Discriminative Inhibitory Control of Cocaine Seeking Involves the Prelimbic Prefrontal Cortex

ARCHIVAL REPORT

Discriminative Inhibitory Control of Cocaine Seeking Involves the Prelimbic Prefrontal Cortex Claudia Mihindou, Karine Guillem, Sylvia Navailles, Caroline Vouillac, and Serge H. Ahmed Background: Recent neuroimaging studies have shown that people with cocaine addiction retain some degree of control over drug craving that correlates with neural activity in the lateral prefrontal cortex (PFC). Here, we report similar findings in a rat model of inhibitory control of cocaine seeking. Methods: Rats actively responding for cocaine were trained to stop responding when presented with a discriminative stimulus that signaled lack of reinforcement. Rats were then tested for inhibitory control of cocaine seeking in novel behavioral contexts and in circumstances when cocaine seeking is particularly intense (e.g., following drug priming). The role of neuronal activity in different subregions of the PFC was assessed using local pharmacologic inactivation and c-Fos immunohistochemistry. Results: Rats progressively acquired the ability to stop cocaine seeking, even during drug intoxication and after a long history of cocaine self-administration. Inhibitory control of cocaine seeking was flexible, sufficiently strong to block cocaine-primed reinstatement, and selectively depended on increased neuronal activity within the prelimbic PFC, which is considered the rodent functional homolog of the human lateral PFC. Conclusions: Parallel evidence in both animal models and humans indicate that recruitment of prefrontal inhibitory control of drug seeking is still functional after prolonged cocaine use. Preclinical investigation of the mechanisms underlying this capacity may contribute to designing new behavioral and/or pharmacologic strategies to promote its use for the prevention of relapse in addiction. Key Words: Behavioral inhibition, cognitive control, craving, prefrontal cortex, relapse, self-regulation

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ecent neuroimaging studies have shown that people with cocaine or tobacco addiction retain some degree of inhibitory control, as they can suppress drug cuesinduced craving when instructed to do so (1–3). In one prominent study, effective cognitive regulation of drug craving was associated with activation of the lateral prefrontal cortex (PFC) (2). The lateral PFC is normally engaged during top-down cognitive control of subcortically mediated emotional and motivational responses in healthy humans (2,4–14). This brain capacity may underlie the efficacy of behavioral therapies of addiction that seek to enhance, through training, use of cognitive control of craving for relapse prevention (2,15,16). Inhibitory control of cocaine-taking or cocaine-seeking behavior has only seldom been studied in animal models of addiction (17). However, available evidence shows that cocaine-exposed animals also retain some degree of inhibitory control over cocaine seeking (18–21). For instance, in one of the first studies directly addressing this problem in animals, cocaine self-administering rats were able to effectively suppress their behavior in response to a specific discriminative cue (19). However, little is known about the strength, generality, flexibility, and neural substrates of inhibitory control of cocaine seeking in animals. From the Universite´ de Bordeaux and the Centre National de la Recherche Scientifique, Institut des Maladies Neurode´ge´ne´ratives, Bordeaux, France. Authors KG and SN Contributed equally to this work. Address correspondence to Serge H. Ahmed, Ph.D., Universite´ BordeauxSegalen, Institut des Maladies Neurode´ge´ne´ratives/CNRS UMR 5293, 146 rue Le´o-Saignat, Bordeaux 33076, France; E-mail: sahmed@ u-bordeaux2.fr. Received May 26, 2012; revised Aug 9, 2012; accepted Aug 9, 2012.

