Michael J. Owens - US grants

This FIRST award utilizes well-documented techniques to investigate the role of corticotropin-releasing factor (CRF) neurons in both the therapeutic actions of benzodiazepines as well as the physiological effects associated with abrupt discontinuation of benzodiazepines. In the last decade considerable evidence from a number of investigators using different experimental approaches has accrued consistent with the hypothesis that CRF mediates the endocrine, behavioral and autonomic responses of mammals to stress. Based upon previous preclinical studies which revealed that exposure to stress and treatment with anxiolytic benzodiazepines produce opposite effects on CRF neurons, this proposal seeks to characterize the dose-response effects of various drugs which are active at the benzodiazepine receptor including agonists, partial agonists, antagonists and inverse agonists for their actions on CRF neurons after both acute and chronic administration. In addition, because benzodiazepine withdrawal is associated with physiological symptoms reminiscent of a classic "stress response", this proposal will seek to investigate whether CRF neurons are involved in the acute drug withdrawal phase. Can intermittent administration of the benzodiazepine antagonist flumazenil during chronic benzodiazepine treatment attenuate drug withdrawal via effects on CRF neurons? Finally, does central administration of a CRF antagonist modify the drug withdrawal phase by altering CRF neuronal activity as measured neurochemically or behaviorally? Currently, the best estimates of CRF neuronal activity are concurrent measurement of CRF concentrations, CRF mRNA expression, and CRF receptor binding in discrete brain regions previously implicated in mediating the actions of CRF. Additional measures of hypothalamic CRF activity will be obtained by measuring plasma ACTH and corticosterone concentrations. These studies will provide further information on the role(s) of CRF in the CNS, and in particular the role of CRF in potentially mediating a portion of the therapeutic (anxiolytic) and deleterious (drug withdrawal) actions of benzodiazepines. Such studies have important implications for the development of novel treatments for anxiety disorders as well as the development of novel agents to ameliorate drug withdrawal symptoms.

There is abundant evidence that both genetic factors and various environmental influences control the development, and ultimately the level of activity, of the adult CNS serotonergic neural systems. There is also unequivocal evidence that both genetic and environmental factors contribute to the vulnerability to many psychiatric disorders, including depression. As noted in the introduction of this CCNMD application, childhood abuse/neglect has been-established as a major risk factor in adult depression These findings suggest that both genetic and environmental influences contribute to individual differences in serotonergic function, and that this impacts upon an individual's vulnerability or resistance to develop depression or other disorders associated with altered CNS 5-HT function. In this project, we investigate the effect of adverse early experience on the development and function of the 5-HT system, utilizing two different animal models. The rodent early life stress model utilizes rats exposed to 180 min of maternal separation on postnatal days 2 to 14 as described in detail in Core B. The primate model also represents an epigenetic model described in detail in Core C; the early life stress is a variable foraging demand (VFD) on the mother reducing the amount of time she has to attend to her infant. The maternal separation and VFD stressors cause persistent changes in the CNS of adult animals as well as documented behavioral alterations. Among the neurochemical consequences of this manipulation in rats are increased hippocampal 5- HT2A receptor density, increased stress-induced HPA axis responsiveness, increased regional CRF concentrations in the CNS, increased CRF mRNA expression, and decreased glucocorticoid receptor density in hippocampus and frontal cortex. The behavioral consequences of the maternal separation stress include increased fear/anxiety behaviors and a pronounced preference for alcohol. All of these neurochemical and behavioral manifestations of maternal separation in adult rats are reversed by chronic treatment with paroxetine, a specific serotonin reuptake inhibitor. VFD monkeys exhibit altered endocrine responses to serotonergic challenge, CSF 5-HIAA alterations, and increased CSF CRF concentrations. Therefore, we propose to characterize the serotonergic systems of both models in detail, including developmental ontogeny, directly testing the role of increased 5-HT2A receptor function in mediating the phenotype, and response to antidepressants. We also propose multiple interactions with the different components of the Center including both basic science and clinical projects as well as all of the Cores. Our pilot data supports the hypothesis that serotonergic dysfunction is a central feature of the phenotypes displayed by each proposed model; and that alterations in markers of serotonergic activity will also be found in the two clinical projects.

