Behavioural Brain Research 258 (2014) 90–96

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Corticotropin-releasing factor receptor type-2 is involved in the cocaine-primed reinstatement of cocaine conditioned place preference in rats Xiaowei Guan a,∗ , Rong Wan b , Chao Zhu b , Shengnan Li b,∗∗ a b

Department of Human Anatomy, Nanjing Medical University, Nanjing 210029, China Department of Pharmacology, Nanjing Medical University, Nanjing 210029, China

h i g h l i g h t s • Changes of CRFR2 expression in mPFC, HP and DS in cocaine-induced and extinct CPP rats. • Effects of local blockade of CRFR2 on reinstatement of cocaine seeking behavior. • CRFR2 is involved in relapse to cocaine-intake in a brain region-specific manner.

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Article history: Received 3 February 2013 Received in revised form 8 October 2013 Accepted 11 October 2013 Available online 19 October 2013 Keywords: Corticotropin-releasing factor type-2 receptor Cocaine Reinstatement Conditioned place preference

a b s t r a c t Here we explored the in vivo role of brain corticotropin-releasing factor receptor type-2 (CRFR2) in cocaine-primed reinstatement of drug seeking. Conditioned place preference (CPP) procedure was used to assess the acquisition, extinction and reinstatement of cocaine-seeking behavior in rats. First, expressions of CRFR2 were shown to be affected in a brain region-specific manner within cocaine-induced CPP and cocaine-extinct CPP models. Bilateral blockade of CRFR2 in the dorsal portion of the medial prefrontal cortex (mPFC), or hippocampus (HP) was partially inhibited, but in the dorsal striatum (DS) did not affect, the cocaine-primed reinstatement of cocaine CPP. © 2013 Elsevier B.V. All rights reserved.

1. Introduction The successful treatment of drug addiction has been challenged by the high rate of relapse. Relapse to cocaine use can be triggered by re-exposure to cocaine, drug-associated cues, or stressors. The molecular mechanisms underlying the compulsive cocaine craving behaviors remain unclear. Corticotropin-releasing factor (CRF)

Abbreviations: Ast2B, astressin2-B; CPP, conditioned place preference; CRF, corticotropin-releasing factor; CRFR1, corticotropin-releasing factor type-1 receptor; CRFR2, corticotropin-releasing factor type-2 receptor; DS, dorsal striatum; HP, hippocampus; mPFC, medial prefrontal cortex. ∗ Corresponding author at: Department of Human Anatomy, Nanjing Medical University, 140 Hanzhong Road, Nanjing 210029, China. Tel.: +86 25 86862019; fax: +86 25 86863050. ∗∗ Corresponding author at: Department of Pharmacology, Nanjing Medical University, 140 Hanzhong Road, Nanjing 210029, China. Tel.: +86 25 86863024; fax: +86 25 86863050. E-mail addresses: [email protected] (X. Guan), [email protected] (S. Li). 0166-4328/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.bbr.2013.10.019

system has been highly implicated in cocaine relapse [1–6]. Among the two types of CRF receptors, the role of CRF receptor type1(CRFR1) in drug abuse has been well studied [7,8], whereas the role of CRFR2 in cocaine abuse is less clear. CRFR2 differs from CRFR1 in its endogenous ligands and brain distribution [9–12]. Therefore, CRFR2 may exhibit different pharmacological profiles and play different roles in cocaine abuse, as compared to CRFR1. Although systemic blockade of CRFR2 activity does not affect the reinstatement of cocaine-seeking behavior after extinction [5], recent studies have suggested that CRFR2 signaling in certain brain areas may contribute to cocaine-induced neuroplasticity, and ultimately lead to cocaine relapse. For instance, selective blockade of CRFR2 activity in ventral tegmental area (VTA) suppressed relapse to cocaine [13], whereas activation of CRFR2 activity in VTA mimicked footshock-induced reinstatement of cocaine seeking behavior [14]. Activation of CRFR2 in prefrontal cortex was reported to potentiate cocaine-induced enhancement of EPSCs [15]. Chronic cocaine administration was able to switch CRFR2-mediated depression to facilitation of glutamatergic transmission in rat lateral septums