0006-3223/$36.00 http://dx.doi.org/10.1016/j.biopsych.2012.08.011

Here, we sought to address these questions in rats with a long history of intravenous cocaine self-administration by using a discriminative inhibitory procedure similar to that developed by Kearns et al. (19). Briefly, while actively engaged in lever pressing for cocaine, rats were required to stop responding when presented with a visual discriminative stimulus (DS) signaling lack of reinforcement (Figure 1A). One important feature of this DS procedure that distinguishes it from classic extinction training (which also recruits inhibitory mechanisms [22]) is that responding for cocaine continues to be reinforced each day before the onset of the nonreinforced period signaled by the DS. As a result, cocaine levels are high immediately before onset of the DS but start to fall during the DS. Previous research has established that increased cocaine seeking occurs precisely when cocaine levels begin to fall below the preferred level maintained during self-administration (23). Thus, during the DS, rats are trained to recruit inhibitory control mechanisms when cocaine seeking is particularly intense. Another important feature of the present discriminative inhibitory control procedure is that no period of cocaine reinforcement followed the DS period to avoid the confounding effect of drug anticipation on cocaine seeking (for a demonstration of this effect, see Figure S1 in Supplement 1).

Methods and Materials See Supplement 1 for more detailed description of methods. Animal Housing and Surgery Male Wistar rats (200–225 g) (Charles River Laboratories, L’Arbresle, France) were housed in groups of two to three and maintained in temperature-controlled vivarium with a 12-hour light-dark cycle. 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 (24). All experiments were carried out in accordance with BIOL PSYCHIATRY 2013;73:271–279 & 2013 Society of Biological Psychiatry

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C. Mihindou et al. Figure 1. Inhibitory control of cocaine seeking. (A) Diagram of the discriminative stimulus (DS) training procedure. During the initial 90-minute period, completion of the fixed-ratio (FR) requirement was reinforced by cocaine (COC). During the final 30-minute DS period (gray box), completion of the FR requirement was no longer reinforced by cocaine. The downward arrow in the upper left indicates when muscimol was infused in the prefrontal cortex inactivation study. (B) Mean (⫾ SEM) DS and pre-DS responses across training sessions (n ¼ 14). *Different from pre-DS responses (Tukey’s Honestly Significant Difference test, p o .01). (C) Final individual percent scores of response inhibition from pre-DS level (i.e., average of last three sessions). (D) Within-session pattern of responses for cocaine during DS training sessions 1 and 10. For comparison, mean responses during active cocaine selfadministration (SA) as measured over the last three sessions before DS training are also shown (n ¼ 14). The gray box indicates the final 30-minute nonreinforced DS period. Note that during active cocaine selfadministration, no DS was presented and responses were reinforced during the final 30-minute period. *Different from pretraining level of responses (Tukey’s Honestly Significant Difference test, p o .01).

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 [de´cret 87-848, 19 October 1987]). Initial Operant Training Rats were first trained to lever press for intravenous cocaine (final dose: .125 mg) under a fixed-ratio (FR) time-out 20-sec (final ratio: 3 or 5) schedule of reinforcement (24). Training sessions began with extension of the left lever, lasted 180 minutes, and were run 6 days per week. Rats were trained for at least 21 sessions before being tested for discriminative inhibitory control of cocaine seeking. In initial experiments, rats were trained under a final FR5 schedule but some rats failed to maintain responding at this FR (i.e., about 15%). To attempt to reduce this attrition, rats were also trained under a mixed FR1 (first 30 minutes)/FR3 (last 150 minutes) schedule in some experiments, but this change had little effect on attrition. Thus, regardless of the FR training, only rats that maintained responding for cocaine despite increased efforts were retained for subsequent DS training. Training for Discriminative Inhibitory Control of Cocaine Seeking During discriminative inhibitory training, daily sessions of selfadministration lasted 120 minutes and were subdivided into two www.sobp.org/journal

components: an initial 90-minute period of cocaine selfadministration followed by a final 30-minute nonreinforcement period signaled by the DS (Figure 1A). The DS consisted of turning on for 30 minutes the house lights of the operant chamber. When the DS was on, completion of the FR was signaled by the 20-sec time-out cue but was no longer reinforced by cocaine. Cocaine-Primed Reinstatement of Cocaine Seeking Cocaine-primed reinstatement of drug seeking was assessed using a within-session reinstatement procedure described previously (24,25). This procedure consisted of an initial 90-minute period of extinction during which responding was not reinforced by cocaine. This extinction period was followed by a maximally effective priming dose of cocaine (i.e., 1 mg, intravenous). Responding remained unrewarded for 45 minutes following drug priming, after which time the session ended. Nonreinforced responses were counted following drug priming and compared with those emitted during the 45-minute control period preceding drug priming (considered as dose 0). The DS was turned on or remained off for 45 minutes during drug priming. Pharmacologic Inactivation of the Prefrontal Cortex The infralimbic (IL) or prelimbic (PL) subdivision of the medial prefrontal cortex (mPFC) was inactivated immediately before onset of the testing session using local bilateral microinfusions of muscimol (i.e., 400 ng per side) (Figure 1A) (26). This dose of

