John F. Neumaier - US grants

DESCRIPTION (Adapted from applicant's abstract): Major depression is hypothesized to involve impaired 5-HT neurotransmission in the brain; this may in part be due to excessive 5-HT1B autoreceptor activity in 5-HTergic axon terminals. These receptors are desensitized by SSRIs, an important class of antidepressant drugs. However, the mechanism underlying this observation is unknown and these receptors are not well understood. I hypothesize that SSRI, by increasing presynaptic 5-HT1B activation by endogenous 5-HT at presynaptic terminals, downregulates 5-HT1B receptor levels. I further propose that 5-HT1B receptor synthesis is downregulated at the mRNA level in dorsal raphe neurons following chronic receptor activation. The goal of this study will be to isolate the regulatory phenomena that apply specifically to 5-HT1B autoreceptors in 5-HTergic neurons (in contrast to the many postsynaptic non-5-HTergic neurons) that also express 5-HT1B receptors. Two approaches will be used: (1) In rat brain, I will investigate whether chronic activation of terminal 5-HT1B autoreceptors reduces 5-HT1B mRNA levels in the dorsal raphe cell lines. This will be achieved by steady infusion of a selective 5-HT1B agonist into discrete forebrain regions innervated by these neurons. I will then compare 5-HT1B mRNA levels between raphe neurons that do or do not project to the site of infusion. I will similarly test the effects of chronic, localized infusion of SSRI on 5-HT1B autoreceptor regulation, (2) The RN46A cell line was recently developed from embryonic rat raphe neurons. It displays 5-HTergic phenotype including 5-HT1B receptor expression. I will characterize the effect of depolarization, hormones, and trophic factors on the levels of 5-HT1B receptor and mRNA in RN46A cells. I will also determine whether SSRI and 5-HT1B agonists downregulate this receptor in these cells as they do in rat brain. Three indices of 5-HT1B levels and activity will be used: mRNA levels in situ hybridization in rat brain, ribonuclease protection assay in cultured neurons), [125I]-cyanopindolol binding to brain sections or RN46A membranes, and 5-HT1B autoreceptor inhibition of 5-HT release (agonist potency determinations using fast cyclic voltammetry in brain slices).

DESCRIPTION: Major depression is a serious mental disorder characterized by cognitive and neurovegetative symptoms although it's pathophysiology is incompletely understood. We have been testing the hypothesis that excessive activity of the 5-HTIB terminal autoreceptors in forebrain projections from dorsal raphe neurons is involved in some of the symptoms of depression and are selectively down regulated by SSRI antidepressants. However, 5-HT1B receptors are also located in many non serotonergic neurons throughout the central nervous system, thus it is crucial to discriminate between presynaptic autoreceptors and heteroreceptors in nonserotonergic neurons. This is a challenging problem, since serotonergic neurons are few in number and project diffusely, making it very difficult to gain experimental access to this specific subpopulation of 5-HTIB receptors. Therefore, we propose to use an innovative new technique to make experimental manipulations in 5-HT1b autoreceptors only in dorsal raphe neurons by using viral mediated gene transfer. We plan to either increase or decrease 5-HTIB mRNA levels in these neurons by injecting replication defective Herpes Simplex Virus carrying 5-HT1b "transgene" cDNA directly into rat dorsal raphe nucleus and carefully validate changes in gene expression and 5-HT1b terminal autoreceptor activity in 5-HT projections to forebrain. In order to consider transgene induced depressive-like symptoms and antidepressant reversal, we will use several behavioral models of depression to determine whether 5-HTIB autoreceptors induce depressive-like behavioral changes. We will test an antisense knockdown RNA, also introduced by viral mediated gene transfer into dorsal raphe, for antidepressant effects. We will also test whether viral mediated gene transfer of the 5-HT1A somatodendritic autoreceptor into dorsal raphe nucleus produces comparable effects. It is our objective that these experiments will shed light on the role of serotonin autoreceptors in depression and its treatment, and will be an opportunity to extend the use of viral mediated gene transfer as a research technique in the study of mental illnesses using animal behavioral models.

DESCRIPTION (provided by applicant): The serotonin system in the brain plays several important roles in modulating the effects of drugs of abuse. In this proposal we will examine the roles of 5-HT1B and 5-HT6 receptors in the rewarding and motor stimulant effects of cocaine and amphetamine in rats, and correlate these to biochemical adaptations that are associated with the progression of drug addiction. We will focus on a critical neuronal reward circuit using cutting edge techniques with molecular and anatomical precision so that we can discern the role of these receptors in this specific context. It is our hypothesis that, in nucleus accumbens projection neurons, 5-HT1B receptors increase and 5-HT6 receptors decrease the behavioral effects of psychostimulants. This proposal is innovative because it focuses on serotonin receptors that have not received much previous attention in relation to drug addiction. The primary strategy that we will use is viral mediated gene transfer to alter the expression of 5-HT1B and 5-HT6 receptors selectively in medium spiny neurons of the nucleus accumbens shell that project to ventral tegmental area. For 5-HT1B receptors, we will study the effect of altered 5-HT1B receptor expression in the efferents of these neurons on cocaine and amphetamine behaviors. Since cocaine has a large, direct effect on serotonin accumulation and low dose amphetamine affects dopamine predominantly, we will be able to assess the role of 5-HT1B receptors in drug reward in general as opposed to for cocaine in particular. We will also use in situ hybridization histochemistry to study the impact of chronic cocaine administration and drug discontinuation on the expression of these two receptors in medium spiny neurons. For the 5-HT6 receptor, we will combine local injections of highly selective 5-HT6 agonists and antagonists into nucleus accumbens shell with systemic cocaine treatment to evaluate the role of 5-HT6 receptors in these neurons on drug reward mechanisms. We have also developed a 5-HT6 viral vector that will allow us to probe the role of these receptors in accumbens neurons with great precision. The overall goal of this proposal is to extend our molecular and behavioral understandings of how serotonin receptors mediate the addictive properties of cocaine and amphetamine, and to determine if these receptors might serve as novel targets for treating addiction pharmacologically.

