People with cocaine addiction retain some degree of prefrontal cortex (PFC) inhibitory control of cocaine craving, a brain capacity that may underlie the efficacy of cognitive behavioral therapy for addiction. Similar findings were recently found in rats after extended access to and escalation of cocaine self-administration. Rats' inhibitory control of cocaine seeking was flexible, sufficiently strong to suppress cocaine-primed reinstatement and depended, at least in part, on neuronal activity within the prelimbic (PL) PFC. Here, we used a large-scale and high-resolution Fos mapping approach to identify, beyond the PL PFC, how top-down and/or bottom-up PFC-subcortical circuits are recruited during inhibition of cocaine seeking. Overall, we found that effective inhibitory control of cocaine seeking is associated with the coordinated recruitment of different top-down cortical–striatal circuits originating from different PFC territories, and of different bottom-up dopamine (DA) and serotonin (5-HT) midbrain subsystems that normally modulate activity in these circuits. This integrated brain response suggests that rats concomitantly engage and experience intricate cognitive and affective processes when they have to inhibit intense cocaine seeking. Thus, even after extended drug use, rats can be successfully trained to engage whole-brain inhibitory control mechanisms to suppress cocaine seeking.

Introduction

Recent neuroimaging research has shown that people with drug addiction retain some degree of prefrontal inhibitory control over drug craving (Kober et al. 2010a, 2010b; Volkow et al. 2010). For instance, when instructed to purposefully resist cue-induced craving, people with cocaine addiction are able to recruit prefrontal cortex (PFC) regions [i.e., dorsomedial (DM), dorsolateral (DL), and ventrolateral prefrontal cortices] to suppress cortical and subcortical brain activity related to drug craving [i.e., ventral striatum, subgenual anterior cingulate (ACC), orbitofrontal cortex (OFC), ventral tegmental area (VTA)]. This functional top-down frontal inhibition of craving-related cortical and subcortical activity may underlie the efficacy of cognitive behavioral therapies for addiction (Carroll and Onken 2005; Sofuoglu et al. 2013).

Like people with cocaine addiction, rats with a history of extended access to cocaine self-administration—which is known to precipitate escalation of drug use and other behavioral changes (Ahmed and Koob 1998; Ahmed et al. 2002; Vanderschuren and Everitt 2004; Hollander et al. 2010; Ahmed 2012)—can be trained to successfully suppress cocaine seeking using an inhibitory discriminative stimulus (DS-) task (Mihindou et al. 2013). Rats' inhibitory control of cocaine seeking was flexible, sufficiently strong to suppress cocaine-primed reinstatement of cocaine seeking and depended on increased neuronal activity within the prelimbic (PL) subdivision of the medial PFC (mPFC), the functional homolog of the human lateral PFC (Vertes 2006). Importantly, reversible inactivation of the PL transiently disinhibited cocaine seeking, thereby reflecting its causal involvement in inhibitory control (Mihindou et al. 2013). This disinhibition was however incomplete, suggesting the involvement of other brain regions and circuits.

Inhibitory control indeed requires the coordinated recruitment of multiple top-down projections from various subdivisions of the PFC (Sofuoglu et al. 2013). These projections are topographically ordered according to specific dorso-ventral, medio-lateral, and/or antero-posterior (AP) gradients within subcortical areas (Voorn et al. 2004; Gabbott et al. 2005; Schilman et al. 2008; Hoover and Vertes 2011; Mailly et al. 2013). Such anatomical heterogeneity has been functionally linked to different cognitive and motivational aspects of goal-directed behaviors (McFarland and Kalivas 2001; McFarland et al. 2003, 2004; Schoenbaum et al. 2003; Voorn et al. 2004; Ishikawa et al. 2008; Peters et al. 2008; Burguiere et al. 2013). PFC executive control also depends on bidirectional interactions with ascending midbrain dopamine (DA) and serotonin (5-HT) systems (Amat et al. 2005; Robbins and Arnsten 2009; Boureau and Dayan 2011; Cools et al. 2011; Jo et al. 2013). Both the VTA and raphe nuclei also display a strong anatomical and neurochemical heterogeneity that plays distinct functional roles in reward- and aversion-related behaviors (Clark et al. 2006; Ikemoto 2007; Halberstadt and Balaban 2008; Lammel et al. 2008, 2012; Schweimer and Ungless 2010; Calizo et al. 2011; Mahler and Aston-Jones 2012; van Zessen et al. 2012; Li et al. 2013).

Whether this coordinated recruitment of different top-down and bottom-up cortical-subcortical circuits is also present during inhibitory control of cocaine seeking in rats is currently unknown. Here, we used a large-scale Fos brain mapping approach to begin to address this question. Brains from a recent study that was originally exclusively focused on the mPFC (Mihindou et al. 2013) were analyzed along their entire AP axis with a 400-µm resolution. These brains belonged to rats with identical pharmacological and learning histories that were tested in 3 different behavioral conditions on the sacrifice day: no inhibition, inhibition, and disinhibition of cocaine seeking. Thus, this follow-up study was designed to advance our knowledge in at least 3 main dimensions. First, it extends analysis to the whole PFC using a higher anatomical resolution, thereby enabling us to study whether the various PFC subdivisions are differentially recruited during inhibition of cocaine seeking in rats. This is important in view of the known functional heterogeneity of the PFC. Second, it extends analysis to a wide range of subcortical brain regions and systems that are differentially connected with the PFC and that are known to influence cocaine seeking. Third, a double-labeling approach for Fos and tyrosine hydroxylase (TH) or tryptophan hydroxylase (TPH) in the VTA and raphe nuclei, respectively, was used to determine the relative contribution of DA and 5-HT neurons during inhibition versus disinhibition of cocaine seeking. Such large-scale Fos brain mapping study will uniquely allow us to see how the recruitment of different top-down and/or bottom-up cortical-subcortical circuits is coordinated during inhibitory control of cocaine seeking.

Materials and Methods

The present follow-up Fos mapping study provides a high-resolution analysis of the entire AP extent of the brains from the same rats used in a recently published study (Mihindou et al. 2013). Fos data from the PL and infralimbic (IL) subregions in the present paper are detailed within their dorso-ventral and/or AP extents, which was not the case in our previous paper where they were averaged across these different levels. The behavioral history and inhibitory control performance of these rats are described in details in the original study and are only described briefly below.

Animals and Surgery

Male Wistar rats (Charles River, L'Arbresle, France, 200–225 g at the beginning of the experiments) were housed in groups of 2 or 3 and maintained in a light- (12-h reverse light-dark cycle) and temperature-controlled vivarium (21 °C). All behavioral testing occurred during the dark phase. Food and water were freely available in the home cages. Rats were surgically prepared with a catheter in the right jugular vein as previously described (Mihindou et al. 2013), and behavioral testing began 7–10 days after surgery. All experiments were carried out in accordance with institutional and international standards of care and use of laboratory animals [UK Animals (Scientific Procedures) Act, 1986; and associated guidelines; the European Communities Council Directive (86/609/EEC, 24 November 1986) and the French Directives concerning the use of laboratory animals (décret 87–848, 19 October 1987)]. All experiments have been approved by the Committee of the Veterinary Services Gironde, agreement number A33-063-922.

Drugs

Cocaine hydrochloride (Coopération Pharmaceutique Française, Melun, France) was dissolved in a solution of NaCl (0.9%) which was filtered through a syringe filter (0.22 µm) and kept at room temperature (RT) in a 500 mL sterile bag (21 ± 2 °C). Drug doses were expressed as the weight of the salt.

Operant Training for Discriminative Inhibitory Control of Cocaine Seeking

Behavioral training took place in operant chambers as previously described (Mihindou et al. 2013). Rats were first trained to lever press for intravenous cocaine (final dose: 0.125 mg) under a fixed-ratio time-out 20-s (final ratio: 5) schedule of reinforcement during daily short-access (3 h) self-administration sessions. After 12 days, rats were further trained during 18 long-access (6 h) self-administration sessions. Previous research has shown that after extended access to cocaine self-administration, rats are more likely to escalate cocaine intake, to work harder and to accept increased costs to seek and/or to obtain the drug (Ahmed 2012). All self-administration sessions began with the extension of the lever and were run 6 days per week during 5 weeks before testing for discriminative inhibitory control of cocaine seeking. After acquisition and stabilization of FR5 responding for cocaine (i.e., no increasing or decreasing trend over 3 consecutive sessions), rats were trained to inhibit cocaine seeking in response to a visual discriminative stimulus (DS-) signaling lack of cocaine reinforcement. Daily sessions lasted 120 min and were subdivided into 2 successive components: an initial 90-min period of regular cocaine self-administration followed by a final 30-min nonreinforcement period signaled by the DS-. The DS- consisted of turning on for 30 min the house light of the operant chamber. When the DS- was on, completion of the FR requirement was signaled by the 20-s time-out cue but was no longer reinforced by cocaine. DS- sessions were run 6 days per week for at least 13 sessions until stabilization of inhibition of cocaine seeking (i.e., no increasing or decreasing trend over 3 consecutive sessions). We previously showed that turning off the DS- resulted in a disinhibition of cocaine seeking (Mihindou et al. 2013).

Experimental Group Assignment for the Fos Mapping Study

At the end of DS- training, rats were allocated to 3 different groups (n = 4 rats/group) with the same level of total cocaine intake and DS- inhibition (Supplementary Fig. 1) before the final test day. To further control for identical levels of cocaine intake at the end of the experiment, brains were processed for deltaFosB immunohistochemistry (Supplementary Fig. 1), a sensitive marker of cumulative exposure to cocaine (Nye et al. 1995; Perrotti et al. 2005). On the final test day, all groups were allowed to self-administer cocaine during the first 90 min, as during a regular DS- training session. Groups only differed regarding to what happened during the last 30 min of the session. In the first group, the DS- was turned on to induce inhibition of cocaine seeking (ON group). In the second group, the DS- was omitted to release inhibition of cocaine seeking (OFF group). The OFF group served as a direct control for inhibition of cocaine seeking in the ON group. Note that this group did not receive cocaine during the period where the DS- was omitted. Finally, in the last group, the DS- was omitted and the cocaine-paired lever was retracted to prevent the expression of uninhibited cocaine seeking (No Lever or NLv group). This last group controlled for eventual neuronal correlates of disinhibited cocaine seeking in the OFF group. All rats were killed immediately after the end of the last 30-min period of the session. This relatively early time point (i.e., 30 min after DS- onset in ON group) was chosen because inhibition of cocaine seeking was immediate after DS- onset (Mihindou et al. 2013) and previous research showed that Fos induction can be seen as early as 15 min (Kovacs 1998) and peak between 30 and 90 min (Frohmader et al. 2010). As previously reported (Mihindou et al. 2013), cocaine seeking in the ON group was inhibited at the onset of, and throughout, DS- presentation. In the OFF group, cocaine seeking was disinhibited by omission of the DS- during the first 5 min of the DS- period, and then progressively decreased toward the end of the DS- period (Supplementary Fig. 1).

