Sheryl Beck - US grants

The long term goal is to reveal the acute and chronic effect(s) of normal and pathological levels of adrenalcortical steroids on hippocampal cell activity. The adrenalcortical steroids have protective actions to maintain homeostasis, yet high chronic levels produce neurotoxic effects. Aldosterone and corticosterone (CT, rat) or cortisol (human) are the primary adrenalcortical steroids. Plasma hormone levels are controlled by the hypothalamic-pituitary-adrenal (HPA) axis. Feedback regulation of the HPA axis is through interaction with CT receptors in the brain; the hippocampus is a primary target. Activation of hippocampal CT receptors adjusts the gain of the HPA axis. Basal activation maintains homeostasis, acute activation increases activity to regain homeostasis. Prolonged excessive CT secretion leads to pathological states such as depression and enhances neurotoxic vulnerability of hippocampal pyramidal cells. Hypothesis 1: The different CT receptor subtypes. i.e.. the mineralocorticoid (MR) and glucocorticoid (GR) receptors, differentially regulate the functioning of hippocampal pyramidal cells under conditions of no stress. acute stress and chronic stress. This regulation occurs through independent mechanisms. i.e., by altering pyramidal cell properties and/or by altering receptor-mediated actions. Previous research has defined CT actions on CA1 pyramidal cells, which provide the final output of the hippocampus. Input from CA3 cells directly influences CA1 activity; CT may alter CA1. output by affecting CA3 pyramidal cell activity. The distribution of MR and GR receptors is different between CA1, and CA3 subfields. CT selectively alters CA3 pyramidal cell morphology leading to cell death. Hypothesis 2: The effects of CT on CA1, and CA3 pyramidal cell activity are different. Defining the actions of CT on CA1, and CA3 cells may provide important information towards differentiating the protective versus destructive actions of CT. To test these hypotheses the following Specific Aims are proposed. CT levels will be controlled by adrenalectomy (ADX) and replacement steroid treatment. SP1: To determine the chronic effects of normal and pathophysiologicaI levels of CT on CA3 hippocampal cell properties. We have previously shown that CT alters CAL. hippocampal pyramidal cell properties. In this application the modulatory actions of CT on CA3 cell properties will be determined and compared to our previous results. SP2: To determine the mechanism of action of the chronic effects of normal and pathophysiological levels of CT on 5-HT neurotransmitter-mediated actions in CA1 and CA3. We have previously shown that CT has different effects on the different 5-HT receptor-mediated responses in CA1. CT effects on 5-HT responses in area CA3 will be measured. Electrophysiology, receptor autoradiography, immunocytochemistry, and Western blot techniques will be used to determine if the modulatory actions of CT are at the receptor, receptor-effector coupling and/or effector level. SP3. To determine if the acute effects of high CT concentrations, that mimic an acute stress response, are dependent upon normal daily activation of MR and GR receptors. The hypothesis that basal activation of MR and circadian activation of GR is required to maintain specific protein products necessary for feedback inhibition will be tested. The effects of acute CT administration on pyramidal cell properties and neurotransmitter mediated responses recorded in areas CA1, and CA3 of Sham, ADX and chronically treated rats with pathophysiological levels of CT will be measured.

