Patricia H. Janak - US grants

DESCRIPTION (provided by applicant): Environmental cues associated with alcohol are more likely to precipitate relapse if experienced in an environmental context associated with past alcohol consumption, as opposed to a context in which alcohol was never consumed. Here we will study contextual modulation of responding to alcohol-conditioned cues in behavioral models of relapse in which rats are trained in a distinctive context to discriminate between one auditory stimulus paired with alcohol (CS+) and a second (CS-) that is never paired with alcohol;conditioned responding is measured as increased entries into the alcohol delivery port during the CS+ relative to the CS-. After extinction of responding in a different context, placement back into the original alcohol training environment enhances CS+-driven alcohol-seeking behavior ('relapse'). Using this paradigm, we propose to investigate the neural circuitry underlying the modulation of cue-induced alcohol-seeking by context using techniques of site-specific microinjections and in vivo electrophysiology. Specifically, we hypothesize that reinstatement of responding to a discrete alcohol-predictive cue requires excitatory projections from the basolateral amygdala (BLA) to the nucleus accumbens core (NACc), whereas the augmentation of this behavior by an alcohol context requires efferents from the ventral hippocampus (vHipp) to the BLA. In Aim 1 we will examine the independent roles of the BLA and NACc, as well as the BLA-NACc pathway, in cue- induced alcohol-seeking. Aim 2 will determine if contextual control over alcohol-seeking occurs via vHipp efferents to the BLA. Aim 3 will further define the neural mechanisms involved by examining neuronal responses to alcohol-predictive cues in the BLA, their modulation by context. Collectively, these studies will refine our understanding of the neural underpinnings of contextual modulation of alcohol-seeking, and may give rise to new therapeutic treatments for relapse in alcoholism. PUBLIC HEALTH RELEVANCE: Exposure to stimuli that have been associated with past alcohol use is thought to contribute to propensity to relapse in human alcoholics. The environment in which alcohol was habitually consumed may be a particularly potent relapse cue. The studies in this proposal are designed to reveal neurobiological mechanisms that underlie the ability of alcohol-associated contexts to trigger relapse.

