ORIGINAL RESEARCH ARTICLE
published: 19 February 2013
doi: 10.3389/fncir.2013.00022
NEURAL CIRCUITS
Distribution and compartmental organization of GABAergic
medium-sized spiny neurons in the mouse nucleus
accumbens
Giuseppe Gangarossa 1,2,3 , Julie Espallergues 1,2,3 , Alban de Kerchove d’Exaerde 4 ,
Salah El Mestikawy 5,6,7 , Charles R. Gerfen 8 , Denis Hervé 9,10,11 , Jean-Antoine Girault 9,10,11 and
Emmanuel Valjent 1,2,3*
1
CNRS, UMR-5203, Institut de Génomique Fonctionnelle, Montpellier, France
Inserm, U661, Montpellier, France
3
Universités de Montpellier 1 & 2, UMR-5203, Montpellier, France
4
Laboratory of Neurophysiology, School of Medicine, Université Libre de Bruxelles, ULB Neuroscience Institute, Brussels, Belgium
5
CNRS, UMR-7224, Paris, France
6
Inserm, U952, Paris, France
7
Université Pierre et Marie Curie, UMR-7224, Paris, France
8
Laboratory of Systems Neuroscience, National Institute of Mental Health, Bethesda, MD, USA
9
Inserm, UMR-S 839, Paris, France
10
Université Pierre et Marie Curie, UMR-S 839, Paris, France
11
Institut du Fer á Moulin, Paris, France
2
Edited by:
Luis De Lecea, Stanford University,
USA
Reviewed by:
Deborah Baro, Georgia State
University, USA
Garret Stuber, University of North
Carolina at Chapel Hill, USA
Marisela Morales, National Institute
on Drug Abuse, National Institutes
of Health, USA
*Correspondence:
Emmanuel Valjent, INSERM, U661,
Universités de Montpellier 1 & 2,
Montpellier, F-34094, France.
CNRS, UMR 5203, Institut de
Génomique Fonctionnelle,
Universités de Montpellier 1 & 2,
141 rue de la Cardonille,
34094 Montpellier, Cedex 05,
France.
e-mail: emmanuel.valjent@
igf.cnrs.fr; emmanuel.valjent@
gmail.com
The nucleus accumbens (NAc) is a critical brain region involved in many reward-related
behaviors. The NAc comprises major compartments the core and the shell, which
encompass several subterritories. GABAergic medium-sized spiny neurons (MSNs)
constitute the output neurons of the NAc core and shell. While the functional organization
of the NAc core outputs resembles the one described for the dorsal striatum, a simple
classification of the NAc shell neurons has been difficult to define due to the complexity
of the compartmental segregation of cells. We used a variety of BAC transgenic mice
expressing enhanced green fluorescence (EGFP) or the Cre-recombinase (Cre) under the
control of the promoter of dopamine D1, D2, and D3 receptors and of adenosine A2a
receptor to dissect the microanatomy of the NAc. Moreover, using various immunological
markers we characterized in detail the distribution of MSNs in the mouse NAc. In addition,
cell-type specific extracellular signal-regulated kinase (ERK) phosphorylation in the NAc
subterritories was analyzed following acute administration of SKF81297 (a D1R-like
agonist), quinpirole (a D2 receptors (D2R)-like agonist), apomorphine (a non-selective
DA receptor agonist), raclopride (a D2R-like antagonist), and psychostimulant drugs,
including cocaine and d-amphetamine. Each drug generated a unique topography and
cell-type specific activation of ERK in the NAc. Our results show the existence of marked
differences in the receptor expression pattern and functional activation of MSNs within the
shell subterritories. This study emphasizes the anatomical and functional heterogeneity of
the NAc, which will have to be considered in its further study.
Keywords: medium-sized spiny neurons, BAC transgenic, nucleus accumbens, dopamine, psychostimulant, ERK
signaling, neural circuits
INTRODUCTION
Located in the rostrobasal forebrain, the nucleus accumbens
(NAc) is a major brain area that processes incentive–reward
responses associated with novel, hedonic, stressful, or aversive
stimuli (Kalivas and Duffy, 1995; Reynolds and Berridge, 2002;
Jensen et al., 2003; Nicola, 2007). Dysfunctions of this structure have been associated with prominent psychiatric disorders
including obsessive-compulsive disorder, depression, and drug
addiction (Nicola, 2007; Sesack and Grace, 2010).
Generally seen as an integral part of the striatal complex, it
is, however, widely accepted that the NAc represents an independent entity that exhibits unique features compared with the dorsal
Frontiers in Neural Circuits
striatum (Herkenham et al., 1984). Using a variety of immunohistochemical markers and tract-tracing approaches, earlier studies
allowed distinguishing three major compartments in the NAc,
the rostral pole, the core and the shell (Zaborszky et al., 1985;
Zahm and Brog, 1992), but also multiple subterritories within
these three compartments (Heimer et al., 1991; Zahm and Brog,
1992; Jongen-Relo et al., 1993, 1994; Groenewegen et al., 1999).
The NAc lacks glutamatergic neurons but is instead mostly
composed of GABAergic medium-sized spiny neurons (MSNs),
the remaining neurons being cholinergic and GABAergic
interneurons (Meredith et al., 1993). The functional organization
of the NAc core MSNs resembles that described for the dorsal
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February 2013 | Volume 7 | Article 22 | 1
Gangarossa et al.
MSNs organization in the NAc
et al., 2010) mice were used as reporter to compare the patterns
of expression in different mouse lines. Male 8–10 week-old mice
were used and maintained in a 12 h light/dark cycle, in stable
conditions of temperature (22◦ C) and humidity (60%), with
food and water ad libitum. For the pharmacological studies only
Drd2-EGFP heterozygous mice were used. All experiments were
in accordance with the guidelines of the French Agriculture and
Forestry Ministry for handling animals (C34-172-13).
striatum. Indeed, NAc core MSNs can be categorized into at least
two different subgroups according to their projections sites. The
MSNs projecting to the ventral tegmental area (VTA) express
exclusively D1 receptors (D1R) resembling therefore the striatonigral MSNs (Gerfen et al., 1990; Schiffmann et al., 1991; Fink et al.,
1992; Gerfen, 1992; Le Moine and Bloch, 1995). However, pallidal
afferents from the NAc appear to differ from the striatopallidal
MSNs of the dorsal striatum since NAc core MSNs innervating the ventral pallidum (VP) express D1R and D2 receptors
(D2R)/adenosine A2a receptors (A2aR) (Lu et al., 1998). Due to
the diversity of their output targets that include VTA, hypothalamus, VP, and brainstem, a simple division into direct and indirect
pathways has been even more difficult to define for the NAc shell
MSNs (Voorn et al., 1989; Zahm and Brog, 1992; Van Dongen
et al., 2008; Humphries and Prescott, 2010; Sesack and Grace,
2010). In addition, a higher proportion of MSNs in the shell
than in the rest of the striatum appears to co-express D1R and
D2R, suggesting that the segregation in two distinct populations
is far from being complete (Le Moine and Bloch, 1996; BertranGonzalez et al., 2008, 2010; Matamales et al., 2009; Durieux et al.,
2011). Moreover, whether D1R- and D2R-expressing MSNs are
randomly distributed or exhibit inhomogeneous distribution patterns in the different subterritories of the NAc shell remains to be
established.
To address these issues, we took advantage of BAC transgenic mice expressing enhanced green fluorescent protein (EGFP)
under the control of the promoter of D1R, Drd1a-EGFP, and
D2R, Drd2-EGFP (Gong et al., 2003; Valjent et al., 2009).
Moreover, the dopamine D3 receptor (D3R) being highly
expressed in the NAc, we also analyzed GFP expression in Drd3Cre crossed with the Rosa26:loxP reporter mouse line. Using a
variety of immunological markers we characterized in detail the
microanatomical distribution of D1R- and D2R-expressing MSNs
in the mouse NAc. We also provide evidence that dopaminergic
agonists and psychostimulant drugs induce specific and topographical patterns of extracellular signal-regulated kinase (ERK)
activation that are closely associated with specific NAc shell
subterritories.
Drd2-EGFP mice were anaesthetized with a mixture of ketamine
(Imalgene 500, 50 mg/ml, Merial), 0.9% NaCl solution
(weight/vol), and xylazine (Rompun 2%, 20 mg/ml, Bayer)
(2:2:1, i.p., 0.1 ml/30 g) and mounted on a stereotaxic apparatus.
The surface of the skull was exposed and a hole was drilled at
the appropriate coordinates. A cannula connected to a Hamilton
0.5 µl microsyringe was stereotaxically lowered to the VTA. The
following coordinates were used: AP = −3.16, L = −0.55, and
V = −4.5 (Franklin and Paxinos, 2007). A volume of 0.25 µl of
6-OHDA∗ HCl (3 µg/µl of free base, dissolved in ascorbic acid
0.02%) was unilaterally injected at a rate of 0.05 µl/min. The
intra VTA microinjection of 6-OHDA was preceded (30 min)
by administration of desipramine (20 mg/kg, i.p.) to avoid
degeneration of noradrenergic fibers. Following injection the
cannula was left in place for another 4 min before retraction.
Mice were allowed to recover for a period of two 2 weeks before
experiments.
MATERIALS AND METHODS
TISSUE PREPARATION AND IMMUNOFLUORESCENCE
ANIMALS
Mice were rapidly anaesthetized with pentobarbital (500 mg/kg,
i.p., Sanofi-Aventis, France) and transcardially perfused with
4% (weight/vol.) paraformaldehyde in 0.1 M sodium phosphate
buffer (pH 7.5). Brains were post-fixed overnight in the same
solution and stored at 4◦ C. Thirty µm-thick sections were
cut with a vibratome (Leica, France) and stored at −20◦ C
in a solution containing 30% (vol/vol) ethylene glycol, 30%
(vol/vol) glycerol, and 0.1 M sodium phosphate buffer, until
they were processed for immunofluorescence. Sections were processed as follows: Day 1: free-floating sections were rinsed in
Tris-buffered saline (TBS; 0.25 M Tris and 0.5 M NaCl, pH 7.5),
incubated for 5 min in TBS containing 3% H2 O2 and 10%
methanol, and then rinsed three times for 10 min each in TBS.
