Andrew Tapper - US grants

DESCRIPTION (provided by applicant): Tobacco use causes five million deaths worldwide annually and is the leading cause of preventable mortality in the world. Nicotine is the addictive component of tobacco that binds to and activates a family of ligand-gated ion channels, nicotinic acetylcholine receptors (nAChRs). Activation of the receptors in the dopaminergic mesocorticolimbic reward pathway is thought to underlie the initiation of addiction whereas signaling through nAChRs in the habenulo-interpeduncular pathway that feeds into the reward pathway is thought to play a key role in eliciting nicotine withdrawal symptoms. One of the challenges in understanding nicotine's effect on nAChRs arises from the existence of multiple nAChR subtypes, each exhibiting unique electrophysiological properties and varying affinities for nicotine. Eleven mammalian neuronal nAChR subunits have been identified (?2-??7, ?9, ?10 and ?2-??4). Five subunits co-assemble to form receptors with the subunit composition of each channel determining its pharmacological and biophysical properties. Chronic nicotine exposure alters the expression of nAChR subtypes, which likely contributes to nicotine dependence; however, the underlying mechanisms regulating these changes remain unclear, although transcriptional mechanisms are not thought to be involved. A growing body of evidence indicates that nicotine and cigarette smoke alters the expression of small 21-24 nucleotide long regulatory molecules, referred to as microRNAs (miRNAs). miRNAs are predicted to regulate the majority of all mammalian protein coding genes, typically by binding to complementary sites in the 3'- untranslated regions (3'-UTRs) of target mRNAs and guiding them to an RNA-induced silencing complex (RISC). To date, one miRNA, miR-1, has been reported to target nAChRs in C. elegans, leading to changes in nAChR function. Little is known regarding miRNA regulation in the context of mammalian nAChRs or nicotine addiction. We have preliminary data from a miRNA analysis using laser-captured material from mice chronically treated with nicotine that suggest: 1) a decrease in expression of two miRNAs, miR-7a and miR-9, in the ventral tegmental area (VTA) and 2) an increase in expression of the same two miRNAs in the medial habenula when compared to saline-treated animals. There was no change in their expression in the interpeduncular nucleus. Interestingly, potential targets of miR-7a and miR-9 include members of the nAChR family as well as other components important in the reward pathway. Our preliminary studies indicate that miR-7a regulates expression of the nAChR ?6 subunit via interactions with a single miR-7a recognition element in the 3'-UTR of the ?6 gene. These data form the basis of this application with the over-arching hypothesis that post-transcriptional regulation of nAChRs by miRNAs modulates nicotine-associated behaviors. The first Aim will test the hypothesis that miR-7a and/or miR-9 modulates nicotine reward-associated behaviors as well as nAChR expression and function. The second Aim will test the hypothesis that miR-7a and/or miR-9 modulates nicotine withdrawal-associated behaviors. The final Aim will test the hypothesis that miR-7a and/or miR-9 interacts with the 3'-UTRs of nAChR subunit genes in a functionally relevant manner. The results from these experiments will provide unique insight to the molecular underpinnings of nicotine addiction. PUBLIC HEALTH RELEVANCE: The goal of the proposed work is to understand the role of small regulatory RNA molecules in nicotine-mediated behaviors. Despite evidence indicating that expression of these molecules is altered by nicotine treatment, little is known regarding their function in this context. The proposed work will substantially close this gap in knowledge.

DESCRIPTION (provided by applicant): The devastating health consequences of tobacco use result in 5 million deaths/year, making smoking the primary cause of preventable mortality in the world. Susceptibility to nicotine addiction has a heritable component, but while some genetic variants that affect nicotine addiction have been identified, they explain only a fraction of the observed heritability. Indeed, it is clear that in addition to genetic predispositions, environmentl influences play a major role in smoking behavior, most importantly, in the initiation of smoking. The work proposed in this application focuses on the hypothesis that environmental exposures not only affect the people experiencing them, but can also have a heritable effect on nicotine-relate behaviors in children. Specifically, epigenetic information can carry environmental information between generations. For example, epidemiological evidence in humans suggests that parental diet and smoking history can affect cardiovascular disease risk in their children. In addition, adolescents whose parents smoke are more likely to smoke themselves and also initiate smoking at an earlier age, although the mechanism underlying this association is unknown. In multiple contexts, is becoming increasingly clear that understanding the effects of ancestral food and drug exposure on offspring metabolism and behavior will be required for a detailed understanding of disease susceptibility. In other words, many diseases with complex genetic bases may also be subject to confounding epigenetic effects in addition to the long sought after genetic predispositions. We recently demonstrated that, in rodents, paternal diet affects metabolic gene expression in offspring. Thus, it is conceivable that paternal exposure to nicotine, the addictive component of tobacco smoke, may have transgenerational effects on gene expression that ultimately affect metabolism and dependence- related behavior. Indeed, in this proposal we provide preliminary evidence that male mice exposed to nicotine father offspring with altered gene expression in the liver and in key brain regions involved in nicotine dependence. We propose to comprehensively characterize this effect from molecular changes in fathers' sperm induced by nicotine, to gross effects on nicotine-related behaviors in children. Together, these experiments constitute an extensive characterization of a novel pathway linking nicotine exposure to phenotype across generations. These results will likely have important implications for the epidemiology of complex diseases in humans.