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[16]. Taken together, these results demonstrate that the roles of CRFR2 in cocaine addiction are likely to be in a brain region-specific manner. We previously explored the roles of CRFR2 in development of cocaine-induced electrophysiological plasticity [17,18]. We found that selective blockade of CRFR2 activity inhibited cocaine withdrawal-enhanced long-term potentiation (LTP) in hippocampus (HP) slices, and also attenuated CRF-enhanced LTP in corticostriatal slices from cocaine withdrawal rats. There is evidence from both human and animal studies that hippocampal and corticostriatal circuits contribute to cocaine addiction [19–21]. Within these circuits, hippocampus (HP), medial prefrontal cortex (mPFC), and dorsal striatum (DS) are three important neural substrates for the development of cocaine relapse [22–26]. We hypothesized that CRFR2 in these brain regions are involved in the development of cocaine relapse. As an extension of our previous observations and to further test this hypothesis, here we assessed whether CRFR2 in HP, mPFC, and DS regions play a role in development of cocaine relapse. Conditioned place preference (CPP) paradigm has been widely used to study the rewarding effects of addictive drugs. In this study, CPP procedure was used to assess the acquisition, extinction and reinstatement of cocaine-seeking behavior in rats. So far, reports on the expression of CRFR2 in cortex remain controversial among studies [12,27–29], and there is little evidence for the presence of CRFR2 in striatum. Thus, the first aim of this study is to examine expressions of CRFR2 in HP, mPFC and DS regions under physiological conditions and during acquisition and extinction of cocaine CPP. Next, the roles of CRFR2 in all three regions in the cocaine-primed reinstatement of cocaine CPP were explored by using a selective CRFR2 antagonist, astressin2-B [30–32].

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Fig. 1. Timeline of the CPP tests and drug treatment.

when we collected samples and after behavioral tests. We discarded the samples and data from the rats present in incorrect injection positions. 2.3. Drug treatment Cocaine hydrochloride (Qinghai Pharmaceutical, China) was dissolved in sterile saline and was injected (10 mg/kg, i.p.) during CPP training (day 1–7) or 10 min before CPP test 3 (as shown Fig. 1). The local activities of CRFR2 were selectively blocked by bilateral intramPFC (0.3 ␮l/side), intra-HP (0.8 ␮l/side) or intra-DS (1 ␮l/side) injections of Astressin2 B (Ast2B) at doses of 0 ng/side (vehicle), 200 ng/side, 400 ng/side, 800 ng/side or 1.6 ␮g/side. On the afternoon of day 15, Ast2B was dissolved in artificial CSF (aCSF, vehicle) and was injected bilaterally over a period of 15 min with Hamilton syringes. The injection needle was kept in place of 1 mm deep from guide cannula for an additional 10 min to allow drug diffusion. 2.4. Conditioned place preference (CPP)

2. Material and methods All experiment procedures were performed in accordance with the Nanjing Medical University Guide for the Care and Use of Laboratory Animals, China, and the National Institute of Health Guide for the Care and Use of Laboratory Animals (NIH Publications No. 80-23). All efforts were made to minimize the number of animals used and to minimize their suffering. 2.1. Animals Male Sprague-Dawley rats (300–330 g, n = 308) were used in this study. Rats were maintained on a reverse 12 h light/dark cycle with ad libitum access to food and water. 2.2. Surgery Rats were anesthetized with chloral hydrate (514 mg/kg, i.p.) and placed in a stereotaxic apparatus. Stainless-steel guide cannula (23-gauge; Plastics One Inc., USA) were bilaterally implanted 1 mm above the hippocampus (HP), the dorsal striatum (DS), or medial prefrontal cortex (mFPC). The coordinates for the CA1 area of HP (Paxinos, 1986) were AP, −4.2 mm from bregma; ML, ±2.2 mm from midline; and DV, −2.5 mm from skull surface. The DS coordinates were AP, +0.7 mm from bregma; ML, ±2.0 mm from midline; and DV, −3.6 mm from skull surface. The implantation of cannulae into mPFC were performed at a 30◦ angle from vertical to avoid the superior sagittal sinus, and the coordinates were AP, +2.2 mm from bregma; ML, ±2.3 mm from midline; and DV, −3.1 mm from skull surface. The cannula was affixed to the skull with screws and dental cement. A stylet was placed into the guide cannula to allow the guide cannula to maintain patency. The rats were allowed to recover for 7 days after surgery. We checked the injection tracks

