Christie D. Fowler, PhD - US grants

DESCRIPTION (provided by applicant): In a number of mammalian species, neurogenesis has been found to continue throughout adulthood in the dentate gyrus of the hippocampus (DG) and subventricular zone (SVZ). Newly proliferated cells have also been found in other brain regions, including the amygdala and hypothalamus. Although a variety of factors have been found to influence adult neurogenesis, the underlying mechanisms remain to be determined. I have recently found that exposure to a male with mating for 48 hrs significantly increases neurogenesis in the DG, SVZ, and amygdala of adult female prairie voles, Microtus ochrogaster (16). Since male exposure increases the level of circulating estrogen (10, 45) and estrogen enhances the expression of brain-derived neurotrophic factor (BDNF) in the brain (37) in female prairie voles, and BDNF treatment enhances neurogenesis in the SVZ in rats (33), I hypothesize that estrogen and BDNF interact to regulate neurogenesis in adult female prairie voles. Three studies are proposed to test this hypothesis by focusing on neurogenesis in the DG. In Specific Aim 1, female voles in different social environments or under different hormonal influences will be examined for the number of proliferating cells in the DG to test the hypothesis that the estrogen surge associated with male exposure is responsible for increasiag neurogenesis. In Specific Aim 2, female voles will receive brain infusions of different doses of BDNF to test the hypothesis that BDNF acts to up-regulate neurogenesis. In Specific Aim 3, double labeling methods will be employed to examine the presence of trkB, the BDNF receptor, on proliferated cells to test the hypothesis that BDNF acts directly to regulate neurogenesis. Taken together, these studies should provide an understanding of the relationship between estrogen and BDNF in the regulation of neurogenesis in female prairie voles, in particular, and will further contribute to our overall understanding of the hormonal and neuronal mechanisms underlying adult neurogenesis.

DESCRIPTION (provided by applicant): Tobacco addiction has a significant negative impact on the health and economic status of the individual and society. Nicotine is considered to be the primary reinforcing component responsible for tobacco addiction in human smokers [2]. Nicotine's main site of action in the brain is the nicotinic acetylcholine receptor (nAChR), which is composed of discrete combinations of various subunits. The a5 subunit has garnered recent attention based on genetic linkage studies suggesting polymorphisms in the a5 nAChR subunit gene (CHRNA5) increase susceptibility to nicotine dependence in human smokers [11, 12]. Further, insertion of this subunit into certain nAChR subtypes has been shown to alter receptor desensitization and activation kinetics in Xenopus oocytes [5]. The objective of this proposal is to determine the role ofaS-containing nAChRs in nicotine reward, dependence and withdrawal in mice. The intravenous self-administration procedure is considered to be the most reliable measure of the reinforcing properties of drugs of abuse. In Specific Aim I, I will examine the role of the a5 subunit in nicotine reward by examining intravenous nicotine self-administration in wildtype (WT) mice and mice with a null mutation of the a5 nAChR subunit gene (a5-/-). The intracranial self-stimulation procedure (ICSS) is considered to be a direct measure of the functioning of the brain's natural reward pathways. Thus, in Specific Aim II, I will investigate the role of a5- containing nAChRs in regulating the stimulatory effects of acutely administered nicotine on brain reward systems by measuring nicotine-induced lowering of ICSS thresholds in WT and a5-/- mice. Finally, in Specific Aim III, I will examine the role of the aS-containing nAChRs in the expression of reward deficits during spontaneous nicotine withdrawal in WT and a5-/- mice;elevation of ICSS thresholds during spontaneous nicotine withdrawal will serve as a measure of the reward deficit associated with nicotine withdrawal. I hypothesize that aS-containing nAChRs play an important role in regulating the actions of nicotine on the brain reward circuits, and thereby regulate nicotine self-administration behavior, the acute reward-enhancing effects of nicotine, and the reward deficit associated with nicotine withdrawal. Together, these data may reveal fundamental insights into the neurobiological mechanisms underlying nicotine reward, dependence and withdrawal. Importantly, these findings could lead to the development of novel therapeutics efficacious for smoking cessation and/or relapse prevention in humans.

