Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2021 Dec 7.
Published in final edited form as: J Neurosci Res. 2020 Jun 7;100(1):35–47. doi: 10.1002/jnr.24640

Dysregulated expression of the alternatively spliced variant mRNAs of the mu opioid receptor gene, OPRM1, in the medial prefrontal cortex of male human heroin abusers and heroin self-administering male rats

Taylor G Brown 1, Jin Xu 1, Yasmin L Hurd 2, Y-X Pan 1
PMCID: PMC8143898  NIHMSID: NIHMS1699817  PMID: 32506472

Abstract

Heroin, a mu agonist, acts through the mu opioid receptor. The mu opioid receptor gene, OPRM1, undergoes extensive alternative splicing, creating an array of splice variants that are conserved from rodent to humans. Increasing evidence suggests that these OPRM1 splice variants are pharmacologically important in mediating various actions of mu opioids, including analgesia, tolerance, physical dependence, rewarding behavior, as well as addiction. In the present study, we examine expression of the OPRM1 splice variant mRNAs in the medial prefrontal cortex (mPFC), one of the major brain regions involved in decision-making and drug-seeking behaviors, of male human heroin abusers and male rats that developed stable heroin seeking behavior using an intravenous heroin self-administration (SA) model. The results show similar expression profiles among multiple OPRM1 splice variants in both human control subjects and saline control rats, illustrating conservation of OPRM1 alternative splicing from rodent to humans. Moreover, the expressions of several OPRM1 splice variant mRNAs were dysregulated in the postmortem mPFCs from heroin abusers compared to the control subjects. Similar patterns were observed in the rat heroin SA model. These finding suggest potential roles of the OPRM1 splice variants in heroin addiction that could be mechanistically explored using the rat heroin SA model.

Keywords: mu opioid receptor, splicing, heroin, OPRM1, medial prefrontal cortex

Introduction

Heroin is a highly addictive drug that has led to the death of numerous people and caused tremendous economic, healthcare and societal costs. The mu opioid receptor gene (OPRM1) is a prominent candidate gene in the underlying neurobiology of heroin addiction. The OPRM1 undergoes extensive alternative splicing, generating multiple splice isoforms or variants that are conserved across species including rodents and humans (Y. X. Pan, 2005; G. W. Pasternak & Pan, 2013). These splice variants can be categorized into three main types based on the predicted receptor structure: 1) Full-length 7 transmembrane (TM) C-terminal variants that are identical except for a different intracellular C-terminal tail due to 3’ splicing; 2) Truncated 6TM variants that lack the first TM due to 5’ splicing; and 3) Truncated single TM variants containing only the first TM due to exon skipping or insertion (G. W. Pasternak & Pan, 2013).

Increasing evidence supports the pharmacological importance of these splice variants. As demonstrated by several in vitro studies, functional differences relate to mu agonist-induced G protein coupling, phosphorylation, internalization and post-endocytic sorting, as well as region- and cell-specific expression for the full-length 7TM C-terminal variants (Abbadie, Gultekin, & Pasternak, 2000; Abbadie, Pan, Drake, & Pasternak, 2000; Abbadie, Pan, & Pasternak, 2000; Abbadie & Pasternak, 2001; Abbadie, Pasternak, & Aicher, 2001; Bolan, Pan, & Pasternak, 2004; Caron et al., 2000; Koch et al., 1998; L. Pan et al., 2005; Y.-X. Pan et al., 2001; Y. X. Pan et al., 1999; D. A. Pasternak et al., 2004; Xu, Lu, Xu, Rossi, et al., 2014). Heterodimeric interaction of the C-terminal tail of mMOR-1D with a gastrin-releasing peptide receptor has also been implicated in morphine-induced itch (X. Y. Liu et al., 2011). Importantly, in vivo functions of several C-terminal variants were recently revealed in C-terminal truncation mouse models with two inbred mouse background (Xu et al., 2017). Particularly, exon 7 (E7)-associated C-terminal truncation in C57BL/6J strain (mE7M-B6) diminished morphine tolerance and reward without altering physical dependence, whereas the E4-associated C-terminal truncation (mE4M-B6) accelerated morphine tolerance and reduced morphine dependence without affecting morphine reward (Puig & Gutstein, 2017; Xu et al., 2017), highlighting the importance of the C-terminal sequences in morphine actions.

The functional relevance of the truncated 6TM variants has also been implicated in the actions of a subset of mu opioid agonists such as heroin (Y.X. Pan et al., 2009; Schuller et al., 1999), as well as a novel class of opioid analgesic such as 3’-iodobenzoyl-6β-naltrexamide (IBNtxA) that are potent against a broad spectrum of pain models without many side-effects associated with traditional opiates(Lu et al., 2015; Lu et al., 2018; Majumdar et al., 2011; Wieskopf et al., 2014). Additionally, in regard to the truncated single TM variants, although unable to bind opioids, they can facilitate expression of the 7TM MOR-1 receptor as a molecular chaperone to enhance morphine analgesia (Xu et al., 2013).

The OPRM1 gene is expressed throughout the brain including the medial prefrontal cortex (mPFC) which is highly involved in decision-making and drug-seeking behaviors that is dysregulated by many abused drugs including heroin (Bossert et al., 2012; Goldstein & Volkow, 2011; Koob & Volkow, 2010; Van den Oever, Spijker, Smit, & De Vries, 2010). Specifically, the mPFC has been documented to be a critical anatomical site for regulating the transition of goal-directed behaviors to habits and for inhibiting impulsive behaviors (Smith & Graybiel, 2013; Smith, Virkud, Deisseroth, & Graybiel, 2012). Intriguingly, forms of heroin relapse such as context-induced reinstatement specifically rely on the mPFC as a switch to promote heroin seeking, which is not seen for cocaine seeking behavior (Bossert et al., 2012; Peters, Pattij, & De Vries, 2013; Rogers, Ghee, & See, 2008). Thus, the mPFC appears to be an important neuroanatomical region relevant to the pathophysiology of heroin abuse. In the present study, we investigated expression of the OPRM1 splice variant mRNAs in the mPFC of human heroin abusers and rats that developed stable heroin seeking behavior using the intravenous heroin self-administration (SA) model. The human and rat OPRM1 genes share many similarities (Figs 1A & 1B and Table 1) so in addition to their gene structure including exon organization, intron size and promoters, their alternative splicing patterns and the resulted splice variants are comparable. Our results showed varied expression levels among the OPRM1 splice variants in the human postmortem mPFC, which was similar to those in the rats, further illustrating conservation of OPRM1 alternative splicing from rodent to humans. Furthermore, the expression of several OPRM1 splice variant mRNAs was dysregulated in the postmortem mPFCs from heroin abusers compared to the control subjects. Similar patterns were observed in the rat heroin SA model. Together, these results suggest the possible involvement of the OPRM1 splice variants in heroin addiction and provide a foundation for such mechanistic studies using the rat heroin SA model.

Fig. 1.

Fig. 1.

Open in a new tab

Schematic of the OPRM1 gene structure and alternative splicing

A. The human OPRM1 gene structure and alternatively spliced variants. Top panel: the human OPRM1 gene structure. Exons and introns are indicated by boxes and horizontal lines, respectively. Intron size is indicated below the introns as kilobases (kb). Promoters are showed by arrows. Exons are numbered based upon the published data. Bottom panel: Alternatively spliced variants. For each variant, exons are joined by tilted lines. Translation start and stop points are shown by bars below and above exon boxes, respectively. The underlined variants with purple letters are studied in the present study. The predicted protein structures of three variant types are shown by inserted cartoons.