CPP was conducted in an apparatus constructed of three chambers (72 cm × 25 cm × 32 cm, Zhenghua Biologic Apparatus, China). The two larger side chambers (30.5 cm × 25 cm × 32 cm each) differ in their walls (black or black with white stripes) and floors (stainless-steel mesh or stainless-steel bars). The smaller middle chamber (11 cm × 25 cm × 32 cm) has gray wall with a smooth PVC floor. The three distinct chambers are separated by removable guillotine doors. Time spent in each chamber was recorded by means of infrared beam crossings which are located in the walls of each chamber. The place preference procedure consisted of three phases: pre-conditioning phase (baseline preference), conditioning (CPP training), and post-conditioning test (CPP test). The detailed CPP timeline of this study is shown in Fig. 1. During pre-conditioning phase (day 0) the rats were free to explore the three chambers for 15 min. Time spent in each chamber was calculated by computer and recorded as baseline data. Rats that spent more than 500 s in one chamber were dismissed from testing. CPP training (day 1–7) was performed for 7 consecutive days with twice daily injections. The first injection was performed in the morning with either administration of cocaine hydrochloride (10 mg/kg, i.p., Qinghai Pharmaceutical, China) or saline (0.5 ml/kg, i.p.), and the rats were confined to one conditioning side chamber (drug-paired chamber) for 45 min after the injection and then returned to their home cages. The second injection was performed in the afternoon with administration of saline (0.5 ml/kg, i.p.), and the rats were confined to the other side chamber (non-drug-paired chamber) for 45 min and then returned to their home cages. In CPP test phase, the rats freely moved throughout the apparatus for 15 min, exactly as in the preconditioning phase. CPP test 1 was carried out on day 8. After the CPP test 1, rats were subjected to extinction of cocaine conditioning. During the extinction period, all rats were free to access the three chambers for 15 min each day without any injections. Following