The 5-hydroxytryptamine (5-HT, serotonin) neurotransmitter system maintains many homeostatic functions; it has also been implicated in the etiology and treatment of neurological and affective disorders, such as depression, anxiety, obesity, anorexia, and Alzheimer's disease. One of the PI's primary areas of interest is to understand the cellular mechanisms underlying the etiology and treatment of affective disorders. In the past the PI has focused on characterizing normal hippocampal neural activity and the changes in that normal physiology by chronic treatment with antidepressants or the "stress" hormone corticosterone. The working hypothesis is that the actions of the drugs or mechanism underlying pathological status is due to differential modification of cellular properties or components of the 5-HT neurotransmitter system. In support of this hypothesis chronic treatment with corticosterone or fluoxetine was found to alter hippocampal pyramidal cell neural activity by changing basic cell properties as well as postsynaptic 5-HT receptor mediated responses. As predicted, the nature of the modulatory effects of the chronic treatments are not the same in the CA1 and CA3 subfields. Another likely target for differential modifications in the 5-HT neurotransmitters system is at the level of the 5-HT cell bodies. The median (MR) and dorsal raphe (DR) are the principal sites where 5-HT cell bodies are located. These two nuclei provide the majority of the 5-HT innervation of the forebrain. Even though they share many of the same features, differences between these two nuclei have been identified. The goal of the experiments outlined in this application is to set up a raphe brain slice preparation maintained in vitro to delineate both the common and disparate features of MR and DR cell neural activity. The development of the raphe brain slice will expand the depth of the PIs research focus. The use of the raphe (5-HT cell body) and hippocampal (5-HT fiber projection area) slice preparations in conjunction will provide a mechanism for a more systematic approach to the analysis of the normal physiology and pathophysiological processes of the 5-HT neurotransmitter system.

Chronic stress causes long term changes in neural circuits that may underlie symptoms of depression. The CA3 subfield of the hippocampus is one of these important neural circuits because it is the induction site for the oscillatory neuronal activity seen during many different behavioral states. This neural activity has been correlated with cognitive functions. The neurons that comprise the circuit are regulated by 5HT projections from the median raphe. Recent evidence indicates that 5HT postsynaptic receptor-effector mechanisms are altered by stress. These postsynaptic changes may be responsible for the long-term alterations in CA3 neural activity following stress. The long term goal is to elucidate the cellular substrates for alterations in CA3 hippocampal function induced by stress. The working hypothesis is that one of the cellular mechanisms underlying stress induced changes in the CA3 hippocampal circuit is a change in the 5HT modulatory input. Intracellular recording techniques in hippocampal brain slice preparations will be used to record CA3 neural activity. Specific aim 1: To characterize postsynaptic 5-HT receptor-mediated responses in CA3 pyramidal cells. Hypothesis: 5-HT concentration response curve characteristics and second messenger systems linked to the 5-HT-1A , 5-HT-4 and 5-HT-7 receptors will not be the same. Specific aim 3: To determine the effects of 5-HT and CA3 oscillatory activity as induced by carbachol. Hypothesis: Selective activation of isolated 5-HT receptors will differentially modulated carbachol induced oscillatory activity. Specific aim 3: To determine the effects of stress on postsynaptic 5-HT receptor-mediated responses, carbachol induced oscillatory activity and 5-HT modulation of carbachol induced oscillatory activity. Two different behavioral stress paradigms will be used, i.e., forced swimming and inescapable restraint as well as a treatment group that has endogenous levels of the stress hormone corticosterone controlled. It is expected that the modulatory effects of the three protocols can be discriminated because the nature and duration of the stressors are vastly different. Together these studies will systematically examine multiple mechanisms for stress induced changes in the neural circuitry of the CA3 subfield. These mechanisms may underlay many of the debilitating symptoms and cognitive deficits seen in depression and stress related disorders. Understanding these mechanisms may also provide important information for the development of new treatments for depression and stress related disorders.

