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. Author manuscript; available in PMC: 2020 Sep 1.
Published in final edited form as: Neurobiol Learn Mem. 2019 Jun 4;163:107031. doi: 10.1016/j.nlm.2019.107031

Neuroplasticity transcript profile of the ventral striatum in the extinction of opioid-induced conditioned place preference

Freddyson J Martínez-Rivera a,1, Namyr A Martínez b,c, Magdiel Martínez b,c, Roxsana N Ayala-Pagán a, Walter I Silva b,c, Jennifer L Barreto-Estrada a,*
PMCID: PMC6689252  NIHMSID: NIHMS1532181  PMID: 31173919

Abstract

Persistent drug-seeking behavior has been associated with deficits in neural circuits that regulate the extinction of addictive behaviors. Although there is extensive data that associates addiction phases with neuroplasticity changes in the reward circuit, little is known about the underlying mechanisms of extinction learning of opioid associated cues. Here, we combined morphine-conditioned place preference (CPP) with real-time polymerase chain reaction (RT-PCR) to identify the effects of extinction training on the expression of genes (mRNAs) associated with synaptic plasticity and opioid receptors in the ventral striatum/nucleus accumbens (VS/NAc). Following morphine extinction training, we identified two animal subgroups showing either extinction (low CPP) or extinction-resistance (high CPP). A third group were conditioned to morphine but did not receive extinction training (sham-extinction; high CPP). RT-PCR results showed that brain derived neurotrophic factor (Bdnf) was upregulated in rats showing successful extinction. Conversely, the lack of extinction training (sham-extinction) upregulated genes associated with kinases (Camk2g), neurotrophins (Ngfr), synaptic connectivity factors (Ephb2), glutamate neurotransmission (Grm8) and opioid receptors (μ1, Δ1). To further identify genes modulated by morphine itself, comparisons with their saline-counterparts were performed. Results revealed that Bdnf was consistently upregulated in the extinction group. Alternatively, widespread gene modulation was observed in the group with lack of extinction training (i.e. Drd2, Cnr1, Creb, μ1, Δ1) and the group showing extinction resistance (i.e. Crem, Rheb, Tnfa). Together, our study builds on the identification of putative genetic markers for the extinction learning of drug-associated cues.

Keywords: Bdnf, Conditioned Place Preference, Genes, Learning, Morphine, Reward

1. Introduction

Drug addiction, characterized by the persistence of maladaptive behaviors, such as compulsion to seek and take the drug (Koob and Volkow 2010), has been associated with deficits in neural circuits that regulate the extinction of addictive behaviors (Goldstein and Volkow 2011; Peters et al. 2009). Given that extinction is a type of learning where conditioned preferences to drug-associated environments weakens by exposure to the cues in the absence of the drug (Heinrichs et al. 2010; Leite-Morris et al. 2014), it is not surprising that some current treatments for addiction in humans are based on extinction processes. In this regard, exposure-based therapies exemplify classical extinction training, which has been suggested as an alternative in preventing relapse in treatment-resistant patients (Myers and Carlezon 2010; Torregrossa and Taylor 2013).

To unravel the biological mechanisms of the extinction learning of drug-associated cues, several studies have correlated neuroplasticity events of the ventral striatum/nucleus accumbens (VS/NAc) with extinction learning (Gass and Chandler 2013; Millan et al. 2011; Peters et al. 2009; Roberts-Wolfe et al. 2018). For instance, circuit-based studies show that AMPA receptor antagonism into VS/NAc combined with crossed inactivation of BLA impaired the extinction of alcohol seeking via BLA glutamatergic inputs (Millan and McNally 2011). Similarly, chemogenetic activation of prefrontal terminals in VS/NAc reduces cocaine relapse after extinction (Augur et al. 2016), whereas pharmacological inactivation of the VS/NAc correlates with activation of the lateral hypothalamus and impaired extinction of alcohol seeking (Millan et al. 2010). On the same line, it is also suggested that accumbal inactivation correlates with dishibition of the ventral pallidum and elevated reward-seeking behaviors (i.e. food and cocaine) (Kalivas 2007; Smith et al. 2009). As for the molecular studies, pharmacological manipulation or overexpression of accumbal AMPA and NMDA receptors facilitate extinction of opioid (Lu et al. 2011), cocaine (Sutton et al. 2003) and alcohol seeking (Millan and McNally 2011). Furthermore, extinction learning has shown to counteract the opioid-associated dendritic plasticity in the VS/NAc (Leite-Morris et al. 2014). Together, although these studies highlight the VS/NAc and its network as a hub for reducing drug-seeking behaviors, the need of more genetic and molecular studies is a prerequisite to understanding the biological mechanisms underlying the extinction of drug-seeking behaviors.