After 15 min incubation in 0.2% Triton X-100 in TBS, sections were rinsed three times in TBS again. Finally, they were
incubated overnight at 4◦ C with the different primary antibodies. For detection of phosphorylated proteins, 50 mM NaF was
Drd2-EGFP (n = 29, Swiss-Webster background, founder
S118), Drd2-Cre (n = 4, C57/Bl6J background, founder ER44),
Drd1a-EGFP (n = 4, Swiss-Webster background, founder X60),
Drd3-Cre (n = 2, C57/Bl6J background, founder KI196), and
Adora2a-Cre (Durieux et al., 2009) (n = 3, C57/Bl6J background)
BAC transgenic mice were used in this study. BAC-EGFP and
BAC-Cre mice were originally generated by GENSAT (Gene
Expression Nervous System Atlas) at the Rockefeller University
(New York, NY) (Gong et al., 2003) except the Adora2a-Cre
(Durieux et al., 2009). Adora2a-Cre mice were used to identify
striatopallidal neurons. Indeed in the striatum, these mice
expressed the Cre recombinase selectively in striatopallidal
neurons but not in other striatal populations (striatonigral
MSNs, GABA, and cholinergic interneurons) or in the presynatic
DA neurons (Durieux et al., 2009). Rosa26:loxP (Srinivas et al.,
2001) and R26R CAG-boosted EGFP:LoxP (RCE:LoxP) (Miyoshi
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DRUGS AND TREATMENT
SKF81297 (5.0 mg/kg, i.p.), quinpirole (1.0 mg/kg, i.p.), apomorphine (3.0 mg/kg, s.c.), and raclopride (0.3 mg/kg, i.p.) were
purchased from Tocris and dissolved in 0.9% (w/v) NaCl (saline).
Cocaine (15 mg/kg, i.p.) and d-amphetamine (10 mg/kg, i.p.)
were purchased from Sigma Aldrich and dissolved in 0.9% (w/v)
NaCl (saline). Mice were habituated to handling and saline injection three consecutive days before the experiment. Drugs were
administrated on day 4. All the mice were injected in the home
cage and perfused 15 min after injection.
6-OHDA LESION
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Gangarossa et al.
MSNs organization in the NAc
included in all buffers and incubation solutions. Slices were
then incubated overnight or 72 h at 4◦ C with the following primary antibodies: chicken, mouse and rabbit anti-GFP (1:500
and 1:1000, respectively, Invitrogen), rabbit anti-vesicular glutamate transporter 1 (VGluT1) or anti-VGluT2 (1:1000), rabbit
anti-calretinin (1:1000, Swant), mouse and rabbit anti-tyrosine
hydroxylase (TH) (1:1000, Millipore), rat anti-dopamine transporter (DAT) (1:1000, Millipore), mouse anti-DARPP-32 (1:1000
gift from P. Greengard), rabbit against diphospho-Thr-202/Tyr204-ERK1/2 (1:400, Cell Signaling Technology). Sections were
rinsed three times for 10 min in TBS and incubated for 45 min
with goat Cy2-, Cy3-, and Cy5-coupled (1:500, Jackson Lab)
and/or goat A488 (1:500, Invitrogen). Sections were rinsed for
10 min twice in TBS and twice in TB (0.25 M Tris) before mounting in 1,4-diazabicyclo-[2.2.2]-octane (DABCO, Sigma-Aldrich).
Confocal microscopy and image analysis were carried out
at the Montpellier RIO Imaging Facility. All images covering
the entire NAc were single confocal sections, acquired using
sequential laser scanning confocal microscopy (Zeiss LSM780)
and stitched together as a single image. Double-labeled images
from each region of interest were also single confocal sections
obtained using sequential laser scanning confocal microscopy
(Zeiss LSM510 META and Zeiss LSM780). Photomicrographs
were obtained with the following band-pass and long-pass filter setting: GFP/Cy2 (band pass filter: 505–530), Cy3 (band
pass filter: 560–615), and Cy5 (long-pass filter 650). GFP-labeled
neurons were pseudocolored cyan or green and other immunoreactive markers were pseudocolored red or magenta. From the
overlap of cyan and red or green and magenta, double-labeled
neurons appeared white. Images used for quantification were
all single confocal sections. The objectives and the pinhole setting (1 airy unit) remain unchanged during the acquisition of
a series for all images. The thickness of the optical section is
∼1.6 µm with a 20× objective and ∼6 µm with a 10× objective. P-ERK-positive cells were quantified in zones or regions
of the same area (630 × 630 µm or 1273 × 1273 µm) in every
shell subterritories delineated in each slice by TH immunoreactivity (Tables A1, A2). A similar analysis was performed to
evaluate the percentage of GFP-positive cells expressing DARPP32 in the different BAC transgenic mice used. Quantification of
immunoreactive cells was performed using the cell counter plugin of the ImageJ software taking as standard reference a fixed
threshold of fluorescence.
DELINEATION OF NAc CORE AND NAc SHELL SUBTERRITORIES
Coronal sections, in which the core-shell boundary was clearly
visible, between bregma 1.34 and 0.98 mm, were selected for
analysis (Franklin and Paxinos, 2007). The NAc core and shell
delineation was done based upon calbindin-D 28 kDa (strongly
enriched in the core compared to the shell) and calretinin
immunostainings (strongly enriched in the shell compared to
the core). In addition to the differential expression of GFP in
the different mouse lines used, the delineation of the substructures of the NAc shell was based upon a combination of markers
including TH, DAT, VGluT1, VGluT2, and calretinin immunoreactivities. The striatal-enriched phosphoprotein DARPP-32 was
used to identify MSNs (Ouimet et al., 1984).
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STATISTICAL ANALYSIS
Data were analyzed using one-way ANOVA, where treatment was
the independent variable, followed by Dunnett’s post hoc test
for specific comparisons. Differences were considered significant
when p < 0.05.
RESULTS
TOPOGRAPHICAL ORGANIZATION OF D1R- AND D2R-EXPRESSING
MSNs IN THE NAc
The analysis of GFP fluorescence in Drd1a-EGFP and Drd2EGFP mice showed a relatively uniform appearance in the
NAc core (Figures 1A, 2A). As previously observed, D1R- and
FIGURE 1 | Topographical distribution of GFP immunofluorescence in
the NAc of Drd1a-EGFP mice. (A) Distribution of GFP in the NAc of
Drd1a-EGFP BAC transgenic mice. Single confocal sections were stitched
together as a single image. Scale bar: 500 µm. DStr, dorsal striatum; aca,
anterior commissure; ICjM, islands of Calleja major; Tu, olfactory tubercles.
(B) Schematic illustration of the different accumbal subterritories analyzed
(see Figure A1). Cyan in the diagram represents distribution of
GFP-expressing cells. A zone homogenously cyan means the GFP is
homogenously distributed. Note that D1R-containing MSNs show a relative
homogeneous distribution. Single scan confocal images stained for GFP
(cyan) and DARPP-32 (red), a marker of MSNs in the dorsal caudomedial (GFP,
B1 ; DARPP-32, B2 ; merge, B3 ) and ventral (GFP, B4 ; DARPP-32, B5 ; merge,
B6 ) part of the shell of Drd1a-EGFP mice. Images shown are representative
of all Drd1a-EGFP BAC transgenic mice analyzed (n = 4). Yellow asterisk
identifies the D2R-expressing MSNs-poor zone. Scale bar: 200 µm.
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February 2013 | Volume 7 | Article 22 | 3
Gangarossa et al.
MSNs organization in the NAc
D2R-expressing MSNs were homogeneously distributed in the
NAc core and all the GFP-positive neurons were DARPP-32immunoreactive MSNs with the exception of ChAT interneurons
identified in Drd2-EGFP mice (data not shown; Bertran-Gonzalez
et al., 2008; Matamales et al., 2009). Altogether, these observations revealed that D1R- and D2R-MSNs in the NAc core lack
anatomical segregation, displaying instead a mixed organization
that resembles that of the dorsal striatum.
On coronal sections used in this study, the core is surrounded on its medial, ventral and lateral sides by the shell
(Figures 1A, 2A). As in the NAc core, no apparent organization
was observed in the NAc shell in Drd1a-EGFP mice (Figure 1A).
All D1R-expressing neurons were DARPP-32-positive in the
medial, ventral and lateral shell (Figure 1B and data not shown)
confirming they are all MSNs (Bertran-Gonzalez et al., 2008).
In contrast, a complex and inhomogeneous distribution of
D2R-expressing MSNs was observed in the shell in Drd2-EGFP
mice (Figures 2, A1). The heterogeneous distribution of D2Rexpressing neurons was particularly evident in the medial and
ventral shell (Figure 2A). Thus, in the dorsal caudomedial part of
the shell, several subterritories have been identified (Figure A1):
(1) the “cone” region (Todtenkopf and Stellar, 2000), (2) a
bundle-shaped area also termed corridors (Seifert et al., 1998),
and (3) a D2R-expressing MSNs-poor zone in the upper part of
caudomedial shell (Figures 2A,B, A1). In the ventral shell, D2Rlacking MSNs areas expressing DARPP-32-positive MSNs were
identified (Figures 2A,B). A similar distribution was observed in
Drd2-Cre mice crossed with the RCE:LoxP reporter line (Miyoshi
et al., 2010) (data not shown).
TOPOGRAPHICAL ORGANIZATION OF A2aR-EXPRESSING MSNs IN
THE NAc
Striatopallidal MSNs of the dorsal striatum express D2R and
A2aR (Gerfen et al., 1990; Schiffmann et al., 1991; Fink et al.,
1992; Le Moine and Bloch, 1995). We therefore analyzed the distribution of A2aR-expressing neurons in the NAc using Adora2aCre mice (Durieux et al., 2009) crossed with the Rosa26:loxP
reporter line. In the NAc core, A2aR-expressing MSNs were
homogeneously distributed (Figure 3A). As observed in Drd2EGFP mice, a heterogeneous distribution of A2aR-expressing
neurons was particularly evident in the medial and ventral
shell (Figure 3A). Thus, GFP-positive cells that co-stained with
DARPP-32 were detected in the bundle-shaped area as well as
in the D2R-expressing MSNs-poor zone in the upper part of the
caudomedial shell identified in Drd2-EGFP mice (Figure 3B). In
the ventral shell, as observed in Drd2-EGFP mice, A2aR-lacking
MSNs areas expressing DARPP-32-positive MSNs were identified
(Figures 3A,B).
