RENEWAL OF EXTINGUISHED COCAINE-SEEKING

Transcription

1 Neuroscience 151 (2008) RENEWAL OF EXTINGUISHED COCAINE-SEEKING A. S. HAMLIN, K. J. CLEMENS AND G. P. McNALLY* School of Psychology, The University of New South Wales, Sydney, New South Wales, 2052, Australia Abstract Rats were trained to self-administer cocaine in a distinctive context (context A). They were then extinguished in a second context (context B) prior to test for cocaineseeking in the original training context, context A (group ABA), context B (group ABB) or no test (group AB0). Group ABA showed renewal of extinguished cocaine-seeking associated with c-fos induction in basolateral amygdala, lateral hypothalamus, and infralimbic prefrontal cortex. Groups ABA and ABB showed test-associated c-fos induction in prelimbic prefrontal cortex, nucleus accumbens (core, shell, rostral pole), striatum, lateral amygdala, perifornical hypothalamus, and ventral tegmental area. Double immunofluorescence revealed that renewal-associated c-fos was expressed in orexin-negative lateral hypothalamic neurons whereas testassociated c-fos was expressed in orexin-positive perifornical hypothalamic neurons. Retrograde tracing from lateral hypothalamus with cholera toxin revealed only sparse duallabeled neurons in basolateral amygdala and infralimbic prefrontal cortex, suggesting that these regions contribute to renewal of cocaine-seeking independently of their projections to lateral hypothalamus. Retrograde tracing from the ventral tegmental area suggested that hypothalamic contributions to cocaine-seeking are likewise independent of projections to the midbrain. These results suggest that renewal of cocaine-seeking depends critically on basolateral amygdala, lateral hypothalamus, and infralimbic prefrontal cortex. Whereas basolateral amygdala and lateral hypothalamus contributions may be common to renewal of extinguished cocaine-, alcohol-, and sucrose-seeking, infralimbic prefrontal cortex contributions appear unique to renewal of cocaineseeking and may reflect the habitual nature of relapse to cocaine IBRO. Published by Elsevier Ltd. All rights reserved. Key words: relapse, orexin, reinstatement, cocaine, rat, tracing. The contexts where drugs are self-administered play an important role in regulating persistent drug-taking and in relapse to such taking after periods of abstinence. The role of contexts is best exemplified by the phenomenon of renewal. Renewal is the term used to describe recovery of extinguished behavior with a change in context Bouton ( and Bolles, 1979). Typically, subjects are trained to seek a *Corresponding author. Tel: ; fax: address: g.mcnally@unsw.edu.au (G. P. McNally). Abbreviations: BLA, basolateral amygdala; CTb, cholera toxin, B subunit; DMH, dorsomedial hypothalamus; f, fornix; ilpfc, infralimbic region, prefrontal cortex; IR, immunoreactive/immunoreactivity; LH, lateral hypothalamus; mt, mammillothalamic tract; NHS, normal horse serum; PB, phosphate buffer; PBS, phosphate buffer saline; PBT-X, phosphate buffer with Triton-X 10; PeF, perifornical hypothalamus; TH, tyrosine hydroxylase; VTA, ventral tegmental area; 3V, third ventricle /08$ IBRO. Published by Elsevier Ltd. All rights reserved. doi: /j.neuroscience reward in one context, context A. This is then extinguished in a second context, context B. When tested in context B, reward-seeking is low (i.e. it has been extinguished). However, when tested in context A reward-seeking returns (i.e. it has been renewed). Renewal has been demonstrated for instrumental responding based on numerous reinforcers, including natural rewards such as food pellets Nakajima ( et al., 2000) or liquid sucrose Hamlin ( et al., 2006 ) as well as drug rewards such as alcohol Hamlin ( et al., 2007; Zironi et al., 2006 ), cocaine (Crombag et al., 2002; Fuchs et al., 2005), heroin (Bossert et al., 2004 ), and a heroin cocaine mixture (speedball) Crombag ( and Shaham, 2002). Renewal shows that extinction is context-specific and that it is lost with a change in context between extinction and test. It is evidence that extinction is not simply erasure of drug-seeking, but additionally involves imposition of a mask on drug-seeking which reduces drug-seeking in the extinction context but not elsewhere Bouton, ( 2002, 2004). Removal of this mask by context change is a source of relapse after behavior change Bouton, ( 2002; Conklin and Tiffany, 2002 ). The neural mechanisms mediating renewal of extinguished drug-seeking remain only poorly understood. Dorsomedial prefrontal cortex, basolateral amygdala (BLA), and dorsal hippocampus have all been implicated in renewal of extinguished cocaine-seeking because infusions of tetrodotoxin into each of these structures prior to testing for ABA renewal prevents the recovery of extinguished cocaine-seeking (Fuchs et al., 2005 ). Recent data suggest that serial interactions between BLA and dorsal hippocampus may be important for renewal of extinguished cocaineseeking (Fuchs et al., 2007 ). The actions of dopamine are also critical to renewal of extinguished cocaine-seeking (Crombag et al., 2002 ). Ventral tegmental area (VTA) has been implicated in renewal of extinguished heroin-seeking (Bossert et al., 2004 ) but its involvement in renewal of cocaine-seeking is unknown. Likewise glutamate actions, including in nucleus accumbens, are important for renewal of extinguished heroin- Bossert ( et al., 2006a ) and sucrose-seeking (Bossert et al., 2006b ) but their involvement in renewal of cocaine-seeking is less well understood. More generally, it is unclear from the available evidence whether common neural mechanisms mediate renewal to seeking different rewards (e.g. cocaine, alcohol, heroin, sucrose), or whether there are distinct neural mechanisms for renewal depending on the specific reward. The experiments reported here had three aims. The first was to use c-fos immunohistochemistry to identify the neural correlates of renewal of extinguished cocaine-seeking. We have used this approach to identify the neural correlates of renewal of extinguished alcohol- (Hamlin

