Elevated BDNF mRNA expression in the medial prefrontal cortex after d -amphetamine reinstated conditioned place preference in rats

Neuroscience 263 (2014) 88–95

ELEVATED BDNF mRNA EXPRESSION IN THE MEDIAL PREFRONTAL CORTEX AFTER D-AMPHETAMINE REINSTATED CONDITIONED PLACE PREFERENCE IN RATS Y.-L. SHEN, a T.-Y. CHANG, b Y.-C. CHANG, a H.-H. TIEN, a F.-C. YANG, a P.-Y. WANG b  AND R.-M. LIAO a,b,c*

extinction may be linked to an increase in BDNF mRNA expression in the mPFC. Ó 2014 IBRO. Published by Elsevier Ltd. All rights reserved.

a Department of Psychology, National Cheng-Chi University, Taipei, Taiwan b Institute of Neuroscience, National Cheng-Chi University, Taipei, Taiwan

Key words: place conditioning, drug seeking, addiction, mesocorticolimbic dopamine, brain-derived neurotrophic factor.

c

Research Center for Mind, Brain and Learning, National Cheng-Chi University, Taipei, Taiwan

INTRODUCTION

Abstract—Drug addiction behavior that is established and maintained by psychostimulants has been shown to be associated with the expression of brain-derived neurotrophic factor (BDNF) in the mesolimbic dopamine (DA) system. Cocaine has been used for most prior studies testing this effect of psychostimulants and therefore relatively little is known about its counterpart amphetamine (AMP). To fill this gap, the present study was designed to test whether BDNF mRNA expression levels in the DA terminal regions were changed specifically by D-AMP-induced conditioned place preference (CPP) followed by drugprimed reinstatement. The dose of D-AMP, 1 mg/kg, was confirmed to significantly induce CPP. Using this dose, a group of rats was initially subjected to D-AMP CPP, which was followed by entry into an extinction protocol with an additional 3-day withdrawal before a drug-primed reinstatement test was carried out. Following extinction of D-AMP CPP, a lower dose of D-AMP, namely 0.75 mg/kg, was able to significantly reinstate CPP. The BDNF mRNA levels in the selected brain areas were determined by real-time polymerase chain reaction (PCR) after the CPP and reinstatement. The BDNF mRNA level in the medial prefrontal cortex (mPFC) was significantly increased after the reinstatement, but not the CPP test. And, none of the other four assessed brain areas showed any change in BDNF mRNA level after D-AMP CPP or reinstatement. These findings support the notion that BDNF is involved in drug-seeking behavior and indicate that D-AMP reinstatement after

Drug addiction is an enduring psychiatric disorder that seems to be derived from an initial phase of voluntary drug-taking, which then leads to a subsequent habit of drug-seeking that is characterized by a loss of control with respect to drug use and a high relapse frequency after withdrawal (Kalivas and O’Brien, 2008; Koob and Le Moal, 2008). Substantial evidence indicates that the mesocorticolimbic dopamine (DA) systems are highly involved in drug addiction behavior based on the findings from rodent models (Di Chiara and Bassareo, 2005; Wise, 2009). Based on a hypothesis that neurotrophins modulate neuronal activity and synaptic plasticity (Poo, 2001; Park and Poo, 2013), a growing literature has supported the idea that brain-derived neurotrophic factor (BDNF), a member of the neurotrophin family, is associated with the behavioral effects of addictive drugs (Thomas et al., 2008; Russo et al., 2009; Ghitza et al., 2010; McGinty et al., 2010). Previous work using psychostimulant-induced behavioral sensitization has showed that BDNF expression in the ventral tegmental area (VTA) is increased by cocaine (Pu et al., 2006). Cocaine-induced psychomotor sensitization can be potentiated when BDNF is locally infused in the VTA or the nucleus accumbens (NAC); furthermore, the development of this locomotion sensitization to cocaine was found to be delayed in BDNF heterozygote knockout mice (Horger et al., 1999). Using a drug selfadministration task, BDNF in the NAC appears to be essential for modulating cocaine-rewarding effects and this seems also to be true for both cue-induced and stressor-induced reinstatement after cocaine withdrawal (Graham et al., 2007). BDNF protein levels in the VTA, NAC and amygdala (AMYG) are significantly increased in a time-dependent manner during withdrawal from cocaine self-administration, which suggests that mesocorticolimbic BDNF is involved in bringing about cocaine relapse (Grimm et al., 2003). Consistent with this finding, local infusion of BDNF in the VTA has been

