Rita J. Valentino - US grants

DESCRIPTION: (Adapted From The Applicant's Abstract) Corticotropin-releasing factor (CRF) and serotonin (5-HT) systems have been independently implicated in stress-related psychiatric disorders. We demonstrate potent effects of CRF on forebrain 5-HT release and activity of putative 5-HT dorsal raphe nucleus (DRN) neurons. Taken with findings of CRF innervation and expression of CRF receptors in the DRN, these results suggest that endogenous CRF acts within the DRN to regulate forebrain 5-HT function. The following AIMS test this hypothesis by characterizing CRF-5-HT interactions at an ultrastructural, electrophysiological and neurochemical level, identifying conditions that engage these interactions, and determining whether CRF effects within the DRN play a role in a behavior associated with depression. 1) Characterize and quantify CRF effects on extracellular 5-HT levels in terminal regions and DRN neuronal activity. Dose-response curves will be generated for CRF effects (administered i.c.v. or intraraphe) on 5-HT release in lateral septum, amygdala and cortex and DRN neuronal activity. The efficacy of CRF antagonists (administered i.c.v., systemically or intraraphe) to prevent these effects will be determined. This AIM will reveal whether CRF acts directly within the DRN to alter forebrain 5-HT release, whether effects on 5-HT release are regionally specific and the CRF receptor subtype involved; 2) Identify physiological conditions during which endogenous CRF impacts on DRN-5-HT systems. The ability of CRF antagonists (administered i.c.v. or intraraphe) to prevent changes in forebrain 5-HT release elicited by different challenges will be determined. This AIM will reveal conditions during which CRF-5-HT interactions are engaged; 3) Characterize cellular substrates for CRF-5-HT interactions in the DRN and identify CRF afferents to the DRN. Interactions between CRF terminals and 5-HT processes in the DRN will be visualized at the ultrastructural level using dual label immunohistochemistry and electron microscopy. Neuronal tract tracing will be used to identify CRF afferents to the DRN. This AIM will reveal potential cellular mechanisms underlying CRF-5-HT interactions and provide a map detailing how CRF systems link to the DRN; 4) Elucidate the role of CRF-5-HT interactions in a behavior associated with depression. The hypothesis that CRF release in the DRN and subsequent inhibition of 5-HT forebrain release underlie behavioral immobility associated with swim stress will be tested. The effects of CRF or CRF antagonists (administered i.c.v. or intraraphe) on behavior in the forced swim test will be determined. This AIM will reveal potential pathophysiological consequences of CRF-5HT interactions.

DESCRIPTION (applicant's abstract): This is a proposal for a Senior Scientist Award (K05) from an investigator who has been funded by a Research Scientist Development Award (K02) for the last 10 years. Prior K02 funding advanced the PI's career by providing time to develop new approaches and foster productive collaborations. At a research level this resulted in expansion from a single to 3 major interrelated NIH-funded research programs. At a technical level, expertise went from predominantly in vivo electrophysiological approaches to including in vivo microdialysis, as well as sophisticated systems and cellular neuroanatomical techniques. At a career level, the PI established a Division of Stress Neurobiology within the Department of Psychiatry [at Hahnemann University]. In February the Division moved to the Children's Hospital of Philadelphia where the PI was offered a prestigious position as a Stokes Investigator and faculty position at the University of Pennsylvania. The proposed research plan integrates aspects of the 3 funded research programs to address the global hypothesis that the impact of corticotropin-releasing factor on biogenic amine systems is integral in the acute stress response and that stress-induced plasticity of these interactions underlies certain stress-related psychopathology. The proposed career development plan will allow the PI to develop and incorporate new cellular approaches into the experimental repertoire of systems approaches currently used to elucidate CRF-biogenic amine interactions and mechanisms underlying stress-induced plasticity. These include cellular and molecular electrophysiological techniques in in vitro slice preparations and the use of viral vectors to manipulate CRF expression in specific CRF afferents to biogenic amine nuclei. These techniques will arm the PI with a range of tools necessary to address research questions on multiple levels (from cellular to systems). A second aspect of career development is to mobilize basic and clinical investigators within the CHOP/ Upenn community who share a common interest in stress in a Center for Stress Neurobiology. Electronic communications, regular research seminars, retreats and shared common resources are used to foster interactions and collaborations among faculty. Because the Center will focus on stress as an etiological factor, rather than on a specific disease, it promises to be an ideal mechanism for understanding and developing new treatments for diverse medical and behavioral disorders. Like the previous K02 award, the K05 award will promote future career development by providing stable salary support and flexibility to enhance technical skills and conceptual bases and promoting leadership.