DESCRIPTION (provided by applicant): The serotonin system in the brain plays an important role in determining the responses to environmental stressors. The interaction between stress and serotonin is complex, however, since acute increases in serotonin can be anxiogenic, while chronic increases can actually reduce anxiety and depressive symptoms, depending on the context and the brain region. Since the 5-HT1B autoreceptor regulates the release and possibly the reuptake of 5-HT at axon terminals, it is strategically placed to modulate extracellular 5-HT in brain regions that mediate fear, anxiety, and stress-induced depression. The effects of 5-HT1B autoreceptors are challenging to study, though, because 5-HT1B heteroreceptors are localized in axon terminals of other neuron types throughout the brain, necessitating the use of anatomically specific techniques. Previously we developed a viral mediated gene transfer strategy to manipulate 5-HT1B autoreceptors in dorsal raphe nucleus and increase the expression of 5-HT1B autoreceptors selectively. We have found that 5-HT1B- overexpressing animals are less anxious when not exposed to environmental stressors, but more anxious when stressed. We have recently constructed a serotonin selective viral vector based on the serotonin transporter promoter to increase expression of 5-HT1B receptors only in serotonergic neurons. We now propose to pursue the function of 5-HT1B autoreceptors in dorsal raphe in four ways. We will examine the neuroanatomical contribution of 5-HT1B autoreceptors to behavior in rostral vs. caudal dorsal raphe, subregions that appear to mediate anxiety and depression, respectively. We will investigate the temporal role of 5-HT1B autoreceptors in modulating fear learning by examining their effects on acquisition, expression, and extinction of conditioned fear. We will study the mechanism by which these autoreceptors regulate extracellular 5-HT levels, and whether they do so by modulating serotonin transporter function. We will examine the modulation of 5-HT1B autoreceptor effects on behavior by stress and the CRF related peptides. These questions will be addressed using a combination of novel molecular, pharmacological, and behavioral strategies that we have developed in our lab, allowing us to isolate the role of 5-HT1B autoreceptors in these animal models of stress-associated illnesses.

[unreadable] DESCRIPTION (provided by applicant): The gene for 5-HT1B serotonin receptors has been linked to alcoholism in humans but there is scant evidence for how these receptors modulate ethanol's reinforcing effects. Since 5-HT1B receptors are expressed in different groups of neurons throughout the brain, they must be studied using techniques that have anatomical and pharmacological specificity. For example, it is hypothesized that the 5-HT1B recaptors in the axon terminals of medium spiny neurons that project from nucleus accumbens shell (NAcc) to ventral tegmental area modulate drug reward by inhibiting GABA release, thereby disinhibiting dopaminergic activity. To investigate this idea, it is necessary to examine.just these 5-HT1B receptors and not those in other neurons that regulate other aspects of behavior. Toward!this aim, we will use targeted manipulation of 5-HT-iBexpression in these NAcc neurons. We will examine this hypothesis by measuring changes in ethanol consumption after manipulating the level of 5-HT1B expression in the NAcc. In Specific Aim 1, we plan to increase 5-HT1B expression using virally-mediated gene transfer and measure the effect on the initiation and maintenance of alcohol drinking behavior. In Specific Aim 2, we will knockdown expression of 5-HT1B receptors in NAcc shell using RNA interference (RNAi) and measure these same behaviors. Thus, we will use novel molecular techniques to manipulate 5-HT1B expression in a discrete group of neurons and measure the resulting changes in free choice ethanol consumption to evaluate how this receptor regulates reward mechanisms in the brain. [unreadable] [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): The gene for 5-HT1B serotonin receptors has been linked to alcoholism in humans but there is scant evidence for how these receptors modulate ethanol's reinforcing effects. Since 5-HT1B receptors are expressed in different groups of neurons throughout the brain, they must be studied using techniques that have anatomical and pharmacological specificity. For example, it is hypothesized that the 5-HT1B recaptors in the axon terminals of medium spiny neurons that project from nucleus accumbens shell (NAcc) to ventral tegmental area modulate drug reward by inhibiting GABA release, thereby disinhibiting dopaminergic activity. To investigate this idea, it is necessary to examine.just these 5-HT1B receptors and not those in other neurons that regulate other aspects of behavior. Toward!this aim, we will use targeted manipulation of 5-HT-iBexpression in these NAcc neurons. We will examine this hypothesis by measuring changes in ethanol consumption after manipulating the level of 5-HT1B expression in the NAcc. In Specific Aim 1, we plan to increase 5-HT1B expression using virally-mediated gene transfer and measure the effect on the initiation and maintenance of alcohol drinking behavior. In Specific Aim 2, we will knockdown expression of 5-HT1B receptors in NAcc shell using RNA interference (RNAi) and measure these same behaviors. Thus, we will use novel molecular techniques to manipulate 5-HT1B expression in a discrete group of neurons and measure the resulting changes in free choice ethanol consumption to evaluate how this receptor regulates reward mechanisms in the brain. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): The serotonin system in the brain plays an important role in determining the responses to environmental stressors. The interaction between stress and serotonin is complex, however, since acute increases in serotonin can be anxiogenic, while chronic increases can actually reduce anxiety and depressive symptoms, depending on the context and the brain region. Since the 5-HT1B autoreceptor regulates the release and possibly the reuptake of 5-HT at axon terminals, it is strategically placed to modulate extracellular 5-HT in brain regions that mediate fear, anxiety, and stress-induced depression. The effects of 5-HT1B autoreceptors are challenging to study, though, because 5-HT1B heteroreceptors are localized in axon terminals of other neuron types throughout the brain, necessitating the use of anatomically specific techniques. Previously we developed a viral mediated gene transfer strategy to manipulate 5-HT1B autoreceptors in dorsal raphe nucleus and increase the expression of 5-HT1B autoreceptors selectively. We have found that 5-HT1B- overexpressing animals are less anxious when not exposed to environmental stressors, but more anxious when stressed. We have recently constructed a serotonin selective viral vector based on the serotonin transporter promoter to increase expression of 5-HT1B receptors only in serotonergic neurons. We now propose to pursue the function of 5-HT1B autoreceptors in dorsal raphe in four ways. We will examine the neuroanatomical contribution of 5-HT1B autoreceptors to behavior in rostral vs. caudal dorsal raphe, subregions that appear to mediate anxiety and depression, respectively. We will investigate the temporal role of 5-HT1B autoreceptors in modulating fear learning by examining their effects on acquisition, expression, and extinction of conditioned fear. We will study the mechanism by which these autoreceptors regulate extracellular 5-HT levels, and whether they do so by modulating serotonin transporter function. We will examine the modulation of 5-HT1B autoreceptor effects on behavior by stress and the CRF related peptides. These questions will be addressed using a combination of novel molecular, pharmacological, and behavioral strategies that we have developed in our lab, allowing us to isolate the role of 5-HT1B autoreceptors in these animal models of stress-associated illnesses.

