β1-Adrenoceptor in the Central Amygdala Is Required for Unconditioned Stimulus-Induced Drug Memory Reconsolidation

β1-Adrenoceptor in the Central Amygdala Is Required for Unconditioned Stimulus-Induced Drug... Background: Drug memories become labile and reconsolidated after retrieval by presentation of environmental cues (conditioned stimulus) or drugs (unconditioned stimulus). Whether conditioned stimulus and unconditioned stimulus retrieval trigger different memory reconsolidation processes is not clear. Methods: Protein synthesis inhibitor or β-adrenergic receptor (β-AR) antagonist was systemically administrated or intra-central amygdala infused immediately after cocaine reexposure in cocaine-conditioned place preference or self-administration mice models. β-ARs were selectively knocked out in the central amygdala to further confirm the role of β-adrenergic receptor in cocaine reexposure-induced memory reconsolidation of cocaine-conditioned place preference. Results: Cocaine reexposure triggered de novo protein synthesis dependent memory reconsolidation of cocaine-conditioned place preference. Cocaine-priming-induced reinstatement was also impaired with post cocaine retrieval manipulation, in contrast to the relapse behavior with post context retrieval manipulation. Cocaine retrieval, but not context retrieval, induced central amygdala activation. Protein synthesis inhibitor or β1-adrenergic receptor antagonist infused in the central amygdala after cocaine retrieval, but not context retrieval, inhibited memory reconsolidation and reinstatement. 1-adr β energic receptor knockout in the central amygdala suppressed cocaine retrieval-triggered memory reconsolidation and reinstatement of cocaine conditioned place preference. β 1-adrenergic receptor antagonism after cocaine retrieval also impaired reconsolidation and reinstatement of cocaine self-administration. Conclusions: Cocaine reward memory triggered by unconditioned stimulus retrieval is distinct from conditioned stimulus retrieval. Unconditioned stimulus retrieval induced reconsolidation of cocaine reward memory depends on β1-adrenergic signaling in the central amygdala. Post unconditioned stimulus retrieval manipulation can prevent drug memory reconsolidation and relapse to cocaine, thus providing a potential strategy for the prevention of substance addiction. Keywords: β-AR, unconditioned stimulus, conditioned stimulus, memory reconsolidation, cocaine Received: September 7, 2017; Revised: October 15, 2017; Accepted: December 1, 2017 © The Author(s) 2017. Published by Oxford University Press on behalf of CINP. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http:// creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, 267 provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com Downloaded from https://academic.oup.com/ijnp/article-abstract/21/3/267/4690578 by Ed 'DeepDyve' Gillespie user on 16 March 2018 268 | International Journal of Neuropsychopharmacology, 2018 Significance Statement It is well known that drug memories become labile and reconsolidated upon retrieval by the presentation of conditioned stimulus (CS) or unconditioned stimulus (US). Whether CS and US retrieval trigger different memory reconsolidation processes is unknown. In this study, we found that US retrieval, but not CS retrieval, triggered memory reconsolidation of cocaine-conditioned place preference dependent on β1-AR and de novo protein synthesis in the central amygdala. Furthermore, cocaine priming-induced reinstatement was impaired with post US retrieval manipulation in contrast to the relapse behavior with post CS retrieval manipu- lation. In cocaine self-administration, 1-AR anta β gonism after US retrieval also impaired reconsolidation and reinstatement. Our study indicates that reconsolidation of cocaine reward memory triggered by US retrieval is distinct from CS retrieval. US retrieval induced reconsolidation of cocaine reward memory depends on 1-adr β energic signaling in the central amygdala. Introduction Drug addiction is commonly considered a learning and memory whether US retrieval and CS retrieval are different in the induc- disorder (Boening, 2001; Hyman, 2005). The reinforcing effects of tion of the memory reconsolidation process. a drug (known as unconditioned stimulus [US]), such as drug- Many studies have demonstrated that β-AR is involved in elicited euphoria, are strongly associated with environmental reconsolidation, because systemic or intra-basolateral amyg- cues (known as conditioned stimulus [CS]) (Hyman and Malenka, dala (BLA) injection of a β-AR antagonist immediately after CS 2001; Shalev et al., 2002; Koob and Volkow, 2016). Consequently, retrieval prevents memory reconsolidation of cocaine-SA or exposure to drug-associated environmental cues can evoke v- ar conditioned place preference (CPP) (Milton et al., 2008 Otis et al., ; ious degrees of psychological dependence or compulsive drug 2013). In the signaling cascades downstream of β-AR, amygdala taking (Cadet et  al., 2014W ; ise and Koob, 2014). A  major chal- protein kinase A and ERK are both involved in the reconsolida- lenge in the treatment of drug addiction is drug relapse, which tion of cocaine-related contextual memories (Wells et al., 2013; can occur even after prolonged abstinence (Meil and See, 1996; Arguello et al., 2014). However, the underlying molecular mecha- Li et al., 2016). Therefore, treatments that weaken the strength of nisms involved in US retrieval-induced drug memory reconsoli- drug-associated memories represent a potential clinical preven- dation remain unknown. tion method for addicted individuals. In this study, we investigated possible differences between Disruption of the association between cues and a drug might US retrieval and CS retrieval-triggered reconsolidation of cocaine attenuate cravings induced by cue reexposure. One way to reward memory and the underlying molecular mechanism of achieve this goal is through interference with the reconsolida- US retrieval-induced memory reconsolidation. Our data suggest tion process (Lee et  al., 2005). Consolidated memory enters an that US is more efficient than CS at triggering a reconsolidation unstable state after memory retrieval, thus rendering a memory process for cocaine reward memory, thus suggesting that a post process temporarily susceptible to disruption by an interven- US retrieval intervention may be a potential approach to prevent tion. Memory then undergoes an additional consolidation-like substance addiction. process and is restabilized as long-term memory (Misanin et al., 1968; Nader et al., 2000aT ; ronson and Taylor, 2007). This dynamic Materials and Methods process is known as memory reconsolidation (Rodriguez-Ortiz and Bermudez-Rattoni, 2007; Jones et al., 2012). Manipulation of Animals memory reconsolidation can update previously learned memo- Six-week-old male C57BL/6J mice or male Sprague-Dawley rats, ries with new information and strengthen or weaken preexist- ing memories (Tronson et al., 2006). In fact, intervention in the weighing about 22 or 280  g, respectively, were purchased from ox/fl fl ox ox/fl fl ox Slaccas Lab Animal. Adrb1 and Adrb2 mice with C57BL/6J reconsolidation process provides a potential way to prevent memory storage in various learning paradigms in animals, background were developed by our laboratory. According to the gene structure and the size of exons, exon of Adrb1 including spatial learning (Przybyslawski and Sara, 1997 Flint ; et  al., 2007), fear conditioning (Debiec and Ledoux, 2004), and (ENSMUSE00000294435) or Adrb2 (ENSMUSE00000399288) can be conditionally removed and will result in no β1-AR or β2-AR cocaine-induced reward memory (Milton and Everitt, 2010). Studies have shown that cue reexposure, as CS retrieval, ren- expression. 5’-loxP site is inserted about 1.4 or 1.2 kb upstream of start codon, where the promoter of Adrb1 or Adrb2 is located. ders memory temporarily susceptible to suppression by post retrieval interventions (Nadel and Land, 2000 Nader and Har ; dt, 3’-loxP site is inserted downstream of 3’UTR. Removal of the flanked exon will result in no protein translation. Mice or rats 2009). In addition to cue reexposure, footshock reexposure can also trigger a reconsolidation process as cue reexposure (Debiec used for experiments were housed with a reversed 12-h-light/- dark cycle and access to food and water available ad libitum. All et al., 2010; Diaz-Mataix et al., 2011Liu et al., ; 2014). Studies have reported that propranolol, the nonselective β-adrenergic recep- animal treatments were strictly in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory tor (β-AR) antagonist, treated after nicotine reexposure inhibits nicotine cravings in rat and human models (Xue et al., 2017a), Animals and were approved by Animal Care and Use Committee of Shanghai Medical College of Fudan University. The male and noncontingent cocaine injection 1 hour before an extinction session decreases cocaine relapse in the tests of reinstatement, mice or rats 8 to 10 weeks old were used for all behavioral tests. ox/fl fl ox Adrb1 mice and subsequent offspring were genotyped using spontaneous recovery, and renewal of cocaine seeking in rats (Luo et al., 2015). Garcinol, a histone acetyltransferase inhibitor, the following primer sets: 5’-CTGTTCGCATCGGAATGAAGC-3’; ox/fl fl ox 5’-TGACGTCATGAACTGGGATTTCAG-3’. Adrb2 mice and impairs cocaine reexposure-induced memory reconsolidation of cocaine self-administration (SA) (Dunbar and Taylor, 2017). subsequent offspring were genotyped using the following primer sets: 5’-GGTTGCACAGCAGCCCTAGAT-3’; 5’-CCGTTATGTG These studies suggest that drug reexposure, as US retrieval, can also trigger drug memory reconsolidation. A key issue is CACCAGACTTTAGG -3’. Downloaded from https://academic.oup.com/ijnp/article-abstract/21/3/267/4690578 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Zhu et al. | 269 CPP Training Session Reagents Mice were confined in one of the conditioning compartments Cocaine hydrochloride (Qinghai Pharmaceutical Firm) was dis- for 30 minutes after injection of cocaine (15 mg/kg i.p.) or the solved in saline at 4 mg/mL for rat cocaine-SA model and 3 other compartment after injection of saline (4 mL/kg i.p.). The mg/mL for mouse cocaine-CPP model. Propranolol, betaxolol, cocaine- or saline-paired training was performed alternatively ICI118,551, and cyclohemixide (Tocris Bioscience) were dissolved in the morning or afternoon and repeated for 3 days. in saline and administered at a dose of 10 mg/kg (i.p.), 5 mg/kg (i.p.), 2 mg/kg (i.p.), and 60 mg/kg (s.c.), respectively. Anisomycin CPP Retrieval Session (Sigma-Aldrich) was dissolved in saline of equal molar of HCl, Mice were injected with a low dose of cocaine (1.5  mg/kg i.p.) diluted with ACSF, and adjusted to pH = 7.4 with NaOH and and kept in their homecage as cocaine reexposure or allowed to administered at 150 mg/kg (i.p.) in mice. Propranolol (6.0 μg/μL), explore the entire apparatus for 5 minutes after injection of sa- betaxolol (10 μg/μL), or ICI 118, 551 (10 μg/μL), cycloheximide line as context reexposure. Immediately after cocaine or context (7.0 μg/μL) was injected into each side of the central amygdala reexposure, mice were infused with β-AR antagonist or protein (CeA) at the velocity of 0.1 μL/min for 5 minutes. Control animals synthesis inhibitor systemically or into the CeA and returned to received an equivalent volume of vehicle. homecage. Cannula Implantation and Drug Delivery CPP Memory Retention Test At 24 hours after memory retrieval, mice were allowed free Mice were anesthetized with 10% chloral hydrate and placed access to the entire apparatus (15 minutes), and the duration in in a stereotaxic apparatus. Pedestal guide cannulas (27 gauge, the cocaine paired side was recorded. RWD Life Science Co., Ltd) were implanted bilaterally 1  mm above the CeA (AP: -1.80  mm; ML: ±2.70  mm; DV: -3.30  mm) CPP Extinction Session (Paxinos and Franklin, 2004). With a 2-week recovery, the be- At 24 hours after memory retention tests, mice were injected havioral tests were performed in the animals. Immediately with saline and immediately confined to the compartment that after memory retrieval of cocaine CPP, the cannula dummy was previously paired with cocaine or saline for 30 minutes al- caps were gently removed. A  34-gauge infusion cannula was ternatively for 3 days. inserted into the guide cannula and infusion began. The mice were restrained in homecage throughout the infusion for Reinstatement Test Session 5 minutes. The infusion cannula was left for an additional After the extinction session, mice received a priming injection 5 minutes to avoid the diffusion of the drug back into guide of saline (4 mL/kg i.p.) and cocaine (15 mg/kg i.p.) on the follow- cannula. ing day and were allowed free access to both compartments for 15 minutes. The amount of time the mice spent in each compart- Viral Constructs and Microinjection ment was recorded. The sessions were taped by a digital video camera, and the time spent in each chamber was recorded by a 12 -1 Titre of AAV9 was exceeding 5 × 10 v.g. mL (Neuron Biotech trained observer blind to the genotype and treatment. The CPP Co., Ltd). To conditional knockout 1-AR in the CeA, β the pack- preference was determined as score with time spent in cocaine aged virus with an EF1α promoter-driven