in
the mpFC and the NAc and on Locomotion in C57 and
DBA Mice
The effects of amphetamine on DA release in the mpFC are
shown in Figure 3. Statistical analyses revealed a significant
strain treatment time interaction (F(1, 180) ¼12.8;
po0.0005). A simple effect analysis showed a significant
effect of time only for amphetamine and a significant
difference between saline and amphetamine. Moreover,
significant differences between C57 and DBA mice chal-
lenged with the psychostimulant were evident. Ampheta-
mine produced a time-dependent increase in DA outflow in
the mpFC of both strains, but DBA mice showed
dramatically higher levels than the C57 background
throughout (20–120 min). Extracellular DA reached
B700% maximal increase (at 40 min) in DBA mice, while
B150% maximal increase was evident (at 40 min) in the
C57 background. No significant differences were evident in
the basal levels between the two backgrounds.
The effects of amphetamine on DA release in the NAc
are shown in Figure 3. Statistical analyses revealed a signifi-
cant strain treatment time interaction (F(1, 144) ¼5.13;
po0.001). A simple effect analysis showed a significant
effect of time only for amphetamine and a significant
difference between saline and amphetamine. Moreover,
significant differences between C57 and DBA mice chal-
lenged with the psychostimulant were evident. Ampheta-
mine produced a time-dependent increase in DA outflow in
the NAc of both strains, but C57 mice showed significantly
higher levels from 20 to 60 min. Extracellular DA reached
B150% maximal increase (at 40 min) in DBA mice, while
B350% maximal increase was evident (at 40 min) in the
C57 background. No significant differences were evident in
basal levels between the two backgrounds.
The effects of amphetamine on locomotor activity in
mice of C57 and DBA strains are shown in Figure 4.
ANOVA showed significant strain treatment interaction
(F(1, 28) ¼7.26; po0.05). Individual between group com-
parisons showed significant differences between mice of
both backgrounds challenged with amphetamine in com-
parison with mice of the same background challenged with
saline. Amphetamine increased locomotor activity in both
backgrounds, but mice of DBA background showed
significantly lower activity scores than the C57 background.
Effects of Selective DA Depletion in the mpFC on DA
Release in the NAc and Locomotion in DBA Mice
Challenged with Amphetamine
Intracortical neurotoxin (6-OHDA) reduced DA levels in the
mpFC by about 90% in comparison with Sham animals,
while nonsignificant changes were evident in the NE content
(Table 1). These results show that our experimental
procedure allows a selective depletion of DA prefrontal
cortical afferents, and is thus suitable for the purposes of
the present study.
The effects of selective DA depletion in the mpFC on DA
release in the NAc are shown in Figure 5. Statistical analyses
revealed significant pretreatment treatment time inter-
action (F(1, 120) ¼10.05; po0.0005). A simple effect analy-
sis revealed a significant effect of time only for
amphetamine and a significant difference between saline
and amphetamine. Moreover, significant differences be-
tween Sham and DA-depleted groups injected with amphe-
tamine were evident. Amphetamine produced a significant
increase in DA release in the NAc of both Sham and DA-
depleted groups, but selective prefrontal cortical DA
depletion led to a clear-cut potentiation of amphetamine-
induced increase in DA outflow.
900
800
700
600
500
400
300
200
100
0
020 6040 80 100 120
900
800
700
600
500
400
300
200
100
0
020 6040 80 100 120
NAc
mpFC
C57 Sal
DBA Sal
C57 Amph
DBA Amph
% of basal DA output% of basal DA output
Time after amphetamine (min)
Time after amphetamine (min)
Figure 3 Extracellular dopamine in the mpFC and NAc of C57BL/6J
(C57) and DBA/2J (DBA) mice (n¼7–11 per group) receiving saline (Sal)
or amphetamine (2.5 mg/kg, i.p.) (Amph). Results are expressed as percent
changes from basal levels. Statistical analyses were carried out on raw data
(mean 7SE). *po0.005 compared with saline; }po0.005 compared with
the other strain.
Prefrontal cortex, genotype, and amphetamine
R Ventura et al
76
Neuropsychopharmacology
A significantly higher DA outflow increase was evident in
DA-depleted mice compared with Sham between 20 and
80 min after injection. Extracellular DA reached a 650%
maximal increase (at 20 min) in DA-depleted animals, while
a 160% maximal increase was evident (at 40 min) in the
Sham group. No significant differences were evident in
basal levels between groups (Figure 5).
The effects of selective DA depletion in the mpFC on
locomotor activity are shown in Figure 6. Statistical analyses
revealed a significant pretreatment treatment interaction
(F(1, 27) ¼7.2; po0.02). Individual comparisons showed
that amphetamine increased locomotor activity in both
groups, but DA-depleted animals exhibited dramatically
higher locomotor activity scores than Sham animals. No
significant differences were evident between DA-depleted
and Sham animals injected with saline.