*Correspondence to: R.-M. Liao, Department of Psychology, National Cheng-Chi University, 64, Sec. 2, Zhinan Road, Taipei 11605, Taiwan. Tel: +886-2-29387397; fax: +886-2-29390644. E-mail address: [email protected] (R.-M. Liao).   Current address: Graduate Institute of Brain and Mind Sciences, National Taiwan University, Taipei, Taiwan. Abbreviations: ANOVA, analysis of variance; AMP, amphetamine; AMYG, amygdala; BDNF, brain-derived neurotrophic factor; CPP, conditioned place preference; DA, dopamine; dHIP, dorsal hippocampus; dSTR, dorsal striatum; LTP, long-term potentiation; mPFC, medial prefrontal cortex; NAC, nucleus accumbens; PCR, polymerase chain reaction; TrKB, tyrosine kinase B; VTA, ventral tegmental area.

0306-4522/13 $36.00 Ó 2014 IBRO. Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.neuroscience.2014.01.015 88

Y.-L. Shen et al. / Neuroscience 263 (2014) 88–95

shown to progressively enhance cocaine-seeking responses after withdrawal (Lu et al., 2004). Interestingly, Berglind et al. (2007) reported that exogenous BDNF infused in the medial prefrontal cortex (mPFC) was able to suppress drug-seeking behavior using a cocaine self-administration model. Thus, although BDNF has been proposed to be involved in drug reward and relapse, the role of BDNF in drugseeking behavior may be more diverse and complex than initially thought and these aspects remain to be elaborated on. In addition to the aforementioned tasks of psychomotor sensitization and self-administration of drug, the conditioned place preference (CPP) paradigm is widely used as a rodent animal model when measuring drug reward and relapse (Sanchis-Segura and Spanagel, 2006; Tzschentke, 2007). In contrast to the extensive literature available on the use of reinstatement procedures in self-administration studies (Shaham et al., 2003), relatively little information is available on using the reinstatement approach for place conditioning (Aguilar et al., 2009). When limited to the investigation of neurotrophic mechanisms, there have been even a less number of studies that have explored the role of BDNF in drug-induced CPP and reinstatement (Ghitza et al., 2010). The development of cocaine CPP was found to be attenuated in heterozygote BDNF knockout mice (Hall et al., 2003), whereas cocaine CPP was increased when BDNF was overexpressed in the NAC via a lentoviral gene delivery (Bahi et al., 2008). Conversely, cocaine CPP was attenuated in the subjects where BDNF expression was decreased in the NAC and VTA (Bahi et al., 2008; Graham et al., 2009). The BDNF has been suggested to be involved in cocaine CPP, yet no study to date has examined the role of BDNF in the amphetamine (AMP)-induced CPP or its reinstatement (Ghitza et al., 2010). Therefore, it is intriguing to examine the changes in BDNF levels after AMP treatment using the CPP behavioral task model that can enhance our understanding of neurotrophin regulation of drug reward by psychostimulants. The current study aimed to measure BDNF mRNA expression changes in selected brain areas after the D-AMP-induced CPP and its reinstatement. The BDNF measurements, assessed by real-time polymerase chain reaction (PCR), were carried out in the samples collected from the mPFC, the dorsal striatum (dSTR), the NAC, the AMYG, and the dorsal hippocampus (dHIP). These areas were selected on the basis of their distinctive roles involved in drug reward systems of the brain.

EXPERIMENTAL PROCEDURES Animal subjects Forty-eight male Wistar rats, weighing 300 ± 25 g at the start of the experiment, were used in this study (BioLASCO Taiwan Co., Ltd, Taipei, Taiwan). The rats were housed individually with free access to food and water and maintained in a 12-h reverse light–dark cycle (light on at 07:30). Upon their arrival in the vivarium, subjects were gentled through daily handling till the start