AAV vector to express paired side minus the time in saline side in each minute (s/min). Cre recombinase and eGFP reporter (AAV-EF1α :eGFP-T2A-Cre) ox/fl fl ox was injected into the CeA of Adrb1 mice. For the micro- Cocaine-SA injection of the virus (Lowery and Majewska, 2010), anesthe- tized mice were positioned in a stereotaxic apparatus with the Cocaine-SA training was in accordance with the method our injection syringe of 36 gauge tips (World Precision Instruments, laboratory previously employed (Wang et  al., 2010 Le et  ; al., Inc.) aimed at the CeA. The intended stereotaxic coordinates 2017). Rats weighing 280 to 320  g were used for surgery. They were: AP: -1.80 mm; ML: ±2.70 mm; DV: -4.30 mm. Then 0.15 were housed in pairs before surgery and singly after. Before μL of the virus was infused into the CeA at 0.05 μL/min. cocaine-SA training session, rats were initially maintained at The needle was left in place for an additional 5 minutes. The 85% of original body weight and trained to lever press under a viral infection area determined by eGFP expression in the CeA fixed-ratio 1 schedule of food pellets in the operant chambers was evaluated after behavioral experiments. In pilot experi- (Med-Associates, Inc). After stable lever press for food pellets ments, expression of eGFP by AAV injection into the CeA was was achieved, rats were anaesthetized with 10% chloral hydrate detectable 7 days after the surgery and lasted for at least 2 and implanted with a single silastic catheter in the right jugular. months. Catheters were flushed every day with 0.1  mL saline solution containing gentamycin (0.5 mg/mL) and heparin (30 U/mL). They Cocaine-CPP were allowed recovery for 7 days before the start of the behav- ioral experiment. A 2-chamber, unbiased CPP paradigm was applied as described previously (Liu et al., 2015). The CPP apparatus was consisted of 2 compartments with distinct floorings and walls. Before each Cocaine SA Training Session session, mice were habituated to the experimental room for at Rats were trained to self-administer i.v. injections of cocaine least 30 minutes for 3 days. (0.75  mg/kg/infusion delivered in 4 seconds) during a 4-h ses- sion daily for 10  days under the FR1 reinforcement schedule. Pre-Test Session Each injection was accompanied with the CS, illumination of Mice were allowed free access to the entire apparatus for 15 the stimulus light, and an audible tone for 20 seconds simul- minutes. Mice with an initial preference (>65% of total time) for taneously. Inactive lever presses were also recorded but had no either chamber were excluded from the experiment. programmed consequences. Downloaded from https://academic.oup.com/ijnp/article-abstract/21/3/267/4690578 by Ed 'DeepDyve' Gillespie user on 16 March 2018 270 | International Journal of Neuropsychopharmacology, 2018 SA Retrieval Session pretreated for heat retrieval and protease digestion. Sections Rats received 4 i.v. cocaine infusions (0.75  mg/kg/infusion) in were then incubated with probes of mouse β1-AR and β2-AR the operant chambers with the withdrawal levers and with no (Adrb1, accession no: NM_001039652.1, target region 1135-2162; light or tone. Immediately after cocaine reexposure, rats were Adrb2, accession no: NM_007420.3, target region 55-962) for 2 injected with β-AR antagonist and returned to the home cage. In hours at 40°C with labeled probe mixture per slide. The nonspe- the control group, rats were only returned to the operant cham- cifically hybridized probe was removed by washing the sections, bers without cocaine infusion. 3 times for 2 minutes each in 1× wash buffer at room tempera- ture, followed by Amplifier 1 for 30 minutes, Amplifier 2 for 30 SA Memory Retention Test minutes at 40°C and Amplifier 3 for 15 minutes at 40°C. Each At 24 hours after memory retrieval, rats were returned to the amplifier was removed by washing with 1× wash buffer for 2 operant chamber for 30 minutes, during which the active lever minutes at room temperature. The slides were incubated with press was accompanied with conditioned cues but no cocaine HRP-C1 or HRP-C2, followed by TSA-fluorophore each. Then the was delivered. The number of active lever presses was counted slides were viewed, analyzed, and photographed with an LSM to show the craving for cocaine and the effect of drug on memory 510 microscope (Zeiss). At least 3 independent experiments reconsolidation of cocaine SA. have been performed and imaged from 3 male C57BL/6J mice. SA Extinction Session Statistical Analysis Rats were reintroduced into an operant chamber similar to the Experimental data were presented as the mean ± SEM and ana- training procedure, except that cocaine was not infused with the lyzed by Sigma plot 12.5. Data of immunofluorescence were active lever press. The rats experienced 4 hours daily extinction non-normally distributed and analyzed by Kruskal-Wallis 1-way sessions for 7 days, during which the active lever press resulted ANOVA on ranks. Data from behavioral tests were analyzed by in an infusion of the same volume of saline with the light and 1-way or 2-way ANOVA with repeated measures followed by the tone cues simultaneously. Extinction training continued until Bonferroni’s posthoc test with sessions or levers as a within- the active lever presses were <10 times in 4 hours for 2 consecu- subjects factor and drug treatment or genotype as a between- tive days. subjects factor. Reinstatement Test Session Rats received a priming injection of 0.9% saline (4 mL/kg i.p.) Results and were returned to the operant chamber. At 24 hours later, rats received 5 mg/kg cocaine injection (i.p.) and were returned Cocaine Reexposure Triggered Protein Synthesis to the operant chamber. The procedure of the reinstatement test Dependent Memory Reconsolidation of Cocaine CPP was the same as the test for memory retention, during which active lever press was accompanied with CS but no cocaine was We first investigated whether cocaine reexposure might trigger delivered. Lever presses during the 60 minutes were counted. reward memory reconsolidation in a similar manner to contex- tual cue reexposure. In this study, cycloheximide (CHX), a protein synthesis inhibitor, was administrated immediately (<5 minutes) Immunohistochemistry and Immunofluorescence after an injection of cocaine (1.5 mg/kg) 1 day after cocaine-CPP The mice were perfused intracardiacally with saline first, then training. Twenty-four hours or 1 hour after cocaine reexposure, with 4% paraformaldehyde in 0.1 M Na HPO /NaH PO buffer a memory retention test was performed (Figure 1A). A signifi- 2 4 2 4 (pH 7.5) and the brains were removed. After post-fixation in cant inhibitory effect of CHX on the preference for the cocaine 4% paraformaldehyde for 4 hours, the samples were stored in paired side in the memory retention test and a treatment-by- 30% sucrose/PBS for 3 days. Brain slices 30  μm thick were incu- session interaction were observed 24 hours later. Bonferroni’s bated in primary antibody (anti-c-Fos 1: 1000, Santa Cruz; anti- posthoc comparison confirmed that CHX administered imme- β1-AR 1:100, Santa Cruz) at 4°C overnight. After being washed diately after cocaine reexposure significantly decreased mem- with PBS 3 times, the slices were incubated with fluorescence ory expression (Figure 1B , F (1, 21) = 17.116, P < .001; treatment × session conjugated secondary antibody at room temperature (1:50 000, Bonferroni posthoc test: t = 4.009, P < .001, vs Veh in test, 2-way Jackson ImmunoResearch) for 1 hour. Then the brain sections RM ANOVA). Treatment with anisomycin, another protein syn- were rinsed in PBS and mounted with antiquenching mounting thesis inhibitor, also significantly decreased the preference for medium (Thermo Fisher Scientific). The sections were visualized the cocaine paired side in the memory retention test (Figure 1C, under a LSM 510 laser confocal fluorescence microscope (Carl F (1, 22) = 5.620, P = .027; Bonferroni posthoc test: t = treatment × session Zeiss) and analyzed by Image-Pro Plus. Labeled cells above the 2.606, P = .014, vs Veh in test, 2-way RM ANOVA). When admin- same threshold determined from control animals were counted istered after saline reexposure, CHX had no effects on memory (Trifilieff et al., 2006). expression of cocaine-CPP (Figure 1D , F (1, 14) = 0.024, treatment × session P = .879, 2-way RM ANOVA). When a retention test was carried out 1 hour post cocaine reexposure, no significant inhibitory High-Resolution Fluorescent in Situ Hybridization effect of CHX on memory expression was detected (Figure 1E, (FISH) by RNAscope F (1, 14) = 0.330, P = .575, 2-way RM ANOVA). Our data treatment × session FISH was performed on the fixed frozen brain tissue follow- suggested that protein synthesis inhibition after cocaine reex- ing the RNAscope procedures (Advanced Cell Diagnostics, posure impaired long-term memory of cocaine-CPP. A number Inc). Hybridization of a probe against the Bacillus subtilis dihy- of studies have demonstrated that protein synthesis inhibitors drodipicolinate reductase gene was used as negative con- are classic amnestic agents that can impair memory reconsoli- trol. In brief, frozen sections (10  µm thick) were cut coronally dation when administered after CS reexposure-induced mem- through the amygdala formation. Sections were thaw-mounted ory retrieval. Thus, the effects of protein synthesis inhibition onto Superfrost Plus microscope slides (Fisher Scientific) and on context retrieval-induced reward memory reconsolidation Downloaded from https://academic.oup.com/ijnp/article-abstract/21/3/267/4690578 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Zhu et al. | 271 Figure  1. Cocaine reexposure induced protein synthesis-dependent memory reconsolidation of cocaine-conditioned place preference (CPP). Mice were trained for 3  days to acquire cocaine-CPP. For cocaine retrieval, cocaine (1.5  mg/kg, i.p.) was injected 24 hours after the last conditioning. The protein synthesis inhibitor was injected after cocaine reexposure. CPP memory retention tests were carried out at the time indicated (A–E). (A) Experimental design. (B–E) Memory retention tests 24 hours (B, C, D) or 1 hour (E) after cocaine or saline reexposure followed by the treatment of cycloheximide (CHX) (60 mg/kg, s.c.) or anisomycin (Ani, 150 mg/kg, i.p.). *P < .05, *** P < .001 vs vehicle-treated group. For context retrieval, a 5-minute reexposure to the conditioned chamber (CPP test1) was performed followed by treatment of protein synthesis inhibitor (F–H). (F) Experimental design. (G–H) Memory retention tests 24 hours after context reexposure followed by the treatment of CHX (60 mg/ kg, s.c.) or Ani (150 mg/kg, i.p.). *P < .05, **P < .01 vs vehicle-treated group. Values in the bar indicate number of mice per group. were also examined. CHX or anisomycin injected immediately to saline priming as a extinction test and cocaine priming as after context reexposure inhibited the preference for cocaine a reinstatement test (Figure 2A). Our data showed that post paired side in the memory retention test 24 hours later (Figure cocaine retrieval treatment of CHX or anisomycin greatly inhib- 1F–H, CHX: F (2, 48) = 3.335, P = .044; Bonferroni post- ited cocaine priming-induced reinstatement (Figure 2B–C, CHX: treatment × session hoc test: t = 2.767, P= .008, vs Veh in test2; Ani: F F (1, 21) = 9.240, P = .006; Bonferroni posthoc test: t = treatment × session treatment × session (2, 38) = 3.744, p = 0.033; Bonferroni posthoc test: t = 2.568, P = .014, 3.453, P = .001, vs Veh in cocaine priming; Ani: F (1, treatment × session vs Veh in test2, 2-way RM ANOVA). Together, our data indicate 22) = 5.152, P = .033; Bonferroni posthoc test: t = 3.483, P = .001, that cocaine reexposure (1.5 mg/kg), as US retrieval, triggered vs Veh in cocaine-priming, 2-way RM ANOVA). Without retrieval, memory reconsolidation of cocaine-CPP in a manner dependent CHX had no effects on reinstatement of cocaine-CPP (Figure 2D , on de novo protein synthesis. F (1, 14) = 0.527, P = .480, 2-way RM ANOVA). However, treatment × session when CHX or anisomycin was injected after context retrieval, the preference for the cocaine paired side was restored after Protein Synthesis Inhibition Post Cocaine cocaine priming (Figure 2E–F , CHX: F (1, 24) = 0.009, treatment × session Retrieval, Not Context Retrieval, Led to Impaired P = .927; Ani: F (1, 19) = 0.363, P = .554, 2-way RM treatment × session Reinstatement ANOVA). These data suggest that treatments post US retrieval, To test whether the amnesia effect of protein synthesis inhibi- not CS retrieval, led to failure of reinstatement of cocaine CPP tor treated after US retrieval was long-lasting or irreversible, we and the amnesia effects induced by the manipulations post US tested cocaine priming-induced reinstatement of cocaine-CPP. retrieval were persistent. Thus, US retrieval might be more effi- With a confined extinction training after memory retrieval and cient than CS retrieval at triggering memory reconsolidation of treatments in Figure 1, the same cohort of mice was subjected cocaine-CPP. Downloaded from https://academic.oup.com/ijnp/article-abstract/21/3/267/4690578 by Ed 'DeepDyve' Gillespie user on 16 March 2018 272 | International Journal of Neuropsychopharmacology, 2018 Figure 2. Protein synthesis inhibition after cocaine retrieval prevented