Immunohistochemistry

Immunohistochemistry experiments were performed as previously described (Mihindou et al. 2013). Briefly, after transcardiac perfusion with 4% formaldehyde (Prolabo, distributed by VWR) in 0.1 M phosphate-buffered (PB, pH 7.4, 37 °C), brains were postfixed overnight and cryoprotected for 48 h at 4 °C in 20% sucrose solution (Sigma-Aldrich, St. Quentin Fallavier, France). Freezing 50-µm-thick coronal sections were cut on a cryostat (Leica CM 3050S) and stored at 4 °C.

For single immunohistochemistry, free-floating sections were incubated with the primary antibody: 48 h at 4 °C in rabbit polyclonal anti-c-Fos antibody (1 : 8000; sc-52, Santa-Cruz Biotechnology, distributed by Clinisciences, France) or overnight at RT in rabbit polyclonal FosB antibody (1 : 500; sc-48, Santa-Cruz Biotechnology). Then, sections were incubated for 2 h at RT with biotinylated goat anti-rabbit IgG (BA-1000: 1 : 200; Vector Laboratories, distributed by Clinisciences, France) and further processed using avidin–biotin–peroxidase complex (1 : 200 for 2 h at RT; ABC Vectastain Elite Kit, PK-6100; Vector laboratories) and 3,3′-diaminobenzidine detection (DAB: 0.05% w/v with 0.003% H2O2; Sigma-Aldrich).

Double immunohistochemistry for Fos and TH or for Fos and TPH were performed on mesencephalic sections. Free-floating sections were incubated overnight at 4 °C with the rabbit polyclonal anti-c-Fos antibody (sc-52, 1 : 6000; see above), then with the secondary biotinylated goat anti-rabbit IgG (BA-1000, 1 : 200) for 2 h at RT. Immunostaining for Fos was processed using avidin–biotin–peroxidase complex (PK-6100, 1 : 200 for 2 h at RT) and DAB detection enhanced by nickel for a dark gray coloration (Peroxidase Substrate Kit SK-4100; Vector laboratories). Then, sections were incubated overnight at RT with either anti-TH mouse monoclonal antibody (MAB318, 1 : 10 000; Chemicon, distributed by Merck Millipore, France) or anti-TPH rabbit polyclonal antibody (AB1541, 1 : 10 000; Chemicon). Sections were subsequently incubated 2 h at RT with their respective secondary biotinylated antibody, that is, goat anti-mouse IgG (BA-9200, 1 : 200; Vector Laboratories) and sheep anti-rabbit IgG (BA-6000, 1 : 200; Vector Laboratories). For Fos/TH labeling, the blocking solution was PBS (0.1 M, pH 7.4) containing 3% normal goat serum with 0.3% triton and antibodies were diluted in PBS containing 1% normal goat serum with 0.3% triton (Sigma-Aldrich). For Fos/TPH labeling, sections were blocked and antibodies were diluted in PBS containing 1% bovine serum albumin with 0.3% triton (Sigma-Aldrich). Immunostaining for TH and TPH was processed using avidin–biotin–peroxidase complex (PK-6100, 1 : 400 for 2 h at RT) and DAB detection for a brown coloration (Peroxidase Substrate Kit SK-4100).

The specificity of the immunostaining was assessed by omission of the primary or the secondary antibody from the protocol. After processing, tissue sections were mounted onto gelatin–alum-coated slides, dehydrated in ascending concentration of ethanol, and coverslipped with Eukitt mounting medium (Sigma-Aldrich).

Immunoreactivity Counting

Our purpose was to generate a large-scale and high-resolution Fos mapping of brain regions recruited during inhibition of cocaine seeking. The experiment was designed to obtain relative differences between behavioral groups in numbers of Fos-expressing neurons in otherwise identically sectioned material. Since a large number of brain regions required evaluation across 3 different behavioral conditions, we chose to use a semiquantitative Fos counting method involving density measurements (Saper 1996) rather than a quantitative method involving stereological estimates (Coggeshall and Lekan 1996).

Single and double immunoreactivity was examined using a Leica DM6000B microscope equipped with a Hamamatsu digital camera ORCA-03G and a motorized stage (x, y, and z) interfaced with an Antec computer with Mercator Pro software (ExploraNova). For counting, the group assignment of each rat was blinded to the observer. Single Fos-positive (Fos+) or deltaFosB+ neurons were identified by a brown oval-shaped nucleus whose staining intensity reached at least twice the background level. For each rat, positive labeled cells were counted in both hemispheres from 2 to 6 sections (50 µm thick, 400 µm apart) covering the AP extent of each region of interest (ROI; Table 1 and Fig. 1). When 2 AP levels were included (one section counted per AP level), they were identified as anterior (a) and posterior (p); when 3 AP levels were included (1 or 2 sections counted per AP level), they were identified as rostral (r), middle (m), and caudal (c) (see Table 1 for the corresponding coordinates of all AP levels counted for each ROI). The shape of the sampled area (square, rectangle, or hand-shaped for the subthalamic nucleus and dorsal raphe) varied depending on the structure being measured. Sampled areas range between 0.158 and 0.486 mm2 (see area of each ROI in the legend of Fig. 1). For each section, the sampled areas were first delineated at low magnification (×2.5) and counting was performed at high magnification (×20). Cell counts from all the sampled areas were normalized by the area counted and expressed as number of Fos+ or deltaFosB+ cells/mm2 (density). DeltaFosB+ cells were counted in the nucleus accumbens (NAc), striatum, tail of VTA (tVTA), and rostromedial tegmental nucleus (RMTg) (Supplementary Fig. 1). In case of double labeling, 3 sections covering the entire AP extent of the VTA and raphe nuclei were used to count Fos+ cells. Colocalization of Fos plus TH or TPH was identified as a gray-black nucleus (indicating Fos immunoreactivity) surrounded by a brown cytoplasm (indicating TH or TPH immunoreactivity) (Supplementary Fig. 2). Cell counts were expressed as the number of total Fos+ and Fos+TH+ or Fos+TPH+ cells/mm2 (see histograms in Figs 7 and 8) or as the percentage of total Fos+ cells/mm2 (see pie charts in Figs 7 and 8). Data from all sections per ROI were averaged for each rat and the mean ± standard error of the mean (SEM) of these values were calculated for each group (n = 4). Some ROIs (all quadrants of the striatum) were counted twice to test for the validity of the counting method and resulted in variability <15% with no statistical difference between counts; results from both counts were then averaged for these ROIs.

Table 1

Brain region patterns of Fos expression induced by discriminative inhibition of cocaine seeking (exposure to the discriminative stimulus DS-) and control behaviors