[unreadable] DESCRIPTION (provided by applicant): This application is the second modified resubmission of the competitive renewal application of NIH MH- 42088, seeking five years of support which would represent years 19-23 of this project on the Psychobiology of Corticotropin-releasing Factor (CRF). In the past 18 years, research supported by this grant has contributed to a significant extent to establishing CRF as a neurotransmitter in extrahypothalamic and hypothalamic brain areas, to elucidating its role in the mammalian stress response, to characterizing its role in the pathogenesis of mood and anxiety disorders and most recently in demonstrating persistent alterations in response to early life trauma. There is also substantial evidence that CRF-containing neural circuits are the target of drugs of abuse such as cocaine, anxiolytics such as benzodiazepines and perhaps of particular clinical relevance that they are particularily responsive to withdrawal from a variety of CNS acting drugs. The current proposal builds upon our previous body of work, as well as proposing some more novel, higher risk research paths. We propose to extend our recent work identifying the Quantitative Trait Loci (QTL) that control hypothalamic CRF gene expression to limbic brain regions, as well as to study gender differences in CRF gene expression regulation. The results of the QTL experiment will impact on the directions taken in both of the other specific aims. In view of the increasing evidence that CRF1 receptor antagonists possess antidepressant properties, we shall determine whether these novel agents share with other antidepressants the now well documented effect of increasing hippocampal neurogenesis after chronic, but not acute, drug administration, as well as assessing effects on other measures of neuroplasticity. In addition we seek to finally characterize the molecular and electrophysiological properties of CRF neurons in hypothalamic and extrahypothalamic brain areas using novel techniques including transgenic mice with fluorescent reporters under control of the CRF expression locus. These studies, taken together, will provide novel information on the neurobiology of CRF systems, with a focus on their putative role in the pathophysiology of depression and related mood and anxiety disorders, in addictive disorders and the potential clinical utility of CRF1 receptor antagonists in the treatment of these devastating illnesses. [unreadable] [unreadable] [unreadable]

PROJECT 4 - Abstract Given the reality that human infants can be exposed to stressors and antidepressant drugs in utero as part of life events or appropriate medical treatment of the mother, the long term developmental consequences on the offspring need to be determined as one aspect of planning and monitoring long term health issues. This project focuses upon the biological consequences (biochemical, molecular, physiological and genetic) of the quality of maternal care interacting with prenatal stress and in utero antidepressant exposure on brain function of offspring when these rats become adults of reproductive age (i.e., does prenatal exposure to these factors alter the long term biology of the brain?). This project will focus on three unique aspects of adult brain function. 1) What are the regional brain neurochemical and molecular changes that result (the markers to be examined have all been previously promulgated, but not tested, to be involved in stress responsivity or the mechanisms of action of antidepressants) from maternal care, prenatal stress exposure, and in utero antidepressant exposure? 2) Are there structural changes in the microvascular system supplying blood to the brain or in the angiogenic growth factors that signal new blood vessel formation and retraction? For example, although.it is well established that CMS plasticity includes changes in synaptic morphology, neuronal connectivity, glial morphology and function as well as changes in vasculature, the effects of maternal care, early life stressors, and in utero antidepressant treatment on the CNS have only been addressed for neurons and glia. 3) We will attempt to identify genetic loci that are epigenetically altered by the quality of maternal care, prenatal stress, and in utero exposure to antidepressants in rat brains. We will use ChlP-on-chip technology to investigate how the quality of maternal care, prenatal stress, and in utero exposure to SSRI's alter the epigenetic profiles (both DNA methylation and histone modification) of the promoter regions across the genome in rat brains at birth and in adulthood. In addition, the mRNA expression profiles from the same brain regions will be evaluated and correlated with the epigenetic profiles. These state-of-the-art techniques can offer unique insight into the long term changes, or lack thereof, produced by differences in maternal care, prenatal stress, and in utero antidepressant exposure.