B. The rat Oprm1 gene structure and alternatively spliced variants. Top panel: the rat Oprm1 gene structure. Exons and introns are indicated by boxes and horizontal lines, respectively. Intron size is indicated below the introns as kilobases (kb). Promoters are showed by arrows. Exons are numbered based upon the published data. Bottom panel: Alternatively spliced variants. For each variant, exons are joined by tilted lines. Translation start and stop points are shown by bars below and above exon boxes, respectively. The underlined variants with purple letters are studied in the present study. The predicted protein structures of three variant types are shown by inserted cartoons.

Table 1.

The human and rat OPRM1 splice variants and their exon composition

Variant type Species Variant name Exon composition
7TM C-terminal variants Human hMOR-1 1a/2/3/4
hMOR-1A 1a/2/3ab
hMOR-1B1 1a/2/3/5a
hMOR-1B2 1a/2/3/5ba
hMOR-1B3 1a/2/3/5cba
hMOR-1B4 1a/2/3/5dcba
hMOR-1B5 1a/2/3/5edcba
hMOR-1O 1a/2/3/O
hMOR-1X 1a/2/3/X
hMOR-1Y 1a/2/3/Y/5cba
hMOR-1H 11ab/1a/2/3/4
hMOR-1i 11ab/1c/1a/2/3/5
Rat rMOR-1 1a/2/3/4
rMOR-1A 1a/2/3ab
rMOR-1B1 1a/2/3/5a
rMOR-1B2 1a/2/3/5ba
rMOR-1C1 1a/2/3/7/8/9a
rMOR-1C2 1a/2/3/7/8/9ba
rMOR-1D 1a/2/3/8/9
rMOR-1P 1a/2/3/15
rMOR-1H1 11ab/1a/2/3/4
rMOR-1H2 11a/1a/2/3/4
rMOR-1i1 11ab/1ba/2/3/4
rMOR-1i2 11a/1ba/2/3/4
rMOR-1i3 11a/1cba/2/3/4
6TM variants Human hMOR-1G1 11ab/2/3/4
hMOR-1G2 11a/2/3/4
hMOR-1K 13/2/3/4
mu3 2/3/4/4a
Rat rMOR-1G1 11ab/2/3/4
rMOR-1G2 11a/2/3/4
1TM variants Human hMOR-1S 1a/4
hMOR-1Z 1a/3/4
SV1 1a/SV-A/2
SV2 1a/SV-AB/2
Rat rMOR-1S 1a/4
rMOR-1Z 1a/3/4
Open in a new tab

Materials and Methods

Animals

All rats were maintained on a 12-hour light/12-hour dark cycle and given ad libitum access to food and water. All animal studies were approved by the Institutional Animal Care and Use Committee (IACUC) of Memorial Sloan-Kettering Cancer Center and Icahn School of Medicine at Mount Sinai.

Rat heroin self-administration (SA) model

A heroin SA paradigm was performed in adult male Long Evans rats (Charles River, Wilmington, MA) as previously described(Ellgren, Spano, & Hurd, 2007; Sillivan et al., 2013; Tomasiewicz et al., 2012). In this study, only male rats were used to be comparable to the human mPFC samples where specimens were only available from male subjects. Briefly, following jugular catheter implantation and recovery, adult Long Even male rats were trained in operant chambers (MED Associates Inc., St. Albans, VT) with two levers; depression of one (the active lever) resulted in the delivery of 30 ug/kg heroin under a fixed-ratio 1 (FR1) schedule of reinforcement, whereas depression of the other (the inactive lever) had no programmed consequences. Rats were given 3 h daily access to IV heroin (30μg/kg/infusion) (n=5) or Saline (n=7) for 10 days. The medial prefrontal cortex (mPFC) was dissected using a 2mm micro-punch 24 h after the last SA session by CO2 followed by decapitation and brains frozen.

Human brain specimens

Fresh frozen human postmortem mPFC samples were obtained from a postmortem collection of Caucasian Swedish and Hungarian subjects processed under conditions that have been previously described (Drakenberg et al., 2006; Jacobs et al., 2013). All the specimens were only available from male subjects. Briefly, these specimens were collected at autopsy within 24 hours after the time of death at the National Institute of Forensic Medicine, Karolinska Institute, Stockholm, Sweden, as well as from the Department of Forensic Medicine at Semmelweis University (Budapest, Hungary) under guidelines approved by the local ethics committees. Tissue punches of the mPFC were taken from 9 male heroin abusers (25.78 ± 0.89 years old, 6.62 brain pH, 7.58 RIN) who died from overdose and10 male control subjects (29.18 ± 2.73 years old, 6.67 ± 0.07 brain pH, 7.71 RIN), who died from myocardial infarction and had negative toxicology for psychoactive substances or a history of psychiatric illness. All subjects were negative for alcohol toxicology.

Reverse transcription and quantitative polymerase chain reaction (RT-qPCR).

Total RNAs were isolated from the human or rat mPFC using miRNeasy Kit (Qiagen) with on-column DNase I treatment and used in RT with Superscript III (Invitrogen) and random primers, as described previously (Xu et al., 2015; Xu, Lu, Xu, Rossi, et al., 2014; Xu et al., 2013). The first-strand cDNAs were used as templates in SYBR green quantitative PCR (qPCR) using HotStart-IT SYBR Green qPCR Master Mix (Affymetrix) and CFX96 Real-Time PCR System (Bio-Rad) to amplify OPRM1 splice variants, as described previously (Xu et al., 2015; Xu, Lu, Xu, Rossi, et al., 2014). Three reference genes for the human samples, succinate dehydrogenase subunit A (SDHA), β2-microglobulin (B2M) and glyceraldehyde 3-phosphate dehydrogenase (G3PDH), and two reference genes for the rat samples, G3PDH and 18S ribosome (18S), were used to calculate normalization factors to obtain ΔC(t) values, where ΔC(t) = C(t)variant – C(t)NF (NF (normalized factor) = (C(t)G3PDH x C(t)SDHA x C(t)β2M)1/3) (Xu, Lu, Xu, Rossi, et al., 2014). We used negative ΔC(t) values (-ΔC(t)) calculated through ΔC(t)/(−1) formula to indicate expression levels. Converting ΔC(t) values to negative ones is commonly used in qPCR to allow visualizing the data in a positive correlation with the expression levels since ΔC(t) values are inversely proportionated with expression levels. mE1/2 amplified with primers from exon 1 to exon 2 represented all 7TM C-terminal variants, including the original mMOR-1. All primers and PCR conditions are listed in Table S1.

Statistics.

The human postmortem and rat tissue samples were not randomized or blinded when experiments were performed. All statistical analysis was carried out using GraphPad Prism 8. A one-way ANOVA or two-way ANOVA was performed with uncorrected Fisher least significant difference (LSD) as described in the figure legend. Data represented the means ± SEM of indicated sample size. Statistical significance was set at p < 0.05.