[unreadable] DESCRIPTION (provided by applicant): Striatum plays an important role in reward-motivated learning such as that associated with habitual use of drugs of abuse or affective disorders. Furthermore, medium spiny neurons in striatum account for the highest expression of 5-HT6 receptors in brain. We have found that locally increased expression of 5-HT6 receptors in rat dorsal striatum using a viral vector impairs learning in a simple operant task using sucrose rewards. This is entirely reversed by a selective 5-HT6 antagonist, suggesting that increased 5-HT6 receptor activity was responsible for the impairment. However, the mechanism of how 5- HT6 receptors modulate striatal function and stimulus-response learning is not known. Therefore, we propose to examine the role of this receptor in striatal function. In the first Aim we will determine whether GABAergic or cholinergic interneurons express 5-HT6 receptors and we will determine the effects of chronic 5-HT6 agonist and antagonist treatments on gene expression in dorsal striatum. We will map changes in cellular activation using immediate early gene expression in animals that overexpress 5-HT6 receptors in dorsomedial striatum. In the second Aim, we will develop viral vectors that express 5-HT6 receptors selectively in neurons of either the direct or indirect pathway; these vector tools will greatly increase our precision in determining how 5-HT6 receptors modulate reward-motivated learning; we will use these tools to investigate which pathways mediate the effects of 5-HT6 receptors on striatal learning. These aims will refine the cellular and mechanistic features of 5-HT6 receptor effects in striatum, so that future studies can focus on the most salient aspects of 5-HT6 receptor regulation of motivated behavior. The ultimate goal of this project is to identify the functional consequences of 5-HT6 receptor manipulations in discrete neural circuits on important psychological processes that are relevant to addiction, cognitive function, and mood regulation; this may ultimately lead to novel therapies for these disorders.Projective Narrative [unreadable] [unreadable] Habit learning is a normal process that depends upon the dorsal striatum that can be subverted in addiction so that compulsive, habitual behaviors persist despite substantial negative consequences. This project is designed to identify the role of striatal 5-HT6 serotonin receptors in reward motivated learning using rat behavioral models, state of the art viral gene transfer strategies, and measurement of gene expression changes. The long-term goal is to identify a new and powerful target for medical treatment of addiction. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): The molecular basis of emotional learning is a rapidly evolving research topic that has crucial implications for the treatment of a variety of mental conditions, including posttraumatic stress disorder, depression, and anxiety disorders. While there is a general understanding of the neurotransmitter systems and brain regions involved in mediating symptoms of these disorders, there is still rather little known about the molecular mediators of emotional learning. This is an important deficit because a better understand of the discrete biological processes involved in problem such as fear conditioning and stress effects on learning and memory could lead to novel therapeutic strategies that will be discovered because of new research into these basic brain mechanisms. The University of Washington has a number of investigators in several departments that are studying several aspects of emotional learning and stress effects on cognition and behavior. We propose to launch a search to recruit a new Investigator at the Rank of Assistant Professor in the Department of Pharmacology to use cutting edge molecular strategies to study the fundamental neural processes that underlie emotional learning. A number of potential technical strategies would meet this expection, such as proteomics, FRET, two photon microscopy, etc, but the search is intended to be open to considering other novel strategies that can be brought to bear on the topic of emotional learning. We expect the new investigator to be an expert in a new technological strategy that will provide synergistic energy to current research efforts at the University of Washington, and that mutually enrichment of the current institutional environment will lead to the development of an exciting new investigator and enhanced research in the laboratories of existing investigators. The new Investigator would join a distinguished Department containing a number of experts in molecular pharmacology, with special emphasis on signal transduction research, and a broader ensemble of behavioral neuroscientists from several departments who are already highly interactive with each other. We expect further collaborative interactions within our institution and between institutions to develop through this recruitment and its related activities. PUBLIC HEALTH RELEVANCE: Emotional learning involves the cognitive and biological processes involved in making associations and developing learned patterns of behavior that are associated with mental conditions associated with fear, depression, stress, and trauma. Identifying specific molecular mechanism associated with emotional learning may identify novel targets for therapeutic intervention in afflicted individuals.

