Circulating plasma and exosome levels of the miR-320 family as a non-invasive biomarker for methamphetamine use disorder.

The neurobiological mechanism underlying methamphetamine (MA) use disorder was still unclear, and no specific biomarker exists for clinical diagnosis of this disorder. Recent studies have demonstrated that microRNAs (miRNAs) are involved in the pathological process of MA addiction. The purpose of this study was to identify novel miRNAs for the diagnosis biomarkers of MA user disorder. First, members of the miR-320 family, including miR-320a-3p, miR-320b, and miR-320c, were screened and analyzed in the circulating plasma and exosomes by microarray and sequencing. Secondly, plasma miR-320 was quantified by real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) in eighty-two MA patients and fifty age-gender-matched healthy controls. Meanwhile, we also analyzed exosomal miR-320 expression in thirty-nine MA patients and twenty-one age-matched healthy controls. Furthermore, the diagnostic power was evaluated using the area under the curve (AUC) of the receiver operating characteristic (ROC) curve. The expression of miR-320 significantly increased in plasma and exosomes of MA patients compared with healthy controls. The AUC of the ROC curves of miR-320 in plasma and exosomes of MA patients were 0.751 and 0.962, respectively. And the sensitivities of miR-320 were 0.900 and 0.846, respectively, whereas the specificities of miR-320 were 0.537 and 0.952, respectively, in plasma and exosomes in MA patients. And the increased plasma miR-320 was positively correlated with cigarette smoking, age of onset, and daily use of MA in MA patients. Finally, cardiovascular disease, synaptic plasticity, and neuroinflammation were predicted to be the target pathways related to miR-320. Taken together, our findings indicated that plasma and exosomal miR-320 might be used as a potential blood-based biomarker for diagnosing MA use disorder.

Differential expression profiling of tRNA-Derived small RNAs and their potential roles in methamphetamine self-administered rats.

Transfer RNA-derived small RNAs (tsRNAs) are a novel class of short, non-coding RNAs that are closely associated with the pathogenesis of various diseases. Accumulating evidence has demonstrated their critical functional roles as regulatory factors in gene expression regulation, protein translation regulation, regulation of various cellular activities, immune mediation, and response to stress. However, the underlying mechanisms by which tRFs & tiRNAs affect methamphetamine-induced pathophysiological processes are largely unknown. In this study, we used a combination of small RNA sequencing, quantitative reverse transcription-polymerase chain reaction (qRT‒PCR), bioinformatics, and luciferase reporter assays to screen the expression profiles and identify the functional roles of tRFs and tiRNAs in the nucleus accumbens (NAc) of methamphetamine self-administration rat models. A total of 461 tRFs & tiRNAs were identified in the NAc of rats after 14 days of methamphetamine self-administration training. Of those, 132 tRFs & tiRNAs were significantly differentially expressed: 59 were significantly upregulated, whereas 73 were significantly downregulated in the rats with methamphetamine self-administration. Decreased expression levels of tiRNA-1-34-Lys-CTT-1 and tRF-1-32-Gly-GCC-2-M2, as well as increased expression levels of tRF-1-16-Ala-TGC-4 in the METH group compared with the saline control were validated by using RT‒PCR. Then, bioinformatic analysis was performed to analyse the possible biological functions of tRFs & tiRNAs in methamphetamine-induced pathogenesis. Furthermore, tRF-1-32-Gly-GCC-2-M2 was identified to target BDNF using the luciferase reporter assay. An altered tsRNA expression pattern was proven, and tRF-1-32-Gly-GCC-2-M2 was shown to be involved in methamphetamine-induced pathophysiologic processes by targeting BDNF. The current study provides new insights for future investigations to explore the mechanisms and therapeutic methods for methamphetamine addiction.

The motivation against change in male methamphetamine users in the compulsory detoxification setting.