Results

Dysregulated expression of OPRM1 splice variant mRNAs in the medial prefrontal cortex of human heroin abusers

To examine expression of OPRM1 splice variant mRNAs in the postmortem mPFC, we used a RT-SYBR Green qPCR approach with primers specific for each splice variants. Given that the original human MOR-1 exon composition (exons 1/2/3/4) is shared with other variants including hMOR-1H and hMOR-1i (Fig. 1A), we used primers in exons 1 and 2 (hE1–2) to amplify entire full-length 7TM variants. We observed that various OPRM1 variants were differentially expressed in the mPFC of normal control subjects (Figs. 2A & 3A and Tables 2 & S2). The expression levels of several variants, such as hMOR-1i, hMOR-1X and hMOR-1B1, were varable among the individual subjects, probably due to varied mRNA stabilities for these variants. Several full-length 7TM C-terminal variants, including hMOR-1B2, hMOR-1O and hMOR-1Y, were expressed at high level, while the expression levels of other variants, such as hMOR-1B1, hMOR-1B5 and hMOR-1i, were quite low. hMOR-1A, hMOR-1X and hMOR-1H had moderate expression level. Both 6TM variants, hMOR-1G1 and hMOR-1G2, had moderate expression, while single TM variants, particularly hMOR-1Z, were lowly expressed. The lower level of hMOR-1Z may be due to nonsense-mediated degradation that targets mRNAs with a stop codon located more than 50 nucleotides upstream of the last exon-exon junction that is served as a pre-mature stop codon (Chang, Imam, & Wilkinson, 2007; Lejeune & Maquat, 2005).

Fig. 2.

Fig. 2.

Open in a new tab

Expression of the OPRM1 alternatively spliced variants in the mPFC of normal human control subjects and saline control rats

A. Expression of the OPRM1 alternatively spliced variants in the mPFC of normal human control subjects. RNAs isolated from the mPFC of normal subjects (n = 10) were used for SYBR Green qPCR. hE1–2 represents all the 7TM C-terminal variants. Expression levels were indicated by -ΔC(t) values calculated through ΔC(t)/(−1), where ΔC(t) = C(t)variant – C(t)NF (NF (Normalized Factor) = (C(t)G3PDH x C(t)SDHA x C(t)β2M)1/3). Converting ΔC(t) values to negative ones through ΔC(t)/(−1) formula is commonly used in qPCR to allow visualizing the data in a positive correlation with the expression levels since ΔC(t) values are inversely proportionated with expression levels. Significant difference was calculated by one-way ANOVA with Fisher LSD, F(13,125) = 74.33, overall p < 0.0001. The results of the multiple comparisons are listed in Table S2.

B. Expression of the OPRM1 alternatively spliced variants in the mPFC of saline control rats (n = 7). rE1–2 represents all the 7TM C-terminal variants. Expression levels were calculated through (ΔC(t)/(−1)), where ΔC(t) = C(t)variant – C(t)NF (NF (Normalized Factor) = (C(t)G3PDH x C(t)18S)1/2). Significant difference was calculated by one-way ANOVA with Fisher LSD, F(12,78) = 125.9, overall p < 0.0001. The results of the multiple comparisons are listed in Table S3.

Fig. 3.

Fig. 3.

Open in a new tab

Heatmap of the expression of the OPRM1 splice variant mRNAs in the mPFC of human heroin abusers and heroin self-administering rats

A. Heatmap of expression of the human OPRM1 splice variant mRNAs in the postmortem mPFC of normal control subjects and heroin abusers. Heatmap was generated using the Heat Map graph of GraphPad 8 from ΔC(t)/(−1) values, where ΔC(t) = C(t)variant – C(t)NF (NF (Normalized Factor) = (C(t)G3PDH x C(t)SDHA x C(t)β2M)1/3). Expression level was indicated by ΔC(t)/(−1) values in order from highest (most dark blue) to lowest (most light blue). ↑ and ↓: significantly increased or decreased expression as compared to control subjects, respectively.

B. Heatmap of expression of the rat Oprm1 splice variant mRNAs in the mPFC of saline control and heroin self-administration rats. Heatmap was generated using the Heat Map graph of GraphPad 8 from ΔC(t)/(−1) values, where ΔC(t) = C(t)variant – C(t)NF (NF (Normalized Factor) = (C(t)G3PDH x C(t)18S)1/2). Expression level was indicated by ΔC(t)/(−1) values in order from highest (most dark blue) to lowest (most light blue). ↑ and ↓: significantly increased or decreased expression as compared to saline control rats, respectively.

Table 2.

Expression of the human OPRM1 alternatively spliced variants in the postmortem medial prefrontal cortex of normal control subjects and heroin abusers.

Human OPRM1 splice variant category Variants ΔC(t) values#
Control subjects Heroin abusers
7TM variants hE1–2 3.31 ± 0.12 3.24 ± 0.18
hMOR-1A 6.95 ± 0.21 8.36 ± 0.56** ↓
hMOR-1B1 9.00 ± 0.53 9.52 ± 0.41
hMOR-1B2 4.44 ± 0.17 6.66 ± 0.25**** ↓
hMOR-1B5 9.61 ± 0.25 10.26 ± 0.38
hMOR-1O 4.69 ± 0.21 4.30 ± 0.12
hMOR-1X 7.38 ± 0.47 5.41 ± 0.18**** ↑
hMOR-1Y 4.48 ± 0.22 4.23 ± 0.19
hMOR-1H 6.59 ± 0.18 5.68 ± 0.28* ↑
hMOR-1i 11.17 ± 0.72 11.24 ± 0.53
6TM variants hMOR-1G1 6.41 ± 0.24 6.00 ± 0.25
hMOR-1G2 5.63 ± 0.19 4.70 ± 0.25* ↑
1TM variants hMOR-1S 9.69 ± 0.24 9.64 ± 0.16
hMOR-1Z 12.58 ± 0.19 12.16 ± 0.33
Open in a new tab
#:

ΔC(t) = C(t)variant – C(t)NF, where NF (Normalized Factor) = (C(t)G3PDH x C(t)SDHA x C(t)β2M)1/3; ↑ & ↓: increased or decreased expression as compared to control subjects, respectively; Two-Way ANOVA with uncorrected Fisher LSD showed a statistically significant effect among the variants, F(13,234) = 153.0, p < 0.0001, between normal control subjects and heroin abusers, F(1,234) = 0.1670, p = 0.6832, and interaction, F(13,234) = 5.129, p < 0.0001. Compared to Control subjects

*:

p < 0.05

**:

p <0.01

****:

p < 0.0001.

In comparing heroin to control subjects, we observed differences in the expression of several OPRM1 variants in the mPFC of heroin abusers. The overall expression levels of the variants in both normal control subjects and heroin abusers are showed in a heatmap made from ΔC(t)/(−1) values, plotting from the highest level (left, dark blue) to the lowest level (right, lightest blue) based on the data of normal control subjects (Fig. 3A). The heatmap displays the wide range of expression levels among the variants in not only normal control subjects but also heroin abusers, from −12 to −3 in ΔC(t)/(−1) values. In 7TM variants, the expression levels of hE1–2 representing all 7TM variants and several 7TM variants including hMOR-1B1, hMOR-1B5, hMOR-1O, hMOR-1Y and hMOR-1i were not changed in heroin abusers, compared to control subjects. However, the expression levels of four 7TM variants were significantly altered in heroin abusers, including increased expression of hMOR-1X and hMOR-1H and decreased expression of hMOR-1A and hMOR-1B2 (Fig. 3A & Table 2). The expression level of hMOR-1G2, a 6TM variant, was also significantly increased in heroin abuser (Fig. 3A & Table 2). Both single TM variants, hMOR-1S and hMOR-1Z, were not changed.