Serotonin is a neurotransmitter in the brain that plays an important role in complex behaviors including addiction. We have previously shown that 5-HT1B receptors alter brain reward mechanisms that play a central role in addiction;these receptors also seem to be involved in the adaptation to stress. Since stress can facilitate drug craving and relapse to drug seeking after abstinence, we will examine the role of this receptor using rodent models of drug addiction. In the initial five years of this grant, we established that the regional expression of 5-HT1B receptors is a critical determinant of their effects on behaviors associated with addiction. An overriding theme in the coming years is to identify the stages at which these receptors play key roles in addiction. We will use strategies that combine molecular and behavioral approaches to investigate the function of 5-HT1B receptors in nucleus accumbens projection neurons, a key component in the brain reward and habit formation systems. We hypothesize that increased 5-HT1B expression is an allostatic adaptation to chronic stress that ameliorates mood disturbance yet facilitates drug addiction. Aim 1 investigates the temporal association between social defeat stress and 5-HT1B gene expression in dorsal and ventral striatum. We will therefore investigate whether experimentally increased 5-HT1B receptor expression using viral mediated gene transfer reduces the impact of social defeat stress on hedonic state, and whether expression knockdown exacerbates stress-induced changes in the same behavior. In Aim 2 we will determine whether increased 5-HT1B expression modulates the motivation to self administer cocaine at several key stages, using animal models that capture the developmental progression inherent to addiction. These two Aims constitute a two-pronged strategy investigating the bidirectional relationship between behavioral stress experience and gene expression that impact hedonic state and the propensity to self-administer cocaine.

DESCRIPTION (provided by applicant): Serotonin-6 (5-HT6) receptors are more heavily expressed in striatal medium spiny neurons than anywhere else in human or rat brain. Therefore, they are positioned to mediate a significant proportion of serotonin's effect on motivated behavior, such as is involved in learning and maintenance of self-administration of natural and drug rewards. We previously established that 5-HT6 receptors interfere with the acquisition of reward motivated learning in both dorsal and ventral striatum. Our core hypothesis is that, since 5-HT6 receptors are expressed in both the direct and indirect pathway medium spiny neurons, these receptors activate both pathways to a similar extent. This opposes the action of dopamine, which activates direct pathway neurons via D1 receptors and inhibits indirect pathway neurons via D2 receptors. Thus, dopamine turn on the on switch and off the off switch, and differential activity in these pathways promotes motivated behavior and procedural learning whereas 5-HT6 receptor activation reduces this differential activity by activating both pathways simultaneously. In order to test this hypothesis we have developed viral vectors that allow us to express transgenes selectively in the direct or indirect pathway medium spiny neurons. These vectors are based on the dynorphin or enkephalin promoter, which differentially target the direct and indirect pathway medium spiny neurons, respectively. We have collected strong evidence that the pDYN and pENK vectors target these pathways selectively. This provides us with key tools to increase or decrease 5-HT6 receptor expression in these pathways differentially and to test the cellular mechanism underlying striatal 5-HT6 receptor actions. The overall plan for this proposal is to use these vectors to disentangle the role of 5-HT6 receptors in direct and indirect pathway neurons of both dorsal and ventral striatum. We will use either overexpression or RNAi knockdown to modulate 5-HT6 receptor expression in either pathway to examine 5-HT6 receptors in discrete subregions of striatum on instrumental learning for a natural reward as well as a drug reward (cocaine). We will also examine the impact of these receptors in each pathway on motivation for cocaine once self-administration is established, and their effect on compulsive drug taking in the face of negative consequences to cocaine taking. The hypothesis, strategy, and tools involved in this project are all highly innovative. The long-term goal of this work is to understand the impact of serotonin on these distinct pathways in drug abuse so that better treatments for addiction can be developed.