[unreadable] DESCRIPTION (provided by applicant): Preventing relapse is a major goal for the treatment of drug addiction. One precipitating factor for relapse is exposure to drug-associated environmental stimuli. The aim of the current application is to investigate novel means for reducing the relapse-triggering power of cocaine-associated stimuli. Our approach is to enhance the learning that occurs during extinction training with the aim that this will decrease the susceptibility to future recovery of the extinguished cocaine-seeking behavior. Specifically, the studies within the current application will use an animal model of relapse to test the hypothesis that deepening the extinction of cocaine-related stimuli can reduce the future ability of these stimuli to produce cocaine-seeking behavior. These studies will capitalize on recent behavioral studies that suggest that presenting reward-related stimuli together, in compound, during extinction will enhance the effectiveness of the extinction training. Therefore, the studies in Aim 1 will test the role of stimulus compounding during extinction on later cue-induced reinstatement and spontaneous recovery of cocaine-seeking by rats. There is also exciting evidence that reward-related stimuli can activate the norepinephrine (NE) system, and that activation of the norepinephrine system may enhance extinction learning. Based on these findings, we will investigate the effects of increasing or decreasing the activity of the norepinephrine system during extinction learning and assess the impact on later cocaine-seeking behavior. These effects will be tested in cue-induced reinstatement in studies in Aim 2. Because there is a need for human addicts to resist the tendency to relapse for extended time periods after treatment, we will also examine the extent to which enhanced extinction learning resulting from activation of the norepinephrine system will withstand the passage of time using tests of spontaneous recovery in studies in Aim 3. The overall hypothesis guiding this work is that the presentation of excitatory stimuli during extinction engages noradrenergic systems that serve to enhance extinction learning, and therefore manipulation of stimulus presentation during extinction learning, or direct pharmacological manipulation of NE during extinction learning, will promote extinction learning. Hence we will use both behavioral and pharmacological approaches to enhance extinction of drug-seeking responses, thereby reducing later drug-seeking. It is anticipated that these initial studies conducted under the R03 mechanism will provide the basis for future in-depth research applications investigating the detailed neural bases of observed positive effects. The overall goal is to find ways to strengthen extinction of cocaine-seeking to reduce the likelihood of future relapse. By determining behavioral and pharmacological ways to enhance the stability and longevity of extinction learning, we hope to provide insight into the development of new therapies for drug addiction that will be less susceptible to relapse. [unreadable] [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): The behavioral effects of alcohol have long been considered to depend on gamma-amino-butyric acid (GABA) neurotransmission. Recently, the GABAA receptor isoform, 1424, has been proposed to mediate effects of alcohol at low-to-moderate concentrations. The current proposal will examine this possibility by testing the hypothesis that the 1424 GABAA receptor mediates aspects of the reinforcing properties of ethanol, thereby critically contributing to voluntary intake of ethanol. We will use viral-mediated RNA interference to knock down expression of the 14 and 4 GABAA receptor subunits in the nucleus accumbens, a brain region involved in processes of reward and reinforcement, to probe the contribution of the 1424 GABAA receptor to ethanol drinking behaviors by rats. Within our experimental aims we will test the contribution of this receptor to oral ethanol consumption, as well as to instrumental responding for ethanol. We will also initiate studies of the mechanism whereby ethanol in the NAc interacts with the 1424 GABAA receptor, using in vitro electrophysiogical techniques. Together, these studies will serve to define a role for two unique subunits of the GABAAR in a primary region of the brain reward circuitry, the NAc, in the reinforcing effects of ethanol. Understanding the neural mechanisms that mediate ethanol's reinforcing effects is critical for the development of treatments to assist in pharmacological therapies for alcohol abuse and alcoholism. PUBLIC HEALTH RELEVANCE The elucidation of the neurotransmitter and receptor systems that support alcohol drinking is critical for understanding how the pharmacological actions of alcohol lead to voluntary intake of alcohol, including under conditions of abuse. If the GABAA receptor studied in the experiments in this proposal is found to contribute to alcohol drinking, then future research on possible pharmaceutical approaches to reduce drinking by interacting with this receptor would be indicated. [unreadable] [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): Habitual behavior does not require an explicit representation of the goal, but is triggered by automatic responses to the stimuli that previously were paired with alcohol reward. The exact time course of this transition has not been defined for alcohol self-administration, however, there are well-defined tests for whether instrumental behavior is goal-directed or habitual that can be used to determine whether or not self-administration is habitual. The current proposal seeks to test the hypothesis that alcohol self-administration after extended training will display the hallmarks of a habitual behavior. As conditioned cues also play a major role in relapse, we will also test the effects of alcohol-predictive cues after extended alcohol self-administration training. The reliance of alcohol-seeking, including that promoted by cues, on dorsal striatal circuitry will be probed. [unreadable] [unreadable] PUBLIC HEALTH RELEVANCE: It has long been hypothesized that alcohol- and drug-seeking becomes habitual over time; this is apparent in our colloquial use of the term `drug habit'. Therefore, understanding the transition of ethanol self-administration from goal-directed to habitual may provide information on the neurobiology that underlies the transition from casual ethanol use to addiction. These results will provide new information on the role of specific neural pathways in alcohol self-administration and increase our understanding of the behavioral mechanisms that underlie its intake. Therefore, these experiments will address an important lack in our basic knowledge of the neurobiology of alcohol addiction that will allow us to better design therapies for alcohol abuse. [unreadable] [unreadable] [unreadable]

Dopamine neurons in the VTA play a very important role in a variety of physiological as well as addictive behaviors. The main goal of the present proposal is to elucidate the relationship between plasticity at excitatory synapses in the ventral tegmental area (VTA)and addictive behaviors such as behavioral sensitization and self-administration of cocaine. During the current funding period (April1st, 2002- February 2006), we have collected evidence that might explain the sequence of events leading from NMDAR activation in the VTA produced by acute cocaine application, to long-term potentiation of VTA neurons that results as a consequence of in vivo cocaine exposure. Further, our preliminary data suggest that long-term changes of excitatory synaptic transmission in the VTA are not only produced by passive cocaine administration (e.g.in vivo cocaine injections), but operant behaviors such as cocaine self-administration. Specific aim 1 will test the hypothesis that in vivo cocaine-induced potentiation involves a direct action of cocaine in the VTA mediated by NMDARs, D5 receptors and the cAMP/PKA-dependent pathway. Specific aim 2 will test the role and time-course of protein synthesis in mediating long-term plasticity at VTA synapses and behavioral sensitization. Finally, specific aim 3 will characterize whether long-term synaptic changes at glutamatergic synapses in the VTA are produced during forced abstinence or extinction of operant responding from either food or cocaine. Taken together, the results from these experiments will likely help us understand the role of plasticity at glutamatergic synapses in the VTA in mediating cocaine-dependent behaviors.