To further visualize expression level patterns, we subsequently quantified the altered expression by normalizing the 2-ΔC(t) values of heroin abusers with those of normal control subjects; expression levels of normal control subjects are provided as 100% (Fig. 4A). Compared to normal control subjects, heroin abusers clearly showed altered expression of several variants, such as hMOR-1B2, hMOR-1G2, hMOR-1H1 and hMOR-1X (Fig. 4A). hMOR-1A and hMOR-1B1 tended to be reduced, but the decrease was not statistically significant. Together, these results suggest dysregulation of OPRM1 alternative splicing in the mPFC of heroin abusers. It should be noted that the statistical significances shown in Fig. 4A were a little different from those in Fig. 3A. For example, hMOR-1A level in heroin abusers was significantly lower than that in control subjects when ΔC(t)/(−1) was used in Fig. 3A and Table 2. However, there was no statistical significance when the normalized 2-ΔC(t) values were use in Fig. 4A. The degree of the statistical differences was also different. For example, the p values for hMOR-1H and hMOR-1G2 levels between control subjects and heroin abusers were < 0.001 and < 0.01, respectively, in Fig. 4A, while their p values in Fig. 3A were < 0.05. These differences are due to the nature of the data used, logarithimic in ΔC(t) vs linear in 2-ΔC(t). Both data formulas are commonly used in qPCR analysis. However, we mainly use ΔC(t)/(−1) data in Fig. 3A and Table 2 to draw our conclusions.

Fig. 4.

Fig. 4.

Open in a new tab

Altered expression of the OPRM1 splice variant mRNAs in the mPFC of human heroin abusers and heroin self-administering rats

A. Altered expression of the human OPRM1 splice variant mRNAs in the postmortem mPFC of normal control subjects and heroin abusers. RNAs isolated from the mPFC of normal subjects (n = 10) and heroin abusers (n = 9) were used for SYBR Green qPCR. Expression level of heroin abusers was normalized with that of normal control subjects by using their 2-ΔC(t) values, so that the expression level in normal control subjects is, as 100%. Two-Way ANOVA with uncorrected Fisher LSD showed a statistically significant effect among the variants, F(13,234) = 3.865, p < 0.0001, between normal control subjects and heroin abusers, F(1,234) = 4.943, p = 0.0272, and interaction, F(13,234) = 3.865, p < 0.0001. Compared to Control subjects, *: p < 0.05; **: p <0.01; ***: p < 0.001; ****: p < 0.0001.

B. Altered expression of the rat Oprm1 splice variant mRNAs in the mPFC of saline control and heroin self-administration rats. RNAs isolated from the mPFC of saline control rats (n = 7) and heroin self-administered rats (n = 5) were used for SYBR Green qPCR. Expression level of heroin self-administered rats was normalized with that of saline control rats by using their 2-ΔC(t) values, so that the expression level in saline control rats is, as always, 100%. Two-Way ANOVA with uncorrected Fisher LSD showed a statistically significant effect among the variants, F(12,129) = 3.217, p = 0.0005, between saline control and heroin self-administered rats, F(1,129) = 2.14e-007, p = 0.9996, and interaction, F(12,129) = 3.217, p < 0.0005. Compared to saline control rats, *: p < 0.05; ***: p < 0.001.

Dysregulated expression of Oprm1 alternative splice variant mRNAs in the medial prefrontal cortex of heroin-self administering rats

Given that OPRM1 alternative splicing has been shown to be conserved from rodents to humans (Y. X. Pan, 2005; G. W. Pasternak & Pan, 2013), we next examined whether expression of Oprm1 splice variants in the mPFC observed in the human heroin abusers could be recapitulated in rats that self-administered heroin. Similar to hE1–2, rE1–2 amplification using primers from exons 1 and 2 represented all full-length 7TM variants and was expressed at the highest level in the mPFC (Figs 2B & 3B).

A differential expression pattern was also observed for the rat Oprm1 splice variants in the mPFC of Saline control rats, consistent with the control human subjects (Figs. 2B & 3B and Tables 3 & S3). The expression patterns of several rat variants including rMOR-1A, rMOR-1H1, rMOR-1i, rMOR-1G1, rMOR-1S and rMOR-1Z, in the saline control rats were similar to their homologs in human normal subjects, in line with the conservation of OPRM1 alternative splicing between rat and human (Figs. 2A & 2B and Tables 2 & 3). However, there were also noted differences for some variants such as rMOR-1B1 and rMOR-1G2. For example, the rat rMOR-1G2 expression was much lower than rMOR-1G1 (Figs. 2B & 3B and Tables 3 & S3), whereas expression of the human hMOR-G1 and hMOR-1G2 was similar at relatively high levels when compared to hE1–2 (Fig. 2A). Additionally, rMOR-1B1 expression was relatively high when compared to rE1–2 (Fig. 2B and Table 3), while hMOR-1B1 expression was much lower relative to hE1–2 (Fig. 2A). These results suggest differences for certain OPRM1 variants between rat and human.

Table 3.

Expression of the human Oprm1 splice variant mRNAs in the medial prefrontal cortex of saline control and heroin self-administration (SA) rats.

Rat OPRM1 splice variant category Variants ΔC(t) values#
Saline control Heroin SA
7TM variants rE1–2 7.48 ± 0.37 7.77 ± 0.25
rMOR-1A 9.91 ± 0.28 12.43 ± 0.39*** ↓
rMOR-1B1 8.59 ± 0.17 9.71 ± 0.39* ↓
rMOR-1C1 15.27 ± 0.17 15.81 ± 0.15
rMOR-1C2 15.31 ± 0.12 15.20 ± 0.34
rMOR-1D 16.52 ± 0.40 15.97 ± 0.34
rMOR-1H1 8.37 ± 0.31 8.63 ± 0.24
rMOR-1H2 13.11 ± 0.33 13.12 ± 0.12
rMOR-1i 13.21 ± 0.21 13.02 ± 0.48
6TM variants rMOR-1G1 10.28 ± 0.36 8.76 ± 0.49** ↑
rMOR-1G2 15.35 ± 0.45 16.05 ± 0.66
1TM variants rMOR-1S 10.83 ± 0.34 11.12 ± 0.22
rMOR-1Z 17.10 ± 0.12 17.11 ± 0.17
Open in a new tab
#:

ΔC(t) = C(t)variant – C(t)NF, where NF (Normalized Factor) = (C(t)G3PDH x C(t)18S)1/2; ↑ & ↓: increased or decreased expression as compared to saline control rats, respectively; Two-Way ANOVA with uncorrected Fisher LSD showed a statistically significant effect among the variants, F(12, 129) = 194.3, p < 0.0001, between normal control subjects and heroin abusers, F(1, 129) = 4.034, p = 0.0467, and interaction, F(12, 129) = 4.005, p < 0.0001. Compared to Control subjects

*:

p < 0.05

**:

p <0.01

***:

p < 0.001.