Region Subregion AP Fos+ cells/mm2
 
NLv OFF ON 
OFC MO (1) +5.16 39.7 ± 13.6 52.9 ± 9.2 54.8 ± 17.7 
+4.68 55.3 ± 22.5 37.9 ± 9.6 68.2 ± 19.2 
+4.20 35.2 ± 11.8 52.0 ± 6.5 43.9 ± 13.9 
VO (2) +5.16 76.6 ± 8.7 31.3 ± 8.9** 65.1 ± 6.7+ 
+4.68 63.0 ± 9.8 28.2 ± 1.6** 56.7 ± 4.4+ 
+4.20 75.2 ± 17.5 32.4 ± 5.7 67.5 ± 9.3 
VLO (3) +5.16 25.4 ± 6.1 15.0 ± 2.4 76.9 ± 14.0**++ 
+4.68 17.4 ± 3.3 27.2 ± 7.5 85.3 ± 22.9*+ 
+4.20 9.7 ± 2.4 16.7 ± 4.4 67.2 ± 20.5*+ 
LO (4) +5.16 15.3 ± 4.9 22.3 ± 5.5 71.7 ± 21.2* 
+4.68 12.5 ± 3.7 26.5 ± 7.0 70.7 ± 14.4**+ 
+4.20 13.9 ± 1.9 21.2 ± 5.6 52.2 ± 7.1**++ 
DLO (5) +5.16 52.3 ± 18.2 76.7 ± 26.1 61.7 ± 17.1 
+4.68 49.3 ± 14.0 62.2 ± 22.7 69.1 ± 9.0 
mPFC Cg1 (6) +3.72 192.7 ± 33.7 46.6 ± 13.1** 161.0 ± 24.5+ 
+3.24 214.8 ± 15.3 61.6 ± 10.0** 174.1 ± 40.9+ 
+2.76 171.6 ± 20.6 61.2 ± 6.1** 163.0 ± 20.8++ 
dPL (7) +3.72 20.8 ± 4.3 14.7 ± 4.2 68.3 ± 20.3+ 
+3.24 24.0 ± 6.6 24.3 ± 4.4 69.0 ± 14.8*+ 
+2.76 19.0 ± 4.2 17.5 ± 4.6 76.9 ± 8.4***+++ 
vPL (8) +3.72 23.3 ± 6.8 20.0 ± 4.8 56.9 ± 6.8*++ 
+3.24 12.5 ± 2.8 17.2 ± 7.0 49.7 ± 3.4**++ 
+2.76 20.4 ± 3.2 21.5 ± 7.0 68.0 ± 3.3***+++ 
IL (9) +3.72 10.5 ± 5.4 9.5 ± 2.6 18.9 ± 0.6 
+3.24 10.0 ± 3.4 11.0 ± 3.9 12.9 ± 4.7 
+2.76 2.0 ± 0.8 7.5 ± 2.1 13.4 ± 3.3* 
NAc Core (10) +2.76/+2.28 26.9 ± 6.8 20.4 ± 4.0 30.4 ± 8.7 
+1.92/+1.56 38.8 ± 9.3 32.4 ± 5.4 30.4 ± 7.1 
+1.20/+0.84 17.9 ± 0.9 13.4 ± 2.5 31.1 ± 4.8*++ 
M shell (11) +2.76/+2.28 17.7 ± 2.3 20.7 ± 5.0 21.4 ± 2.5 
+1.92/+1.56 12.7 ± 2.6 11.7 ± 2.5 28.4 ± 4.9*+ 
+1.20/+0.84 21.4 ± 5.3 15.4 ± 5.1 19.4 ± 6.4 
L shell (12) +1.92/+1.56 10.3 ± 4.5 10.3 ± 4.2 11.1 ± 3.0 
+1.20/+0.84 10.3 ± 3.3 30.8 ± 6.1* 15.0 ± 2.4 
Striatum DM (13) +1.92/+1.56 4.0 ± 0.2 3.2 ± .9 10.0 ± 2.2*+ 
+1.20/+0.84 5.8 ± 1.0 3.6 ± 0.8 7.5 ± 1.3 
+0.48/+0.12 5.4 ± 1.4 4.5 ± 1.7 6.7 ± 0.9 
VM (14) +1.92/+1.56 13.0 ± 1.2 7.5 ± 1.8 13.1 ± 1.5+ 
+1.20/+0.84 5.5 ± 0.6 2.8 ± 0.4 7.5 ± 1.0++ 
+0.48/+0.12 7.6 ± 1.1 6.6 ± 1.4 9.3 ± 1.1 
DL (15) +1.92/+1.56 4.3 ± 0.7 4.0 ± 0.4 8.9 ± 1.7*+ 
+1.20/+0.84 6.4 ± 0.6 3.6 ± 0.8 6.9 ± 1.1 
+0.48/+0.12 6.3 ± 1.1 5.6 ± 2.7 9.4 ± 2.4 
VL (16) +1.92/+1.56 10.1 ± 1.4 7.5 ± 1.8 13.1 ± 1.5 
+1.20/+0.84 12.3 ± 2.0 6.0 ± 1.7 14.1 ± 2.9 
+0.48/+0.12 13.3 ± 2.8 7.2 ± 2.0 14.8 ± 2.1 
Central (17) +1.92/+1.56 5.9 ± 1.3 5.7 ± 1.7 23.5 ± 7.5+ 
+1.20/+0.84 5.8 ± 1.3 6.3 ± 2.9 18.4 ± 4.5* 
+0.48/+0.12 8.9 ± 1.4 5.4 ± 1.0 22.1 ± 4.2*++ 
Septum Medial (18) + 1.56 32.9 ± 6.0 30.1 ± 5.8 34.1 ± 2.3 
+ 1.20 36.5 ± 3.9 11.0 ± 3.0* 30.4 ± 8.4 
+ 0.84 29.2 ± 7.9 5.5 ± 1.8* 27.4 ± 0.7+ 
Lateral (19) + 1.56 11.9 ± 6.6 11.0 ± 2.6 7.3 ± 4.5 
+ 1.20 5.5 ± 2.4 5.5 ± 2.4 9.1 ± 4.8 
+ 0.84 1.8 ± 1.8 4.6 ± 2.7 6.4 ± 3.1 
Lateral hypothalamus (20) 2.28 18.8 ± 6.0 19.1 ± 5.1 15.3 ± 4.7 
3.00 19.1 ± 6.5 21.0 ± 5.5 46.3 ± 6.4*+ 
3.84 23.4 ± 6.3 66.5 ± 14.3* 73.0 ± 5.5* 
Lateral habenula (21) 3.00 18.9 ± 7.3 18.9 ± 4.4 30.9 ± 14.6 
3.48 16.9 ± 5.5 20.6 ± 7.4 98.9 ± 7.4***+++ 
3.84 32.9 ± 8.6 30.4 ± 11.8 42.3 ± 13.7 
STN (22) 3.48 31.7 ± 5.2 28.5 ± 10.0 59.6 ± 6.6+ 
3.84 36.7 ± 3.9 36.9 ± 8.9 79.8 ± 3.5**++ 
VTA Medial (23) 4.92 29.9 ± 11.8 40.8 ± 12.4 109.5 ± 11.6**++ 
5.28 17.9 ± 11.6 47.2 ± 7.1 64.3 ± 6.6* 
5.64 27.9 ± 13.1 29.9 ± 15.7 28.9 ± 13.3 
Lateral (24) 4.92 13.9 ± 3.1 34.4 ± 4.8* 15.4 ± 5.0+ 
5.28 7.0 ± 5.0 35.8 ± 8.6* 11.0 ± 5.0 
5.64 8.5 ± 4.6 12.4 ± 1.7 7.5 ± 3.5 
tVTA (25) 6.36 130.2 ± 17.5 213.7 ± 13.5* 140.6 ± 22.0+ 
6.72 107.8 ± 28.5 201.9 ± 22.5* 110.8 ± 16.4+ 
RMTg (26) 7.80 111.4 ± 23.0 146.6 ± 15.5 71.8 ± 4.0+ 
Raphe Median (27) 7.44 12.7 ± 3.5 12.7 ± 3.5 5.5 ± 1.8 
7.80 10.9 ± 2.1 14.5 ± 3.0 3.6 ± 2.1+ 
Dorsal (28) 6.96 40.7 ± 5.7 76.4 ± 8.4** 51.1 ± 4.3+ 
7.44 39.9 ± 8.0 94.8 ± 12.4** 48.1 ± 4.3+ 
7.80 29.6 ± 2.9 91.4 ± 12.3** 32.5 ± 8.5++ 
Region Subregion AP Fos+ cells/mm2
 
NLv OFF ON 
OFC MO (1) +5.16 39.7 ± 13.6 52.9 ± 9.2 54.8 ± 17.7 
+4.68 55.3 ± 22.5 37.9 ± 9.6 68.2 ± 19.2 
+4.20 35.2 ± 11.8 52.0 ± 6.5 43.9 ± 13.9 
VO (2) +5.16 76.6 ± 8.7 31.3 ± 8.9** 65.1 ± 6.7+ 
+4.68 63.0 ± 9.8 28.2 ± 1.6** 56.7 ± 4.4+ 
+4.20 75.2 ± 17.5 32.4 ± 5.7 67.5 ± 9.3 
VLO (3) +5.16 25.4 ± 6.1 15.0 ± 2.4 76.9 ± 14.0**++ 
+4.68 17.4 ± 3.3 27.2 ± 7.5 85.3 ± 22.9*+ 
+4.20 9.7 ± 2.4 16.7 ± 4.4 67.2 ± 20.5*+ 
LO (4) +5.16 15.3 ± 4.9 22.3 ± 5.5 71.7 ± 21.2* 
+4.68 12.5 ± 3.7 26.5 ± 7.0 70.7 ± 14.4**+ 
+4.20 13.9 ± 1.9 21.2 ± 5.6 52.2 ± 7.1**++ 
DLO (5) +5.16 52.3 ± 18.2 76.7 ± 26.1 61.7 ± 17.1 
+4.68 49.3 ± 14.0 62.2 ± 22.7 69.1 ± 9.0 
mPFC Cg1 (6) +3.72 192.7 ± 33.7 46.6 ± 13.1** 161.0 ± 24.5+ 
+3.24 214.8 ± 15.3 61.6 ± 10.0** 174.1 ± 40.9+ 
+2.76 171.6 ± 20.6 61.2 ± 6.1** 163.0 ± 20.8++ 
dPL (7) +3.72 20.8 ± 4.3 14.7 ± 4.2 68.3 ± 20.3+ 
+3.24 24.0 ± 6.6 24.3 ± 4.4 69.0 ± 14.8*+ 
+2.76 19.0 ± 4.2 17.5 ± 4.6 76.9 ± 8.4***+++ 
vPL (8) +3.72 23.3 ± 6.8 20.0 ± 4.8 56.9 ± 6.8*++ 
+3.24 12.5 ± 2.8 17.2 ± 7.0 49.7 ± 3.4**++ 
+2.76 20.4 ± 3.2 21.5 ± 7.0 68.0 ± 3.3***+++ 
IL (9) +3.72 10.5 ± 5.4 9.5 ± 2.6 18.9 ± 0.6 
+3.24 10.0 ± 3.4 11.0 ± 3.9 12.9 ± 4.7 
+2.76 2.0 ± 0.8 7.5 ± 2.1 13.4 ± 3.3* 
NAc Core (10) +2.76/+2.28 26.9 ± 6.8 20.4 ± 4.0 30.4 ± 8.7 
+1.92/+1.56 38.8 ± 9.3 32.4 ± 5.4 30.4 ± 7.1 
+1.20/+0.84 17.9 ± 0.9 13.4 ± 2.5 31.1 ± 4.8*++ 
M shell (11) +2.76/+2.28 17.7 ± 2.3 20.7 ± 5.0 21.4 ± 2.5 
+1.92/+1.56 12.7 ± 2.6 11.7 ± 2.5 28.4 ± 4.9*+ 
+1.20/+0.84 21.4 ± 5.3 15.4 ± 5.1 19.4 ± 6.4 
L shell (12) +1.92/+1.56 10.3 ± 4.5 10.3 ± 4.2 11.1 ± 3.0 
+1.20/+0.84 10.3 ± 3.3 30.8 ± 6.1* 15.0 ± 2.4 
Striatum DM (13) +1.92/+1.56 4.0 ± 0.2 3.2 ± .9 10.0 ± 2.2*+ 
+1.20/+0.84 5.8 ± 1.0 3.6 ± 0.8 7.5 ± 1.3 
+0.48/+0.12 5.4 ± 1.4 4.5 ± 1.7 6.7 ± 0.9 
VM (14) +1.92/+1.56 13.0 ± 1.2 7.5 ± 1.8 13.1 ± 1.5+ 
+1.20/+0.84 5.5 ± 0.6 2.8 ± 0.4 7.5 ± 1.0++ 
+0.48/+0.12 7.6 ± 1.1 6.6 ± 1.4 9.3 ± 1.1 
DL (15) +1.92/+1.56 4.3 ± 0.7 4.0 ± 0.4 8.9 ± 1.7*+ 
+1.20/+0.84 6.4 ± 0.6 3.6 ± 0.8 6.9 ± 1.1 
+0.48/+0.12 6.3 ± 1.1 5.6 ± 2.7 9.4 ± 2.4 
VL (16) +1.92/+1.56 10.1 ± 1.4 7.5 ± 1.8 13.1 ± 1.5 
+1.20/+0.84 12.3 ± 2.0 6.0 ± 1.7 14.1 ± 2.9 
+0.48/+0.12 13.3 ± 2.8 7.2 ± 2.0 14.8 ± 2.1 
Central (17) +1.92/+1.56 5.9 ± 1.3 5.7 ± 1.7 23.5 ± 7.5+ 
+1.20/+0.84 5.8 ± 1.3 6.3 ± 2.9 18.4 ± 4.5* 
+0.48/+0.12 8.9 ± 1.4 5.4 ± 1.0 22.1 ± 4.2*++ 
Septum Medial (18) + 1.56 32.9 ± 6.0 30.1 ± 5.8 34.1 ± 2.3 
+ 1.20 36.5 ± 3.9 11.0 ± 3.0* 30.4 ± 8.4 
+ 0.84 29.2 ± 7.9 5.5 ± 1.8* 27.4 ± 0.7+ 
Lateral (19) + 1.56 11.9 ± 6.6 11.0 ± 2.6 7.3 ± 4.5 
+ 1.20 5.5 ± 2.4 5.5 ± 2.4 9.1 ± 4.8 
+ 0.84 1.8 ± 1.8 4.6 ± 2.7 6.4 ± 3.1 
Lateral hypothalamus (20) 2.28 18.8 ± 6.0 19.1 ± 5.1 15.3 ± 4.7 
3.00 19.1 ± 6.5 21.0 ± 5.5 46.3 ± 6.4*+ 
3.84 23.4 ± 6.3 66.5 ± 14.3* 73.0 ± 5.5* 
Lateral habenula (21) 3.00 18.9 ± 7.3 18.9 ± 4.4 30.9 ± 14.6 
3.48 16.9 ± 5.5 20.6 ± 7.4 98.9 ± 7.4***+++ 
3.84 32.9 ± 8.6 30.4 ± 11.8 42.3 ± 13.7 
STN (22) 3.48 31.7 ± 5.2 28.5 ± 10.0 59.6 ± 6.6+ 
3.84 36.7 ± 3.9 36.9 ± 8.9 79.8 ± 3.5**++ 
VTA Medial (23) 4.92 29.9 ± 11.8 40.8 ± 12.4 109.5 ± 11.6**++ 
5.28 17.9 ± 11.6 47.2 ± 7.1 64.3 ± 6.6* 
5.64 27.9 ± 13.1 29.9 ± 15.7 28.9 ± 13.3 
Lateral (24) 4.92 13.9 ± 3.1 34.4 ± 4.8* 15.4 ± 5.0+ 
5.28 7.0 ± 5.0 35.8 ± 8.6* 11.0 ± 5.0 
5.64 8.5 ± 4.6 12.4 ± 1.7 7.5 ± 3.5 
tVTA (25) 6.36 130.2 ± 17.5 213.7 ± 13.5* 140.6 ± 22.0+ 
6.72 107.8 ± 28.5 201.9 ± 22.5* 110.8 ± 16.4+ 
RMTg (26) 7.80 111.4 ± 23.0 146.6 ± 15.5 71.8 ± 4.0+ 
Raphe Median (27) 7.44 12.7 ± 3.5 12.7 ± 3.5 5.5 ± 1.8 
7.80 10.9 ± 2.1 14.5 ± 3.0 3.6 ± 2.1+ 
Dorsal (28) 6.96 40.7 ± 5.7 76.4 ± 8.4** 51.1 ± 4.3+ 
7.44 39.9 ± 8.0 94.8 ± 12.4** 48.1 ± 4.3+ 
7.80 29.6 ± 2.9 91.4 ± 12.3** 32.5 ± 8.5++ 

