Todd D. Gould, M.D. - US grants

DESCRIPTION (provided by applicant): The combined lifetime population incidence of mood disorders, including bipolar disorder and unipolar depression, is over 15%. Many of these individuals do not receive optimized pharmacological treatment, which is influenced by the fact that the ability to predict patients who will respond to a particular drug is limited. The therapeutic efficacy of lithium for the treatment of mood disorders is well characterized. It is commonly used as a mood stabilizer, and in some patients as an antidepressant or adjunct antidepressant. However, despite extensive clinical use, it remains unclear exactly by what mechanism lithium exerts its therapeutic effects, and which patients can be optimally treated with lithium. This lack of knowledge is related to two specific clinical problems: 1) a restricted capacity to apply pharmacogenetic approaches to study genes associated with lithium response as well as predict prior to treatment which patients will respond to lithium, and 2) an inability to use a hypothesis driven approach to improve upon lithium's side- effect profile and narrow dose range with second-generation compounds. This research proposal is designed to characterize an animal model that will allow future studies to experimentally address the above problems. Inbred and outbred mice are commonly utilized in the field of behavioral pharmacology to evaluate effects of biological and genetic variations in behavioral models of medication responses. These differential behavioral patterns that manifest in different mouse strains have assisted in the characterization of essential neural processes and response to medications that are influenced by strain-dependent inheritable traits. However, despite over 50 years of clinical use and an unidentified mechanism of action, the use of mouse strain differences has not yet been fully employed in the study of the mood stabilizer- and antidepressant-like effects of lithium. The objective in this R21 application is to identify, and characterize behaviorally and pharmacologically, strains of mice that manifest distinct responses to lithium treatment in behavioral models. The Specific Aims are to: 1) Identify strains of mice with differential antidepressant-like effects of lithium, 2) Identify lithium pharmacokinetic correlates to the differential behavioral effects of lithium, and 3) Assess the mood stabilizer-like actions of lithium in individual strains by contrasting strain variation in models of antimanic vs. antidepressant efficacy. The mouse strain variations in lithium response characterized through the proposed research will likely lead directly to studies that will help define the target, and the underlying genetics, of lithium response. This new knowledge would ultimately lead to further optimization of treatment for patients who suffer from mood disorders. PUBLIC HEALTH RELEVANCE: Despite extensive clinical use of lithium for the treatment of mood disorders it remains unclear which patients will respond best to lithium and by what mechanism lithium exerts its therapeutic effects. It has therefore not been possible to improve upon lithium's side- effect profile and narrow dose range by developing novel medications that share the therapeutic target of lithium. This research proposal is designed to characterize a model that will assist future studies in experimentally determining the clinically relevant targets and underlying genetics of human response to lithium.

DESCRIPTION (provided by applicant): Suicide has a devastating impact on victims, their extended families, and public health in the United States, as well as throughout the world. Few treatments have been shown to reduce risk. An exception, supported by extensive clinical evidence, is that lithium is effective in reducing the risk of both attempted and completed suicide. However, the mechanisms underlying lithium's antisuicidal actions are not yet known, limiting the development of improved prevention approaches. We intend to use the mouse as a model organism to elucidate molecular pathways by which lithium interacts with biological and behavioral factors associated with suicide in humans. However, rather than attempting the infeasible task of modeling suicide in mice, we will focus on approaches that assess mouse behavior in tests relevant to well validated endophenotypes (deconstructed components of complex behavioral phenotypes) associated with suicide including aggression and impulsivity. These endophenotypes will be used in combination with human genetic, biochemical, and pharmacological findings in suicide research to provide construct-valid animal models. Toward this end, clinical studies have implicated polymorphisms in a number of genes, including neuronal nitric oxide synthase (NOS1), with measures of impulsivity, aggression, and suicide. Similarly, the results of extensive research have implicated deficits in serotonin (5-HT) neurotransmission in the etiology of suicidal behavior as well as increased impulsivity and aggression. Data from preclinical and human genetic studies indicate that lithium may exert some of its mood stabilizing effects through inhibition of the enzyme glycogen synthase kinase-3! (GSK-3!). Intriguingly, emerging basic science evidence links NOS1 function, 5-HT neurotransmission, and GSK-3! activity suggesting that they may be causally linked in the pathophysiological processes relevant to the