DESCRIPTION (provided by applicant): 5-HT1B receptors are expressed in diverse neuron types throughout the brain where they act as inhibitory receptors on presynaptic terminals. When expressed by serotonergic neurons they are autoreceptors whereas in other types of neurons they act as heteroreceptors. It has been difficult to sort out the role of 5-HT1B autoreceptors and heteroreceptors because they are intermixed in most brain regions, even though they are regulating the release of different neurotransmitters. Furthermore, constitutive knockout of 5-HT1B receptors have a complex phenotype that may reflect developmental compensations predominantly, instead of informing about the role of these receptors in adult brain function. Therefore, two types of conditional expression are needed as tools to investigate the contribution of 5-HT1B receptors in different neurons to complex emotional behavior: temporal and phenotype specificity of gene knockout. This proposal intends to solve this problem by creating a new transgenic mouse that will allow conditional and cell type-specific deletion or protection of 5-HT1B receptor expression using available Cre and Flp driver lines. The proposed transgenic mouse is innovative for several reasons. 1. Conditional, cell type-specific expression of Cre will delete the gene in the targeted neurons. 2. Conditional, cell type-specific expression of Flp will excise a loxP site thereby selectively protecting the 5-HT1B gene in those neurons while remaining 5-HT1B receptors elsewhere can be subsequently knocked out by Cre. 3. The targeting construct is designed to minimize the chances of baseline reduction in 5-HT1B expression prior to deletion (i.e. hypomorphism). 4. This strategy can be applied to many different situations to target (or preserve) 5-HT1B receptors with any available Cre and Flp driver lines. For this revised R21 proposal we will focus our characterization on the conditional knockout of 5-HT1B autoreceptors just in serotonergic neurons. In Aim 1 we will construct the targeting construct, express it via homologous recombination, derive transgenic mice, and characterize the behavioral phenotype of the mice. In Aim 2 we will examine the impact of the selective knockout of 5-HT1B autoreceptors (in serotonin neurons) on serotonin transporter function and conditioned fear, an animal model relevant to a number of psychiatric disorders. This will allow us to probe our hypothesis that 5-HT1B autoreceptors are the primary site of 5-HT1B-mediated reductions in fear behavior definitively. In the future it will be possible to examine the site of action of 5-HT1B drugs by using a wide range of other Cre and Flp driver lines to investigate other important neurobiological problems including models of drug addiction, regulation of eating behaviors, and control of respiration in animal models of Sudden Infant Death Syndrome.

DESCRIPTION (provided by applicant): While a great deal has been learned about the neuropharmacology of addiction in recent years, we still have no biological treatments that can prevent or reverse the adaptations in brain function that highjack the mechanisms involved in normal motivated behavior. Brain regions such as the striatum are key targets for developing novel treatments for addiction, but it is important that these treatments do not disrupt normal information processing involved in routine motivated behaviors. This has been a particular problem with dopaminergic drugs, but other neurotransmitters such as serotonin are also centrally involved in the biology of addiction. One potential target is the 5-HT6 serotonin receptor, which is heavily expressed in striatum and affects procedural learning that is involved in drug reward and habit formation. Additionally, the 5-HT6 receptor is the only serotonin receptor localized to the primary neuronal cilium, a cellular organelle present on most neurons that has a critical role in brain development and normal cognitive function that has not yet been thoroughly characterized. Since cilia contain a number of cellular signaling systems that modulate gene transcription and plasticity without interfering with synaptic function directly, thi suggests that exploring the significance of 5-HT6 localization in cilia is a crucial next step. Ciliary localization is thought to be governed by a discrete consensus sequence in the third intracellular loop of GPCRs including 5-HT6 and a small set of other proteins. This R21 project has two aims. The first aim will focus on making site direct mutations in 5-HT6 receptors that will prevent them from localizing to cilia without perturbing their molecular functions otherwise; we will then investigate the signaling consequences of activating 5-HT6 receptors that are or are not localized in cilia. We will perform most of these experiments in primary cultured striatal neurons prepared from 5-HT6 knockout mice, providing a strong basis for reaching conclusions about their function. We will examine the effects of 5-HT6 receptors on cilia and dendritic morphology, cyclic AMP production, DARPP32 hosphorylation, and gene expression using PCR arrays focusing on key pathways predicted to be sensitive to 5-HT6 and cilia function. In the second aim we will test whether ciliary localization of 5-HT6R receptors is critical to the well- established behavioral effects of expressing 5-HT6 receptors in dorsomedial striatum using instrumental learning as the key behavioral outcome. We will perform these experiments both in rats (where the effect is well established) and in wild-type and 5-HT6 knockout mice, where the effects of normal or mutant 5-HT6 receptors can be studied in the presence and absence of endogenous 5-HT6 receptors. These experiments will create a platform for determining whether targeting cell signaling mechanisms in the cilia will offer new opportunities for developing therapeutics for addiction and other compulsive disorders.