7. Project Summary/Abstract Nicotine contained in tobacco is considered to be the primary reinforcing component responsible for tobacco addiction in human smokers[1]. The positive effects of nicotine involve the brain¿s mesolimbic reward pathways[2,3], whereas the aversive effects appear to be mediated by the habenulo-interpeduncular pathway[4]. Defining the mechanisms that regulate the motivational signals to consume nicotine is essential to understanding the components that contribute to tobacco addiction in human smokers. The PI¿s past experiences have defined the function of the habenulo-interpeduncular pathway with respect to inhibition of intake at higher doses of nicotine[4]. Through this research, the PI has developed and validated a mouse model for nicotine self-administration, learned molecular techniques for genotyping and assessing mRNA expression in tissue, and assisted with the development and validation of lentiviral vectors, all of which provide a basis for the research proposed. The immediate goals of this proposal are twofold: (1) to further define the mechanisms of the habenulo-interpeduncular pathway and its afferents/efferents that regulate an inhibitory motivational signal to limit nicotine consumption, and (2) to provide an essential training experience for career and professional development to ensure success of the PI as an independent academic scientist. With regard to the first goal, the PI will apply the in vivo mouse neurooptogenetic technique to more clearly define the specific role of the pathways, neurotransmitters and receptors involved in an inhibitory motivational signal. The lightactivated inhibitory halorhodopsin pump (eNpHR3.0) will be injected into select brain regions to permit transfection of cholinergic neurons/axons in BAC transgenic choline acetyltransferase (ChAT)::Cre mice. Subsequently, the effect of light-mediated activation of this pump on intravenous nicotine self-administration behavior will be examined. It should be noted that this proposal is highly innovative by combining two novel techniques ¿ mouse intravenous nicotine self-administration and optogenetics ¿ to define the neuronal processes regulating nicotine¿s actions. The PI will receive further assistance/training from Dr. Garret Stuber, Dr. Peter Kalivas, and Dr. David Fitzpatrick. Neurotransmitter and receptor involvement in signal processing and protein changes with acute and chronic nicotine self-administration will also be examined. The overall hypothesis of this study is that the posterior septum transmits an inhibitory motivational signal to the medial habenula (MHb) and that this signal is then relayed to the interpeduncular nucleus (IPN) and then to the dorsal tegmental nucleus (DTN) to inhibit behavioral self-administration of nicotine. It is further hypothesized that cholinergic neurons expressing the alpha-5 nicotinic acetylcholine receptor (nAChR) subunit modulate information processing within the IPN-DTN pathway and that information is subsequently relayed from the DTN to the ventral tegmental area. Experiments outlined in this proposal will test these hypotheses and may thereby provide valuable insights into the mechanisms underlying the nicotine habit in human tobacco smokers. Further, the excellent environment at The Scripps Research Institute will provide the equipment, space, facilities, and scientific support necessary to complete the studies for the K99 phase. With this training, the PI may then begin to address long-term goals of elucidating the mechanisms by which nicotine modulates brain function and by which neuronal activity alters behavior to promote or inhibit further consumption of the drug. As such, R01 grant submissions during the independent phase may focus on other nAChR subunits and signaling mechanisms (dopamine, serotonin, enkephalin, etc.) in the MHb-IPN pathway for their involvement in nicotine reinforcement, dependence and withdrawal. These findings may then provide a basis for further identification of novel drug targets with the goal of developing more efficacious smoking cessation therapeutics. With regard to career development, the mentored career training in this proposal will be equally essential to ensure the PI¿s future success as an independent research investigator. First, the PI will obtain didactic instruction by attending a relevant course at either Cold Springs Harbor or Woods Hole and will conduct intensive reading of the literature to be discussed with the mentor and/or advisory committee members. Second, the PI will be trained in laboratory management and grant preparation and management. In addition to writing a complete and thorough R01 grant proposal, this training will promote knowledge of the preparation of budgets, tracking of expenditures, maintaining direction and preparation of progress reports. Further instruction will be obtained by attending an NIH Regional Seminar on Program Funding and Grants Administration offered by the NIH Office of Extramural Research. Third, the interactions with the mentor and newly-formed advisory committee will provide necessary feedback regarding progress and presentation skills, as well as input relative to job searches and transitioning to independence. These individuals have a great wealth of knowledge of the drug addiction field, hold numerous R01 and other grants, serve on NIH and non-NIH grant review committees, serve as editors on journals, and have been involved in faculty hiring. In addition, the PI will attend career development seminars offered by the Career and Postdoctoral Services office and Network for Women in Science at Scripps Florida, and will enroll in the Responsible Conduct of Research course. Therefore, with the hands-on, didactic and mentored training components, this plan will substantially enhance the PI¿s research skills in a new technique (optogenetics), permit her to use this technique under novel, innovative conditions (in conjunction with mouse self-administration), and advance her professional knowledge to ensure success and scientific independence to attain R01 funding in the early independent years as tenure-track faculty.