reinstatement for cocaine- conditioned place preference (CPP). After memory retention test, the confined extinction was carried out for 3 days followed by saline and cocaine priming-induced reinstatement tests. (A) Experimental design. Cycloheximide (CHX) or ani- somycin was injected systemically after cocaine or context retrieval. (B–C) Reinstatement tests of cocaine-CPP with post cocaine retrieval treatment of CHX (60 mg/kg, s.c.) or anisomycin (Ani, 150 mg/kg, i.p.). **P < .01 vs vehicle-treated group. (D) Reinstatement tests of cocaine-CPP with treatment of CHX (60 mg/kg, s.c.) after saline reex- posure. (E–F) Reinstatement tests of cocaine-CPP with post context-retrieval treatment of CHX (60 mg/kg, s.c.) or anisomycin (Ani, 150 mg/kg, i.p.). Values in the bar indicate number of mice per group. Cocaine Retrieval Induced Neuronal Activation in (2, 17) = 9.138, P = .002; Bonferroni posthoc test: = 4.185, t P = .002 cocaine vs saline, t = 2.582, P = .038, context vs saline; BLA: F (2, 17) = the CeA 15.228, P < .001; Bonferroni posthoc test: = 5.518, t P < .001, cocaine The above data indicated that the memory reconsolidation pro- vs saline, t = 2.256, P = .037, context vs saline, =2.978, t P = .016, cess induced by cocaine retrieval was distinct from that induced cocaine vs context; supplementary Figure 1, PrL-PFC: (2, F 10) = by context retrieval. We next investigated brain nuclei activation 25.126, P < .001; Bonferroni posthoc test: =7.029, t P < .001, cocaine induced by cocaine reexposure or context reexposure. The optoge- vs saline, t = 2.927, P = .045, context vs saline, = 4.483, t P = .004, netic and pharmacogenetic studies have revealed that, during cocaine vs context; IL-PFC: F (2, 9) = 3.346, P = .082; AcbC: F (2, 10) = learning, c-Fos-positive neurons encode and store information 16.642, P < .001; Bonferroni posthoc test: = 5.768, t P < .001, cocaine (Liu et al., 2012; Liu et al., 2014b). c-Fos, the immediate-early gene, vs saline, t = 2.928, P = .045, context vs saline, = 3.152, t P = .031, is induced in specific brain regions during neuronal activity asso- cocaine vs context, 1-way ANOVA). In addition, c-Fos positive cells ciated with behavioral tasks (Morgan et al., 1987; Kang et al., 2001). significantly increased in the CeA only after cocaine reexposure, Maximum expression levels of c-Fos in rodents were observed however, no significant increase of c-Fos positive cells in the CeA from 1 to 3 hours after sensory stimuli or behavioral tasks, return- was detected after context reexposure (Figure 3B , CeA: F (2, 17) = ing to baseline values at 6 hours (Xiu et al., 2014 Barr ; os et al., 7.068, P = .006; Bonferroni posthoc test: =3.624, t P = .006, cocaine vs 2015). We measured the c-Fos expression levels 1 hour after mem- saline, t =2.785, P = .025, cocaine vs context, 1-way ANOVA). These ory retrieval (Figure 3A). The levels of c-Fos immunoreactivity in results suggest that cocaine retrieval induced full activation of the the brain sections of the control group (injected with saline at amygdala, and the CeA played a distinct role from other subnuclei homecage) were used to determine the threshold for c-Fos posi- of the amygdala in cocaine retrieval triggered memory reconsoli- tive cell counts for all groups. Our data showed that cocaine and dation of cocaine-CPP. context reexposure both induced a significant increase in c-Fos We then explored the distribution of mRNAs encoding for positive cell counts in the lateral amygdala (LA), the BLA, the pre- β1-AR and β2-AR protein in the amygdala by performing high- limbic prefrontal cortex (PrL-PFC), and nucleus accumbens core resolution FISH on brain slices. Adrb1 and Adrb2 mRNAs were (AcbC). c-Fos positive cell counts in the BLA, PrL-PFC, and AcbC were also significantly elevated in the cocaine reexposure group present in the LA, BLA, and CeA (Figure 3C). No FISH signal could compared with the context reexposure group (Figure 3B , LA: F be detected using the negative control probe. Downloaded from https://academic.oup.com/ijnp/article-abstract/21/3/267/4690578 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Zhu et al. | 273 Figure 3. Cocaine retrieval-induced neuronal activation in the central amygdala (CeA). One hour after memory retrieval, mice were decapitated, and we examined the activation of brain regions induced by context or cocaine reexposure. (A) Experimental design. (B) Immunofluorescence staining of c-Fos (red) in the CeA, basolateral amygdala (BLA), and lateral amygdala (LA). *P < .05, **P < .01, and ***P < .001 vs control group. Scale bar: 100  µm. (C) mRNA expression of Adrb1 and Adrb2 in the CeA, BLA, and LA. Scale bar: 50 µm. The inhibition of CPP memory reconsolidation was observed β1-Adrenergic Blockade in the CeA Impaired Cocaine Retrieval-Induced, Not Context Retrieval-Induced, by CHX infusion in the CeA immediately after cocaine retrieval Memory Reconsolidation and the Subsequent (Figure 4A-4C, F (1, 18) = 11.010, P = .004; Bonferroni treatment × session Reinstatement of Cocaine-CPP posthoc test: t = 3.928, P < .001, vs Veh in test, 2-way RM ANOVA). Cocaine priming-induced reinstatement was also inhibited As c-Fos expression significantly increased in the CeA after after CPP extinction in the same cohort of mice (Figure 4D , cocaine reexposure, but not context reexposure, it is critical to F (1, 18) = 4.449, P = .049; Bonferroni posthoc test: t = confirm the role of the CeA in cocaine retrieval triggered mem- treatment × session ory reconsolidation. 3.233, P = .003, vs Veh in cocaine priming, 2-way RM ANOVA). The Downloaded from https://academic.oup.com/ijnp/article-abstract/21/3/267/4690578 by Ed 'DeepDyve' Gillespie user on 16 March 2018 274 | International Journal of Neuropsychopharmacology, 2018 Figure 4. Protein synthesis inhibition or β1-AR blockade in the central amygdala (CeA) after cocaine retrieval impaired cocaine-conditioned place preference (CPP) memory reconsolidation and reinstatement. The 3-day cocaine-CPP training was performed 2 weeks after cannula implantation in CeA of C57 mice. The protein syn- thesis inhibitor was infused in the CeA after cocaine reexposure (A–D). (A) Experimental design. (B) Representative image of implanted cannula traces of drug infu- sion site in the CeA. (C) Memory retention tests of cocaine-CPP 24 hours after the cocaine retrieval followed by infusion of CHX (3.5 μg/side). (D) Reinstatement tests of cocaine-CPP with infusion of cycloheximide (CHX) after cocaine retrieval (3.5 μg/side). **P < .01 and ***P < .001 vs vehicle-treated group. After cocaine-CPP training, β-blocker was infused in the CeA after cocaine retrieval (E–G). (E) Memory retention tests of cocaine-CPP 24 hours after cocaine retrieval followed by propranolol injec- tion (10 mg/kg, i.p.). (F) Memory retention tests of cocaine-CPP 24 hours after cocaine retrieval followed by infusion of β-blocker (Prop: 3 μg/side; Bet: 5 μg/side; ICI: 5 μg/side). *P < .05 and **P < .01 vs vehicle-treated group. (G) Reinstatement tests of cocaine-CPP with post cocaine retrieval infusion of β-blocker in the CeA. *P < .05 vs vehicle-treated group. After cocaine-CPP training, CHX or betaxolol was infused in the CeA after context retrieval (H–L). (H) Experimental design. (I) Memory retention tests of cocaine-CPP 24 hours after context retrieval followed by infusion of CHX (CHX: 3.5 μg/side). (J) Reinstatement tests of cocaine-CPP with post context retrieval infusion of CHX. (K) Memory retention tests of cocaine-CPP 24 hours after context retrieval followed by infusion of betaxolol (Bet: 5 μg/side). (L) Reinstatement tests of cocaine-CPP with post context retrieval infusion of betaxolol. Values in the bar indicate number of mice per group. above data suggest the CeA was critically involved in cocaine preference for the cocaine paired side (Figure 4E, F treatment × session retrieval-induced memory reconsolidation of cocaine-CPP. (1, 25) = 5.935, P = .022; Bonferroni posthoc test: = 3.285, t P = .002, It has been hypothesized that -adr β energic antagonism vs Veh in test, 2-way RM ANOVA). To test the role of -AR in the β impairs memory reconsolidation via deactivation of second CeA in cocaine retrieval-induced memory reconsolidation, we messengers that initiate gene transcription and translation of injected a β-blocker in the CeA after cocaine retrieval. Our data new proteins (Johansen et al., 2011). Our results suggest that - β showed that betaxolol, a 1 subtype-selecti β ve antagonist, as well AR was involved in and required for cocaine retrieval -induced as propranolol, infused in the CeA after cocaine retrieval signifi- reward memory reconsolidation, because systemic injection of cantly decreased the preference for the cocaine paired side in propranolol after cocaine retrieval significantly impaired the the memory retention test, but ICI 118, 551, a 2-AR selecti β ve Downloaded from https://academic.oup.com/ijnp/article-abstract/21/3/267/4690578 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Zhu et al. | 275 antagonist, did not show the inhibitory effects on CPP expression suggest that 1-AR acti β vation in the CeA was specifically involved (Figure 4F, F (3, 39) = 3.837, P = .017; Bonferroni posthoc in US retrieval-induced, but not CS retrieval-induced, memory treatment × session test: t = 3.677, P = .003, Prop vs Veh in test, = 2.905, t P = .029, Bet vs reconsolidation of cocaine-CPP, and the amnesia effect induced Veh in test, 2-way RM ANOVA). In addition, β 1-AR blockade in the by β1-AR blockade in the CeA after US-retrieval was persistent. CeA post cocaine retrieval impaired cocaine priming-induced reinstatement (Figure 4G , F (3, 39) = 3.204, P = .034; treatment × session β1-AR Deletion in the CeA Impaired Cocaine Bonferroni posthoc test: = t 2.821, P = .037, Prop vs Veh in Cocaine- Retrieval-Induced Memory Reconsolidation and priming, t = 2.884, P = .031, Bet vs Veh in Cocaine-priming, 2-way Reinstatement of Cocaine-CPP RM ANOVA). When CHX or betaxolol was infused in the CeA after context-retrieval (Figure 4H), memory reconsolidation and rein- Furthermore, the role of β1-AR in cocaine retrieval-induced statement were not significantly changed (Figure 4I-L, CHX/CPP memory reconsolidation was further examined by β1-AR knock- test 2: F (2, 38) = 0.426, P = .656; CHXreinstatement, out in the CeA (Figure 5A-B). An AAV encoding Cre recombin- treatment × session F (1, 19) = 0.073, P = .789; betaxolol / CPP test 2: ase (AAV-EF1α:eGFP-T2A-Cre) was bilaterally infused into the treatment × session ox/fl fl ox F (2, 32) = 0.118, P = .889; betaxolol/reinstatement: CeA of Adrb1 mice to generate focal homozygous dele- treatment × session F (1, 16) = 0.356, P = .559, 2-way RM ANOVA). Our data tions of β1-AR in the CeA. As shown in Figure 5C, β1-AR focal treatment × session Figure 5. β1-AR deletion in the central amygdala (CeA) impaired cocaine retrieval-induced memory reconsolidation and reinstatement of cocaine- conditioned place ox/fl fl ox preference (CPP). (A) Experimental design. AAV-EF1α:eGFP-2A-Cre was injected in CeA of Adrb1 mice 3 weeks before cocaine-CPP training. (B) Generation of β1-AR ox/fl fl ox CKO mice using the Cre-loxp system. The Adrb1 mice contain loxp sites flanking the region of the exon to be deleted. The loxp sites can be recognized by Cre re- ox/fl fl ox combinase, resulting in the deletion of the exon of Adrb1. (C) Immunofluorescence staining of β1-AR (red) in the CeA of Adrb1 mice injected with AAV-EF1α:eGFP- ox/fl fl ox T2A-Cre. Scale bar: 100 μm. (D) β1-AR mRNA levels significantly decreased 3 weeks after AAV injection in the CeA of Adrb1 mice. Number of mice per group is indicated in the bar. *P < .05 vs wild-type mice. Memory retention tests (E) and reinstatement tests (F) of cocaine-CPP with cocaine retrieval in β1-AR CKO mice. ***P < .001 vs wild-type group. Memory retention tests (G) and reinstatement tests (H) of cocaine-CPP in β1-AR CKO mice without cocaine retrieval. Values in the bar indicate number of mice per group. Downloaded from https://academic.oup.com/ijnp/article-abstract/21/3/267/4690578 by Ed 'DeepDyve' Gillespie user on 16 March 2018 276 | International Journal of Neuropsychopharmacology, 2018 knockout was confirmed by eGFP expression combined with β2-AR conditional knockout mice (supplementary Figure 2D , CPP β1-AR immunostaining. The deletion of Adrb1 was also con- test: F (1, 22) = 2.546, P = .125; reinstatement: F treatment × session treatment × firmed by examination of its mRNA expression levels 3 weeks (1, 22) = 2.304, P = .143, 2-way RM ANOVA). Our data suggest session after surgery (Figure 5D , t(5) = 3.731, P = .0136, 2-tailed Student’s that β1-AR in the CeA was required for US retrieval-triggered t test). After 3 weeks of AAV infection, daily conditioning and memory reconsolidation of cocaine-CPP. CPP tests were performed. The mice with selective deletion of β1-AR in the CeA showed a significantly decreased preference β1-Adrenergic Blockade Impaired Cocaine for the cocaine paired side 24 hours after cocaine retrieval com- Retrieval-Induced Memory Reconsolidation and pared with wild-type mice (Figure 5EF , (1, 33) = 9.288, genotype × session Reinstatement of Cocaine-SA P = .005; Bonferroni posthoc test: t = 4.652, P < .001, vs wild type in test, 2-way RM ANOVA). CPP expression was not inhibited in To further determine whether the treatment combined with