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of behavioral experiment. All experimental sessions were conducted during the light portion of the cycle. The temperature of the colony was maintained at 23 ± 1 °C throughout the experiment. All procedures were conducted in accordance NIH Guide for the Care and Use of Laboratory Animals and approved by an institutional review committee. Apparatus used for place conditioning The two equal-sized chambers (36 L  36 W  61 H cm each) separated by a removable partition were used for the CPP apparatus. One chamber was painted gray on the wall and had a wire-meshed floor and the other was painted with black and white vertical stripes (4 cm each) and had a bar grid floor made of stainless steel rods (0.2 cm each) running in parallel. A second removable partition that had a small opening (8  12 cm) allowed the rat to move freely between chambers during the preference tests. The apparatus was located in a semisoundproof room with a medium roof light illumination (160 lux). The construction of CPP apparatus used in this study showed no unconditioned preference for either side chamber, that the untrained or drug-naı¨ ve rats spent an average about 50% of time on each chamber before conditioning. Behavioral measurement In the Experiment 1, as in a regular CPP task, rats were placed in one of the chambers and allowed to move freely into either of the compartments for 10 min as the pre-conditioning session of CPP (Day 1). Upon completion of the trial, the subject was removed from the apparatus and returned its home cage. The chambers were then completely cleaned before the next rat’s trial. The amount of time the rat spent in each chamber was measured by a video-camera attached to a video mouse trace system (SINGA Technology Co., Taipei, Taiwan). The designation of the drug-paired chamber was counterbalanced for each group, so that half of the subjects were assigned to initially preferred chamber as the drug-paired side, and the others half of the subjects were assigned to initially non-preferred chamber for drug-paired side. Following the unbiased assignment protocol, the means of time spent in the two chambers were closer to 50% of session time before drug pairing association. And, no significant difference was revealed by any tested group (p > 0.05, by a paired t-test). In the morning of the conditioning phase (Days 2–4), the rats within both the control and drug groups were placed into a conditioning chamber (as the paired side) for 30 min after the i.p. injection of D-AMP sulfate (Sigma Chemical Co., St. Louis, MO, USA) or saline at the dose of 0, 0.1, 0.5 and 1.0 mg/kg (n = 5, n = 6, n = 5, and n = 7, respectively). Four hours later, in the afternoon, the subjects of each group received vehicle infusion before being placed into the other conditioning chamber (as the unpaired side) for another 30 min. The protocol for conditioning sessions conducted twice a day with an interval of 4 h was adopted by a pilot work from this laboratory and referred

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from the reports from us (e.g. Shen et al., 2011) and others (e.g. Der-Avakian et al., 2007; Cruz et al., 2008, 2010). The post-conditioning test (Day 5) was conducted 24 h after the last conditioning day. During this session, an identical procedure to that of the preconditioning session was run in order to determine the CPP induced by D-AMP. In the Experiment 2, a separate group of rats were initially subjected to acquire CPP induced by D-AMP of 1.0 mg/kg following the protocol described above. After the post-conditioning test, a modified 8-day extinction protocol was applied from Days 6 to 13 (Cruz et al., 2008). On Days 6, 8, 10 and 12, each rat was confined in the conditioning compartment in a similar manner to that of the conditioning days, but without receiving any injections. On the alternative days (Day 7, 9, 11 and 13), the rat was run through a procedure similar to that of the post-conditioning test. Behavioral data was collected throughout these 4 days and used to determine the effect of extinction on D-AMP CPP. Subsequent to the 8-day extinction, the rat was kept in the homecage for 3 days (Days 14–16) in order to simulate a period of drug withdrawal. On the Day 17, the subjects were randomly assigned into three groups for the reinstatement CPP test of the dose–response effects of D-AMP given at 0, 0.5 and 0.75 mg/kg (n = 8, n = 7, and n = 10, respectively). For each of reinforcement test, rats were administered either saline or D-AMP via i.p. injection immediately before being placed into the CPP apparatus. This drug treatment was conducted in a holding desk just outside the CPP test room. The procedure of reinstatement test was the same as that of post-conditioning CPP test as described above. BDNF mRNA assay The animals were sacrificed by rapid decapitation right after the completion of behavioral test for D-AMP CPP or drug-primed reinstatement. The brains were removed and embedded in Tissue-Tek embedding medium OCT (VWR Scientific Products, Bridgeport, NJ, USA ) and frozen in melting isopentane. Brain sections (60-lmthick each) were prepared by a cryostat (Leica, 3050S, Nussloch, Germany). Twenty samples from each brain region, which were punched out using a blunt-end syringe needle of 18-gage, were collected from the coronal sections. The maximum anterior-to-posterior extension was about +3.72 to +2.52 mm for the mPFC, +2.04 to +1.44 mm for dSTR and NAC, and 2.92 to 3.24 mm for the dHIP and AMYG (Paxinos and Watson, 2005). Fig. 1 depicts the coronal sections of the rat brain with the regions of interest where the brain tissues were punched from for BDNF mRNA assay marked. Total RNA was extracted using the RNeasy Mini Kit (Qiagen, Valencia, CA, USA). The RNA was treated with DNase and converted to cDNA using oligo-d(T)15 (Invitrogen) and SuperScript III reverse transcriptase (Invitrogen, Carlsbad, CA, USA) as described previously (Chou et al., 2013). Real-time PCR reactions were performed using a 7300 Real-Time PCR System