The fact that VS/NAc receives projections from brain regions that promote extinction of drug-associated cues (Millan et al. 2011; Peters et al. 2009) supports the idea that a genetic profile of this region could reveal modulation in the expression of genes associated with neuroplasticity events underlying contextual learning, which are relevant for extinction (Liang et al. 2011). Specifically, our study searched for a transcript (mRNA) profile in the VS/NAc, with the aim to reveal modulation of genes responsible for extinction of opioid-induced conditioned place preference (CPP). Using the RT2 Profiler PCR Array for Synaptic Plasticity transcripts (Qiagen, Hilden, NRW) and Taqman analyses for opioid receptor transcripts, we identified differential expression of genes associated with neurotrophic factors, neurotransmitter systems and synaptic connectivity, among others, in rats that were either exposed to extinction training or to sham-extinction.

2. Materials and Methods

2.1. Subjects

Forty-one (41) adult male Sprague–Dawley rats (~350g; Envigo Laboratories, Indianapolis, IN) were individually housed with food and water available ad libitum (12:12 hour light/dark cycle; 64°F, 30% humidity). The behavioral experiments were performed during the light phase of the cycle, and all procedures were in accordance with the Institutional Animal Care and Use Committee of the University of Puerto Rico, Medical Sciences Campus, and the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC).

2.2. Drug

As previously described (Heinrichs et al. 2010; Martinez-Rivera et al. 2016; Mueller et al. 2002), animals received subcutaneous injections of morphine sulfate (Sigma–Aldrich, St. Louis, MO; 5mg/kg) dissolved in saline (0.09%; 0.2 ml/100g of body weight), whereas control animals received saline injections.

2.3. Conditioned Place Preference (CPP)

Apparatus

The CPP was similarly performed as previously described (Martinez-Rivera et al. 2015; Martinez-Rivera et al. 2016). Briefly, the CPP apparatus consists of an acrylic chamber (42cm long × 30cm high × 42cm high) separated into two compartments with an entry containing a removable guillotine door. Compartments consisted of grated-texture flooring with black and white checker walls, or smooth flooring with black and white lined walls. The removable guillotine door was used to isolate the rats within specific sides. All CPP protocols were performed at semi-darkness conditions (~10 lux). Behavioral data was acquired using the Any-Maze tracking system (Stoelting Co., Wood Dale, IL).

Conditioning

On days 1 (habituation) and 2 (baseline), animals were allowed to move freely between compartments for 20 min, and the preferred and non-preferred sides were identified during baseline. During the conditioning phase, the experimental rats were injected with morphine and restricted to the non-preferred (NP) side (drug-paired side) or injected with saline and restricted to the preferred (P) side (saline-paired side). The restriction was done for 45 min. A total of 4 alternated injections of either morphine or saline were administered during the conditioning sessions (days 3-10). Control animals received daily saline injections. The next day, after the last conditioning session (day 11, conditioning test), rats were given a drug-free test of 20 min in which they could move freely between both compartments of the CPP chamber. The amount of time spent (%) in the drug-paired side was calculated as an index of conditioning.

Extinction

The extinction protocol was followed as previously described (Heinrichs et al. 2010; Leite-Morris et al. 2014). Briefly, saline injected or morphine-conditioned rats were restricted to the previous drug-paired side or saline side for 45 min for 4 days (days 12-15), in the absence of the drug. On the other hand, animals in the sham-extinction group (no extinction training) were kept in their home cages (with another Plexiglas cage on top as a lid; no food/water available during sessions) and next to the CPP cages where the other rats received extinction training (Fig. 1A). The next day, after the last extinction session (day 16), rats were given an extinction test, and could move freely between compartments. The amount of time spent (%) in the previous drug-paired side was measured as an index of extinction. Rats that did not change their preference or increased the time for the drug-paired side after extinction sessions were classified as extinction resistant. In summary, the outcome of our extinction protocol allowed us to categorized animals based on their extinction training (setting) and learning (rate) as follows: morphine/extinction, morphine/extinction-resistant and morphine/sham-extinction (Fig. 1 and S1). Similarly, the saline counterparts were classified as: saline-extinction and saline/sham-extinction. After the extinction test, animals were genetically profiled.