TOPOGRAPHICAL ORGANIZATION OF D3R-EXPRESSING MSNs IN
THE NAc
Unlike the dorsal part of the striatum, the NAc appears to
be the area where the D3R is expressed at the highest level
(Sokoloff et al., 1990; Bouthenet et al., 1991; Diaz et al., 1995;
Le Moine and Bloch, 1996). We therefore investigated the distribution of D3R-expressing cells in the NAc by assessing the
distribution of GFP-positive neurons in Drd3-Cre mice (http://
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FIGURE 2 | Topographic distribution of GFP immunofluorescence in
the NAc of Drd2-EGFP mice. (A) Distribution of GFP in the NAc of
Drd2-EGFP BAC transgenic mice. Single confocal sections were stitched
together as a single image. Scale bar: 500 µm. DStr, dorsal striatum; aca,
anterior commissure; Tu, olfactory tubercles. (B) Schematic illustration of
the different accumbal subterritories analyzed (see Figure A1). Cyan in
the diagram represents distribution of GFP-expressing cells. A zone
homogenously cyan means the GFP is homogenously distributed. White
means lack of GFP expression throughout the zone and dots indicate
that few GFP cells are detected throughout the zone. Note that
D2R-expressing MSNs exhibit inhomogeneous distributions in the NAc
shell. In the caudomedial NAc shell (1) a bundle-shaped area and (2) a
D2R-expressing MSNs-poor zones (white) in the upper part of the
caudomedial shell (cyan) can be visualized. In the ventral shell, two
zones lacking D2R-expressing MSNs (white) can be delineated. Single
scan confocal images stained for GFP (cyan) and DARPP-32 (red), a
marker of MSNs, in the dorsal caudomedial (GFP, B1 ; DARPP-32, B2 ;
merge, B3 ) and ventral (GFP, B4 ; DARPP-32, B5 ; merge, B6 ) part of the
shell of Drd2-EGFP mice. Images shown are representative of all
Drd2-EGFP BAC transgenic mice analyzed (n = 5). Yellow asterisk
identifies the D2R-expressing MSNs-poor zone. Yellow arrowheads
identify the bundle-shaped area. Scale bar: 200 µm.
www.gensat.org/cre.jsp) crossed with the Rosa26:loxP reporter
line (Srinivas et al., 2001). Only few scattered GFP-positive
cells that co-stained with DARPP-32 were detected in the NAc
core of Drd3-Cre mice (Figure 4A). GFP-immunoreactive cells
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MSNs organization in the NAc
FIGURE 3 | Topographic distribution of GFP immunofluorescence in
the NAc of Adora2a-Cre mice crossed with the Rosa26:loxP
reporter line. (A) Single confocal sections were stitched together as
a single image. Scale bar: 500 µm. DStr, dorsal striatum; aca, anterior
commissure; ICjM, islands of Calleja major; Tu, olfactory tubercles.
(B) Schematic illustration of the accumbal subterritories analyzed (see
Figure A1). Cyan in the diagram represents distribution of
GFP-expressing cells. A zone homogenously cyan means the GFP is
homogenously distributed. White means lack of GFP expression
throughout the zone and dots indicate that few GFP cells are
detected throughout the zone. Single scan confocal images showing
double stained for GFP (cyan) and DARPP-32 (red) in the dorsal
caudomedial (GFP, B1 ; DARPP-32, B2 ; merge, B3 ) and ventral (GFP,
B4 ; DARPP-32, B5 ; merge, B6 ) part of the shell of Adora2a-Cre mice.
Images shown are representative of all Adora2a-Cre BAC transgenic
mice analyzed (n = 3). Yellow asterisk identifies the D2R-expressing
MSNs-poor zone. Yellow arrowheads identify the bundle-shaped area.
Scale bar: 200 µm.
were absent in the lateral part of the shell and the lateral
half of the ventral part (Figures 4A,B). D3R-expressing neurons were heterogeneously distributed in the medial part of the
shell and in the medial half of the ventral part of the NAc
shell (Figure 4A). Thus, the highest density of D3R-expressing
cells was confined to the caudomedial shell (Figures 4A,B)
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FIGURE 4 | Topographic distribution of GFP immunofluorescence in
the NAc of Drd3-Cre mice crossed with the Rosa26:loxP reporter
line. (A) Single confocal sections were stitched together as a single
image. Scale bar: 500 µm. DStr, dorsal striatum; aca, anterior
commissure; ICjM, islands of Calleja major; Tu, olfactory tubercles.
Note that GFP-positive cells are restricted to the caudomedial part of
the NAc shell. Only sparse GFP-expressing cells were detected in
the NAc core. (B) Schematic illustration of the accumbal subterritories
analyzed (see Figure A1). Cyan in the diagram represents the
distribution of GFP-expressing cells. A zone homogenously cyan
means the GFP is homogenously distributed. White means lack of
GFP expression throughout the zone and dots indicate that few GFP
cells are detected throughout the zone. Single scan confocal images
showing double stained for GFP (cyan) and DARPP-32 (red) in the
dorsal caudomedial (GFP, B1 ; DARPP-32, B2 ; merge, B3 ) and ventral
(GFP, B4 ; DARPP-32, B5 ; merge, B6 ) part of the shell of Drd3-Cre
mice. Images shown are representative of all Drd3-Cre BAC
transgenic mice analyzed (n = 2). Yellow asterisk identifies the
D2R-expressing MSNs-poor zone. Yellow arrowheads identify the
bundle-shaped area. Scale bar: 200 µm.
while only few and scattered GFP-immunoreactive neurons were
observed in the bundle-shaped area (Figures 4A,B). Doubleimmunofluorescence revealed that the majority of D3R-positive
cells were MSNs since they co-localized with DARPP-32
(Figure 4B).
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MSNs organization in the NAc
CHARACTERIZATION OF GFP-EXPRESSING NEURONS IN THE
CAUDOMEDIAL PART OF THE NAc SHELL
The bundle-shaped area and the D2R-expressing MSNs-poor
zone of the upper part of the caudomedial shell displayed the
most heterogeneous distribution pattern of MSNs in the mouse
NAc. In an attempt to better characterize the accumbal circuitry
in relationship to D1R-, D2R-, A2aR-, and D3R-expressing MSNs,
we identified DARPP-32-positive MSNs in Drd1a-EGFP, Drd2EGFP, Adora2a-Cre, and Drd3-Cre mice in these two subterritories and calculated the percentage of DARPP-32-immunostained
neurons expressing GFP in each line.
In the bundle-shaped area, 72 ± 2% and 66 ± 3% of the
DARPP-32 immunoreactive neurons were GFP-positive in Drd1aEGFP and Drd2-EGFP mice, respectively (Figure 5A). Although
these numbers were obtained from different mice, the proportion of MSNs expressing D1R, D2R, or both was therefore roughly
estimated from these data by adding the percentage of DARPP-32positive neurons, which were GFP-positive in Drd1a-EGFP and
Drd2-EGFP mice. This estimation is based on the assumption
that every MSN express either D1R, D2R, or both, as previously shown (Matamales et al., 2009). The summed percentages
FIGURE 5 | Expression of D1R-, D2R-, A2aR-, and D3R-containing MSNs
in subterritories of the caudomedial NAc shell. (A) Percentage of
DARPP-32-positive neurons expressing GFP in Drd1a-EGFP, Drd2-EGFP,
and Drd3-Cre mice in bundle-shaped area of the caudomedial NAc shell.
Quantifications were obtained from several images. Total number of MSNs
counted: n = 950 MSNs in Drd1a-EGFP (eight hemispheres from four mice),
n = 898 MSNs in Drd2-EGFP (eight hemispheres from four mice), and
n = 629 MSNs in Drd3-Cre (eight hemispheres from two mice). The table
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obtained in Drd1a-EGFP and Drd2-EGFP mice exceeding 100%
were taken as an indication of co-expression. This estimation
revealed a high degree of D1R/D2R co-localization (38%) (72%
of DARPP-32/D1R + 66% of DARPP-32/D2R = 138%) in this
NAc shell subterritory while 34% (72% of DARPP-32/D1R—38%
of DARPP-32/D2R/D1R = 34%) and 28% (66% of DARPP32/D2R—38% of DARPP-32/D2R/D1R = 28%) of MSNs could
express only D1R and D2R, respectively (Figure 5A). Moreover,
we found that only a small proportion of the MSNs (∼10%)
located in this area expressed D3R (Figure 5A).
We performed the same analysis in the D2R-expressing MSNspoor zone in the upper part of the caudomedial shell (Figure 5B).
In this subregion, 75% of DARPP-32 immunoreactive neurons
were found to be the GFP-positive in Drd1a-EGFP. However,
the almost complete absence of D2R-expressing MSNs (∼10%)
raised the intriguing hypothesis that this subterritory is composed almost exclusively of D1R-containing MSNs. In the dorsal striatum D2R-containing MSNs co-express the adenosine
A2aR (Schiffmann et al., 1991). We therefore analyzed the
expression of GFP in Adora2-Cre mice (Durieux et al., 2009)
crossed with the Rosa26:loxP reporter line (Srinivas et al., 2001).
summarizes the surface area analyzed for each mouse line. (B) Percentage of
DARPP-32-positive neurons expressing GFP in Drd1a-EGFP, Adora2a-Cre, and
Drd3-Cre mice in the D2R-expressing MSNs-poor zone of the caudomedial
NAc shell. Quantifications were obtained from several images. Total number
of MSNs counted: n = 329 MSNs in Drd1a-EGFP (six hemispheres from
three mice), n = 349 MSNs in Adora2-Cre (six hemispheres from three mice),
and n = 365 MSNs in Drd3-Cre (eight hemispheres from two mice). The
table summarizes the surface area analyzed for each mouse line.
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MSNs organization in the NAc
Surprisingly, we found that in the D2R-expressing MSNspoor zone, 93% of DARPP-32-positive neurons were GFPimmunoreactive in Adora2a-Cre mice (Figure 5B). Using the
same approach described above and if we assume that all MSNs
of this subterritory express either D1R or A2aR or both, it can be
estimated that 68% co-express both receptors (75% of DARPP32/D1R + 93% of DARPP-32/A2aR = 168%), 7% (75% of
DARPP-32/D1R—68% of DARPP-32/D1R/A2aR = 7%) of the
MSNs express only D1R and 25% (93% of DARPP-32/A2aR—
68% of DARPP-32/D1R/A2aR = 25%) express only A2aR. In
addition, we also found that 56% of the DARPP-32-positive cells
contained D3R (Figure 5B). However, all these calculations represent lower limits and should be taken with caution since it is
not known whether some MSNs do not express either of these
receptors.