Aims: The study was designed to develop a measurement for the motivation for and against change in methamphetamine users in the compulsory detoxification setting. Design: This is a cross-sectional study. Setting: The study was carried out in a compulsory detoxification center for male drug users in China. Participants: A total of 228 male methamphetamine users who had undergone the program for at least 30 days. Measurements: The motivation for/against change relating to compulsory detoxification was carried out using the Likert scale. A series of questionnaires were filled out by the participants, including the Egna Minnen Beträffande Uppfostran for rearing style, the Barratt Impulsiveness Scale-11, the adult ADHD self-report scale, and the Pittsburgh sleep quality index. Participants were also asked to recall the withdrawal symptoms before the program and to rate their current craving levels. Findings: Motivations were grouped into three factors, namely, the expectation to use drugs upon the completion of the program (factor 1), the disagreement with the compulsory setting (factor 2), and the motivation to quit drug use (factor 3). Cronbach's alpha values were 0.8037, 0.8049, and 0.6292, respectively. The structural equation model showed that the overall motivation was characterized by motivation against change rather than that for change. The overall motivation was also directly affected by the current craving level and indirectly affected by the severity of addiction, paternal authoritarian upbringing style, and ADHD traits. Conclusion: This study provided a measurement of motivation for and against change in subjects with drug misconduct and suggested that the motivation against change may disclose more psychological barriers than the motivation for change.

Association of GABA receptor delta subunit gene variations with increased risk of methamphetamine dependence.

OBJECTIVE: Evidence reveals that γ-aminobutyric acid (GABA) receptors are involved in the development of methamphetamine (METH) dependence. The GABA receptor delta subunit gene (GABRD) might be a good candidate gene for METH dependence. In a case-control study, we investigated the association between the single nucleotide polymorphisms (SNPs) in GABRD and METH dependence in a Chinese Han population. METHODS: A total of 300 METH dependent patients and 300 age and sex matched normal control subjects were recruited. Four SNPs (rs13303344, rs4481796, rs2376805, and rs2229110) in GABRD were determined with the TaqMan genotyping assay. The association of the SNPs with METH dependence was assessed. RESULTS: Only the allele frequency of rs2376805 significantly differed between the patients and controls (P = 0.030). The G allele frequency of rs2376805 was higher in the METH dependent group than in the controls (odds ratio = 1.332, 95 % CI: 1.028-1.724). This association was found in females but not in males. In females, the frequencies of genotype and allele at rs2376805 significantly differed between the patients and controls (P = 0.025, 0.022, respectively); the rs2376805 G allele may also be a risk factor for METH dependence (odds ratio = 1.548, 95 % CI: 1.063-2.257). The haplotype ACGT frequency significantly differed between the patients and controls in total subjects (P = 0.008, odds ratio = 1.815, 95 % CI: 1.183-2.782), as well as in females (P = 0.005, odds ratio = 2.702, 95 % CI: 1.313-5.562). In females only, the METH craving score was significantly lower in patients harboring the G allele at rs2376805 than in those harboring the homozygous AA genotype (P = 0.044). CONCLUSION: The preliminary results indicate that GABRD rs2376805 is associated with METH dependence, especially in females.

Association between rs1799971 in the mu opioid receptor gene and methadone maintenance treatment response.

OBJECTIVE: Genetic variations can affect individual response to methadone maintenance treatment (MMT) for heroin addiction. The A118G variant (rs1799971) in the mu opioid receptor gene (OPRM1) is a potential candidate single nucleotide polymorphism (SNP) for personalized MMT. This study determined whether rs1799971 is related to MMT response or dose. METHODS: We recruited 286 MMT patients from a Han Chinese population. The rs1799971 genotype was determined via TaqMan genotyping assay. The genetic effect of this SNP on MMT response or dose was evaluated using logistic regression. A meta-analysis was performed to merge all available data to evaluate the role of rs1799971 in MMT using RevMan 5.3 software. RESULTS: No statistical significance was observed in the association between the OPRM1 rs1799971 and MMT response or dose in our Chinese cohort. Meta-analysis indicated that the OPRM1 A118G variation was not significantly associated with MMT response or dose requirement. CONCLUSION: The results suggest that rs1799971 in OPRM1 might not play a critical role alone in influencing MMT response or dose.

Corrigendum: Increased Expression of Plasma miRNA-320a and let-7b-5p in Heroin-Dependent Patients and Its Clinical Significance.

[This corrects the article DOI: 10.3389/fpsyt.2021.679206.].

Increased Expression of Plasma miRNA-320a and let-7b-5p in Heroin-Dependent Patients and Its Clinical Significance.