Figure 1. Extinction of morphine conditioned place preference.

Figure 1.

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(A) Diagram showing the protocol for morphine conditioning and extinction. (B) A schematic drawing showing the saline-treated (left panel) and morphine-treated (right panel) animal groups throughout the CPP protocol. (C) A pre-conditioning test (baseline) established the non-preferred (drug-paired) side for each animal. After conditioning training, a post-conditioning preference test (conditioning test) was performed. An extinction test was performed to calculate the rate of extinction learning. Two subgroups were observed and separated from the extinction-trained rats, the morphine/extinction and morphine/extinction-resistant groups. **p< 0.01. Saline-extinction: n= 7; Saline/sham-extinction: n= 7; Morphine/extinction: n= 9; Morphine/extinction-resistant: n= 9; Morphine/sham-extinction: n= 9. Data is shown as mean and SEM.

2.4. RT-PCR analyses

Brain dissection

Following the extinction test, rats were sacrificed by decapitation, and the brains were dissected as previously described (Martinez-Rivera et al. 2015). Briefly, the VS/NAc (coordinates AP; +2.76 to +0.70) (Paxinos and Watson 2007) was bilaterally dissected and stored in RNAlater solution (Sigma–Aldrich, St. Louis, MO) for RT-PCR analysis (4-5 animals per group).

RNA Isolation and cDNA synthesis

Total RNA was isolated and purified from bilateral VS/NAc using the TRIzol-RNeasy Hybrid Protocol following manufacturer’s instructions (Life Technologies, Carlsbad CA and Qiagen, Hilden, NRW). RNA integrity number (RIN) and concentration were evaluated using the Agilent 2100 Bioanalyzer (Agilent Technologies, San Clara, CA). Consistent with other studies (Boone et al. 2015; Torres Mena et al. 2014; Wang et al. 2010), our RIN values ranged between 4 to 6, with an average of 5. Further genomic DNA elimination and first-strand cDNA synthesis were performed using RT2 First Strand Kit according to the manufacturer’s instructions (Qiagen, Hilden, NRW). Subsequently, the cDNA was diluted with nuclease-free water (Life Technologies, Carlsbad, CA) to 50 ng/μL.

RT2 Profiler™ PCR Array for Synaptic Plasticity

This kit profiled the expression of 86 synaptic plasticity genes plus five housekeeping genes for normalization purposes. The housekeeping genes include: (β-actin (Actb), β-2 microglobulin (β2m), hypoxanthine phosphoribosyltransferase 1 (Hprt1), Lactate dehydrogenase A (Ldha) and ribosomal protein, large, P1 (Rplp1). In the case of sham-extinction rats, Rplp1 and Hprt1 were excluded for normalization, since these genes were modulated. In addition, this profile was customized (CAPR12675-PARN-126Z, Modified array; Qiagen, Hilden, NRW) for the addition of dopamine receptors (D1 and D2). Real Time PCR amplification mixtures (25 μl/well) contained 100 ng template cDNA, RT2 SYBR Green ROX qPCR Mastermix buffer (Qiagen, Hilden, NRW) and nuclease-free water (Life Technologies, Carlsbad, CA). Reactions were run on an ABI PRISM 7500 Fast Real-Time PCR System (Applied-Biosystems, Foster City, CA). The cycling conditions comprised 10 min polymerase activation at 95°C, followed by 40 cycles at 95°C for 15 s, and finally at 60°C for 60 s. All values were reported after quality control requirements (i.e., genomic DNA, reverse transcription efficiency, and positive PCR controls).