IMMUNOCHEMICAL CHARACTERIZATION OF GFP-EXPRESSING
NEURONS IN THE CAUDOMEDIAL PART OF THE NAc SHELL
The caudomedial part of the NAc shell exhibits inhomogeneous distribution patterns of various markers (Herkenham
et al., 1984). We next assessed whether GFP distribution in
Drd2-EGFP, Adora2a-Cre, and Drd3-Cre mice corresponded
to cytoarchitecturally and cytochemically defined subterritories of the caudomedial part of the shell. Confirming previous studies, we observed that vesicular glutamate transporters
1 (VGluT1) and 2 (VGluT2) showed a complementary distribution in the NAc shell defining two well-separated neuronal circuits (Figures 6, 7) (Hartig et al., 2003). Thus, VGluT1
immunoreactivity was enriched in the bundle-shaped area where
few D3R-expressing MSNs and an estimated high degree of
D1R/D2R co-localization have been observed (Figure 6). In contrast, this subterritory was devoid of VGluT2-positive terminals and calretinin immunoreactivity (Figures 7A,B). On the
other hand, in D2R-expressing MSNs-poor zone located in the
upper part of the caudomedial shell, VGluT2-, and calretininpositive fibers were dense whereas VGluT1 staining was weak
(see the asterisks in Figures 6, 7). Interestingly, the pattern
of TH immunoreactivity revealed a mosaic heterogeneity that
resembled that of the VGluT2/calretinin distribution (Figure 8A).
Therefore, the bundle-shaped area was identified as TH/DATpoor area while the D2R-poor zone was stained by a dense
plexus of TH/DAT fibers arising from the VTA (Figures 8B,C).
It should be noted here that the remaining TH staining visible in the NAc shell following 6-OHDA-induced VTA lesion
corresponded most likely to noradrenaline fibers arising from
the locus coeruleus because they were devoid of DAT labeling
(Figure 8C).
FIGURE 6 | Distribution pattern of vesicular transporter 1 (VGluT1) in
the caudomedial NAc shell. Distribution of VGluT1 (red) and GFP (cyan)
immunofluorescence in the NAc of Drd2-EGFP, Drd3-Cre/Rosa26:loxP
and Adora2a-Cre/Rosa26:loxP double transgenic mice. Images are single
confocal sections. Yellow asterisk indicates the D2R-expressing MSNs-poor
zone. Yellow arrowheads indicate the bundle-shaped area. Scale bar:
250 µm. v, ventricles.
(quinpirole, 1 mg/kg), a non-selective dopamine receptor agonist
(apomorphine, 3 mg/kg), and a D2R-like antagonist (raclopride,
0.3 mg/kg) in the core and in the various subterritories previously identified in the ventral and caudomedial part of the NAc
shell.
NAc core
TOPOGRAPHICAL AND CELL-TYPE REGULATION OF ERK
PHOSPHORYLATION IN THE NAc
Activated by a variety of therapeutic agents or drugs of abuse
in physiological and pathological contexts, the ERK pathway has
been proposed to play a critical role in the molecular mechanisms involved in dopamine-controlled striatal plasticity (Girault
et al., 2007). Using Drd2-EGFP mice, we next analyzed the
pattern of ERK phosphorylation following the administration
of a D1R-like agonist (SKF81297, 5 mg/kg), a D2R-like agonist
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As previously reported (Bertran-Gonzalez et al., 2008), vehicletreated mice showed sparse P-ERK positive neurons that were
only D2R-negative (Figures 9, 10A). Double fluorescence analysis
of mice perfused 15 min after SKF81297 administration revealed
that ERK phosphorylation occurred exclusively in D2R-negative
neurons (Figures 9, 10A). In contrast, mice injected with quinpirole showed an inhibition of the basal ERK phosphorylation
(Figures 9, 10A). Although apomorphine treatment did not significantly increase the total number of P-ERK-positive neurons,
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FIGURE 7 | Distribution patterns of vesicular transporter 2 (VGluT2)
and Calretinin in the caudomedial NAc shell. Distribution of VGluT2
(red) (A) or calretinin (red) (B) and GFP (cyan) fluorescence in the
NAc of Drd2-EGFP, Drd3-Cre/Rosa26:loxP and Adora2a-Cre/Rosa26:loxP
it induced areas of intense ERK phosphorylation in the neuropil (Figures 9, 10A). Finally, raclopride-treated mice showed a
robust increase of ERK phosphorylation that occurred essentially
in D2R-negative MSNs and to a smaller extent in D2R-expressing
MSNs (Figures 9, 10A).
NAc ventral and caudomedial shell
Only few scattered P-ERK positive cells were detected in the
ventral shell of vehicle-treated mice (Figures 10B,C, 11A). An
inhomogeneous distribution was observed in the caudomedial
part: basal ERK phosphorylation was observed in both D2Rpositive and -negative MSNs in the bundle-shaped area enriched
in D1R/D2R-co-expressing cells (Figures 10D–F, 12A), whereas
P-ERK immunoreactivity was detected mostly in D1R-expressing
neurons of the D2R-MSNs-poor zone and in the rest of the
caudomedial shell. SKF81297 administration markedly increased
the number of P-ERK-positive neurons in all subterritories of
the ventral and the caudomedial shell with the exception of
the D2R-expressing MSNs-poor zone (Figures 10B–F, 11A,B,
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double transgenic mice. Images are single confocal sections. Yellow
asterisk indicates the D2R-expressing MSNs-poor zone. Yellow
arrowheads indicate the bundle-shaped area. Scale bar: 250 µm.
v, ventricles.
and 12A). P-ERK immunoreactivity was detected only in D2Rnegative cells, except in the bundle-shaped area where it was
found in both cell types (Figure 10). Although less pronounced,
a similar pattern of ERK activation was observed with apomorphine (Figures 10B–F, 11). The main difference concerned the
cell-type specificity of ERK activation, which was restricted to
D2R-negative cells in the bundle-shaped area (Figure 10E). As
previously observed in the dorsal striatum (Gangarossa et al.,
2012) and in the NAc core, quinpirole failed to induce ERK phosphorylation in any subterritory of the caudomedial and ventral
shell analyzed (Figures 10B–F, 11). Finally, mice treated with
raclopride also displayed a specific pattern of ERK activation:
raclopride increased ERK phosphorylation in both D2R-negative
and positive cells in the bundle-shaped area of the caudomedial shell whereas no change was found in the D2R-expressing
MSNs-poor zones (Figures 10B,E, 11). P-ERK immunoreactivity
was also slightly increased in the “cone” and ventral shell where
a significant effect was observed in D2R-containing neurons
(Figures10B,F, 12B).
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FIGURE 8 | Distribution patterns of tyrosine hydroxylase (TH) and
dopamine transporter (DAT) in the caudomedial NAc shell. (A)
Distribution of TH (red) and GFP (cyan) fluorescence in the NAc of Drd2-EGFP
and Drd3-Cre/Rosa26:loxP double transgenic mice. Yellow asterisk identifies
the D2R-expressing MSNs-poor zone. Images are single confocal sections.
Yellow arrowheads identify the bundle-shaped area. Scale bar: 250 µm.
(B) Triple immunostaining for GFP (green), TH (red), and DAT (blue) allowed
SPECIFIC TOPOGRAPHICAL AND CELL-TYPE REGULATION OF
PSYCHOSTIMULANT-INDUCED ERK PHOSPHORYLATION IN THE NAc
Acute cocaine treatment increases ERK phosphorylation in
D1R-containing MSNs in the dorsal striatum and the NAc
(Bertran-Gonzalez et al., 2008). Because of the inhomogeneous
distribution of D1R- and D2R-expressing output neurons in the
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the identification of DA neurons on the unlesioned side of Drd2-EGFP mice.
Note the lack of staining in the VTA on the lesioned side. Images are single
confocal sections. Scale bar: 500 µm. (C) The same triple immunostaining
performed at the caudomedial NAc shell level revealed the absence of DAT
and a strong reduction of immunoreactive terminals in the D2R-expressing
MSNs-poor zone (yellow asterisk). Images are single confocal sections.
Scale bar: 250 µm.
NAc, we examined the patterns of P-ERK-positive neurons taking into account the accumbal subterritories. A single injection
of cocaine (15 mg/kg) or d-amphetamine (10 mg/kg) increased
the number of P-ERK-positive neurons in D1R-containing
MSNs in the NAc core (Figure 13A). In contrast, a more
complex pattern of cocaine-induced ERK phosphorylation was
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MSNs organization in the NAc
MSNs-poor zone while d-amphetamine induced a weak increase
(Figures 13E,G).
DISCUSSION
Recent advances in technologies for the identification of specific cell types, including BAC transgenic mice expressing
fluorescent reporter or the Cre recombinase, allow a more
comprehensive understanding of the involvement of D1R- and
D2R-expressing MSNs in various physiological and pathological conditions. Although, potential caveats or difficulties in using
such approaches have to be taken into account (i.e., incomplete
and/or ectopic expression depending on the insertion site of
the transgenes) (Gong and Yang, 2005), a careful identification
and characterization of the mouse line that produces expression patterns matching that of the endogenous gene should avoid
overstated conclusions. By using various BAC transgenic mice
(Drd1a-EGFP, Drd2-EGFP, Drd3-Cre, and Adora2-Cre) combined with several immunohistochemical markers, we reveal a
high level of heterogeneity of the NAc shell cellular organization. In the NAc core as in the adjacent dorsal striatum, D1Rand D2R-expressing MSNs appear to be randomly distributed.
In contrast, in the ventral and the caudomedial part of the NAc
shell, D1R- and D2R-expressing MSNs exhibit an inhomogeneous distribution. Identified patterns are closely associated with
specific accumbal subterritories previously delineated by specific neurochemical markers. Figure A1 summarizes the main
differences between the subterritories. Moreover, our results support the hypothesis that the heterogeneous composition of the
NAc can be functionally important as illustrated by the distinct patterns of ERK activation triggered by pharmacological
treatments.
THE BUNDLE-SHAPED AREA
FIGURE 9 | Regulation of ERK phosphorylation in the NAc core. Double
immunofluorescence for P-ERK (magenta) and GFP (green) in the NAc Core
of Drd2-EGFP mice treated with vehicle, SKF81297 (5 mg/kg), quinpirole
(1 mg/kg), apomorphine (3 mg/kg), and raclopride (0.3 mg/kg). Scale bar:
200 µm. High magnification of the area delineated by the white dashed
square. Yellow arrowheads identify D2R-expressing MSNs that contain
P-ERK. Images are single confocal sections. Scale bar: 50 µm. Note that
apomorphine-induced ERK phosphorylation in clusters that resemble the
striosomal compartment.
observed within the NAc shell subterritories (Figures 13B–G).