2 660 A. S. Hamlin et al. / Neuroscience 151 (2008) et al., 2007) and sucrose-seeking (Hamlin et al., 2006). Identification of the neural correlates of renewal of extinguished cocaine-seeking would permit comparison with our recent results and provide important insights into whether there are common or unique correlates for renewal of seeking different rewards. In those studies, ABA renewal of extinguished alcohol- and sucrose-seeking was associated with significant induction of c-fos protein in BLA, nucleus accumbens shell, and lateral hypothalamus (LH). c-fos induction was elevated in several other structures including medial prefrontal cortex and nucleus accumbens core, but it was also elevated in these structures among rats tested in the extinction context (group ABB), suggesting that these structures may be necessary but not sufficient for renewal of extinguished reward-seeking. Neisewander et al. (2000) showed that re-exposure to a cocaine-associated context after forced abstinence in a second, different context, was associated with significant c-fos protein induction in the BLA, hippocampal CA1 region, dentate gyrus, nucleus accumbens shell and core, and anterior cingulate. However, because this study employed forced abstinence rather than extinction of drugseeking, it is unclear which, if any, of these correlates would also be associated with renewal of extinguished cocaine-seeking. The second aim of the experiments reported here was to study the possible role of hypothalamic orexin neurons in renewal of extinguished cocaine-seeking by studying their recruitment during renewal. Recent evidence implicates orexin in multiple aspects of reward processing and seeking. For example, exposure to food, opiate, psychostimulant, or ethanol-related contexts or stimuli induces c-fos in hypothalamic orexin neurons (Harris et al., 2005; Dayas et al., 2007; Hamlin et al., 2007). The midbrain VTA is one efferent of hypothalamic orexin neurons and orexin actions there contribute to synaptic and behavioral responsiveness to psychostimulants and opiates (Borgland et al., 2006; Narita et al., 2006). Importantly, orexin has been implicated in other forms of recovery of extinguished drugseeking. Systemic injections of orexin receptor antagonist SB prevent cue-induced reinstatement of extinguished alcohol-seeking (Lawrence et al., 2006) and stress-induced reinstatement of extinguished cocaineseeking (Boutrel et al., 2005). However, to date only a single study has assessed c-fos induction in hypothalamic orexin neurons during renewal of extinguished drug-seeking and this was based on an alcohol reinforcer (Hamlin et al., 2007). There are no available data on the recruitment of orexin neurons during renewal of extinguished cocaine-seeking. The final aim of these experiments was to combine c-fos immunohistochemistry with retrograde tracing to begin to identify possible neuroanatomical pathways recruited during renewal of extinguished cocaine-seeking. There has been little attention to date on identifying the pathways, as opposed to structures, implicated in renewal of extinguished reward-seeking (but see Fuchs et al., 2007), and to the best of our knowledge, there have been no studies combining tract tracing with activity marker expression during recovery of extinguished drug-seeking. The focus here was on determining which, if any, forebrain afferents to LH or VTA were recruited, as indexed by c-fos induction, during renewal of extinguished cocaine-seeking. We focused on afferents to VTA and LH because recent research has suggested an important role for LH in mediating the behavioral effects of drug-associated contexts (Harris et al., 2006) and the VTA has been implicated in renewal of extinguished opiate-seeking (Bossert et al., 2004). To achieve these aims we trained rats to self-administer i.v. cocaine in one context, context A. Cocaine-seeking was then extinguished in a second context, context B. On test in the first experiment, rats were placed in either context B (group ABB) or context A (group ABA). An additional control group was included: group AB0. Group AB0 received identical training and extinction as groups ABA and ABB but they were never tested. Group AB0 therefore controls for several variables (e.g. cocaine history; training history; transport; handling etc) which may confound interpretation of patterns of c-fos induction. In the second experiment we employed only group ABA and applied the tracer cholera toxin B subunit (CTb) to either the LH or the VTA prior to cocaine self-administration training in context A. Rats were subsequently extinguished in context B, and tested for renewal of cocaine-seeking in context A. Subjects EXPERIMENTAL PROCEDURES Subjects were experimentally naive male Long-Evans rats ( g) obtained from a commercial supplier (Monash Animal Services, Gippsland, Victoria, Australia). After arrival and prior to surgery, rats were housed in groups of eight in plastic cages maintained on a reverse 12-h light/dark cycle (lights on at 19:00 h). After surgery, rats were housed individually in plastic cages maintained on a reverse 12-h reverse light/dark cycle (lights on at 19:00 h). Food and water were freely available prior to surgery and during recovery. During sucrose training rats were allowed 1 h access to food and water following daily training sessions. At the conclusion of the first day of cocaine training, water was freely available and food was restricted to 1 h per day. The experiments were designed to minimize the number of animals used and their suffering. The procedures were approved by the Animal Care and Ethics Committee at the University of New South Wales and conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Publications No ) revised Surgery Rats were injected intraperitoneally with 1.3 ml/kg of the anesthetic ketamine (Ketapex; Apex Laboratories, Sydney, Australia) at a concentration of 100 mg/ml and with 0.3 ml/kg of the muscle relaxant xylazine (Rompun; Bayer, Sydney, NSW, Australia) at a concentration of 20 mg/ml followed by 0.1 ml of 50 mg/ml carprofen administered s.c. as a preoperative analgesic. Surgery was conducted to implant a chronic indwelling catheter into the right jugular vein, terminating proximal to the right atrium of the heart and secured with sutures. Catheters consisted of 100 mm of Tygon Micro Bore tubing (ID 0.02 in., OD 0.06 in., Small Parts, FL, USA) attached to a back-mount cannula connector pedestal (Plastics One, VA, USA) secured in place with sutures. Catheters were