DESCRIPTION (provided by applicant): The Alcohol Center for Translational Genetics (ACTG) will identify novel proteins as targets for therapeutics for alcohol use disorders and will determine mechanisms by which they act to regulate excessive ethanol intake. Candidate proteins will be evaluated in ethanol self-administration procedures that model excessive binge drinking in humans, motivation to drink ethanol, and relapse. The anatomical focus will be on 3 brain regions, the nucleus accumbens, the amygdala, and the ventral tegmental area, which all play important roles in ethanol consumption and relapse. Based on findings during the current funding cycle that identified H-Ras/PI3 kinaseAKT/mTORC1 signaling as a key regulator of ethanol consumption, all Research Components will include experiments that test the relationship of the novel proteins with this pathway. Three research projects will focus on proteins new to alcohol research: SGK1, GSK-3 and others whose translation is regulated by mTORC1 (Component 4); PKM? and its direct substrates (Component 5); and orexin/hypocretin receptors (Component 6). An Administrative Core (Component 1) will manage ACTG functions. An Animal Behavior Core (Component 2) will perform studies of intermittent ethanol access in rats and mice and will provide assistance in rat operant self-administration procedures. A Vector and Imaging Core (Component 3) will provide state-of-the art services to generate viral vectors for transgenic expression or gene silencing, and to analyze transcript and protein abundance by laser capture microdissection, high resolution immunofluorescence, and quantitative fluorescent in situ hybridization for detecting mRNAs in dendrites. Two Pilot projects are planned. The first will test the hypothesis that delta opioid receptor mediated inhibition of GABA release decreases alcohol consumption in anxious alcoholics. The second will determine whether up-regulation of NMDA receptor activity, induced by excessive ethanol consumption, facilitates long-term potentiation in the dorsomedial striatum, and thereby enhances ethanol drinking and seeking. Collectively, the ACTG provides a unique opportunity for integrated study of novel proteins that may lead to the development of new treatments for alcohol use disorders in humans.

The Animal Behavior Core will provide services, technical assistance and training needed for behavioral testing of mice and rats to satisfy the experimental aims of the investigators within the Research Components of the ACTG. A primary goal of the Animal Behavior Core is to standardize the methods and the analyses for alcohol-related behavioral testing for mice and rats. Core personnel will conduct all homecage drinking studies in mice and rats, and other ethanol-related behavioral tests, as required by the Research Components. The core will instruct research personnel in other behavioral procedures as needed. In addition, the Core will directly compare the rat operant self-administration procedures currently in use by Center Investigators. By conducting a considerable proportion of the behavioral testing within the Core, we ensure that the procedures are performed, and the data are analyzed, in a consistent manner, allowing for maximal comparability of the effects of different experimental manipulations across center projects. This model of centralized behavioral testing by the Animal Behavior Core worked extraordinarily well in the first funding period, allowing for smooth, efficient completion of many behavioral studies. The overall goal of the Animal Behavior Core is to assist Research Components in experiments that test hypotheses regarding the role of novel signaling molecules in alcohol drinking.

The Pilot Project Component is designed to provide a flexible means for developing and exploring new research activities or directions, and unique opportunities that can evolve into independently funded research projects. During Years 6-10 we propose funding two pilot projects per year with a budget of about $50,000 per project. The expected duration of these projects will be 1 year; projects can be extended to two years upon successful competitive renewal. The Center Scientific Director will manage this Component. Pilot Project applications will be solicited annually from EGCRC and UCSF investigators. Each proposal will be evaluated for scientific merit, innovation, and for relatedness to the Center's overall goals by at least two members of the ACTG Program Advisory Board, the Scientific Director, and the Center Director. Recommendations for funding will be considered for approval by the Center Steering Committee. Two Pilot Projects have been proposed and awarded funding for Year 06.Project 7A (A. Kayser, Director) will examine whether the Delta Opioid Receptor (DOR) agonist, ALK33, can be a potential drug to treat alcohol intake and craving in a preclinical human trial. Experiments in human subjects are also aimed to conducted fMRI studies to determine whether the increase the connectivity between the medial prefrontal cortex and brain regions associated with reward and anxiety.