etiology and treatment of psychiatric diseases such as suicide, where impulsivity and aggression play a role. Thus, our Specific Aims are to: 1) Identify the effects of lithium on behavior in mice with genetically- and pharmacologically-induced decreases in 5-HT levels;2) Identify the effects of lithium on behavior in mice with genetically- and pharmacologically-mediated deficiencies in nitric oxide synthase 1 (NOS1) activity;3) Evaluate the role of glycogen synthase kinase-3!, a direct target of lithium, in modifying behaviors mediated by decreased 5-HT and NOS1 function. These studies will capitalize on current knowledge of lithium pharmacology and use mouse genetic knockouts and pharmacological approaches to dissect the molecular and neurobiological mechanisms whereby lithium may modify impulsive and aggressive behavior as well identify points of interaction between lithium and biological markers known to be associated with suicide. Public Health Relevance: The data derived rom these studies should promote the development of improved pharmacological interventions to modify aggressive and impulsive behaviors thereby decreasing the risk of suicide across all diagnostic categories. PUBLIC HEALTH RELEVANCE: A completed suicide has a devastating impact on families, society, and public health. Few treatments have been shown to result in reduced risk;however, lithium treatment is effective, for unknown reasons, in reducing the risk of both attempted and completed suicide. This application proposes experiments in the context of endophenotype strategies that will reveal molecular mechanisms whereby lithium acts to exert its therapeutics effects.

DESCRIPTION (provided by applicant: It is clear that sex differences are a critical consideration to understanding the pathophysiology of mood disorders. While it is hypothesized as likely that susceptibility genes and hormones interact to modify the development of mood disorders, there exists limited current experimental evidence to support this. Numerous recent human genetic studies indicate a robust association between polymorphisms in CACNA1C, a gene that codes for the 11 subunit of the CaV1.2 L-type calcium channel, and a diagnosis of bipolar disorder or depression. We have recently identified sex differences in both humans and mice, which collectively indicate that CACNA1C genotype influences resilience to mood-related behavioral changes primarily in females, but the factors underlying these sex differences are unknown. The objective of this application is to use our mouse model to further assess the interaction between sex and Cacna1c genotype leading to the development of depression-related behavior, and to define the hormonal mechanisms that determine female-specific behavioral effects in Cacna1c haploinsufficient mice. Sex differences in the adult brain can be established early in development as a result of an organizational process mediated by gonadal hormones. The same gonadal hormones act on the adult brain and their actions may be constrained by earlier organizational processes or may, on their own, mediate sex differences in physiology and behavior. Our central hypothesis is that gonadal hormones interact with Cacna1c genotype during the early neonatal organizational period to modulate depression-related behavior at adulthood, and that decreased expression of Cacna1c will result in female mice that are resilient to the effects of chronic stress. Our specific aims are to: 1) Identify the role of activational effects of gonadal hormones in determining female-specific learned helplessness behavior in Cacna1c haploinsufficiency, 2) Identify the role of organizational effects of gonadal hormones in determining female-specific learned helplessness behavior in Cacna1c haploinsufficiency, and 3) Define the interaction between Cacna1c haploinsufficiency and sex in modifying behavior and brain plasticity in the chronic unpredictable stress model of depression. Our studies will improve understanding of the role of the Cacna1c gene in regulating behavior and other brain functions in a context relevant to the pathophysiology of mood disorders. Completion of the experiments describe in this application will provide critical support for the hypothesis that gonadal hormones and genetic risk factors interact at sensitive developmental time periods to modulate the risk to develop a mood disorder. Public Health Relevance: Once we complete these experiments it will then be possible to translate our findings to additional animal models and ultimately to humans, elucidating mechanisms and possible drug targets, and developmental time points for intervention in patients who suffer from mood disorders and who may carry CACNA1C risk alleles. PUBLIC HEALTH RELEVANCE: Recent human genetic studies indicate a robust association between polymorphisms in CACNA1C, a gene that codes for the 11 subunit of the CaV1.2 L-type calcium channel, and a diagnosis of bipolar disorder or depression. We have recently identified sex differences in both humans and mice, which collectively indicate that CACNA1C genotype influences mood-related behavioral changes primarily in females, but the factors underlying these sex differences are unknown. Completion of the experiments described in this application will provide critical support for the hypothesis that gonadal hormones and genetic risk factors interact at sensitive developmental time periods to modulate the risk to develop a mood disorder.