Numbers of Fos+ nuclei are shown for each brain region examined from rats that did not inhibit cocaine seeking (No Lever, NLv group), rats that disinhibited cocaine seeking (DS- off, OFF group) and rats that inhibited cocaine seeking (DS- on, ON group). Data are expressed as the mean (±SEM) of Fos+ cells per mm2 of tissue sampled (n = 4 rats/group). Fos counts were averaged from the left and right hemispheres where 2–3 antero-posterior (AP) levels were analyzed for each brain region (AP values in mm from bregma are indicated in italics; 2 values indicate that 2 sections were counted per AP level in the NAc and striatum). The number in parentheses associated to each subregion refers to the location of the corresponding counting area illustrated in Figure 1. OFC, orbitofrontal cortex; MO, medial orbitofrontal cortex; VO, ventral orbitofrontal cortex; VLO, ventrolateral orbitofrontal cortex; LO, lateral orbitofrontal cortex; DLO, dorsolateral orbitofrontal cortex; mPFC, medial prefrontal cortex; Cg1, anterior cingulate cortex; dPL, dorsal prelimbic cortex; vPL, ventral prelimbic cortex; IL, infralimbic cortex; NAc, nucleus accumbens; M shell, medial part of the nucleus accumbens shell; L shell, lateral part of the nucleus accumbens shell; DM, dorsomedial striatum; VM, ventromedial striatum; DL, dorsolateral striatum; VL, ventrolateral striatum; STN, subthalamic nucleus; VTA, ventral tegmental area; tVTA, tail of the ventral tegmental area; RMTg, rostromedial tegmental nucleus.

*P < 0.05, **P < 0.01, ***P < 0.001 different from NLv group; +P < 0.05, ++P < 0.01, +++P < 0.001 different from OFF group, Tukey's test.

Figure 1.

Schematic representation of the brain regions analyzed. Numbers above each coronal section represent the distance from bregma in millimeters. Numbers in the black squares represent the regions analyzed as follows (area in square millimetre for each AP level analyzed): 1, medial orbitofrontal cortex (0.359); 2, ventral orbitofrontal cortex (0.359); 3, ventrolateral orbitofrontal cortex (0.359); 4, lateral orbitofrontal cortex (0.359); 5, dorsolateral orbitofrontal cortex (0.251); 6, anterior cingulate cortex (0.486); 7, dorsal prelimbic cortex (0.349); 8, ventral prelimbic cortex (0.349); 9, infralimbic cortex (0.251); 10, nucleus accumbens core (0.251); 11, medial part of the nucleus accumbens shell (0.251); 12, lateral part of the nucleus accumbens shell (0.158); 13, dorsomedial quadrant of the striatum (0.486); 14, ventromedial quadrant of the striatum (0.486); 15, dorsolateral quadrant of the striatum (0.486); 16, ventrolateral quadrant of the striatum (0.486); 17, central quadrant of the striatum (0.486); 18, medial septum (0.137); 19, lateral septum (0.137); 20, lateral hypothalamus (0.359); 21, lateral habenula (0.251); 22, subthalamic nucleus (0.393 ± 0.025 and 0.442 ± 0.019 for the anterior and posterior level, respectively); 23, medial ventral tegmental area (0.251); 24, lateral ventral tegmental area (0.251); 25, tail of the ventral tegmantal area (0.179); 26, rostromedial tegmental nucleus (0.359); 27, medial raphe (0.137); 28, dorsal raphe (0.291 ± 0.030, 0.581 ± 0.040, 0.869 ± 0.041 at the rostral, middle, and caudal levels, respectively). Drawings are adapted from Paxinos and Watson atlas (Paxinos and Watson 2007).

Figure 1.

Schematic representation of the brain regions analyzed. Numbers above each coronal section represent the distance from bregma in millimeters. Numbers in the black squares represent the regions analyzed as follows (area in square millimetre for each AP level analyzed): 1, medial orbitofrontal cortex (0.359); 2, ventral orbitofrontal cortex (0.359); 3, ventrolateral orbitofrontal cortex (0.359); 4, lateral orbitofrontal cortex (0.359); 5, dorsolateral orbitofrontal cortex (0.251); 6, anterior cingulate cortex (0.486); 7, dorsal prelimbic cortex (0.349); 8, ventral prelimbic cortex (0.349); 9, infralimbic cortex (0.251); 10, nucleus accumbens core (0.251); 11, medial part of the nucleus accumbens shell (0.251); 12, lateral part of the nucleus accumbens shell (0.158); 13, dorsomedial quadrant of the striatum (0.486); 14, ventromedial quadrant of the striatum (0.486); 15, dorsolateral quadrant of the striatum (0.486); 16, ventrolateral quadrant of the striatum (0.486); 17, central quadrant of the striatum (0.486); 18, medial septum (0.137); 19, lateral septum (0.137); 20, lateral hypothalamus (0.359); 21, lateral habenula (0.251); 22, subthalamic nucleus (0.393 ± 0.025 and 0.442 ± 0.019 for the anterior and posterior level, respectively); 23, medial ventral tegmental area (0.251); 24, lateral ventral tegmental area (0.251); 25, tail of the ventral tegmantal area (0.179); 26, rostromedial tegmental nucleus (0.359); 27, medial raphe (0.137); 28, dorsal raphe (0.291 ± 0.030, 0.581 ± 0.040, 0.869 ± 0.041 at the rostral, middle, and caudal levels, respectively). Drawings are adapted from Paxinos and Watson atlas (Paxinos and Watson 2007).

Data Analysis

All data were subjected to mixed analyses of variance, followed by appropriate post hoc comparisons using the Tukey's Honestly Significant Difference test. Comparisons of percent scores of response inhibition to the control level of 100% were performed using Student's t-test. Correlation analyses were performed between the number of lever presses during the DS- period on the test session from rats of the OFF and ON groups (n = 8 rats) and 1) the number of Fos+ cells/mm2 in all ROIs and 2) the number of Fos+TH+ or Fos+TPH+ cells/mm2 in the VTA and raphe nuclei, respectively. In some brain regions, we also analyzed the effect size of the relevant groups by calculating the Hedge's g (Hentschke and Stuttgen 2011). Statistical analyses were run using Statistica, version 7.1 (Statsoft, Inc., Maisons-Alfort, France).

Results

All Fos data reported in the present paper are from the same rats tested for behavior in the study by Mihindou et al. (2013).

Region-Dependent Patterns of Fos Expression Associated with Inhibition and Disinhibition of Cocaine Seeking

Table 1 summarizes the data for each group and each brain region at all AP levels examined. Two main patterns of Fos expression were observed and are referred to as “inhibition” and “disinhibition” patterns (Fig. 2A). Representative pictures of single Fos labeling are shown in Figure 2B,C and significant results are compiled in Figures 36. The inhibition pattern is defined as a significant increase (or decrease) in Fos+ cells in the ON group compared with the OFF and/or NLv group (Fig. 2A). The disinhibition pattern is defined as statistically significant change (increase or decrease) in Fos+ cells in the OFF group compared with the ON and/or NLv group (Fig. 2A). Another pattern named “common effect” (Fig. 2A) shows similar changes in Fos expression in the ON and OFF groups that differ significantly from the NLv group and may reflect the fact that levers are retracted only in the NLv group. Finally, a “no-effect” pattern (Fig. 2A) shows no significant difference in Fos expression between the 3 groups. The inhibition (Fig. 3) and disinhibition (Fig. 4) patterns were observed in restricted subregions of the cortical and striatal complexes and paralleled by corresponding patterns in distinct monoaminergic subnuclei of the midbrain (Fig. 6). Only the inhibition pattern was further observed in hypothalamic, epithalamic, and subthalamic nuclei (Fig. 5). These 2 patterns may provide distinct and overlapping neural correlates for the inhibition (ON group) and disinhibition (OFF group) of cocaine seeking. Representative pictures of double labeling for Fos and TH in the VTA or Fos and TPH in raphe nuclei are shown in Supplementary Figure 2 and data are summarized in Figures 7 and 8, respectively. Figure 9 provides an overall picture of the Fos-activated brain regions in the ON and OFF conditions and the corresponding hypothetical brain circuits recruited during inhibition and disinhibition of cocaine seeking. Two criteria were used to define a circuit. A set of Fos-activated brain regions is considered to represent a circuit: 1) if they display a similar inhibition or disinhibition pattern and 2) if they are known to be anatomically and/or functionally connected (see Discussion).