mPFC

+ 3.72 mm

dSTR NAC + 2.04 mm

dHIP

AMYG

- 2.92 mm

Fig. 1. Schematic representation of the areas of the medial prefrontal cortex (mPFC), dorsal striatum (dSTR), nucleus accumbens (NAC), dorsal hippocampus (dHIP) and amygdala (AMYG) where tissues were punched to carry out the BDNF mRNA assays. The darkened circles identify the regions where the tissue punch samples were taken. Only the rostral faces of each coronal section are shown. The stereotaxic drawings and coordinates are modified from Paxinos and Watson (2005).

(Applied Biosystems, Foster City, CA, USA), SYBR Green Master Mix (Applied Biosystems) and the gene-specific primers: BDNF-F:50 -GCCCAACGA AGA AAACCATAAG-3’; BDNF-R:50 -GTTTGCGGCATCCAGG TAATT-30 ; GAPDH-F: 50 -TACATGGCCTCCAAGGAGTA AGAA-30 ; GAPDH-R: 50 -GGATGG AAATTGTGAGGGA GA TG-30 . A two-step PCR reaction was carried out with denaturation at 95 °C for 15 s and annealing/extension combined at 60 °C for one minute for a total of 40 cycles. The mRNA expression level of each target gene were compared to GAPDH and quantified by subtraction: Cttarget  CtGAPDH = DCt. A difference of one PCR cycle equates to a twofold change in mRNA expression level. The uniqueness of the amplicons was assessed using their dissociation curves. Statistical analysis All data are expressed as means ± SEM’s and were analyzed using SPSS software (v. 18). Behavioral data

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were the times spent in each chamber as the dependent variable. The CPP was verified by the time difference between two pairing chambers on each of the test sessions for each group via a one-sample t test, while the dose–response effects of D-AMP on the CPP performance and reinstatement experiments were examined by a mixed two-way analysis of variance (ANOVA) with a between-subject factor of the dose and a within-subject factor of the pairing chamber. The conditioning or reinstatement dose effects of D-AMP on the expression level of BDNF mRNA in each brain area were verified by a one-way ANOVA. A post hoc Tukey HSD test or a simple main effect was adopted as appropriate. The acceptable significance level was set at p 6 0.05.

RESULTS Fig. 2 shows the dosage effect of D-AMP-induced CPP and the test results of BDNF mRNA for place

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conditioning in Experiment 1. CPP was examined by comparing the times the animals stayed in the drugpaired vs. drug-unpaired chamber on the test day. As shown in Fig. 2A, the results of a two-way ANOVA showed a significant dosage-by-pairing interaction, [F(3, 19) = 3.16, p < 0.05]. Neither the main effect test for dose nor that for pairing was significant (p > 0.05). The simple main effect comparisons revealed a significant time difference between the paired and unpaired chambers when the rats were treated with D-AMP given at 1.0 mg/kg, [F(1, 19) = 13.47, p < 0.01]; no such effect was found for the other three dosage groups (p > 0.05). The expression levels of BDNF mRNA in the mPFC, dSTR, NAC, dHIP and AMYG after the D-AMP CPP test are shown in Fig. 2B. Using a oneway ANOVA, it was found that there was no significant difference in relation to dose detectable for any of the brain areas tested (p > 0.05). The results of Experiment 2 regarding the drug-primed reinstatement of D-AMP-induced CPP and expressions of BDNF mRNA are shown in Fig. 3. Before extinction, the subjects acquired D-AMP CPP as indexed by the time difference between two pairing chambers on the

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conditioning dose of d-AMP (mg/kg) 0.020

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saline d-AMP (0.1) d-AMP (0.5) d-AMP (1.0)

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reinstatement dose of d-AMP (mg/kg) 0.020

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FC STR NAC HIP YG d AM d mP Fig. 2. Data of Experiment 1, (A) the dose effects of D-amphetamineinduced conditioned place preference. The time in seconds spent in the drug-paired and drug-unpaired chambers for each of four groups that received dose of 0, 0.1, 0.5 and 1.0 mg/kg of D-amphetamine, respectively, during the conditioning sessions. ⁄⁄p < 0.01: difference between the paired and unpaired bars using a simple main effect comparison. (B) BDNF mRNA expressions measured in the brain areas as indicated after the D-amphetamine-induced conditioned place preference.