? DESCRIPTION (provided by applicant): Currently available antidepressants work well for only a subset of individuals with depression and other stress-related disorders. Therefore, identifying new targets for treating depression is an urgent health imperative. The Lateral Habenula (LHb) is a small, bilateral structure that appears to play a critical role as a relay node connecting several key brain regions that are involved in mood control such as the dorsal raphe nucleus (DRN), ventral tegmental area (VTA), and the rostromedial tegmental nucleus (RMTG). The detailed neurochemistry of the LHb is poorly understood. We know that LHb plays a critical role in mediating negative or aversive emotional states relating to mood, reward, and motivation. Pilot studies in humans and animal models suggest that the LHb may be an important target for treating refractory depression. For example, we found that the expression and activation of hM4Di, a Gi-coupled inhibitory DREADD, in LHb produced an antidepressant effect in the forced swim test model of antidepressant action. The primary technical strategy for this project will be the use of intersectional transgene expression in rats using two types of viral vectors: AAV-DIO-transgene vectors (injected into LHb) and CAV2-Cre vectors (injected into one of the projection target regions-DRN, VTA or RMTG). Together these vectors produce transgene expression only in neurons of the desired pathway. This strategy will be used to manipulate the activity of these discrete pathways (with DREADDs) as well as to interrogate mRNA translation in each (with RiboTag). Aim 1 will test which of these LHb pathways is responsible for modulating immobility in the forced swim test. We predict that modulating the LHb to DRN pathway will alter immobility, and that activating the Gi-coupled DREADD with the ligand clozapine-N-oxide will reduce immobility whereas activating the Gs-coupled DREADD will exacerbate immobility. Aim 2 will assess the contribution of these pathways to different domains of stress-related emotional states such as anhedonia (saccharin preference), anxiety (open field), and emotional learning (conditioned place aversion) using DREADDs that activate or inhibit these pathways selectively. Aim 3 will assess how LHb pathways alter the emergence of behavioral vulnerability or resilience in response to repeated social defeat stress. Aim 4 will use RiboTag, an epitope-tagged ribosomal protein, to selectively immunopurify the polyribosomes from the transduced pathways followed by RNAseq and validation of significantly different mRNAs. This will allow us to evaluate the gene expression phenotypes of neurons in each pathway, and to investigate how LHb neurons respond to stress exposure as well as antidepressant treatment. It will be a powerful method for identifying regulatory networks and potential nodes of control that can promote resilience to stress. Together, these experiments will provide a great deal of new information about the functional organization of LHb, its role in animal models of stress, and may help to identify new molecular targets in LHb that can leveraged in the treatment of stress disorders.

Project Summary Repeated, uncontrollable stress is a key contributor to depression and anxiety disorders, including posttraumatic stress disorder. Stress causes a series of psychological and physiological adaptations that can be chronic and sustained, and likely contribute to the vulnerability to subsequent bouts of depression and anxiety. On the other hand, some individuals can be exposed to stress repeatedly and appear to be relatively resilient. Given that the serotonin system plays a key role in stress responses and the pharmacological treatment of stress disorders, we presume that the adaptations associated with vulnerability and resilience are represented in cellular and molecular plasticity in the raphe neurons of individuals following exposure to stress, especially in pathways that are involved in signal transduction. This project will use mouse behavioral and molecular strategies to investigate these adaptations and how resilience might be promoted while vulnerability might be prevented or reversed. Aim 1 will examine how modulating signal transduction pathways within serotonergic neurons might alter the behavioral phenotype of animals exposed to repeated social defeat stress. We proposed to express DREADDs that activate Gi or Gs signaling pathways selectively in serotonergic neurons (ePet1-Cre mice). The effects of DREADD activation prior to repeated social defeat on stress responsiveness in behavioral assays of depression-like and anxiety-like behaviors. This Aim will provide new mechanistic and therapeutic clues regarding how plasticity in the serotonin system confers vulnerability or resilience to stress. In Aim 2 we will evaluate whether DREADD-mediated inhibition of serotonergic neurons in the interval after repeated social defeat stress can reverse the enhanced vulnerability to subsequent stressors, using corticosterone responses to restraint and immobility in the forced swim test to assess different domains of stress reactivity. Aim 3 will selectively interrogate the translation of mRNA by selectively expressing ?RiboTag? in the serotonergic neurons of ePET1-Cre mice. RiboTag is an epitope-tagged ribosomal protein that allows for antibody-mediated pull down only of polysomes from target cells derived from a complex cell homogenate (in this case a fresh midbrain tissue punch). Enrichment of mRNA from serotonergic neurons will allow us to perform RNA-seq to investigate the totality of actively translated mRNA at key points during and after stress exposure. We will focus on mRNAs associated with excitability, signal transduction pathways, and cytokine responses, and use pathway analysis to identify co-regulation of mRNA translation in response to stress; this is intended to provide new mechanistic information and new targets for therapeutic development.

? DESCRIPTION (provided by applicant): The practice of psychiatry is increasingly becoming an evidence-based science that is built upon our evolving understanding of the biological underpinnings of mental illness accompanied by the establishment of effective means of delivering high quality care. Resident psychiatrists learn best practices during their training, bu the pipeline of physicians trained to lead mental health research programs in the future is in jeopardy. Multiple barriers interfere with the likelihood that a psychiatrist will choose to become a researcher including recruitment, retention, advancement, and financial counter-incentives. We intend to address these barriers at the University of Washington with an R25-supported Psychiatry Resident Research Program (PRRP). Our proposal is to recruit four residents into our research track every year. The PRRP will emphasize three areas of excellence within our Department: Neuroscience, Health Services, and Addiction Psychiatry, but participating residents may also continue to choose from among a wider range of mentors for their research experience. We will build this program based on already successful existing components including our Neuroscience Research Track and our Psychiatry-Primary Care T32 Program. The PRRP will be overseen by an internal organizing committee and will be guided by an external advisory committee. We are well positioned for success because our starting point is based on a vibrant, diverse, and highly competitive residency program that has strong leadership and institutional commitment. Even though we already have seven residents active in the Research Pathway, the PRRP will allow us to attract more research-oriented residents to our residency. PRRP participants will begin the process of selecting a mentor during their first or second year and will gradually expand research time each year; the typical participant will have 80% research time available for 16 months, scheduled in a flexible manner that fits their research training needs. A The curriculum will meet the individual's needs and consist of group tutorials, advanced coursework, grant writing course, structured education in the responsible conduct of research, research seminars and conferences, and scientific and career guidance from a mentoring committee. We will emphasize creating a smooth transition from resident to faculty status by encouraging participants to apply for positions on our existing NIH T32 grants and VA research fellowships, apply for Institutional KL2 awards and NIH K08 awards, and secure PHS loan repayment awards. We will expand our applicant pool by reaching out to medical students from under- represented groups and those with a demonstrated commitment to research. Success of the program will be demonstrated by fully filling the PRRP class each year, attracting a diverse group of participants, increasing the rate of successful transition to fellowship and career development experiences, and ultimately launching more psychiatrists into research careers. Additional benefits will include the positive impact on the academic culture of our entire residency and influencing research oriented medical students to become psychiatrists.