In the ventral part of the shell, cocaine increased ERK phosphorylation only in the zones lacking D2R-expressing MSNs
while d-amphetamine also increased it in the surrounding
area (Figures 13B,C,G). The analyses performed in the caudomedial part of the shell revealed that cocaine and damphetamine triggered almost similar patterns of ERK activation (Figures 13D–G). Thus, increased ERK phosphorylation
restricted to D2R-negative MSNs was observed in the bundleshaped area and the “cone” following administration of cocaine or
d-amphetamine (Figures 13D,F,G). On the other hand, cocaine
at this dose failed to activate ERK in the D2R-expressing
Frontiers in Neural Circuits
Generally seen as an integral part of the striatal complex, the
identification of several anatomical features has led to propose
that the NAc was an independent functional entity. Beyond the
well-known NAc core and shell compartmentalization, selective
markers and tract–tracing studies performed in rats allowed the
identification of multiple accumbal shell subterritories (Zahm
and Brog, 1992). Among them, cell clusters (Herkenham et al.,
1984) or “corridors” (Seifert et al., 1998) have been identified in the caudomedial shell in the border region between the
core and shell. Characterized by an enrichment of opioid receptors and low staining for acetylcholinesterase, substance P, and
enkephalin (Herkenham et al., 1984; Voorn et al., 1989), the
bundle-shaped area is also avoided by terminals originating from
the ventral subiculum (Groenewegen et al., 1987), the infralimbic
cortex (Berendse et al., 1992) and the paraventricular thalamic
nucleus that are identified by VGluT2/calretinin immunoreactivity (Hartig et al., 2003). In addition, dopaminergic projections
from the VTA also poorly innervate the bundle-shaped area as
demonstrated by the weak density of dopamine, TH and DAT
immunoreactive terminals (Voorn et al., 1989; Jansson et al.,
1999, and present study). The paucity of these extrinsic afferent projections within the bundle-shaped area largely contributed
to put forward the hypothesis that this accumbal subterritory
would constitute a way-station favoring intrinsic information
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FIGURE 10 | Topographical and cell-type specific regulation of P-ERK
by SKF81297, quinpirole, apomorphine, and raclopride in the NAc.
Quantification of P-ERK-positive cells among GFP-negative (white bars) and
GFP-positive (green bars) neurons in the NAc core (A), in the ventral NAc
shell [(B) D2R-expressing MSNs-poor zone and (C) area surrounding the
D2R-expressing MSNs-poor zone] and in the caudomedial NAc shell [(D)
D2R-expressing MSNs-poor zone; (E) bundle-shaped area; and (F) cone] in
processing (Herkenham et al., 1984). However, the demonstration
of a dense plexus of VGluT1 immunopositive fibers arising from
the prelimbic cortex and the caudal parvicellular basal amygdaloid nucleus (Berendse et al., 1992; Wright and Groenewegen,
1996; Wright et al., 1996; Hartig et al., 2003) strongly supports the idea that MSNs located in the bundle-shaped area
could also integrate and process specific cortical and subcortical
information.
Our study clearly points out that the bundle-shaped area also
displays several specific features regarding the distribution pattern of D1R- and D2R-expressing MSNs. Thus, this area can
be identified by an enrichment of GFP immunofluorescence in
Drd2-EGFP mice and a low number of D3R-containing MSNs
(10% of all DARPP-32 immunoreactive cells). Interestingly, our
estimated percent of D1R/D2R co-expression of 38% was roughly
2-fold higher than our previous evaluation in the whole NAc
shell (Bertran-Gonzalez et al., 2008; Matamales et al., 2009).
Although informative, these estimations should be taken with
caution since numbers were obtained from different mice and
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Drd2-EGFP mice, 15 min after vehicle (Veh), SKF81297 (SKF), quinpirole (Qui),
apomorphine (Apo), and raclopride (Rac) administration. Green squares in
diagrams indicate the region analyzed. Data are number of cells per area (see
Table A1) and are expressed as means ± SEM (3–5) and were analyzed using
one-way ANOVA (see Table A3 for F values). Dunnett’s test: ∗ p < 0.05,
∗∗ p < 0.01, ∗∗∗ p < 0.001 veh vs. drugs in Drd2-EGFP negative, ◦ p < 0.05,
◦◦◦ p < 0.001 veh vs. drugs in Drd2-EGFP positive.
calculations were based on the assumption that every MSN
express either D1R or D2R or both in the case of the bundleshaped area and either D1R or A2aR or both in the case of
the D2R-expressing MSNs-poor zone in the caudomedial shell.
Because recent studies suggest that MSNs co-expressing both
receptors display unique signaling properties (Perreault et al.,
2010, 2011), the bundle-shaped area would therefore represent an ideal anatomical substrate where D1R-D2R heteromersdependent signaling could preferentially take place. In light of
these observations, it is interesting to note that, because of the
low TH and DAT expression, previous studies proposed that
dopaminergic transmission in the bundle-shaped area results
from a non-synaptic, volume transmission type of DA communication (Garris et al., 1994; Jansson et al., 1999). Further studies
will be therefore necessary to determine whether DA-dependent
signaling in the bundle-shaped area results in a prolonged action
of DA as a consequence of a slow diffusion into the extracellular space following DA release from the rich surrounding DA
networks.
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FIGURE 11 | Regulation of ERK phosphorylation in the ventral NAc
shell. (A) Double immunofluorescence for P-ERK (magenta) and GFP
(green) in the NAc ventral shell of Drd2-EGFP mice treated with vehicle,
SKF81297 (5 mg/kg), apomorphine (3 mg/kg), and raclopride (0.3 mg/kg).
Scale bar: 100 µm. High magnification of the area delineated by the white
dashed square. Yellow arrowheads indicate D2R-expressing MSNs that
contain P-ERK. Images are single confocal sections. Scale bar: 50 µm.
(B) P-ERK immunoreactivity (red) was detected together with DARPP-32
(blue) and GFP (green) immunoreactivities in the D2R-expressing
MSNs-poor zone located in the ventral shell of Drd2-EGFP mice treated
with SKF81297 (5 mg/kg) in a triple fluorescence analysis. Images are single
confocal sections. Note that all P-ERK-positive cells co-localize with
DARPP-32. Scale bar: 50 µm.
D2R-EXPRESSING MSNs-POOR ZONES
Another level of compartmentalization of the NAc shell results
from the existence of D2R-expressing MSNs-poor zones. Such
areas located in the ventral shell have been identified. These
zones contain neither A2aR nor D3R as demonstrated by the
absence of GFP in the Adora2-Cre and Drd3-Cre mice, respectively. Therefore, the high number of DARPP-32-immunoreactive
cells suggests that these D2R-expressing MSNs-poor zones are
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FIGURE 12 | Regulation of ERK phosphorylation in the caudomedial
NAc shell. (A) Triple immunofluorescence for P-ERK (red), GFP (green), and
DARPP-32 (blue) in the caudomedial NAc shell of Drd2-EGFP mice treated
with vehicle and SKF81297 (5 mg/kg) (A) and raclopride (B). Images are
single confocal sections. Scale bar: 200 µm. High magnification of the
area delineated by the white squares. Scale bar: 100 µm. Yellow asterisk
indicates the D2R-expressing MSNs-poor zone. Yellow arrowheads indicate
P-ERK/GFP/DARPP-32 positive neurons.
composed almost exclusively of D1R-contaning MSNs. It must
be noted that the other histochemical markers used in previous
or present studies did not allow the identification of this specific
shell subterritory. Whether these clusters exhibit other particular
features remains to be determined.
The second D2R-expressing MSNs-poor zone is located in the
upper part of the caudomedial part of the shell. Identified as
VGluT2-, calretinin-, TH/DAT-rich zone, this small area receives
massive inputs from the paraventricular thalamic nucleus, the
infralimbic cortex, and the VTA (Herkenham et al., 1984;
Berendse et al., 1992; Groenewegen et al., 1999; Jansson et al.,
1999; Hartig et al., 2003). Surprisingly, while D1R-expressing
neurons represented 75% of DARPP-32-positive neurons, 93% of
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FIGURE 13 | Continued
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FIGURE 13 | Topographical and cell-type specific regulation of P-ERK
by cocaine and d-amphetamine in the NAc. (A–F) Quantification of
P-ERK positive cells among GFP-negative (white bars) and GFP-positive
(green bars) neurons in the NAc core (A), in the ventral NAc shell
[(B) D2R-expressing MSNs-poor zone and (C) area surrounding the
D2R-expressing MSNs-poor zone] and in the caudomedial NAc shell [(D)
bundle-shaped area; (E) D2R-expressing MSNs-poor zone; and (F) cone]
in Drd2-EGFP mice, 15 min after vehicle (Veh), cocaine (Coc), and
d-amphetamine (d-amph) administration. Note that cocaine- and
d-amphetamine-induced ERK activation is restricted to D1R-expressing
the MSNs in this area were GFP-positive in Adora2a-Cre mice suggesting (1) the existence of MSNs co-expressing D1R and A2aR
and (2) the lack of co-localization between A2aR and D2R, which
is normally observed in the dorsal striatum and others accumbal
regions. These observations have important functional implications since the reciprocal antagonistic interactions between
A2aR and D2R should not occur. This contrasts with the dorsal striatum, in which the co-expressed D2R and A2aR interact
either directly, to form heteromers, or indirectly, at the level of
adenylyl cyclase, to trigger the activation of specific signaling
cascades (Ferre et al., 2011). Interestingly, half of the DARPP-32containing neurons of this subterritory also express D3R suggesting that MSNs located in this area display a high degree of D1R,
A2aR, and D3R co-expression. Whether this accumbal subterritory constitutes a “hot spot” where these receptors could interact
and form functional D1R-D3R and A2aR-D3R heteromeric complexes will require further investigations (Torvinen et al., 2005;
Fiorentini et al., 2008; Marcellino et al., 2008).
FUNCTIONAL ASPECTS OF MSNs DISTRIBUTION IN THE NAc: IMPACT
ON ERK ACTIVATION
The pharmacological, physiological and pathological regulation
of the ERK pathway in striatal and accumbal MSNs has been
extensively studied (Girault et al., 2007; Santini et al., 2008;
Gangarossa et al., 2012). Our present findings demonstrate the
existence of a topographical and cell-type specific regulation of
the ERK cascade signaling in the NAc in response to SKF81297,
quinpirole, and apomorphine. As in the dorsal striatum, stimulation of D2R by quinpirole administration inhibited basal ERK
phosphorylation in D1R-containing MSNs most likely as a result
of DA release inhibition through the activation of D2 autoreceptors (Mercuri et al., 1997; Centonze et al., 2002; Gangarossa et al.,
2012).