[unreadable] DESCRIPTION (provided by applicant): The cellular mechanisms underlying stress induced behaviors and the development and treatment of stress related psychiatric disorders most likely involve both the 5-HT cell body containing nuclei of the raphe as well as their projection areas in the limbic forebrain. The median (MR) and dorsal raphe (DR) are the principal sites where 5-HT cell bodies are located and they provide the majority of the 5-HT innervation of the forebrain. Approximately 50% of the neurons in the MR and DR are non-5-HT containing. The MR and DR projections terminate in many of the same limbic brain areas, but also have exclusive projections to other brain areas. The anatomy of the different subfields of the DR and within the MR is not the same. These distinctions have important implications in terms of regional selectivity of the subfields of the DR and the MR in controlling different emotional behaviors. The long term goal is to delineate both the common and disparate features of MR and DR cell neural activity. This information will provide a foundation for understanding the factors that differentially regulate 5-HT and non-5-HT physiology and may underlie some of the functional differences. The overall hypothesis is that there are discrete regional cellular differences in the 5-HT and non-5-HT containing neurons of the different subfields of the DR and MR. The excitatory neurotransmitter glutamate, the inhibitory neurotransmitter GABA and the stress hormone CRF are all posed to have selective and regionally distinct modulatory roles on DR and/or MR activity. In this application, the goal is to regionally map the differences in basic cellular characteristics (Specific Aim 1), excitatory glutamatergic (Specific Aim 2) and inhibitory GABAergic neural activity (Specific Aim 3) and their modulation by 5-HT1A, 5-HT1B, and CRF receptor activation. Selective alteration of these responses will be measured in 2 animal models, i.e., swim stressed rats and the 5-HT1B knockout mouse (Specific Aim 4). Whole cell recording techniques, immunohistochemistry, aRNA amplification and histological procedures will be used in the different subfields of the DR and MR in 5-HT and non-5-HT containing neurons. The clinical relevance of these studies is in supplying important information that will be crucial for understanding how the 5-HT neurotransmitter system regulates stress and emotions and how it may be involved in the etiology and treatment of mood disorders such as anxiety and depression. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): This application addresses broad Challenge Area (15) Translational Science, Gene x environment x development (GxExD) studies of brain function and mental disorders. Gene x environment epidemiology and psychiatry research studies have only recently been used and the findings of these studies have come into the limelight of the etiology of psychiatric disorders. Most research does not combine the examination of all three factors, i.e., genes, environment and development, but usually includes the examination of gene x environment, gene x development, or development x environment, often producing inconsistent findings. In many cases the genetic disposition is understood and development in terms of how it influences brain structure, but how the environment alters brain function to interact with the genetic and development factors is unknown. The challenge, then is to determine how the genetic predisposition, development of the relevant neural circuitry of the nervous system and the environment interact to alter brain function to produce the psychiatric disorder. In this application, using an animal model, we propose to examine all three factors using genetic, behavioral, electrophysiological and morphological/anatomical techniques. The disorder under investigation is anxiety. Anxiety can be defined as a state of cognitive and behavioral preparedness that an organism mobilizes in response to a future or distant potential threat. In its non-pathological form anxiety can be divided into two categories: 1) state anxiety, which is an acute adaptive response of heightened vigilance and arousal that enables an organism to navigate an unfamiliar environment of unknown danger, and 2) trait anxiety which is a measure of an individual's baseline reactivity or tendency to generate anxious responses. In its pathological form, anxiety is a maladaptive state that impairs the ability of an organism to respond optimally to its environment. Anxiety disorders are highly prevalent and are associated with high levels of morbidity and mortality as well as high cost. It is estimated that anxiety disorders may affect up to 20% of the population at some point in their lifetime with an annual estimated cost of $44 billion dollars in the United States alone. The mean age of onset for an anxiety disorder is 11. This early onset is consistent with the finding that individual levels of trait anxiety are established at an early age and are fairly constant over a lifetime. Thus, both trait anxiety and anxiety disorders are likely to be determined by early developmental processes or events that affect the way an individual's brain is "wired". Prior to the publication of landmark studies like those by Caspi et al demonstrating how early maltreatment interacts with the monoamine oxidase A genotype to yield antisocial behavior and how the 5-HTT transporter interacts with adverse early adult events to increase risk of depression, our understanding of how early environmental conditions might differentially impact the expression of underlying genetic risk had been limited, as these two fields of inquiry had been the purvey of two separate disciplines, i.e., genetic and epidemiological. Neither approach alone is capable of providing a full explanation of the environmental and genetic contributions to behavior. This is because genes do not generate behaviors directly. Rather, they act in developmental pathways that first generate neurons and then circuits and finally systems that mediate behavioral responses. The genetic program therefore unfolds in a predictable manner that samples the surrounding environment and is in turn shaped by it. In such a model, one would predict that the effect of any given environment will depend on the developmental program that is unfolding at the time. Thus, gene x environment interactions are perhaps more appropriately conceived of as gene x environment x development, with some time periods being more susceptible to environmental manipulation than others. We will test this hypothesis with the use of a genetically manipulated animal model, the 5-HT1A autoreceptor KO mouse, to determine the critical period for its influence on the serotonergic raphe nuclei in producing anxiety and the potential impact of environmental stress to amplify the genetic effect. PUBLIC HEALTH RELEVANCE: Recently attention has been focused on how genetic variation interacts with the environment and development. The goal of the proposed research is to determine how lack of the 5-HT1A receptor interacts with the development of a crucial neural circuit of the raphe and an environmental stressor. This research is relevant to public health because knowledge of the early neurodevelopmental modifications that lead to the future occurrence of anxiety/depression will define therapeutic targets for early intervention which may prevent the development of mental illness in adulthood.