Taqman RT-PCR

Because opioid receptors (ORs) are highly expressed in the VS/NAc (Lutz and Kieffer 2013), transcript expression of ORs (mu/μ1; MOR, kappa/K1; KOR and delta/Δ1; DOR) was also evaluated. Given that the RT2 Profiler™ PCR Array for Synaptic Plasticity did not provide reactions for opioid receptors, multiplex Taqman analysis was performed. Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH) Fam-labeled probe was used for normalization. Real Time PCR amplification mixtures (20 μl/well) contained 100 ng template cDNA, TaqMan® Gene Expression ROX Master Mix (Applied-Biosystems, Foster City, CA), nuclease-free water (Life Technologies, Carlsbad, CA) and the respective gene probes. Specifically, gene probes were: mu-opioid receptor (MOR; 1467 bp; NM_001038597.2; Rn01430371_m1), kappa-opioid receptor (KOR; 1358 bp; NM_017167.2; Rn01448892_m1), delta-opioid receptor (DOR; 1418 bp; NM_012617.1; Rn00561699_m1), and GAPDH (1306 bp; NM_017008.4; Rn01775763_g1). Reactions were run on an ABI PRISM 7500 Fast Real-Time PCR System (Applied-Biosystems, Foster City, CA). The cycling conditions comprised 10 min polymerase activation at 95°C, followed by 40 cycles at 95°C for 15 s, and finally at 60°C for 60 s.

2.5. Statistical analyses

Data is presented as mean ± standard error of the mean (SEM) and statistical significance was established as p ≤ 0.05. For CPP and extinction tests, two-way repeated measures ANOVA followed by Tukey post hoc analysis was employed. For gene comparisons among the experimental groups (morphine/extinction, morphine/extinction-resistant and morphine/sham-extinction), two- and one-way ANOVAs followed by Tukey post hoc analyses were employed for 2−ΔΔCt measures (fold changes) (Livak and Schmittgen 2001; Schmittgen and Livak 2008). This method normalizes the expression of targeted genes by housekeeping genes and by their saline counterparts. Therefore, once we manually normalized each gene, this method provides for direct gene expression comparison between the experimental groups. Similarly, to compare opioid receptors among these animal groups, two-way ANOVA followed by Tukey post hoc analyses was applied to 2−ΔΔCt values (fold changes) (Livak and Schmittgen 2001; Schmittgen and Livak 2008) that were manually calculated.

To identify genes mainly modulated by morphine exposure itself, morphine-treated groups were compared to their saline counterparts. For this purpose, we used the integrated web-based software package for RT2 Profiler™ PCR Array (3.5; Qiagen, Hilden, NRW), which automatically performs 2−ΔΔCt calculations from uploaded raw threshold cycle (Ct) data. At this point, fold change values greater or lesser than 1 indicates a fold-increase or fold-decrease of the experimental group over its saline counterpart. The software also provided Student’s t-test analyses for statistical significance.

3. Results

3.1. Conditioning and extinction of morphine place preference

To test for extinction learning, rats were first conditioned to express morphine place preference over 8 days followed by another 4 days of extinction or sham-extinction training (Fig. 1A). As index of learning, the time spent in the drug-paired side was measured during the baseline, conditioning and extinction tests. The extinction learning criteria was based on the decreased percent time that animals spent on the drug side from conditioning to extinction tests. Two-way repeated measures ANOVA revealed significant differences of time (CPP phases: baseline, conditioning, extinction) and groups (morphine and saline); interaction: F(8, 72)= 5.95; p< 0.001; main effects of time: F(2, 72)= 6.39; p< 0.001 and groups: F(4, 36)= 83.43; p< 0.001).

Similar to our previous findings (Martinez-Rivera et al. 2016), animals expressed morphine-CPP as compared to either their baseline (Tukey post hoc; p’s< 0.01) or their respective saline group during the conditioning test (Fig. 1C, Tukey post hoc; p’s< 0.01). Regarding extinction learning, post hoc analyses showed that animals in the morphine/extinction group significantly reduced the preference for the morphine side when compared to the conditioning test, indicating that rats extinguished the preference for the morphine-paired side (Fig. 1C and S1; p= 0.001). In contrast, rats in the morphine/extinction-resistant and morphine/sham-extinction groups retained the preference for the morphine-paired side as compared to their respective saline groups (morphine/extinction-resistant vs. saline-extinction, p= 0.003; morphine/sham-extinction vs. saline/sham-extinction, p= 0.0005). Also, when compared to the morphine/extinction group, both morphine/extinction-resistant (p= 0.001) and morphine/sham-extinction (p= 0.0001) groups retained higher place preference for the morphine-paired side. In fact, the morphine/extinction-resistant and the morphine/sham-extinction rats behaved similarly, when compared to the time spent in the morphine-paired side during the conditioning versus extinction tests, p= 1.00 and 0.99, respectively.