US β1-AR CKO mice with saline reexposure (Figure 5G , F retrieval has translational potential, we used a cocaine-SA proce- genotype × ses- (1, 22) = 0.009, P = .923, 2-way RM ANOVA). Together, these dure in this study. With a 10-day cocaine-SA training, stable lever sion results suggest that β1-adrenergic signaling in the CeA was pressing (approximately 40 times within 4 hours) was developed required for cocaine retrieval-triggered memory reconsolidation in all rats (supplementary Figure 3). Then the rats were injected of cocaine-CPP. In addition, β1-AR deletion in the CeA impaired with a β-blocker immediately after i.v. cocaine injections with- cocaine priming-induced reinstatement (Figure 5F , cocaine out levers and cues. Memory retention tests were performed reexposure: F (1, 33) = 6.011, P = .020; Bonferroni post- 24 hours later (Figure 6A). The data showed that administra- genotype × session hoc test: t = 4.382, P < .001 vs WT in cocaine priming; Figure 5H, tion of propranolol immediately after cocaine retrieval signifi- saline reexposure: F (1, 22) = 0.532, P = .473, 2-way cantly decreased lever presses for cocaine (Figure 6B , F genotype × session treatment × lever RM ANOVA). Furthermore, β2-AR was also deleted in the CeA (1, 23) = 4.315, P = .049; Bonferroni posthoc test: t = 3.316, P = ox/fl fl ox in Ardb2 mice (supplementary Figure 2A–C, t(6) = 3.865, P .002, vs Veh in active lever press, 2-way RM ANOVA). When pro- = .008, 2-tailed Student’s t test), and no significant suppression pranolol was injected after saline reexposure, the lever presses of memory reconsolidation or reinstatement was detected in for cocaine were not significantly different from those of control Figure 6. β1-AR blockade post cocaine retrieval impaired memory reconsolidation and reinstatement of cocaine-self-administration (SA). With 10-day cocaine-SA training, 4 i.v. infusions of cocaine (0.75 mg/kg/infusion) were given as cocaine retrieval 24 hours after cocaine-SA training, followed by the treatment of β-blocker. The cocaine-SA memory retention test was carried out at the time indicated. After the memory retention test, the extinction training without cocaine delivery was carried out for 7 days followed by the reinstatement tests. (A) Experimental design. (B,E) Counts of lever presses in cocaine-SA memory retention tests 24 hours after cocaine retrieval followed by treatment of propranolol (Prop: 10 mg/kg i.p.) or betaxolol (Bet: 5 mg/kg i.p.). **P < .01 vs vehicle-treated group. (C–D, F–G) Counts of lever presses in cocaine-SA reinstatement tests with treatment of propranolol or betaxolol after cocaine retrieval ***P < .001 vs vehicle-treated group. Values in the bar indicate number of rats per group. Downloaded from https://academic.oup.com/ijnp/article-abstract/21/3/267/4690578 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Zhu et al. | 277 group in the memory retention tests (supplementary Figure 4A, retrieval) was critical to induce a labile state of reward memory F (1, 14) = 1.638, P = .221, 2-way RM ANOVA). To address leading to de novo protein synthesis-dependent memory re- treatment × lever whether β-blocker treatment after cocaine retrieval might affect consolidation. It is also possible that the dose of propranolol cocaine relapse, we tested reinstatement after extinction train- or protein synthesis inhibitor did not produce an adequate ing in the same cohort of rats (Figure 6A). Extinction training long-lasting effect on memory reconsolidation. The result that was performed after the memory retention test in all groups a higher dose of cocaine may not induce memory restabilization to obtain a low number of lever presses in response to cues indicates that there is a nonmonotonic relationship between US (extinction criteria: no more than 10 active lever presses for the reexposure and memory restabilization, which needs further last 2 days). One day after the extinction session, rats showed investigation. no spontaneous recovery with saline-priming (Figure 6C , In the same cohort of mice, our data showed that reinstate- F (1, 23) = 0.434, P = .516, 2-way RM ANOVA). Then, in ment was impaired by post US retrieval treatment, but not post treatment × lever reinstatement tests with cocaine priming, propranolol injection CS retrieval treatment, thus indicating that memory impairment after cocaine retrieval significantly inhibited drug seeking be- induced by pharmacological treatment after US retrieval was havior (Figure 6D , F (1, 23) = 6.121, P = .021; Bonferroni persistent. The finding that treatment after US retrieval impairs treatment × lever posthoc test: t = 3.728, P < .001, vs Veh in active lever press, reconsolidation is consistent with studies showing that aniso- 2-way RM ANOVA). A significant inhibitory effect on memory mycin injection in the BLA after US retrieval of fear memory can reconsolidation was also detected by administration of betaxo- impair multiple CS-US associations to the same US. Similarly, in lol, but not ICI 118, 551, after cocaine retrieval (Figure 6E, Bet: studies of fear memory reconsolidation, memory retrieval by a F (1, 14) = 5.327, P = .037; Bonferroni posthoc test: t = 3.602, single CS leads to a selective amnesia for that specific CS when treatment × lever P = .001, vs Bet in active lever press; supplementary Figure 4B , ICI: multiple CSs are associated with the US. It is likely targeting of a F (1, 21) = 0.505, P = .485, 2-way RM ANOVA). Treatment different component of the association that underlies the differ - treatment × lever of betaxolol after cocaine retrieval also suppressed relapse to ence. In addition, the pharmacological blockade after contextual cocaine in the reinstatement test (Figure 6F–G , saline prim- reexposure impaired long-term memory as indicated by CPP ing: F (1, 14) = 0.257, P = .620; cocaine priming: test, while memories reinstated after extinction session as indi- treatment × lever F (1, 14) = 10.898, P = .005, Bonferroni posthoc test: t = cated by cocaine priming-induced reinstatement test, suggest- treatment × lever 4.976, P < .001, vs Veh in active lever press, 2-way RM ANOVA). ing that memories were possibly not erased by pharmacological Our data suggest that activation of β-AR, particularly β1-AR, intervention but were temporally inhibited. It might be that CS was required for US retrieval-induced memory reconsolidation induces memory trace partially reactivated, and systemic treat- of cocaine-SA. In agreement with the results of cocaine-CPP, ment after CS retrieval partially inhibited memory reconsolida- this impaired memory reconsolidation of cocaine-SA by β1-AR tion. Thus, manipulation post US retrieval may destabilize more blockade during the time window after US retrieval also led to memory trace of CS-US associations than CS retrieval. Our data decreased relapse to cocaine. suggest that US retrieval might be more efficient than CS re- trieval, and manipulations after US retrieval might have an ad- vantage for treating substance addiction by preventing drug Discussion relapse. In this study, memory reconsolidation of cocaine-CPP was The present results demonstrated that cocaine triggered pro- tein synthesis-dependent memory reconsolidation of cocaine- impaired by systemic treatment or CeA infusion of protein syn- thesis inhibitor or β-blocker after US-retrieval, but was intact CPP or cocaine-SA. The drug memory reconsolidation triggered by cocaine reexposure, not by context reexposure, was found to with local injection in the CeA after CS-retrieval. In addition, immunofluorescence staining showed that c-Fos expression be mediated by β1-AR in the CeA. Furthermore, the manipula- tion post cocaine retrieval also decreased cocaine relapse in the significantly increased in the CeA after cocaine reexposure, but not context reexposure. These data suggest that CeA might be reinstatement tests, in contrast to the drug-seeking behavior observed in the cocaine priming-induced reinstatement test involved in the US, but not CS, -induced memory reconsolida- tion process. with treatment after context retrieval. These findings demon- strate that US retrieval triggered memory reconsolidation pro- Our current study suggested that -adr β energic activation might participate in the US retrieval-triggered memory recon- cess is distinct from the CS retrieval, or US retrieval is more efficient than CS retrieval, thus offering a potential manipu- solidation process, adding new evidence to previous findings, and demonstrates that memory reconsolidation triggered by US lation strategy by which the persistent inhibition of addiction memory might be obtained. retrieval is also dependent on -AR mediated signaling. β Previous research has revealed that activation of -AR r β ecruits ERK and/ In this study, we demonstrated that a low dose of cocaine (1.5 mg/kg) was sufficient to trigger reconsolidation of reward or mammalian target of rapamycin signaling, thereby facili- tating protein synthesis-dependent long-term potentiation memory, because protein synthesis inhibitors or β-blockers, the classic amnestic agents for CS retrieval-induced memory recon- (Gelinas and Nguyen, 2005; Gelinas et al., 2007). In this study, systemic or intra-CeA injection of propranolol and a protein solidation (Nader et al., 2000bDe ; biec et al., 2002; De Jaeger et al., 2014), blocked cocaine reexposure-induced memory recon- synthesis inhibitor impaired reconsolidation of cocaine reward memory, thus suggesting that -b βlockers or protein synthesis solidation of cocaine-CPP or cocaine-SA. The similar US retrieval procedure was also performed with lower dose of nicotine or co- inhibitors might induce a common cellular process respon- sible for the failure of US retrieval-induced memory reconsoli- caine injection (compared with the dose for training) as UCS re- trieval (Luo et al., 2015 Xue et al., ; 2017b). Furthermore, we tested dation. However, these 2 manipulations may also potentially drive behavior through 2 independent cellular processes, other doses of cocaine. Cocaine-induced memory reconsolida- tion at a dose of 3 mg/kg, but not 5 mg/kg or more, could be given that a recent study has shown that CHX, not propran- olol, blocks cue-induced memory reconsolidation of cocaine-SA inhibited by propranolol (data not shown). In this study, the ap- propriate dose of cocaine for US retrieval (1.5 mg/kg for cocaine- (Dunbar and Taylor, 2016). β1-AR and β2-AR are 2 subtypes of β-AR that are distributed widely in the central nervous system. CPP memory retrieval; 4 i.v. injections for cocaine-SA memory Downloaded from https://academic.oup.com/ijnp/article-abstract/21/3/267/4690578 by Ed 'DeepDyve' Gillespie user on 16 March 2018 278 | International Journal of Neuropsychopharmacology, 2018 Although β1-AR are in much higher levels than 1-AR within β Supplementary Material forebrain structures, isoproterenol, which has equal affinity for Supplementary data are available at International Journal of both subtypes, induces much greater adenylyl cyclase activity Neuropsychopharmacology online. upon stimulation of β 2-AR than β1-AR. Then effects of both β1- AR and β2-AR on memory reconsolidation were examined in this study by using pharmacological treatment or conditional Acknowledgments knockout strategy, respectively. Our data suggested that 1-AR, β This research was supported by grants from the National Natural rather than β2-AR, in the CeA was required for reconsolida- Science Foundation of China (grant nos. 31430033, 91632307, and tion of cocaine reward memory induced by cocaine retrieval. 31421091 to L.M.; 31371136, 31571036, and 31771176 to X.L.) and However, previous work has demonstrated that postretrieval the Ministry of Science and Technology (grant no. 2015CB553500 long-term memory of cocaine-CPP might be mediated by 2-AR β and 2014CB942801 to L.M.). but not β 1-AR (Bernardi et al., 2009). The disagreement between our findings and these previous results might be due to differ - ences in experimental designs and targeted brain regions. The Statement of Interest negative results of ICI 118,551 infusion or β2-AR conditional None. knockout in the CeA could also attribute to its low expression levels. 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Neuropsychopharmacol encoding nicotine-associated memories inhibits nicotine 38:753–762. preference and relapse. Biol Psychiatr y. Downloaded from https://academic.oup.com/ijnp/article-abstract/21/3/267/4690578 by Ed 'DeepDyve' Gillespie user on 16 March 2018 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png International Journal of Neuropsychopharmacology Oxford University Press

β1-Adrenoceptor in the Central Amygdala Is Required for Unconditioned Stimulus-Induced Drug Memory Reconsolidation

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Abstract