DESCRIPTION (provided by applicant): Depression afflicts approximately 16 percent of the world population at some point in their lives. Although antidepressant medications are available and useful, many patients remain treatment-refractory, and currently used drugs take several weeks to be effective. A recent exciting development is the finding that ketamine, which is widely used as an anesthetic in surgical settings, has efficacy as a rapidly acting antidepressant in treatment resistant patients. A single intravenous (i.v.) administration of a sub-anesthetic dose of ketamine results in prompt improvement in mood in depressed individuals, and the beneficial effect is sustained for about a week. Despite these promising results, ketamine's potential as a long-term antidepressant medication is limited due to its addictive nature, anesthetic properties, capacity to produce dissociative effects even when administered at low doses, and the invasiveness of its most common route of administration (i.v.). The proposed project takes advantage of ketamine's likely mechanism of action (inhibition of the NMDA receptor) but targets a site of the receptor (the glycineB receptor), which is less likely to precipitate adverse effects. Specifically, we will examine 4-chlorokynurenine (4-Cl-KYN), a brain-permeable pro-drug of the selective glycineB receptor antagonist 7-chlorokynurenic acid (7-Cl-KYNA). Notably, 4-Cl-KYN is currently under development for the treatment of neuropathic pain and, in a randomized dose escalation study in healthy human volunteers, was well tolerated and showed good oral bioavailability. Our long-term goal is to pursue the use of 4-Cl-KYN for the treatment of major depressive disorders in humans. Here, we will use mice to test the antidepressant-like properties and the side effect profile of 4-Cl-KYN. We will first, in Specific Aim #1, define the range of 4-Cl-KYN action on depression-related behaviors. These studies will evaluate dose- response relationships and include tests to predict rapid therapeutic action and to validate the glycineB receptor as a target for clinical intervention. In Specific Aim #2, we will define biochemical and antidepressant-like effects of prolonged treatment with 4-Cl-KYN. We will measure behaviors, and the levels of both the precursor (4-Cl-KYN) and the effective compound (7-Cl-KYNA), in the brain of mice following various dosing schedules (continuous/chronic vs. intermittent, and peripheral vs. oral administration). The goal here is to address clinically relevant questions regarding prolonged efficacy, and possible tolerance or sensitization phenomena related to sustained glycineB receptor blockade. Finally, in Specific Aim #3, we will compare the side effect profiles of 4-Cl-KYN and ketamine, using treatment paradigms selected from Aim #2 and assessing behavior in tests that predict abuse and psychotomimetic potential. We anticipate that 4-Cl-KYN will prove superior to ketamine on several important experimental measures, and that successful completion of the project will allow us, to the extent possible using preclinical methods, to realistically evaluate the potential of 4-Cl-KYN as a treatment option for depression in humans.