Heroin use disorder is a chronic and relapsing disease that induces persistent changes in the brain. The diagnoses of heroin use disorders are mainly based on subjective reports and no valid biomarkers available. Recent researches have revealed that circulating miRNAs are useful non-invasive biomarkers for diagnosing brain diseases such as Alzheimer's disease, multiple sclerosis, schizophrenia, and bipolar disorder. However, studies on circulating miRNAs for the diagnosis of heroin use disorders are rarely reported. In this study, we investigated the differential expression of plasma miRNAs in 57 heroin-dependent patients. Based on literature research and microarray analysis, two candidate miRNAs, miR-320a and let-7b-5p, were selected and analyzed by quantitative real-time RT-PCR. The results showed miR-320a and let-7b were significantly upregulated in plasma of the heroin-dependent patients compared to that in healthy controls. The area under curves (AUCs) of receiver operating characteristic (ROC) curves of miR-320a and let-7b-5p were 0.748 and 0.758, respectively. The sensitivities of miR-320a and let-7b-5p were 71.9 and 70.2%, while the specificities of miR-320a and let-7b-5p were 76.1 and 78.3%, respectively. The combination of these two miRNAs predicted heron dependence with an AUC of 0.782 (95% CI 0.687-0.876), with 73.7% sensitivity and 82.6% specificity. Our findings suggest a potential use for circulating miRNAs as biomarkers for the diagnosis of heroin abuse.

Association study of genetic polymorphisms in GABRD with treatment response and dose in methadone maintenance treatment.

Aim: This study determined if gene variants in the GABA receptor delta subunit (GABRD) are associated with treatment response and dose in methadone maintenance treatment (MMT) for heroin addiction. Materials & methods: A total of 286 MMT patients were recruited and divided into response and nonresponse groups based on retention time in therapy. A total of 177 responders were classified into low dose and high dose subgroups according to the stabilized methadone dose. Four (single nucleotide polymorphisms) SNPs (rs13303344, rs4481796, rs2376805 and rs2229110) in GABRD were genotyped using the TaqMan SNP assay. Logistic regression was used to assess the genetic effects of the SNPs in MMT. Results: No significant associations were observed between the SNPs and treatment response or dose, except the frequency of haplotype ACGC at the four SNPs significantly differed between responders and nonresponders. Conclusion: The results indicated that GABRD variants may play a small role in modulating methadone treatment response.

Lay abstract This study determined if gene variants in the GABA receptor delta subunit (GABRD) are associated with treatment response and dose in methadone maintenance treatment (MMT) for heroin addiction. A total of 286 MMT patients were recruited and divided into response and nonresponse groups. A total of 177 responders were classified into low and high dose subgroups. Four single nucleotide polymorphisms (SNPs) (rs13303344, rs4481796, rs2376805 and rs2229110) in GABRD were genotyped and assessed the genetic effects of the SNPs in MMT. No significant associations were observed between the SNPs and treatment response or dose, except the frequency of haplotype ACGC significantly differed between responders and nonresponders. The results indicated that GABRD variants may play a small role in MMT, which may help provide a foundation for personalized solutions for MMT.

Association between GABA receptor delta subunit gene polymorphisms and heroin addiction.

Evidence suggests that γ-aminobutyric acid (GABA) receptors are involved in the development of drug dependence. Considering its exclusively extrasynaptic localization, GABA receptor delta subunit (GABRD) is likely involved in heroin addiction. The purpose of this study was to explore the association between the single nucleotide polymorphisms (SNPs) of GABRD and heroin addiction. Genotyping of five SNPs (rs13303344, rs4481796, rs2376805, rs2229110, and rs41307846) in GABRD gene was performed by using TaqMan SNP assay. The association between heroin addiction and these SNPs was assessed in 446 heroin dependent patients and 400 normal control subjects of male Han Chinese origin. Only the genotype and allele frequencies at rs13303344 differed significantly between the cases and controls (nominal P values were 0.028 and 0.019, respectively). The C allele of rs13303344 was associated with an increased risk of heroin addiction (OR = 1.281, 95 % CI: 1.042-1.575). After Bonferroni correction, the association lost significance. The frequencies of the haplotype C-C-A and A-C-A at GARBD (rs13303344-rs4481796- rs2376805) differed significantly between the cases and controls. The heroin craving score was significantly higher in patients with CC/AC genotypes at rs13303344 than in those with the AA genotype (nominal P = 0.017). The results suggest that GABRD rs13303344 may contribute to the susceptibility to heroin addiction and is associated with the drug cravings of heroin dependent patients.