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Results Rats (n ¼ 14) progressively learned to inhibit cocaine seeking with repeated DS training sessions [DS  session: F(9,117) ¼ 17.6, p o .01] (Figure 1B). During session 1, responding during the DS dramatically rose above the pre-DS level, showing that rats first reacted to lack of reinforcement by intensely seeking the drug. A within-session analysis of session 1 revealed that increased cocaine seeking was particularly evident during the first 5 minutes of the DS and then progressively returned to both the pre-DS level and the level of responding during active cocaine self-administration (Figure 1D). However, with repeated training, responding gradually decreased and fell below the pre-DS level from session 4 onward, suggesting inhibition of cocaine seeking by the DS (Figure 1B). During the last three sessions, suppressed responding during the DS was stable and was about fourfold lower than pre-DS level. All rats showed inhibitory control of cocaine seeking during the DS, as final individual percent scores of inhibition (i.e., averaged over last three training sessions) were all

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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 the Student t test. Statistical analyses were run using Statistica, version 7.1 (Statsoft Inc., Maisons-Alfort, France).

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c-Fos Immunohistochemistry Following transcardiac perfusion with 4% formaldehyde in .1 mol/L sodium phosphate buffer, brains were postfixed and cryoprotected in 20% sucrose solution. Freezing 50-mm-thick sections were cut on a cryostat and stored at 41C in .1 mol/L phosphate-buffered saline containing sodium azide. Free-floating sections were first incubated for 48 hours at 41C with rabbit polyclonal anti-c-Fos antibody (1:8000; sc-52, Santa-Cruz, Biotechnology, Santa Cruz, California), then for 2 hours at room temperature with a biotinylated goat anti-rabbit immunoglobulin G (1:200; Vector Laboratories, Burlingame, California). Tissue sections were processed further using ABC Vectastain Elite kit (Vector Laboratories, Burlingame, California) and 3,3’-diaminobenzidine detection. Fos immunoreactivity was counted in both hemispheres from three sections per structure and per animal covering the anteroposterior extent of the target locations of cannulae probes for PL and IL inactivation. Cell counts were normalized by the area counted and expressed as number of Fos-labeled cells/mm2 (density) (31,32).

clearly below 100% (range: 5.4%–70.6%; mean: 24.0 ⫾ 4.4%) (Figure 1C). Finally, a within-session time course analysis of session 10 revealed that inhibition of cocaine seeking was immediate (i.e., maximal during the first 5 minutes) and stable throughout the DS [F(1,13) ¼ 87.2, p o .01] (Figure 1D). Note that though suggestive, these data are not sufficient to show that the DS has acquired inhibitory effects during training. Direct evidence for these effects is shown below by omitting the DS. To assess whether rats’ inhibitory ability can be retained following extensive exposure to cocaine, a subgroup of eight rats from the above experiment were given 30 additional daily sessions of cocaine self-administration and then retested for discriminative inhibitory control. In total, these rats were exposed to 61 days of cocaine self-administration, 21 before the first DS training and 61 before the second DS training (including the first 10 DS training sessions), during which they took a large amount of cocaine. Additional exposure to cocaine selfadministration did not change the ability of rats to inhibit cocaine seeking (Figure 2A, B). In fact, rats relearned to inhibit cocaine seeking in response to the DS faster than initially [drug day: F(1,6) ¼ 21.0, p o .01; drug day  DS session: F(9,54) ¼ 4.2, p o .01], suggesting a memory effect (Figure 2A).