Following selective D1R stimulation, the most striking difference concerned the cell-type selectivity. Our data indicate that
following SKF81297 administration ERK activation occurred in
both D2R-positive and negative neurons in the bundle-shaped
area. The most parsimonious explanation is that D2R-expressing
MSNs in which ERK activation occurred could also contain
D1R, a hypothesis supported by the high degree of D1R/D2R
co-localization (38%) in this zone and indirectly by our results
obtained with apomorphine. In that case, ERK phosphorylation occurred exclusively in D2R-negative MSNs suggesting that
when both D1R and D2R are stimulated in the MSNs coexpressing them, ERK activation does not occur. In line with
this hypothesis, a recent study showed that the co-activation of
both receptors within the dopamine D1R-D2R heteromers by the
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MSNs. Yellow asterisk indicates the D2R-expressing MSNs-poor zone.
Green squares in diagrams indicate the region analyzed. Data are
number of cells per area (see Table A2) and are expressed as means
± SEM (4–5) and were analyzed using one-way ANOVA (see Table A3
for F values). Dunnett’s test: ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001 veh vs.
drugs in Drd2-EGFP negative. (G) Double immunofluorescence for P-ERK
(magenta) and GFP (green) in various subterritories of the NAc
shell in Drd2-EGFP mice treated with vehicle, cocaine (15 mg/kg) and
d-amphetamine (10 mg/kg). Images are single confocal sections.
Scale bar: 100 µm.
selective D1R-D2R heteromer agonist SKF83959 failed to increase
ERK phosphorylation in the NAc (Perreault et al., 2012).
In the dorsal striatum, the blockade of D2R by haloperidol or raclopride activates ERK selectively in D2R-expressing
striatopallidal MSNs (Bertran-Gonzalez et al., 2008, 2009). Our
study reveals different principles of regulation in the NAc. Thus,
raclopride-induced ERK phosphorylation was observed exclusively in D1R-expressing MSNs in the NAc core and in both
D2R and D1R-MSNs in the NAc shell. In the dorsal striatum,
haloperidol-induced ERK activation in D2R-expressing MSNs
involved A2aR (Bertran-Gonzalez et al., 2009). Given that raclopride produces a marked increase in extracellular adenosine in
the NAc (Nagel and Hauber, 2004), it is tempting to speculate
that similarly to the dorsal striatum, A2aR contributes to ERK
activation in D2R-containing MSNs of the NAc shell following
raclopride administration. On the other hand, the increase of
ERK phosphorylation in D1R-expressing MSNs could result from
the ability of raclopride to enhance DA release in the NAc core and
shell (Aragona et al., 2008).
As previously reported cocaine- and d-amphetamine-induced
ERK phosphorylation in the NAc was always restricted to D1Rexpressing MSNs (Bertran-Gonzalez et al., 2008; Gerfen et al.,
2008). Our study highlights an additional level of complexity
since we show here that psychostimulants trigger specific patterns of ERK activation, which vary in the accumbal subterritories
analyzed. In the ventral shell, contrasting with d-amphetamine,
cocaine administration induced a small increase in the number
of ERK-positive cells, which was restricted to the D2R-expressing
MSNs-poor zone. A subterritory-specific ERK phosphorylation
was also observed in the caudomedial NAc shell. Thus, ERK
activation is restricted to the bundle-shaped area and surrounding zones but absent from the D2R-expressing MSNs-poor zone
located in the upper part of the caudomedial shell. Several mechanisms could explain why psychostimulant drugs trigger compartmentalized patterns of ERK phosphorylation. First, the segregated
activation of ERK could be directly linked to the various combinatorial expressions of D1R, D2R, A2aR, and D3R within the
different accumbal subterritories. Thus, MSNs located in the
D2R-expressing MSNs-poor zone, which display a high degree
of D1R, A2aR, and D3R expression would have distinct signaling
properties than MSNs co-expressing only D1R and D2R. Second,
the inhomogeneous release of DA in the NAc shell following psychostimulants administration could be also an important factor
that would drive this specific pattern of ERK phosphorylation
(Aragona et al., 2008). Interestingly, recent studies revealed that in
the NAc shell, different populations of DA neurons might release
glutamate eliciting therefore excitatory postsynaptic responses
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in MSNs innervated by these DA neurons (Stuber et al., 2010;
Tecuapetla et al., 2010). Whether those particular DA neurons
also participate in cocaine-induced ERK regulation will require
further investigations. Finally, given that the glutamatergic transmission largely contributes to psychostimulant-evoked ERK activation in the NAc (Valjent et al., 2000, 2005; Pascoli et al.,
2011), it is tempting to speculate that specific inputs arising from
distinct cortical, subcortical, and thalamic areas play also a critical role in the establishment of the compartmentalized ERK
phosphorylation induced by cocaine and d-amphetamine.
In conclusion, we demonstrate that the inhomogeneous distribution of D2R-expressing MSNs allows defining subterritories in
the NAc shell, which exhibit particular neurochemical and inputsspecific features. Combined with our in vivo functional signaling
analysis, our study highlights the importance to precisely determine the neuronal populations in which signaling pathways are
activated in order to better understand how they are regulated and
what their corresponding functions are.
ACKNOWLEDGMENTS
REFERENCES
adenosine receptor: selective coexpression with D2 dopamine
receptors in rat striatum. Brain Res.
Mol. Brain Res. 14, 186–195.
Fiorentini, C., Busi, C., Gorruso,
E., Gotti, C., Spano, P., and
Missale, C. (2008). Reciprocal
regulation of dopamine D1
and D3 receptor function and
trafficking
by
heterodimerization. Mol. Pharmacol. 74,
59–69.
Franklin, K., and Paxinos, G. (2007).
The Mouse Brain in Stereotaxic
Coordinates, 3rd Edn. Amsterdam:
Elsevier.
Gangarossa, G., Perroy, J., and Valjent,
E. (2012). Combinatorial topography and cell-type specific regulation of the ERK pathway by
dopaminergic agonists in the mouse
striatum. Brain Struct. Funct. doi:
10.1007/s00429-012-0405-6. [Epub
ahead of print].
Garris, P. A., Ciolkowski, E. L., Pastore,
P., and Wightman, R. M. (1994).
Efflux of dopamine from the synaptic cleft in the nucleus accumbens
of the rat brain. J. Neurosci. 14,
6084–6093.
Gerfen, C. R. (1992). The neostriatal
mosaic: multiple levels of compartmental organization. Trends
Neurosci. 15, 133–139.
Gerfen, C. R., Engber, T. M., Mahan, L.
C., Susel, Z., Chase, T. N., Monsma,
F. J. Jr., et al. (1990). D1 and D2
dopamine receptor-regulated gene
expression of striatonigral and striatopallidal neurons. Science 250,
1429–1432.
Gerfen, C. R., Paletzki, R., and
Worley, P. (2008). Differences
between dorsal and ventral striatum in Drd1a dopamine receptor
Aragona, B. J., Cleaveland, N. A.,
Stuber, G. D., Day, J. J., Carelli,
R. M., and Wightman, R. M.
(2008). Preferential enhancement of
dopamine transmission within the
nucleus accumbens shell by cocaine
is attributable to a direct increase
in phasic dopamine release events.
J. Neurosci. 28, 8821–8831.
Berendse, H. W., Galis-De Graaf, Y.,
and Groenewegen, H. J. (1992).
Topographical organization and
relationship with ventral striatal
compartments of prefrontal corticostriatal projections in the rat.
J. Comp. Neurol. 316, 314–347.
Bertran-Gonzalez, J., Bosch, C.,
Maroteaux, M., Matamales, M.,
Herve, D., Valjent, E., et al. (2008).
Opposing patterns of signaling
activation in dopamine D1 and
D2 receptor-expressing striatal
neurons in response to cocaine
and haloperidol. J. Neurosci. 28,
5671–5685.
Bertran-Gonzalez, J., Hakansson, K.,
Borgkvist, A., Irinopoulou, T.,
Brami-Cherrier, K., Usiello, A., et al.
(2009). Histone H3 phosphorylation is under the opposite tonic
control of dopamine D2 and adenosine A2A receptors in striatopallidal
neurons. Neuropsychopharmacology
34, 1710–1720.
Bertran-Gonzalez, J., Hervé, D.,
Girault, J. A., and Valjent, E.
(2010). What is the degree of
Segregation between Striatonigral
and Striatopallidal Projections?
Front. Neuroanat. 4:136. doi:
10.3389/fnana.2010.00136
Bouthenet, M. L., Souil, E., Martres,
M. P., Sokoloff, P., Giros, B., and
Schwartz, J. C. (1991). Localization
Frontiers in Neural Circuits
of dopamine D3 receptor mRNA
in the rat brain using in situ
hybridization histochemistry: comparison with dopamine D2 receptor
mRNA. Brain Res. 564, 203–219.
Centonze, D., Usiello, A., Gubellini,
P., Pisani, A., Borrelli, E., Bernardi,
G., et al. (2002). Dopamine
D2
receptor-mediated
inhibition of dopaminergic neurons
in mice lacking D2L receptors.
Neuropsychopharmacology
27,
723–726.
Diaz, J., Levesque, D., Lammers,
C. H., Griffon, N., Martres, M.
P., Schwartz, J. C., et al. (1995).
Phenotypical characterization of
neurons expressing the dopamine
D3 receptor in the rat brain.
Neuroscience 65, 731–745.
Durieux, P. F., Bearzatto, B., Guiducci,
S., Buch, T., Waisman, A., Zoli,
M., et al. (2009). D2R striatopallidal neurons inhibit both locomotor and drug reward processes. Nat.
Neurosci. 12, 393–395.
Durieux, P. F., Schiffmann, S. N.,
and de Kerchove D’Exaerde, A.
(2011). Differential regulation of
motor control and response to
dopaminergic drugs by D1R and
D2R neurons in distinct dorsal
striatum subregions. EMBO J. 31,
640–653.
Ferre, S., Quiroz, C., Orru, M., Guitart,
X., Navarro, G., Cortes, A., et al.
(2011). Adenosine A(2A) receptors and A(2A) receptor heteromers
as key players in striatal function. Front. Neuroanat. 5:36. doi:
10.3389/fnana.2011.00036
Fink, J. S., Weaver, D. R., Rivkees,
S. A., Peterfreund, R. A., Pollack,
A. E., Adler, E. M., et al. (1992).
Molecular cloning of the rat A2
This work was supported by Inserm and grants from ATIPAvenir (Inserm), Sanofi-Aventis R&D, and from the Agence
Nationale de la Recherche (ANR-2010-JCJC-1412) to Emmanuel
Valjent. Research in Jean-Antoine Girault and Denis Hervé lab
was supported by grants from the Fondation pour la recherche
médicale (FRM), the Agence nationale de la recherche (ANRBLAN08-1_346422), European Union Framework program 7
(FP7, SynSys), and the European research council (ERC). AdKdE
is a Research Associate of the FRS-FNRS (Belgium) and is supported by FRS-FNRS (Belgium), FER from ULB, Action de
Recherche Concertée from the CFWB. Julie Espallergues was a
recipient of a postdoctoral fellowship from UM1. We are grateful to Laurent Fagni and Julie Perroy (Institut de Génomique
Fonctionnelle) for providing some transgenic mice used in this
study. We thank Fredéric Gallardo (IGF) and Natacha Roblot,
Rachida Boukhari, and Yohann Bertelle (IFM) for animal care,
breeding and genotyping.
www.frontiersin.org
coupling of dopamine- and cAMPregulated phosphoprotein-32 to
activation of extracellular signalregulated kinase. J. Neurosci. 28,
7113–7120.
