Methamphetamine (METH) is a powerful psychostimulant with a high abuse liability. Due to its potent and long lasting effects in the central nervous system (CNS), METH addiction is a major and growing public health concern. Dextro-METH or the racemic mixture of the two isomers can be consumed through oral, nasal, and anal administrations, or injected intravenously or subcutaneously, and there is no FDA approved therapeutic for the treatment of METH addiction. METH abuse causes many deleterious physiological effects, such as anxiety, mood disturbances, and visual and auditory hallucinations. In addition, neuroimaging studies have demonstrated altered structural and functional changes in the brain associated with emotion and memory, as well as reduced motor speed and impaired verbal learning. Current literature implicates microglia, the resident macrophages of the CNS, as major mediators of the neurological side effects. Upon activation, microglia undergo a morphological change to an amoeboid shape with increased capacity for migration, phagocytosis and cytokine production. Although the initial microglial activation is poorly understood, METH-induced microgliosis precedes dopaminergic neurotoxicity, and drugs that prevent glial activation are candidate therapies for METH addiction. Microglia express purinergic receptors, ligand-gated ion channels, which have been implicated in a variety of chronic inflammatory and neurodegenerative processes. In particular, P2X7R is activated by pathological concentrations of ATP. I show the concurrent increases in P2X7R expression with Iba-1, a marker of microglial activation after chronic METH treatment in vivo. I confirmed the METH-induced increases in P2X7R protein and mRNA expression in the Embryonic Stem cell derived microglia (ESdM) cell line. siRNA knockdown of P2X7R in ESdM significantly reduced the METH-induced TNF-α secretion, compared to scrambled siRNA. Furthermore, I demonstrated METH-induced microglial migration and phagocytosis is blocked by pretreatment with a P2X7R antagonist, A-438079. When administered in vivo, the P2X7R antagonist A-438079 did not affect the locomotor activity of mice, as determined by ambulation, stereotypy and total activity at lower doses (5, 10 mg/kg) but it did decrease METH-induced stereotypy and ambulation at the higher dose tested (50mg/kg). The 10mg/kg dose of A-438079 was effective at blocking METH-induced expression of conditioned place preference, a behavioral assay that measures the appetitive value of a compound. This data suggests that P2X7R antagonism may be useful as a therapy for METH addiction. P2X7R activation is known to activate the inflammasome complex and lead to the generation and shedding of extracellular vesicles (EVs). EVs are classified as exosomes or microvesicles, and contain protein, mRNA and miRNA that alter the biological activity of target cells. I used electron microscopy, nanosight quantification, and confocal imaging techniques to confirm that METH causes the increase in primary human microglia-derived exosomes, which are around 100nm in size. Compared to control exosomes, METH causes significant decreases in packaged miR-124, mir-186 and miR-9, as analyzed by qPCR. These miRNAs are associated with many neuroimmune and neurodegenerative disorders, and indicate a possible mechanism for METH-dependent increased P2X7R expression seen in previous analyses. Taken together, these studies highlight the importance of P2X7R in METH-induced microgliosis. Our findings indicate that purinergic mechanisms contribute to altered microglia effector functions during stimulant abuse, and that modulating the glial response during addiction may have potential therapeutic implications. / Biomedical Sciences

Master of Science / Department of Psychological Sciences / Mary Cain / Research has demonstrated that an enriched rearing environment improves learning in many tasks. However, growing evidence suggests that an enriched environment may not provide the same benefits during a fear conditioning paradigm. In fact, it appears that an isolated rearing environment may facilitate acquisition of fear to an aversive stimulus. The neural mechanisms responsible for this disparity in fear learning among differentially reared animals are currently unknown. The NR2B subunit of the NMDA receptor has been shown to be involved in the acquisition of fear and influenced by differential rearing, making it a prime candidate to begin investigating these underlying neural mechanisms. Therefore, this study assessed the expression of the NR2B subunit in brain regions important for the acquisition of fear (amygdala and hippocampus) among differentially reared rats. Rats were reared in an enriched, an isolated, or a standard condition for 30 days. They received four tone-footshock pairings, after which their brains were removed and expression of the NR2B subunit was quantified in the basolateral amygdala (BLA), central nucleus of the amygdala (ACe), and the CA3 region of the hippocampus. Analyses found that the isolated rats began to acquire fear to the aversive stimulus faster than the enriched and standard housed rats. However, the isolated rats showed the least amount of NR2B expression in the BLA while there were no rearing differences in expression within the ACe or the CA3. The results from this study provide further insight to the importance of the rearing environment in learning and memory, especially the learning of fear, and its central neural basis.