3 A. S. Hamlin et al. / Neuroscience 151 (2008) filled with 10 IU/ml heparinized saline and closed with a dust cap. Catheter patency was maintained by a daily i.v. flush of 0.2 ml of antibiotic (cephazolin sodium, 100 g/ml) in 100 IU/ml of heparinized saline. Immediately following jugular catheterization rats were placed in a stereotaxic apparatus (Model 900, Kopf, Tujunga, CA, USA), and the incisor bar maintained at 3.3 mm below horizontal to achieve a flat skull position. A 30-gauge needle attached to a 1 l Hamilton syringe was lowered into the right LH (n 5) (A-P: 2.3; M-L: 1.8; D-V: 8.6) or the right A10 dopamine cell group consisting of the VTA, paranigral, and interfascicular nuclei (n 5) (A-P: 5.5; M-L: 0.5; DV 8.2) and 40 nl of 1% low salt CTb (List Biological Laboratories, Campbell, CA, USA) was injected using a manual stereotaxic injector (Stoelting, Wood Dale, IL, USA). Injections were conducted over 1 min and the needle was left in place for 10 min prior to removal to allow for diffusion and to reduce spread up the injection tract. Immediately after surgery, rats were injected intraperitoneally with 0.3 ml of a 300 mg/ml solution of procaine penicillin, s.c. with 0.1 ml of a 100 mg/ml cephazolin sodium, and s.c. with 5 mg/kg carprofen. Rats were allowed 3 days to recover from surgery, during which time they were handled and weighed daily. Behavioral apparatus Training, extinction, and test were conducted in two sets of four chambers which differed in their visual (brightly lit versus dark), tactile (Perspex versus grid floors), and olfactory (rose oil versus peppermint essence) properties. For all chambers (24 cm [length] 30 cm [width] 21 cm [height]), the front and rear walls as well as the hinged door were constructed of clear Perspex and the end walls were made of stainless steel. In all chambers, two nose-poke holes, containing a white cue-light, were located on one side wall of the chamber 3 cm above a grid floor. A recessed magazine was located behind a4cm 4 cm opening in the center of the same wall, between the two nose-pokes. Responding on one (active) nose-poke resulted in delivery of i.v. cocaine (experiment 1: 1.0 mg/kg/infusion 3 s, infusion volume of 53 l 1 mg/kg; experiment 2: 0.67 mg/kg/infusion 4 s, infusion volume of 71 l 1 mg/kg) followed by a 24 s timeout period during which the nose-poke light was extinguished and no events were scheduled. Responding on the other (inactive) nose-poke had no programmable consequences. In one set of chambers the floors consisted of stainless steel rods, 4 mm in diameter, spaced 15 mm apart (center to center). These chambers were located in sound- and light-attenuating cabinets equipped with fans providing constant ventilation and low level background noise. The doors to these cabinets were ajar. There was no illumination other than that provided by the white cue light recessed in the nose pokes and the fluorescent light in the room in which the cabinets were held. One milliliter of a dilute rose oil essence was placed in the bedding beneath these chambers. In the second set of four chambers the floors consisted of Perspex. These chambers were located in sound- and light-attenuating cabinets equipped with fans providing constant ventilation. There was no illumination other than that provided by the white cue light recessed in the nose pokes. One milliliter of a dilute peppermint essence was placed in the bedding beneath these chambers. These two sets of chambers were fully counterbalanced to serve as contexts A and B. Behavioral testing procedure On the first day rats received two daily sessions of 20 min magazine training, one in context A, the other in context B. During these sessions there were 10 non-contingent deliveries of 0.15 ml of sucrose solution at time intervals variable around a mean of 1.2 min. From the second magazine training session onwards rats were connected to the swivel assembly via the spring connector. Magazine training was followed by a single 60 min session of training, on a FR-1 schedule for 0.15 ml of sucrose in context A. Cocaine self-administration on a FR-1 schedule of reinforcement for 2 h sessions commenced in context A the following day. There were 7 days of self-administration training in experiment 1 and 10 days of training in experiment 2. Training was followed by four (experiment 1) or six (experiment 2) days of extinction training in context B. For extinction training, all aspects of self-administration remained the same except for the removal of the cocaine syringe from the pump. The procedures for experiment 1 and 2 differed slightly in terms of dose of cocaine, days of training and extinction. We reduced the dose of i.v. cocaine and extended self-administration training in experiment 2 to ensure that rats which had received CTb injections into VTA or LH acquired the same stable levels of self-administration as rats in experiment 1. This was necessary because we had been unable to find any prior reports using CTb tracer with cocaine-self administration. This in turn required an additional 2 days of extinction training to ensure that cocaineseeking had been extinguished to the same levels as experiment 1. It is worth emphasizing that the procedures for tests were identical in the two experiments and that these procedural variations had no impact on terminal levels of cocaine-seeking during self-administration training and extinction or on the magnitude of ABA renewal and c-fos expression. On the test day in experiment 1, rats were allocated to three groups. All rats were transported to the laboratory. Group ABA (n 6) were placed in context A for 60 min. Group ABB (n 5) were placed in context B for 60 min. The procedure for test for groups ABA and ABB was the same as extinction. Group AB0 (n 5) was not tested. Rather, they remained in the holding area outside the test room. At the conclusion of the test, rats remained in the holding area outside the test room for 60 min prior to perfusion. On the test day in experiment 2, rats were all allocated to group ABA (n 5 VTA CTb; n 5 LH CTb). The procedure for