MicroRNAs Are Involved in the Development of Morphine-Induced Analgesic Tolerance and Regulate Functionally Relevant Changes in Serpini1.

Long-term opioid treatment results in reduced therapeutic efficacy and in turn leads to an increase in the dose required to produce equivalent pain relief and alleviate break-through or insurmountable pain. Altered gene expression is a likely means for inducing long-term neuroadaptations responsible for tolerance. Studies conducted by our laboratory (Tapocik et al., 2009) revealed a network of gene expression changes occurring in canonical pathways involved in neuroplasticity, and uncovered miRNA processing as a potential mechanism. In particular, the mRNA coding the protein responsible for processing miRNAs, Dicer1, was positively correlated with the development of analgesic tolerance. The purpose of the present study was to test the hypothesis that miRNAs play a significant role in the development of analgesic tolerance as measured by thermal nociception. Dicer1 knockdown, miRNA profiling, bioinformatics, and confirmation of high value targets were used to test the proposition. Regionally targeted Dicer1 knockdown (via shRNA) had the anticipated consequence of eliminating the development of tolerance in C57BL/6J (B6) mice, thus supporting the involvement of miRNAs in the development of tolerance. MiRNA expression profiling identified a core set of chronic morphine-regulated miRNAs (miR\u27s 27a, 9, 483, 505, 146b, 202). Bioinformatics approaches were implemented to identify and prioritize their predicted target mRNAs. We focused our attention on miR27a and its predicted target serpin peptidase inhibitor clade I (Serpini1) mRNA, a transcript known to be intricately involved in dendritic spine density regulation in a manner consistent with chronic morphine\u27s consequences and previously found to be correlated with the development of analgesic tolerance. In vitro reporter assay confirmed the targeting of the Serpini1 3′-untranslated region by miR27a. Interestingly miR27a was found to positively regulateSerpini1 mRNA and protein levels in multiple neuronal cell lines. Lastly, Serpini1 knockout mice developed analgesic tolerance at a slower rate than wild-type mice thus confirming a role for the protein in analgesic tolerance. Overall, these results provide evidence to support a specific role for miR27a and Serpini1 in the behavioral response to chronic opioid administration (COA) and suggest that miRNA expression and mRNA targeting may underlie the neuroadaptations that mediate tolerance to the analgesic effects of morphine

A Behavior-Genetic Strategy to Identify Genes and Genes Networks Involved in Opiate Tolerance and Addiction.

Morphine is an extremely efficient analgesic drug however when used repetitively can have two major detrimental side effects: tolerance and addiction. Understanding the molecular mechanisms that lead to these side effects may ultimately help develop improved therapeutics without tolerance and addiction liability. The goal of this dissertation was to discover candidate genes involved in morphine tolerance and addiction. To accomplish this goal a specific behavior genetics approach in conjunction with microarray analysis was used to identify high-value candidate genes. The genes were further investigated by bioinformatics analysis, rtPCR, and in vivo shRNA knockdown. To identify genes associated with tolerance two inbred genotypes which differ in morphine-related behaviors (C57BL/6J (B6), DBA2/J (D2)) and two reciprocal congenic genotypes (B6D2, D2B6) in which the proximal region of CH10 was introgressed into opposing backgrounds were used. The proximal region of CH10 has been implicated in morphine related behaviors. Tolerance following therapeutically-relevant doses of morphine developed most rapidly in the B6s followed by the B6D2s and did not develop in the D2s and only slightly in the D2B6s thus indicating a strong influence of the proximal region of CH10 in the development of tolerance. Gene expression profiling identified 81, 96, 106, and 82 genes involved in the development of tolerance in the periaquaductal gray (PAG), prefrontal cortex (PFC), temporal lobe (TL) and ventral striatum (VS) respectively. Bionformatics analysis highlighted genes associated with microRNA (miRNA) mechanisms in response to chronic administration. In vivo knockdown of Dicer1, the miRNA processing gene, in the PFC of the B6 genotype verified Dicer1's role in the development of tolerance. Gene expression profiling identified 33 miRNAs involved in the development of tolerance. B6 and D2 genotypes were also used to identify candidate genes in morphine addiction. Gene expression profiling identified total of 271 and 178 candidate genes in animals actively self-administering morphine, in the VS and ventral midbrain (VMB), respectively. Bionformatics analysis highlighted genes associated with miRNA mechanisms and synaptic plasticity. These results indicate a significant role of miRNAs and neuroadaptation genes in tolerance and addiction. Ultimately, the candidate genes could be pharmacological targets to counteract tolerance and decrease addiction liability.Includes 7 tables in .doc and .xls formats. Word 97-2003 Document. Excel 97-2003 Worksheet