0.000

FC STR NAC HIP YG d AM d mP Fig. 3. Data for Experiment 2, (A) the dose effects of D-amphetamine (0, 0.5, 0.75 mg/kg) on reinstatement of the extinguished conditioned place preference. ⁄⁄⁄p < 0.001, difference between the paired and unpaired bars using a simple main effect comparison. (B) BDNF mRNA expression measured in the brain areas as indicated after the drug-primed reinstatement test of the D-amphetamine-induced conditioned place preference. ⁄⁄p < 0.01, post hoc Tukey HSD test.

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post-conditioning test: 113.855 ± 20.94 s, t(24) = 5.438, p < 0.001. Following the 8-day extinction, the CPP was diminished as determined by a lack of significant difference on the time spent in two pairing chambers on Day 13: 42.786 ± 25.14 s, t(24) = 1.702, p = 1.102. An additional analysis was conducted by using a one-way ANOVA repeated measure for confirming the significance of CPP extinction, F(1, 24) = 4.763, p < 0.05. In considering the subjects being randomly assigned to three groups for the reinstatement test, the results of CPP for each group was also analyzed by t test on examining the difference time spent in two pairing chambers during the post-conditioning test [82.576 ± 33.12 s, t(7) = 2.49, p < 0.05, for the saline control; 165.89 ± 52.16 s, t(6) = 3.18, p < 0.05, for the group of 0.5 mg/kg D-AMP; 102.45 ± 25.65 s, t(9) = 3.99, p < 0.01, for the group of 0.75 mg/kg D-AMP]. And, on the last day of the extinction (Day 13), no significant CPP effect was detected for any of these three group: 10.06 ± 37.96 s for the saline control, t(7) = 0.27; 67.63 ± 49.23 s for the group of 0.5 mg/kg D-AMP, t(6) = 1.37; 67.57 ± 42.58 s for the group of 0.75 mg/kg D-AMP, t(9) = 1.59; all p > 0.05). The dose effects of D-AMP when used to reinstate the extinguished CPP are shown in Fig. 3A. Results of a two-way ANOVA showed a significant main effect of pairing and a significant dose-by-pairing interaction [F(1, 22) = 6.75, p < 0.05; F(2, 22) = 3.40, p = 0.05, respectively], but no main effect of dose (p > 0.05). The simple main effect of dose revealed that a significant time difference between the two pairing chambers appeared when D-AMP was given at 0.75 mg/kg, F(2, 22) = 15.15, p < 0.001. No such effect was seen in the group primed by the saline or the group primed with D-AMP at 0.5 mg/kg (both p > 0.05). When the expression levels of BDNF mRNA after the drug reinstatement test were examined (Fig. 3B), the results of the one-way ANOVA’s revealed that there was only a significant increment in BDNF mRNA level in the mPFC [F(2, 22) = 5.56, p < 0.05] and not in the other areas. Furthermore, the post hoc comparisons also showed a significant difference between the saline group and the group primed by D-AMP at 0.75 mg/kg (p < 0.01).

DISCUSSION The present results confirm a significant CPP induced by when the conditioning dose was 1.0 mg/kg, but not 0.1 and 0.5 mg/kg (Experiment 1). Via a drug-primed reinstatement test (Experiment 2), D-AMP injected at the dose of 0.75 mg/kg, but not 0.5 mg/kg, significantly reinstated the extinguished CPP. Biochemically, BDNF mRNA levels were significantly increased only in the mPFC after the reinstatement test. That a dose of 1 mg/kg of D-AMP injected via the i.p. route significantly produced CPP in the present study is consistent with previous reports (Liao et al., 1998; Rademacher et al., 2006; Shen et al., 2006). Recent studies have applied CPP to test the reinstatement of drug, mostly for cocaine and morphine (Aguilar et al., 2009). Only a few studies reported on the reinstatements