DESCRIPTION (provided by applicant): Mesolimbic dopamine is considered to have a major role in Pavlovian reward learning. Pavlovian reward learning processes underlie many aspects of drug seeking behavior and may play a critical role in drug relapse. Hence our overall goal is to better understand the basic neurobehavioral mechanisms of reward learning. Here we seek to better define that role using optogenetic tools that will allow precise control of dopamine neuron activity during behavior. We will use the Th:Cre rat, a transgenic rat expressing Cre recombinase under control of a tyrosine hydroxylase (Th) promoter that allows for gene expression limited to dopamine neurons in the VTA following infusion of cre-dependent viruses expressing either channelrhodopsin or halorhodopsin. We can then deliver light through optical fibers surgically implanted into the VTA in the behaving rat to activate or inhibit activity of DA neurons. The proposed studies are based upon the notion that 1) dopamine neuron activity during the receipt of unexpected reward drives new learning about cues that predict the availability that reward, and that 2) decreases in dopamine neuron activity during expected reward may contribute to extinction of responding to reward-predictive cues. Thus we propose to extend preliminary findings to show that increases in dopamine neuron activity induced by optical stimulation functions as a positive prediction error and can cause learning. We also will test the hypothesis that suppression of dopamine neuron activity by optical stimulation functions as a negative prediction error and decreases learned responding. Additionally, we will test the hypothesis that these effects are mediated by dopamine neurons that project to the nucleus accumbens. Hence, using well-characterized Pavlovian conditioning procedures in combination with state-of- the-art behavioral optogenetics, the current aims are designed to expand our understanding of causal roles for VTA DA neuron activity in reward learning.

Alcohol use disorders (AUDS) impact millions of individuals and constitute one of the most serious public health problems worldwide. Despite its devastating impact on society, only a few effective medications are currently available. Orexins/hypocretins are hypothalamic peptides that act via orexin receptors that have been linked to regulating feeding and sleep. In addition, the orexin system has been shown to play an important role in alcohol self-administration and in stress-induced reinstatement. These effects are thought to be mediated by orexin containing neurons that project from the lateral hypothalamus to the ventral tegmental area (VTA) and nucleus accumbens (NAc) or from the perifornical regions to the amygdala respectively. However, the role of orexins through orexin receptors and their signaling pathway in driving ethanol-mediated behaviors is less explored. The overall goal of this research project, component 6 of the NIAAA-Gallo Center application is to determine the role of orexins, orexin receptors and their downstream signaling pathways in different brain regions in ethanol-mediated behaviors. The Center provides a unique opportunity to apply a multidisciplinary approach that integrates behavioral, biochemical and electrophysiological techniques in the latest animal models of binge drinking, ethanol self-administration and stress-induced reinstatement. The research aims to understand the role of orexin-mediated synaptic and cellular mechanisms in the central amygdala, VTA and NAc using behavioral models of alcohol addiction. In Aim 6.1, we will determine whether Ox-Rs in the central amygdala drive stress-induced reinstatement of ethanol seeking. In Aim 6.2, we will apply electrophysiological and biochemical techniques to dissect the signaling pathways underlying orexins effects in the central amygdala. In Aim 6.3, we will determine the mechanism of action of orexins in the VTA and the NAc in regulating binge ethanol consumption and ethanol self-administration. Orexins/hypocretins play a crucial role in addiction, the sleep-wake cycle, motivation and stress, and this provides a strong rationale for our proposed studies, the experiments outlined have been designed specifically to lead to clinical studies aimed at determining the efficacy of orexin receptor antagonists in human subjects with AUDs.

Project Summary/Abstract While considerable effort has been focused upon the neurobiological circuits mediating appetitive, or reward seeking, aspects on alcohol self-administration, our understanding of the circuits that mediate the consummatory aspects ? the actual alcohol intake itself ? is more limited. Therefore, the studies proposed in this application seek to define the neural circuits that control alcohol intake. These studies build on past work supporting a role for the nucleus accumbens and the amygdala central nucleus both in both natural feeding and in alcohol intake. The contribution of these regions and selected afferents and efferents will be determined using optogenetics to activate or suppress neuronal populations or specific neural projections, and using calcium imaging to query the activity of these neural elements, during cue-elicited and spontaneous alcohol intake. In addition, these studies will examine the impact of prior alcohol dependence on the role of the accumbens and the amygdala central nucleus given the strong evidence for alcohol- induced neuroadaptations in these extended amygdala regions. These studies represent important steps towards specification of consummatory circuits whose function may change after chronic alcohol representing a possible neurobiological mediator of excessive or escalated alcohol intake.