3.2. Extinction of morphine place preference on synaptic plasticity genes in the VS/NAc

To identify specific genes that could be differentially modulated among extinction, extinction-resistant and sham-extinction groups, we used the 2−ΔΔCt method (Livak and Schmittgen 2001; Schmittgen and Livak 2008) to compare the fold change value of 86 genes among morphine-treated groups. A two-way ANOVA that compared genes versus morphine-treated groups revealed a significant main (group) effect (F(2, 1032)= 4.55; p= 0.010). A Tukey post hoc analysis showed that expression of Brain-derived neurotrophic factor (Bdnf) significantly increased in the morphine/extinction group as compared to the morphine/sham-extinction (Fig. 2A, p= 0.0001), suggesting an important role of this gene in extinction learning. In contrast, morphine/sham-extinction animals showed increased upregulation of Nerve growth factor receptor (Ngfr; Fig. 2B) as compared with both morphine/extinction (p= 0.026) and morphine/extinction-resistant (p= 0.004) groups. Also, when one-way ANOVA’s were applied individually to each gene, we found a significant main effect in Calcium/calmodulin-dependent protein kinase II gamma (Camk2g; F(2, 12)= 8.75; p= 0.004), Ephrin type-B receptor 2 (Ephb2; F(2, 12)= 4.40; p= 0.03) and Glutamate receptor metabotropic 8 (Grm8; F(2, 12)= 3.62; p= 0.05). Tukey post hoc analyses showed that Camk2g was upregulated in the morphine/sham-extinction group, as compared to both morphine/extinction and morphine/extinction-resistant groups (Fig. 2C), whereas Ephb2 and Grm8 were upregulated in the morphine/sham-extinction, as compared only with morphine/extinction-resistant (Fig. 2D–E). These results might suggest that in the absence of extinction training, upregulation of particular genes occurs in the VS/NAc for the support of maladaptive drug-seeking behaviors.

Figure 2. Extinction-dependent modulation of synaptic plasticity genes.

Figure 2.

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(A) The morphine/extinction group showed a fold-change increase of Bdnf, as compared to the morphine/sham-extinction group. The morphine/sham-extinction group showed increased expression of Ngfr (B) and Camk2g (C), as compared to both morphine/extinction and morphine/extinction-resistant. Similarly, the lack of extinction training increased the expression of Ephb2 (D) and Grm8 (E), as compared only to morphine/extinction-resistant. *p<0.05; **p<0.01. Morphine/extinction: n= 5; Morphine/extinction-resistant: n= 5; Morphine/sham-extinction: n= 5. Data is shown as mean and SEM.

To identify genes mainly modulated by morphine exposure, animal groups treated with morphine were compared to their saline counterparts. In this regard, when morphine/extinction and saline/extinction groups were compared, three (3) genes were upregulated (Fig. 3A and S2). These were: Bdnf, which confirm our finding that Bdnf has a putative role in extinction learning, Glutamate receptor, ionotropic-AMPA 4 (Gria4) and Reelin (Reln). On the other hand, comparison between morphine/extinction-resistant and saline/extinction groups might reveal genes that promote extinction failure. In this category, we found two (2) upregulated genes (Fig. 3B and S2): cAMP-responsive element modulator (Crem) and Ras homolog enriched in brain (Rheb), while Tumor necrosis factor alpha (Tnfa) was downregulated. When the morphine/sham-extinction and saline/sham-extinction groups were compared, a total of twenty-four (24) genes were modulated (Fig. 3C and S3). Sixteen (16) of these genes were upregulated, whereas eight (8) were downregulated. Interestingly, some of the upregulated genes included: Camk2g, Ephb2, and Grm8, all of them confirming our finding of having a role in the absence of extinction training (see Fig. 2).

Figure 3. Volcano plots for extinction and sham-extinction groups.

Figure 3.