DESCRIPTION (provided by applicant): The male brain contains significant concentrations of 17b-estradiol (E2), due to conversion from testosterone by aromatase, and estrogen receptors that upon activation initiate a number of signaling pathways implicated in the treatment of depression. However, the effects of E2 on male behavior in animal models of depression and anxiety have received only limited consideration. Similarly, the potential beneficial effects of E2 in male humans have been studied only to a limited degree, in part due to its potential side effects including feminization. As such, brain-selective E2 therapy could, for some patients, be an improvement over existing treatments, which result in inadequate treatment for the majority of patients. Therefore, there is a huge unmet need for a new and safer E2 therapy for preclinical studies, as well as human male proof of concept and future clinical treatment studies. Herein, we propose to test, in animal models of depression and anxiety, 10b,17b- dihydroxyestra-1,4-dien-3-one (DHED)-an innovative compound classified according to its unique mechanism of action as a brain-selective bioprecursor prodrug of E2. Based on encouraging preliminary data, we aim at establishing the possible benefits of brain-selective E2 in the treatment of depression and anxiety in males. Our central hypothesis is that brain selective E2 treatment wil result in antidepressant- and anxiolytic-like effects in male mice. This hypothesis is supported by our studies where administration of DHED to male and female rodents produced significant E2 levels in the brain. Further, our preliminary data indicates that administration of DHED has antidepressant- and anxiolytic-like effects in female mice and is likely to have similar effects in male mice. In the first Specific Aim, we will define the range of brain-selective E2 treatment with DHED on depression- and anxiety-like behaviors in male mice. These studies will encompass acute and chronic administrations, dose-response curves, and studies involving gonadally intact and orchidectomized males. We hypothesize that E2 formed from the systemically administered DHED will result in robust antidepressant- and anxiolytic-like effects in male mice and that E2 will reverse deleterious effects of orchidectomy on depression and anxiety outcomes. In the second Specific Aim, we will identify, through bioanalytical methods, brain-selective DHED distribution and bioactivation to E2 that associate with behaviors and intracellular signaling pathway activity. We will focus on the measurement of E2 in target (CNS) and non- target (peripheral) tissues and in the circulation after acute and chronic systemic administration. We wil correlate all behavioral outcomes in Specific Aim 1 with brain levels of E2, which will be the first study ever in males to link brain concentration of E2 to anxiety- and depression-related behavioral outcomes. Successful completion of the proposed experiments will allow us to conclude whether to move forward with brain-selective E2 therapy as a potential treatment of depression and anxiety in men, and provide proof-of-concept to support future rodent and human testing of brain selective E2 in various other CNS diseases in males.

DESCRIPTION (provided by applicant): One of the most consistent findings to have emerged from psychiatric disorder genome wide association studies (GWAS) is with CACNA1C, a gene that codes for the alpha1 subunit of a voltage-dependent L-type calcium channel. Consistent with the NIMH Research Domain Criteria (RDOC) initiative, the biological implications of CACNA1C function are relevant to a diagnosis of bipolar disorder, depression, and schizophrenia. However, in spite of strong genetic data implicating sequence variations in CACNA1C as a risk factor, it is not known how genetic variants located within the gene modify risk. All GWAS-identified SNPs in CACNA1C are located within a single large intron 3 and do not lead directly to changes in the sequence of the coded protein. The central hypothesis guiding the present research effort, supported by our preliminary data, is that specific genetic variation in CACNA1C intron 3 modifies regulatory functions that can be bioinformatically predicted and experimentally validated. Our hypothesis is based on our bioinformatic analyses of the regions surrounding associated human SNPs and preliminary data from our in vitro functional validation of a subset of the in silico predictions. This proposal proposes a multiple methodology strategy, consistent with studies already underway in the laboratory. We will, in Specific Aim #1, use a bioinformatics approach to define sequences in human CACNA1C that are likely to harbor regulatory elements. We predict that the human CACNA1C gene harbors putative regulatory elements containing alleles in linkage disequilibrium (LD) with GWAS identified SNPs. Specific Aim #2 proposes to test human candidate regions in reporter vector systems to assess regulatory activity. We will clone putative regulatory elements into reporter vectors to assess their function as modifiers of transcription in vitro, fine map the location of these regulatory elements, and evaluate these elements for putative TF binding using co-transfection and TF-specific gel-shift assays. We predict that psychiatric condition-associated SNPs (or genetic variations inherited with them) will result in allele-specific changes in CACNA1C gene expression and/or altered function through cis-acting regulatory elements and that we will identify proteins that interact with such regions. As these in vitro and cell-based assays incompletely predict endogenous activity, we will map the presence of human regulatory domains in the mouse (Specific Aim #3). We will locate the corresponding regions in the mouse Cacna1c gene, validate their activity in vitro, and assess in vivo TF binding during different developmental stages. Our intent is to plan future in vivo studies with inbred or transgenic mice harboring similar genetic variations. Overall, our studies will improve basic knowledge of CACNA1C regulation, and significantly progress understanding of the mechanism by which CACNA1C gene intronic variation may modify risk for developing psychiatric disorders.