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the last extinction trial, CPP test 2 was carried out on the morning of day 15. On day 15, all rats spent the same amount of time in the drug-paired chamber as they did in the non-drug-paired chamber, indicating complete extinction was established. On the afternoon of day 15, rats were bilaterally injected with Ast2B into the HP, the DS, or the mPFC regions. On the morning of day 16 (about 12 h after the intracranial injection), all rats received a priming injection of cocaine (10 mg/kg, i.p.). CPP test 3 was carried out immediately after the prime injection. The CPP score was defined as the ratio of the time spent in the drug-paired chamber minus the time spent in non-drug-paired chamber. Some rats received the same drug treatment paradigm as shown in Fig. 1, but did not experience CPP training and tests. 2.5. Western blot Rats with CPP were decapitated 30 min following behavioral tests on day 8, day 15 and day 16. Rats without CPP procedure were decapitated on day 8, day 15 and day 16. HP (CA1 mainly), mPFC and DS were collected immediately. Total protein was extracted respectively from HP, DS and mPFC using RIPA lysis buffer (Beyotime, China). Proteins were separated by 10% SDS-PAGE and electrophoretically transferred onto PVDF membranes. The transferred membranes were blocked with 5% non-fat dry milk and 0.1% Tween 20 in 10 mM Tris–HCl (TBST buffer, pH 7.5) for 2 h at room temperature and subsequently incubated with a goat polyclonal antibody against CRFR2 (1:400 dilution; Santa Cruz, USA) at 4 ◦ C overnight. The next day, the membranes were incubated with an HRP-conjugated secondary antibody (1:4000, Santa Cruz, USA) at room temperature for 1.5 h. The blots were visualized using an ECL kit (Beyotime, China) and exposed to X-ray film (Kodak, USA). Since the molecular weight of CRFR2 might be close to that of loading controls used in this study, we used stripping buffers (Beyotime, China) to remove the CRFR2 antibody from the blotted membranes in order to re-probe for the loading controls on the same membranes. In brief, the membranes were incubated for 10 min at room temperature on a shaker with stripping buffers and then washed three times for 5 min each in a TBST buffer. Then the procedure from blocking to exposing was repeated as described above. In this study, beta-actin was used as a loading control. Values for CRFR2 protein levels were calculated using Image J software (NIH, USA) and normalized to beta-actin. Western blots were repeated at least four times in different rats. 2.6. Reverse transcriptase-polymerase chain reaction (RT-PCR) Rats with CPP were decapitated 30 min following behavioral tests on day 8, day 15 and day 16. HP (CA1 mainly), mPFC and DS were collected immediately. Total RNA was extracted by the Trizol method (Invitrogen, USA), precipitated with isopropanol, and treated with DNase I (New England Biolabs, USA). The quality and quantity of RNA samples were assessed by using a NanoDrop 1000 UV spectrophotometer (Midland, Canada). cDNA for total RNA were reversely transcribed with an Omniscript RT kit (QIAGEN, USA). Template (1 ␮l) was amplified by PCR with platinum Taq polymerase (1 unit; Promega, USA) in 20 ␮l total reaction volume containing 0.5 ␮mol of each specific PCR primer as follows: CRFR2, 5 -CCT GCT GCA ACT CAT CGA CC-3 and 5 -AAG AGC CAC TTG CGC AGA TG-3 ; GAPDH, 5 -TCA TCC CTG CAT CCA CTG GT-3 and 5 -TCC TCA GTG TAG CCC AGG AT-3 . GAPDH was used as a loading control. All the primers were blasted (http://blast.ncbi.nlm.nih.gov/blast.cgi) to ensure that all these primers were selective for CRFR2 and GAPDH. Each PCR cycle consisted of 20 s at 94 ◦ C, 20 s at 55 ◦ C, and 30 s at 72 ◦ C. PCR amplification was carried out for 30 cycles. After amplification, the products were separated on a 2% agarose gel containing 0.025%

ethidium bromide. Bands were then visualized under UV illumination, and gels were photographed with the BioDoc-It imaging system (Ultra-Violet Products Ltd., USA). Values for CRFR2 mRNA levels were calculated using Image J software (NIH, USA) and normalized to GAPDH. RT-PCR was repeated at least four times in different rats. 2.7. Immunofluorescence Rats with CPP were anesthetized 30 min after the end of the CPP test 1 (on day 8), and perfused with saline followed by ice-cold 4% paraformaldehydev(PFA) in a PBS buffer (pH 7.4). The brains were removed and post-fixed in the same 4% PFA for 2 h, and then were put in 30% sucrose overnight and cut on cryostat to form frozen coronal sections (15 ␮m). The immunofluorescent procedure was adapted from protocols previously described [33]. In brief, freefloating frozen sections were blocked with 5% fetal bovine serum in PBS for 1 h and were incubated at 4 ◦ C overnight in PBS, 4% Triton X-100 and 1% fetal bovine serum containing the following primary antibodies: a goat anti-CRF2R polyclonal antibody (1:100 dilution; Santa Cruz, USA) and a mouse anti-neuronal class III ␤-tublin polyclonal antibody (Tju1; 1:250 dilution; Beyotime, China). Then, the sections were incubated with the TRITC-labeled rabbit anti-goat secondary antibody and FITC-labeled goat-anti mouse secondary antibody (1:400 dilution respectively; Santa Cruze, USA) at 37 ◦ C for 1 h. Negative control sections were performed by replacing the primary antibodies with PBS buffer. The positive cells of immunoreactive products were counted by software (Image J software, USA). Here, Tju1 was served as a marker for staining neurons in the brain sections. The percentage of CRF2R-positive cells was calculated by the number of CRF2R-positive cells minus the number of the Tju1positive cells in the same section. The experiments were repeated at least three times in different rats’ brains, and four fields were tested in one brain section. 2.8. Data analysis All results were presented as mean ± SD. For the western blot and RT-PCR experiments, levels of CRFR2 protein and mRNA served as the ratio of the normalized value of CRFR2 expression to the average value of CRFR2 expression in control groups. For the study of CRFR2 expression, we used the data from the 7-day salineadministrated rats as our normal control, and did not set up saline extinction control further. Western blot data of CRFR2 expression were analyzed by two-way ANOVA with a Student–Newman–Keuls multiple comparisons test. Immunofluorescence data of CRFR2 positive cells were analyzed by student t-test. The CPP score was the ratio of time spent in the drug-paired chamber to time spent in the non-drug-paired chamber. Data of CPP tests behavior were analyzed by ANOVA with a Student–Newman–Keuls multiple comparisons test. In all cases, significance was set at p < 0.05. 3. Results 3.1. Expressions of CRFR2 protein and mRNA in HP, mPFC and DS regions in cocaine-induced and cocaine-extinct CPP rat models As shown in Fig. 2A, rats spent a similar amount of time in drugpaired and non-drug-paired chambers in the pre-conditioning phase (D0). After 8-day cocaine administration with CPP training, rats got a higher CPP score than that measured on D0, suggesting the acquisition of cocaine-induced CPP in rats (p < 0.01 vs. D0). In contrast, the saline-treated rats did not show place preference (p > 0.05 vs. D0) on day 8 (D8), and hence were used as normal control. On D8, levels of CRFR2 protein (F = 87.367 for mPFC, F = 12.920 for HP, and F = 54.658 for DS, p < 0.01) as well as mRNA (F = 224.31