Effects of Amphetamine Applied to mpFC or to NAc on
Dialysate DA Levels
Amphetamine infused through the microdialysis probe
produced a clear-cut increase of extracellular DA in both
the mpFC (F(5, 24) ¼7.67; po0.0005) and the NAc
(F(5, 30) ¼92.2; po0.0005). In particular, the psychostimu-
lant produced a dose-related parallel percent increase from
the basal levels in the two brain areas, thus indicating a
similar effect of local infusion in the mpFC and the NAc
(Figure 7).
DISCUSSION
The present results show that mesocortical DA controls the
genotype-dependent effects of systemic amphetamine on
mesoaccumbems DA release and on locomotion.
500
400
200
300
100
0
Sal Amph
C57
DBA
crossings
*#
*
Figure 4 Effects of amphetamine (2.5 mg/kg, i.p.) (Amph) or saline (Sal)
on locomotor activity (mean crossings 7SE) of naive C57BL/6J (C57) and
DBA/2J (DBA) mice. *po0.01 in comparison with saline (Sal); #po0.01in
comparison with the other strain.
Table 1 DA and NE Tissue Levels (ng/g Wet Weight) in mpFC of
Sham and DA-depleted DBA/2J Mice
NE DA
Sham 790 728 191 717
DA depleted 739 749 53 78*
*po0.005 in comparison with Sham.
900
800
700
600
500
400
300
200
100
0
020 6040 80 100 120
Sham Sal
DA depl Sal
Sham Amph
DA depl Amph
% of basal DA output
Time after amphetamine (min)
Figure 5 Effects of mpFC dopamine depletion (DA depl) on
extracellular dopamine in the NAc of animals (n¼10–12 per group)
receiving saline (Sal) or amphetamine (2.5 mg/kg, i.p.) (Amph). Results are
expressed as percent changes from basal levels. Statistical analyses were
carried out on raw data (mean 7SE). *po0.05 compared with saline;
}po0.05 compared with the Sham group.
500
400
200
300
100
0
Sal Amph
DBA Sham
DBA DA depl
crossings
*#
*
Figure 6 Effects of amphetamine (2.5 mg/kg, i.p.) (Amph) or saline (Sal)
on locomotor activity (mean crossings 7SE) of DBA/2J (DBA) mice
either bearing a selective DA depletion in the mpFC (DA depl) or sham
lesioned (Sham). *po0.01 in comparison with saline (Sal); #po0.01 in
comparison with DA depl.
Prefrontal cortex, genotype, and amphetamine
R Ventura et al
77
Neuropsychopharmacology
First, in response to systemic amphetamine challenge,
DBA mice show higher mesocortical DA activation accom-
panied by a weak dopaminergic accumbal response, while
the opposite occurs in the C57 background, a finding that
supports the hypothesis of an inverse relationship between
mesocortical and mesoaccumbens DA functioning. Indeed,
we observed dramatic differences in the effects of systemic
amphetamine on DA outflow in the mpFC of the two
backgrounds. In fact, at all time points, DBA mice showed
significantly higher DA responses than C57 mice. The effect
of amphetamine on DA outflow lasted up to 120 min in DBA
mice, while in C57 mice DA levels were no different from
saline at 100 min postinjection. Moreover, amphetamine
produced B700% maximal increase in DA release in the
mpFC of DBA mice and B150% maximal increase in DA in
the C57 strain.
Instead, amphetamine produced a higher DA release in
the NAc of C57 strain than DBA mice, confirming previous
results (Zocchi et al, 1998). In fact, the C57 strain showed
B350% increase, while in DBA mice B150% maximal
outflow occurred. It should be pointed out that DBA mice
showed moderately higher DA basal levels in the mpFC than
C57 (+24%) that were conceivably too weak to lead to
parallel lower DA basal levels in the NAc.
Consistent with the effects produced in the NAc, systemic
amphetamine challenge induced a stronger increase of
locomotor activity in C57 mice than the DBA background,
in accordance with the consolidated literature (see for
review Puglisi-Allegra and Cabib, 1997).
Second, prefrontal cortical DA depletion in DBA mice
produced a dramatic increase in the accumbal DA response
to the psychostimulant in comparison with the Sham group.
These results are in agreement with the view that increased
DA release in the mpFC has an inhibitory effect on
accumbal DA release (Deutch et al, 1990; Le Moal and
Simon, 1991; Piazza et al, 1991; Thompson and Moss, 1995;
Doherty and Gratton, 1996; Karreman and Moghaddam,
1996; Harden et al, 1998; Jentsch et al, 1998).
Since dopamine exerts a tonic stimulatory control on
GABA interneurons in the prefrontal cortex (Penit-Soria
et al, 1987; Pirot et al, 1992), it may be that 6-OHDA-
induced DA depletion decreases the activity of GABA
interneurons. This reduction could, in turn, trigger an
increased activity of efferents to the NAc by removing the
inhibition of pyramidal cells (Bunney and Aghajanian, 1976;
Ferron et al, 1984).
To our knowledge, the present results provide the first
demonstration of potentiation accumbal extracellular DA
induced by acute amphetamine challenge by prefrontal DA
depletion in rodents.