the test was the same as group ABA in experiment 1. Immunohistochemistry At 2 h after being placed in the testing chambers (or 2 h following transport to the testing rooms for AB0 rats) rats were deeply anesthetized with sodium pentobarbital (100 mg/kg i.p.) and perfused transcardially with 50 ml of 0.9% saline, containing 1% sodium nitrite and heparin (5000 i.u./ml), followed by 500 ml of 4% paraformaldehyde in 0.1 M phosphate buffer (PB), ph 7.4. Brains were postfixed for 1 h in the same fixative and placed in 20% sucrose solution overnight. Brains were blocked using a matrix aligned to the atlas of Paxinos and Watson (1997) and 40 m coronal sections were cut using a cryostat (Microm HM560, Microm International, Waldorf, Germany). Four serially adjacent sets of sections were obtained from each brain and stored in 0.1% sodium azide in 0.1 M phosphate buffer saline (PBS) ph 7.2. One series of sections was used to reveal c-fos in combination with tyrosine hydroxylase (TH) (experiment 1) or anti-choleragenoid, for CTb (experiment 2) using two-color peroxidase immunohistochemistry. Free floating sections were washed repeatedly in 0.1 M PB (ph 7.4), followed by two 30 min washes in 50% ethanol, the second of which contained 3% H 2 O 2, and were then incubated in 5% normal horse serum (NHS) in PB (ph 7.4) for 30 min. Sections were incubated in rabbit antiserum against c-fos (1:5000; c-fos (4), sc-52, Santa Cruz Biotechnologies, Santa Cruz, CA, USA), which was mixed with either a sheep antiserum against TH (1:3000; AB1542, Chemicon, Temecula, CA, USA) or goat anti-choleragenoid (1:5000; List Biological Laboratories). These primary antibodies were diluted in 0.1 M PB (ph 7.4) containing 2% NHS and 0.2% Triton X-100 (PBT-X), and incubations were for 48 h at 4 C, with gentle agitation. After washing off unbound primary antibodies, sections were incubated overnight at room temperature in biotinylated donkey anti-rabbit IgG (1:1000; Jackson Immunoresearch Labo-

4 662 A. S. Hamlin et al. / Neuroscience 151 (2008) ratories, West Grove, PA, USA) diluted in 2% NHS PBT-X. Sections were then incubated for 2 h at room temperature in ABC reagent (Vector Elite kit: 6 l/ml avidin and 6 l/ml biotin; Vector Laboratories, Burlingame, CA, USA). Black immunoreactive (IR) nuclei labeled for c-fos were revealed by a nickel-intensified diaminobenzidine reaction, with peroxide being generated by glucose oxidase. To do this sections were washed in PB, followed by 0.1 M acetate buffer (ph 6.0), and then incubated for 15 min in 0.1 M acetate buffer (ph 6.0) containing 2% nickel sulfate, 0.025% 3,3- diaminobenzidine, 0.004% ammonium chloride, and 0.02% D- glucose. The peroxidase reaction was started by adding 0.2 l/ml glucose oxidase and stopped using acetate buffer (ph 6.0). Brain sections were then washed in PB and processed again, in a similar manner using biotinylated donkey anti-sheep IgG (1:1000; Jackson Immunoresearch Laboratories) but without nickel-intensification to localize IR for TH or CTb, revealed as a brown reaction product. Sections were mounted onto gelatin-treated slides, dehydrated, cleared in histolene, and coverslipped with DePeX. A second series of sections through the LH was selected from each rat from experiment 1 and two-color immunofluorescence was used to reveal c-fos and orexin. Free floating sections were washed repeatedly in 0.1 M PBS (ph 7.2), followed by a2h incubation in PBS (ph 7.2) containing 10% NHS and 0.5% Triton X-100. Sections were then incubated in goat antiserum against c-fos (1:2000; Santa Cruz Biotechnologies), mixed with rabbit antiserum against orexin (1:20,000; Phoenix Pharmaceuticals, Belmont, CA, USA). These primary antibodies were diluted in 0.1 M PBS (ph 7.2) containing 0.1% sodium azide, 2% NHS and 0.2% Triton X-100, and incubations were for 48 h at room temperature, with gentle agitation. After washing off unbound primary antibodies, sections were then incubated for 4 h at room temperature in biotinylated donkey anti-sheep IgG (1:1000; Jackson Immunoresearch Laboratories), and donkey anti-rabbit FITC (1: 200; Jackson Immunoresearch Laboratories), diluted in 2% NHS PBT-X. After washing off unbound secondary antibodies, sections were further incubated for 1 h in streptavidin CY3 (1:2000; Sigma). Sections were mounted onto chrome alum/gelatin-treated slides and coverslipped with buffered glycerol (ph 8.6). Neuronal counting Counts of neurons IR for c-fos were conducted through the rostro-caudal extent of each brain region of interest by an observer unaware of group allocations. All sections counted were 160 m apart. Chemoarchitecture was revealed by TH labeling which aided in the identification of anatomical boundaries. The brain regions analyzed were: prelimbic and infralimbic cortex over four sections beginning at 3.20 mm; nucleus accumbens (rostral pole) over three sections beginning at 2.70 mm; nucleus accumbens core and shell subnuclei over six sections beginning at 1.70 mm; dorsal striatum over four sections beginning at 1.70 mm; anterior cingulate over four sections beginning at 1.20 mm; BLA over six sections beginning at 1.80 mm; dorsomedial, perifornical, and LH over six sections beginning at 2.80 mm; substantia nigra, VTA, paranigral, and interfascicular nucleus over six sections beginning at 5.20 mm. All coordinates given are distance from bregma according to the rat brain atlas of Paxinos and Watson (1997). To determine the distribution of c-fos-ir neurons relative to orexin containing neurons in the hypothalamus eight-bit monochrome images were taken of c-fos and orexin over six sections of the hypothalamus beginning at 2.80 mm from bregma (Olympus BX51 camera running ImagePro software). Images were exported into ImageJ software (NIH), where image stacks of corresponding c-fos and orexin were combined. Single-labeled c-fos or dual-labeled c-fos/orexin IR neurons were identified by alternating between the c-fos and orexin images and determining if the c-fos-ir neuron was localized to an orexin-ir positive or negative neuron. The hypothalamus was further divided into three regions for analysis based on structural landmarks. The medial area that contains the orexin neurons of the dorsomedial hypothalamus (DMH) was