In comparing the mPFC of saline and heroin SA rats, a pattern similar to the human OPRM1 splice variants was evident where the rat Oprm1 variants displayed a broad range of expression patterns from −17 to −7 in ΔC(t)/(−1) values in both the saline and heroin self-administration (SA) groups (Fig. 3B). Compared to the saline group, three rat Oprm1 variants, rMOR-1A, rMOR-1B1 and rMOR-1G1, had altered expressions in heroin-SA rats, while other variants were not significantly changed (Fig. 3B). rMOR-1A expression in heroin-SA rats was much lower than that in saline control rats. A similar reduction was seen for hMOR-1A in heroin abusers. rMOR-1B1 was also reduced in heroin-SA rats, similar to hMOR-1B2 in heroin abusers. The expression of rMOR-1G1, a 6TM variant, was greatly upregulated in the mPFC of heroin-SA rats, a similar scenario seen in hMOR-1G2 expression in the mPFC of heroin abusers. The plot by using normalized 2-ΔC(t) values in Fig. 4B showed similar results to those by using ΔC(t)/(−1) in Fig. 3B with some discrepancies in statistical analysis, a similar observation seen in the human variants (Figs. 3A & 4A). For example, rMOR-1B1 expression in heroin-SA rats was significantly decreased as compared to saline control rats when ΔC(t)/(−1) was used in Fig. 3B and Table 3. However, there was no statistical significance when the normalized 2-ΔC(t) values were use in Fig. 4B. As stated above, these differences are due to the nature of the data used, logarithimic in ΔC(t) vs linear in 2-ΔC(t) and we use ΔC(t)/(−1) data in Fig. 3B and Table 3 for making our conclusion.

Discussion

The present study reveals for the first time differential expression of OPRM1 splice variants in the mPFC of both human and rat, confirming that alternative splicing of the Oprm1 detected in the mouse brain (Xu, Lu, Xu, Rossi, et al., 2014) is indeed conserved in humans. Also, we show altered expressions of several OPRM1 splice variants in heroin abusers as compared to control subjects, suggesting that the dysregulation of OPRM1 alternative splicing in human is correlated with heroin exposure. Moreover, we demonstrate that these changes are due to heroin since rats that self-administered heroin had similar expression alterations of the Oprm1 splice variants as human heroin abusers. These findings further support the conservation of the OPRM1 alternative splicing in the mPFC between rat and human.

In the current study, we examined the expression of 13 human OPRM1 variants and 12 rat variants (Figs. 1–3). Of 9 variants shared by both human and rat, including hMOR-1A/rMOR-1A, hMOR-1B1/rMOR-1B1, hMOR-1H/rMOR-1H1 or rMOR-1H2, hMOR-1i/rMOR-1i1, hMOR-1O/rMOR-1C1 or rMOR-1C2 (human exon O is a homolog of rat exon 7), hMOR-1G1/rMOR-1G1, hMOR-1G2/rMOR-1G2, hMOR-1S/rMOR-1S and hMOR-1Z/rMOR-1Z, we observed similar expression among 6 variants, including hMOR-1A/rMOR-1A, hMOR-1H/rMOR-1H1, hMOR-1i/rMOR-1i1, hMOR-1G1/rMOR-1G1, hMOR-1S/ rMOR-1S and hMOR-1Z/rMOR-1Z, while other 3 variants, including hMOR-1B1/rMOR-1B1, hMOR-1O/rMOR-1C1 or rMOR-1C2 and hMOR-1G2/rMOR-1G2, had divergent expression patterns between human and rat (Figs. 2 & 3 and Tables 2 & 3).

Furthermore, the expression of the Oprm1 variants in heroin-SA rats shared several similarities with those in human heroin abusers. First, the decreased expression of rMOR-1A in heroin SA rats matched that of hMOR-1A in heroin abusers. Second, the increased expression of rMOR-1G1 in heroin SA rats was similar to that of hMOR-1G2 in heroin abusers. Both human and rat MOR-1G1 and MOR-1G2 are generated by the same splicing mechanisms except for two alternative 5’ donor sites in exon 11 that define exons 11a and 11b in MOR-1G1 and MOR-1G2, respectively (Fig. 1). Both MOR-1G1 and MOR-1G2 predict a 6TM receptor and an increase of either rMOR-1G1 or hMOR-1G2 leads to an increase of the 6TM variant. Third, the decreased expression of rMOR-1B1 in heroin SA rats was similar to that of hMOR-1B2 in heroin abusers. The trend decrease of hMOR-1B1 also mimicked the decreased rMOR-1B1, although it was not statistically significant due to the large variations among the subjects. The rat Oprm1 has two MOR-1Bs, including rMOR-1B1 and rMOR-1B2, which are homologs of hMOR-1B1 and hMOR-1B2 in human, while the human OPRM1 has additional 3 MOR-1Bs (Fig. 1). These MOR-1Bs result from alternative splicing through different 3’ acceptor sites in exon 5, which is conserved between rat and human. Both rMOR-1B1 and hMOR-1B2 are exon 5-associated C-terminal variants. Finally, the expressions of several variants, including hMOR-1O/rMOR-1C1 or rMOR-1C2, hMOR-1i/rMOR-1i1, hMOR-1S/rMOR-1S and hMOR-1Z/rMOR-1Z were not changed in both heroin-SA rats and heroin abusers.

On the other hand, there were some differences between rat and human.. In addition to the differences in expression of the OPRM1 variants in normal controls mentioned above, the unchanged expression of rMOR-1H1 in heroin SA rats did not resemble the increased expression of hMOR-1H in heroin abusers. Despite these differences, the findings highlight that the rat heroin SA model does mimic molecular alterations evident in human heroin abusers and thus is an important tool to explore the neurobiological mechanisms underlying heroin addiction.

Several of the current findings raise questions regarding the potential roles of the altered OPRM1 splice variant expression in heroin addiction. For example, does the altered OPRM1 variant expression facilitate the rewarding properties of heroin that can accelerate acquisition or maintain the cycle of abuse? The actions of heroin are primarily mediated through mu opioid receptor. While there exist multiple OPRM1 full-length 7TM C-terminal splice variants that share the same binding pocket (G. W. Pasternak & Pan, 2013), they have different intracellular C-termini generated by alternative 3’ splicing. Presumably, heroin binds to these 7TM C-terminal variants with similar affinity, raising questions regarding the roles of these 7TM C-terminal variants in heroin addiction. We previously demonstrated that exon 7-associated variants had biased signaling toward β-arrestin2 when compared to G protein coupling and truncation of the C-termini encoded by exon 7 in C57BL/6J mice led to reduced morphine reward, as determined by conditioned place preference (Xu et al., 2017). In our current study, we did not observe significant changes of a human exon O (human exon 7 homolog)-associated variant, hMOR-1O, and two rat exon 7-associated variants, rMOR-1C1 and rMOR-1C2 in the mPFC, raising questions whether their expression levels are altered in other brain regions relevant to heroin abuse. Yet, we observed significant changes in other 7TM variants, including hMOR-1B2, hMOR-1X, hMOR-1H1, and rMOR-1A. It will be important to further explore the role of exon 7-associated 7TM variants or other 7TM variants in heroin addiction-related behaviors using the rat model.