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muscimol has been frequently used in previous research on PFC functions in rats (27–29). Comparable doses of muscimol were also shown to suppress neuronal firing for at least 3 hours, with a limited diffusion range (26,30). The effects of muscimol on responding during the DS were compared with the effects of vehicle and with baseline responding as measured during the last 3 training days before cortical microinfusions. In addition, since mPFC inactivations were expected to disinhibit cocaine seeking during the DS, the effects of muscimol were also compared with uninhibited cocaine seeking produced by omitting the DS during the final 30-minute nonreinforcement period. This test was conducted on a separate day following cortical microinfusions.

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Figure 2. Effects of prolonged exposure to cocaine self-administration on inhibitory control of cocaine seeking. (A) Mean (⫾ SEM) percent inhibition from prediscriminative stimulus (pre-DS) level across DS sessions after 21 or 61 days of cocaine self-administration (n ¼ 8). *Different from 21 days (Tukey’s Honestly Significant Difference test, p o .01); #different from 100% (t test, p o .01). (B) Final individual scores of response inhibition as a function of days of cocaine exposure. (C) Mean (⫾ SEM) DS (open circles) and pre-DS (closed circles) responses across training sessions (n ¼ 18). *Different from pre-DS responses (Tukey’s Honestly Significant Difference test, p o .01). (D) Final individual percent scores of response inhibition from pre-DS level (i.e., average of last three sessions).

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Following 61 days of cocaine self-administration, percent scores of inhibition were significantly different from 100% earlier than following 21 days (t test, p o .05) (i.e., from DS session 3 vs. 6). Regardless of cocaine exposure, all final individual scores of inhibition were well below 100% (Figure 2B). In addition, they were positively correlated across number of days of cocaine exposure (r ¼ .80, p o .05) (Figure 2B), showing that individual differences in inhibitory control were stable. To assess whether further exposure to cocaine selfadministration could alter inhibitory control of cocaine seeking, a separate group of rats (n ¼ 18) was given 12 3-hour sessions followed by 19 6-hour sessions of cocaine self-administration before being trained for inhibitory control of cocaine seeking (33). Despite this relatively high level of initial drug exposure, rats learned to inhibit cocaine seeking within 10 DS training sessions (Figure 2C). Final individual percent scores of inhibition were all below 100% (range: 9.9%–99.2%; mean: 39.4 ⫾ 5.5%) (Figure 2D). The rat with the inhibition score of 99.2% eventually inhibited its behavior with additional training sessions (i.e., to 49.1%). To probe the strength of inhibitory control of cocaine seeking, a separate group of rats (n ¼ 13) previously trained under the DS procedure were tested for cocaine-primed reinstatement of cocaine seeking in presence (ON) or absence (OFF) of the DS. Cocaine-primed reinstatement of cocaine seeking is a wellvalidated animal model of craving and/or relapse (24,34). Before drug reinstatement testing, rats showed strong inhibitory control of cocaine seeking in response to the DS [DS: F(1,12) ¼ 188.1, p o .01] (Figure 3A). During reinstatement testing, the DS dramatically influenced the priming effects of cocaine on extinguished cocaine seeking [DS: F(2,24) ¼ 30.5, p o .01]. As expected, when the DS was OFF, cocaine (1 mg, intravenous)

