Brandon J. Henderson, Ph.D. - US grants

DESCRIPTION (provided by applicant): Alpha6* (alpha6-containing) nicotinic acetylcholine receptors (nAChRs) play an important role in nicotine addiction. Alpha6* nAChRs are found in just a few regions of the mammalian brain (the mesolimbic and nigrostriatal dopaminergic pathways) and mediate reward and motor control. Aim 1 develops and exploits a cell line expressing alpha6beta2beta3 nAChRs without the use of chimeric or concatameric subunits, which have both been required for the successful expression of this subtype in cell lines. Successful expression of alpha6beta2beta3 nAChRs will be aided by expressing two point mutations in the beta2 subunit, which have been found to enhance export from the endoplasmic reticulum (ER). In addition, a fluorescent marker for ER exit sites (Sec24D) will be used to identify cells with abundant ER exit sites which are a marker for high levels of alpha6beta2beta3 nAChR expression on the plasma membrane. Functional alpha6beta2beta3 will be characterized using electrophysiological techniques. A distinguishing factor that governs functional properties of nAChRs is the subunit stoichiometry of the assembled pentamers. Aim 2 therefore determines the subunit stoichiometry of alpha6beta2beta3 nAChRs using a novel technique involving zero-mode waveguides (ZMWs). ZMWs allow the single-molecule analysis of a single membrane receptor by seeding cells on nanofabricated apertures ~100 nm diameter. Combining fluorescent nAChR subunits and photobleaching techniques will allow the quantification of specific subunits in receptor assemblies. It is well known that some nAChRs (e.g., alpha4beta2) are upregulated when chronically stimulated by nicotine. This change in receptor number has been implicated as one of the mechanisms that is critical for nicotine addiction. To determine how alpha6* nAChRs may play a role in nicotine addiction, Aim 3 determines how these receptors are changed when exposed to nicotine by using mice that have fluorescent alpha6 nAChR subunits. The mice will be chronically exposed to nicotine and quantitative fluorescence will document the change in receptor number. The understanding of how nicotine, in comparison to other drugs (i.e., acetylcholine, epibatidine), modifies the functio and receptor levels of alpha6* nAChRs will provide novel insights regarding the mechanisms that mediate nicotine addiction. Characterization of alpha6beta2beta3 nAChRs, determination of alpha6beta2beta3 nAChR stoichiometry, and determination of upregulation of alpha6* nAChRs will advance the understanding of nicotine addiction and will further the development of novel smoking cessation therapeutics.

? DESCRIPTION (provided by applicant): Currently, it is unknown how menthol may play a role in the addiction to nicotine. Despite this, it is clear that people who smoke menthol cigarettes exhibit poorer quit rates compared to those who smoke non- menthol cigarettes. I have a unique opportunity at Caltech to gain understanding of menthol's effects as I have access to unique tools that will facilitate the in vivo characterization of menthol's effect on the addiction to nicotine. I plan to combine multiple in vivo techniques to look specifically at how menthol alters nicotine reward, dopamine release, and neurobiology. These include: 1) behavioral assays with conditioned place preference and self-administration; 2) in vivo and ex vivo voltammetry assays; 3) electrophysiology using mouse brain slices; and 4) microscopy to study changes in neurobiology. I, along with NIDA, hold a strong interest in menthol and how menthol plays a role in nicotine addiction. I have preliminary data showing that menthol enhances nicotine reward-related behavior in mice in a conditioned place preference assay. Despite this, there is much that we need to know concerning menthol. This proposal aims to address many of the concerns issued recently by NIDA concerning menthol. The assays (behavior, voltammetry, microscopy, and electrophysiology) are planned to be synergistic. Doses of nicotine and menthol found to be efficacious in behavior assays will be used in both microscopy and electrophysiology assays. Through these proposed assays, my goal is to collect data that will further the aims of NIDA by providing the basic research that will improve public health and encourage new science that will reduce tobacco dependence. To accomplish this proposed research plan, I will pursue two years of additional postdoctoral training in animal behavioral assays and fast scan cyclic voltammetry under the supervision and direction of my mentors. Henry Lester, PhD (Caltech), my primary mentor is an expert in neuroscience, neurobiology, and biophysics. Marina Picciotto, PhD (Yale) and Nii Addy, PhD (Yale) are both experts in neuropharmacology, rodent behavior, rodent genetics, and mechanisms in reward, learning, and memory. At Caltech and Yale, I will have all the necessary guidance, tools, and resources to be adequately trained in the new assays and successfully complete this project. With the skills attained from my additional training, I will transition to an academic faculty position as a independent investigator.