In addition, selective DA depletion in the mpFC leads to
increased effects of amphetamine challenge on locomotor
activity in DBA mice, which parallels the increased DA
response in the NAc. The behavioral effects of prefrontal
cortical DA depletion are in agreement with previous results
obtained in rat (Pycock et al, 1980; Bjijou et al, 2002), but
not with others (King et al, 1997). It is worth noting that
selective DA depletion of pFC has been shown to increase
the effects of systemic cocaine on DA outflow in the NAc as
well as on locomotion, thus leading to sensitization-like
effects in rats receiving acute psychostimulant challenge
(Beyer and Steketee, 1999).
Third, the results from reverse microdialysis experiments
support the fact that the low DA activation in the NAc of the
DBA background following systemic amphetamine depends
on an inhibitory action outside the accumbens. Indeed, the
local infusion of amphetamine produces a comparable
increase in extracellular DA in both the mpFC and the NAc,
indicating that the effects of amphetamine on nerve
terminals in the two brain areas are similar in potency.
Therefore, the different responses by mpFC and NAc to
systemic drug administration cannot be ascribed to
differences in transporter-related mechanisms in the two
brain areas (Moron et al, 2002), but involve an active
inhibitory mechanism. In the light of the results obtained by
the experiment on mesocortical DA depletion described
above, this inhibitory role can be ascribed to prefrontal
cortical DA transmission.
Our present results add new evidence, indicating a major
role of prefrontal cortical DA transmission in the effects of
psychostimulants on mesoaccumbens DA release and on
behavioral output. Moreover, they indicate that individual
differences in susceptibility to the addictive properties of
psychostimulants depend on genotype-controlled balance
between drug-induced mesocortical and mesoaccumbens
DA response.
In fact, mice of the C57 strain that respond with higher
mesoaccumbens dopamine release and lower prefrontal DA
release to amphetamine challenge than mice of the DBA
background are also more responsive to the stimulating
effects of amphetamine than DBA mice. In the C57
background, a dramatically less pronounced effect of the
psychostimulant on DA release in the mpFC is likely to
produce a low inhibitory action on DA outflow in the NAc
and, therefore, a more pronounced accumbal DA release
and behavioral effects of amphetamine. Consistent with
these findings, selective DA depletion in the mpFC of the
DBA background makes these animals similar to C57 in
terms of the effects of amphetamine on both accumbal DA
release and behavioral output.
500
400
200
300
100
0
0 5 10 100 1000
mpFC
NAc
% of basal DA output
Amphetamine (microM)
Figure 7 Effects of local amphetamine infusion (1, 5, 10, 100, 1000 mM)
on extracellular dopamine in the mpFC and the NAc of DBA/2J mice.
Results are expressed as percent changes from basal levels. Statistical
analyses were carried out on raw data. All data are mean 7SE (n¼6 per
group). *po0.01 in comparison with basal values.
Prefrontal cortex, genotype, and amphetamine
R Ventura et al
78
Neuropsychopharmacology
In conclusion, the present results indicate a genetic
control over the balance between mesocortical and meso-
accumbens DA response to amphetamine that determines
the behavioral outcome following challenge with the
psychostimulant.
These data have a greater clinical relevance, since
imbalance between mesocortical and mesolimbic DA system
has been proposed as a major etiological factor in psychoses
(Deutch et al, 1990; Karreman and Moghaddam, 1996; Egan
and Weinberger, 1997; Di Chiara et al, 1999) and in drug
addiction (Piazza and Le Moal, 1996).
ACKNOWLEDGEMENTS
We thank Dr E Catalfamo and Dr C Castellano (CNR) for
guidance in implantation and lesion experiments. This
research has been supported by Ministero della Ricerca
Scientifica e Tecnologica (COFIN 2001), Universita
`‘La
Sapienza’ Ateneo (1999–2001), Ministero della Salute
(Progetto Finalizzato RF00.96P 2001-2003).
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    • "Furthermore, because mesocortical dopamine function and 531 responsiveness of mesolimbic dopamine neurons to stressors and drugs of abuse are inversely 532 related (Jackson and Moghaddam, 2001; Ventura et al., 2004; Scornaiencki et al., 2009; Pokinko 533 et al., 2015), it has been suggested that the role of mPFC dopamine on PPI is mediated by 534 changes in ventral striatal dopamine function (Bubser and Koch, 1994; Koch and Bubser, 1994; 535 Ellenbroek et al., 1996; Grant et al., 2007; Flores, 2011). However, there are no differences in Jackson and Moghaddam, 2001; Ventura et al., 2004; Grant et al., 2007; 544 Scornaiencki et al., 2009; Pokinko et al., 2015). "
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    • "Presuming that the reduced expression reflects decreased glutamatergic tone at NMDA receptors, it may represent an additional mechanism underlying changes to cognition and psychostimulant responses induced by adolescent defeat, which were previously attributed to decreased mPFC DA activity [17] . Specifically , rats defeated in adolescence show deficits in adult working memory tasks [12] along with enhanced conditioned place preference and locomotion responses to amphetamine [14,16], similar to effects of pharmacologically reducing mPFC DA function [48][49][50][51]. However, these behaviors are also modulated by NMDA receptors in the mPFC. "
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