NMDA Receptor-Dependent Cholinergic Modulation of Mesolimbic Dopamine Cell Bodies: Neurochemical and Behavioral Studies.

Substance abuse disorders are devastating, costly, and difficult to treat. Identifying the neurochemical mechanisms underlying reinforcement promises to provide critical information in the development of effective treatments. Several lines of evidence suggest that striatal dopamine (DA) release serves as a teaching signal in reinforcement learning, and that shifts in DA release from the primary reward to reward-predicting stimuli play a critical role in the self-administration of both natural and non-natural rewards. However, far less is known about the reinforcing effects of motivationally neutral sensory stimuli, or how these signals can facilitate self-administration behavior. Thus, we trained rats ( n = 7) to perform a visual stimulus-induced instrumental task, which involved lever pressing for activation of a stimulus light. We then microinfused vehicle (phosphate buffered saline), carbachol (acetylcholine receptor agonist), or carbachol in the presence of an N-methyl-d-aspartate (NMDA) receptor-specific drug (NMDA itself, or the antagonist, AP5) into the ventral tegmental area (VTA). This enabled us to directly evaluate how chemical modulation of dopamine cell bodies affects the instrumental behavior, as well as the nature of extracellular dopamine transients recorded in the nucleus accumbens shell (NAc shell) using fast-scan cyclic voltammetry (FSCV). Intra-VTA infusion of carbachol enhanced the magnitude and frequency of dopamine transients in the NAc shell and potentiated active lever responding without altering inactive lever responding, as compared to infusion of vehicle. Coinfusion of carbachol with AP5 abolished dopamine transients recorded in the NAc and attenuated active lever responding without altering inactive lever responding. Finally, coadministration of carbachol and NMDA into the VTA restored both lever pressing and dopaminergic signals recorded in the striatum. Together, these results suggest that acetylcholine and glutamate synergistically act at dopamine cells in the VTA to modulate VTA-NAc shell dopaminergic output, and this underlies motivation to lever press for a motivationally neutral visual stimulus.

Hypermethylation of the κ1 opioid receptor promoter in Chinese heroin and methamphetamine addicts.

Heroin and methamphetamine (METH) addiction continues to be a major social, economic and therapeutic problem worldwide. The opioid pathway may mediate the effects of addictive drugs. However, the potential correlation between the κ1 opioid receptor (OPRK1) and drug addiction has not yet been characterized. The aim of the present study was to investigate the potential association between methylation of the OPRK1 promoter and substance abuse. Bisulfite pyrosequencing technology was used to determine the levels of OPRK1 promoter methylation in 60 drug abusers (30 heroin and 30 METH addicts) and 52 controls, observed to exhibit no significant differences in age or gender. The results indicated that levels of OPRK1 promoter methylation were significantly higher in drug addicts when compared with controls (P=2.43×10-4). Significant correlations between OPRK1 promoter methylation and the length and frequency of drug use were also observed in male heroin addicts (length: r=0.661, P=0.007; frequency: r=-0.684, P=0.005). In addition, a luciferase reporter gene assay indicated that the OPRK1 promoter fragment was able to regulate gene expression (fold change between two groups >32.12, P≤0.0001). In conclusion, results of the present study indicate that methylation of the OPRK1 promoter contributes to the pathophysiology of drug addiction.

Dopamine receptor D4 promoter hypermethylation increases the risk of drug addiction.