? DESCRIPTION (provided by applicant): Currently, it is unknown how menthol may play a role in the addiction to nicotine. Despite this, it is clear that people who smoke menthol cigarettes exhibit poorer quit rates compared to those who smoke non- menthol cigarettes. I have a unique opportunity at Caltech to gain understanding of menthol's effects as I have access to unique tools that will facilitate the in vivo characterization of menthol's effect on the addiction to nicotine. I plan to combine multiple in vivo techniques to look specifically at how menthol alters nicotine reward, dopamine release, and neurobiology. These include: 1) behavioral assays with conditioned place preference and self-administration; 2) in vivo and ex vivo voltammetry assays; 3) electrophysiology using mouse brain slices; and 4) microscopy to study changes in neurobiology. I, along with NIDA, hold a strong interest in menthol and how menthol plays a role in nicotine addiction. I have preliminary data showing that menthol enhances nicotine reward-related behavior in mice in a conditioned place preference assay. Despite this, there is much that we need to know concerning menthol. This proposal aims to address many of the concerns issued recently by NIDA concerning menthol. The assays (behavior, voltammetry, microscopy, and electrophysiology) are planned to be synergistic. Doses of nicotine and menthol found to be efficacious in behavior assays will be used in both microscopy and electrophysiology assays. Through these proposed assays, my goal is to collect data that will further the aims of NIDA by providing the basic research that will improve public health and encourage new science that will reduce tobacco dependence. To accomplish this proposed research plan, I will pursue two years of additional postdoctoral training in animal behavioral assays and fast scan cyclic voltammetry under the supervision and direction of my mentors. Henry Lester, PhD (Caltech), my primary mentor is an expert in neuroscience, neurobiology, and biophysics. Marina Picciotto, PhD (Yale) and Nii Addy, PhD (Yale) are both experts in neuropharmacology, rodent behavior, rodent genetics, and mechanisms in reward, learning, and memory. At Caltech and Yale, I will have all the necessary guidance, tools, and resources to be adequately trained in the new assays and successfully complete this project. With the skills attained from my additional training, I will transition to an academic faculty position as a independent investigator.

Project Summary/Abstract There is a fundamental gap in the understanding of how tobacco flavorants alter nicotine addiction. Since electronic nicotine delivery systems (ENDS) offer a multitude of flavors, there is a critical need to under- stand how these flavors alter nicotine addiction. Until this knowledge gap is closed, we face the risk of in- creased smoking initiation, decreased cessation, and a cumulative effect of a growing population of lifelong smokers in America. Our overall goal is to identify a biomarker to test which flavorants enhance nicotine addic- tion and abuse liability. This is a major priority of the FDA-CTP. We hypothesize that ?4?6?2 nicotinic acetylcholine receptor (nAChR) upregulation is a suitable bi- omarker for identifying flavorants that enhance nicotine addiction. The rationale behind this comes from the applicant?s previous findings that only ?4?6?2 nAChRs are upregulated by concentrations of nicotine that are sufficient to elicit reward-related behavior in animal models. Therefore, ?4?6?2 nAChRs present a potential biomarker that is more sensitive when compared to other nAChR subtypes. Furthermore, the applicant has shown that an enhancement in nicotine reward by menthol, the most popular tobacco flavorant, is accompa- nied by enhancements in ?4?6?2 nAChR upregulation. This strong preliminary data suggests that these meth- ods can be utilized to develop ?4?6?2 nAChR upregulation as a novel biomarker to examine other flavorants for their role in enhancing nicotine addiction and abuse liability. This novel biomarker will be validated by exam- ining two chemicals used in green apple and fruit flavorants (farnesol and farnesene) in three specific aims: 1) characterization of flavorants? effect (farnesol and farnesene) on nicotine reward-related behavior; 2) character- ization of flavorants? effect on nicotine-induced ?4?6?2 nAChR upregulation; and 3) characterization of flavor- ants? effect on nicotine-induced changes in dopamine neuron excitability. In the first aim, a mouse conditioned place preference assay identical to one used to report menthol-induced enhancements in nicotine reward will determine if the flavorants farnesene and farnesol enhance nicotine reward. These assays will be supplement- ed with mouse self-administration assays utilizing e-VapeTM technology. The second aim will use the brains from Aim 1 mice to examine ?4?6?2 nAChR upregulation and provide a direct correlation between enhance- ments in nicotine reward and ?4?6?2 nAChR upregulation. This will be a critical feature to validate our bi- omarker assay. Aim 3 will provide validation measures that will supplement upregulation using electrophysio- logical methods to examine changes in dopamine neuron excitability. This approach is innovative, in the appli- cant's opinion, because it establishes a direct correlation between nicotine reward and nAChR upregulation in an in vivo model. This is complemented by the use of a novel mouse expressing ?4-mCherry and ?6-GFP nA- ChR subunits that allow analysis of upregulation without the use of antibodies. The proposed research is signif- icant, because it presents a novel biomarker method to identify tobacco flavorants that pose a risk to nicotine addiction and abuse liability. This would contribute significantly to several of the FDA-CTP research priorities.