formed by the area between the third ventricle (3V) and the medial edge of the mammillothalamic tract (mt), the central region that contains the orexin neurons of the perifornical hypothalamus (PeF) from the mt past the lateral edge of the fornix (f) (approximately half the width of the f past the lateral edge of the f) and the lateral regions that contains the orexin neurons of the LH by the remaining area extending to the to the lateral edge of the internal capsule (Fig. 2). Data analysis The number of responses was recorded during training, extinction, and test. These data were analyzed by means of planned orthogonal contrasting testing procedure that preserved the factorial design. A multivariate approach to repeated measures was adopted (O Brien and Kaiser, 1985). The type I error rate ( ) was controlled at 0.05 for each contrast tested. The means of the total counts of c-fos-ir neurons in each brain region was analyzed using ANOVA. RESULTS Experiment 1: Neural correlates of renewal of extinguished cocaine-seeking Behavior. Fig. 1A shows the mean and standard error of the mean (S.E.M.) responses at the end of selfadministration training (left panel), during extinction training (middle panel), and on test (right panel). Inspection of the figure confirms the acquisition, extinction, and ABA renewal of cocaine-seeking. There was significantly more responding on the active than inactive nose-poke at the end of self-administration training (F(1, 15) 27.9, P 0.05). During extinction training, there was significantly more responding on the active than inactive nose-poke, averaged across days (F(1, 15) 16.3; P 0.05). There was a significant decrease in overall levels of responding across days of extinction training (F(1, 15) 22.2; P 0.05). There was a significant interaction so that the decrease in responding on the active nose-poke was greater than the inactive nose-poke across the course of extinction training (F(1, 15) 23.2; P 0.05). For test, there was an overall significant difference in level of responding between group ABA and group ABB (F(1, 9) 20.6; P 0.05). There was also an overall significant difference in responding on the active versus inactive nose-poke (F(1, 9) 25.0; P 0.05). Finally, there was a significant interaction, so that the difference in responding between group ABA versus group ABB was significantly greater for active than inactive nose-poking (F(1, 9) 22.0; P 0.05). This confirms the ABA renewal of extinguished cocaine-seeking. Immunohistochemistry. c-fos-ir was examined in 31 brain regions. Quantification was performed in serially adjacent series of tissue sections taken from the same animals, in which c-fos-ir was combined with either TH or orexin-ir. In the analyses described below, renewal-associated c-fos induction refers to significant group differences in c-fos expression so that group ABA group

5 A. S. Hamlin et al. / Neuroscience 151 (2008) Fig. 1. (A) Mean ( S.E.M.) responses on active and inactive nose-poke during training, and extinction of instrumental responding and on test. Rats were trained in context A, extinguished in context B, and tested in context A (groups ABA) or B (groups ABB). Return to the original training context after extinction (group ABA) resulted in renewal of cocaine-seeking. (B) Effect of ABA renewal of cocaine-seeking on c-fos-ir in the infralimbic cortex, BLA and LH. Bars show the mean ( S.E.M.) number of c-fos-ir neurons counted in each of the treatment groups A specific induction of c-fos that was associated with ABA renewal was detected in the infralimbic cortex, BLA and LH (* ABA versus ABB, P 0.05). (C) Photomicrographs showing TH-IR and c-fos-ir neurons in representative AB0, ABB, and ABA rats in the infralimbic cortex and the BLA. Scale bar 200 m. ABB group AB0. Test-associated c-fos induction refers to significant group differences in c-fos expression so that that group ABA group ABB group AB0. The accumbens was separated into three main subdivisions: shell, core, and rostral pole (Zahm and Brog, 1992). The accumbens shell was further subdivided into its dorsomedial and ventral regions for analysis. All four accumbal subregions analyzed showed significant test-associated c-fos induction (P 0.05) (Table 1). A similar pattern of c-fos induction was detected in the four dorsal striatal regions analyzed (Table 1). We next examined some of the major efferents and afferents of the accumbens including amygdala, hypothalamus and cortex. There was significant renewal-associated c-fos expression in BLA (P 0.05) (Fig. 1). There was significant test-associated c-fos expression in lateral amygdala (P 0.05). There were no other differences between groups in amygdala (Table 1). Hypothalamus was divided into DMH, PeF, and LH regions for analysis. There was significant renewal-associated c-fos in LH (Fig. 1B) (P 0.05) and significant testassociated Fos in PeF and DMH (P 0.05) (Table 1).

6 664 A. S. Hamlin et al. / Neuroscience 151 (2008) Table 1. Mean S.E.M. numbers of c-fos immunoreactive nuclei Structure AB0 ABB ABA Prefrontal cortex Prelimbic a 170 (31.2) 517 (63.1) 568 (45.2) Anterior cingulate a 134 (23.8) 439 (73.8) 484 (47.5) Accumbens Rostral pole a 45 (10.8) 115 (15.7) 150 (24.0) Dorsomedial shell a 97 (10.9) 174 (24.1) 205 (23.8) Ventral shell a 83 (14.2) 161 (24.4) 194 (21.3) Core a 203 (42.0) 356 (54.2) 397 (60.7) Striatum Dorsomedial a 34 (8.2) 137 (31.3) 168 (29.9) Ventromedial a 41 (6.2) 169 (35.2) 181 (47.1) Dorsolateral a 3 (0.5) 49 (16.9) 42 (9.0) Ventrolateral a 5 (1.5) 29 (7.1) 29 (9.5) Amygdala LA a 8 (2.2) 17 (2.1) 24 (2.1) AStr 15 (3.3) 17 (2.5) 19 (1.5) CeC 15 (2.1) 14 (1.1) 17 (2.1) CeL 46 (6.1) 33 (4.9) 39 (8.8) CeM 13 (3.8) 23 (4.4) 17 (2.3) BNST BSTLD 37 (5.6) 53 (5.3) 57 (10.2) BSTMA 93 (15.7) 137 (16.2) 108 (10.7) BSTV 14 (9.1) 48 (15.0) 45 (19.3) Hypothalamus PVN 27 (7.2) 47 (15.7) 59 (10.8) Dorsomedial a 36 (9.1) 79 (13.1) 80 (26.4) Perifornical a 40 (10.3) 118 (19.5) 139 (22.8) Midbrain SN 9 (5.7) 36 (15.7) 23 (8.4) VTA a 8 (2.2) 25 (7.6) 31 (6.1) PN a 5 (1.8) 16 (6.0) 18 (5.1) IF a 13 (4.4) 49 (14.1) 56 (7.4) a Structures showing a test effect (ABB/ABA AB0). Abbreviations: AStr, amygdalostriatal transition zone; BSTLD, lateral dorsal bed nucleus of the stria