Clinton Kilts - US grants

The amygdala is a collection of anatomically, biochemically and functionally heterogeneous nuclei and is a major component of the limbic system. This proposal will test two general hypotheses: that different amygdaloid nuclei have markedly different roles in mediating the actions of antipsychotic drugs and; that the dopamine (DA) neurons terminating in these nuclei, and their postsynaptic elements, exhibit characteristics unusual to neurons of other DA systems. While DA will be the primary neurochemical focus, emphasis will be placed on the role of interactions between DA and norepinephrine (NE) and DA and neurotensin in the expression of antipsychotic drug effects on DA synthesis or turnover in the individual amygdaloid nuclei and other brain nuclei. The role of end-product inhibition and presynaptic DA autoreceptors in regulating the synthesis of DA in mesoamygdaloid neurons will be determined, and the functional significance of conventional postsynaptic DA mechanisms (e.g., adenylate cyclase) will also be assessed. The biochemically estimated in vivo synthesis and turnover of DA in amygdaloid neurons and midbrain cell bodies will be examined as relates to the compensatory response to DA receptor blockade by acute and chronic administration of "typical" and "atypical" antipsychotics. The role of selective dosage regimen and drug subclass differences on NE and neurotensin mechanisms in influencing drug effects on DA function will be determined, and the significance of static or dynamic (drug-induced) bilateral asymmetries of amygdaloid DA, NE or neurotensin will be examined. The tools to be used include tissue micropunching from coronal brain slices, sensitive trace enrichment HPLC with electrochemical detection, in vitro adenylate cyclase and radioligand binding techniques optimized for microdeterminations and radioimmunoassay of neurotensin. To complement these methods, site specific microinjections, selective brain lesions and the administration of antipsychotics with differentiated neuropharmacological properties will be used. From these experiments a further understanding of the role of DA, perhaps as a function of interrelated neuromodulators, in the neurobiology and pharmacotherapy of schizophrenia will be obtained.

DESCRIPTION: (Verbatim from the Applicant's Abstract) The complex syndrome of schizophrenia has been related theoretically and empirically to an elementary dysfunction in stimulus filtering/selective attention and the control of behavior by context. Using artificial selection pressure on a behavioral phenotype (Latent Inhibition of LI) that reflects these key psychophysiological constructs of schizophrenia, we have generated a novel animal (rat) behavioral model of enduring cognitive behavioral deficits associated with schizophrenia, and their pharmacological corollaries. This application for renewed funding is a plan for model testing focusing on the neural system, pharmacologic, and neurotransmitter correlates of schizophrenia. Hypotheses to be tested include that selection for low LI phenotype results in a sensitive and specific analogue of the pharmacotherapy of schizophrenia; that selection-induced deficits in LI is a developmentally delayed and enduring trait; that selection for deficits in LI is associated with hemispherically lateralized alterations in limbic-striatal networks and of intrinsic dopamine (DA) and serotonin neuronal populations; and that selection for deficits in LI is related to alterations in brain neurotensin (NT) mechanisms and their response to antipsychotic drugs. Hypothesis testing will involve the following specific aims: Specific aim #1 would develop a third replicate of LI selection and characterize behaviorally the response of LI to artificial selection. Behavioral testing would define the age dependence of the response to selection and further evaluate deficits in the prepulse inhibition (PPI) of an acoustic startle response as a genetically correlated trait. Specific aim #2 would define the impact of a genetically selected deficit in LI on the effects of antipsychotic drugs on stimulus filtering ability. The effects on LI of typical and atypical antipsychotics, nonantipsychotics, and DA receptor agonists would be compared in the Low, High, and Control lines. Specific aim #3 would elucidate3 the neural pathway and neurotransmitter correlates of LI selection to explore the mechanisms of schizophrenia and antipsychotic drug action. Mitochondrial markers of synaptic activity and in vivo microdialysis estimates of DA and serotonergic neuronal activity would define the neural correlates of disrupted stimulus processing and the restorative effects of antipsychotic drugs. Specific aim #4 would determine the role of altered brain NI mechanisms in disrupted stimulus processing and the differential effects of antipsychotic drugs on disrupted versus normal stimulus filtering. Line differences in NT gene and peptide expression would be defined. The shorter term goals of this plan are to use behavioral genetic techniques to define the mechanisms of differential stimulus processing and the deficit state and genetic determinants of antipsychotic drug effects on this operation, with the longer term objective of developing a unique genetic model of the pharmacology and mechanisms of schizophrenia.