Background: Drug memories become labile and reconsolidated after retrieval by presentation of environmental cues (conditioned stimulus) or drugs (unconditioned stimulus). Whether conditioned stimulus and unconditioned stimulus retrieval trigger different memory reconsolidation processes is not clear. Methods: Protein synthesis inhibitor or β-adrenergic receptor (β-AR) antagonist was systemically administrated or intra-central amygdala infused immediately after cocaine reexposure in cocaine-conditioned place preference or self-administration mice models. β-ARs were selectively knocked out in the central amygdala to further confirm the role of β-adrenergic receptor in cocaine reexposure-induced memory reconsolidation of cocaine-conditioned place preference. Results: Cocaine reexposure triggered de novo protein synthesis dependent memory reconsolidation of cocaine-conditioned place preference. Cocaine-priming-induced reinstatement was also impaired with post cocaine retrieval manipulation, in contrast to the relapse behavior with post context retrieval manipulation. Cocaine retrieval, but not context retrieval, induced central amygdala activation. Protein synthesis inhibitor or β1-adrenergic receptor antagonist infused in the central amygdala after cocaine retrieval, but not context retrieval, inhibited memory reconsolidation and reinstatement. 1-adr β energic receptor knockout in the central amygdala suppressed cocaine retrieval-triggered memory reconsolidation and reinstatement of cocaine conditioned place preference. β 1-adrenergic receptor antagonism after cocaine retrieval also impaired reconsolidation and reinstatement of cocaine self-administration. Conclusions: Cocaine reward memory triggered by unconditioned stimulus retrieval is distinct from conditioned stimulus retrieval. Unconditioned stimulus retrieval induced reconsolidation of cocaine reward memory depends on β1-adrenergic signaling in the central amygdala. Post unconditioned stimulus retrieval manipulation can prevent drug memory reconsolidation and relapse to cocaine, thus providing a potential strategy for the prevention of substance addiction. Keywords: β-AR, unconditioned stimulus, conditioned stimulus, memory reconsolidation, cocaine Received: September 7, 2017; Revised: October 15, 2017; Accepted: December 1, 2017 © The Author(s) 2017. Published by Oxford University Press on behalf of CINP. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http:// creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, 267 provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com Downloaded from https://academic.oup.com/ijnp/article-abstract/21/3/267/4690578 by Ed 'DeepDyve' Gillespie user on 16 March 2018 268 | International Journal of Neuropsychopharmacology, 2018 Significance Statement It is well known that drug memories become labile and reconsolidated upon retrieval by the presentation of conditioned stimulus (CS) or unconditioned stimulus (US). Whether CS and US retrieval trigger different memory reconsolidation processes is unknown. In this study, we found that US retrieval, but not CS retrieval, triggered memory reconsolidation of cocaine-conditioned place preference dependent on β1-AR and de novo protein synthesis in the central amygdala. Furthermore, cocaine priming-induced reinstatement was impaired with post US retrieval manipulation in contrast to the relapse behavior with post CS retrieval manipu- lation. In cocaine self-administration, 1-AR anta β gonism after US retrieval also impaired reconsolidation and reinstatement. Our study indicates that reconsolidation of cocaine reward memory triggered by US retrieval is distinct from CS retrieval. US retrieval induced reconsolidation of cocaine reward memory depends on 1-adr β energic signaling in the central amygdala. Introduction Drug addiction is commonly considered a learning and memory whether US retrieval and CS retrieval are different in the induc- disorder (Boening, 2001; Hyman, 2005). The reinforcing effects of tion of the memory reconsolidation process. a drug (known as unconditioned stimulus [US]), such as drug- Many studies have demonstrated that β-AR is involved in elicited euphoria, are strongly associated with environmental reconsolidation, because systemic or intra-basolateral amyg- cues (known as conditioned stimulus [CS]) (Hyman and Malenka, dala (BLA) injection of a β-AR antagonist immediately after CS 2001; Shalev et al., 2002; Koob and Volkow, 2016). Consequently, retrieval prevents memory reconsolidation of cocaine-SA or exposure to drug-associated environmental cues can evoke v- ar conditioned place preference (CPP) (Milton et al., 2008 Otis et al., ; ious degrees of psychological dependence or compulsive drug 2013). In the signaling cascades downstream of β-AR, amygdala taking (Cadet et  al., 2014W ; ise and Koob, 2014). A  major chal- protein kinase A and ERK are both involved in the reconsolida- lenge in the treatment of drug addiction is drug relapse, which tion of cocaine-related contextual memories (Wells et al., 2013; can occur even after prolonged abstinence (Meil and See, 1996; Arguello et al., 2014). However, the underlying molecular mecha- Li et al., 2016). Therefore, treatments that weaken the strength of nisms involved in US retrieval-induced drug memory reconsoli- drug-associated memories represent a potential clinical preven- dation remain unknown. tion method for addicted individuals. In this study, we investigated possible differences between Disruption of the association between cues and a drug might US retrieval and CS retrieval-triggered reconsolidation of cocaine attenuate cravings induced by cue reexposure. One way to reward memory and the underlying molecular mechanism of achieve this goal is through interference with the reconsolida- US retrieval-induced memory reconsolidation. Our data suggest tion process (Lee et  al., 2005). Consolidated memory enters an that US is more efficient than CS at triggering a reconsolidation unstable state after memory retrieval, thus rendering a memory process for cocaine reward memory, thus suggesting that a post process temporarily susceptible to disruption by an interven- US retrieval intervention may be a potential approach to prevent tion. Memory then undergoes an additional consolidation-like substance addiction. process and is restabilized as long-term memory (Misanin et al., 1968; Nader et al., 2000aT ; ronson and Taylor, 2007). This dynamic Materials and Methods process is known as memory reconsolidation (Rodriguez-Ortiz and Bermudez-Rattoni, 2007; Jones et al., 2012). Manipulation of Animals memory reconsolidation can update previously learned memo- Six-week-old male C57BL/6J mice or male Sprague-Dawley rats, ries with new information and strengthen or weaken preexist- ing memories (Tronson et al., 2006). In fact, intervention in the weighing about 22 or 280  g, respectively, were purchased from ox/fl fl ox ox/fl fl ox Slaccas Lab Animal. Adrb1 and Adrb2 mice with C57BL/6J reconsolidation process provides a potential way to prevent memory storage in various learning paradigms in animals, background were developed by our laboratory. According to the gene structure and the size of exons, exon of Adrb1 including spatial learning (Przybyslawski and Sara, 1997 Flint ; et  al., 2007), fear conditioning (Debiec and Ledoux, 2004), and (ENSMUSE00000294435) or Adrb2 (ENSMUSE00000399288) can be conditionally removed and will result in no β1-AR or β2-AR cocaine-induced reward memory (Milton and Everitt, 2010). Studies have shown that cue reexposure, as CS retrieval, ren- expression. 5’-loxP site is inserted about 1.4 or 1.2 kb upstream of start codon, where the promoter of Adrb1 or Adrb2 is located. ders memory temporarily susceptible to suppression by post retrieval interventions (Nadel and Land, 2000 Nader and Har ; dt, 3’-loxP site is inserted downstream of 3’UTR. Removal of the flanked exon will result in no protein translation. Mice or rats 2009). In addition to cue reexposure, footshock reexposure can also trigger a reconsolidation process as cue reexposure (Debiec used for experiments were housed with a reversed 12-h-light/- dark cycle and access to food and water available ad libitum. All et al., 2010; Diaz-Mataix et al., 2011Liu et al., ; 2014). Studies have reported that propranolol, the nonselective β-adrenergic recep- animal treatments were strictly in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory tor (β-AR) antagonist, treated after nicotine reexposure inhibits nicotine cravings in rat and human models (Xue et al., 2017a), Animals and were approved by Animal Care and Use Committee of Shanghai Medical College of Fudan University. The male and noncontingent cocaine injection 1 hour before an extinction session decreases cocaine relapse in the tests of reinstatement, mice or rats 8 to 10 weeks old were used for all behavioral tests. ox/fl fl ox Adrb1 mice and subsequent offspring were genotyped using spontaneous recovery, and renewal of cocaine seeking in rats (Luo et al., 2015). Garcinol, a histone acetyltransferase inhibitor, the following primer sets: 5’-CTGTTCGCATCGGAATGAAGC-3’; ox/fl fl ox 5’-TGACGTCATGAACTGGGATTTCAG-3’. Adrb2 mice and impairs cocaine reexposure-induced memory reconsolidation of cocaine self-administration (SA) (Dunbar and Taylor, 2017). subsequent offspring were genotyped using the following primer sets: 5’-GGTTGCACAGCAGCCCTAGAT-3’; 5’-CCGTTATGTG These studies suggest that drug reexposure, as US retrieval, can also trigger drug memory reconsolidation. A key issue is CACCAGACTTTAGG -3’. Downloaded from https://academic.oup.com/ijnp/article-abstract/21/3/267/4690578 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Zhu et al. | 269 CPP Training Session Reagents Mice were confined in one of the conditioning compartments Cocaine hydrochloride (Qinghai Pharmaceutical Firm) was dis- for 30 minutes after injection of cocaine (15 mg/kg i.p.) or the solved in saline at 4 mg/mL for rat cocaine-SA model and 3 other compartment after injection of saline (4 mL/kg i.p.). The mg/mL for mouse cocaine-CPP model. Propranolol, betaxolol, cocaine- or saline-paired training was performed alternatively ICI118,551, and cyclohemixide (Tocris Bioscience) were dissolved in the morning or afternoon and repeated for 3 days. in saline and administered at a dose of 10 mg/kg (i.p.), 5 mg/kg (i.p.), 2 mg/kg (i.p.), and 60 mg/kg (s.c.), respectively. Anisomycin CPP Retrieval Session (Sigma-Aldrich) was dissolved in saline of equal molar of HCl, Mice were injected with a low dose of cocaine (1.5  mg/kg i.p.) diluted with ACSF, and adjusted to pH = 7.4 with NaOH and and kept in their homecage as cocaine reexposure or allowed to administered at 150 mg/kg (i.p.) in mice. Propranolol (6.0 μg/μL), explore the entire apparatus for 5 minutes after injection of sa- betaxolol (10 μg/μL), or ICI 118, 551 (10 μg/μL), cycloheximide line as context reexposure. Immediately after cocaine or context (7.0 μg/μL) was injected into each side of the central amygdala reexposure, mice were infused with β-AR antagonist or protein (CeA) at the velocity of 0.1 μL/min for 5 minutes. Control animals synthesis inhibitor systemically or into the CeA and returned to received an equivalent volume of vehicle. homecage. Cannula Implantation and Drug Delivery CPP Memory Retention Test At 24 hours after memory retrieval, mice were allowed free Mice were anesthetized with 10% chloral hydrate and placed access to the entire apparatus (15 minutes), and the duration in in a stereotaxic apparatus. Pedestal guide cannulas (27 gauge, the cocaine paired side was recorded. RWD Life Science Co., Ltd) were implanted bilaterally 1  mm above the CeA (AP: -1.80  mm; ML: ±2.70  mm; DV: -3.30  mm) CPP Extinction Session (Paxinos and Franklin, 2004). With a 2-week recovery, the be- At 24 hours after memory retention tests, mice were injected havioral tests were performed in the animals. Immediately with saline and immediately confined to the compartment that after memory retrieval of cocaine CPP, the cannula dummy was previously paired with cocaine or saline for 30 minutes al- caps were gently removed. A  34-gauge infusion cannula was ternatively for 3 days. inserted into the guide cannula and infusion began. The mice were restrained in homecage throughout the infusion for Reinstatement Test Session 5 minutes. The infusion cannula was left for an additional After the extinction session, mice received a priming injection 5 minutes to avoid the diffusion of the drug back into guide of saline (4 mL/kg i.p.) and cocaine (15 mg/kg i.p.) on the follow- cannula. ing day and were allowed free access to both compartments for 15 minutes. The amount of time the mice spent in each compart- Viral Constructs and Microinjection ment was recorded. The sessions were taped by a digital video camera, and the time spent in each chamber was recorded by a 12 -1 Titre of AAV9 was exceeding 5 × 10 v.g. mL (Neuron Biotech trained observer blind to the genotype and treatment. The CPP Co., Ltd). To conditional knockout 1-AR in the CeA, β the pack- preference was determined as score with time spent in cocaine aged virus with an EF1α promoter-driven AAV vector to express paired side minus the time in saline side in each minute (s/min). Cre recombinase and eGFP reporter (AAV-EF1α :eGFP-T2A-Cre) ox/fl fl ox was injected into the CeA of Adrb1 mice. For the micro- Cocaine-SA injection of the virus (Lowery and Majewska, 2010), anesthe- tized mice were positioned in a stereotaxic apparatus with the Cocaine-SA training was in accordance with the method our injection syringe of 36 gauge tips (World Precision Instruments, laboratory previously employed (Wang et  al., 2010 Le et  ; al., Inc.) aimed at the CeA. The intended stereotaxic coordinates 2017). Rats weighing 280 to 320  g were used for surgery. They were: AP: -1.80 mm; ML: ±2.70 mm; DV: -4.30 mm. Then 0.15 were housed in pairs before surgery and singly after. Before μL