DESCRIPTION (provided by applicant): Chronic exposure to tobacco smoke accounts for ~6 million deaths per year making health complications from smoking the primary cause of preventable mortality in the world. Nicotine, the addictive component of tobacco smoke, initiates dependence by binding to and activating neuronal nicotinic acetylcholine receptors (nAChRs), yet nicotine replacement therapy and existing smoking cessation aids that target abundant nAChR subtypes are minimally effective. Thus, a better understanding of neural circuits and neurotransmitter systems that contribute to nicotine withdrawal syndrome is needed to facilitate the development of new therapies for smoking cessation. Recent studies have identified the habenulo-interpeduncular (Hb-IPN) axis as a potentially important circuit that is hypothesized to mediate the aversive effects of high doses of nicotine. In addition, this pathway is involved in physical withdrawal symptoms in rodents. However, the role of the Hb-IPN circuit in affective withdrawal and the neurotransmitter systems underlying the manifestation of these symptoms are unknown. Our data indicate that the IPN may be a neuro-anatomical substrate for anxiety during nicotine withdrawal. In addition, preliminary data indicate that the stress peptide, corticotropin releasing factor (CRF), and its cognate receptors (CRF receptors) within the IPN play a significant role in nicotine withdrawal-induced anxiety in a mouse model of nicotine dependence. These observations provide the foundation for this application. We propose to test the hypothesis that chronic nicotine engages CRF signaling within the IPN, initiating the anxiogenic effects of nicotine withdrawal. In Aim 1 we propose to use pharmacological and viral mediated gene delivery approaches to determine if CRF receptor signaling in the Hb-IPN axis is critical for the expression of anxiety during nicotine withdrawal. In Aim 2, we will use a biophysical approach to determine how chronic nicotine and withdrawal modulates neuronal excitability within the IPN. It is anticipated that elucidating the circuitry and mechanistic detais of the anxiogenic effects of nicotine withdrawal will provide new insights into novel therapeutic strategies to facilitate smoking cessation.

Project Summary/Abstract Chronic exposure to tobacco smoke accounts for ~6 million deaths per year making health complications from smoking the primary cause of preventable mortality in the world. Current smoking cessation aids including nicotine replacement therapy and medication that target nicotinic receptors are minimally effective. Thus, a better understanding of brain regions, neural circuits, and neurotransmitter systems that underlie nicotine withdrawal syndrome is needed to facilitate the development of new therapies for smoking cessation. While dopaminergic (DAergic) neurons in the ventral tegmental area (VTA) that project to the nucleus accumbens are critical for nicotine reward and reinforcement, recent studies have identified the habenulo-interpeduncular (Hb- IPN) axis as a critical circuit in nicotine withdrawal symptoms. In particular, the IPN is implicated in increased anxiety during nicotine withdrawal in rodents and receives inputs from cholinergic/glutamatergic medial habenula neurons associated with affective behavior. Interestingly, we have identified a novel circuit consisting of VTA neurons that project to the IPN, the mesointerpeduncular pathway. Our data indicate that DA from VTA afferents may innervate the IPN to modulate neuronal activity and anxiety-like behavior via DA receptor signaling. In addition, DA receptor expression in the IPN appears to be regulated by chronic nicotine exposure. These observations provide the foundation for this application. We propose to test the hypothesis that VTA DAergic neurons that project to the IPN modulate anxiety and are critical for increased anxiety during nicotine withdrawal. In Aim 1, we propose to use molecular and biophysical approaches to test the hypothesis that DA controls IPN neuronal activity through activation of DA receptors that are regulated by nicotine. In Aim 2, we will use pharmacology, optogenetics, and biophysical approaches to test the hypothesis that the IPN receives DA input from the VTA. Finally, in Aim 3 we will use pharmacology, optogenetics, and behavior to test the hypothesis that the mesointerpeduncular circuit modulates anxiety-like behavior at baseline and during nicotine withdrawal. It is anticipated that understanding the role of the mesointerpeduncular circuit in anxiety and nicotine withdrawal will provide novel strategies to alleviate anxiety and nicotine withdrawal symptoms.

Project Summary/Abstract Identifying factors that may predispose individuals to developing addiction is a critical component in understanding the basis of the disease. The most common personality traits associated with addiction include heightened novelty seeking and preference, which are associated with increased exploratory behavior in response to novel stimuli and more interaction with novel stimuli compared to familiar stimuli when given a choice, respectively. Genetic studies have linked novelty seeking with the dopamine (DA) neurotransmitter system, as well as genes involved in serotonin (5-hydroxytryptophan, 5HT) signaling. However, the interaction between 5HT and DA in brain areas and neuronal circuits involved in novelty seeking and preference are poorly understood. Recently, we have shown that the interpeduncular nucleus (IPN) is critically involved in novelty seeking and preference. Specifically, activation of IPN GABAergic neurons acts as a brake to reduce exploration of novel stimuli as they become familiar. Our published and preliminary data indicate that the IPN receives DAergic and 5HTergic inputs from the ventral tegmental area (VTA) and median raphe (MR), respectively. We hypothesize that these two IPN afferents interact to affect novelty seeking and preference through modulation of familiarity signaling. Aim 1 will combine optogenetics and behavior in mice to test the hypothesis that activation of a VTA?IPN DAergic circuit prevents familiarity signaling to increase novelty seeking and motivation to explore novel stimuli. Aim 2 will use a similar approach to determine how 5HT receptor signaling through serotonergic input from the MR to the IPN may influence the behavioral response to novel and familiar stimuli. Finally, Aim 3 will use a biophysical and optogenetic approach to determine how 5HT and DA interact in the IPN and how this controls novelty preference. The results from the proposed experiments should yield significant insight into circuits critical for novelty seeking and preference and elucidate new mechanisms underlying behavioral traits associated with addiction.