DESCRIPTION (provided by applicant): Anxiety is normal and adaptive. In its pathological form anxiety impairs the ability to optimally respond to the environment and can be debilitating. Anxiety states begin early in life; normal trait anxiety symptoms emerge around age 6, and anxiety disorders may present as early as age 11. Approximately 8% of 13-18 year olds exhibit pathological anxiety (NIMH). The incidence and etiology of anxiety suggest that early life is a critical time for the development of anxiety disorders. Serotonin (5-HT, 5-hydroxytryptamine) is one of the key neurotransmitters involved in anxiety disorders. Direct dysregulation of the midbrain 5-HT system early in development, but not in adulthood, results in a more anxious phenotype in rodents. The 5-HT system is connected to many forebrain areas also implicated in mediating anxiety, leading to the hypothesis that alterations are also occurring in these areas. Our recent studies in mice show that the cellular characteristics, synaptic activity, and 5-HT receptor-mediated responses of 5-HT neurons are immature and develop during the first several weeks of postnatal life. In contrast characteristics recorded from 5-HT1A receptor and Pet-1 knockout mice, that exhibit anxiety phenotype, were similar to each other, immature with enhanced excitability. Unfortunately, the development of raphe-limbic system circuitry is not well understood. Our long- term goal is to elucidate the changes at the cellular level within the 5-HT-limbic system that underlie the development of normal and pathological anxiety. In this proposal our current goal is to focus on the 5-HT- mPFC connection. Our hypothesis is that there is a critical period during the first few weeks of life in which 5- HT innervation of mPFC coordinates the morphological and physiological development of its local circuits. The following Specific Aims (SA) will test this hypothesis. SP1: To determine the developmental profile of layer 5 pyramidal neurons in the mPFC in concert with responses elicited by activation of 5-HT fibers Transgenic Pet-1 ChR2 mice for optogenetic stimulation will be used to test the hypothesis that projection fibers to the mPFC mature between P12-P21 leading to the release of both glutamate and 5-HT and that mPFC neurons mature physiologically between P4 and P21. SP2: To determine the developmental profile of layer 5 pyramidal cells in the mPFC in 5-HT1A and Pet-1 knockout mice. The hypothesis is that the neural development of the layer 5 pyramidal neurons will be disrupted leading to neurons with immature phenotypes. Aim 3: To determine the consequences of losing 5-HT neural activity on behavior and 5-HT-mPFC neural development. We hypothesize that a lack of 5-HT neuron activity in the dorsal raphe between P12-P21 will lead to increased anxiety-related behavior in pups and a higher anxiety in adults. The raphe and mPFC neurons will have immature phenotypes. Mice engineered with a synthetic pharmacologically activated inhibitory receptor located only in dorsal and median raphe neurons will be used. The new technology provides an innovative approach that will provide information at a functional level of 5-HT action on developing circuits. PUBLIC HEALTH RELEVANCE: The cellular mechanisms underlying stress induced behaviors and the development and treatment of stress related psychiatric disorders most likely involve both the 5- hydroxytryptamine (5-HT) cell body containing nuclei of the raphe as well as their projection areas in the limbic forebrain. This project is investigating the postnatal development of the raphe and one its projection areas, the medial prefrontal cortex. This research is relevant to public health because stress at an early age is known to result in mood disorders as adults; knowledge about the neural circuitry of the raphe-limbic system and its development will provide information about where modifications may occur to produce mood disorders.