of the virus was infused into the CeA at 0.05 μL/min. cocaine-SA training session, rats were initially maintained at The needle was left in place for an additional 5 minutes. The 85% of original body weight and trained to lever press under a viral infection area determined by eGFP expression in the CeA fixed-ratio 1 schedule of food pellets in the operant chambers was evaluated after behavioral experiments. In pilot experi- (Med-Associates, Inc). After stable lever press for food pellets ments, expression of eGFP by AAV injection into the CeA was was achieved, rats were anaesthetized with 10% chloral hydrate detectable 7 days after the surgery and lasted for at least 2 and implanted with a single silastic catheter in the right jugular. months. Catheters were flushed every day with 0.1  mL saline solution containing gentamycin (0.5 mg/mL) and heparin (30 U/mL). They Cocaine-CPP were allowed recovery for 7 days before the start of the behav- ioral experiment. A 2-chamber, unbiased CPP paradigm was applied as described previously (Liu et al., 2015). The CPP apparatus was consisted of 2 compartments with distinct floorings and walls. Before each Cocaine SA Training Session session, mice were habituated to the experimental room for at Rats were trained to self-administer i.v. injections of cocaine least 30 minutes for 3 days. (0.75  mg/kg/infusion delivered in 4 seconds) during a 4-h ses- sion daily for 10  days under the FR1 reinforcement schedule. Pre-Test Session Each injection was accompanied with the CS, illumination of Mice were allowed free access to the entire apparatus for 15 the stimulus light, and an audible tone for 20 seconds simul- minutes. Mice with an initial preference (>65% of total time) for taneously. Inactive lever presses were also recorded but had no either chamber were excluded from the experiment. programmed consequences. Downloaded from https://academic.oup.com/ijnp/article-abstract/21/3/267/4690578 by Ed 'DeepDyve' Gillespie user on 16 March 2018 270 | International Journal of Neuropsychopharmacology, 2018 SA Retrieval Session pretreated for heat retrieval and protease digestion. Sections Rats received 4 i.v. cocaine infusions (0.75  mg/kg/infusion) in were then incubated with probes of mouse β1-AR and β2-AR the operant chambers with the withdrawal levers and with no (Adrb1, accession no: NM_001039652.1, target region 1135-2162; light or tone. Immediately after cocaine reexposure, rats were Adrb2, accession no: NM_007420.3, target region 55-962) for 2 injected with β-AR antagonist and returned to the home cage. In hours at 40°C with labeled probe mixture per slide. The nonspe- the control group, rats were only returned to the operant cham- cifically hybridized probe was removed by washing the sections, bers without cocaine infusion. 3 times for 2 minutes each in 1× wash buffer at room tempera- ture, followed by Amplifier 1 for 30 minutes, Amplifier 2 for 30 SA Memory Retention Test minutes at 40°C and Amplifier 3 for 15 minutes at 40°C. Each At 24 hours after memory retrieval, rats were returned to the amplifier was removed by washing with 1× wash buffer for 2 operant chamber for 30 minutes, during which the active lever minutes at room temperature. The slides were incubated with press was accompanied with conditioned cues but no cocaine HRP-C1 or HRP-C2, followed by TSA-fluorophore each. Then the was delivered. The number of active lever presses was counted slides were viewed, analyzed, and photographed with an LSM to show the craving for cocaine and the effect of drug on memory 510 microscope (Zeiss). At least 3 independent experiments reconsolidation of cocaine SA. have been performed and imaged from 3 male C57BL/6J mice. SA Extinction Session Statistical Analysis Rats were reintroduced into an operant chamber similar to the Experimental data were presented as the mean ± SEM and ana- training procedure, except that cocaine was not infused with the lyzed by Sigma plot 12.5. Data of immunofluorescence were active lever press. The rats experienced 4 hours daily extinction non-normally distributed and analyzed by Kruskal-Wallis 1-way sessions for 7 days, during which the active lever press resulted ANOVA on ranks. Data from behavioral tests were analyzed by in an infusion of the same volume of saline with the light and 1-way or 2-way ANOVA with repeated measures followed by the tone cues simultaneously. Extinction training continued until Bonferroni’s posthoc test with sessions or levers as a within- the active lever presses were <10 times in 4 hours for 2 consecu- subjects factor and drug treatment or genotype as a between- tive days. subjects factor. Reinstatement Test Session Rats received a priming injection of 0.9% saline (4 mL/kg i.p.) Results and were returned to the operant chamber. At 24 hours later, rats received 5 mg/kg cocaine injection (i.p.) and were returned Cocaine Reexposure Triggered Protein Synthesis to the operant chamber. The procedure of the reinstatement test Dependent Memory Reconsolidation of Cocaine CPP was the same as the test for memory retention, during which active lever press was accompanied with CS but no cocaine was We first investigated whether cocaine reexposure might trigger delivered. Lever presses during the 60 minutes were counted. reward memory reconsolidation in a similar manner to contex- tual cue reexposure. In this study, cycloheximide (CHX), a protein synthesis inhibitor, was administrated immediately (<5 minutes) Immunohistochemistry and Immunofluorescence after an injection of cocaine (1.5 mg/kg) 1 day after cocaine-CPP The mice were perfused intracardiacally with saline first, then training. Twenty-four hours or 1 hour after cocaine reexposure, with 4% paraformaldehyde in 0.1 M Na HPO /NaH PO buffer a memory retention test was performed (Figure 1A). A signifi- 2 4 2 4 (pH 7.5) and the brains were removed. After post-fixation in cant inhibitory effect of CHX on the preference for the cocaine 4% paraformaldehyde for 4 hours, the samples were stored in paired side in the memory retention test and a treatment-by- 30% sucrose/PBS for 3 days. Brain slices 30  μm thick were incu- session interaction were observed 24 hours later. Bonferroni’s bated in primary antibody (anti-c-Fos 1: 1000, Santa Cruz; anti- posthoc comparison confirmed that CHX administered imme- β1-AR 1:100, Santa Cruz) at 4°C overnight. After being washed diately after cocaine reexposure significantly decreased mem- with PBS 3 times, the slices were incubated with fluorescence ory expression (Figure 1B , F (1, 21) = 17.116, P < .001; treatment × session conjugated secondary antibody at room temperature (1:50 000, Bonferroni posthoc test: t = 4.009, P < .001, vs Veh in test, 2-way Jackson ImmunoResearch) for 1 hour. Then the brain sections RM ANOVA). Treatment with anisomycin, another protein syn- were rinsed in PBS and mounted with antiquenching mounting thesis inhibitor, also significantly decreased the preference for medium (Thermo Fisher Scientific). The sections were visualized the cocaine paired side in the memory retention test (Figure 1C, under a LSM 510 laser confocal fluorescence microscope (Carl F (1, 22) = 5.620, P = .027; Bonferroni posthoc test: t = treatment × session Zeiss) and analyzed by Image-Pro Plus. Labeled cells above the 2.606, P = .014, vs Veh in test, 2-way RM ANOVA). When admin- same threshold determined from control animals were counted istered after saline reexposure, CHX had no effects on memory (Trifilieff et al., 2006). expression of cocaine-CPP (Figure 1D , F (1, 14) = 0.024, treatment × session P = .879, 2-way RM ANOVA). When a retention test was carried out 1 hour post cocaine reexposure, no significant inhibitory High-Resolution Fluorescent in Situ Hybridization effect of CHX on memory expression was detected (Figure 1E, (FISH) by RNAscope F (1, 14) = 0.330, P = .575, 2-way RM ANOVA). Our data treatment × session FISH was performed on the fixed frozen brain tissue follow- suggested that protein synthesis inhibition after cocaine reex- ing the RNAscope procedures (Advanced Cell Diagnostics, posure impaired long-term memory of cocaine-CPP. A number Inc). Hybridization of a probe against the Bacillus subtilis dihy- of studies have demonstrated that protein synthesis inhibitors drodipicolinate reductase gene was used as negative con- are classic amnestic agents that can impair memory reconsoli- trol. In brief, frozen sections (10  µm thick) were cut coronally dation when administered after CS reexposure-induced mem- through the amygdala formation. Sections were thaw-mounted ory retrieval. Thus, the effects of protein synthesis inhibition onto Superfrost Plus microscope slides (Fisher Scientific) and on context retrieval-induced reward memory reconsolidation Downloaded from https://academic.oup.com/ijnp/article-abstract/21/3/267/4690578 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Zhu et al. | 271 Figure  1. Cocaine reexposure induced protein synthesis-dependent memory reconsolidation of cocaine-conditioned place preference (CPP). Mice were trained for 3  days to acquire cocaine-CPP. For cocaine retrieval, cocaine (1.5  mg/kg, i.p.) was injected 24 hours after the last conditioning. The protein synthesis inhibitor was injected after cocaine reexposure. CPP memory retention tests were carried out at the time indicated (A–E). (A) Experimental design. (B–E) Memory retention tests 24 hours (B, C, D) or 1 hour (E) after cocaine or saline reexposure followed by the treatment of cycloheximide (CHX) (60 mg/kg, s.c.) or anisomycin (Ani, 150 mg/kg, i.p.). *P < .05, *** P < .001 vs vehicle-treated group. For context retrieval, a 5-minute reexposure to the conditioned chamber (CPP test1) was performed followed by treatment of protein synthesis inhibitor (F–H). (F) Experimental design. (G–H) Memory retention tests 24 hours after context reexposure followed by the treatment of CHX (60 mg/ kg, s.c.) or Ani (150 mg/kg, i.p.). *P < .05, **P < .01 vs vehicle-treated group. Values in the bar indicate number of mice per group. were also examined. CHX or anisomycin injected immediately to saline priming as a extinction test and cocaine priming as after context reexposure inhibited the preference for cocaine a reinstatement test (Figure 2A). Our data showed that post paired side in the memory retention test 24 hours later (Figure cocaine retrieval treatment of CHX or anisomycin greatly inhib- 1F–H, CHX: F (2, 48) = 3.335, P = .044; Bonferroni post- ited cocaine priming-induced reinstatement (Figure 2B–C, CHX: treatment × session hoc test: t = 2.767, P= .008, vs Veh in test2; Ani: F F (1, 21) = 9.240, P = .006; Bonferroni posthoc test: t = treatment × session treatment × session (2, 38) = 3.744, p = 0.033; Bonferroni posthoc test: t = 2.568, P = .014, 3.453, P = .001, vs Veh in cocaine priming; Ani: F (1, treatment × session vs Veh in test2, 2-way RM ANOVA). Together, our data indicate 22) = 5.152, P = .033; Bonferroni posthoc test: t = 3.483, P = .001, that cocaine reexposure (1.5 mg/kg), as US retrieval, triggered vs Veh in cocaine-priming, 2-way RM ANOVA). Without retrieval, memory reconsolidation of cocaine-CPP in a manner dependent CHX had no effects on reinstatement of cocaine-CPP (Figure 2D , on de novo protein synthesis. F (1, 14) = 0.527, P = .480, 2-way RM ANOVA). However, treatment × session when CHX or anisomycin was injected after context retrieval, the preference for the cocaine paired side was restored after Protein Synthesis Inhibition Post Cocaine cocaine priming (Figure 2E–F , CHX: F (1, 24) = 0.009, treatment × session Retrieval, Not Context Retrieval, Led to Impaired P = .927; Ani: F (1, 19) = 0.363, P = .554, 2-way RM treatment × session Reinstatement ANOVA). These data suggest that treatments post US retrieval, To test whether the amnesia effect of protein synthesis inhibi- not CS retrieval, led to failure of reinstatement of cocaine CPP tor treated after US retrieval was long-lasting or irreversible, we and the amnesia effects induced by the manipulations post US tested cocaine priming-induced reinstatement of cocaine-CPP. retrieval were persistent. Thus, US retrieval might be more effi- With a confined extinction training after memory retrieval and cient than CS retrieval at triggering memory reconsolidation of treatments in Figure 1, the same cohort of mice was subjected cocaine-CPP. Downloaded from https://academic.oup.com/ijnp/article-abstract/21/3/267/4690578 by Ed 'DeepDyve' Gillespie user on 16 March 2018 272 | International Journal of Neuropsychopharmacology, 2018 Figure 2. Protein synthesis inhibition after cocaine retrieval prevented reinstatement for cocaine- conditioned place preference (CPP). After memory retention test, the confined extinction was carried out for 3 days followed by saline and cocaine priming-induced reinstatement tests. (A) Experimental design. Cycloheximide (CHX) or ani- somycin was injected systemically after cocaine or context retrieval. (B–C) Reinstatement tests of cocaine-CPP with post cocaine retrieval treatment of CHX (60 mg/kg, s.c.) or anisomycin (Ani, 150 mg/kg, i.p.). **P < .01 vs vehicle-treated group. (D) Reinstatement tests of cocaine-CPP with treatment of CHX (60 mg/kg, s.c.) after saline reex- posure. (E–F) Reinstatement tests of cocaine-CPP with post context-retrieval treatment of CHX (60 mg/kg, s.c.) or anisomycin (Ani, 150 mg/kg, i.p.). Values in the bar indicate