DESCRIPTION: (Applicant's Abstract) Drug craving or intense wanting associated with cocaine (crack) dependence represents a generally recognized cause of the chronically relapsing nature of cocaine abuse. A major premise of this R21 proposal is that knowing where in the human brain the correlates of cocaine craving are localized would provide essential direction to the heretofore unsuccessful development of treatments aimed at curbing drug craving. This research plan would use guided imagery as individualized internal cues for the provocation of drug craving and control conditions and Positron Emission Tomography (PET) to map their neural correlates. The proposed experiments would test the following hypotheses: that drug craving associated with cocaine versus nicotine dependence involves the activation of similar neural pathways; that important sex differences exist in the neural correlates of cocaine craving in dependent individuals; and that the subjective states of drug craving and drug "liking" are associated with distinct brain activation maps. Hypothesis testing will involve the following specific aims: Specific aim #1 would compare the neural correlates of human drug craving associated with crack cocaine versus nicotine dependence. Task-related changes in regional Cerebral Blood Flow (rCBF) would be compared in cocaine-dependent who are not nicotine users and nicotine-dependent men who are not cocaine users. Drug craving, and the control conditions (anger, neutral episodic memory recall), would be induced by guided imagery of personalized scripts. Specific aim #2 would examine sex differences in the neural correlates of cocaine craving associated with crack cocaine dependence. A group of cocaine-dependent women, well matched to the men studied in aim #1, would be submitted to the identical PET neuroactivation analysis of internally generated cocaine craving. Specific aim #3 would compare the neural correlates of the urge for cocaine use in cocaine-dependent and occasional, non-dependent crack users. A group of occasional crack users, well matched to the cocaine-dependent men studied in aim #1, would be submitted to an identical PET neuroactivation analysis of the subjective states of drug craving and drug liking in dependent and non-dependent cocaine users, respectively. The design of the proposed studies involve the use of multiple control conditions to isolate the neural correlates of cue-induced cocaine craving and the use of both subjective and objective measures of cue response.

[unreadable] DESCRIPTION (provided by applicant): Cocaine dependence (CD) is defined by the loss of control over drug seeking and use behavior despite clear adverse consequences of drug use. The conceptual framework of the proposed project asserts that cocaine addiction represents a disorder of cognitive control of behavior with definable neural correlates. The objective of this proposal is to use event-related fMRI to test hypotheses related to a neural model of the effect of CD on context-modulated cognitive control. Aim 1 of the project would use a Go-NoGo task involving human faces to assess the behavioral and neural correlates of the effect of CD on response inhibition. Aims 2 and 3 would assess the specific impact of conditioned cocaine cues on cognitive control. Studies would assess the effect of CD on the behavioral, neural, psychophysiological and cognitive correlates of attempts to volitionally regulate the drug craving response to mental imagery of cocaine cues (Aim 2). Comparisons at early (7-21 days) vs. prolonged (60-75 days) periods of cocaine abstinence would test further the neural model. Additional studies would use a cocaine counting word Stroop task (cocStroop) to define the behavioral and neural correlates of cognitive interference by cocaine cues (Aim 3). Aim 4 would use a Prisoners Dilemma game variation as a naturalistic social model to define the neural substrates of a CD-related abnormality in the expression and regulation of social aggression. Aim 5 would determine whether impairments in cognitive control of behavior represent vulnerability factors for cocaine addiction. The behavioral and neural correlates of response inhibition, cognitive interference and social aggression would be assessed in an "at risk" sample of cocaine-naive, same-sex siblings of CD probands. The long-term goal of these studies is to develop a novel understanding of the processes related to the acquisition, maintenance and social consequences of cocaine addiction. [unreadable] [unreadable]

Childhood abuse and parental neglect are increasingly linked to adverse outcomes in adulthood, yet the specific neurobiological events that transduce early trauma to a myriad of problems in later life remain largely unknown. Patterns of anatomical connectivity within the brain are the foundations of its functional organization. By close integration with the proposed fMRI studies of Project 0012 and the proposed Primate Core, this project would apply cutting edge MRI technology and approaches to a characterization of the impact of childhood abuse/early trauma, and its developmental course, on brain structure and connectivity. The proposed project would test an overarching hypothesis of childhood abuse as a precipitant of neurodevelopmental abnormalities. Specific hypotheses to be tested include: that childhood abuse/early trauma is associated with a disorganization of extrinsic connectivity of brain networks subserving the stress response and hippocampal function (Aim 1), and salience attribution, mood regulation and emotion processing (Aim 2); and that early trauma is associated with deficits in the developmental maturation of the brain at multiple levels reflected in the volume, white matter integrity and extrinsic connectivity of brain areas (Aim 3). For Aim 1, four groups of women reflecting the different association possibilities of histories of childhood abuse and major depression (ie., +/+, +/-, -/+, -/-) would be compared by the results of fractional anisotropy (FA) brain maps derived from diffusion tensor imaging (DTI) and by baseline functional connectivity (FC) brain maps derived from low frequency time course correlation analyses of resting state images. For Aim 2, group differences in anatomic and functional connectivity for neural circuits involved in a negative mood state, and emotion and incentive salience processing would be defined using the task-related activation volumes identified in Project 0012 to define ROI and seed clusters in FA and FC analyses, respectively. For Aim 3, longitudinal MRI imaging in monkeys subjected to an intermittent maternal separation paradigm would define the developmental impact of early trauma on brain structural volumes and their extrinsic connectivity (DTI). This knowledge would frame the neurobiological impact of early trauma, provide a reference for understanding its role in the diathesis of subsequent mood and anxiety disorders, and drug abuse and dependence, and aid the design of treatment plans seeking to limit the adverse consequences of childhood abuse.