D-AMP

of drug CPP using AMP and its derivatives including d, l-AMP (Cruz et al., 2008, 2010), methamphetamine (Li et al., 2002; DeMarco et al., 2009; Qi et al., 2009; Do Couto et al., 2011; Yu et al., 2011; Abulseoud et al., 2012; Berry et al., 2012; Subiah et al., 2012) and 3,4-methylenedioxymethylamphetamine (MDMA) (DazaLosada et al., 2011; Vidal-Infer et al., 2012). To our knowledge, the present work is the first in reporting drug-primed CPP reinstatement using D-AMP. It has been emphasized that the CPP paradigm is useful when investigate the underlying neurobiological mechanisms of drug addiction and when evaluating potential antirelapse drug treatment protocols (Aguilar et al., 2009; Napier et al., 2013). Our findings show that the level of BDNF mRNA was significantly increased in the mPFC after the D-AMP CPP reinstatement, and no similar effect was observed for the other four areas being tested. Furthermore, the increase in BDNF mRNA in the mPFC was absent after the initial acquisition and performance of the D-AMP CPP. These results primarily highlight that the involvement of the mPFC in the drug-seeking stage is more critical than that in the drug-taking stage of the CPP paradigm. The involvement of the mPFC in drug-seeking behavior has been argued by previous CPP studies of pharmacological approach (Sanchez et al., 2003), lesion (Zavala et al., 2003; Hsu and Packard, 2008) or neurochemical assay (Qi et al., 2009). While these findings indicate the importance of mPFC in the drugprimed CPP reinstatement, this brain region may not necessarily be involved in drug-context association during the initial stage of CPP. For example, the acquisition of AMP CPP has been found not to be affected by the mPFC lesion (Tzschentke and Schmidt, 1998). In the absence of previous work examining BDNF mRNA levels using the drug-primed CPP reinstatement paradigm, comparing the relevant data collected from other behavioral tasks in relation to drug reinstatement should be informative. Hearing et al. (2008) reported that BDNF mRNA levels were greater in the dorsal mPFC of the rat re-exposed to the self-administration chamber previously associated with cocaine infusion as reinforcement. Interestedly, this increase of BDNF mRNA level was observed in the subjects after 14 days of abstinence in the home cage rather than 22 h. The environmental re-exposure test set up after abstinence for subjects without lever availability is thought to induce a kind of context-conditioned effect of drug reward. This operant behavior condition without the lever availability is simulated partially, if not totally, by the CPP test. These contradictory results after abstinence for 22 h and for 14 days as described above seem to agree with the present findings; where there was a lack of change in mPFC BDNF mRNA levels during the CPP test, but there was a significant increase during the reinstatement test. Regarding the psychostimulant-induced behavioral sensitization paradigm, Fanous et al. (2011) showed that a challenge dose of D-AMP of 1 mg/kg had a sensitization effect on locomotor activity and increased mPFC BDNF expression. Although there have been

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0.0006406 ± 0.0003140 0.0010411 ± 0.0003047 0.0106821 ± 0.0024598 0.0140316 ± 0.0045225 0.0001398 ± 0.0000992 0.0002984 ± 0.0002055 0.0000128 ± 0.0000057 0.0000312 ± 0.0000082 Data indicate mean ± SEM.

0.0050150 ± 0.0008472 0.0067664 ± 0.0009213 (n = 5)

Saline (n = 5)