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Fig. 2. Expression of CRFR2 protein and mRNA in mPFC, HP and DS regions. (A) Acquisition and extinction of cocaine CPP. CPP score is presented as the ratio of the time spent in the drug-paired chamber to the time spent in non-drug-paired chamber. D0, day 0; D8, day 8; D15, day 15. **p < 0.01 vs. D0; ## p < 0.01 vs. D8. (B and C) Levels of CRFR2 protein and mRNA in mPFC, HP and DS regions in rats with CPP procedure. (D) Levels of CRFR2 protein in mPFC, HP and DS regions in rats without CPP procedure. The top pictures represent CRFR2 band. Beta-actin and GAPDH are served as loading controls. The intensity of CRFR2 band is firstly normalized by beta-actin or GAPDH. The levels of CRFR2 protein or mRNA are presented as fold of normalized CRFR2 protein levels in COC or CET rats over that in SAL rats. The bottom bars show the relative levels of CRFR2 protein. n = 3–4 rats per group. **p < 0.01 vs. SAL; ## p < 0.01 vs. COC. SAL, saline-treated rats; COC, cocaine CPP-acquired rats; CET, cocaine CPP-extinguished rats; CCE, chronic cocaine-exposed rats; CCA, chronic cocaine-absent rats.

for mPFC, F = 74.042 for HP, and F = 44.337 for DS) were both significantly increased in mPFC, HP or DS regions in cocaine-treated rats (COC, Fig. 2B, C), as compared to those in saline control (SAL). On day 15 (D15), after 8-day extinction training of cocaine treatment, rats with a cocaine-treated history showed similar CPP score as that measured in pre-conditioning phase (p > 0.05), indicating that the cocaine-induced CPP had diminished in rats (CET). At this point of time, expressions of CRFR2 protein and mRNA in mPFC and DS returned to normal levels (p > 0.05 vs. SAL), but remained at an up-regulated level in HP (p < 0.01 vs. SAL) in cocaine-experienced rats. Levels of CRFR2 protein in mPFC, HP or DS regions in rats that received the same drug treatment but without a CPP procedure were examined in this study. As shown in Fig. 2D, expression of CRFR2 protein in mPFC, HP or DS regions in rats without CPP procedure showed similar changes to that in rats with CPP procedure. The CRFR2 protein level in HP increased in rats following repeated cocaine exposure (p < 0.01 vs. SAL), and this increase persisted even following an 8-day abstinence from cocaine injection (F = 131.05, p < 0.01 vs. SAL). The CRFR2 protein levels in mPFC (F = 165.65) and DS (F = 405.53) regions were also up-regulated in chronically cocaine-exposed rats (p < 0.01 vs. SAL), but returned to normal level on day 15 following an 8-day abstinence from cocaine injection (p > 0.05 vs. SAL).