MODEL DEVELOPMENT AND PHENOTYPING CORE PROJECT ABSTRACT In the United States, 70% of deaths annually can be attributed to chronic conditions. One of the most frequent and debilitating is pain, which can either occur as a symptom of chronic illness or as a primary problem. According to the recent Institute of Medicine (IOM) report on Relieving Pain in America (2011), chronic pain is a public health epidemic affecting more than 116 million Americans and costing more than $600 billion per year in healthcare expenses and lost work productivity?despite advances in pharmacological treatment, most people do not obtain adequate pain relief. Recently, various types of self-management strategies have been tested for chronic pain management, including cognitive behavioral therapy (CBT), non-pharmacologic treatments (e.g., heat, cold, acupuncture, etc.) and physical activity. However, much like pharmacogenomic influences on individual response to drug treatment, self-management intervention trials have demonstrated mixed results in that some, but not all, study participants respond or participate. This could be due to many factors, including the omics mechanisms that underlie an individual's resilience, motivation and/or capability to engage in self-management behaviors that provide a symptom benefit. Moreover, the omics mechanisms underlying the relative success or failure of self-management interventions on an individual level have been understudied. Guided by an adapted National Institutes of Health Symptom Science (NIHSS) model, we propose to develop the University of Maryland, Baltimore (UMB) Omics Associated with Self-management Interventions for Symptoms (OASIS) Center. The science of the Center will focus on our hypothesis that genomic, transcriptomic, epigenomic and proteomic outcomes (hereafter referred to as ?omics?), mediated by psychosocial factors and sex differences and/or moderated by the environment, predict individual resilience, motivation and capability to engage in and participate in self-management behaviors (physical activity) and response to interventions designed to improve chronic pain. The Model Development and Phenotyping Core (MDPC) will provide specific technologies including rodent behavioral testing instruments for nociception, neuromuscular function, sensory and motor fiber function, activity, cognition, depression, and anxiety. Clinical testing instruments include a Medoc Pathway (thermal sensory testing), Medoc pressure algometer, Neurometer (sensory fiber function), von Frey filaments, weighted probes, grip dynamometer, balance, and gait analysis. The MDPC will also provide training and support for the proper handling and husbandry of animals, use of the instruments, and analysis of the data. In consultation with the MDPC Directors, the pilot PIs will refine their research plans and study designs, select appropriate behavioral assays, and develop optimal experimental protocols to maximize the quality of their study results. The goal of the MDPC is to augment the success of the pilot projects by ensuring optimal model selection, assay selection, and data interpretation/analysis. To achieve the MDPC goals, we propose the following specific aims, including (1) Provide expertise in and support for the development and behavioral phenotyping of animal models of chronic pain, (2) Provide expertise in and support for the development of clinical experimental pain models, clinical behavioral testing, and phenotyping of chronic pain patients and (3) Provide instruments, space, and resources that can be used by the OASIS pilots and future studies of self-management of chronic pain.