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(A) Morphine/extinction vs. saline-extinction. (B) Morphine/extinction-resistant vs. saline-extinction. (C) Morphine/sham-extinction vs. saline/sham-extinction. The vertical lines correspond to 1-fold up (to the right) and down (to the left), respectively. Horizontal lines represent a p-value of 0.05. Red dots above and to the right of the horizontal and vertical lines, respectively, represent a significant gene upregulation. Green dots above and to the left of the horizontal and vertical lines, respectively, represent a significant gene downregulation. Saline-extinction: n=5; Saline/sham-extinction: n= 4; Morphine/extinction: n= 5; Morphine/extinction-resistant: n= 5; Morphine/sham-extinction: n= 5.

3.3. Extinction of morphine place preference on transcripts for opioid receptors in the VS/NAc

Because opioid receptors (ORs) are highly expressed in the VS/NAc (Lutz and Kieffer 2013), transcript expression of ORs (mu/μ1; MOR, kappa/K1; KOR and delta/Δ1; DOR) was also evaluated (Fig. 4). For this, we compared the same morphine and saline groups (samples) using the 2 −ΔΔCt method (Livak and Schmittgen 2001; Schmittgen and Livak 2008). First, gene expression values were normalized by the reference gene (GAPDH) and by the correspondent saline-treated group. A two-way ANOVA revealed a significant interaction between gene expression and extinction subgroups (F(8, 61)= 15.13; p< 0.001), and a significant main effect of gene (F(2, 61)= 3.49; p= 0.036) and extinction subgroups (F(4, 61)= 35.74; p< 0.001; Fig. 4). Tukey post-hoc analysis revealed an upregulation of MOR’s and DOR’s in morphine/sham-extinction when compared to either the morphine/extinction (MOR: p= 0.0001; DOR: p= 0.0007) or morphine/extinction-resistant groups (MOR: p= 0.0001; DOR: p= 0.003), suggesting that upregulation of these two types of ORs in the VS/NAc might reveal absence of extinction training. Also, in the absence of extinction training, MOR’s and DOR’s were upregulated when morphine and saline groups were compared (morphine/sham-extinction vs. saline/sham-extinction; MOR: p= 0.0001; DOR: p= 0.0007).

Figure 4. Extinction-dependent modulation of gene transcripts for opioid receptors.

Figure 4.

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Animals in the morphine/sham-extinction group showed a fold-change increase in the expression of transcripts for mu (μ1 and delta (Δ1) opioid receptors, as compared to both morphine/extinction and morphine/extinction-resistant. **p< 0.01. Morphine/extinction: n= 5; Morphine/extinction-resistant: n= 5; Morphine/sham-extinction: n= 5. Data is shown as mean and SEM.

4. Discussion

To overcome addiction, extinction-based therapy has been suggested as a non-invasive alternative for drug addicts. However, the effectiveness of this approach is questionable, in part due to recurrent relapsing episodes and the lack of information regarding neurobiological mechanisms of extinction (Hutton-Bedbrook and McNally 2013). To build on previous extinction studies, we profiled synaptic plasticity genes during the process of extinction learning of morphine-CPP. We found different sets of accumbal genes that were grouped based on the capacity of extinction learning (extinction and resistant), whereas another set of genes reflected the presence or absence (sham) of extinction training (Fig. 5). Also, ingenuity pathway analyses revealed specific biological processes/networks and potential up- and down-stream targets associated with these extinction profiles (Fig. 6A–C). In the following section, we will discuss the potential role of the identified genes, according to their respective extinction settings.

Figure 5. Venn diagram summarizing extinction-dependent genes that were modulated in morphine-treated rats.

Figure 5.

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The name and number of genes are shown in each extinction learning phenotype. Morphine/extinction (3 genes: Bdnf, Gria 4, Reln); Morphine/extinction-resistant (3 genes: Crem, Rheb, Tnfa); Morphine/sham-extinction (27 genes-see figure). Shared genes between Morphine/extinction and Morphine/sham-extinction groups are also shown (1 gene; Gria4). ↑= upregulation and ↓= downregulation.

Figure 6. Extinction-dependent network of genes in morphine-treated rats.

Figure 6.