cocaine-induced CPP rats. Thus, these sub-regions were selected to be the infusion sites for Ast2B in this study. As shown in Fig. 3 B, rats that received cocaine CPP training acquired CPP behavior on D8 (p < 0.01 vs. D0). These cocaineinduced CPP behaviors were extinct after 8-day extinction training on D15 (p > 0.05 vs. D0). On day 16 (D16), a single cocaine-priming injection successfully reinstated cocaine CPP in these rats, as indicated by a higher CPP score than that measured after cocaine extinction (p < 0.01 vs. D15). Injection of vehicle (0 ng/side) into the brain did not affect reinstatement of cocaine CPP in rats. However, bilateral infusion of Ast2B at every dose of 200 ng, 400 ng, 800 ng, and 1.6 ␮g per side into mPFC, or at a high dose of 1.6 ␮g per side into HP prior to cocaine priming significantly attenuated the cocaine-primed reinstatement of CPP in cocaine-experienced rats (p < 0.01 vs. 0 ng/side on D16, as shown in Fig. 3C). The pretreatment of bilateral infusions of Ast2B at every dose into DS, or at lower doses (200–800 ng/side) into HP did not alter cocaine-primed reinstatement of CPP (p > 0.05 vs. 0 ng/side on D16). The infusions of Ast2B at every dose into HP, mPFC or DS did not affect CPP in saline-treated rats (p > 0.05 vs. D0, Fig. S2), suggesting that blockade of CRFR2 in these brain regions did not affect animal CPP behavior under physiological conditions.

3.2. Effects of blocking CRFR2 in HP, mPFC and DS on cocaine-primed reinstatement of CPP

Emerging evidence shows that corticotropin-releasing factor receptor type-2 (CRFR2) in the brain plays an important regulatory role in the development of drug abuse [13,14,16]. In this study, we examined the expressions of CRFR2 protein and mRNA in the medial prefrontal cortex (mPFC), hippocampus (HP) and dorsal striatum (DS) regions in cocaine-induced and cocaine-extinct CPP rat models. Then, we investigated the roles of CRFR2 in these brain regions in the reinstatement of cocaine CPP. We found that dynamic changes of CRFR2 expression occurred in mPFC, HP and DS regions during the acquisition and extinction of cocaine CPP. First, there was an increase in CRFR2 mRNA and protein in mPFC, HP and DS in cocaine CPP rats, as compared to controls. Subsequently, following extinction training from cocaine

To investigate the in vivo roles of CRFR2 in cocaine-primed reinstatement of cocaine seeking, we injected Ast2B into mPFC, HP or DS to block CRFR2 activity. Five doses (0 ng, 200 ng, 400 ng, 800 ng and 1.6 ␮g per side) of Ast2B were used in this study. To observe the sub-regions of mPFC, HP or DS where changes in CPRR2 protein expression mainly occurred during acquisition of cocaine CPP, immunostainings for CRFR2 were performed in SAL and COC rats with CPP procedure. As shown in Fig. 3A and Fig. S1, the number of CRFR2-positive neurons mainly increased in the dorsal portion of mPFC, CA1 region of HP, and the anterior portion of DS in

4. Discussion

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Fig. 3. Effects of pretreatments with Ast2B into mPFC, HP and DS on cocaine-primed reinstatement of cocaine CPP. (A) Immunostaining for CRFR2 protein in mPFC, HP and DS regions in rats with CPP procedure and anatomical location of drug injection sites. The dark spots show intra-injection sites. Scale bar, 24 ␮m. SAL, saline-treated rats; COC, cocaine CPP-acquired rats. (B) Expression of CPP scores on day 0 (D0), D8, D15 and D16. On D15, rats receive an intra-dorsal mPFC, HP CA1 or DS injection of vehicle (aCSF). On D16, rats receive a single cocaine injection to reinstate the extinguished cocaine CPP. **p < 0.01 vs. D0. (C) Curve of Ast2B dose-CPP response. On D15, rats receive bilateral intra-dorsal mPFC, HP CA1 or DS injection of Ast2B at doses of 0, 200, 400, 800, and 1600 ng per side. On D16, CPP test 3 is performed to observe the effects of local Ast2B infusion on cocaine-primed reinstatement of CPP. n = 6–9 rats per group. $$ p < 0.01 vs. vehicle (0 ng/side).