terminalis; BSTMA, medial bed nucleus of the stria terminalis; BSTV, ventral bed nucleus of the stria terminalis; CeC, capsular division of the central amygdala; CeL, lateral division of the central amygdala; CeM, medial division of the central amygdala; IF, interfascicular nucleus; LA, lateral amygdala; PN, paranigral nucleus; PVN, paraventricular hypothalamus; SN, substantia nigra. There were no differences between groups in paraventricular hypothalamus. To study the distribution of c-fos relative to orexin neurons in hypothalamus we used double immunofluorescence. The mean (S.E.M.) numbers of orexin-ir, single labeled c-fos-ir, and dual-labeled c-fos/ orexin-ir neurons are presented in Table 2 and representative photomicrographs as well distributions are shown in Fig. 2. For both DMH and PeF there was significant testassociated c-fos induction in orexin neurons (P 0.05). By contrast, for LH, there were only minimal numbers of duallabeled c-fos/orexin neurons and there were no differences between groups. This indicates that the majority of renewal-associated c-fos expression in LH was restricted to orexin-negative lateral hypothalamic neurons. Indeed, there was a moderate positive correlation (r 0.65) between single c-fos labeled LH neurons and nose-poking on test (Fig. 2). There was significant renewal-associated c-fos induction in the infralimbic region, prefrontal cortex (ilpfc) (P 0.05) (Fig. 1). By contrast there was significant testassociated c-fos induction in the prelimbic and anterior cingulate regions of the PFC (P 0.05). In midbrain, there was no evidence for renewal-associated c-fos induction in any region examined. There was, however, significant testassociated c-fos induction in VTA, paranigral, and interfascicular regions (P 0.05). Three structures BLA, LH, and ilpfc showed renewal-associated c-fos induction. We have previously shown for sucrose (Hamlin et al., 2006) and alcohol (Hamlin et al., 2007) that renewal-associated c-fos induction is not caused by the increased nose-poking observed in group ABA. Nonetheless, we also determined for cocaine whether renewal-associated c-fos was due to re-exposure to the training context (i.e. context A) or to the nose-poking elicited by that re-exposure. We studied c-fos expression in BLA, LH, and ilpfc of two further groups: group ABA- Response (n 3) and group ABA No Response (n 3). These groups were trained and extinguished in an identical manner to groups ABA and also returned to context A for 1 h on test. However, the nose-poke manipulanda had been removed from the contexts prior to test for group ABA No Response (to prevent nose-poking) and was present for group ABA-Response. One brain was lost during perfusion for group ABA No Response. Regardless, for BLA (group ABA-Response mean 249; group ABA No Response mean 243), LH (group ABA-Response mean 58; group ABA No Response mean 62) and ilpfc (group ABA-Response mean 196; group ABA No Response mean 173) both groups showed equivalent levels of c-fos induction, despite group ABA- Response engaging in high levels of nose-poking (mean active 27.9) and group ABA No Response engaging in Table 2. Mean S.E.M. numbers of orexin, total c-fos, single c-fos, c-fos/orexin double labeled neurons, and the percentage of orexin neurons expressing c-fos in the DMH, PeF, and LH following cocaine renewal AB0 ABB ABA Dorsomedial Orexin ve 77 (5.8) 66 (6.0) 69 (4.3) c-fos ve 36 (9.2) 101 (17.1) 139 (37.2) c-fos ve/orexin ve 34 (8.2) 79 (15.8) 112 (33.4) c-fos ve/orexin ve 2 (1.0) 22 (5.4) 27 (5.0) % Orexin ve/c-fos ve 2 (1.2) 33 (6.9) 40 (7.9) Perifornical Orexin ve 334 (24.0) 292 (18.9) 333 (16.3) c-fos ve 40 (10.4) 134 (18.9) 193 (33.3) c-fos ve/orexin ve 31 (7.7) 77 (17.8) 117 (25.3) c-fos ve/orexin ve 9 (5.2) 57 (10.2) 76 (11.4) % Orexin ve/c-fos ve 3 (2.1) 19 (2.7) 23 (3.1) Lateral Orexin ve 118 (8.8) 128 (9.4) 132 (5.5) c-fos ve 21 (3.6) 44 (6.2) 77 (12.3) c-fos ve/orexin ve 20 (3.4) 40 (6.5) 71 (12.0) c-fos ve/orexin ve 1 (0.5) 4 (1.1) 5 (1.3) % Orexin ve/c-fos ve 1 (0.5) 3 (0.8) 4 (1.0)

7 A. S. Hamlin et al. / Neuroscience 151 (2008) none. This is consistent with our previous findings that nose-poking is not causal to c-fos expression under the conditions studied here (Hamlin et al., 2006, 2007). Experiment 2: Retrograde tracing from LH and VTA during renewal of cocaine-seeking Behavior. Fig. 3A shows the mean and S.E.M. responses at the end of self-administration training (left panel), during extinction training (middle panel), and on test (right panel). Inspection of the figure confirms the acquisition, extinction, and ABA renewal of cocaine-seeking. There was significantly more responding on the active than inactive nose-poke at the end of self-administration training (F(1, 9) 19.3; P 0.05). During extinction training, the analyses confirmed that, averaged across days, there was significantly more responding on the active than inactive nose-poke (F(1, 9) 13.0; P 0.05). There was also a significant decrease in overall levels of responding across days of extinction training (F(1, 9) 19.8; P 0.05). The interaction between the decrease in responding on the active versus the inactive nose-poke across the course of extinction training approached, but did not reach, significance (F(1, 9) 3.8; P 0.05). To test for the presence of ABA renewal we compared responding during test with responding during the last day of extinction. There was an overall significant difference in level of responding between the last day of extinction in context B and the test in context A (F(1, 9) 15.8; P 0.05). There was an overall significant difference in responding on the active versus inactive nose-poke averaged across both days (F(1, 9) 28.4; P 0.05). There was also a significant interaction, so that the difference in responding between the last day of extinction in context B and test in context A was significantly greater for active than inactive nose-poking (F(1, 9) 15.3; P 0.05). This confirms ABA renewal of extinguished cocaine-seeking. Injection sites and distribution of retrograde labeled neurons. Forty-nanoliter injections of the retrograde tracer CTb were directed at VTA (Fig. 3B) or LH (Fig. 3C). Injections targeting VTA resulted in a dense region of CTb staining