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boosted cocaine-seeking responses (i.e., by about fourfold) above the dose 0 level (Figure 3B). This priming effect was almost totally abolished when the DS was ON (Figure 3B). To assess the specificity of the antipriming effect of DS, we measured cocaine-induced locomotion during the reinstatement test in presence or absence of the DS. Twelve rats from the above experiment were thus retested for reinstatement in a different set of operant chambers equipped with infrared beams to measure locomotion. These new chambers (context B) were also located in an experimental room that was different from the initial training room (context A). Before reinstatement retesting, we checked the efficacy of the DS in the new context by testing rats under the standard DS procedure. Normal conditioned inhibition is generally sensitive to context (22). We found that the change of context resulted in a partial and transient decrease in inhibitory control [DS  context: F(5,22) ¼ 5.8, p o .01] (Figure 3C). During the first day in context B, responding during the DS was significantly inhibited from pre-DS level but less than in the initial training context. However, rats recovered their initial level of response inhibition during the second day in context B (Figure 3C). As expected, during reinstatement retesting in context B, cocaine priming reinstated cocaine seeking [F(2,22) ¼ 18.3, p o .01] when the DS was OFF but not when it was turned ON (Figure 3D). In contrast, the presence or absence of the DS had no effect on cocaine-induced locomotion during reinstatement [F(2,22) ¼ 18.3, p o .01] (Figure 3E). Thus, rats’ inhibitory control mechanisms are context-sensitive, behaviorally specific, and sufficiently strong to oppose the priming effects of cocaine. Interestingly, measurement of locomotion following full recovery of inhibition in context B (i.e., on the second day of DS testing preceding reinstatement retesting) also revealed

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Figure 3. Inhibitory control of cocaine-primed reinstatement of cocaine seeking. (A) Baseline level of inhibitory control of cocaine seeking before drug reinstatement (n ¼ 13). *Different from prediscriminative stimulus (preDS) responses (Tukey’s Honestly Significant Difference [HSD] test, p o .01). (B) Effects of DS on cocaineprimed reinstatement of cocaine seeking. The DS remained off (OFF) or was turned on (ON) during drug priming. *Different from dose 0 and condition ON (Tukey’s HSD test, p o .01). (C) Effects of switch from context A (ConA) to context B on inhibitory control of cocaine seeking. Rats (n ¼ 12) were tested in context B for 2 days (ConB1 and ConB2) before drug reinstatement. *Different from pre-DS responses; #different from DS responses in context A (Tukey’s HSD test, p o .01). (D) Effects of DS on cocaine-primed reinstatement of cocaine seeking. The DS remained off or was turned on during drug priming. *Different from dose 0 and condition ON (Tukey’s HSD test, p o .01). (E) Effects of DS on cocaine-induced locomotion during drug reinstatement. *Different from dose 0 (Tukey’s HSD test, p o .01). (F) Within-session pattern of crossovers during the second day in context B. *Different from last 5-minute pre-DS interval (Tukey’s HSD test, p o .01).

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C. Mihindou et al. that inhibition of cocaine seeking during the DS was associated with a large, though transient, rebound increase in locomotion (Figure 3F). Since rats develop focused behavioral stereotypies during cocaine self-administration (personal observation) (35) that can compete with the expression of locomotion (36,37), we hypothesize that this rebound hyperactivity reflects a release of competition from stereotypies. Regardless of the underlying mechanisms, however, this rebound increase in locomotion during the DS shows that rats can inhibit cocaine seeking even when otherwise hyperactive. To further investigate the response specificity of rats’ inhibitory control mechanisms, a separate group of 12 rats were first trained to inhibit responses on the left lever during the DS (right lever retracted throughout the session). Once robust inhibition of left lever responses was evident, we tested whether this inhibition could be transferred to right lever responses (left lever retracted throughout the session). A total of three transfer tests was conducted, interspersed by rebaselining sessions with the left lever. Response change had no impact on pre-DS responding but selectively affected DS responding (Figure 4A) [DS  lever: F(3,33) ¼ 7.20, p o .01]. During the first transfer session, DS responding was disinhibited, showing no immediate transfer of inhibition across different responses. However, with repeated testing, rats quickly learned to inhibit right lever responses during the DS (Figure 4A). During the third transfer test, the level of inhibition of right lever responses was similar to that of left lever responses. Finally, once rebaselined for inhibition of left lever responses, rats were tested in context B (see above). Once again, this change of context resulted in a partial decrease in inhibitory control (Figure 4B) [DS  context: F(1,11) ¼ 22.75, p o .01]. A separate group of brain-cannulated rats (n ¼ 18) was tested to investigate the role of the PL (n ¼ 10) and IL (n ¼ 8) subdivisions of the mPFC in inhibitory control of cocaine seeking (for histological verification, see Figure 5A, B). Responding during the DS period was influenced by treatment conditions [DS  treatment for