Other OPRM1 variants are also of interest to explore in future studies. For example, our early studies showed that exon 11-associated 6TM variants involved heroin analgesia (Ying Xian Pan et al., 2009). Disruption of all the 6TM variants in an exon 11-KO mouse significantly reduced heroin analgesia. Although analgesic action is distinct from reward, the increased expression of a human 6TM variant, hMOR-1G2, and a rat 6TM variant, rMOR-1G1, raises intriguing question regarding the role of these 6TM variants in heroin reward and addiction.

Two distinct promoters, exon 1 and exon 11 promoters, control expressions of all the OPRM1 splice variants. The expression levels of both hE1–2 and rE1–2 were not significantly changed, suggesting that the overall transcription level by exon 1 promoter is not altered. The transcription controlled by exon 11 promoter seems also not altered since several exon 11-associated variants, including hMOR-1i and hMOR-1G1 in human and rMOR-1H1, rMOR-1H2, rMOR-1i and rMOR-1G2 in rat, had no changes in their expression. However, altered expressions with either decreasing or increasing in several individual splice variants, as noted above, suggest alterations in alternative splicing mechanisms. It will be interesting to further explore the mechanisms underlying the altered alternative splicing.

There are several limitations and points of consideration in the interpretation of the current study. First, similar to most neurobiological studies, we used bulk tissue from the mPFC, which reflects a heterogenous pool of different types of neurons and other cells such as microglial. Therefore, we lack the anatomical resolution to provide insights as to OPRM1 variants in specific cell populations. Future studies using single cell molecular strategies (e.g., qPCR or single cell sequencing) may provide a more complete assessment of variant expression. Also, the current study was conducted only using samples from the mPFC. It will be interesting to examine OPRM1 expression in other mesocorticolimbic brain regions highly implicated in addiction such as the nucleus accumbens and ventral tegmental area to determine whether there is brain-region specificity of the variants altered in human heroin abusers. Additionally, the use of the rat heroin SA model will allow the possibility to follow the time course of the OPRM1 variant alterations following acute use, various stages of long-term and abstinence periods and during drug-seeking conditions. Finally, the current study used the mPFC samples only from male subjects or male rats mainly due to the limitation of the human postmortem brain availability. Sex differences were observed in expression of Oprm1 splice variants and various opioid responses such as analgesia and addiction(Becker & Chartoff, 2019; A. Liu et al., 2018; Mogil & Bailey, 2010; Verzillo, Madia, Liu, Chakrabarti, & Gintzler, 2014). It will be interesting to explore if sex differences exist in the expression of OPRM1 splice variants in the mPFC of normal human subjects and saline control rats, as well as heroin abusers and heroin-SA rats if the samples are available.

Individual genetics may also contribute to differences in OPRM1 variants. It has been shown that a single nucleotide polymorphism (A118G) of the human OPRM1 gene is associated with heroin addiction in some human populations (Bond et al., 1998; Drakenberg et al., 2006). In an A112G mouse model that mimics human A118G, the expression of mu opioid receptor in some brain regions was reduced (Wang, Huang, Ung, Blendy, & Liu-Chen, 2012), and DAMGO-stimulated [35S]GTPγS binding in the ventral tegmental area was decreased (Wang, Huang, Blendy, & Liu-Chen, 2014). Furthermore, heroin self-administration behavior was also altered in the A112G mice (Zhang et al., 2015). A genetic study revealed association of an intronic single nucleotide polymorphism with the severity of heroin intake in male Chinese (Xu, Lu, Xu, Pan, et al., 2014). Interestingly, this SNP had a significant impact on OPRM1 alternative splicing (Xu, Lu, Xu, Pan, et al., 2014).

In conclusion, the current study shows similar expression profiles among multiple OPRM1 splice variant mRNAs in the mPFC of male human subjects and rats, further emphasizing conservation of OPRM1 alternative splicing. Furthermore, the expression of several OPRM1 splice variant mRNAs were dysregulated in the mPFCs from heroin abusers compared to the control subjects that were mimicked in the rat heroin SA model. These findings bring attention to dysregulation of specific OPRM1 splice variants in a critical mesocorticolimbic brain region of human heroin abusers that we demonstrate is directly linked to heroin intake as verified by the rat heroin SA model. It provides an important foundation for future mechanistic studies to furnish neurobiological insights to their role in heroin addiction vulnerability and perpetuation of the disorder.

Supplementary Material

Table S1
Table S2
Table S3
jnr24640-sup-0004
jnr24640-sup-0005

Significance Statement.

The present study reveals dysregulation of the OPRM1 splice variants in the mPFC of both male human heroin abusers and heroin self-administered male rats, illustrating conservation of the dysregulation and the usefulness of the rat heroin self-administration model and providing new mechanisms underlying heroin addiction.

Acknowledgments

Support:

This work was supported, in part, by grants from the National Institute on Drug Abuse of the National Institutes of Health, DA042888, DA046714 and DA07242, the Mayday Foundation and the Peter F. McManus Charitable Trust to YXP, DA15446 to YLH, and a core grant from the National Cancer Institute (CA008748) to Memorial Sloan Kettering Cancer Center.

Footnotes

Conflict of Interest Statement:

YXP is a co-scientific founder of Sparian Biosciences.

Data Accessibility Statement

The data that support the findings of this study are available on request from the corresponding authors.