? DESCRIPTION (provided by applicant): Depression afflicts approximately 16% of the world population. Although antidepressant medications are available, many patients remain treatment-refractory, and currently used drugs take several weeks to be effective. A recent finding is that the non-competitive NMDA receptor antagonist ketamine has rapid antidepressant efficacy in treatment-resistant patients. Despite these promising results, ketamine's potential as a long-term antidepressant medication is limited due to its addictive nature, anesthetic properties, and capacity to produce dissociative effects even at low doses. Similar to many existing psychotropic drugs, the full clinical actions of ketamine may be due to more than one target. Ketamine is rapidly and stereospecifically metabolized to various metabolites that have distinct biological activities. These metabolites may be responsible for either the therapeutic or the side effects of ketamine when it is utilized as an antidepressant. Our preliminary data indicate that some metabolites exert antidepressant-like actions and increase AMPA excitatory post-synaptic current frequency in stratum radiatum interneurons, indicative of increases in glutamate release from CA3 Schaffer collateral inputs. The central hypothesis guiding the proposed studies is that a ketamine metabolite or metabolites independently exert clinically relevant actions that substantially explain ketamine's clinical profile. Here, we will use mice to test the antidepressant-like properties and the side effect profile of these compounds. We will first, in Specific Aim #1, define the range of ketamine metabolite's actions on ketamine-sensitive tests related to depression. In addition to utilizing tests to predict rapid and sustained therapeutic antidepressant action in both male and female mice, these studies will assess different endophenotypes associated with depression including helplessness and anhedonia. Quantifying plasma and brain levels at time points relevant to our behavioral studies will permit us to determine the extent to which ketamine's behavioral effects are associated with brain concentrations of its metabolites. In Specific Aim #2, we will assess whether ketamine metabolites account for the side effects of ketamine. We will determine the effects of metabolites in behavioral tests that predict stimulant effects, as well as abuse and psychotomimetic potential. In Specific Aim #3, we will determine the pharmacological activity of ketamine metabolites relevant to their antidepressant actions. We will use whole-cell patch-clamp electrophysiology to determine the cellular mechanisms that underlie the antidepressant actions of ketamine metabolites. Using behavioral approaches we will assess the contribution of identified mechanisms. A comprehensive understanding of how the therapeutic actions of ketamine are exerted is imperative for the development of improved pharmacotherapies that will effectively reproduce the therapeutic benefit of ketamine, but without the unwanted side effects. Completion of the proposed experiments will provide a strong scientific framework to better understand these properties.

Research in the field of neuroimmunology, including work from our group, has shown that peripheral and systemic inflammation is transduced into the central nervous system (CNS) by several mechanisms resulting in neuroinflammatory processes characterized by increased expression of cytokines and other inflammatory mediators. These mechanisms have been shown to affect neuroendocrine function, monoaminergic neurotransmission and neuronal plasticity negatively impacting brain function and behavior. These processes have been associated with the high incidence of emotional and cognitive impairments in people suffering from chronic inflammatory diseases such as autoimmune, allergic and systemic inflammatory diseases. Despite the broad use of current anti-inflammatory drugs to treat these conditions, they are ineffective in ameliorating the neurobehavioral component, which has been related to the persistence of neuroinflammation once it is triggered from the periphery. Therefore, there is the need to find new treatment options targeting these neuroinflammatory processes. It has been long known that the anesthetic drug ketamine has anti-inflammatory properties in surgical settings. Using rodent models, it has been shown that KET reduces inflammatory molecules in the brain triggered by immune and psychogenic stressors, offering hope for the use of KET as an anti-neuroinflammatory drug in humans. However, the dissociative, addictive, and sedative effects are limitations for the development of KET based therapies. Our preliminary studies reveal that a metabolite of KET, (2R, 6R)-hydroxynorketamine (HNK), which lacks the sedative and addictive effects of KET, possesses powerful anti-neuroinflammatory properties in the mouse model of peripheral administration of bacterial lipopolysaccharides (LPS). Notably, our preliminary studies also revealed that HNK administered alone induces sustained transcription in the brain of the cytokine IFN-g, raising the possibility that HNK mediates these effects through the production of this cytokine. The present application will comprehensively assess the anti-neuroinflammatory properties of HNK and test the role of IFN-g in mediating HNK effects in two models of peripherally-induced neuroinflammation using LPS or bacterial superantingens (SEA). In specific aim #1 we will characterize the dose, timing and effects of HNK on CNS inflammatory processes, as well as identify the cellular source of IFN-g production in the CNS induced by HNK. We will also evaluate whether metabolism of KET to HNK is required for sustained anti-neuroinflammatory effects of KET. In specific aim #2 we will utilize mice lacking IFN-g, as well as pharmacological blockade of IFN-g using neutralizing antibodies, to test the role of this cytokine in the anti-neuroinflammatory effects of HNK. We will also test if this cytokine is required for HNK antidepressant actions in the LPS model. This aim will also employ telemetry to monitor activity, temperature and EEG power bands. If successful, these exploratory/developmental studies will provide the basis for a follow-up R01 application to determine HNK-induced IFN-g anti-neuroinflammatory mechanisms.