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(A) Network of genes associated in morphine/extinction (score of 9; network associated to behavior, cell-to-cell signaling and interaction and nervous system development and function), (B) morphine/extinction-resistant (score of 9; network associated to cell-mediated immune response, cellular movement, hematological system development and function (C) morphine/sham-extinction groups (score of 25; network associated to behavior, cell-to-cell signaling and interaction, cell morphology, nervous system development and function). All measures were obtained using Ingenuity Pathway Analysis (IPA). Direct interactions are represented as solid lines, whereas indirect interactions appear as dotted lines. Upregulated and downregulated genes are represented by green and red lines, respectively.

4.1. Genes associated with extinction

In our study, Bdnf was found to be the strongest candidate gene associated with the extinction of morphine-CPP. Rats showing extinction of morphine-CPP (lower CPP values at extinction test) revealed a Bdnf significant fold increase (2 −ΔΔCt) over morphine-conditioned animals that underwent sham extinction. Bdnf has been extensively related to extinction learning and LTP (Barker et al. 2015; McGinty et al. 2010). Likewise, increased Bdnf in prefrontal and hippocampal regions have led to extinction facilitation in fear-conditioned rats (Peters et al. 2010; Rosas-Vidal et al. 2014). In addiction studies, BDNF infusion into infralimbic (IL) prefrontal cortex facilitated extinction of cocaine-CPP (Otis et al. 2014), whereas Bdnf upregulation in the BLA was associated with extinction facilitation of morphine-CPP (Wang et al. 2015). Accordingly, morphine-CPP reduced Bdnf expression in the ventral tegmental area (VTA) (Koo et al. 2012). Other studies showed that morphine-CPP was enhanced when the Bdnf receptor (TrkB) was knocked down in medium spiny neurons (MSNs) of the VS/NAc (Koo et al. 2014), and that BDNF infusion into the VS/NAc facilitates extinction of cocaine self-administration (Bobadilla et al. 2018). In the current study, we found upregulation of Bdnf transcripts in the VS/NAc of rats showing extinction of morphine-CPP, suggesting that Bdnf signaling in this brain region could be a key player for extinction of opioid-associated behaviors. Alternatively, because the VS/NAc expresses low Bdnf levels (Conner et al. 1997; Li et al. 2013), it is possible that our results are also due to changes in VS/NAc afferents such as the hippocampus, a brain region typically known for its high levels of Bdnf (Conner et al. 1997; Li et al. 2013). Interestingly, we observed that rats in the morphine/extinction group significantly increased hippocampal BDNF protein expression, as measured by Western blot analysis (see Fig. S4). In addition, it was not surprising to observe upregulation of Gria4 when the morphine/extinction group was compared to its saline counterpart, given that glutamatergic neurotransmission is critical for extinction learning (Millan et al. 2011; Millan and McNally 2011). Reln was another modulated gene in the morphine/extinction group when compared to its saline counterpart. Although its role in opioid addiction remains undetermined, it has been associated with reduced cocaine sensitization (Teixeira et al. 2011). Together, our results suggest that extinction learning activates neuronal BDNFergic circuits in processes of morphine-induced CPP.

4.2. Genes associated with extinction resistance

When compared with their saline counterparts (saline-extinction) rats exhibiting extinction resistant (morphine/extinction-resistant), showed gene modulation in Crem, Rheb and Tnfa. It is noteworthy to point out that animals in the morphine/extinction-resistant group did not show differences to morphine/extinction or morphine/sham-extinction. This result highlights the complexity of a group that could have the potential of carrying genetic markers of extinction resistance.

Although the role of Crem in addiction seems to rely on polymorphisms (Miller et al. 2018), our finding of Crem upregulation is consistent with others showing that exposure to stress, stimulants or morphine withdrawal, induces Crem expression in the reward circuit (Ammon-Treiber and Hollt 2005; McClung and Nestler 2008). As for Rheb upregulation, it is in accordance with results showing that mice lacking Rheb in the striatum decreased morphine analgesia, tolerance and dependence (Lee et al. 2011), suggesting its possible association with opioid-seeking behaviors and extinction resistance. Conversely, our study showed downregulation of Tnfa in extinction-resistant rats. In this regard, others have shown that systemic (Niwa et al. 2007) or intra-accumbal (Wu et al. 2014) Tnfa injections attenuate morphine-CPP and sensitization, respectively. Therefore, in our study, Tnfa modulation in extinction-resistant animals suggests that low-expression levels of this gene might interfere with its role for the facilitation of extinction learning.