treatment, the expressions of CRFR2 returned to normal levels in mPFC and DS, but remained at an elevated level in HP. These results suggest that CRFR2 expression during acquisition and extinction of cocaine CPP may be exhibited in a region-specific manner, and such region-specific changes of CRFR2 might perform different contributing mechanisms underlying cocaine abuse. Repeated cocaine exposure also produced an increase in CRFR2 protein in all three regions in rats without CPP training. Furthermore, this increase was still evident in HP following an 8-day abstinence from any drug injection, suggesting changes of CRFR2 expression in cocaine CPP and cocaine-extinct CPP rat models might be due to cocaine treatment rather than the CPP paradigm. In order to determine whether these changes of CRFR2 expression in three regions contribute to the reinstatement to cocaine seeking behavior, we injected asstressin2-B (Ast2B) bilaterally into mPFC, HP or DS prior to one cocaine-priming challenge. To decide the injection sites, immunostaining for CRFR2 in three regions were performed in cocaine CPP rats and controls. Our results show that the increase in CRFR2 protein following cocaine treatment occurred mainly in the dorsal portion of mPFC rather than its ventral portion. Dorsal and ventral mPFC have been thought to play different roles in drug abuse [34–37]. It has been reported that acute inactivation of dorsal but not ventral mPFC neurons attenuated cocaine priming-, cocaine cue-, or stress-induced reinstatement of cocaine seeking [34,38,39]. Based on these findings and our immunostaining results, we injected Ast2B mainly into the dorsal portion of mPFC. We found that Ast2B at every dose used here could significantly attenuate cocaine-primed reinstatement of cocaine seeking, although the CPP score was still much higher than baseline. This finding is in line with our previous in vitro observation that inhibiting CRFR2 reduce urocortin 2 (a selective CRFR2 agonist) enhanced LTP in corticostriatal slices from cocaine withdrawal rats [18]. However, it should be noted that there was no differences in CRFR2 levels in rat mPFC following extinction training when rats

received Ast2B injections. Mechanisms through which Ast2B modulate cocaine-primed reinstatement of cocaine seeking behavior remains to be further investigated. Drug-primed reinstatement of CPP may be partially due to a recall of drug-associated memory or renewed incentive effects of the priming drug [40,41]. It has been shown that brain CRFR2 signaling may affect neuronal activity that are involved in rewarding, learning and arousal responses [42,43]. mPFC is an important substrate for development of reward effects in drug abuse, and lesions in mPFC have been reported to inhibit cocaine-primed reinstatement of CPP [38,44,45]. We hypothesized that an increase in CRFR2 expression in mPFC following cocaine exposure results in neuroadaptional and neurochemical changes in mPFC. Long-term changes in mPFC by cocaine exposure may lead to dysfunctional goal-directed behavior and impair decisionmaking, which contribute to relapse to drug seeking [6,45,46]. Although there was no increase in CRFR2 levels in mPFC during extinction phase of CPP in rats, Ast2B given at this point of time could inhibit CRFR2 activity, somehow partially reverse cocaine exposure-induced neural changes in mPFC, and therefore attenuate cocaine-primed reinstatement of cocaine-seeking behavior. Drug-related memory is an important mechanism for relapse to drugs [47]. The Hippocampus, due to its role in memory, is thought to be key brain region involved in cocaine abuse [19,20]. Disruption of neural activities in HP was reported to inhibit cocaine CPP [48], suggesting HP plays an important role in the expression of CPP behavior. In the current study, CRFR2 proteins and mRNA in HP (mainly CA1 region) were kept at higher levels in cocaineinduced and cocaine-extinct rats, as compared to controls. Infusion of Ast2B at a high dose of 1.6 ␮g/side, but no lower than 800 ng/side, into the HP CA1 region was able to partially attenuate cocaineprimed reinstatement. CRFR2 is highly expressed in HP [28]. Most of the mechanisms related to drug addiction in HP, such as synaptic modulation, glutamate receptors, and dendritic morphology, likely involve CRFR2 [49–51]. The molecular mechanisms for how