that encapsulated VTA, paranigral, and interfascicular nuclei, which spread throughout the rostro-caudal extent of VTA in one hemisphere. Analysis of forebrain sections ranging from 4.2 to 6.0 mm from bregma (Paxinos and Watson, 1997) revealed a pattern of retrograde labeled neurons consistent with previous studies (Phillipson, 1979; McDonald et al., 1991; Wright et al., 1996; Geisler and Zahm, 2005, 2006; Geisler et al., 2007). Retrograde labeled neurons were detected throughout forebrain including prefrontal cortex (anterior cingulate, orbital, prelimbic, infralimbic and the dorsal tenia tecta), claustrum, septum, preoptic, habenula and hypothalamus. Based on the distribution of c-fos induction in LH following renewal of cocaine-seeking, CTb injections into LH were directed at rostral regions lateral to the f. These resulted in a distribution of retrograde labeled neurons similar to previous studies (Risold et al., 1997; Petrovich et al., 2001, 2005; Yoshida et al., 2006) and included prefrontal cortex (anterior cingulate, orbital, prelimbic, and infralimbic), nucleus accumbens BNST, central amygdala, septum, habenula, and VTA. Distribution of c-fos-positive VTA and LH afferents. Analysis of VTA and LH afferents recruited during renewal focused on regions where experiment 1 detected test- or renewal-associated induction of c-fos. Renewal of cocaine-seeking induced c-fos protein in a number of regions that project to VTA or LH, however, only low numbers of these neurons were found in VTA or LH afferents. c-fos-ir was detected in regions of the forebrain that project to VTA including PFC and hypothalamus. In PFC, large numbers of c-fos-ir neurons and VTA afferents were observed in anterior cingulate, orbital, prelimbic and infralimbic cortices. However, in these regions, c-fos-ir neurons were negative for cytoplasmic labeling of CTb (Fig. 3D). Similarly, in hypothalamus, significant numbers of c-fos- and CTb-IR were observed in PeF and LH regions but no c-fos/ctb double-labeled neurons were found (Fig. 3F; Table 3). c-fos-ir was detected in many forebrain regions projecting to LH including PFC, nucleus accumbens, central amygdala as well as the VTA. However, again, there was no double-labeling of c-fos and CTb detected in these regions. DISCUSSION We studied the neural correlates of the renewal of extinguished cocaine-seeking. Rats were trained to self-administer cocaine in context A. This responding was then extinguished in a second, different context, context B. Finally, rats were tested for cocaine-seeking in context A (group ABA) or context B (group ABB). Return to the original training context renewed extinguished cocaine-seeking. This confirms previous reports of ABA renewal of extinguished cocaine-seeking (e.g. Fuchs et al., 2005, 2007) and confirms the important role for contexts in determining relapse to drug-seeking (Bouton, 2002). Renewal-associated and test-associated Fos expression Examination of c-fos induction revealed two patterns. The first, renewal-associated c-fos, was observed in three structures: BLA, LH, and ilpfc. The presence of renewalassociated c-fos in BLA is consistent with the well-documented role for BLA in renewal (Fuchs et al., 2005, 2007) as well as cue-induced relapse to drug-seeking (e.g. Fuchs and See, 2002; Kantak et al., 2002; McLaughlin and See, 2003; Yun and Fields, 2003). BLA has long been implicated in mediating the behavioral impact of environmental stimuli on instrumental responding (Alderson et al., 2000; Balleine et al., 2003; Cador et al., 1989; Corbit and Balleine, 2005; Everitt et al., 1989; Whitelaw et al., 1996). The role of LH in renewal is less well understood. To the best of our knowledge there has been no examination of the effects of LH manipulations (e.g. lesion; reversible inactivation) on renewal of extinguished reward-seeking. Nonetheless, LH

8 666 A. S. Hamlin et al. / Neuroscience 151 (2008) Fig. 2. (A) Low-powered photomicrograph showing the distribution of orexin neurons and the divisions of the hypothalamus used for analysis. The hypothalamus was divided into three regions based on structural landmarks (Paxinos and Watson, 2006). The medial area that contains the orexin neurons of the DMH was formed by the area between the 3V and the medial edge of the mt, the central region that contains the orexin neurons of the PeF from the mt past the lateral edge of the f (approximately half the width of the f past the lateral edge of the f) and the lateral regions that contains the orexin neurons of the LH by the remaining area extending to the to the lateral edge of the internal capsule. Scale bar 200 m. (B) Percentage of orexin neurons expressing c-fos in the dorsomedial, perifornical, and LH. Bars show the mean ( S.E.M.) percentage of orexin neurons that expressed c-fos for each of the treatment groups. Being placed in either environment on test increased the percentage of orexin neurons that expressed c-fos in both the DMH and PeF (ABB/ABA versus AB0, * P 0.05). (C) Illustration showing the distribution of single labeled orexin (green

9 A. S. Hamlin et al. / Neuroscience 151 (2008) and BLA are the only structures to show renewal-associated c-fos induction across three different reinforcers (cocaine, alcohol, sucrose) (Hamlin et al., 2006, 2007). This suggests that LH, like BLA, may have a fundamental role in renewal and identifies this role as worthy of further examination. Renewal-associated c-fos was detected in ilpfc. We did not detect this previously in our studies using sucrose (Hamlin et al., 2006) or alcohol (Hamlin et al., 2007) reinforcers. Moreover, Fuchs et al. (2005) reported that renewal of cocaine-seeking was attenuated by reversible inactivation of prelimbic but not ilpfc. The infralimbic region plays an important role in masking pavlovian appetitive responding after extinction training (Rhodes and Killcross, 2004, 2007), but its role in instrumental conditioning may be distinct. The available evidence suggests that infralimbic and prelimbic cortex serve complementary roles in instrumental responding. The prelimbic region mediates dominance over behavior by voluntary or goal-directed responding whereas ilpfc mediates dominance over behavior by habit (Killcross and