This project responds to several sections in Center for Tobacco Products Research Plan. Nicotine dependence (Tobacco Use Disorder, DSM-5, 305.1, and Tobacco Withdrawal, 292.0) is the tobacco-related disorder that underlies all other tobacco-related diseases. Establishing a biomarker for nicotine dependence in animals, and then exploiting this biomarker for information on menthol's actions, will contribute importantly to tobacco control research. A biomarker relevant to distinct components of human nicotine dependence must be localized at the level of brain region, of individual cell types, and of axonal vs. somatodendritic compartments. The biomarker must develop during maintained exposure to nicotine. Furthermore, this biomarker should immediately be exploited for studies on menthol. The biomarker chosen will consist of detecting the total level of beta2 nicotinic acetylcholine receptor (nAChR) subunits (intracellular plus plasma membrane). Mouse brain is an entirely appropriate model system. The approach develops, and then begins to exploit, a production-level assay for determining the upregulation of beta2* nAChRs in mouse brain. Maintained exposure to nicotine produces upregulation of beta2* nAChRs, and such upregulation is hypothesized to be both necessary and sufficient for some early stages of nicotine dependence.
Aim 1 develops a rather efficient, quantitative, but anatomically low-resolution approach, refining an immunoblot technique ("western blots") for quantifying the amount of beta2 subunits in various regions of mouse brain. The approach will lead to valuable data.
Aim 2 develops a higher-resolution, semiquantitative approach that identifies the neuronal cell types in which upregulation occurs, as well as the subcellular regions (axonal vs. somatodendritic) in which upregulation occurs.
Aim 2 employs a recently developed strain of beta2-GFP knock-in mice. Most of Aim 2 uses direct fluorescence for the GFP-labeled beta2 subunit;immunofluorescence is used to enhance sensitivity.
Aim 2 is not on the production pathway for further work, but the data of Aim 2 are the minimum necessary for understanding and validating the beta2 biomarker.
Aim 3 uses these tools to test an important hypothesis: that menthol upregulates nAChRs even when delivered intravenously. The underlying assumption is that menthol increases tobacco dependence via events at the level of neurons, in addition to possible increased absorption of nicotine through airway epithelium or possible decreased nicotine catabolism. The overall results of this project will be a production-level procedure that can be used to assess levels in the beta2 subunit biomarker as a function of key experimental variables that interest the Center for Tobacco Products. Furthermore Aim 3 will use the beta2 biomarker to begin answering the important question, where does menthol act? FDA requires this key information in order to evaluate / measure menthol. Future projects can test additional variables including flavorings, additional possible addictive components such as cotinine, and other compounds in tobacco smoke. The beta2 biomarker assay will also be appropriate for mice engineered to possess alterations in accessory proteins. Therefore this project responds to the RFA with high impact, high significance, and a highly appropriate approach.
Nicotine dependence is the tobacco-related disorder that underlies all other tobacco-related diseases. Establishing a biomarker for nicotine dependence in animals, and then exploiting this biomarker for information on menthol's actions, will contribute importantly to tobacco control research.