References

  1. Abbadie C, Gultekin SH, & Pasternak GW (2000). Immunohistochemical localization of the carboxy terminus of the novel mu opioid receptor splice variant MOR-1C within the human spinal cord. Neuroreport, 11, 1953–1957. [DOI] [PubMed] [Google Scholar]
  2. Abbadie C, Pan YX, Drake CT, & Pasternak GW (2000). Comparative immunohistochemical distributions of carboxy terminus epitopes from the mu-opioid receptor splice variants MOR-1D, MOR-1 and MOR-1C in the mouse and rat CNS. Neuroscience, 100(1), 141–153. [DOI] [PubMed] [Google Scholar]
  3. Abbadie C, Pan YX, & Pasternak GW (2000). Differential distribution in rat brain of mu opioid receptor carboxy terminal splice variants MOR-1C-like and MOR-1-like immunoreactivity: Evidence for region-specific processing. Journal of Comparative Neurology, 419(2), 244–256. [DOI] [PubMed] [Google Scholar]
  4. Abbadie C, & Pasternak GW (2001). Differential in vivo internalization of MOR-1 and MOR-1C by morphine. Neuroreport, 12, 3069–3072. [DOI] [PubMed] [Google Scholar]
  5. Abbadie C, Pasternak GW, & Aicher SA (2001). Presynaptic localization of the carboxy-terminus epitopes of the [mu] opioid receptor splice variants MOR-1C and MOR-1D in the superficial laminae of the rat spinal cord. Neuroscience, 106(4), 833–842. [DOI] [PubMed] [Google Scholar]
  6. Becker JB, & Chartoff E (2019). Sex differences in neural mechanisms mediating reward and addiction. Neuropsychopharmacology, 44(1), 166–183. doi: 10.1038/s41386-018-0125-6 [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Bolan EA, Pan YX, & Pasternak GW (2004). Functional analysis of MOR-1 splice variants of the mouse mu opioid receptor gene Oprm. Synapse, 51(1), 11–18. [DOI] [PubMed] [Google Scholar]
  8. Bond C, LaForge KS, Tian M, Melia D, Zhang S, Borg L, … Yu L (1998). Single-nucleotide polymorphism in the human mu opioid receptor gene alters beta-endorphin binding and activity: possible implications for opiate addiction. Proc.Natl.Acad Sci U.S.A, 95(16), 9608–9613. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Bossert JM, Stern AL, Theberge FR, Marchant NJ, Wang HL, Morales M, & Shaham Y (2012). Role of projections from ventral medial prefrontal cortex to nucleus accumbens shell in context-induced reinstatement of heroin seeking. J Neurosci, 32(14), 4982–4991. doi:32/14/4982 [pii]; 10.1523/JNEUROSCI.0005-12.2012 [doi] [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Caron MG, Bohn LM, Barak LS, Abbadie C, Pan Y-X, & Pasternak GW (2000). Mu opioid receptor spliced variant, MOR1D, internalizes after morphine treatment in HEK-293 cells. Soc.Neurosci., 26, 783–783. [Google Scholar]
  11. Chang YF, Imam JS, & Wilkinson MF (2007). The nonsense-mediated decay RNA surveillance pathway. Annu.Rev Biochem, 76, 51–74. doi: 10.1146/annurev.biochem.76.050106.093909 [doi] [DOI] [PubMed] [Google Scholar]
  12. Drakenberg K, Nikoshkov A, Horvath MC, Fagergren P, Gharibyan A, Saarelainen K, … Hurd YL (2006). Mu opioid receptor A118G polymorphism in association with striatal opioid neuropeptide gene expression in heroin abusers. Proc Natl Acad Sci U S A, 103(20), 7883–7888. doi:0600871103 [pii];10.1073/pnas.0600871103 [doi] [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Ellgren M, Spano SM, & Hurd YL (2007). Adolescent cannabis exposure alters opiate intake and opioid limbic neuronal populations in adult rats. Neuropsychopharmacology, 32(3), 607–615. doi:1301127 [pii];10.1038/sj.npp.1301127 [doi] [DOI] [PubMed] [Google Scholar]
  14. Goldstein RZ, & Volkow ND (2011). Dysfunction of the prefrontal cortex in addiction: neuroimaging findings and clinical implications. Nat Rev Neurosci, 12(11), 652–669. doi:nrn3119 [pii]; 10.1038/nrn3119 [doi] [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Koch T, Schulz S, Schroder H, Wolf R, Raulf E, & Hollt V (1998). Carboxyl-terminal splicing of the rat mu opioid receptor modulates agonist-mediated internalization and receptor resensitization. J Biol.Chem, 273(22), 13652–13657. [DOI] [PubMed] [Google Scholar]
  16. Koob GF, & Volkow ND (2010). Neurocircuitry of addiction. Neuropsychopharmacology, 35(1), 217–238. doi:npp2009110 [pii]; 10.1038/npp.2009.110 [doi] [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Lejeune F, & Maquat LE (2005). Mechanistic links between nonsense-mediated mRNA decay and pre-mRNA splicing in mammalian cells. Curr Opin Cell Biol, 17(3), 309–315. [DOI] [PubMed] [Google Scholar]
  18. Liu A, Zhang H, Qin F, Wang Q, Sun Q, Xie S, … Lu Z (2018). Sex Associated Differential Expressions of the Alternatively Spliced Variants mRNA of OPRM1 in Brain Regions of C57BL/6 Mouse. Cell Physiol Biochem, 50(4), 1441–1459. doi: 10.1159/000494644 [DOI] [PubMed] [Google Scholar]
  19. Liu XY, Liu ZC, Sun YG, Ross M, Kim S, Tsai FF, … Chen ZF (2011). Unidirectional cross-activation of GRPR by MOR1D uncouples itch and analgesia induced by opioids. Cell, 147(2), 447–458. doi:S0092–8674(11)01064–6 [pii]; 10.1016/j.cell.2011.08.043 [doi] [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Lu Z, Xu J, Rossi GC, Majumdar S, Pasternak GW, & Pan YX (2015). Mediation of opioid analgesia by a truncated 6-transmembrane GPCR. J Clin Invest, 125(7), 2626–2630. doi:81070 [pii]; 10.1172/JCI81070 [doi] [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Lu Z, Xu J, Xu M, Rossi GC, Majumdar S, Pasternak GW, & Pan YX (2018). Truncated mu-Opioid Receptors With 6 Transmembrane Domains Are Essential for Opioid Analgesia. Anesth Analg, 126(3), 1050–1057. doi: 10.1213/ANE.0000000000002538 [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Majumdar S, Grinnell S, Le R,V, Burgman M, Polikar L, Ansonoff M, … Pasternak GW (2011). Truncated G protein-coupled mu opioid receptor MOR-1 splice variants are targets for highly potent opioid analgesics lacking side effects. Proc Natl Acad Sci U S A, 108(49), 19778–19783. doi:1115231108 [pii]; 10.1073/pnas.1115231108 [doi] [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Mogil JS, & Bailey AL (2010). Sex and gender differences in pain and analgesia. Prog Brain Res, 186, 141–157. doi: 10.1016/B978-0-444-53630-3.00009-9 [DOI] [PubMed] [Google Scholar]
  24. Pan L, Xu J, Yu R, Xu MM, Pan YX, & Pasternak GW (2005). Identification and characterization of six new alternatively spliced variants of the human mu opioid receptor gene, Oprm. Neuroscience, 133(1), 209–220. [DOI] [PubMed] [Google Scholar]
  25. Pan Y-X, Xu J, Mahurter L, Bolan EA, Xu MM, & Pasternak GW (2001). Generation of the mu opioid receptor (MOR-1) protein by three new splice variants of the Oprm gene. Proc.Natl.Acad.Sci.U.S.A, 98(24), 14084–14089. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Pan YX (2005). Diversity and complexity of the mu opioid receptor gene: alternative pre-mRNA splicing and promoters. DNA Cell Biol, 24(11), 736–750. [DOI] [PubMed] [Google Scholar]
  27. Pan YX, Xu J, Bolan EA, Abbadie C, Chang A, Zuckerman A, … Pasternak GW (1999). Identification and characterization of three new alternatively spliced mu opioid receptor isoforms. Mol.Pharmacol, 56, 396–403. [DOI] [PubMed] [Google Scholar]