Project Summary/Abstract There is a fundamental gap in the understanding of how electronic nicotine delivery systems (ENDS) al- ter the adolescent brain. Adolescents are a high-risk population in regards to nicotine-containing products as prenatal or early exposure triggers significant changes in the prefrontal cortex. With the growing popularity of ENDS among American adolescents, there is a critical need to understand how ENDS devices alter neurobiol- ogy to trigger addiction to nicotine. This is especially true given that ENDS are unique from combustible ciga- rettes given the multitude of flavors and different nicotine formulations that are specific to only ENDS e-liquids. Additionally, pod-based ENDS (i.e., Juul) contain a significantly higher concentration of nicotine compared to combustible cigarettes and tank-based ENDS. Until this knowledge gap is closed, we face the risk of increased smoking initiation, decreased cessation, and a cumulative effect of a growing population of lifelong smokers in America. Our overall goal is to identify the key changes in neurobiology, specific to ENDS, in an adolescent mouse model system that regulates nicotine reward and reinforcement. To address this, we will utilize a novel contingent nicotine self-administration assay system that allows us to use the same e-liquid tanks and Pods popular with adolescent ENDS users in a mouse model system. This will provide high translational value as we can directly assess how ENDS directly alter neurobiology and neurophysiology. We hypothesize that directly linking self-administration behavior to nAChR upregulation and changes in neurophysiology will identify brain regions and cell-types that are critical for the initiation of nicotine addiction and continued reinforcement. The rationale behind this comes from the applicant?s previous success in corre- lating nicotine reward to nAChR upregulation. We will identify ENDS-specific changes in neurobiology with three specific aims. First, we will utilize e-Vape nicotine self-administration assays in mice to examine initiation and nicotine reinforcement of ENDS to examine the impact of nicotine dose, formulation, and flavors on vap- ing-related behavior. Second, we will use the brains from the first aim to examine nAChR upregulation and provide a direct link between self-administration behavior and nAChR upregulation. Third, we will examine changes in neurophysiology via electrophysiology and fast-scan cyclic voltammetry. This approach is innovative, in the applicant's opinion, because it establishes a direct correlation be- tween vaping-related self-administration and nAChR upregulation as well as changes in neurophysiology in an in vivo model and utilizes the exact same ENDS and e-liquids popular with human adolescent vapers. This is complemented by the use of a novel mouse expressing ?4-mCherry and ?6-GFP nAChR subunits that allow analysis of upregulation without the use of antibodies. The proposed research is significant, because it will dramatically increase our knowledge of how ENDS contribute to addictive behavior. This would contribute sig- nificantly to several of the priorities and interests of NIDA.