DESCRIPTION (provided by applicant): A 40-bp variable number (3-13) of tandem repeats (VNTR) polymorphism exists in the 3' untranslated region (UTR) of the dopamine transporter (DAT) gene (dat1). The 10-repeat VNTR (hDAT10), has been associated with attention deficit hyperactivity disorder (ADHD) and nonresponse of ADHD to methylphenidate (MPH). The mechanisms underlying these genotype-phenotype correlations are unknown. This exploratory R21 proposal seeks to use in vitro molecular genetic studies to guide in vivo positron emission tomography (PET) and functional magnetic resonance imaging (fMRI) approaches to clarifying the role of this genetic polymorphism in the pathophysiology and pharmacotherapy of ADHD. Specifically, in vitro gene transfection/expression experiments would define the influence of dat1 VNTR genotype (hDATI0, hDAT9, hDAT0 and hDAT) on DAT binding and function, and pharmacological regulation by MPH. In vivo [18F]FECNT PET studies would compare brain regional DAT binding potential and MPH dose-dependent DAT occupancy in sib pairs discordant for ADHD and dat1 VNTR genotype. Finally, in vivo fMRI studies would define the effect of hDAT10 on the neural correlates of the effect of MPH administration on cognitive impairments associated with ADHD. These studies would test the following specific hypotheses: that the 10- repeat VNTR of dat1 is associated with (1) increases in DAT number and/or function, and attenuated MPH inhibition of DAT in in vitro model cell systems; with (2) increases in the in vivo regional brain DAT binding potential and diminished MPH dose-dependent occupancy of human brain DAT; and, with (3) a blunted effect of MPH administration on the cognitive task-related activation of a prefrontal-parietal cortical circuit. This exploratory proposal would novelly use in vivo imaging approaches to explore genotype-phenotype correlations and partner in vitro and in vivo approaches, and is thus well suited to the goals of the R21 mechanism to foster the exploration of the use of approaches new to a area. By uniquely defining the impact of the dat1 VNTR polymorphism on DAT, these findings will generate knowledge of how genes confer vulnerability to ADHD and medication nonresponse.

[unreadable] DESCRIPTION (provided by applicant): Consistent findings from both animal models and human studies of drug addiction support the roles of stress as a precipitant of relapse and as a vulnerability factor. The conceptual framework of this revised proposed research plan remains based on the contention that drug addiction is a stress-related disorder with definable neural events that link early life and acute stress to both the development of drug addiction and its chronically relapsing nature. The neural correlates of the effects of acute (i.e., stress experience mental imagery) and early (i.e., childhood abuse) stressors on motivation for relapse to drug seeking behavior and on frontal cortical functions that represent opponent processes to addiction would be defined using functional magnetic resonance imaging (fMRI) in cocaine-dependent men and women. Aim 1 of the project would use z-SAGA fMRI to define those neural activations that link an acute stressor (stress mental imagery) to an enhanced motivation for drug use in cocaine-dependent men and women without early trauma histories, and compare the functional anatomy of stress-induced and conditioned drug cue (drug use imagery)-induced cocaine craving in a within-subjects design. A hypothesis that acute stressors and conditioned cocaine cues activate distinct drug craving-related neural substrates in addicted individuals would be tested. Aim 2 would compare cocaine- dependent women and men with childhood abuse histories to those without (Aim 1) to define the effect of early life stress on the neural correlates of stress-induced, and drug cue-induced, cocaine craving. A hypothesis that childhood abuse is associated with exaggerated drug incentive and related neural responses to acute stress, as well as conditioned drug cues, in cocaine-dependent individuals would be tested. Aim 3 would compare groups of non-drug- dependent men and women with and without early life trauma histories on measures of impulse regulation (stop task), and reward valuation and decision making (reward task). A hypothesis that the increased risk for drug abuse and addiction associated with childhood abuse is related to deficits in the prefrontal cortical regulation of cognitive behaviors central to the addiction process would be tested. Sex differences in the roles of stress in cocaine addiction would be assessed with the expectation that women exhibit exaggerated roles compared to men. By integrating the Substance Abuse Treatment Program at the Atlanta VAMC with the childhood abuse research team of the Emory Conte Center for the Neurobiology of Mental Disorders, we propose to identify the neural processing mechanisms that couple acute and early life stress to an increased acquisition and maintenance of drug abuse and addiction. [unreadable] [unreadable] [unreadable]