Figure 2.

Observed Fos expression patterns and their significance. (A) Representative histograms show patterns of increased Fos expression but the same reasoning also applies to patterns of decreased Fos expression. The inhibition pattern shows significant changes in Fos expression in the ON group compared with NLv and OFF groups, as illustrated in the medial nucleus accumbens shell (B). This pattern is associated with the ability of rats in the ON group to inhibit cocaine seeking. The disinhibition pattern shows significant changes in Fos expression in the OFF group compared with NLv and/or ON groups, as illustrated in the lateral nucleus accumbens shell (C). A difference from both the NLv and ON groups may reflect the expression of cocaine seeking, while a difference from the ON group may specifically reflect a disinhibited response to cocaine. Pattern of common effects in the ON and OFF groups versus NLv group and pattern showing no difference between groups were also observed. (B and C) Photomicrographs of Fos labeling in the medial (Msh; B) and lateral (Lsh; C) nucleus accumbens shell of representative rats from the NOL, OFF, and ON groups showing an inhibition (B) and a disinhibition (C) pattern, respectively. Fos-immunoreactive nuclei (indicated by black arrows) were counted within the squared area depicted on the coronal sections taken at 1.56 (B) and 0.84 (C) mm from bregma, which correspond, respectively, to the middle Msh and caudal Lsh (×10 magnification). ICjM: islands of Calleja; c: extension of the anterior commissure. Scale bar: 200 µm.

Figure 2.

Observed Fos expression patterns and their significance. (A) Representative histograms show patterns of increased Fos expression but the same reasoning also applies to patterns of decreased Fos expression. The inhibition pattern shows significant changes in Fos expression in the ON group compared with NLv and OFF groups, as illustrated in the medial nucleus accumbens shell (B). This pattern is associated with the ability of rats in the ON group to inhibit cocaine seeking. The disinhibition pattern shows significant changes in Fos expression in the OFF group compared with NLv and/or ON groups, as illustrated in the lateral nucleus accumbens shell (C). A difference from both the NLv and ON groups may reflect the expression of cocaine seeking, while a difference from the ON group may specifically reflect a disinhibited response to cocaine. Pattern of common effects in the ON and OFF groups versus NLv group and pattern showing no difference between groups were also observed. (B and C) Photomicrographs of Fos labeling in the medial (Msh; B) and lateral (Lsh; C) nucleus accumbens shell of representative rats from the NOL, OFF, and ON groups showing an inhibition (B) and a disinhibition (C) pattern, respectively. Fos-immunoreactive nuclei (indicated by black arrows) were counted within the squared area depicted on the coronal sections taken at 1.56 (B) and 0.84 (C) mm from bregma, which correspond, respectively, to the middle Msh and caudal Lsh (×10 magnification). ICjM: islands of Calleja; c: extension of the anterior commissure. Scale bar: 200 µm.

Figure 3.

Inhibition pattern of Fos expression in the cortical and striatal macrosystems. Data are the mean (± s.e.m.) density of Fos-positive (Fos+) cells in restricted subregions of the cortex and the striatum in rats exposed to the DS- (ON), not exposed to the DS- with (OFF) or without levers out (NLv). In the illustrated subregions, Fos expression is significantly increased in the ON group compared with the OFF (+P < 0.05, ++P < 0.01, +++P < 0.001; Tukey's test) and/or NLv (*P < 0.05, **P < 0.01, ***P < 0.001) groups (n = 4 rats/group). c dPL: caudal part of the dorsal prelimbic cortex; c vPL, caudal part of the ventral prelimbic cortex; c IL, caudal part of the infralimbic cortex; c VLO, caudal part of the ventrolateral orbitofrontal cortex; c LO, caudal part of the lateral cortex; r DM, rostral part of the dorsomedial quadrant of the striatum; r DL, rostral part of the dorsolateral quadrant of the striatum; c CS, caudal part of the central quadrant of the striatum; m Msh, middle part of the medial nucleus accumbens shell; c core, caudal part of the nucleus accumbens core.

Figure 3.

Inhibition pattern of Fos expression in the cortical and striatal macrosystems. Data are the mean (± s.e.m.) density of Fos-positive (Fos+) cells in restricted subregions of the cortex and the striatum in rats exposed to the DS- (ON), not exposed to the DS- with (OFF) or without levers out (NLv). In the illustrated subregions, Fos expression is significantly increased in the ON group compared with the OFF (+P < 0.05, ++P < 0.01, +++P < 0.001; Tukey's test) and/or NLv (*P < 0.05, **P < 0.01, ***P < 0.001) groups (n = 4 rats/group). c dPL: caudal part of the dorsal prelimbic cortex; c vPL, caudal part of the ventral prelimbic cortex; c IL, caudal part of the infralimbic cortex; c VLO, caudal part of the ventrolateral orbitofrontal cortex; c LO, caudal part of the lateral cortex; r DM, rostral part of the dorsomedial quadrant of the striatum; r DL, rostral part of the dorsolateral quadrant of the striatum; c CS, caudal part of the central quadrant of the striatum; m Msh, middle part of the medial nucleus accumbens shell; c core, caudal part of the nucleus accumbens core.

Figure 4.

Disinhibition pattern of Fos expression in the cortical and striatal macrosystems. Data are the mean (± SEM) density of Fos+ cells in restricted subregions of the cortex and the striatum in rats exposed to the DS- (ON), not exposed to the DS- with (OFF) or without levers out (NLv). In the illustrated subregions, Fos expression in the OFF group is significantly different from the ON (+P < 0.05, ++P < 0.01; Tukey's test) and/or NLv (*P < 0.05, **P < 0.01) groups (n = 4 rats/group). c Cg1, caudal part of the anterior cingulate cortex; m VO, middle part of the ventral orbitofrontal cortex; m VM, middle part of the ventromedial quadrant of the striatum; c MS, caudal part of the medial septum; c Lsh, caudal part of the lateral nucleus accumbens shell.

Figure 4.

Disinhibition pattern of Fos expression in the cortical and striatal macrosystems. Data are the mean (± SEM) density of Fos+ cells in restricted subregions of the cortex and the striatum in rats exposed to the DS- (ON), not exposed to the DS- with (OFF) or without levers out (NLv). In the illustrated subregions, Fos expression in the OFF group is significantly different from the ON (+P < 0.05, ++P < 0.01; Tukey's test) and/or NLv (*P < 0.05, **P < 0.01) groups (n = 4 rats/group). c Cg1, caudal part of the anterior cingulate cortex; m VO, middle part of the ventral orbitofrontal cortex; m VM, middle part of the ventromedial quadrant of the striatum; c MS, caudal part of the medial septum; c Lsh, caudal part of the lateral nucleus accumbens shell.

Figure 5.

Inhibition pattern of Fos expression in hypothalamic, epithalamic and subthalamic nuclei. Data are the mean (± SEM) density of Fos+ cells in restricted subregions of hypothalamic, epithalamic and subthalamic nuclei in rats exposed to the DS- (ON), not exposed to the DS- with (OFF) or without levers out (NLv). In all illustrated subregions, Fos expression is significantly increased in the ON group compared with the OFF (+P < 0.05, ++P < 0.01, +++P < 0.001; Tukey's test) and/or NLv (*P < 0.05, **P < 0.01, ***P < 0.001) groups (n = 4 rats/group). m LH, middle part of the lateral hypothalamus; c LH, caudal part of the lateral hypothalamus; m LHb, middle part of the lateral habenula; p STN, posterior part of the subthalamic nucleus.

Figure 5.

Inhibition pattern of Fos expression in hypothalamic, epithalamic and subthalamic nuclei. Data are the mean (± SEM) density of Fos+ cells in restricted subregions of hypothalamic, epithalamic and subthalamic nuclei in rats exposed to the DS- (ON), not exposed to the DS- with (OFF) or without levers out (NLv). In all illustrated subregions, Fos expression is significantly increased in the ON group compared with the OFF (+P < 0.05, ++P < 0.01, +++P < 0.001; Tukey's test) and/or NLv (*P < 0.05, **P < 0.01, ***P < 0.001) groups (n = 4 rats/group). m LH, middle part of the lateral hypothalamus; c LH, caudal part of the lateral hypothalamus; m LHb, middle part of the lateral habenula; p STN, posterior part of the subthalamic nucleus.

Figure 6.

Inhibition and disinhibition patterns of Fos expression in midbrain nuclei. Data are the mean (±SEM) density of Fos+ cells in restricted subregions of midbrain nuclei of rats exposed to the DS- (ON), not exposed to the DS- with (OFF) or without levers out (NLv). Inhibition patterns are observed in the M VTA with a significant increase in Fos expression in the ON group compared with the OFF (++P < 0.01; Tukey's test) and NLv (**P < 0.01) groups (n = 4 rats/group). Disinhibition patterns are shown in other brain regions with a significant increase in Fos expression for the OFF group compared with the ON (+P < 0.05, ++P < 0.01) and/or NLv (*P < 0.05, **P < 0.01) groups (n = 4 rats/group). r M VTA, rostral part of the medial ventral tegmental area; r L VTA, rostral part of the lateral ventral tegmental area; c DR, caudal part of the dorsal raphe; p MR, posterior part of the medial raphe; p tVTA, posterior part of the tail of the ventral tegmental area; RMTg, rostromedial tegmental nucleus.

Figure 6.

Inhibition and disinhibition patterns of Fos expression in midbrain nuclei. Data are the mean (±SEM) density of Fos+ cells in restricted subregions of midbrain nuclei of rats exposed to the DS- (ON), not exposed to the DS- with (OFF) or without levers out (NLv). Inhibition patterns are observed in the M VTA with a significant increase in Fos expression in the ON group compared with the OFF (++P < 0.01; Tukey's test) and NLv (**P < 0.01) groups (n = 4 rats/group). Disinhibition patterns are shown in other brain regions with a significant increase in Fos expression for the OFF group compared with the ON (+P < 0.05, ++P < 0.01) and/or NLv (*P < 0.05, **P < 0.01) groups (n = 4 rats/group). r M VTA, rostral part of the medial ventral tegmental area; r L VTA, rostral part of the lateral ventral tegmental area; c DR, caudal part of the dorsal raphe; p MR, posterior part of the medial raphe; p tVTA, posterior part of the tail of the ventral tegmental area; RMTg, rostromedial tegmental nucleus.