  28. Pan YX, Xu J, Xu M, Rossi GC, Matulonis JE, & Pasternak GW (2009). Involvement of exon 11-associated variants of the mu opioid receptor MOR-1 in heroin, but not morphine, actions. Proceedings of the National Academy of Sciences, 106(12), 4917–4922. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Pan YX, Xu J, Xu M, Rossi GC, Matulonis JE, & Pasternak GW (2009). Involvement of exon 11-associated variants of the mu opioid receptor MOR-1 in heroin, but not morphine, actions. Proc.Natl.Acad.Sci.U.S.A, 106(12), 4917–4922. doi:0811586106 [pii]; 10.1073/pnas.0811586106 [doi] [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Pasternak DA, Pan L, Xu J, Yu R, Xu MM, Pasternak GW, & Pan YX (2004). Identification of three new alternatively spliced variants of the rat mu opioid receptor gene: dissociation of affinity and efficacy. J Neurochem, 91(4), 881–890. [DOI] [PubMed] [Google Scholar]
  31. Pasternak GW, & Pan YX (2013). Mu opioids and their receptors: evolution of a concept. Pharmacol Rev, 65(4), 1257–1317. doi:65/4/1257 [pii]; 10.1124/pr.112.007138 [doi] [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Peters J, Pattij T, & De Vries TJ (2013). Targeting cocaine versus heroin memories: divergent roles within ventromedial prefrontal cortex. Trends in Pharmacological Sciences, 34(12), 689–695. doi:S0165–6147(13)00188–0 [pii]; 10.1016/j.tips.2013.10.004 [doi] [DOI] [PubMed] [Google Scholar]
  33. Puig S, & Gutstein HB (2017). A “tail” of opioid receptor variants. J Clin Invest, 127(4), 1221–1224. doi: 10.1172/JCI93582 [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Rogers JL, Ghee S, & See RE (2008). The neural circuitry underlying reinstatement of heroin-seeking behavior in an animal model of relapse. Neuroscience, 151(2), 579–588. doi:S0306–4522(07)01301–2 [pii]; 10.1016/j.neuroscience.2007.10.012 [doi] [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Schuller AG, King MA, Zhang J, Bolan E, Pan YX, Morgan DJ, … Pintar JE (1999). Retention of heroin and morphine-6 beta-glucuronide analgesia in a new line of mice lacking exon 1 of MOR-1. Nat.Neurosci, 2(2), 151–156. [DOI] [PubMed] [Google Scholar]
  36. Sillivan SE, Whittard JD, Jacobs MM, Ren Y, Mazloom AR, Caputi FF, … Hurd YL (2013). ELK1 transcription factor linked to dysregulated striatal mu opioid receptor signaling network and OPRM1 polymorphism in human heroin abusers. Biol Psychiatry, 74(7), 511–519. doi: 10.1016/j.biopsych.2013.04.012 [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Smith KS, & Graybiel AM (2013). A dual operator view of habitual behavior reflecting cortical and striatal dynamics. Neuron, 79(2), 361–374. doi:S0896–6273(13)00490-X [pii]; 10.1016/j.neuron.2013.05.038 [doi] [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Smith KS, Virkud A, Deisseroth K, & Graybiel AM (2012). Reversible online control of habitual behavior by optogenetic perturbation of medial prefrontal cortex. Proc Natl Acad Sci U S A, 109(46), 18932–18937. doi:1216264109 [pii]; 10.1073/pnas.1216264109 [doi] [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Tomasiewicz HC, Jacobs MM, Wilkinson MB, Wilson SP, Nestler EJ, & Hurd YL (2012). Proenkephalin mediates the enduring effects of adolescent cannabis exposure associated with adult opiate vulnerability. Biol Psychiatry, 72(10), 803–810. doi:S0006–3223(12)00409-X [pii]; 10.1016/j.biopsych.2012.04.026 [doi] [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Van den Oever MC, Spijker S, Smit AB, & De Vries TJ (2010). Prefrontal cortex plasticity mechanisms in drug seeking and relapse. Neurosci Biobehav.Rev, 35(2), 276–284. doi:S0149–7634(09)00182–1 [pii]; 10.1016/j.neubiorev.2009.11.016 [doi] [DOI] [PubMed] [Google Scholar]
  41. Verzillo V, Madia PA, Liu NJ, Chakrabarti S, & Gintzler AR (2014). Mu-opioid receptor splice variants: sex-dependent regulation by chronic morphine. Journal of Neurochemistry, 130(6), 790–796. doi: 10.1111/jnc.12768 [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Wang YJ, Huang P, Blendy JA, & Liu-Chen LY (2014). Brain region- and sex-specific alterations in DAMGO-stimulated [(35) S]GTPgammaS binding in mice with Oprm1 A112G. Addict Biol, 19(3), 354–361. doi: 10.1111/j.1369-1600.2012.00484.x [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Wang YJ, Huang P, Ung A, Blendy JA, & Liu-Chen LY (2012). Reduced expression of the mu opioid receptor in some, but not all, brain regions in mice with OPRM1 A112G. Neuroscience, 205, 178–184. doi: 10.1016/j.neuroscience.2011.12.033 [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Wieskopf JS, Pan YX, Marcovitz J, Tuttle AH, Majumdar S, Pidakala J, … Mogil JS (2014). Broad-spectrum analgesic efficacy of IBNtxA is mediated by exon 11-associated splice variants of the mu-opioid receptor gene. Pain. doi:S0304–3959(14)00333–9 [pii]; 10.1016/j.pain.2014.07.014 [doi] [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Xu J, Faskowitz AJ, Rossi GC, Xu M, Lu Z, Pan YX, & Pasternak GW (2015). Stabilization of morphine tolerance with long-term dosing: Association with selective upregulation of mu-opioid receptor splice variant mRNAs. Proc Natl Acad Sci U S A, 112(1), 279–284. doi:1419183112 [pii]; 10.1073/pnas.1419183112 [doi] [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Xu J, Lu Z, Narayan A, Le Rouzic VP, Xu M, Hunkele A, … Pan YX (2017). Alternatively spliced mu opioid receptor C termini impact the diverse actions of morphine. J Clin Invest, 127(4), 1561–1573. doi: 10.1172/JCI88760 [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Xu J, Lu Z, Xu M, Pan L, Deng Y, Xie X, … Pan YX (2014). A Heroin Addiction Severity-Associated Intronic Single Nucleotide Polymorphism Modulates Alternative Pre-mRNA Splicing of the mu Opioid Receptor Gene OPRM1 via hnRNPH Interactions. J Neurosci, 34(33), 11048–11066. doi:34/33/11048 [pii]; 10.1523/JNEUROSCI.3986-13.2014 [doi] [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Xu J, Lu Z, Xu M, Rossi GC, Kest B, Waxman AR, … Pan YX (2014). Differential expressions of the alternatively spliced variant mRNAs of the micro opioid receptor gene, OPRM1, in brain regions of four inbred mouse strains. PLoS One, 9(10), e111267. doi: 10.1371/journal.pone.0111267 [doi];PONE-D-14–33518 [pii] [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Xu J, Xu M, Brown T, Rossi GC, Hurd YL, Inturrisi CE, … Pan YX (2013). Stabilization of the mu-opioid receptor by truncated single transmembrane splice variants through a chaperone-like action. J Biol Chem, 288(29), 21211–21227. doi:M113.458687 [pii]; 10.1074/jbc.M113.458687 [doi] [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Zhang Y, Picetti R, Butelman ER, Ho A, Blendy JA, & Kreek MJ (2015). Mouse model of the OPRM1 (A118G) polymorphism: differential heroin self-administration behavior compared with wild-type mice. Neuropsychopharmacology, 40(5), 1091–1100. doi: 10.1038/npp.2014.286 [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Table S1
NIHMS1699817-supplement-Table_S1.xlsx (13.9KB, xlsx)
Table S2
NIHMS1699817-supplement-Table_S2.xlsx (10.5KB, xlsx)
Table S3
NIHMS1699817-supplement-Table_S3.xlsx (10.4KB, xlsx)
jnr24640-sup-0004
NIHMS1699817-supplement-jnr24640-sup-0004.docx (1.7MB, docx)
jnr24640-sup-0005
NIHMS1699817-supplement-jnr24640-sup-0005.docx (1.7MB, docx)

RESOURCES