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Figure 4. Response specificity of inhibitory control of cocaine seeking. (A) Effects of response switch on inhibitory control of cocaine seeking. Rats (n ¼ 12) were first trained to inhibit left lever responses (L) and then tested on three occasions for inhibition of right lever responses (R1 to R3). *Different from prediscriminative stimulus (pre-DS) responses; #different from DS left responses (Tukey’s Honestly Significant Difference test, p o .01). (B) Effects of context change on inhibitory control of cocaine seeking. *Different from pre-DS responses; #different from DS responses in context A (ConA) (Tukey’s Honestly Significant Difference test, p o .01). ConB, context B.

IL- or PL-cannulated rats, respectively: F(3,21) ¼ 28.6, p o .01; F(3,27) ¼ 23.5, p o .01]. As expected, during baseline or following vehicle microinfusions, IL- and PL-cannulated rats showed strong inhibitory control in response to the DS (Figure 5C, D). This inhibitory control was directly imputable to the DS, since its omission (OFF) resulted in an immediate loss of inhibition in both groups (Figure 5C, D). Transient bilateral inactivation of PL, but not IL, cortex with muscimol disinhibited cocaine seeking compared with baseline or vehicle (Figure 5C, D). This disinhibition was behaviorally specific, as PL inactivation neither affected pre-DS responding for cocaine (Figure 5D) nor rebounded hyperactivity during the DS (Figure 5F). Interestingly, however, PL inactivationinduced disinhibition of cocaine seeking was lower than the loss of inhibition produced by DS omission (OFF) (Figure 5D). This difference is largely because of the transient disinhibitory effects of PL inactivation compared with the more prolonged effects produced by DS omission (OFF) (Figure S2 in Supplement 1). Finally, though IL inactivation did not reduce inhibitory control of cocaine seeking (Figure 5C), it selectively abolished rebound hyperactivity during the DS, showing that it was pharmacologically effective (Figure 5E). Finally, neuronal activity in IL and PL during inhibitory control of cocaine seeking was assessed using c-Fos immunohistochemistry. This experiment was conducted in 12 rats previously trained to inhibit cocaine seeking after 19 6-hour sessions of cocaine selfadministration. They were split into three subgroups (n ¼ 4 each) with identical level of inhibitory control [DS: F(1,9) ¼ 20.70, p o .01; group or group  DS: F o 1]. On the sacrifice day, all subgroups were allowed to self-administer cocaine during the first 90 minutes, as during regular DS training. During the last 30 minutes of the session, the first group was presented with the DS (DS ON) and was sacrificed thereafter. This relatively early time point for c-Fos induction (i.e., 30 minutes after DS onset) was chosen because inhibition of cocaine seeking was immediate after DS onset (Figure 1D) and because previous research showed that c-Fos induction can be seen as early as 15 minutes (38) and can peak between 30 and 90 minutes (39). The second group was treated as the first subgroup except that the DS was omitted (DS OFF). Finally, the last subgroup was treated as the DS OFF subgroup except that the lever was retracted during the last 30-minute period (NO lever), thereby preventing the expression of uninhibited cocaine seeking. This last subgroup controlled for eventual neuronal correlates of disinhibited cocaine seeking in the DS OFF group. As expected, the DS ON subgroup showed strong inhibition of cocaine seeking compared with the DS OFF subgroup, which showed disinhibited cocaine seeking [group  DS: F(1,6) ¼ 12.79, p o .01] (Figure 6A). Consistent with the brain inactivation study, inhibition of cocaine seeking in the DS ON subgroup was associated with a large and selective increase in c-Fos expression in the PL (Figure 6B, D) but not in the IL (Figure 6B, C) [group: F(2,18) ¼ 14.83, p o .001; group  brain region: F(2,18) ¼ 8.06, p o .01]. Overall, regardless of the subgroup, c-Fos expression was slightly, though not statistically, higher in the PL than the IL, a finding consistent with previous research (40,41).