Cortical and Striatal Macrosystems

As previously reported (Mihindou et al. 2013), the inhibition pattern was primarily observed in the PL. Specifically, we show here that this pattern was homogeneously observed throughout the entire dorso-ventral and AP extents of the PL (group × dorso-ventral × AP: F4,18 = 0.05, P = 0.99; Table 1 and Fig. 3). The presentation of the DS- in the ON group was associated with a large increase in Fos expression in the dorsal PL (group: F2,9 = 5.78 and 7.11, P < 0.05 at the rostral and middle levels, respectively, and F2,9 = 31.33, P < 0.001 at the caudal level) and ventral PL (group: F2,9 = 10.77, P < 0.01 at the rostral level and F2,9 = 17.82 and 31.36, P < 0.001 at the middle and caudal levels, respectively). Although this increase was previously shown to be specific to the PL but not IL (Mihindou et al. 2013), a more thorough examination of Fos pattern in the AP extent of the IL revealed a significant increase in Fos expression at the caudal level of this subregion in the ON group compared with the NLv group (group: F2,9 = 6.27, P < 0.05; Fig. 3). The size of this effect was relatively large (Hedge's g = 1.51). The inhibition pattern was further observed throughout the entire AP extent of the ventrolateral OFC (VLO; group: F2,9 = 13.77, P < 0.01 at the rostral level and F2,9 = 6.85 and 6.62, P < 0.05 at the middle and caudal levels, respectively) and at the middle and caudal levels of the lateral OFC (LO; group: F2,9 = 10.25 and 14.59, respectively, P < 0.01; Table 1 and Fig. 3).

In the striatal complex, inhibition of cocaine seeking in the ON group was associated with increased Fos expression in confined subregions of the striatum and NAc. A significant increase in Fos expression in the ON group compared with the OFF and NLv groups was restricted to the rostral level of the DM and DL quadrants of the striatum (group: F2,9 = 6.86 and 6.08, respectively, P < 0.05; Table 1 and Fig. 3) and the caudal part of the central striatum (group: F2,9 = 7.09, P < 0.05; Table 1 and Fig. 3). In the NAc, the inhibition pattern was specifically observed at the middle level of the medial shell (group: F2,9 = 7.08, P < 0.05; Table 1 and Fig. 3) and at the caudal level of the core (group: F2,9 = 8.34, P < 0.01; Table 1 and Fig. 3).

Fewer cortical and striatal brain regions displayed a disinhibition pattern. Lower Fos expression in the OFF group compared with the ON and NLv groups was observed at the rostral, middle, and caudal AP levels of the ACC (Cg1; group: F2,9 = 9.29, 9.41, and 12.64, respectively, P < 0.01; Table 1 and Fig. 4), at the rostral and middle levels of the ventral OFC (VO; group: F2,9 = 8.33 and 8.81, respectively, P < 0.01; Table 1 and Fig. 4) and at the caudal level of the medial septum (MS; group: F2,9 = 7.92, P < 0.05; Table 1 and Fig. 4). Lower Fos expression in the OFF group compared with the ON group was observed at the rostral and middle levels of the ventromedial (VM) striatum (group: F2,9 = 5.99, P < 0.05 and 10.98, P < 0.01, respectively; Table 1 and Fig. 4) but did not reach significance in the AP extent of the ventrolateral (VL) striatum (Table 1; Fig. 4). Lower Fos expression in the OFF group compared with the NLv group was observed at the middle level of the MS (group: F2,9 = 5.66, P < 0.05; Table 1). Interestingly, an inverted pattern was observed at the posterior level of the lateral NAc shell, with a significant increase in Fos expression in the OFF group compared with the NLv group (group: F2,9 = 6.49, P < 0.05; Table 1 and Fig. 4).

Hypothalamic, Epithalamic and Subthalamic Nuclei

Analysis of Fos expression at 3 different AP levels of the lateral hypothalamus (LH) revealed 3 distinct patterns (Table 1). First, the inhibition pattern appeared only at the middle level of the LH (group: F2,9 = 6.04, P < 0.05; Fig. 5). Second, no difference between groups was seen at the rostral level (group: F2,9 = 0.49, P = 0.63 ; Table 1). Third, the “common effect” pattern (see Fig. 2A) was observed at the caudal level with a significant and similar increase in Fos expression in the ON and OFF groups compared with the NLv group (group: F2,9 = 7.95, P < 0.05; Fig. 5). A strong inhibition pattern was restricted to the middle level of the lateral habenula (LHb; group: F2,9 = 46.11, P < 0.001; Table 1 and Fig. 5) and the posterior level of the subthalamic nucleus (STN; group: F2,9 = 17.36, P < 0.001; Table 1 and Fig. 5). A significant increase in Fos expression in the ON group compared with the OFF group was also observed at the anterior level of the STN (group: F2,9 = 5.12, P < 0.05; Table 1).

Midbrain Nuclei

Analysis of Fos expression in the VTA showed strong AP differences between groups within the medio-lateral extent of the VTA (group × medio-lateral × AP: F4,18 = 3.87, P < 0.05; Table 1, Fig. 6). At the rostral level, an inhibition pattern was observed in the medial VTA (MVTA; group: F2,9 = 13.11, P < 0.01; Table 1 and Fig. 6), whereas a disinhibition pattern was seen in the lateral VTA (LVTA; group: F2,9 = 6.78, P < 0.05; Table 1 and Fig. 6). A significant increase in Fos expression in the ON group compared with the NLv group was found at the middle, but not caudal, MVTA while a significant increase in Fos expression in the OFF group compared with the NLv group was found at the middle, but not caudal, LVTA (Table 1). Analysis of Fos/TH immunolabeling showed that the increased Fos expression in the rostral MVTA of the ON group resulted in a proportional increase in both TH+ and TH-negative (TH) cells (Fig. 7A; see representative pictures in Supplementary Fig. 2A). At the middle level, however, a significant decrease in the proportion of Fos+TH+ cells was observed in the OFF group (see pie charts in Fig. 7A: P < 0.05 versus ON and NLv groups). In the LVTA, the increase in Fos labeling in the OFF group is mainly located in TH cells (Fig. 7B). Indeed, the proportion of Fos+TH+ cells was significantly decreased in the OFF group compared with NLv (see pie charts: Tukey's test, P < 0.05 at the 3 AP levels) and/or ON (P < 0.05 at the caudal level) groups. A significant positive correlation was found between the number of lever presses during the DS- period on the test day for the ON and OFF groups and the number of Fos+TH+ cells in the caudal LVTA (r = −0.74, P < 0.05; data not shown).

Figure 7.

Expression pattern of total Fos+ cells and Fos+ cells in tyrosine hydroxylase-positive (TH+) cells in the medial (A) and lateral (B) ventral tegmental area (VTA) of rats exposed to the DS- (ON), not exposed to the DS- with (OFF) or without levers out (NLv). Histogram data are expressed as the mean (±SEM) density of total Fos+ cells (left x-axis for black histograms) or the mean (±SEM) density of Fos+TH+ cells (right x-axis for white histograms; n = 4 rats/group). Pie charts represent the relative proportion of TH+ and TH cells expressing Fos in percentage of total Fos+ cells for each group and at each antero-posterior level examined. Labeled percentages are for TH+Fos+ cells (white portion). **P < 0.01 versus NLv group and ++P < 0.01 versus OFF group for Total Fos+ cells/mm2; °P < 0.05, °°P < 0.01 versus NLv group and §P < 0.05, §§P < 0.01 versus OFF group for Fos+TH+ cells/mm2 and pie charts, Tukey's test.

Figure 7.

Expression pattern of total Fos+ cells and Fos+ cells in tyrosine hydroxylase-positive (TH+) cells in the medial (A) and lateral (B) ventral tegmental area (VTA) of rats exposed to the DS- (ON), not exposed to the DS- with (OFF) or without levers out (NLv). Histogram data are expressed as the mean (±SEM) density of total Fos+ cells (left x-axis for black histograms) or the mean (±SEM) density of Fos+TH+ cells (right x-axis for white histograms; n = 4 rats/group). Pie charts represent the relative proportion of TH+ and TH cells expressing Fos in percentage of total Fos+ cells for each group and at each antero-posterior level examined. Labeled percentages are for TH+Fos+ cells (white portion). **P < 0.01 versus NLv group and ++P < 0.01 versus OFF group for Total Fos+ cells/mm2; °P < 0.05, °°P < 0.01 versus NLv group and §P < 0.05, §§P < 0.01 versus OFF group for Fos+TH+ cells/mm2 and pie charts, Tukey's test.

In the tVTA, Fos expression paralleled the disinhibition pattern of the LVTA at both AP levels (group: F2,9 = 6.40 and 5.40, P < 0.05 at the anterior and posterior levels, respectively; Table 1 and Fig. 6). In the RMTg, Fos expression was significantly lower in the ON group compared with the OFF group (group: F2,9 = 10.84, P < 0.01; Table 1, Fig. 6). A positive correlation between the number of lever presses during the DS- period on the test day for the ON and OFF groups, and the number of Fos+ cells was found in the posterior tVTA (r = 0.73, P < 0.05; data not shown).

In the dorsal raphe (DR), a disinhibition pattern was observed at all AP levels (group: F2,9 = 8.33, 11.06, 15.76, P < 0.01 at the rostral, middle, and caudal levels, respectively; Table 1, Fig. 6) with higher Fos+ cells in the OFF group compared with both NLv and ON groups. In the median raphe (MR), a significant decrease in the number of Fos+ cells in the ON group compared with the OFF group was found at the posterior level (group: F2,9 = 5.25, P < 0.05; Table 1, Fig. 6). Analysis of Fos/TPH immunolabeling in the DR indicated that the increased Fos expression in the OFF group resulted from an increased proportion of Fos+TPH cells at the rostral level (see representative pictures in Supplementary Fig. 2B and pie chart in Fig. 8A: P < 0.05 versus NLv and ON groups) while it resulted from a similar increase in both Fos+TPH+ and Fos+TPH cells at the middle and caudal levels (Fig. 8A). In the MR, however, the decrease in total Fos+ cells in the ON group resulted from a selective deactivation of TPH cells since Fos was expressed in TPH+ cells only at both AP levels (see pie charts in Fig. 8B: P < 0.05 versus NLv and OFF groups). A positive correlation between the number of lever presses during the DS- period on the test day for the ON and OFF groups and the number of Fos+ cells was found at the rostral DR (r = 0.91, P < 0.001), caudal DR (r = 0.80, P < 0.05), and anterior MR (r = 0.88, P < 0.01).

Figure 8.