Heroin and methylamphetamine (METH) are two addictive drugs that cause serious problems for society. Dopamine receptor D4 (DRD4), a key receptor in the dopaminergic system, may facilitate the development of drug addiction. The aim of the present study was to investigate the association between the promoter methylation level of DRD4 gene and drug addiction. Bisulfite pyrosequencing technology was used to measure the methylation levels of DRD4 promoter in 60 drug addicts and 52 matched controls. Significantly higher levels of DRD4 CpG1 and CpG4 methylation were detected in METH and heroin drug addicts compared with controls (P<0.05). Male METH addicts exhibited significantly higher DRD4 CpG1, CpG2 and CpG4 methylation levels compared with sex-matched controls (P<0.05). In heroin addicts, a positive correlation was observed between depression-dejection and DRD4 CpG5 methylation (r=0.537, P=0.039) whereas there was a negative correlation between drug usage frequency and CpG1 methylation (r=-0.632, P=0.011). In METH addicts, methylation levels were not significantly associated with depression-dejection and drug usage frequency. In addition, luciferase assays demonstrated that the target sequence of the DRD4 promoter upregulates gene expression. The results of the present study suggest that DNA methylation of DRD4 may be responsible for the pathophysiology of drug addiction.

Biochemical Diagnosis in Substance and Non-substance Addiction.

An optimal biochemical marker for addiction would be some easily traced molecules in body specimens, which indicates indulgent addictive behaviors, or susceptibility to certain addictive stimuli. In this chapter, we discussed existing literature about possible biomarkers, and classified them into three categories: origin forms and metabolites of substances, markers from biochemical responses to certain addiction, and genetic and epigenetic biomarkers suggesting susceptibility to addiction. In every category, we examined studies concerning certain type of addiction one by one, with focuses mainly on opiates, psychostimulants, and pathological gambling. Several promising molecules were highlighted, including those of neurotrophic factors, inflammatory factors, and indicators of vascular injury, and genetic and epigenetic biomarkers such as serum miRNAs. DNA methylation signatures and signal nucleotide polymorphism of candidate gene underlying the addiction.

Association between genetic variations of NMDA receptor NR3 subfamily genes and heroin addiction in male Han Chinese.

Growing amounts of evidence suggest that N-Methyl-d-aspartate (NMDA) receptor mediated glutamate neurotransmission may be involved in the pathophysiology of drug dependence. The NMDA receptor consists of three subfamilies (NR1, NR2, and NR3). The ability of subunit NR3 to negatively modulate the NMDA receptor function makes it an attractive candidate gene of heroin addiction. The purpose of this study is to explore the association between four single nucleotide polymorphisms (SNPs) of NR3 gene and heroin addiction. Genotyping of two SNPs (rs3739722 and rs17189632) in GRIN3A and two SNPs (rs4807399 and rs2240158) in GRIN3B was performed using TaqMan SNP genotyping method. The association between heroin addiction and these SNPs was assessed among 332 male heroin dependent patients and 400 male normal control subjects. The results showed the genotype and allele frequencies of rs17189632 and rs2240158 were significantly different between the cases and the controls (nominal P values were 0.0284, 0.0136 for rs17189632; 0.0048, 0.0013 for rs2240158, respectively). After Bonferroni correction, rs2240158 of GRIN3B was still found to be associated with heroin addiction. The frequencies of haplotype C-A at GRIN3A (rs3739722-rs17189632) and of C-C and C-T at GRIN3B (rs4807399-rs2240158) differed significantly between the cases and the controls. The genotype and allele distributions of rs3739722 and rs4807399 were not significantly different between in the cases and in the controls (P>0.05). These results suggest that GRIN3A rs17189632 and GRIN3B rs2240158 may contribute to the susceptibility of heroin addiction.

A significant association between BDNF promoter methylation and the risk of drug addiction.

As a member of the neurotrophic factor family, brain derived neurotrophic factor (BDNF) plays an important role in the survival and differentiation of neurons. The aim of our work was to evaluate the role of BDNF promoter methylation in drug addiction. A total of 60 drug abusers (30 heroin and 30 methylamphetamine addicts) and 52 healthy age- and gender-matched controls were recruited for the current case control study. Bisulfite pyrosequencing technology was used to determine the methylation levels of five CpGs (CpG1-5) on the BDNF promoter. Among the five CpGs, CpG5 methylation was significantly lower in drug abusers than controls. Moreover, significant associations were found between CpG5 methylation and addictive phenotypes including tension-anxiety, anger-hostility, fatigue-inertia, and depression-dejection. In addition, luciferase assay showed that the DNA fragment of BDNF promoter played a key role in the regulation of gene expression. Our results suggest that BDNF promoter methylation is associated with drug addiction, although further studies are needed to understand the mechanisms by which BDNF promoter methylation contributes to the pathophysiology of drug addiction.