DESCRIPTION (provided by applicant): Corticotropin-releasing factor (CRF) and the serotonergic (5-HT)-dorsal raphe nucleus (DRN) system have been independently implicated in the acute response to stress and in stress-related psychiatric disorders (e.g., anxiety, depression). During the last few years, we provided evidence that these two systems are anatomically and functionally linked in the response to stress. Thus, we demonstrated that CRF regulates DRN neuronal activity and 5-HT release in forebrain targets in response to acute stress. Moreover, we demonstrated that repeated stress results in cross-adaptation of the DRN-5-HT system to CRF and subsequent stress. The guiding hypothesis of this competing renewal is that CRF neuromodulation of the DRN-5-HT system underlies certain components of the behavioral limb of the acute stress response and that stress-induced plasticity in this function of CRF underlies psychiatric symptoms associated with repeated or chronic stress. The following AIMs integrate diverse approaches to test this hypothesis. AIM 1: will use in vivo microdialysis and electrophysiology to elucidate specific neuronal and neurochemical consequences of selectively activating CRF-R1 vs. CRF-R2 receptors within the DRN. AIM 2: will identify the anatomical substrates and circuitry by which CRF impacts on the DRN-5-HT system using immunohistochemistry, in situ hybridization and electron microscopy. AIM 3: will identify behavioral consequences of selectively activating CRF-R1 vs. CRF-R2 receptors within the DRN. AIM 4: will examine stress-induced adaptation of the DRN-5-HT system with regard to a) the role of CRF, b) the underlying mechanisms and c) whether neurochemical adaptation is causal to behavioral adaptation (using in vivo microdialysis, behavior and receptor autoradiography). Our previous work introduced the innovative idea that CRF-5-HT interactions are a pathophysiological focus of psychiatric disorders and a target of novel psychotherapeutic agents. The proposed studies will thoroughly characterize these interactions from a cellular to behavioral level. As such, they will enhance our understanding of the role of stress in psychopathology and indicate novel targets for the treatment of stress-related psychiatric disorders.

DESCRIPTION (provided by applicant): Social stress has adverse consequences for physical and mental health throughout life and its impact may be particularly relevant during adolescence as this is a time of substantial growth and reorganization of brain circuits. To date, most basic research into the behavioral and physiological consequences of social stress has focused on adult male rodents. The proposed research uses a model of rat social stress, "the resident- intruder model", to characterize the neurobehavioral responses to, and consequences of, social stress at different stages of adolescent development compared to adulthood. This research also takes on the important challenge of investigating sex differences in the response to social stress or its consequences. The research program proposed here is based on preliminary data indicating that the defensive subordinate response to an aggressive conspecific develops throughout adolescence into adulthood. Temporally correlated to this is a shift away from active coping strategies in response to subsequent challenges. These age-related behavioral differences are associated with distinct changes in locus coeruleus (LC) neuronal activity. Therefore, this proposal tests the central hypothesis that social stress in early adolescence disrupts the ontogeny of specific neural pathways important for regulating the development of subordinate defensive behaviors and that this disruption has effects on behavior and cognition in adolescence that endure into adulthood. This hypothesis will be tested in three specific Aims. Aim 1 will identify and compare neural circuits engaged by social stress at different stages of adolescence and in adulthood using a functional neuroanatomy approach and examining changes in peptide systems related to social behavior and stress. Aim 2 will characterize and compare responses of LC neurons to the social stress at different stages of adolescence and in adulthood. Aim 3 will examine specific behavioral and cognitive consequences of social stress during adolescence and evaluate the endurance of these effects. Because some of the neural substrates being investigated in Aims 1 and 2 have been implicated in the behavioral endpoints being tested in AIM 3, the studies are organized to reveal how the neural substrates and circuits involved in the initial response to social stress (the defensive behaviors) at a particularly time in development play a role in determining the subsequent acute and long-term consequences of social stress. Together, the proposed studies will provide critical information about developmental and sex differences in defensive behaviors and in the consequences of social stress. Given the impact of social stress on mental health these studies will advance our understanding of the development of psychiatric disorders in humans and on the neurobiological basis for gender differences in the onset and etiology of these diseases.) PUBLIC HEALTH RELEVANCE: This proposal uses a rat model of social stress to elucidate the neurobiological mechanisms by which social stress occurring at specific times in adolescence shapes present and future behavioral responses to stress, social interactions and/or cognitive function. By performing studies in both male and female rats, the basis for sex differences in vulnerability to and the developmental trajectory of psychiatric disorders will be identified.

