Integrated miRNA/mRNA-Seq of the Habenulo-Interpeduncular Withdrawal Circuit

Nicotine dependence is responsible for perpetuating the adverse health effects due to tobacco use, the leading cause of preventable death worldwide. Nicotine is an agonist for nicotinic acetylcholine receptors, which are enriched in the habenulo-interpeduncular withdrawal circuit. Drugs of abuse, including nicotine, induce stable neuroadaptations, requiring protein synthesis through regulation of transcription factors, epigenetic mechanisms, and non-coding RNAs. It also been shown that miRNAs in brain are regulated by nicotine and that miRNA dysregulation contributes to brain dysfunction, including drug addiction. While much is known about the neurocircuitry responsible for the behaviors associated with nicotine reward or withdrawal, the underlying mechanisms of how changes in behavior are induced are less clear. Using miRNA- and mRNA-Seq, we demonstrate that there are widespread changes in both miRNA and mRNA expression in the IPN and MHb during acute nicotine withdrawal. Conserved, differentially expressed miRNAs were predicted to target inversely regulated mRNAs. This dataset represents a valuable resource, identifying a multitude of miRNAs/genes, which upon further study may reveal new mechanisms underlying the neuroadaptations of nicotine dependence and the symptoms of nicotine withdrawal. Male C5BL/6J mice were treated with chronic nicotine (NIC) or control tartaric acid solution (TA) orally. Additional nicotine withdrawal groups were treated with nicotine and then spontaneously withdrawn for 48 hours (NAWD) or 4 weeks (NLWD). Mice were sacrificed for tissue collection and the interpeduncular nucleus (IPN) and medial habenula (MHb) were extracted by tissue punch. Each sample contained tissue pooled from 4 animals. Total RNA was isolated and small RNAs were separated by denaturing gel electrophoresis. Separate sequencing libraries were generated for miRNAs and mRNAs from the same total RNA samples. Stranded mRNA libraries were generated using an Illumina kit by manufacturer’s instructions. miRNAs and mRNAs were sequenced and differential expression and gene ontology analyses were performed.