D-AMP

dHIP NAC dSTR mPFC

Table 1. BDNF mRNA expressions after acute D-AMP treatment of 1 mg/kg

several other studies that have also used the repeated stimulant injection regiment and reported an increase in BDNF mRNA levels in the mPFC (Fumagalli et al., 2007) and other brain areas (Meredith et al., 2002), it should be noted that the BDNF mRNA levels were measured in systems where drug withdrawal and sensitization were absent in these cases. Based on the current findings, it is reasonable to infer that the induction of drug-seeking behavior is attributable to an elevation in BDNF expression in the mPFC. It has been suggested that underlying cellular mechanisms of this phenomenon may be related to BDNF being able to regulate long-term potentiation (LTP) of the mPFC neurons after the withdrawal from repeated cocaine (Lu et al., 2010). Thus the up-regulated mPFC BDNF level, in response to repeated cocaine treatment, facilitates activity-induced LTP of the excitatory synapses of the mPFC neurons. This LTP facilitation, via suppression of intrinsic GABAergic neurotransmission, is then linked to cocaine-induced behavioral sensitization as measured by locomotor activity. It is intriguing to verify these findings that can be generalized to drug reinstatement, as tested by self-administration or CPP paradigm. In contrast to the elevated mPFC BDNF mRNA after D-AMP CPP reinstatement, the BDNF mRNA levels in the mPFC remained unchanged after the initial CPP in the present study. Accumulating evidence shows the BDNF is closely associated with drug reward (Ghitza et al., 2010; McGinty et al., 2010), but, strictly, this has not been confirmed for the acquisition and performance of CPP induced by AMP. Nonetheless, the formation of contextual stimuli and the rewarding effect of D-AMP in the CPP task has been shown to be accompanied by increased tyrosine kinase B (TrKB) receptor expression in several terminal areas of the mesolimbic DA systems (Rademacher et al., 2006; Shen et al., 2006). Unfortunately, expression of the TrK receptor in the mPFC was not assessed in either one of these two studies. Even though the TrKB receptor is known to bind BDNF as a ligand, there needs to be a cautious interpretation of any changes in TrKB receptor expression when investigating BDNF level alterations. It is known that the distribution of BDNF mRNA in terms of levels in the brain is region specific. Conner et al. (1997) reported that some regions of the brain in normal adult rats, including the NAC and the caudateputamen, contain no BDNF mRNA or BDNF immunereactive cells/fibers. The AMYG and the hippocampal formation also contain no BDNF mRNA or BDNF immunereactive cells, but do contain a high density of immunelabeled fibers. This pattern is region specific with respect to some subareas within the AMYG and hippocampal formation. In contrast, the mPFC has been shown to express relatively high amounts of BDNF mRNA and protein. Our results from the vehicle controls are consistent with the distribution pattern of BDNF mRNA as described above. However, it was unexpected to observe the BDNF mRNA levels unchanged after D-AMP induced CPP in this study. To explain this negative result, it should be noted that the brain tissues were collected immediately after the post-conditioning

AMYG

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CPP test in this study. BDNF mRNA expression may be gradually increased over a number of hours after CPP is performed or for that matter any other classical conditioning behavioral task. For example, BDNF mRNA levels in the mPFC and the basolateral AMYG have been found to be elevated after olfactory fear learning when the brain samples were collected 2 h after the conditioned behavior test (Jones et al., 2011). Also, from association conditioning aspect, the limited number of drug-paired sessions conducted in the conditioning stage may result in an undetectable BDNA mRNA change. A question may be raised in concerning the present findings of the increased BDNF mRNA levels in the mPFC observed in the CPP reinstatement test but not in the initial CPP performance test. The subjects received (priming) drug injections in the reinstatement test, whereas those with the initial CPP performance did not have any injection before the test. It is then arguable that the increment of mPFC BDNF mRNA could be the result from an acute injection of D-AMP given before the reinstatement test. An additional experiment was conducted on verifying this speculation. A separate group of ten rats were recruited and raised in the condition similar to those subjects in the Experiment 2. For each subject, the brain samples were collected 30 min after an acute i.p. injection of D-AMP of 1 mg/kg or saline, and BDNF mRNA were then analyzed. As shown in Table 1, the results indicate that there was no significant difference between the drug treatment and saline control in any of the five brain areas tested. Thus, it is unlikely that the discrepancy described above was the cause of BDNF mRNA in the mPFC differentially expressed in the initial CPP performance and the drugprimed reinstatement. These results are consistent with those from a previous study showing that acute D-AMP (5 mg/kg) treatment did not significantly alter BDNF mRNA levels in most of forebrain areas including the mPFC when compared to controls (Meredith et al., 2002). Together, it is most likely that both the drug treatment and behavioral manipulation are essential for the detection of changes of BDNF mRNA expression.

CONCLUSIONS This study is the first to report drug-primed reinstatement of CPP induced by D-AMP. BDNF mRNA levels in the mPFC were dose-dependently increased after drugprimed reinstatement of D-AMP CPP. The present results support the hypothesis that BDNF mRNA levels in the mPFC are critical in drug-seeking stage of the CPP paradigm.

CONFLICT OF INTEREST Authors declare no conflict of interest. Acknowledgments—This study was supported in part by grants, NSC 99-2410-H-004-090-MY2 and NSC 101-2410-H-004-084MY3 to RML, from National Science Council, Taiwan.

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(Accepted 8 January 2014) (Available online 15 January 2014)