The purpose of this Core is to provide a common set of state-of-the-art biochemical and[unreadable] genetic analyses to CIDAR researchers relevant to testing various hypotheses related to[unreadable] prediction of antidepressant response. The laboratories of the Core will provide assessments of[unreadable] hypothalamic-pituitary-adrenal (HPA) axis function, interleukin-6 and C-reactive protein (C-RP),[unreadable] antidepressant drug concentrations, and detailed genetic analyses. The latter includes fine[unreadable] mapping of candidate genes, choosing "tagging SNPs" as well as genetic data management.[unreadable] Particular emphasis will be placed on development of a dense polymorphism map for each[unreadable] candidate gene to identify the degree of linkage disequilibrium in different ethnic groups. These[unreadable] data will be provided to the statistical modeling core. Having these determinations performed by a[unreadable] central facility using a common set of high quality assay modalities will provide consistency across[unreadable] all of the subjects enrolled in this 5 year study.

Until now, determining whether patients were receiving an adequate dose of a given antidepressant medication was based almost exclusively upon the experience of the prescribing physician. Recently in vivo imaging technology with PET (positron emission tomography) and SPECT (single photon emission computed tomography) are emerging as new tools for assessment and optimization of pharmacological treatment (e.g. monitor adequacy of dosing), psychiatric medication development, and basic understanding of the pathophysiology of psychiatric illness. However, these techniques are associated with limited availability and significant financial costs that preclude the availability of this technology to the vast majority of clinicians. The goal of this project is to provide a valid and simpler alternative (an assay using a human blood sample) to PET imaging to furnish similar in vivo molecular site occupancy data. Briefly, it now appears that most SSRI antidepressants block approximately 80% of their target serotonin transporters (SERT) at standard clinical doses. This data suggests that appropriate clinical dosing might best be determined by assessing brain SERT occupancy. We have developed a unique method in which we are able to measure the magnitude of 5-HT, NE or dopamine (DA) transporter occupancy in antidepressanttreated patients by exposing cells transfected with the human SERT, NET or DAT to the patients'serum after steady-state is attained. Following validation of this technique using concomitant PET imaging we will determine what magnitude of serotonin and/or norepinephrine uptake blockade is required for an optimal treatment response. Simply stated, if a patient has not responded to a standard dose of an SSRI or SNRI, is it because they have not yet achieved a substantial occupancy of the SERT and/or norepinephrine (NET) transporter? These data may be extremely valuable in monitoring patient compliance, the need for dosage adjustment and, in the case of adequate occupancy without therapeutic response, information that provides a rational decision to switch medication class or initiate other treatment options.

DESCRIPTION (provided by applicant): Untreated bipolar disorder has severe consequences for the mother and there is increasing utilization of atypical antipsychotics as mood stabilizers and adjuncts to antidepressant therapy, with an associated increase in fetal exposure to these medications. Because of large differences in the pharmacokinetics of many drugs between humans and rodents, we have demonstrated that behaviorally active doses of many psychiatric drugs in laboratory animals do not reflect/model actual human clinical exposure and we have published on the need for clinically relevant and sustained drug administration in animal studies to mimic human exposures. Using a rat model, this study provides rigorous control of clinically relevant fetal exposure and will assess behavioral, gene expression and epigenomic changes in the offspring with an outcome that can inform clinical risks as well as identifying which medication may be the most appropriate clinical choice. Specific Aim 1 will provide animals with clinically relevant and verified drug exposure throughout gestation for the most commonly utilized atypical antipsychotics in the Emory University Women's Mental Health Program: quetiapine/norquetiapine, olanzapine, and risperidone/paliperidone. Specific Aim 2 will provide a behavioral assessment of development (motor and cognitive) prior to weaning and as juveniles (postnatal days 23-35) with the hypothesis that prenatal exposure to atypical antipsychotic drugs alters normal development compared to vehicle controls. Specific Aim 3 will utilize our skills in gene expression (transcriptome) and epigenetic analysis (whole genome methylation patterns) in two brain regions (frontal cortex and hippocampus) likely to be important targets for healthy neurobiological functioning and relevant to the behavioral studies in specific aim 2. We will also assay leukocytes collected from these offspring which might provide biomarkers as windows into the brain. These data will provide important basic science information regarding medication effects on the developing brain which can translate, based upon known function(s) of specific genes, into areas of clinical assessment that may be most closely watched by pediatricians following these children. The focus of these studies on a single class of medications, with generally similar clinical efficacy, may directly guide physicians and patients in determining which medication may provide the least risk.