Nicotine Alters Gene Regulatory Networks in Dopaminergic Neurons in the Ventral Tegmental Area
Keller, Renee F
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Maternal smoking during pregnancy is associated with developmental, cognitive, and behavioral disorders, including a predisposition to drug abuse later in life. Studies have shown that nicotine, the primary addictive and psychoactive component of tobacco, modulates gene expression in many brain regions implicated in the reward pathway. Dopaminergic (DA) neurons originating from the ventral tegmental area (VTA) of the brain are activated by nicotine and form the natural reward pathway that is known to contribute to addiction. In recent years, microRNAs (miRNAs) have been found to post-transcriptionally regulate gene expression with the ability to target multiple genes. Dysfunction in regulation mechanisms has been associated with various disorders, including addiction. First, we sought to investigate the altered expression of addiction-related miRNAs in DA and non-DA neurons from the VTA following perinatal nicotine exposure. We employed patch clamping to identify and harvest DA and non-DA neurons in the VTA from rats perinatally exposed to nicotine and used single-cell RT-qPCR to investigate expression changes. Next, we conducted a large-scale study using microarrays in order to identify mRNA and miRNA expression differences in DA neurons. We used these results to create miRNA-gene prediction networks and to identify gene regulatory networks that are significantly altered by genes and miRNAs following perinatal nicotine exposure. Then, we expanded the microarray to include non-DA samples in order to compare differential expression and nicotine-modulated regulatory pathways of DA and non-DA neurons in the VTA in order to examine alterations in the area based on neuron type that were due to perinatal nicotine exposure. Lastly, we investigated neuron populations in the sub-regions of the VTA, which display altered neural activity patterns following perinatal nicotine exposure. Using reverse transcription quantitative polymerase chain reaction (RT-qPCR) and immunohistochemistry (IHC) to investigate the number of GABA, DA, and glutamate neurons, we found that the middle region of the VTA contained a more diverse population of neurons and the bottom region showed a higher expression of DA markers. Our investigations have shown that perinatal nicotine exposure modulates genes and miRNAs that dysregulate neurogenesis, neuron development, synapse formation and connectivity, which are associated with the conditions associated with maternal smoking during pregnancy.