number of mice per group. Cocaine Retrieval Induced Neuronal Activation in (2, 17) = 9.138, P = .002; Bonferroni posthoc test: = 4.185, t P = .002 cocaine vs saline, t = 2.582, P = .038, context vs saline; BLA: F (2, 17) = the CeA 15.228, P < .001; Bonferroni posthoc test: = 5.518, t P < .001, cocaine The above data indicated that the memory reconsolidation pro- vs saline, t = 2.256, P = .037, context vs saline, =2.978, t P = .016, cess induced by cocaine retrieval was distinct from that induced cocaine vs context; supplementary Figure 1, PrL-PFC: (2, F 10) = by context retrieval. We next investigated brain nuclei activation 25.126, P < .001; Bonferroni posthoc test: =7.029, t P < .001, cocaine induced by cocaine reexposure or context reexposure. The optoge- vs saline, t = 2.927, P = .045, context vs saline, = 4.483, t P = .004, netic and pharmacogenetic studies have revealed that, during cocaine vs context; IL-PFC: F (2, 9) = 3.346, P = .082; AcbC: F (2, 10) = learning, c-Fos-positive neurons encode and store information 16.642, P < .001; Bonferroni posthoc test: = 5.768, t P < .001, cocaine (Liu et al., 2012; Liu et al., 2014b). c-Fos, the immediate-early gene, vs saline, t = 2.928, P = .045, context vs saline, = 3.152, t P = .031, is induced in specific brain regions during neuronal activity asso- cocaine vs context, 1-way ANOVA). In addition, c-Fos positive cells ciated with behavioral tasks (Morgan et al., 1987; Kang et al., 2001). significantly increased in the CeA only after cocaine reexposure, Maximum expression levels of c-Fos in rodents were observed however, no significant increase of c-Fos positive cells in the CeA from 1 to 3 hours after sensory stimuli or behavioral tasks, return- was detected after context reexposure (Figure 3B , CeA: F (2, 17) = ing to baseline values at 6 hours (Xiu et al., 2014 Barr ; os et al., 7.068, P = .006; Bonferroni posthoc test: =3.624, t P = .006, cocaine vs 2015). We measured the c-Fos expression levels 1 hour after mem- saline, t =2.785, P = .025, cocaine vs context, 1-way ANOVA). These ory retrieval (Figure 3A). The levels of c-Fos immunoreactivity in results suggest that cocaine retrieval induced full activation of the the brain sections of the control group (injected with saline at amygdala, and the CeA played a distinct role from other subnuclei homecage) were used to determine the threshold for c-Fos posi- of the amygdala in cocaine retrieval triggered memory reconsoli- tive cell counts for all groups. Our data showed that cocaine and dation of cocaine-CPP. context reexposure both induced a significant increase in c-Fos We then explored the distribution of mRNAs encoding for positive cell counts in the lateral amygdala (LA), the BLA, the pre- β1-AR and β2-AR protein in the amygdala by performing high- limbic prefrontal cortex (PrL-PFC), and nucleus accumbens core resolution FISH on brain slices. Adrb1 and Adrb2 mRNAs were (AcbC). c-Fos positive cell counts in the BLA, PrL-PFC, and AcbC were also significantly elevated in the cocaine reexposure group present in the LA, BLA, and CeA (Figure 3C). No FISH signal could compared with the context reexposure group (Figure 3B , LA: F be detected using the negative control probe. Downloaded from https://academic.oup.com/ijnp/article-abstract/21/3/267/4690578 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Zhu et al. | 273 Figure 3. Cocaine retrieval-induced neuronal activation in the central amygdala (CeA). One hour after memory retrieval, mice were decapitated, and we examined the activation of brain regions induced by context or cocaine reexposure. (A) Experimental design. (B) Immunofluorescence staining of c-Fos (red) in the CeA, basolateral amygdala (BLA), and lateral amygdala (LA). *P < .05, **P < .01, and ***P < .001 vs control group. Scale bar: 100  µm. (C) mRNA expression of Adrb1 and Adrb2 in the CeA, BLA, and LA. Scale bar: 50 µm. The inhibition of CPP memory reconsolidation was observed β1-Adrenergic Blockade in the CeA Impaired Cocaine Retrieval-Induced, Not Context Retrieval-Induced, by CHX infusion in the CeA immediately after cocaine retrieval Memory Reconsolidation and the Subsequent (Figure 4A-4C, F (1, 18) = 11.010, P = .004; Bonferroni treatment × session Reinstatement of Cocaine-CPP posthoc test: t = 3.928, P < .001, vs Veh in test, 2-way RM ANOVA). Cocaine priming-induced reinstatement was also inhibited As c-Fos expression significantly increased in the CeA after after CPP extinction in the same cohort of mice (Figure 4D , cocaine reexposure, but not context reexposure, it is critical to F (1, 18) = 4.449, P = .049; Bonferroni posthoc test: t = confirm the role of the CeA in cocaine retrieval triggered mem- treatment × session ory reconsolidation. 3.233, P = .003, vs Veh in cocaine priming, 2-way RM ANOVA). The Downloaded from https://academic.oup.com/ijnp/article-abstract/21/3/267/4690578 by Ed 'DeepDyve' Gillespie user on 16 March 2018 274 | International Journal of Neuropsychopharmacology, 2018 Figure 4. Protein synthesis inhibition or β1-AR blockade in the central amygdala (CeA) after cocaine retrieval impaired cocaine-conditioned place preference (CPP) memory reconsolidation and reinstatement. The 3-day cocaine-CPP training was performed 2 weeks after cannula implantation in CeA of C57 mice. The protein syn- thesis inhibitor was infused in the CeA after cocaine reexposure (A–D). (A) Experimental design. (B) Representative image of implanted cannula traces of drug infu- sion site in the CeA. (C) Memory retention tests of cocaine-CPP 24 hours after the cocaine retrieval followed by infusion of CHX (3.5 μg/side). (D) Reinstatement tests of cocaine-CPP with infusion of cycloheximide (CHX) after cocaine retrieval (3.5 μg/side). **P < .01 and ***P < .001 vs vehicle-treated group. After cocaine-CPP training, β-blocker was infused in the CeA after cocaine retrieval (E–G). (E) Memory retention tests of cocaine-CPP 24 hours after cocaine retrieval followed by propranolol injec- tion (10 mg/kg, i.p.). (F) Memory retention tests of cocaine-CPP 24 hours after cocaine retrieval followed by infusion of β-blocker (Prop: 3 μg/side; Bet: 5 μg/side; ICI: 5 μg/side). *P < .05 and **P < .01 vs vehicle-treated group. (G) Reinstatement tests of cocaine-CPP with post cocaine retrieval infusion of β-blocker in the CeA. *P < .05 vs vehicle-treated group. After cocaine-CPP training, CHX or betaxolol was infused in the CeA after context retrieval (H–L). (H) Experimental design. (I) Memory retention tests of cocaine-CPP 24 hours after context retrieval followed by infusion of CHX (CHX: 3.5 μg/side). (J) Reinstatement tests of cocaine-CPP with post context retrieval infusion of CHX. (K) Memory retention tests of cocaine-CPP 24 hours after context retrieval followed by infusion of betaxolol (Bet: 5 μg/side). (L) Reinstatement tests of cocaine-CPP with post context retrieval infusion of betaxolol. Values in the bar indicate number of mice per group. above data suggest the CeA was critically involved in cocaine preference for the cocaine paired side (Figure 4E, F treatment × session retrieval-induced memory reconsolidation of cocaine-CPP. (1, 25) = 5.935, P = .022; Bonferroni posthoc test: = 3.285, t P = .002, It has been hypothesized that -adr β energic antagonism vs Veh in test, 2-way RM ANOVA). To test the role of -AR in the β impairs memory reconsolidation via deactivation of second CeA in cocaine retrieval-induced memory reconsolidation, we messengers that initiate gene transcription and translation of injected a β-blocker in the CeA after cocaine retrieval. Our data new proteins (Johansen et al., 2011). Our results suggest that - β showed that betaxolol, a 1 subtype-selecti β ve antagonist, as well AR was involved in and required for cocaine retrieval -induced as propranolol, infused in the CeA after cocaine retrieval signifi- reward memory reconsolidation, because systemic injection of cantly decreased the preference for the cocaine paired side in propranolol after cocaine retrieval significantly impaired the the memory retention test, but ICI 118, 551, a 2-AR selecti β ve Downloaded from https://academic.oup.com/ijnp/article-abstract/21/3/267/4690578 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Zhu et al. | 275 antagonist, did not show the inhibitory effects on CPP expression suggest that 1-AR acti β vation in the CeA was specifically involved (Figure 4F, F (3, 39) = 3.837, P = .017; Bonferroni posthoc in US retrieval-induced, but not CS retrieval-induced, memory treatment × session test: t = 3.677, P = .003, Prop vs Veh in test, = 2.905, t P = .029, Bet vs reconsolidation of cocaine-CPP, and the amnesia effect induced Veh in test, 2-way RM ANOVA). In addition, β 1-AR blockade in the by β1-AR blockade in the CeA after US-retrieval was persistent. CeA post cocaine retrieval impaired cocaine priming-induced reinstatement (Figure 4G , F (3, 39) = 3.204, P = .034; treatment × session β1-AR Deletion in the CeA Impaired Cocaine Bonferroni posthoc test: = t 2.821, P = .037, Prop vs Veh in Cocaine- Retrieval-Induced Memory Reconsolidation and priming, t = 2.884, P = .031, Bet vs Veh in Cocaine-priming, 2-way Reinstatement of Cocaine-CPP RM ANOVA). When CHX or betaxolol was infused in the CeA after context-retrieval (Figure 4H), memory reconsolidation and rein- Furthermore, the role of β1-AR in cocaine retrieval-induced statement were not significantly changed (Figure 4I-L, CHX/CPP memory reconsolidation was further examined by β1-AR knock- test 2: F (2, 38) = 0.426, P = .656; CHXreinstatement, out in the CeA (Figure 5A-B). An AAV encoding Cre recombin- treatment × session F (1, 19) = 0.073, P = .789; betaxolol / CPP test 2: ase (AAV-EF1α:eGFP-T2A-Cre) was bilaterally infused into the treatment × session ox/fl fl ox F (2, 32) = 0.118, P = .889; betaxolol/reinstatement: CeA of Adrb1 mice to generate focal homozygous dele- treatment × session F (1, 16) = 0.356, P = .559, 2-way RM ANOVA). Our data tions of β1-AR in the CeA. As shown in Figure 5C, β1-AR focal treatment × session Figure 5. β1-AR deletion in the central amygdala (CeA) impaired cocaine retrieval-induced memory reconsolidation and reinstatement of cocaine- conditioned place ox/fl fl ox preference (CPP). (A) Experimental design. AAV-EF1α:eGFP-2A-Cre was injected in CeA of Adrb1 mice 3 weeks before cocaine-CPP training. (B) Generation of β1-AR ox/fl fl ox CKO mice using the Cre-loxp system. The Adrb1 mice contain loxp sites flanking the region of the exon to be deleted. The loxp sites can be recognized by Cre re- ox/fl fl ox combinase, resulting in the deletion of the exon of Adrb1. (C) Immunofluorescence staining of β1-AR (red) in the CeA of Adrb1 mice injected with AAV-EF1α:eGFP- ox/fl fl ox T2A-Cre. Scale bar: 100 μm. (D) β1-AR mRNA levels significantly decreased 3 weeks after AAV injection in the CeA of Adrb1 mice. Number of mice per group is indicated in the bar. *P < .05 vs wild-type mice. Memory retention tests (E) and reinstatement tests (F) of cocaine-CPP with cocaine retrieval in β1-AR CKO mice. ***P < .001 vs wild-type group. Memory retention tests (G) and reinstatement tests (H) of cocaine-CPP in β1-AR CKO mice without cocaine retrieval. Values in the bar indicate number of mice per group. Downloaded from https://academic.oup.com/ijnp/article-abstract/21/3/267/4690578 by Ed 'DeepDyve' Gillespie user on 16 March 2018 276 | International Journal of Neuropsychopharmacology, 2018 knockout was confirmed by eGFP expression combined with β2-AR conditional knockout mice (supplementary Figure 2D , CPP β1-AR immunostaining. The deletion of Adrb1 was also con- test: F (1, 22) = 2.546, P = .125; reinstatement: F treatment × session treatment × firmed by examination of its mRNA expression levels 3 weeks (1, 22) = 2.304, P = .143, 2-way RM ANOVA). Our data suggest session after surgery (Figure 5D , t(5) = 3.731, P = .0136, 2-tailed Student’s that β1-AR in the CeA was required for US retrieval-triggered t test). After 3 weeks of AAV infection, daily conditioning and memory reconsolidation of cocaine-CPP. CPP tests were performed. The mice with selective deletion of β1-AR in the CeA showed a significantly decreased preference β1-Adrenergic Blockade Impaired Cocaine for the cocaine paired side 24 hours after cocaine retrieval com- Retrieval-Induced Memory Reconsolidation and pared with wild-type mice (Figure 5EF , (1, 33) = 9.288, genotype × session Reinstatement of Cocaine-SA P = .005; Bonferroni posthoc test: t = 4.652, P < .001, vs wild type in test, 2-way RM ANOVA). CPP expression was not inhibited in To further determine whether the treatment combined with US β1-AR CKO mice with saline reexposure (Figure 5G , F retrieval has translational potential, we used a cocaine-SA proce- genotype × ses- (1, 22) = 0.009, P = .923, 2-way RM ANOVA). Together, these dure in this study. With a 10-day cocaine-SA training, stable lever sion results suggest that β1-adrenergic signaling in the CeA was pressing (approximately 40 times within 4 hours) was developed required for cocaine retrieval-triggered memory reconsolidation in all rats (supplementary Figure 3). Then the rats were injected of cocaine-CPP. In addition, β1-AR deletion in the CeA impaired with a β-blocker immediately after i.v. cocaine injections with- cocaine priming-induced reinstatement (Figure 5F , cocaine out levers and cues. Memory retention tests were performed reexposure: F (1, 33) = 6.011, P = .020; Bonferroni post- 24 hours later (Figure 6A). The data showed that administra- genotype × session hoc test: t = 4.382, P < .001 vs WT in cocaine priming; Figure 5H, tion of propranolol immediately after cocaine retrieval signifi- saline reexposure: F (1, 22) = 0.532, P = .473, 2-way cantly decreased lever presses for cocaine (Figure 6B , F genotype × session treatment × lever RM ANOVA). Furthermore, β2-AR was also deleted in the CeA (1, 23) = 4.315, P = .049; Bonferroni posthoc test: t = 3.316, P = ox/fl fl ox in Ardb2 mice (supplementary Figure 2A–C, t(6) = 3.865, P .002, vs Veh in active lever press, 2-way RM ANOVA). When pro- = .008, 2-tailed Student’s t test), and no significant suppression pranolol was injected after saline reexposure, the lever presses of memory reconsolidation or reinstatement was detected in for cocaine were not significantly different from those of control Figure 6. β1-AR blockade post cocaine retrieval impaired memory reconsolidation and reinstatement of cocaine-self-administration (SA). With 10-day cocaine-SA training, 4 i.v. infusions of cocaine (0.75 mg/kg/infusion) were given as cocaine retrieval 24 hours after cocaine-SA training, followed by the treatment of β-blocker. The cocaine-SA memory retention test was carried out at the time indicated. After the memory retention test, the extinction training without cocaine delivery was carried out for 7 days followed by the reinstatement tests. (A) Experimental design. (B,E) Counts of lever presses in cocaine-SA memory retention tests 24 hours after cocaine retrieval followed by treatment of propranolol (Prop: 10 mg/kg i.p.) or betaxolol (Bet: 5 mg/kg i.p.). **P < .01 vs vehicle-treated group. (C–D, F–G) Counts of lever presses in cocaine-SA reinstatement tests with treatment of propranolol or betaxolol after cocaine retrieval ***P < .001 vs vehicle-treated group. Values in the bar indicate number of rats per group. Downloaded from https://academic.oup.com/ijnp/article-abstract/21/3/267/4690578 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Zhu et al. | 277 group in the memory retention tests (supplementary Figure 4A, retrieval) was critical to induce a labile state of reward memory F (1, 14) = 1.638, P = .221, 2-way RM ANOVA). To address leading to de novo protein synthesis-dependent memory re- treatment × lever whether β-blocker treatment after cocaine retrieval might affect consolidation. It is also possible that the dose of propranolol cocaine relapse, we tested reinstatement after extinction train- or protein synthesis inhibitor did not produce an adequate ing in the same cohort of rats (Figure 6A). Extinction training long-lasting effect on memory reconsolidation. The result that was performed after the memory retention test in all groups a higher dose of cocaine may not induce memory restabilization to obtain a low number of lever presses in response to cues indicates that there is a nonmonotonic relationship between US (extinction criteria: no more than 10 active lever presses for the reexposure and memory restabilization, which needs further last 2 days). One day after the extinction session, rats showed investigation. no spontaneous recovery with saline-priming (Figure 6C , In the same cohort of mice, our data showed that reinstate- F (1, 23) = 0.434, P = .516, 2-way RM ANOVA). Then, in ment was impaired by post US retrieval treatment, but not post treatment × lever reinstatement tests with cocaine priming, propranolol injection CS retrieval treatment, thus indicating that memory impairment after cocaine retrieval significantly inhibited drug seeking be- induced by pharmacological treatment after US retrieval was havior (Figure 6D , F (1, 23) = 6.121, P = .021; Bonferroni persistent. The finding that treatment after US retrieval impairs treatment × lever posthoc test: t = 3.728, P < .001, vs Veh in active lever press, reconsolidation is consistent with studies showing that aniso- 2-way RM ANOVA). A significant inhibitory effect on memory mycin injection in the BLA after US retrieval of fear memory can reconsolidation was also detected by administration of betaxo- impair multiple CS-US associations to the same US. Similarly, in lol, but not ICI 118, 551, after cocaine retrieval (Figure 6E, Bet: studies of fear memory reconsolidation, memory retrieval by a F (1, 14) = 5.327, P = .037; Bonferroni posthoc test: t = 3.602, single CS leads to a selective amnesia for that specific CS when treatment × lever P = .001, vs Bet in active lever press; supplementary Figure 4B , ICI: multiple CSs are associated with the US. It is likely targeting of a F (1, 21) = 0.505, P = .485, 2-way RM ANOVA). Treatment different component of the association that underlies the differ - treatment × lever of betaxolol after cocaine retrieval also suppressed relapse to ence. In addition, the pharmacological blockade after contextual cocaine in the reinstatement test (Figure 6F–G , saline prim- reexposure impaired long-term memory as indicated by CPP ing: F (1, 14) = 0.257, P = .620; cocaine priming: test, while memories reinstated after extinction session as indi- treatment × lever F (1, 14) = 10.898, P = .005, Bonferroni posthoc test: t = cated by cocaine priming-induced reinstatement test, suggest- treatment × lever 4.976, P < .001, vs Veh in active lever press, 2-way RM ANOVA). ing that memories were possibly not erased by pharmacological Our data suggest that activation of β-AR, particularly β1-AR, intervention but were temporally inhibited. It might be that CS was required for US retrieval-induced memory reconsolidation induces memory trace partially reactivated, and systemic treat- of cocaine-SA. In agreement with the results of cocaine-CPP, ment after CS retrieval partially inhibited memory reconsolida- this impaired memory reconsolidation of cocaine-SA by β1-AR tion. Thus, manipulation post US retrieval may destabilize more blockade during the time window after US retrieval also led to memory trace of CS-US associations than CS retrieval. Our data decreased relapse to cocaine. suggest that US retrieval might be more efficient than CS re- trieval, and manipulations after US retrieval might have an ad- vantage for treating substance addiction by preventing drug Discussion relapse. In this study, memory reconsolidation of cocaine-CPP was The present results demonstrated that cocaine triggered pro- tein synthesis-dependent memory reconsolidation of cocaine- impaired by systemic treatment or CeA infusion of protein syn- thesis inhibitor or β-blocker after US-retrieval, but was intact CPP or cocaine-SA. The drug memory reconsolidation triggered by cocaine reexposure, not by context reexposure, was found to with local injection in the CeA after CS-retrieval. In addition, immunofluorescence staining showed that c-Fos expression be mediated by β1-AR in the CeA. Furthermore, the manipula- tion post cocaine retrieval also decreased cocaine relapse in the significantly increased in the CeA after cocaine reexposure, but not context reexposure. These data suggest that CeA might be reinstatement tests, in contrast to the drug-seeking behavior observed in the cocaine priming-induced reinstatement test involved in the US, but not CS, -induced memory reconsolida- tion process. with treatment after context retrieval. These findings demon- strate that US retrieval triggered memory reconsolidation pro- Our current study suggested that -adr β energic activation might participate in the US retrieval-triggered memory recon- cess is distinct from the CS retrieval, or US retrieval is more efficient than CS retrieval, thus offering a potential manipu- solidation process, adding new evidence to previous findings, and demonstrates that memory reconsolidation triggered by US lation strategy by which the persistent inhibition of addiction memory might be obtained. retrieval is also dependent on -AR mediated signaling. β Previous research has revealed that activation of -AR r β ecruits ERK and/ In this study, we demonstrated that a low dose of cocaine (1.5 mg/kg) was sufficient to trigger reconsolidation of reward or mammalian target of rapamycin signaling, thereby facili- tating protein synthesis-dependent long-term potentiation memory, because protein synthesis inhibitors or β-blockers, the classic amnestic agents for CS retrieval-induced memory recon- (Gelinas and Nguyen, 2005; Gelinas et al., 2007). In this study, systemic or intra-CeA injection of propranolol and a protein solidation (Nader et al., 2000bDe ; biec et al., 2002; De Jaeger et al., 2014), blocked cocaine reexposure-induced memory recon- synthesis inhibitor impaired reconsolidation of cocaine reward memory, thus suggesting that -b βlockers or protein synthesis solidation of cocaine-CPP or cocaine-SA. The similar US retrieval procedure was also performed with lower dose of nicotine or co- inhibitors might induce a common cellular process respon- sible for the failure of US retrieval-induced memory reconsoli- caine injection (compared with the dose for training) as UCS re- trieval (Luo et al., 2015 Xue et al., ; 2017b). Furthermore, we tested dation. However, these 2 manipulations may also potentially drive behavior through 2 independent cellular processes, other doses of cocaine. Cocaine-induced memory reconsolida- tion at a dose of 3 mg/kg, but not 5 mg/kg or more, could be given that a recent study has shown that CHX, not propran- olol, blocks cue-induced memory reconsolidation of cocaine-SA inhibited by propranolol (data not shown). In this study, the ap- propriate dose of cocaine for US retrieval (1.5 mg/kg for cocaine- (Dunbar and Taylor, 2016). β1-AR and β2-AR are 2 subtypes of β-AR that are distributed widely in the central nervous system. CPP memory retrieval; 4 i.v. injections for cocaine-SA memory Downloaded from https://academic.oup.com/ijnp/article-abstract/21/3/267/4690578 by Ed 'DeepDyve' Gillespie user on 16 March 2018 278 | International Journal of Neuropsychopharmacology, 2018 Although β1-AR are in much higher levels than 1-AR within β Supplementary Material forebrain structures, isoproterenol, which has equal affinity for Supplementary data are available at International Journal of both subtypes, induces much greater adenylyl cyclase activity Neuropsychopharmacology online. upon stimulation of β 2-AR than β1-AR. Then effects of both β1- AR and β2-AR on memory reconsolidation were examined in this study by using pharmacological treatment or conditional Acknowledgments knockout strategy, respectively. Our data suggested that 1-AR, β This research was supported by grants from the National Natural rather than β2-AR, in the CeA was required for reconsolida- Science Foundation of China (grant nos. 31430033, 91632307, and tion of cocaine reward memory induced by cocaine retrieval. 31421091 to L.M.; 31371136, 31571036, and 31771176 to X.L.) and However, previous work has demonstrated that postretrieval the Ministry of Science and Technology (grant no. 2015CB553500 long-term memory of cocaine-CPP might be mediated by 2-AR β and 2014CB942801 to L.M.). but not β 1-AR (Bernardi et al., 2009). The disagreement between our findings and these previous results might be due to differ - ences in experimental designs and targeted brain regions. The Statement of Interest negative results of ICI 118,551 infusion or β2-AR conditional None. knockout in the CeA could also attribute to its low expression levels. Future studies should determine the precise cellular and β-adrenoceptor-mediated signaling mechanisms of this References phenomenon. The amygdala plays an essential part in processing both fear - Arguello AA, Hodges MA, Wells AM, Lara H, 3rd, Xie X, Fuchs ful and rewarding environmental stimuli (Janak and Tye, 2015). RA (2014) Involvement of amygdalar protein kinase A, but The amygdala is comprised of multiple interconnected nuclei not calcium/calmodulin-dependent protein kinase II, in the such as the BLA, LA, and CeA. The BLA has the status of a “cor - reconsolidation of cocaine-related contextual memories in tical-level structure” that contains most of glutamatergic princi- rats. Psychopharmacology 231:55–65. pal neurons (Heimer, 2003). The BLA is critically involved in CS Balleine BW, Killcross S (2006) Parallel incentive processing: retrieval-induced reconsolidation process of drug reward mem- an integrated view of amygdala function. Trend Neurosci ories (Milton and Everitt, 2010 T; orregrossa and Taylor, 2013), and 29:272–279. the BLA neuronal activity is critical for both CS and US exposure- Barros VN, Mundim M, Galindo LT, Bittencourt S, Porcionatto triggered reconsolidation of nicotine reward memories (Xue et M, Mello LE (2015) The pattern of c-Fos expression and its al., 2017b). In this study, we showed the similar results that both refractory period in the brain of rats and monkeys. Front Cell CS retrieval and US retrieval induced BLA activation, while US Neurosci 9:72. retrieval induced greater c-Fos expression than CS retrieval did, Bernardi RE, Ryabinin AE, Berger SP, Lattal KM (2009) Post- indicating that US retrieval may reactivate more memory traces retrieval disruption of a cocaine conditioned place prefer - than CS retrieval did in BLA. 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International Journal of NeuropsychopharmacologyOxford University Press

Published: Mar 1, 2018

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