Summary The 2017 Gordon Research Conference on Catecholamines will take place at Sunday River in Newry, ME in August 2017. This small conference, held every other year, is an ideal forum for exploring new developments in the field of catecholamines. It is also an excellent opportunity for junior investigators and trainees to interact with established investigators in a relaxed atmosphere. The evaluations of recent conferences highlight the strong impact this meeting typically has on emerging as well as established scientists. As the funds allocated by the Gordon Conference organization are limited, we are requesting funding from other sources. We are hoping that the results of our requests enable us to support speakers and discussion leaders as well as junior investigators to participate in the meeting. The conference includes two plenary talks, by Dr. Marina Wolf and Dr. Amy Arnsten. The preliminary program includes a large number of junior scientists and women, and the sessions planned include: 1) Substance Abuse, 2) Neurophysiology and Neurotransmission, 3) Neurodegeneration, 4) Cognition, 5) Stress/Affective Disorders, 6) Circuits and Molecules, and 7) Learning and Motivation. The themes covered in the 2017 Gordon Conference on Catecholamines are highly relevant to understanding the neurobiological bases of drug addiction as well as neurological diseases of aging and neuropsychiatric disorders. This forum will provide a unique opportunity to discuss recent advances in the understanding of how catecholamine systems may contribute to the pathophysiology and treatment of those conditions. The fact that a large proportion of the participants are junior investigators and a significant number will be graduate students and postdocs ensure that this conference may have an impact on how future generations of neuroscientists view these issues.

Project Summary Alcohol use disorders take a heavy toll on personal, social, and economic facets of our society. Thus we seek to better understand the underlying brain mechanisms for alcohol's effects. The central nucleus of the amygdala (CeA) is a brain region that plays a vital role in alcohol intake, yet the means by which neurons in this region contribute to alcohol intake are not clear. The studies in this application propose to address this gap in a focused set of studies designed to expand our understanding of CeA circuit mechanisms in alcohol-related behaviors. Studies proposed will use in vivo electrophysiological recording to assess the neural correlates in the CeA during alcohol self- administration in rat models of drinking. Further, using electrophysiology and opto- and chemo- genetics, additional studies will test the hypothesis that a major excitatory input to the CeA, the insula, and a major output region, the parabrachial nucleus, are key players in a larger CeA circuit for alcohol self-administration. These candidate key regions will be compared with other regions, such as the paraventricular nucleus of the thalamus and the BNST. The role of each of these circuits in intake of natural reward will also be assessed to understand possible distinctions in mechanisms of alcohol action. These studies represent important steps towards an understanding of the neurobiology of this devastating health disorder.

Project summary Mesolimbic dopamine, along with its ventral striatal targets, plays a major role in Pavlovian reward learning. Pavlovian reward learning processes underlie many aspects of drug seeking behavior and play a critical role in drug relapse. Instrumental learning processes also form a fundamental element of drug seeking behavior, and may depend on nigrostriatal dopamine innervation of dorsal striatum. Here we seek to understand how mesolimbic and nigrostriatal dopamine action within striatal targets mediates select components of reward-seeking behavior, and to test whether interactions between these systems contribute to normal and pathological reward-seeking behavior. We focus on dopamine because both natural and drug rewards activate dopamine neurons, and, as confirmed in our recent findings, dopamine neuron activation, when substituted for reward, drives specific forms of associative learning implicated in addictive behavior. Here, using the Th:Cre transgenic rat to limit channelrhodopsin expression to dopamine neurons, we take advantage of the ability to selectively activate mesolimbic dopamine neurons to better understand their impact on behavior, neural activity, and integration of ventral and dorsal striatal systems. We test whether mesolimbic dopamine neuron reward-related activation recruits more dorsal striatal circuits during specific forms of learning. A prominent hypothesis posits that reward seeking depends over time on more dorsal striatal circuits as behavioral control becomes habitual. Thus, we also ask whether activating mesolimbic dopamine systems can hasten development of habitual responding for natural or drug reward. In addition, we will determine neural signals mediating behaviors conditioned by mesolimbic dopamine activation in order to reveal neural changes in downstream neuronal populations that mediate the performance of these dopamine-mediated learned behaviors. These studies will provide new information on the separate and interactive contributions of ventral and dorsal striatal circuits to reward seeking behavior that can be initiated by dopamine, and are relevant for our understanding of behavioral disorders involving overeating and substance use disorders.