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hippocampal CRFR2 modulates cocaine addiction and relapse need to be further investigated. In this study, the injection site in the DS was much more medial and anterior, at which area we previously had recorded CRFR2depended changes in corticostriatal LTP from cocaine-withdrawal rats [18]. DS is known as an important region for habit forming and expectancy-mediated actions [52–54]. Cocaine exposure induced an increase in CRFR2 expression and might contribute to the formation and development of cocaine addiction. In this study, however, no effect was found on cocaine-primed reinstatement of CPP by infusion of Ast2B at every dose into DS. One possible reason for these results might be due to the animal behavioral models used here. CPP is a commonly used animal model to study relapse to drug abuse. However, some positive results could not be detected by CPP paradigm due to its methodological limitations [55]. So, results obtained by CPP models often need to be confirmed by other animal models, such as self-administration model. Another possibility is that CRFR2 in DS may not be involved in drug priminginduced relapse, but contribute to other types of relapses, such as cue- or stressor-induced relapse to drugs. Future studies are needed to determine functional roles of CRFR2 alteration in DS in cocaine addiction and relapse. In summary, the current study shows dynamic changes in CRFR2 expression in mPFC, HP and DS regions during the acquisition and extinction of cocaine CPP. Local blockade of CRFR2 in mPFC or HP attenuated, but in DS did not alter, cocaine-primed reinstatement of cocaine CPP. These findings suggest that CRFR2 may be involved in relapse to cocaine intake in a region-specific manner, and CRFR2 may be a useful target for managing cocaine relapse. Acknowledgements This work was supported by the National Natural Science Foundation of China (No. 81000572 and No. 81373467) and the Natural Science Foundation of Jiangsu Province of China (No. 10KJB180005). The authors would like to thank Dr. Lei Li, Department of Human Anatomy, Nanjing Medical University, for stereotaxic surgical assistance and Dr. Yun Guan, Department of Anesthesiology/CCM, Johns Hopkins University, for editing the manuscript. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.bbr.2013.10.019. References [1] Blacktop JM, Seubert C, Baker DA, Ferda N, Lee G, Graf EN, et al. Augmented cocaine seeking in response to stress or CRF delivered into the ventral tegmental area following long-access self-administration is mediated by CRF receptor type 1 but not CRF receptor type 2. J Neurosci 2011;31:11396–403. [2] Buffalari DM, Baldwin CK, Feltenstein MW, See RE. Corticotrophin releasing factor (CRF) induced reinstatement of cocaine seeking in male and female rats. Physiol Behav 2011;105:209–14. [3] Erb S, Salmaso N, Rodaros D, Stewart J. A role for the CRF-containing pathway from central nucleus of the amygdala to bed nucleus of the stria terminalis in the stress-induced reinstatement of cocaine seeking in rats. Psychopharmacology 2001;158:360–5. [4] Graf EN, Hoks MA, Baumgardner J, Sierra J, Vranjkovic O, Bohr C, et al. Adrenal activity during repeated long-access cocaine self-administration is required for later CRF-Induced and CRF-dependent stressor-induced reinstatement in rats. Neuropsychopharmacology 2011;3:1444–54. [5] Lu L, Liu D, Ceng X. Corticotropin-releasing factor receptor type 1 mediates stress-induced relapse to cocaine-conditioned place preference in rats. Eur J Pharmacol 2001;415:203–8. [6] Wang B, Shaham Y, Zitzman D, Azari S, Wise RA, You ZB. Cocaine experience establishes control of midbrain glutamate and dopamine by corticotropinreleasing factor: a role in stress-induced relapse to drug seeking. J Neurosci 2005;25:5389–96. [7] Gurkovskaya O, Goeders NE. Effects of CP-154,526 on responding during extinction from cocaine self-administration in rats. Eur J Pharmacol 2001;432:53–6.

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