Expression pattern of total Fos+ cells and Fos+ cells in tryptophan hydroxylase-positive (TPH+) cells in the dorsal (A) and median (B) raphe of rats exposed to the DS- (ON), not exposed to the DS- with (OFF) or without levers out (NLv). Histogram data are expressed as the mean (±SEM) density of total Fos+ cells (left x-axis for black histograms) or the mean (±SEM) density of Fos+TPH+ cells (right x-axis for white histograms; n = 4 rats/group). Pie charts represent the respective proportion of TPH+ and TPH cells expressing Fos in percentage of total Fos+ cells for each group and at each antero-posterior level examined. Labeled percentages are for TPH+Fos+ cells (white portion) **P < 0.01 versus NLv group and +P < 0.05, ++P < 0.01 versus OFF group for Total Fos+ cells/mm2; °P < 0.05 versus NLv group and §P < 0.05 versus OFF group for Fos+TPH+ cells/mm2 and pie charts, Tukey's test.

Figure 8.

Expression pattern of total Fos+ cells and Fos+ cells in tryptophan hydroxylase-positive (TPH+) cells in the dorsal (A) and median (B) raphe of rats exposed to the DS- (ON), not exposed to the DS- with (OFF) or without levers out (NLv). Histogram data are expressed as the mean (±SEM) density of total Fos+ cells (left x-axis for black histograms) or the mean (±SEM) density of Fos+TPH+ cells (right x-axis for white histograms; n = 4 rats/group). Pie charts represent the respective proportion of TPH+ and TPH cells expressing Fos in percentage of total Fos+ cells for each group and at each antero-posterior level examined. Labeled percentages are for TPH+Fos+ cells (white portion) **P < 0.01 versus NLv group and +P < 0.05, ++P < 0.01 versus OFF group for Total Fos+ cells/mm2; °P < 0.05 versus NLv group and §P < 0.05 versus OFF group for Fos+TPH+ cells/mm2 and pie charts, Tukey's test.

Discussion

Our large-scale Fos brain mapping study reveals that effective inhibitory control of cocaine seeking in rats is not limited to the neuronal activation of the PL subdivision of the PFC (Mihindou et al. 2013) but is associated with the coordinated recruitment of different top-down cortical-subcortical circuits originating from different PFC territories, and of different bottom-up DA and 5-HT midbrain subsystems that normally modulate activity in these circuits. This integrated brain response likely reflects the fact that rats from the ON group are concomitantly engaging and experiencing intricate cognitive and affective processes when they have to inhibit cocaine seeking in response to the DS-. For instance, they may engage cognitive processes involved in selective attention, context monitoring, interoception, and behavioral inhibition. They may also experience affective reactions due to lack of reinforcement, like frustration and/or aversion. In contrast, our study also reveals that disinhibition of cocaine seeking (OFF group) was generally associated with a de-activation of brain regions involved in behavioral inhibition and activation of brain regions involved in reward seeking and motivation.

Fos Pattern Associated with Inhibition of Cocaine Seeking

Top-Down Cortical Networks

Different functional subdivisions of the PFC, projecting to different striatal territories, were selectively Fos-activated during inhibition of cocaine seeking. First, we previously reported that the PL subregion of the mPFC was Fos-activated during inhibition of cocaine seeking (Mihindou et al. 2013). Here, we confirm and extend this finding to the entire AP and dorso-ventral extent of the PL (Gabbott et al. 2005), thereby further suggesting that the PL is the functional homolog of the human lateral PFC which is also activated during inhibitory control of drug craving (Kober et al. 2010b; Volkow et al. 2010). Second, a small restricted population of IL neurons, only at the caudal level, was also recruited during inhibition of cocaine seeking. There was no change of Fos expression at other AP levels in the IL which confirms previous research (Mihindou et al. 2013). Third, beyond the mPFC, there was also a robust increase in Fos expression in the OFC, specifically in the VLO and LO subregions. Recruitment of these different PFC subdivisions was associated with a topographic Fos activation of their corresponding striatal territories, including the NAc core, the DM and DL striatum (mostly PL territories), the NAc shell (IL territory) and the central striatum (OFC territory) (Voorn et al. 2004; Gabbott et al. 2005; Schilman et al. 2008). The co-activation of these different cortico-striatal pathways likely reflects the co-engagement of different psychological processes during inhibition of cocaine seeking, including cue-induced suppression of prepotent responses (PL-NAc core pathway; (Christakou et al. 2004)), inhibition of unreinforced actions (IL-NAc shell pathway; (Peters et al. 2008)) and flexible, context-dependent decision-making (lateral OFC–central striatum pathway; (Remijnse et al. 2006; Burke et al. 2009)). The strong Fos activation of the STN may reflect the recruitment of the inhibitory-response-control system involving the OFC–STR–STN circuit (Eagle and Baunez 2010) and promote accuracy of stopping at the onset of DS- presentation (Eagle et al. 2008). Interestingly, Fos activation of both the IL and the NAc shell during inhibition of cocaine seeking further supports a role for this pathway in inhibiting drug seeking both during discriminative control procedures (Ishikawa et al. 2008; Mihindou et al. 2013) and reinstatement procedures (Peters et al. 2008).

Bottom-Up Dopamine and Serotonin Systems

Overall, Fos activation of ascending DA and 5-HT brain systems during inhibition of cocaine seeking was consistent with their role in modulating information flow within cortico-striatal circuits (Robbins and Arnsten 2009). There was, however, a significant degree of specificity in Fos activation of these monoaminergic systems, at both the cellular and anatomical level, as detailed below.

Only the medial VTA was strongly Fos-activated during inhibition of cocaine seeking. When looking closer at the type of Fos-activated cells, we found that Fos was homogeneously expressed within TH+ (30–40%) and TH cells (60–70%) throughout the VTA (Fig. 7). Fos+TH neurons may correspond to GABA mesoaccumbens neurons, while Fos+TH+ neurons may mainly correspond to DA mesocortical neurons. Both types of neurons are preferentially activated by aversive stimuli (Lammel et al. 2008, 2011; Brischoux et al. 2009). Activation of GABA mesoaccumbens neurons contribute to suppression of firing in DA mesoaccumbens neurons in response to aversive events and drive conditioned aversion (Cohen et al. 2012; Tan et al. 2012). Activation of DA mesocortical neurons may indirectly influence accumbal synaptic plasticity by modulating either pyramidal neurons that project to the NAc or pyramidal neurons that project to GABA mesoaccumbens neurons (Carr et al. 1999; Carr and Sesack 2000). These findings suggest that inhibition of cocaine seeking is likely an aversive experience. This interpretation is further supported by Fos activation of the LHb that is known to send aversion-related information to both DA and GABA neurons in the VTA (Omelchenko et al. 2009; Stamatakis and Stuber 2012).

The VTA also receives strong afferents from the LH that could participate in Fos activation of the medial VTA (Mahler et al. 2012). Both middle and caudal levels of the LH were Fos-activated during inhibition of cocaine seeking. At the middle level of the LH (i.e., orexinergic field), orexin neurons encode discrete and contextual cues associated with reward seeking (Harris et al. 2005; Richardson and Aston-Jones 2012; Sartor and Aston-Jones 2012). Under the influence of the massive mPFC projections (Gabbott et al. 2005), the selective Fos activation of the middle LH may encode the contextual monitoring of DS- presentation that can be relayed indirectly to the VTA through the LHb to promote inhibition of cocaine seeking (Fadel and Deutch 2002; Poller et al. 2013). At the caudal level of the LH, Fos activation could reflect the recruitment of orexin neurons from the perifornical area that regulate arousal and stress in response to DS- and/or lack of reinforcement (Estabrooke et al. 2001; Winsky-Sommerer et al. 2004; Harris and Aston-Jones 2006).

In the raphe, inhibition of cocaine seeking mainly recruited 5-HT neurons as a result of deactivation of non-TPH, presumably GABA, neurons. Indeed, the remaining Fos activation in the MR was restricted to TPH+ cells and paralleled by a larger proportion of Fos+TPH+ cells in the DR (∼55%; Fig. 8). The parallel recruitment of 5-HT neurons with VTA DA neurons is consistent with their cooperative role in attributing affective valence (reward versus punishment) and in energizing action (invigoration versus inhibition) (Boureau and Dayan 2011; Cools et al. 2011). 5-HT neurons encode low salience stimuli or lack of reward, thus contributing to limit ineffective responding (Ranade and Mainen 2009; Li et al. 2013). Besides, Fos activation of 5-HT neurons may reflect the recruitment of cognitive inhibitory mechanisms through afferents to the mPFC that modulate decision-making and impulsivity (Clarke et al. 2004; Chudasama and Robbins 2006) and through afferents to the OFC that promote behavioral flexibility and reinforcer devaluation (Roberts 2011; West et al. 2013). Notably, the maintenance of elevated DR activity and 5-HT levels in target regions is correlated with successful withholding of responses (Miyazaki et al. 2012).

Fos Pattern Associated with Disinhibition of Cocaine Seeking

Overall, disinhibition of cocaine seeking was associated with disengagement of cortico-striatal circuits and of ascending modulatory DA and 5-HT systems that were active during inhibition of cocaine seeking. More interestingly, few brain regions were selectively activated or de-activated during disinhibition of cocaine as described below.

Top-Down Cortical Networks

There was a large and selective de-activation of Fos expression in the Cg1 in rats from the OFF group. Since the Cg1 is involved in context and/or performance monitoring, its de-activation probably indicates that cognitive monitoring is suspended during disinhibition of cocaine seeking (MacDonald et al. 2000; Kerns et al. 2004; Hyman et al. 2012). De-activation of the Cg1 may in turn explain the neural de-activation observed in downstream brain regions or circuits that are directly involved in behavioral inhibition. For instance, it may drive the disengagement of the VO-VM/VL striatal pathway (Ongur and Price 2000; Eagle and Robbins 2003; Mailly et al. 2013) and of the MS (Gong et al. 1995). By relaying stress and contextual information to the mPFC (Gaykema et al. 1990; Luo et al. 2011), the septum is known to promote neural processes underlying behavioral inhibition (Gorenstein and Newman 1980; Highfield et al. 2000).

Bottom-Up Dopamine and Serotonin Systems

During disinhibition of cocaine seeking, there was a parallel Fos activation of the lateral VTA and lateral NAc shell. Fos expression was mostly found in TH cells (∼85%) throughout the medio-lateral VTA (Fig. 7) and was mirrored by a larger proportion of Fos+TPH cells in the DR (∼65%) and MR (∼80%; Fig. 8). The main activation of these presumed GABA cells within both DA and 5-HT systems may promote termination of reward-related behaviors (Halberstadt and Balaban 2008; van Zessen et al. 2012). Indeed, after an initial disinhibition of cocaine seeking during DS- omission, rats progressively stop responding within a 10-min time scale of nonreinforced lever presses (Supplementary Fig. 1). In support of this hypothesis, the tVTA/RMTg was also robustly Fos-activated during disinhibition of cocaine seeking. This activation may occur concomitantly with VTA GABA neurons contributing to the inhibition of mesoaccumbens DA neurons, thereby regulating motor response outputs (Jhou et al. 2009; Kaufling et al. 2010; Barrot et al. 2012; Pan et al. 2013).