? DESCRIPTION (provided by applicant): Tobacco addiction imposes a significant negative impact on the health and economic status of the individual and society. The main psychoactive component in tobacco responsible for addiction is nicotine, which may also be consumed in alternate forms (e.g., vaporized liquid in e-cigarettes) with similar abuse potential. Unfortunatel, the vast majority of currently available pharmacotherapeutics for nicotine dependence are marginally effective in promoting long-term abstinence. Thus, a pressing need exists to identify novel targets for therapeutic development through innovative approaches/perspectives. Interestingly, the cerebrospinal fluid (CSF)-neuronal interface is beginning to emerge as a critical regulator of neural function and pathology. The choroid plexus and CSF provide a rich source of signaling molecules, and recent reports have uncovered the presence of a multitude of functional microRNAs (miRNAs). Past investigations into epigenetic regulation of gene expression have focused on signaling within the neurons themselves. However, we contend that this focus needs to be expanded to recognize the importance of intercellular communication via the extracellular environment. In this proposal, we will investigate circulating miRNAs from the CSF and establish their role in mediating gene expression in the habenula. Further, we predict that this will result in altered behavioral responses and consumption of nicotine. By identifying these novel extracellular mechanisms mediating nicotine reinforcement and reward, we hope to ascertain important insights into the persistence of the tobacco habit in human smokers/nicotine users. Moreover, identified circulating miRNAs have the potential to serve as biomarkers for nicotine addiction. It should also be noted that the choroid plexus is considered a main entry point for viral access into the brain via the CSF, and as such, an increased understanding of these processes may have broad implications for the pathogenesis of other human conditions, such as HIV/AIDS. In conclusion, findings from these investigations have to potential to significantly advance the field of epigenetic regulation of substance abuse, and in doing so, may induce a paradigm shift from an intracellular focus on neuronal function in addictive processes to recognize the importance of extracellular mechanisms. Through these efforts, we may achieve our overarching goal of identifying novel targets for the development of more efficacious therapeutics to treat nicotine dependence.

Given the recent increase in cannabis use in the US, there is an urgent need to more clearly understand the effects of cannabinoids on central and peripheral signaling mechanisms. The main psychoactive component in cannabis, ?9-tetrahydrocannabinol (THC), has been shown to act directly on the cannabinoid receptors. These receptors are expressed in both central and peripheral cellular populations that have been shown to secrete extracellular vesicles, including the choroid plexus, neurons, glia, and adipocytes. The main goals of this proposal are to investigate the effects of THC on extracellular vesicle signaling in the brain and blood and to identify a panel of biomarkers related to THC use. To further validate the selectivity of the biomarker profile, RNA expression patterns will be compared in the presence of another drug commonly co-used with cannabis, nicotine. Thereafter, we will determine whether THC's effects on extracellular vesicle density and RNA cargo can be attributed to release from dopaminergic cells in the ventral tegmental area, a brain region shown to mediate the reinforcing and rewarding properties of drugs of abuse. By systematically examining changes in extracellular vesicle signaling with THC exposure and relating to drug co-use conditions, these studies seek to reveal important insights into central and peripheral extracellular signaling dynamics. Further, given that peripheral miRNAs have been proposed as biomarkers of disease state, and THC-containing cannabinoids are often used by patients, such as individuals with cancer, findings may further inform on the relevance of other proposed biomarker profiles for clinical applications. Finally, with this foundation, cannabinoid-associated extracellular signaling factors may also lead to the identification of extracellular mechanisms underlying drug dependence and thus potential novel targets for therapeutic development to treat dependence.

PROJECT SUMMARY The Society for Research on Nicotine and Tobacco (SRNT) is the only international scientific membership society dedicated exclusively to the field of nicotine and tobacco research. SRNT has more than 1,200 members from over 40 countries, with approximately 80 percent of those members being affiliated with U.S. research, advocacy, pharmaceutical and related organizations. The overall objective of the SRNT Annual Meeting is to stimulate advancement in the field of nicotine and tobacco research through presentation and discussion. It is the hallmark conference in the field where investigators, advocates, and policymakers can rapidly ?get smart? on the latest evolutions in the field, novel products in the marketplace, and effective interventions and policies to reduce tobacco use. The SRNT Annual Meeting is a unique venue for presentation of novel findings from tobacco regulatory science research projects and networking across scientists in this emerging field. The aims of this conference grant are to: 1) ensure that tobacco regulatory science content is presented in ways that are maximally informative to FDA and to researchers in the field; and 2) to highlight the work of trainees and early career investigators in tobacco regulatory science via special oral sessions, professional development forums, travel awards, and other recognition. To meet both aims, SRNT?s Program Committee will meet regularly throughout the year to plan the content and format of the SRNT 2021 Virtual Annual Meeting, aligning the presentation of scientific content with research priorities in the Tobacco Regulatory Science Program (TRSP) and with FDA?s information requests via the public docket. The Committee will curate paper sessions on specific topics (e.g., flavors, nicotine reduction) that highlight advances in the area, foster scientific discussion and debate, and stimulate collaboration across researchers. The conference grant will also support 10 travel awards for trainees and early career investigators presenting on content related to the FDA and NIH Tobacco Regulatory Science Program research priorities. These interactions will facilitate the development of novel questions and approaches to tobacco regulatory science research.