SUMMARY Major depressive disorder (MDD) afflicts ~16% of the world population. Despite the availability of several classes and types of antidepressant medications, patients typically take many weeks, if not months, to respond to these drugs, and the majority never attain sustained remission of their symptoms. A remarkable development for the pharmacological treatment of MDD is the finding that the non-competitive N-methyl-D-aspartate receptor (NMDAR) antagonist, ketamine, is an effective, rapidly acting antidepressant in treatment-refractory patients. During our previous funding cycle, we began exploring the role of ketamine?s metabolites in both the therapeutic and adverse effects of ketamine. We identified behavioral, synaptic, and neurochemical effects of the (2R,6R)- hydroxynorketamine (HNK) metabolite. In contrast to ketamine, (2R,6R)-HNK has low affinity for the NMDAR, which is consistent with its reduced adverse effects as measured in preclinical studies. We have also found that (2R,6R)-HNK enhances excitatory synaptic transmission in the hippocampus through a concentration- dependent, NMDAR activity-independent increase in glutamate release probability. Our long-term goal is to elucidate the biological activities of (2R,6R)-HNK, as well as ketamine?s eleven additional HNK metabolites, and utilize our findings to develop novel, effective compounds for the treatment of depression. The central hypothesis is that HNKs exert an acute, synapse-selective form of presynaptic plasticity that leads to a sustained strengthening of mood-relevant circuits. In Specific Aim #1 we will use slice electrophysiology to resolve the synaptic actions of (2R,6R)-HNK, and identify the mechanism(s) by which (2R,6R)-HNK acutely enhances the probability of synaptic glutamate release. We hypothesize that (2R,6R)-HNK acts through a presynaptic cAMP- BDNF-dependent mechanism to promote glutamate release. In Specific Aim #2 we will use in vivo fiber photometry assessments of neuronal activity to determine the synaptic effects of (2R,6R)-HNK on hippocampal circuitry, specifically the Schaffer collateral synapses in the CA1 region of the hippocampus. These experiments will determine (2R,6R)-HNK?s synaptic action in an intact circuit. Finally, in Specific Aim #3 we will define, in vitro and in vivo, the relative synaptic and behavioral potencies for all 12 HNKs produced via ketamine metabolism. These experiments will define structure-activity relationships at the level of synaptic function, which will allow us to refine the structure of the HNKs, in order to optimize their antidepressant and pharmacokinetic activity. Overall, our work thus far strongly implicates an immediate drug effect on presynaptic plasticity, which when the mechanism underlying this action is clarified, will open up new avenues for novel antidepressant drug discovery based upon this mechanism. The completion of our proposed experiments will have implications for the understanding of rapid-acting antidepressant drug pharmacology, development of novel and innovative therapies, and the future treatment of depression.