A Behavior-Genetic Strategy to Identify Genes and Genes Networks Involved in Opiate Tolerance and Addiction.

Morphine is an extremely efficient analgesic drug however when used repetitively can have two major detrimental side effects: tolerance and addiction. Understanding the molecular mechanisms that lead to these side effects may ultimately help develop improved therapeutics without tolerance and addiction liability. The goal of this dissertation was to discover candidate genes involved in morphine tolerance and addiction. To accomplish this goal a specific behavior genetics approach in conjunction with microarray analysis was used to identify high-value candidate genes. The genes were further investigated by bioinformatics analysis, rtPCR, and in vivo shRNA knockdown. To identify genes associated with tolerance two inbred genotypes which differ in morphine-related behaviors (C57BL/6J (B6), DBA2/J (D2)) and two reciprocal congenic genotypes (B6D2, D2B6) in which the proximal region of CH10 was introgressed into opposing backgrounds were used. The proximal region of CH10 has been implicated in morphine related behaviors. Tolerance following therapeutically-relevant doses of morphine developed most rapidly in the B6s followed by the B6D2s and did not develop in the D2s and only slightly in the D2B6s thus indicating a strong influence of the proximal region of CH10 in the development of tolerance. Gene expression profiling identified 81, 96, 106, and 82 genes involved in the development of tolerance in the periaquaductal gray (PAG), prefrontal cortex (PFC), temporal lobe (TL) and ventral striatum (VS) respectively. Bionformatics analysis highlighted genes associated with microRNA (miRNA) mechanisms in response to chronic administration. In vivo knockdown of Dicer1, the miRNA processing gene, in the PFC of the B6 genotype verified Dicer1's role in the development of tolerance. Gene expression profiling identified 33 miRNAs involved in the development of tolerance. B6 and D2 genotypes were also used to identify candidate genes in morphine addiction. Gene expression profiling identified total of 271 and 178 candidate genes in animals actively self-administering morphine, in the VS and ventral midbrain (VMB), respectively. Bionformatics analysis highlighted genes associated with miRNA mechanisms and synaptic plasticity. These results indicate a significant role of miRNAs and neuroadaptation genes in tolerance and addiction. Ultimately, the candidate genes could be pharmacological targets to counteract tolerance and decrease addiction liability.Includes 7 tables in .doc and .xls formats. Word 97-2003 Document. Excel 97-2003 Worksheet

Neuroplasticity, axonal guidance and micro-RNA genes are associated with morphine self-administration behavior

Neuroadaptations in the ventral striatum (VS) and ventral midbrain (VMB) following chronic opioid administration are thought to contribute to the pathogenesis and persistence of opiate addiction. In order to identify candidate genes involved in these neuroadaptations, we utilized a behavior-genetics strategy designed to associate contingent intravenous drug self-administration with specific patterns of gene expression in inbred mice differentially predisposed to the rewarding effects of morphine. In a Yoked-control paradigm, C57BL/6J mice showed clear morphine-reinforced behavior, whereas DBA/2J mice did not. Moreover, the Yoked-control paradigm revealed the powerful consequences of self-administration versus passive administration at the level of gene expression. Morphine self-administration in the C57BL/6J mice uniquely up- or down-regulated 237 genes in the VS and 131 genes in the VMB. Interestingly, only a handful of the C57BL/6J self-administration genes (\u3c3%) exhibited a similar expression pattern in the DBA/2J mice. Hence, specific sets of genes could be confidently assigned to regional effects of morphine in a contingent- and genotype-dependent manner. Bioinformatics analysis revealed that neuroplasticity, axonal guidance and micro-RNAs (miRNAs) were among the key themes associated with drug self-administration. Noteworthy were the primary miRNA genes H19 and micro-RNA containing gene (Mirg), processed, respectively, to mature miRNAs miR-675 and miR-154, because they are prime candidates to mediate network-like changes in responses to chronic drug administration. These miRNAs have postulated roles in dopaminergic neuron differentiation and mu-opioid receptor regulation. The strategic approach designed to focus on reinforcement-associated genes provides new insight into the role of neuroplasticity pathways and miRNAs in drug addiction. © 2012 The Authors, Addiction Biology © 2012 Society for the Study of Addiction