Girault, J. A., Valjent, E., Caboche,
J., and Herve, D. (2007). ERK2:
a logical AND gate critical for
drug-induced plasticity? Curr. Opin.
Pharmacol. 7, 77–85.
Gong, S., and Yang, X. W. (2005).
Modification of bacterial artificial
chromosomes (BACs) and preparation of intact BAC DNA for generation of transgenic mice. Curr. Protoc.
Neurosci. Chapter 5:Unit 5.21. doi:
10.1002/0471142301.ns0521s31
Gong, S., Zheng, C., Doughty, M. L.,
Losos, K., Didkovsky, N., Schambra,
U. B., et al. (2003). A gene expression atlas of the central nervous
system based on bacterial artificial chromosomes. Nature 425,
917–925.
Groenewegen, H. J., Vermeulen-Van
Der Zee, E., Te Kortschot, A., and
Witter, M. P. (1987). Organization
of the projections from the subiculum to the ventral striatum in
the rat. A study using anterograde transport of Phaseolus vulgaris leucoagglutinin. Neuroscience
23, 103–120.
Groenewegen, H. J., Wright, C. I.,
Beijer, A. V., and Voorn, P. (1999).
Convergence and segregation of
ventral striatal inputs and outputs. Ann. N.Y. Acad. Sci. 877,
49–63.
Hartig, W., Riedel, A., Grosche, J.,
Edwards, R. H., Fremeau, R. T.
Jr., Harkany, T., et al. (2003).
Complementary distribution of
vesicular glutamate transporters 1
and 2 in the nucleus accumbens
February 2013 | Volume 7 | Article 22 | 15
Gangarossa et al.
of rat: relationship to calretinincontaining extrinsic innervation
and
calbindin-immunoreactive
neurons. J. Comp. Neurol. 465,
1–10.
Heimer, L., Zahm, D. S., Churchill,
L., Kalivas, P. W., and Wohltmann,
C. (1991). Specificity in the projection patterns of accumbal core
and shell in the rat. Neuroscience 41,
89–125.
Herkenham, M., Edley, S. M., and
Stuart, J. (1984). Cell clusters in the
nucleus accumbens of the rat, and
the mosaic relationship of opiate
receptors,
acetylcholinesterase
and subcortical afferent terminations.
Neuroscience
11,
561–593.
Humphries, M. D., and Prescott, T.
J. (2010). The ventral basal ganglia, a selection mechanism at
the crossroads of space, strategy,
and reward. Prog. Neurobiol. 90,
385–417.
Jansson, A., Goldstein, M., Tinner, B.,
Zoli, M., Meador-Woodruff, J. H.,
Lew, J. Y., et al. (1999). On the
distribution patterns of D1, D2,
tyrosine hydroxylase and dopamine
transporter immunoreactivities in
the ventral striatum of the rat.
Neuroscience 89, 473–489.
Jensen, J., McIntosh, A. R., Crawley, A.
P., Mikulis, D. J., Remington, G., and
Kapur, S. (2003). Direct activation
of the ventral striatum in anticipation of aversive stimuli. Neuron 40,
1251–1257.
Jongen-Relo, A. L., Groenewegen, H. J.,
and Voorn, P. (1993). Evidence for
a multi-compartmental histochemical organization of the nucleus
accumbens in the rat. J. Comp.
Neurol. 337, 267–276.
Jongen-Relo, A. L., Voorn, P., and
Groenewegen, H. J. (1994).
Immunohistochemical
characterization of the shell and core
territories of the nucleus accumbens in the rat. Eur. J. Neurosci. 6,
1255–1264.
Kalivas, P. W., and Duffy, P. (1995).
Selective activation of dopamine
transmission in the shell of the
nucleus accumbens by stress. Brain
Res. 675, 325–328.
Le Moine, C., and Bloch, B. (1995).
D1 and D2 dopamine receptor gene
expression in the rat striatum: sensitive cRNA probes demonstrate
prominent segregation of D1 and
D2 mRNAs in distinct neuronal
populations of the dorsal and ventral striatum. J. Comp. Neurol. 355,
418–426.
Le Moine, C., and Bloch, B. (1996).
Expression of the D3 dopamine
receptor in peptidergic neurons of
Frontiers in Neural Circuits
MSNs organization in the NAc
the nucleus accumbens: comparison with the D1 and D2 dopamine
receptors.
Neuroscience
73,
131–143.
Lu, X. Y., Ghasemzadeh, M. B., and
Kalivas, P. W. (1998). Expression of
D1 receptor, D2 receptor, substance
P and enkephalin messenger RNAs
in the neurons projecting from the
nucleus accaumbens. Neuroscience
25, 767–780.
Marcellino, D., Ferre, S., Casado, V.,
Cortes, A., Le Foll, B., Mazzola,
C., et al. (2008). Identification
of dopamine D1-D3 receptor heteromers. Indications for a role of
synergistic D1-D3 receptor interactions in the striatum. J. Biol. Chem.
283, 26016–26025.
Matamales, M., Bertran-Gonzalez, J.,
Salomon, L., Degos, B., Deniau,
J. M., Valjent, E., et al. (2009).
Striatal medium-sized spiny neurons: identification by nuclear
staining and study of neuronal
subpopulations in BAC transgenic
mice. PLoS ONE 4:e4770. doi:
10.1371/journal.pone.0004770
Mercuri, N. B., Saiardi, A., Bonci, A.,
Picetti, R., Calabresi, P., Bernardi,
G., et al. (1997). Loss of autoreceptor function in dopaminergic
neurons from dopamine D2 receptor deficient mice. Neuroscience 79,
323–327.
Meredith, G. E., Pennartz, C. M., and
Groenewegen, H. J. (1993). The cellular framework for chemical signalling in the nucleus accumbens.
Prog. Brain Res. 99, 3–24.
Miyoshi, G., Hjerling-Leffler, J.,
Karayannis, T., Sousa, V. H., Butt,
S. J., Battiste, J., et al. (2010).
Genetic fate mapping reveals that
the caudal ganglionic eminence
produces a large and diverse
population of superficial cortical interneurons. J. Neurosci. 30,
1582–1594.
Nagel, J., and Hauber, W. (2004).
Reverse microdialysis of a dopamine
D2 receptor antagonist alters extracellular adenosine levels in the rat
nucleus accumbens. Neurochem. Int.
44, 609–615.
Nicola, S. M. (2007). The nucleus
accumbens as part of a basal
ganglia action selection circuit.
Psychopharmacology (Berl.) 191,
521–550.
Ouimet, C. C., Miller, P. E., Hemmings,
H. C. Jr., Walaas, S. I., and
Greengard, P. (1984). DARPP32, a dopamine- and adenosine
3’:5’-monophosphate-regulated
phopshoprotein
enriched
in
dopamine-innervated
brain
regions. III. Immunocytochemical
localization. J. Neurosci. 4, 111–124.
Pascoli, V., Besnard, A., Herve, D.,
Pages, C., Heck, N., Girault, J.
A., et al. (2011). Cyclic adenosine monophosphate-independent
tyrosine phosphorylation of NR2B
mediates cocaine-induced extracellular signal-regulated kinase
activation. Biol. Psychiatry 69,
218–227.
Perreault, M. L., Fan, T., Alijaniaram,
M., O’Dowd, B. F., and George,
S. R. (2012). Dopamine D1-D2
receptor heteromer in dual phenotype GABA/glutamate-coexpressing
striatal medium spiny neurons:
regulation of BDNF, GAD67 and
VGLUT1/2. PLoS ONE 7:e33348.
doi: 10.1371/journal.pone.0033348
Perreault, M. L., Hasbi, A., Alijaniaram,
M., Fan, T., Varghese, G., Fletcher,
P. J., et al. (2010). The dopamine
D1-D2 receptor heteromer localizes
in dynorphin/enkephalin neurons: increased high affinity state
following amphetamine and in
schizophrenia. J. Biol. Chem. 285,
36625–36634.
Perreault, M. L., Hasbi, A., O’Dowd,
B. F., and George, S. R. (2011).
The dopamine d1-d2 receptor heteromer in striatal medium spiny
neurons: evidence for a third distinct neuronal pathway in Basal
Ganglia. Front. Neuroanat. 5:31. doi:
10.3389/fnana.2011.00031
Reynolds, S. M., and Berridge, K.
C. (2002). Positive and negative
motivation in nucleus accumbens
shell: bivalent rostrocaudal gradients for GABA-elicited eating, taste
“liking”/“disliking” reactions, place
preference/avoidance, and fear.
J. Neurosci. 22, 7308–7320.
Santini, E., Valjent, E., and Fisone,
G. (2008). Parkinson’s disease:
levodopa-induced dyskinesia and
signal transduction. FEBS J. 275,
1392–1399.
Schiffmann, S. N., Jacobs, O., and
Vanderhaeghen, J. J. (1991). Striatal
restricted adenosine A2 receptor
(RDC8) is expressed by enkephalin
but not by substance P neurons: an
in situ hybridization histochemistry
study. J. Neurochem. 57, 1062–1067.
Seifert, U., Hartig, W., Grosche, J.,
Bruckner, G., Riedel, A., and Brauer,
K. (1998). Axonal expression sites
of tyrosine hydroxylase, calretininand
calbindin-immunoreactivity
in striato-pallidal and septal
nuclei of the rat brain: a doubleimmunolabelling study. Brain Res.
795, 227–246.
Sesack, S. R., and Grace, A. A.
(2010). Cortico-Basal Ganglia
reward network: microcircuitry.
Neuropsychopharmacology
35,
27–47.
www.frontiersin.org
Sokoloff, P., Giros, B., Martres, M. P.,
Bouthenet, M. L., and Schwartz,
J. C. (1990). Molecular cloning
and characterization of a novel
dopamine receptor (D3) as a target for neuroleptics. Nature 347,
146–151.
