2022SCK8MW - A systematic analysis of neuronal activity alterations during chronic cigarette smoke and e-cigarette vapor exposure and withdrawal in the mouse

Pathologies related to tobacco smoke are the leading preventable cause of death worldwide. Tobacco consumption leads to nicotine addiction, making smoking cessation challenging for most individuals. Recently, e-cigarettes (eCigs) have been introduced as a less harmful means of nicotine delivery than traditional cigarettes (Cigs). Though less harmful than Cigs, e-Cigs are still associated with some pathologies and, due to their novelty, long-term health effects are still unknown. Moreover, their use among adolescents and young adults is growing and leads to nicotine addiction, which predisposes to subsequent use of Cigs. The goal of this project is to obtain a characterization of the neurophysiological response to chronic Cig smoke (CigS) and eCig vapor (eCigV) in mice, both during exposure and withdrawal. The project specific objectives are to obtain: 1. a quantitative behavioral characterization of mice during exposure to and withdrawal from chronic Cig smoke (CigS) or eCig vapor (eCigV). 2. a global mapping of the activated neuronal populations during exposure to and withdrawal from chronic CigS or eCigV. 3. an evaluation of the functional changes in the striatal medium spiny neurons (MSNs) of the direct and indirect pathway during withdrawal from chronic CigS or eCigV. More in detail: 1. The effects of chronic CigS and eCigV exposure will be studied through innovative technologies to provide the most reliable information. Besides classical tests for mouse behaviors, animal behavior will be tracked with cameras during exposure and in their home cage: deep-learning approaches will be employed to track behavior and quantitatively recording the animal habits, locomotion, social interactions, and stereotypies. 2. We will visualize c-Fos, a marker of neuronal activity, in the entire mouse brain throughout the exposure and withdrawal periods. Moreover, persistent genetic tagging of the neuronal ensemble active during early withdrawal will be induced in animals through “TRAP” (Targeted Recombination in Active Populations), causing fluorescent expression in active cells at the selected time point. After sacrifice, the animal brains will be optically cleared and imaged in their entirety through Light-Sheet Fluorescence Microscopy, to identify brain areas and neuronal populations involved in addictive processes. 3. Transgenic animals expressing calcium sensitive fluorescent proteins in striatal MSNs will be implanted with a miniaturized endoscopic head mounted microscope. The head mounted microscopes will record the activity of neurons of both the direct and indirect pathways, which will be compared to the mouse behavior, simultaneously recorded through an external camera throughout the CigS or eCigV exposure and withdrawal periods. Overall, in this project we propose to study and compare with unprecedented depth the behavioral and functional effects of chronic CigS and eCigV exposure and withdrawal.