Opiate drugs are the leading treatment for severe or chronic pain in the USA despite their extremely addictive properties. Chronic opiate exposure induces unique neuroadaptations in the mesocorticolimbic system, particularly in ventral tegmental area (VTA) dopamine (DA) neurons. For example, opiates reduce VTA DA neuron soma size, a change correlated with increased DA activity and reward tolerance. Prevention of this morphological change is sufficient to rescue the morphine-induced changes to behavior, suggesting its direct involvement in addiction-related processes. To better understand the circuit-based consequences of morphine-induced neuroadaptations, we compared the morphology of VTA DA neurons that project to the nucleus accumbens (NAc) and medial prefrontal cortex (mPFC) in sham and morphine treated mice. Chronic morphine treatment significantly altered the soma size of NAc m.shell- and PFC- projecting VTA DA neurons, but conversely had no effect on NAc l.shell projecting VTA DA. While VTA DA structural and functional plasticity are central to morphine reward and addiction, the molecular responses driving these neuroadaptations remain elusive. To date, studies of drug-induced changes in VTA gene expression have been limited to the homogenization of the entire VTA, which includes GABAergic and dopaminergic (tyrosine hydroxylase (TH)-positive) neurons. While several candidate genes have been identified with this approach, it is unknown whether these changes occur specifically in DA neurons and contribute to the structural neuroadaptations. To determine morphine-induced gene expression changes specifically in VTA DA neurons, we utilized Translating Ribosome Affinity Purification (TRAP). We crossed DA- (TH- or dopamine transporter (DAT)-Cre) driver lines with Rosa26 EGFP-L10a mice, thereby allowing for isolation of mRNA from VTA DA neurons. In both DA-specific lines (THL10a-GFP and DATL10a-GFP), we found significant enrichment of DA-specific markers and depletion of GABAergic markers in DA-specific IP fractions compared to input controls, consistent with successful purification. We then completed RNA sequencing on samples from DATL10a-GFP as an unbiased approach to identify changes that occur specifically in VTA DA cells. Using differential gene expression (DEG) analysis, we first identified 4,499 significantly enriched/depleted genes in sham-treated VTA DA-specific IP compared to sham-input (VTA DA transcriptome). In the following DEG analyses, we identified 410 significant morphine regulated genes in whole VTA input, and 393 significant morphine regulated genes in VTA DA-specific IP. We then validated select candidate genes in separate DATL10a-GFP TRAP samples using RT-PCR. Overall, the results of this dissertation identified projection-specificity of morphine-induced changes in VTA DA neuron morphology and identified morphine-induced gene expression changes specifically in VTA DA neurons. These findings are critical in driving our understanding of morphine-induced adaptations in specific VTA DA circuits as well as to identify novel mechanisms that underlie opiate-induced neuroadaptations in the VTA in order to develop innovative targets for improved therapeutics.
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