Srinivas, S., Watanabe, T., Lin, C. S.,
William, C. M., Tanabe, Y., Jessell,
T. M., et al. (2001). Cre reporter
strains produced by targeted insertion of EYFP and ECFP into the
ROSA26 locus. BMC Dev. Biol. 1:4.
doi: 10.1186/1471-213X-1-4
Stuber, G. D., Hnasko, T. S., Britt,
J. P., Edwards, R. H., and Bonci,
A. (2010). Dopaminergic terminals in the nucleus accumbens
but not the dorsal striatum corelease glutamate. J. Neurosci. 30,
8229–8233.
Tecuapetla, F., Patel, J. C., Xenias, H.,
English, D., Tadros, I., Shah, F., et al.
(2010). Glutamatergic signaling by
mesolimbic dopamine neurons in
the nucleus accumbens. J. Neurosci.
30, 7105–7110.
Todtenkopf, M. S., and Stellar, J.
R. (2000). Assessment of tyrosine
hydroxylase immunoreactive innervation in five subregions of the
nucleus accumbens in rats treated
with repeated cocaine. Synapse 38,
261–270.
Torvinen, M., Marcellino, D., Canals,
M., Agnati, L. F., Lluis, C., Franco,
R., et al. (2005). Adenosine A2A
receptor and dopamine D3 receptor
interactions: evidence of functional
A2A/D3 heteromeric complexes.
Mol. Pharmacol. 67, 400–407.
Valjent, E., Bertran-Gonzalez, J., Herve,
D., Fisone, G., and Girault, J. A.
(2009). Looking BAC at striatal signaling: cell-specific analysis in new
transgenic mice. Trends Neurosci.
32, 538–547.
Valjent, E., Corvol, J. C., Pages,
C., Besson, M. J., Maldonado,
R., and Caboche, J. (2000).
Involvement of the extracellular signal-regulated kinase cascade
for cocaine-rewarding properties.
J. Neurosci. 20, 8701–8709.
Valjent, E., Pascoli, V., Svenningsson, P.,
Paul, S., Enslen, H., Corvol, J. C.,
et al. (2005). Regulation of a protein
phosphatase cascade allows convergent dopamine and glutamate signals to activate ERK in the striatum.
Proc. Natl. Acad. Sci. U.S.A. 102,
491–496.
Van Dongen, Y. C., Mailly, P., Thierry,
A. M., Groenewegen, H. J., and
Deniau, J. M. (2008). Threedimensional
organization
of
dendrites and local axon collaterals
of shell and core medium-sized
spiny projection neurons of the rat
February 2013 | Volume 7 | Article 22 | 16
Gangarossa et al.
nucleus accumbens. Brain Struct.
Funct. 213, 129–147.
Voorn, P., Gerfen, C. R., and
Groenewegen, H. J. (1989).
Compartmental
organization
of the ventral striatum of the rat:
immunohistochemical
distribution of enkephalin, substance P,
dopamine, and calcium-binding
protein. J. Comp. Neurol. 289,
189–201.
Wright, C. I., Beijer, A. V., and
Groenewegen, H. J. (1996). Basal
amygdaloid complex afferents to
the rat nucleus accumbens are compartmentally organized. J. Neurosci.
16, 1877–1893.
Frontiers in Neural Circuits
MSNs organization in the NAc
Wright, C. I., and Groenewegen, H.
J. (1996). Patterns of overlap and
segregation between insular cortical, intermediodorsal thalamic
and basal amygdaloid afferents in
the nucleus accumbens of the rat.
Neuroscience 73, 359–373.
Zaborszky, L., Alheid, G. F., Beinfeld,
M. C., Eiden, L. E., Heimer,
L., and Palkovits, M. (1985).
Cholecystokinin innervation of the
ventral striatum: a morphological
and radioimmunological study.
Neuroscience 14, 427–453.
Zahm, D. S., and Brog, J. S. (1992). On
the significance of subterritories
in the “accumbens” part of the rat
ventral striatum. Neuroscience 50,
751–767.
Conflict of Interest Statement: The
authors declare that the research
was conducted in the absence of any
commercial or financial relationships
that could be construed as a potential
conflict of interest.
Received: 06 December 2012; paper
pending published: 28 December 2012;
accepted: 02 February 2013; published
online: 19 February 2013.
Citation: Gangarossa G, Espallergues J,
de Kerchove d’Exaerde A, El Mestikawy
S, Gerfen CR, Hervé D, Girault J-A
www.frontiersin.org
and Valjent E (2013) Distribution
and compartmental organization of
GABAergic medium-sized spiny neurons
in the mouse nucleus accumbens. Front.
Neural Circuits 7:22. doi: 10.3389/fncir.
2013.00022
Copyright © 2013
Gangarossa,
Espallergues, de Kerchove d’Exaerde,
El Mestikawy, Gerfen, Hervé, Girault
and Valjent. This is an open-access
article distributed under the terms of the
Creative Commons Attribution License,
which permits use, distribution and
reproduction in other forums, provided
the original authors and source are credited and subject to any copyright notices
concerning any third-party graphics etc.
February 2013 | Volume 7 | Article 22 | 17
Gangarossa et al.
MSNs organization in the NAc
APPENDIX
FIGURE A1 | Schematic representation of the subterritories of the caudomedial NAc shell analyzed. Each region was defined based on a review of
previous anatomical and neurochemical studies. See text for more detailed description.
Table A1 | Measurement of the surface areas of the NAc subregions analyzed in Figure 10.
Area (mm2 )
SEM (±)
N
Veh
SKF
Qui
Apo
Rac
0.3119
0.0061
4
0.3190
0.0074
5
0.3363
0.0107
3
0.3259
0.0102
4
0.3273
0.0059
4
F -value
Area (mm2 )
SEM (±)
N
F(4, 15) = 1.158; NS
Veh
SKF
Qui
Apo
Rac
0.0618
0.0014
4
0.0595
0.0057
5
0.0497
0.0076
3
0.0535
0.0013
4
0.0541
0.0048
4
F -value
Area (mm2 )
SEM (±)
N
F(4, 15) = 1.002; NS
Veh
SKF
Qui
Apo
Rac
0.2403
0.0068
4
0.2428
0.0080
5
0.2747
0.0090
3
0.2653
0.0080
4
0.2588
0.0102
4
F -value
Area (mm2 )
SEM (±)
N
F(4, 15) = 2.825; NS
Veh
SKF
Qui
Apo
Rac
0.0509
0.0040
4
0.0467
0.0064
5
0.0385
0.0055
3
0.0466
0.0016
4
0.0458
0.0035
4
F -value
F(4, 15) = 0.7322; NS
(Continued)
Frontiers in Neural Circuits
www.frontiersin.org
February 2013 | Volume 7 | Article 22 | 18
Gangarossa et al.
MSNs organization in the NAc
Table A1 | Continued
Veh
SKF
Qui
Apo
Rac
Area (mm2 )
0.0373
0.0366
0.0372
0.0425
0.0363
SEM (±)
0.0030
0.0010
0.0023
0.0016
0.0019
N
4
5
3
4
4
F -value
F(4, 15) = 1.682; NS
Veh
SKF
Qui
Apo
Rac
Area (mm2 )
0.2021
0.2046
0.1949
0.2106
0.2034
SEM (±)
0.0126
0.0067
0.0149
0.0018
0.0057
N
4
5
3
4
4
F(4, 15) = 0.3593; NS
F -value
Data are expressed as means ± SEM (n = 3–5) and were analyzed using one-way ANOVA followed by the Dunnett’s post hoc test. NS: not significant.
Table A2 | Measurement of the surface areas of the NAc subregions analyzed in Figure 13.
Area (mm2 )
SEM (±)
N
Veh
Coc
d-amph
0.3275
0.0078
4
0.3088
0.0064
5
0.3304
0.0050
4
F -value
(mm2 )
Area
SEM (±)
N
F(2, 10) = 3.429; NS
Veh
Coc
d-amph
0.0482
0.0041
4
0.0489
0.0046
5
0.0549
0.0049
4
F -value
Area (mm2 )
SEM (±)
N
F(2, 10) = 0.6231; NS
Veh
Coc
d-amph
0.2836
0.0122
4
0.2635
0.0080
5
0.2716
0.0031
4
F -value
Area (mm2 )
SEM (±)
N
F(2, 10) = 1.405; NS
Veh
Coc
d-amph
0.0464
0.0050
4
0.0474
0.0041
5
0.0519
0.0033
4
F -value
(mm2 )
Area
SEM (±)
N
F(2, 10) = 0.4495; NS
Veh
Coc
d-amph
0.0336
0.0040
4
0.0318
0.0018
5
0.0304
0.0004
4
F -value
(mm2 )
Area
SEM (±)
N
F(2, 10) = 0.4061; NS
Veh
Coc
d-amph
0.2016
0.0167
4
0.2129
0.0056
5
0.2107
0.0030
4
F -value
F(2, 10) = 0.3741; NS
Data are expressed as means ± SEM (n = 4–5) and were analyzed using one-way ANOVA followed by the Dunnett’s post hoc test. NS: not significant.
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Gangarossa et al.
MSNs organization in the NAc
Table A3 | F -values corresponding to the statistical analysis of the results presented in Figures 10 and 13.
Drd2-negative cells
Drd2-positive cells
Figure 10A
F(4, 15) = 21.02, p < 0.001
F(4, 15) = 6.30, p < 0.01
Figure 10B
F(4, 15) = 80.37, p < 0.001
F(4, 15) = 0.12, NS
Figure 10C
F(4, 15) = 44.27, p < 0.001
F(4, 15) = 9.74, p < 0.001
Figure 10D
F(4, 15) = 4.78, p < 0.05
F(4, 15) = 0.15, p NS
Figure 10E
F(4, 15) = 20.48, p < 0.001
F(4, 15) = 29.08, p < 0.001
Figure 10F
F(4, 15) = 18.30, p < 0.001
F(4, 15) = 99.33, p < 0.001
Figure 13A
F(2, 10) = 55.35, p < 0.001
F(2, 10) = 0.90, NS
Figure 13B
F(2, 10) = 75.93, p < 0.001
F(2, 10) = 1.57, NS
Figure 13C
F(2, 10) = 151.20, p < 0.001
F(2, 10) = 2.56, NS
Figure 13D
F(2, 10) = 19.90, p < 0.001
F(2, 10) = 1.20, NS
Figure 13E
F(2, 10) = 4.30, p < 0.05
F(2, 10) = 3.90, NS
Figure 13F
F(2, 10) = 19.90, p < 0.001
F(2, 10) = 2.32, NS
NS: not significant.
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