Potential Limitations and Alternative Interpretations

First, only rats from the ON group were exposed to the visual DS- on the final test day. It is thus possible that parts of the selective brain responses to the DS- reported here were due to its purely visual sensory and/or unconditioned arousing properties, and not to its conditioned behavioral significance. This is unlikely, however, because rats were repeatedly exposed to the DS-, making it a highly familiar stimulus at the final test. In addition, Fos activation in response to the DS- was mainly observed in brain regions that do not normally respond to pure sensory stimuli (Downar et al. 2002; Bastle et al. 2012).

Second, the brain activity seen in the OFF group, where the DS- stimulus is omitted during a period of cocaine unavailability, may not only reflect disinhibition of cocaine seeking, as assumed here, but also new learning, specifically extinction learning. Both the ON and OFF groups have learned that the DS- signals cocaine unavailability. When the DS- is presented to the ON group, no new learning is required because rats from this group are expecting that they will not receive cocaine. In contrast, in the OFF group, the period of cocaine unavailability is no longer signaled and, as a result, rats from this group should expect cocaine. Not receiving cocaine is thus now a surprising event that should spur extinction learning. This hypothesis is consistent with the burst of responding seen in the OFF group (Supplementary Fig. 1) which resembles a classic extinction burst. However, this hypothesis is only partially supported by our Fos data. Although some brain regions involved in new learning, such as the VTA, are indeed selectively Fos-activated in the OFF group (compared with both the NLv and ON groups), extinction learning-related brain regions, such as the PL, are not (Nic Dhonnchadha et al. 2012). Clearly, further research involving additional control groups will be needed to dissect the respective role of these different psychological processes (i.e., extinction learning and disinhibition) in the behavior of the OFF group.

Finally, brain regions that were robustly activated during inhibition of cocaine seeking were also previously shown to be involved in triggering cocaine seeking. This was the case for the PL, NAc core, LH, and VTA (McFarland et al. 2003, 2004; Harris et al. 2005; Hamlin et al. 2008; Marchant et al. 2009). Regarding the PL, this apparent contradiction may be resolved by postulating that the PL has a more general function in controlling cocaine seeking than inhibition or excitation per se. For instance, the PL plays a fundamental role in context monitoring (Chatham et al. 2012)—a function that is both involved in the inhibition (Mihindou et al. 2013) and activation of cocaine seeking in rats (Bouton 2002). Regarding the other brain regions, Fos activation of the NAc, LH, and VTA during reinstatement (Mahler and Aston-Jones 2012) or inhibition of cocaine seeking (present study) may reflect the recruitment of distinct neuronal ensembles (Pennartz et al. 1994; McFarland and Kalivas 2001). According to this idea, inputs from the PL may activate a particular subset of neurons in the NAc, LH, and VTA, which may reciprocally influence each other to activate or inhibit cocaine seeking. For instance, a dorso-ventral compartmentalization of medial NAc shell projections to different neurochemical populations of the LH can either promote (ventral) or prevent (dorsal) drug seeking (Marchant et al. 2009; McNally 2014). In the VTA (and raphe nuclei), our high-resolution Fos analysis and double-labeling approach allowed us to reveal that the involvement of the VTA in inhibition and disinhibition of cocaine seeking actually rely on the recruitment of distinct neuronal ensembles made of aminergic and/or nonaminergic neurons. This suggests that the implementation of a specific neurochemical DA/5-HT balance by direct projections from the VTA and raphe nuclei throughout the PFC–NAc–LH pathways may help translating contextual information to promote or inhibit cocaine seeking (Goto and Grace 2008; Boureau and Dayan 2011).

Concluding Remarks

The present findings describe a specific pattern of activation/deactivation of restricted brain regions during inhibition and disinhibition of cocaine seeking (Fig. 9). It is proposed that the coordinated recruitment of cortical-subcortical “executive” and “limbic” circuits together with the ascending DA/5-HT “motivational drive” (Dalley et al. 2004; Kehagia et al. 2010) may converge on striatal domains for integration of cognitive and reward/aversion processes that promote appropriate goal-directed behavioral output, that is, effective discriminative inhibition of cocaine seeking. Overall, this study shows that, even after extended cocaine use, rats can be successfully trained to engage whole-brain inhibitory control mechanisms to inhibit intense cocaine seeking, as was recently shown in drug abusers (Kober et al. 2010a, 2010b; Volkow et al. 2010). Studying the factors that facilitate the use of this cognitive capacity may prove particularly helpful for relapse prevention.

Figure 9.

Summary of Fos-activated brain regions associated with inhibition (ON) and disinhibition (OFF) of cocaine seeking. Hypothetical brain circuits are depicted by arrows between the main interconnected brain regions but intermediate structures could also be involved. Although not illustrated here, some subcortical structures (i.e., LH, VTA, DR) also send projections to the PFC and may control PFC neuronal activity. Left diagram: inhibition of cocaine seeking may result in activation of circuits involved in attention (1: PL-DStr) and cognitive flexibility (1: PL-core), suppression of unreinforced action (2: IL-shell), affective switching (3: VLO/LO-CStr), impulse control (4: mPFC-Str/STN), and aversion (5: LHb-MVTA-MShell/mPFC). Fos was homogeneously expressed in both aminergic and nonaminergic cells of the VTA and DR, except in the MR where the remained Fos expression was selectively observed in 5-HT cells. This Fos pattern may encode negative salience triggered by the DS- presentation and promote immediate inhibition of cocaine seeking. Right diagram: disinhibition of cocaine seeking may result in activation of circuits involved in reward (6: LVTA-LShell) and aversion processing (tVTA/RMTg-MVTA) together with the disengagement of circuits involved in impulse control (4: mPFC-Str) and arousal/stress/interoception processes (7: Cg1-MS-LH). In the VTA and raphe subnuclei, Fos was more abundantly expressed in nonaminergic cells (GABA and/or glutamate) compared with aminergic cells (dotted line arrows) which may signal unexpected lack of reinforcement and promote the progressive loss of responding. Color intensity (dark, intermediate, and light) indicates the relative changes (higher, similar, and lower, respectively) in Fos expression with respect to the NOL group. Cg1, anterior cingulate cortex; PL, prelimbic cortex; IL, infralimbic cortex; VO, ventral orbitofrontal cortex; VLO, ventrolateral orbitofrontal cortex; LO, lateral orbitofrontal cortex; DStr, dorsal striatum; CStr, central striatum; VStr, ventral striatum; Msh, medial nucleus accumbens shell; Lsh, lateral nucleus accumbens shell; core, nucleus accumbens core; MS, medial septum; STN, subthalamic nucleus; mLH, cLH, middle and caudal lateral hypothalamus; MVTA, medial ventral tegmental area; LVTA, lateral ventral tegmental area; LHb, lateral habenula; tVTA/RMTg, tail of the VTA and rostromedial tegmental nucleus; DR, dorsal raphe; MR, median raphe. Glu, glutamate; DA, dopamine; 5-HT, serotonin.

Figure 9.

Summary of Fos-activated brain regions associated with inhibition (ON) and disinhibition (OFF) of cocaine seeking. Hypothetical brain circuits are depicted by arrows between the main interconnected brain regions but intermediate structures could also be involved. Although not illustrated here, some subcortical structures (i.e., LH, VTA, DR) also send projections to the PFC and may control PFC neuronal activity. Left diagram: inhibition of cocaine seeking may result in activation of circuits involved in attention (1: PL-DStr) and cognitive flexibility (1: PL-core), suppression of unreinforced action (2: IL-shell), affective switching (3: VLO/LO-CStr), impulse control (4: mPFC-Str/STN), and aversion (5: LHb-MVTA-MShell/mPFC). Fos was homogeneously expressed in both aminergic and nonaminergic cells of the VTA and DR, except in the MR where the remained Fos expression was selectively observed in 5-HT cells. This Fos pattern may encode negative salience triggered by the DS- presentation and promote immediate inhibition of cocaine seeking. Right diagram: disinhibition of cocaine seeking may result in activation of circuits involved in reward (6: LVTA-LShell) and aversion processing (tVTA/RMTg-MVTA) together with the disengagement of circuits involved in impulse control (4: mPFC-Str) and arousal/stress/interoception processes (7: Cg1-MS-LH). In the VTA and raphe subnuclei, Fos was more abundantly expressed in nonaminergic cells (GABA and/or glutamate) compared with aminergic cells (dotted line arrows) which may signal unexpected lack of reinforcement and promote the progressive loss of responding. Color intensity (dark, intermediate, and light) indicates the relative changes (higher, similar, and lower, respectively) in Fos expression with respect to the NOL group. Cg1, anterior cingulate cortex; PL, prelimbic cortex; IL, infralimbic cortex; VO, ventral orbitofrontal cortex; VLO, ventrolateral orbitofrontal cortex; LO, lateral orbitofrontal cortex; DStr, dorsal striatum; CStr, central striatum; VStr, ventral striatum; Msh, medial nucleus accumbens shell; Lsh, lateral nucleus accumbens shell; core, nucleus accumbens core; MS, medial septum; STN, subthalamic nucleus; mLH, cLH, middle and caudal lateral hypothalamus; MVTA, medial ventral tegmental area; LVTA, lateral ventral tegmental area; LHb, lateral habenula; tVTA/RMTg, tail of the VTA and rostromedial tegmental nucleus; DR, dorsal raphe; MR, median raphe. Glu, glutamate; DA, dopamine; 5-HT, serotonin.

Supplementary Material

Supplementary Material can be found at http://www.cercor.oxfordjournals.org/ online.

Funding

This work was supported by the French Research Council (CNRS), the French National Agency (ANR-2010-BLAN-1404-01 to S.H.A.), the Fondation NRJ, the Université Bordeaux Segalen, and the Conseil Régional d'Aquitaine (Convention Neurocampus 11004375/11004699 to S.H.A.).

Notes

We thank Ourida Gaucher and Samya Aribi for administrative assistance, Sandra Dovero, Evelyne Doudnikoff, and Matthieu Bastide for technical advice and Erwan Bezard and Etienne Gontier for giving us access to their laboratory facility. Finally, we also thank the reviewer for their helpful and constructive criticism. Conflict of Interest: None declared.

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