4.3. Genes associated with lack of extinction training

In the absence of extinction training, we found six upregulated genes in the morphine/sham-extinction group, as compared to both morphine/extinction and morphine/extinction-resistant groups. Specifically, two opioid receptors: MORs and DORs, and four synaptic plasticity genes: Camk2g, Ngfr, Ephb2 and Grm8, were modulated. Given that opioid receptors mediate the rewarding effects of morphine (Lutz and Kieffer 2013), this finding might suggest that the absence of extinction training activates these two types of opioid receptors for the maintenance of drug-seeking behavior (Billa et al. 2010; Di Chiara and Imperato 1988). Other upregulated genes that might enhance drug-seeking in the absence of extinction training are: Camk2g, Ngfr and Grm8. For instance, morphine-CPP (Narita et al. 2004) and oxycodone self-administration (Zhang et al. 2015) induced Camk2g upregulation. Similarly, Ngfr has been associated with enhanced morphine-CPP, through modulation of delta opioid receptors in the central amygdala (Bie et al. 2012). Regarding Grm8, it is associated with decreased NMDA receptor function (Ambrosini et al. 1995; Lea et al. 2002), which might account for impairment of the extinction of drug-associated cues (Lu et al. 2011). In contrast, expression of Ephb2 in the hippocampus has been associated with decreased opioid tolerance (Huroy et al. 2016).

When compared with their saline counterpart, the morphine/sham-extinction group revealed twenty-four additional genes associated with absence of extinction training (see Figs. 3, 5 and S2). It is noteworthy to mention that fewer modulated genes were found in the VS/NAc of rats that underwent extinction training, as compared to those in the sham-extinction group. This can suggest that high levels of gene activity are actively involved in biological process that could be induced by morphine incubation or withdrawal in the absence of extinction training. In such a way, enhanced drug-craving behaviors typical of withdrawal periods (Koob and Volkow, 2010), might precede modulation of a diverse group of genes associated with the lack of extinction training of drug-associated cues.

For instance, upregulation of Camk2g, Ephb2 and Grm8 confirmed our findings associating these genes with the absence of extinction training when the morphine-treated groups were compared. Similarly, upregulation of other genes that has also been associated with drug-seeking behaviors include Akt1, cannabinoid receptor 1 (Cnr1), Gaba receptors (Gabra5) and metabotropic glutamate receptors (mGluR1). Among the downregulated genes, we found dopamine receptor-2 (Drd2), NMDA glutamate receptor (Grin1) and Map-kinase (Mapk1); all of them associated to counteract drug-seeking behaviors.

4.4. Conclusion

Our results show modulated genes in the VS/NAc that could be associated with synaptic plasticity processes in different morphine-extinction phenotypes. This represents a novel molecular profile for genetic targeting during extinction of drug-associated cues. However, it is important that future studies correlate mRNA levels with protein abundance by measuring protein expression levels and/or pharmacological manipulation. Because rodent models of extinction of drug seeking resembles exposure-based therapies in humans (Myers and Carlezon 2010), it is possible that, when combined with targeted modulation of gene products, it might represent an effective future approach to reduce the symptoms of addiction (Everitt 2014; Myers and Carlezon 2010; Torregrossa and Taylor 2013).

Supplementary Material

1

Highlights.

  • Extinction learning induced Bdnf upregulation in the ventral striatum

  • Extinction resistance was associated with selective gene targets

  • Extinction training counteracts the withdrawal-associated plasticity

  • The lack of extinction training induced a widespread modulation of genes

Acknowledgments

This work was supported by NIGMS R25 GM061838 (to FJM-R and NAM), and NIH/NIMHD Grants G12 RR003051 and G12 MD007600 (to JLB-E). Authors want to thank Ryan D. Oyola-Rivera, Mario E. Lloret-Torres and Maria E. Santiago-Gascot for technical support. We also want to thank Gregory J. Quirk for his mentorship and support as well as to Angélica B. Rolón-Barreto, Kelvin Quiñones-Laracuente and Maria Diehl for reviewing and editing the manuscript.

Footnotes

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Financial disclosures

The authors reported no biomedical financial interests or potential conflicts of interest.

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Supplementary Materials

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