Contribution of an SFK-Mediated Signaling Pathway in the Dorsal Hippocampus to Cocaine-Memory Reconsolidation in Rats.

Environmentally induced relapse to cocaine seeking requires the retrieval of context-response-cocaine associative memories. These memories become labile when retrieved and must undergo reconsolidation into long-term memory storage to be maintained. Identification of the molecular underpinnings of cocaine-memory reconsolidation will likely facilitate the development of treatments that mitigate the impact of cocaine memories on relapse vulnerability. Here, we used the rat extinction-reinstatement procedure to test the hypothesis that the Src family of tyrosine kinases (SFK) in the dorsal hippocampus (DH) critically controls contextual cocaine-memory reconsolidation. To this end, we evaluated the effects of bilateral intra-DH microinfusions of the SFK inhibitor, PP2 (62.5 ng per 0.5 µl per hemisphere), following re-exposure to a cocaine-associated (cocaine-memory reactivation) or an unpaired context (no memory reactivation) on subsequent drug context-induced instrumental cocaine-seeking behavior. We also assessed alterations in the phosphorylation state of SFK targets, including GluN2A and GluN2B N-methyl-D-aspartate (NMDA) and GluA2 α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor subunits at the putative time of memory restabilization and following PP2 treatment. Finally, we evaluated the effects of intra-DH PEAQX (2.5 µg per 0.5 µl per hemisphere), a GluN2A-subunit-selective NMDAR antagonist, following, or in the absence of, cocaine-memory reactivation on subsequent drug context-induced cocaine-seeking behavior. GluN2A phosphorylation increased in the DH during putative memory restabilization, and intra-DH PP2 treatment inhibited this effect. Furthermore, PP2-as well as PEAQX-attenuated subsequent drug context-induced cocaine-seeking behavior, in a memory reactivation-dependent manner, relative to VEH. These findings suggest that hippocampal SFKs contribute to the long-term stability of cocaine-related memories that underlie contextual stimulus control over cocaine-seeking behavior.

Correlation Equation for Predicting the Single-Collector Contact Efficiency of Colloids in a Horizontal Flow.

The single-collector contact efficiency (η0) for physicochemical colloid filtration under horizontal flow in saturated porous media was calculated using trajectory analysis in three dimensions. Past studies have developed correlation equations for colloids with densities close to that of water, such as bacteria and latex particles. A new correlation equation was developed for predicting η0 based on a large number of trajectory simulations to account for higher-density particles representative of metal colloids. The correlation equation was developed by assuming Brownian diffusion, interception, and gravitational sedimentation contributed to η0 in an additive manner. Numerical simulations for colloid trajectory analysis used for calculating η0 were based on horizontal flow around a collector under the action of van der Waals attractive forces, gravity, and hydrodynamic forces as well as Brownian motion. The derived correlation equation shows excellent agreement with existing correlation equations for particles with density close to that of water. However, the correlation equation presented in this study shows that η0 of high-density colloids, such as metal particles, transported under horizontal flow deviates from that predicted by existing correlations for colloids larger than 4 µm and under low approach velocities. Simulations of trajectory paths show that a significantly reduced contact of high-density colloids larger than 4 µm in size with a collector is due to gravity forces causing trajectory paths to deviate away from the underside of collectors. The new correlation equation is suitable for predicting the single collector efficiency of large particles (several hundred nanometers to several micrometers) and with a large amount of density transport in the horizontal flow mode but is unsuitable for particles with a quite small size (several to tens of nanometers) and for the particle with a large amount of density flow in the vertical flow mode. The trajectory analysis was conducted for particles under favorable deposition conditions.

A heroin addiction severity-associated intronic single nucleotide polymorphism modulates alternative pre-mRNA splicing of the µ opioid receptor gene OPRM1 via hnRNPH interactions.