Discussion Recent neuroimaging research has shown that people with drug addiction can, under some circumstances, recruit lateral PFC-mediated inhibitory control mechanisms to suppress drug craving (1,3). Here, we report that rats also retain some degree of www.sobp.org/journal

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inhibitory control over cocaine seeking, even after a long history of cocaine self-administration. Though there is some individual variation in inhibition levels, virtually all rats are able to stop responding for cocaine, even when highly motivated to seek the drug (first 5 minutes after DS onset) and otherwise behaviorally hyperactive. Most importantly, rats can even resist cocaineprimed reinstatement of cocaine seeking, demonstrating that this priming effect can be controlled through behavioral means—a conclusion consistent with other recent research (24,42). Recruitment of inhibitory control mechanisms to suppress cocaine seeking is response- and context-specific, suggesting a significant degree of cognitive flexibility. Finally, such flexible inhibitory control of cocaine seeking selectively depends on neuronal activity within the PL, but not the IL, subdivision of the medial PFC. The latter functional dissociation adds to growing evidence suggesting that the rodent prelimbic cortex likely represents a functional homolog of the human lateral PFC (43), which is consistently activated during voluntary inhibitory control of emotions and motivational impulses in people (2,4– 14). Overall, the present findings parallel those from recent neuroimaging research on inhibitory control of craving in addiction and show that rats also retain some degree of inhibitory control over craving-like behavior, even after prolonged drug use. Preclinical investigation of the underlying mechanisms may contribute to designing new ways to exploit www.sobp.org/journal

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Figure 5. Differential involvement of the prelimbic (PL) and the infralimbic (IL) subdivision of the prefrontal cortex in inhibitory control of cocaine seeking. (A) Coronal sections showing the location of all microinjection tips in the PL (black circle) and the IL (gray circle). The numbers indicate millimeters anterior to bregma (illustration reprinted from [71] with permission from Elsevier, copyright 2007). (B) Photomicrographs of unstained coronal sections representing cannulas and microinjection tips in the PL and the IL. Mean (⫾ SEM) discriminative stimulus (DS) and pre-DS responses in IL-cannulated (C) (n ¼ 8) and PL-cannulated rats (D) (n ¼ 10) during baseline (BL) (last three DS sessions before brain microinfusions), DS omission (OFF), or following vehicle (VEH) and muscimol microinfusions (MUS). Mean (⫾ SEM) DS and pre-DS crossovers in IL-cannulated (E) and PL-cannulated rats (F) during baseline (last three sessions before brain microinfusions), DS omission, or following vehicle and muscimol microinfusions. *Different from pre-DS performance; # different from DS performance during baseline and following vehicle (Tukey’s Honestly Significant Difference test, p o .01).

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this capacity, pharmacologically and/or behaviorally, to prevent relapse (2,15). The functional dissociation between the PL and the IL reported here confirms previous neuroanatomical (44,45), electrophysiological (46,47), and behavioral studies (28–30,48–55), suggesting that the PL versus the IL would implement different, sometimes opposite, psychological functions. Specifically, it confirms previous studies that have specifically implicated the PL, but not the IL, in nondrug-related behavioral inhibition (28,30,46,47,54,56–58). For instance, inactivation of the PL, but not the IL, was recently shown to impair inhibitory control in the stop-signal task in rats (28,59). However, this dissociation seems to contradict other work showing that inactivation of the PL abolishes drug, cue-, or context-primed reinstatement of cocaine seeking (60–65). Thus, it seems that the PL is involved in both excitatory (or disinhibitory) and inhibitory control of cocaine seeking. One way to resolve this apparent paradox is to postulate that the PL has a more general function in the control of cocaine seeking than merely excitation or inhibition. One such function would be context monitoring (30,66), as context can control both the inhibition (present study) and activation of cocaine seeking in rats (22). Thus, by compromising context monitoring, inactivation of the PL could block the recruitment of both inhibitory and excitatory control of cocaine seeking. Intriguingly, the function of the human dorsolateral prefrontal cortex was also recently