Abstract Cocaine addiction involves the loss of control over drug taking so that individuals take more drug over time and can have prolonged vulnerability to relapsing to drug seeking, even after extended periods of abstinence. The progressive molecular and synaptic adaptations in neurons in the CNS that underlie these changes are not well understood, but can be studied using animal models based on extended cocaine self-administration followed by abstinence in rats. These models have shown that the nucleus accumbens core (NAcC), a small but critical brain region in ventral striatum, is implicated in compulsive cocaine taking and relapse to cocaine seeking after extended abstinence. The NAcC receives and integrates afferent information from many different brain regions and has two main output projections, the direct and indirect pathways; these pathways tend to oppose one another functional, and we predict that adaptations in signaling processes within these neurons are critical determinants affecting the relapse to drug seeking. By understanding the adaptations in cell signaling in these NAcC output neurons following extensive cocaine exposure and abstinence, we hope to contribute to novel treatment strategies for reducing the potential for relapse to drug seeking. We propose to investigate the time-dependent increase in drug seeking during abstinence known as the ?incubation of craving?. We will use several innovative tools. First, we will use an intersectional viral vector approach to introduce DREADDs and other transgenic proteins to perturb and study direct and indirect pathway neurons selectively during incubation. By injecting AAV vectors with floxed and inverted transgenes into NAcC, we can activate transgene expression selectively in the direct or indirect pathway neurons by injecting the ventral tegmental area or ventral pallidum with CAV2-Cre, which is retrogradely transported to the cell bodies in NAcC. Second, we will use engineered ?DREADD? receptors, a technology that we helped to establish for use in rat brain during complex behavioral experiments. DREADDs will allow us to activate Gs or Gi signaling pathways selectively in either direct or indirect pathway neurons during either repeatedly during cocaine taking or during early or late forced abstinence, thereby assessing how these canonical second messenger pathways modulate the plasticity involved in escalation or incubation. Third, we will utilize RiboTag technology to immunopurify polyribosomes selectively from direct or indirect pathway neurons and investigate the changes in RNA translation in these opposing pathways during abstinence and incubation of craving, both in cell bodies and in the synapses where activity dependent changes in local protein translation has been described. By perturbing and measuring signaling pathways in specified neurons, we hope to develop new strategies for ameliorating the adaptations associated with compulsive drug use and relapse to seeking.

There is an epidemic of opioid overdoses associated with the use of illicit drugs such as heroin and the misuse of prescribed narcotic pain medications. Chronic use of opioids, even when under medical supervision, is associated with the development of tolerance and escalating risk of withdrawal. The opioid withdrawal syndrome is a serious medical problem that can become a major reason why addicts keep using opioid drugs. Currently the only treatments to prevent the withdrawal syndrome are either continuing to take opioids (including substitution drugs such as methadone) or symptomatic management of the many severe problems such as diarrhea, vomiting, dehydration, cramping, muscle pain, insomnia, irritability, and anxiety. Addicted individuals often prefer to continue drugs rather than face withdrawal. While the sudden reduction in mu opioid receptor activation is the proximal cause of withdrawal, surprisingly little is known about how downstream physiological processes contribute to the syndrome. Being ?dope-sick? has many attributes of a severe inflammatory state. Thus, it is not surprising that evidence is accumulating that both opioid tolerance and especially acute withdrawal produce a neuroinflammatory state that is a major contributor to the symptoms experienced. We propose the hypothesis that microglia, the resident immune cells in the brain, become activated during opioid withdrawal and that the inflammatory cascades mediated by these cells lead to much of the withdrawal syndrome. We will test this idea using two strategies. First, we will measure the RNAs in microglia cells that are actively being translated to make protein (as indicators of the biological pathways that are activated in these cells during withdrawal). We will investigate mice given escalating doses of morphine followed by precipitated withdrawal and then use RiboTag, a new technology for retrieving the RNA from specific cell types, to interrogate the sequential changes occurring during microglial activation from opioid withdrawal. Second, we will assess whether the withdrawal syndrome can be prevented by inhibiting microglial activation using engineered ?DREADD? receptors to inhibit microglia during withdrawal. These experiments will utilize state of the art transgenic strategies that are established in our lab and will allow us unprecedented precision in investigating and modulating microglia during acute opioid withdrawal. The ultimate goal of this proposal is to identify novel molecular targets in microglia that can prevent the inflammation associated with withdrawal, leading to the development of new treatments to mitigate opioid withdrawal. This will make withdrawal itself safer and potentially contribute to the motivation of addicted individuals to discontinue opioid use.

