Effects of Chronic Nicotine Exposure and 24h Withdrawal on Synaptosomal Protein Expression in the Mouse Ventral Hippocampus

Abstract

Despite cigarette smoking being the leading cause of preventable death in the United States 20% of Americans continue to smoke. We now know that 80% of smokers desire to quit; however, less than 5% of them are successful. The low success rate of smoking cessation is partially due to current pharmacotherapies only addressing a subset of nicotine withdrawal symptoms, which is the primary driver of smoking relapse. A prominent, yet unaddressed, symptom of nicotine withdrawal is affective dysfunction. Affective symptoms, such as increased anxiety and reduced emotional regulation, are underpinned by discrete disruption of select brain circuits. For example, the ventral hippocampus (VHIPP) has a well-described role sub-serving the interface between contextual learning and affective responding, suggesting it as a potential circuitry target for examination of molecular alterations during nicotine withdrawal. Therefore, we explored this by treating 18-week-old mice for two weeks with either saline, nicotine (18 mg/kg/day), or 24h withdrawal from nicotine (18 mg/kg/day) via osmotic minipumps (Alzet 2002 model). Following treatment, the subjects were sacrificed and the VHIPP was collected and synaptosomes were prepared. These samples were analyzed via liquid chromatography-mass spectrometry to quantify changes in synaptosomal protein expression. These results were then analyzed using Enrichr pathway and transcriptional factor databases. Our results indicate changes in the axon guidance, vesicle-mediated transport, and transmission across chemical synapses pathways in both nicotine versus saline and withdraw versus saline synaptosomal protein comparisons. However, upon qPCR analysis mRNA expression of key genes associated with the pathways of interest remained unchanged for each treatment group. Further experiments will utilize western blotting to validate changes in total and phosphorylated proteins of interest in the aforementioned pathways.