Identification of candidate genes and gene networks specifically associated with analgesic tolerance to morphine

Chronic morphine administration may alter the expression of hundreds to thousands of genes. However, only a subset of these genes is likely involved in analgesic tolerance. In this report, we used a behavior genetics strategy to identify candidate genes specifically linked to the development of morphine tolerance. Two inbred genotypes [C57BL/6J (B6), DBA2/J (D2)] and two reciprocal congenic genotypes (B6D2, D2B6) with the proximal region of chromosome 10 (Chr10) introgressed into opposing backgrounds served as the behavior genetic filter. Tolerance after therapeutically relevant doses of morphine developed most rapidly in the B6 followed by the B6D2 genotype and did not develop in the D2 mice and only slightly in the D2B6 animals indicating a strong influence of the proximal region of Chrl0 in the development of tolerance. Gene expression profiling and pattern matching identified 64,53,86, and 123 predisposition genes and 81, 96,106, and 82 tolerance genes in the periaqueductal gray (PAG), prefrontal cortex, temporal lobe, and ventral striatum, respectively. A potential gene network was identified in the PAG in which 19 of the 34 genes were strongly associated with tolerance. Eleven of the network genes were found to reside in quantitative trait loci previously associated with morphine-related behaviors, whereas seven were predictive of tolerance (morphine-naive condition). Overall, the genes modified by chronic morphine administration show a strong presence in canonical pathways representative of neuroadaptation. A potentially significant role for the micro-RNA and epigenetic mechanisms in response to chronic administration of pharmacologically relevant doses of morphine was highlighted by candidate genes Dicer and H19. Copyright © 2009 Society for Neuroscience

Neuroplasticity, axonal guidance and micro-RNA genes are associated with morphine self-administration behavior

Neuroadaptations in the ventral striatum (VS) and ventral midbrain (VMB) following chronic opioid administration are thought to contribute to the pathogenesis and persistence of opiate addiction. In order to identify candidate genes involved in these neuroadaptations, we utilized a behavior-genetics strategy designed to associate contingent intravenous drug self-administration with specific patterns of gene expression in inbred mice differentially predisposed to the rewarding effects of morphine. In a Yoked-control paradigm, C57BL/6J mice showed clear morphine-reinforced behavior, whereas DBA/2J mice did not. Moreover, the Yoked-control paradigm revealed the powerful consequences of self-administration versus passive administration at the level of gene expression. Morphine self-administration in the C57BL/6J mice uniquely up- or down-regulated 237 genes in the VS and 131 genes in the VMB. Interestingly, only a handful of the C57BL/6J self-administration genes (\u3c3%) exhibited a similar expression pattern in the DBA/2J mice. Hence, specific sets of genes could be confidently assigned to regional effects of morphine in a contingent- and genotype-dependent manner. Bioinformatics analysis revealed that neuroplasticity, axonal guidance and micro-RNAs (miRNAs) were among the key themes associated with drug self-administration. Noteworthy were the primary miRNA genes H19 and micro-RNA containing gene (Mirg), processed, respectively, to mature miRNAs miR-675 and miR-154, because they are prime candidates to mediate network-like changes in responses to chronic drug administration. These miRNAs have postulated roles in dopaminergic neuron differentiation and mu-opioid receptor regulation. The strategic approach designed to focus on reinforcement-associated genes provides new insight into the role of neuroplasticity pathways and miRNAs in drug addiction. © 2012 The Authors, Addiction Biology © 2012 Society for the Study of Addiction