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BIOL PSYCHIATRY 2013;73:271–279 277 Figure 6. Selective activation of the prelimbic (PL) during inhibitory control of cocaine seeking. (A) Mean (⫾ SEM) discriminative stimulus (DS) and pre-DS responses. (B) Photomicrographs of c-Fos immunoreactivity in the prefrontal cortex of representative rats from the DS turned off (OFF) and DS turned on (ON) subgroups. Top sections correspond to bregma 3.2 mm and illustrate the boundaries of the PL and infralimbic (IL) subregions. Bottom panels show Fos-immunoreactive nuclei (indicated by black arrows) within the PL at 10  magnification. Mean (⫾ SEM) density of c-Fos labeled cells in the IL (C) and the PL (D). The DS was turned on or off during the last 30-minute period in the DS ON group (n ¼ 4) and the DS OFF group (n ¼ 4), respectively. The lever was retracted during the last 30-minute period for the NO lever (NOL) group (n ¼ 4), which explains why this group did not respond during the DS. *Different from pre-DS level; #different from the DS ON group (Tukey’s Honestly Significant Difference test, p o .01). fmi, forceps minor of the corpus callosum.

reinterpreted in terms of context monitoring to explain its activation during both inhibitory and excitatory control of behaviors (67). Regardless of the specific psychological functions involved, the present study nevertheless provides evidence for strong and flexible inhibitory control of cocaine seeking in rats that depends on the functional PFC homolog of the human lateral PFC. There are several potential limitations to the present study. First, extrapolations from animals to humans must be made with caution. Unlike people, rats cannot be specifically instructed to inhibit their motivational impulses to use drugs. As a result, there is some uncertainty about what exactly rats are inhibiting during the DS (e.g., drug motivation and/or motor output) and how they achieve this successful inhibition. An important challenge for future research will be to develop more specific inhibitory control training procedures. Second, reversible neuronal inactivation of the PL was evident only during the first 5 minutes of the DS and was highly variable across individuals (Figure S2 in Supplement 1). Since muscimol was infused 90 minutes before the presentation of the DS, this short-term and variable effect may indicate that muscimol action was beginning to wane when inhibitory control was recruited, at least in some rats. Alternatively, this limited effect may also suggest that other brain regions are involved in inhibitory control of cocaine seeking. Third, the lack of effects of IL inactivation on inhibitory control reported here contrasts with a previous study in rats showing that IL inactivation alone reinstated cocaine seeking after extinction (68). This lack of effects may be due to the nonspecific motor deficits induced by the relatively high dose of muscimol used here. This dose of muscimol tended, indeed, to decrease

nonsignificantly pre-DS responding below the vehicle condition. Future research with lower doses of muscimol will be useful to better rule out a role of the IL in discriminative inhibitory control of cocaine seeking. The discrepancy between the two studies could also be due to the recruitment of different cortical inhibitory control mechanisms by different training procedures (i.e., DS vs. extinction training). This possibility is consistent with the observation that reinstatement of cocaine seeking can be induced by cocaine priming after extinction (69), while it is blocked after presentation of the DS. Fourth, the finding that rats retained the ability to stop cocaine seeking after prolonged cocaine exposure seems to contradict some (70), but not all (19), previous animal studies. In one study, about 30% of rats appeared unable to stop cocaine seeking (70). However, in this study, drug anticipation was not ruled out when inhibition of cocaine seeking was required, making any interpretation of the behavior of these rats in terms of loss of inhibitory control uncertain. This work was supported by the French Research Council, the French National Agency, the Fondation Nouvelle Radio Jeunes (NRJ), the Universite´ Bordeaux-Segalen, and the Conseil Regional d’Aquitaine. We thank Stephane Lelgouach for animal care, Pierre Gonzalez for technical assistance, Marie-He´le`ne Bruye`res and Ourida Gaucher for administrative assistance, Christian Darrack for his help with data extraction, and Alain Labarriere for housekeeping assistance. We also thank Sandra Dovero for technical advice and Erwan Bezard and Etienne Gontier for giving us access to their laboratory facility. We finally thank Hanna Pickard for English www.sobp.org/journal

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