Single nucleotide polymorphisms (SNPs) in the OPRM1 gene have been associated with vulnerability to opioid dependence. The current study identifies an association of an intronic SNP (rs9479757) with the severity of heroin addiction among Han-Chinese male heroin addicts. Individual SNP analysis and haplotype-based analysis with additional SNPs in the OPRM1 locus showed that mild heroin addiction was associated with the AG genotype, whereas severe heroin addiction was associated with the GG genotype. In vitro studies such as electrophoretic mobility shift assay, minigene, siRNA, and antisense morpholino oligonucleotide studies have identified heterogeneous nuclear ribonucleoprotein H (hnRNPH) as the major binding partner for the G-containing SNP site. The G-to-A transition weakens hnRNPH binding and facilitates exon 2 skipping, leading to altered expressions of OPRM1 splice-variant mRNAs and hMOR-1 proteins. Similar changes in splicing and hMOR-1 proteins were observed in human postmortem prefrontal cortex with the AG genotype of this SNP when compared with the GG genotype. Interestingly, the altered splicing led to an increase in hMOR-1 protein levels despite decreased hMOR-1 mRNA levels, which is likely contributed by a concurrent increase in single transmembrane domain variants that have a chaperone-like function on MOR-1 protein stability. Our studies delineate the role of this SNP as a modifier of OPRM1 alternative splicing via hnRNPH interactions, and suggest a functional link between an SNP-containing splicing modifier and the severity of heroin addiction.

Cocaine seeking and taking: role of hippocampal dopamine D1-like receptors.

Despite the well-documented involvement of dopamine D1-like receptor stimulation in cocaine-induced goal-directed behaviours, little is known about the specific contribution of D1-like receptor populations in the dorsal hippocampus (DH) to drug context-induced cocaine-seeking or drug-reinforced instrumental behaviours. To investigate this question, rats were trained to lever press for un-signalled cocaine infusions in a distinct context followed by extinction training in a different context. Cocaine-seeking behaviour (non-reinforced lever responding) was then assessed in the previously cocaine-paired and extinction contexts. SCH23390-induced D1-like receptor antagonism in the DH, but not the overlying trunk region of the somatosensory cortex, dose-dependently inhibited drug context-induced cocaine-seeking behaviour, without altering cocaine-reinforced instrumental responding, cocaine intake, food-reinforced instrumental responding, or general motor activity, relative to vehicle treatment. These findings suggest that D1-like receptor stimulation in the DH is critical for the incentive motivational effects and/or memory of cocaine-paired contextual stimuli that contribute to drug-seeking behaviour.

Involvement of amygdalar protein kinase A, but not calcium/calmodulin-dependent protein kinase II, in the reconsolidation of cocaine-related contextual memories in rats.

RATIONALE: Contextual control over drug relapse depends on the successful reconsolidation and retention of context-response-cocaine associations in long-term memory stores. The basolateral amygdala (BLA) plays a critical role in cocaine memory reconsolidation and subsequent drug context-induced cocaine-seeking behavior; however, less is known about the cellular mechanisms of this phenomenon. OBJECTIVES: The present study evaluated the hypothesis that protein kinase A (PKA) and calcium/calmodulin-dependent protein kinase II (CaMKII) activation in the BLA is necessary for the reconsolidation of context-response-cocaine memories that promote subsequent drug context-induced cocaine-seeking behavior. METHODS: Rats were trained to lever-press for cocaine infusions in a distinct context, followed by extinction training in a different context. Rats were then briefly re-exposed to the previously cocaine-paired context or an unpaired context in order to reactivate cocaine-related contextual memories and initiate their reconsolidation or to provide a similar behavioral experience without explicit cocaine-related memory reactivation, respectively. Immediately after this session, rats received bilateral microinfusions of vehicle, the PKA inhibitor, Rp-adenosine 3',5'-cyclic monophosphorothioate triethylammonium salt (Rp-cAMPS), or the CaMKII inhibitor, KN-93, into the BLA or the posterior caudate putamen (anatomical control region). Rats were then tested for cocaine-seeking behavior (responses on the previously cocaine-paired lever) in the cocaine-paired context and the extinction context. RESULTS: Intra-BLA infusion of Rp-cAMPS, but not KN-93, following cocaine memory reconsolidation impaired subsequent cocaine-seeking behavior in a dose-dependent, site-specific, and memory reactivation-dependent fashion. CONCLUSIONS: PKA, but not CaMKII, activation in the BLA is critical for cocaine memory re-stabilization processes that facilitate subsequent drug context-induced instrumental cocaine-seeking behavior.