PROJECT SUMMARY/ABSTRACT Compulsive behaviors are prominent, disabling, and often treatment-resistant symptoms of several neuropsychiatric disorders, including obsessive compulsive disorder (OCD). Dysfunction within fronto- subcortical brain structures is thought to underlie compulsive behaviors, but the precise circuits involved remain unknown. Currently, chronic (> 4 weeks) treatment with serotonin reuptake inhibitors (SRIs) provides the only effective pharmacological monotherapy for compulsive behaviors; yet, approximately 50% of OCD patients do not respond to SRIs. We have shown that serotonin 1B receptors (5-HT1BRs) regulate the expression of compulsive behaviors. 5-HT1BRs are located on axon terminals of serotonin-containing neurons (presynaptic), and neurons containing other neurotransmitters (postsynaptic)(3), where they inhibit neurotransmitter release when activated. Furthermore, 5-HT1BRs signal through both a canonical G protein- mediated pathway, and a noncanonical G protein-independent pathway. Canonical 5-HT1BR-mediated Gi- signaling requires direct interaction of 5-HT1BRs with glycogen synthase kinase-3 beta (GSK3?). On the other hand, the intracellular scaffolding protein beta arrestin-2 (?-arrestin2) mediates noncanonical 5-HT1BR signaling. We recently found that activation of 5-HT1BRs within the orbitofrontal cortex (OFC) is necessary and sufficient to induce compulsive behaviors in mice. The OFC contains both pre- and postsynaptic 5-HT1BRs, including those on dorsal raphe-OFC and basolateral amygdala (BLA)-OFC projections, respectively. We propose to develop and use a two virus, in vivo CRISPR-Cas9 system to dissect the role of 5-HT1BR density and canonical versus noncanonical signaling within these two circuits in modulating compulsive behaviors. In Specific Aim 1, we will either overexpress or knockout 5-HT1BR expression within these two projections. We will infuse 1) a Cre recombinase (Cre) dependent adeno-associated virus (AAV) which expresses either 5- HT1BR, or the S. aureus Cas9 (SaCas9) gene plus a guide sequence against 5-HT1BR, into the dorsal raphe or BLA, and 2) a Cre-expressing retrograde canine adenovirus (CAV2-cre) into the OFC. In Specific Aim 2, we will also use the two virus, in vivo CRISPR-Cas9 system, but will infuse a Cre-dependent AAV expressing SaCas9 and a guide sequence against either ?-arrestin2 or GSK3? into the dorsal raphe or BLA. Mice will be evaluated for 5-HT1BR agonist-induced compulsive behaviors in the open field, a delayed alternation task, and an operant paradigm assessing both acquisition and persistence of habitual lever pressing. The proposed work could establish a novel in vivo CRISPR-Cas9 system for manipulating gene expression within specific neural circuits, and could lead to innovative therapeutic strategies for treating compulsions.

The current epidemic of opioid overdoses has been propelled by both illicit and prescribed narcotic pain medications. Extensive opioid use and repeated abstinence increases the likelihood of severe withdrawal and perpetuates the vulnerability to relapse via means of negative reinforcement. The negative emotional valence of withdrawal can last long after the initial, dramatic physical signs, involving a protracted negative emotional state, drug craving, and a high likelihood of relapse. These combined symptoms are commonly referred to as being ?dope sick?. Addicted individuals often prefer to continue drugs rather than face withdrawal. Being ?dope-sick? has many attributes of a severe inflammatory state and this led us to investigate the involvement of microglia, the innate immune cells that reside in the brain, in opioid tolerance and withdrawal, and this was supported by a CEBRA R21 grant (R21-DA044757). That CEBRA R21 grant resulted in our findings of dramatic changes in ribosome-bound mRNAs?the ?translatome??in microglia using RNA sequencing of RiboTag purified microglial RNAs. Those results provided us with the leads that form that basis for this proposal. Many of the changes related to cyclic AMP signaling and its downstream targets, and experimental chemogenetic stimulation of Gi/o signaling was found to actually worsen opioid withdrawal. With the understanding that glia are partners in plasticity, we suspect that the relapsing/remitting nature of opioid dependence serves to prime and condition microglia, shifting the impact from tempering withdrawal during initial stages to exacerbating withdrawal and opioid seeking after multiple cycles of tolerance and withdrawal. Thus, investigations into the role of glia in withdrawal may provide new therapeutic avenues. We propose three Aims using fentanyl and a recently developed transgenic mouse that allows conditional and microglia-specific Cre and TdTomato expression without disrupting microglia function. In Aim 1 we will analyze the trajectory of the changing microglial translatome after one vs. five cycles of opioid dependence and spontaneous withdrawal. In Aim 2 we will examine the physical and behavioral consequences of one vs. five cycles of opioid dependence and withdrawal, to explore the idea that intermittent cycles of dependence and withdrawal exacerbate the negative consequences of withdrawal. We will then investigate the hypothesis that the purinergic receptors P2Y12 and P2X7 are involved in microglial responses during initial and delayed phases of opioid withdrawal. In Aim 3 we will use an in vitro brain slice model with 2- photon confocal imaging of microglia. We will study the microstructure and motility of microglia using time- lapse microscopy. We will measure real-time changes in cyclic AMP using a FRET-based biosensor and calcium dynamics with GCaMP6. These three Aims integrate the temporal, behavioral, and molecular consequences of microglial engagement during opioid dependence and withdrawal.