Coutureau, 2003; Coutureau and Killcross, 2003). Induction of c-fos in ilpfc during renewal could indicate that relapse is dominated by habit (Tiffany, 1990). This explains why levels of responding during renewal are unaffected by inactivation of ilpfc (Fuchs et al., 2005) ilpfc inactivation reinstates goaldirected control over otherwise habitual behavior (Killcross and Coutureau, 2003; Coutureau and Killcross, 2003). Inactivation of ilpfc does not attenuate levels of responding during renewal of instrumental responding; it switches renewal from habit to voluntary or goal-directed cocaineseeking. Nonetheless, these data leave unanswered the important question of the neural circuits through which BLA, LH, and ilpfc contribute to renewal of cocaine-seeking. The simplest possibility is that renewal depends on BLA and/or ilpfc afferents to LH. There was no evidence for this possibility. There was significant retrograde labeling of ilpfc from LH but only sparse dual-labeled c-fos/ctb neurons. For BLA there was only sparse retrograde labeling from LH. Generally, there were few dual-labeled c-fos/ CTb neurons throughout the forebrain. This could indicate involvement of trans-synaptic projections, but it also raises the possibility that LH afferents during renewal arise from brainstem. The second pattern of expression detected here, testassociated c-fos, was observed in numerous structures, including anterior cingulate, entire nucleus accumbens, dorsal striatum, and midbrain dopamine regions. Testassociated Fos expression implies that these structures do not discriminate between renewal to cocaine-seeking (group ABA) and the potential to renew to cocaine-seeking (group ABB). This Fos expression could reflect the effects of being placed in a context on test, although the patterns of Fos expression are different to those induced by context exposure per se (Hamlin et al., 2006; Neisewander et al., 2000). It might also reflect the potential for recovery of extinguished cocaine-seeking which is shared between groups ABA and ABB. These structures may be necessary, but not sufficient, for renewal of cocaine-seeking. Additional activity, namely that provided by BLA, LH, or ilpfc may be required to realize this potential and renew cocaine-seeking. Hypothalamus, orexin, and renewal Orexin has been implicated in synaptic and behavioral responsiveness to psychostimulants and opiates (Borgland et al., 2006; Narita et al., 2006) as well as in reinstatement of extinguished alcohol- (Lawrence et al., 2006) and cocaine-seeking (Boutrel et al., 2005). It has been suggested that distinct populations of hypothalamic orexin neurons serve different functions in reward-seeking (Harris and Aston-Jones, 2006). Specifically, LH orexin neurons are important for reward-seeking whereas PeF orexin neurons are important for stress or arousal (Harris and Aston-Jones, 2006). This suggestion was based on c-fos induction in LH orexin neurons by drug- and foodassociated stimuli versus c-fos induction in PeF orexin neurons by foot shock. We found no renewal-associated c-fos induction in LH orexin neurons. Rather, renewalassociated c-fos was restricted to non-orexin LH neurons. This finding underscores the need to understand nonorexin LH contributions to renewal. Regardless of the reasons for this discrepancy in LH (e.g. conditioned place preference versus self-administration; self- versus experimenter-administered cocaine), there was evidence for c- Fos induction in PeF orexin neurons (see also Dayas et al., 2007). This was due specifically to being tested because it was common between groups ABA and ABB and was not observed in a group with the same pharmacological and behavioral history but never tested (group AB0). We suggest that PeF orexin neurons may motivate general activity or exploration required for cocaine-seeking rather than motivating cocaine-seeking itself (Scammell and Saper, 2005). A second interesting feature emerges from comparison with our recent work, using the same designs, on renewal of sucrose- (Hamlin et al., 2006) and alcohol-seeking (Hamlin et al., 2007). Levels of responding during renewal of extinguished sucrose-, alcohol- or cocaine-seeking were similar, whereas distributions of c-fos in orexin and nonorexin neurons were different. For LH, there were large numbers of dual-labeled c-fos/orexin-ir neurons during renewal of alcohol-seeking which correlated positively with magnitude of behavioral renewal. By contrast, there were few such dual-labeled neurons during renewal of either circles), single labeled c-fos (red dots), and dual-labeled c-fos/orexin neurons (stars) in a representative hypothalamic section from an ABA, ABB, and an AB0 rat. (D) Photomicrograph showing orexin (green cytoplasm) and c-fos (red nuclear) dual-immunofluorescence in the PeF. Arrows show dual-labeled c-fos/orexin-ir neurons. Arrowheads show single-labeled c-fos-ir neurons. Scale bar 50 m. (E) Effect of ABA renewal of cocaineseeking on the number of single labeled c-fos-ir neurons in the LH and a scatterplot showing the correlation between active nose-poke responding and the number of single labeled c-fos-ir neurons in the LH. Abbreviation: ic, internal capsule.

10 668 A. S. Hamlin et al. / Neuroscience 151 (2008) Fig. 3. (A) Mean ( S.E.M.) responses on active and inactive nose-poke during training, and extinction of instrumental responding and on test. Rats were trained in context A, extinguished in context B, and tested in context A. Return to the original training context after extinction resulted in renewal of cocaine-seeking. Photomicrographs of representative 40 nl deposits of CTb in the VTA (B) and LH (C) and c-fos-ir and CTb retrograde labeled neurons after ABA renewal of cocaine-seeking (D G). D and F show c-fos-ir and retrograde-labeled neurons in the infralimbic prefrontal cortex (D) and the LH (F) following application of CTb directed at the VTA. E and G show c-fos-ir and retrograde-labeled neurons in the infralimbic prefrontal cortex (E) and the accumbens shell (G) following application of CTb directed at the LH. No double-labeling of c-fos and CTb was observed in any of these brain regions. Scale bar 100 m. Abbreviations: AcbSh, nucleus accumbens shell; PN, paranigral nucleus.