The preclinical study of methamphetamine self-administration and the underlying molecular biology and proteomics changes in the reward system

Drug addiction is a chronic, relapsing brain disorder where users continue to seek out and take drugs despite the adverse consequences. Addiction is a complex brain disorder that combines changes in gene expression, protein expression, synaptic plasticity, and neurotransmitter release. The changes to these systems allow the brain to remodel in a process known as neuroplasticity which is thought to drive repeated drug-seeking and taking behaviour. In New Zealand, as many as 2.8% of people aged 15-64 have tried amphetamine-like substances at some stage in their life, which is one of the highest rates in the world (UNODC 2010). Methamphetamine is among the most highly addictive drugs in the world and strongly activates the natural reward system in the brain. Although much research has been done into the changes induced by methamphetamine, the molecular underpinnings are still incompletely understood. In addition, the molecular changes that correlate with different stages of the addiction cycle (binge, maintenance, withdrawal and relapse) are also not fully understood. Self-administration is a model of drug addiction with excellent face validity to human intake and changes that occur within the reward system allow researchers to gain useful insight into what happens in the human brain. A greater understanding of the molecular changes that occur with self-administered drugs will lead to better design of therapeutics for treating drug addiction in the future. This thesis research examined methamphetamine self-administration in rats and the effect on the dopaminergic system following 14 days of abstinence. It used an approach that combined the investigation of a single protein (dopamine transporter), a global proteomics approach and a global gene expression approach. There was no change in dopamine transporter function or expression in the dorsal striatum and nucleus accumbens, two brain regions essential for the development and expression of drug addiction behaviours. This result is in contrast to findings that used “binge” injections which lead to extensive neurotoxicity, suggesting that methamphetamine self-administration does not lead to dopamine terminal degeneration. Global shotgun proteomics was performed in synaptosomes prepared from the dorsal striatum of rats trained for methamphetamine self-administration. This study identified 27 differentially-expressed proteins from purified synaptosomes that were involved in mitochondria, synaptic vesicles, cytoskeletal rearrangement, cell signalling and neuroprotection. To complement this work, a global gene expression study was performed in the ventral tegmental area, which is a region of the brain that contains dopaminergic cell bodies. Gene expression microarrays identified 49 differentially-expressed genes and 71 differentially-expressed microRNA following methamphetamine self-administration. These genes were involved in endosomes, cell signalling, neurite growth, neuroprotection, RNA-processing and transcriptional regulation. In addition, 11 of the mRNA changes have predicted binding sites for the differentially-expressed microRNA. One of the differentially-expressed microRNA, miR-145, appears to be enriched in both mRNA and proteomics datasets, suggesting that it plays a significant role in the cellular effects of methamphetamine across multiple brain regions. The understanding of the cellular processes altered with long-term methamphetamine exposure that persist into abstinence will lead to the better development of pharmacotherapies to treat drug relapse, which occurs in as many as 80% of psychostimulant addicts (Shippenberg et al. 2007). To this end, a novel kappa-opioid receptor agonist was tested for its effect on drug-seeking behaviour. The agonist, KMS, significantly reduced cocaine-seeking behaviour and may be a useful drug to test with methamphetamine in the future. This thesis reports a number of novel proteins, genes and miRNA previously not associated with methamphetamine and contributes to the extensive knowledge that has been generated regarding methamphetamine-induced neural changes. The application of large scale “omics” datasets to a highly relevant behavioural model should lead to important novel mediators of the addiction process, which should be translatable to the human experience.