DESCRIPTION (provided by applicant): Proper urinary function requires that the brain process sensory information from the bladder and respond by initiating signals that coordinate bladder activity with appropriate cognitive and behavioral responses so that micturition occurs in an appropriate and safe environment and remains separate from other behaviors. Disordered central processing of bladder information can result in voiding dysfunctions as well as sensations of urgency and pain as is seen with interstitial cystitis. Despite the importance of understanding how the brain processes bladder information to achieve these functions, research in this area has been limited. The overall goal of this research is to elucidate how the brain processes bladder information and coordinates the visceral and cognitive/behavioral aspects of micturition. This will be done using state-of-the- art neuronal recording techniques that can directly quantify activity of neurons in a pontine micturition circuit along with cortical electroencephalographic (EEG) activity and determine how neuronal activity corresponds with urodynamic measures using in vivo cystometry in unanesthetized rats. Multichannel electrodes will record single unit activity from neurons in Barrington's nucleus (the pontine micturition center) and/or the locus coeruleus (LC), a brain region that is thought to initiate arousal in response to bladder pressure increases via its cortical projections. Cortical EEG will be simultaneously recorded with activity from these neurons during cystometric measures of urodynamic endpoints such as bladder pressure and micturition. Aim 1 will determine how Barrington's nucleus neuronal activity, LC neuronal activity and cortical EEG co-vary with bladder pressure and micturition under normal conditions. Aim 2 will examine this in 3 models of urinary dysfunction a) partial bladder outlet obstruction, b) social stress-induced voiding dysfunction and c) a neurogenic model of interstitial cystitis. Aim 3 will test the hypothesis that corticotropin-releasing factor (CRF) and/or glutamate neurotransmission in the LC are integral for relaying bladder information to the cortex in normal and/or pathological conditions. Although it is well recognized that the brain regulates visceral and behavioral components of the micturition reflex and can play an integral role in diseases of urinary function, this will be the irst study to elucidate the precise relationship between brain neuronal activity and bladder activity in unanesthetized rats in normal and disease states. As such, this research will lead to new perspectives on the etiology of urinary disorders and guide novel therapeutic directions.

Project Summary/Abstract Stress is implicated in cognitive dysfunctions that characterize many psychiatric diseases, including depression, substance abuse and post-traumatic stress disorder. One mechanism by which stressors impact on cognition is through their engagement of monoamine systems that regulate activity of the prefrontal cortex (PFC) that governs executive functions. The locus-coeruleus (LC)-norepinephrine (NE) system, is a major stress response system that initiates arousal and modulates cognitive flexibility in response to stressors through its regulation of the PFC. Stress-induced engagement of the LC-NE system is mediated by CRF neurotransmission. It has been hypothesized that stress-induced impairments in executive function arise from excessive LC-NE drive to the medial PFC (mPFC) and resulting inhibition of mPFC function. This important hypothesis has never been directly tested. Although the activating effects of stressors and CRF on activity of single LC neurons have been well described, a major gap exists in our understanding of how these single cellular events translate to changes in cortical activity that then govern executive functions such as cognitive flexibility. To address this question, this research will quantify network activity in response to CRF or stress in a circuit linking the LC with the mPFC and the orbitofrontal cortex (OFC), PFC subregions that have been implicated in different cognitive processes. Network activity is the synchronization of neural oscillations that supports communication between brain regions and is recorded by local field potentials (LFPs). The following Aims will quantify LC-PFC network dynamics, including their strength, connectivity and directionality by recording LFPs in these regions in response to stress and CRF and determining how these effects translate to changes in cognitive flexibility. Because stress-related psychiatric diseases are more prevalent in females and female LC neurons are more sensitive to CRF compared to males, this research will also reveal the role of sex as a determinant of stress effects. Aim 1 will identify the effects of activating CRF1 or Gs-protein-related signaling in LC neurons on network activity within the LC-PFC circuit. These effects will be examined at rest and during performance of tasks that test cognitive flexibility. Aim 2 will identify the effects of acute and repeated social stress (resident-intruder stress) on LC-PFC network activity, and will dissect the role of CRF in the LC in these effects. Aim 3 will determine whether repeated social stress has enduring effects on LC-PFC network activity that translate to changes in cognitive flexibility. Together these studies take a network approach to elucidate how cellular effects of stress on LC neurons are amplified to cortical circuits to affect cognitive flexibility, an important attribute of executive function that is impaired in multiple stress-related psychiatric disorders.