This application, in response to RFA: MH-03-008 (National Cooperative Drug Discovery Groups for the Treatment of Mood Disorders or Nicotine Addiction, NC 336-MD/NA), proposes the creation of"The Emory-GSK-NIMH Collaborative Mood Disorders Initiative." This unique opportunity to accererate antidepressant drug development brings together expertise of three complementary research groups: the Emory University School of Medicine Department of Psychiatry and Behavioral Sciences, the Mood and Anxiety Disorders Program at NIMH and the Center for Excellence in Drug Discovery (CEDD) in Psychiatry of GlaxoSmithKline (GSK), one of the largest multinational pharmaceutical companies. The two major goals of the current application are the development of innovative new models for basic and clinical research in mood disorders and the intensive scrutiny of 5 novel GSK antidepressant candidates in preclinical and clinical paradigms. In addition to an Administrative and Animal/Assay Core, 7 research projects are proposed. Of these, two are based in the intramural NIMH program; Neurogenesis, Synaptic Plasticity and Signal Transduction (Husseni Manji, M.D., PI) and Clinical Models to Assess Novel Antidepressants (Dennis S. Charney, M.D., PI). The remainder are based at Emory University led by established investigators including Jay M. Weiss, Ph.D. (Animal Models of Depression), Michael Davis, Ph.D. (Animal Models of Fear and Anxiety), Clinton D. Kilts, Ph.D. and Mark Goodman, Ph.D., (Functional Brain Imaging, with a focus on new PET ligand development), Michael J. Owens, Ph.D. and Charles B. Nemeroff, M.D., Ph.D. (Ex Vivo Assessment of Neurotransmitter Receptor and Transporter Occupancy of Antidepressants), and Andrew H. Miller, MD (Cytokine Induced Depression: A Rhesus Monkey Model). In conjunction with GSK, 5 novel GSK antidepressant candidates and others that become available via GSK Drug Discovery within the lifetime of the grant will be intensively scrutinized to synergize with the in-house GSK effort. This proposal encompasses virtually all of the major goals outlined in the RFA, namely, development of new neurochemical tools including novel PET ligands, exploration of new models of drug development and facilitation of a partnership between academia, NIMH and industry.

The premise of this revised project remains that the development of medications targeting the brain CRF-\ receptor is dependent on the availability of a novel, validated CRFi receptor PET ligand. Supporting applications of such a ligand include testing mechanism of action hypotheses in which the dose- and timeeffect relationships of candidate CRF^ receptor antagonists are defined by the percent occupancy of the brain CRF, receptor. Dose finding studies related to therapeutic and side effects as a function of percent occupancy would be enabled and guide Phase II trials. Assessing whether major depression or anxiety disorders, or CRF, receptor gene variation, are associated with altered in vivo brain CRF^ receptor availability represent examples of the application of such a PET ligand to test models of pathophysiology and pharmacogenetics, respectively. We propose to build on the ongoing efforts of the CRF! receptor PET ligand development initiative funded as a supplement to the current U19 award. Revised Project 3 remains as an iterative plan of molecular design, chemistry, and in vitro and in vivo candidate evaluation based on two distinct but paralleling approaches starting from common pyridopyrazinone CRF-\ receptor antagonists. Proposed approaches represent planned modifications of the pyridopyrazinone starting molecule, and the specific characterization of the effect of manipulating the out-of-plane aryl ring on CRF1 receptor binding affinity. Five specific aims are proposed to yield 11C- and 18F-labeled versions of brain CRF^ receptor PET ligands resulting from the interactions between a proposed program of chemical synthesis and the step-wise evaluation of candidate CRFi receptor PET ligands. Candidate evaluation would involve a 9-step hierarchical model with well-defined criteria as branch points that spans transfected cell systems, rodents, and in vitro and in vivo assessments in non-human primates to optimize the target ligand properties of CRFi receptor affinity, lipophilicity and metabolic stability. Evolving structure-activity relationship (SAP) data would refine synthetic chemistry schemes to generate novel candidates for re-entry into the evaluation scheme, and would evolve a molecular modeling approach in collaboration with the QSAR molecular modeling/drug discovery program of the joint Chemistry/Pharmacology Emory initiative. End stage candidate evaluation would involve in vivo microPET imaging in monkeys to establish the feasibility and models for quantitating brain emission data, evaluation of dose-and time-dependent brain CRF, receptor occupancy for GSK-008, and organ dosimetry. The final stage of testing in support of an IND application for human use would be enabled by the GSK partnership in this cooperative agreement. The goals of this proposed project 3 are to characterize the interactions of GSK-008 with the primate brain CRF^ receptor, and to provide a valuable novel, valid PET ligand to the field of CRF research.