Intravenous morphine self-administration alters accumbal microRNA profiles in the mouse brain

Abstract

A significant amount of evidence indicates that microRNAs (miRNAs) play an important role in drug addiction. The nucleus accumbens (NAc) is a critical part of the brain's reward circuit and is involved in a variety of psychiatric disorders, including depression, anxiety, and drug addiction. However, few studies have examined the expression of miRNAs and their functional roles in the NAc under conditions of morphine addiction. In this study, mice were intravenously infused with morphine (0.01, 0.03, 0.3, 1 and 3 mg/kg/infusion) and showed inverted U-shaped response. After morphine self-administration, NAc was used to analyze the functional networks of altered miRNAs and their putative target mRNAs in the NAc following intravenous self-administration of morphine. We utilized several bioinformatics tools, including Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway mapping and CyTargetLinker. We found that 62 miRNAs were altered and exhibited differential expression patterns. The putative targets were related to diverse regulatory functions, such as neurogenesis, neurodegeneration, and synaptic plasticity, as well as the pharmacological effects of morphine (receptor internalization/endocytosis). The present findings provide novel insights into the regulatory mechanisms of accumbal molecules under conditions of morphine addiction and identify several novel biomarkers associated with morphine addiction.

Keywords: bioinformatics; microRNA; morphine; nerve regeneration; neural regeneration; nucleus accumbens; self-administration.

Figure 1

Schematic diagram of intravenous self-administration procedure.

Figure 2

Mice that intravenously self-administered morphine exhibited general drug-seeking behavior. Either food pellets or intravenous morphine infusions was earned as rewards during the consecutive self-administration sessions. (A) Over five consecutive food-training sessions, the number of active lever presses to acquire food pellets gradually increased during the 1-hour sessions. Additionally, the mice underwent two additional food-training sessions following the catheter implantation surgery to reinstate their memory of the procedure. After food training, we conducted 6–9 consecutive self-administration. (B) Number of morphine infusions earned and lever presses performed to receive a morphine infusion (0.3 mg/kg/infusion) during consecutive self-administration sessions. (C) Number of morphine infusions earned and lever presses performed to receive a morphine infusion (0.03 mg/kg/infusion) during consecutive self-administration sessions. (D) Number of morphine infusions earned and lever presses performed to receive a morphine infusion (1 mg/kg/infusion) during consecutive self-administration sessions. (E) Number of morphine infusions earned and lever presses performed to receive a morphine infusion (0.1 mg/kg/infusion) during consecutive self-administration sessions. (F) Number of morphine infusions earned and lever presses performed to receive a morphine infusion (3 mg/kg/infusion) during consecutive self-administration sessions. All data are reported as the mean ± SE (n = 8–9 mice per dose of morphine). FT: Food training; Re-FT: re-food training.

Figure 3

A typical inverted U-shaped curve in the dose-response test was generated from mice that intravenously self-administered morphine. (A) Dose-response tests for morphine self-administration revealed relatively high numbers of infusions at unit doses of 0.03 and 0.3 mg/kg/infusions and low numbers of infusions at unit doses of 0.01, 1, and 3 mg/kg/infusions. The numbers of morphine infusions earned over the last 3 sessions were averaged for each dose. (B) The number of lever presses for morphine infusions over the last 3 sessions were averaged for each dose. (C) Total amount of morphine infused at each dose. All data are reported as mean ± SE (n = 8–9 mice per dose of morphine). All data are expressed as the mean ± standard error for each group, and analyzed with one-way analysis of variance.

Figure 4

Accumbal miRNAs clustered after intravenous self-administration of morphine and saline. A heat map was created using significantly altered miRNAs according to the microarray data (n = 2); miRNA expression in the nucleus accumbens of the intravenous morphine self-administration and age-matched drug-naïve control groups were compared. The red color indicates an increased expression level and the green color indicates a decreased expression level.

Figure 5

Representative miRNA/target interaction networks in the NAccreated using CyTargetLinker following morphine self-administration. Based on the miRNA array data, we divided the responsive miRNAs into increased miRNAs and decreased miRNAs groups. After that, we used CyTargetLinker to show the interaction between responsive miRNAs and predicted target gene interaction after morphine self-administration. (A) Increased miRNAs-mRNA and (B) decreased miRNAs-mRNA showed diverse patterns of networks.

Figure 6

Classification of predicted target genes by morphine-responsive miRNAs according to the KEGG pathway analysis. A total of 62 altered miRNAs were analyzed based on the target prediction algorithms of the bioinformatics database. KEGG pathways targeted by (A) upregulated and (B) downregulated miRNAs in the NAc. The vertical axis represents the pathway categories and the horizontal axis represents pathway enrichment calculated as –log [modified Fisher's exact P-value].