Jeffrey G. Tasker - US grants

93015308 Tasker The hypothalamus is the brain structure that through its link with the pituitary gland, is responsible for controlling most of the organism's basic reproductive and homeostatic functions. Pituitary hormones are released into the blood circulation on command from the hypothalamus and are carried to endocrine and muscle targets, where they cause steroid secretion or muscle contraction. As might be expected from the wide range of functions of pituitary hormones, there is a diversity of hormone- secreting systems in the hypothalamus, each activated under different conditions and with different release profiles. Dr. Tasker is interested in determining whether the distinct hypothalamic secretory systems interact to alter one another's secretion. For example, the hormone systems responsible for lactation and for the body's stress response are both located in the paraventricular nucleus of the hypothalamus. Empirical evidence points to an interaction between these two systems since emotional stress can suppress a mother's ability to provide milk to her young, and breast feeding provided the mother with a temporary feeling of calm and well being. Dr. Tasker will employ electrophysiological and neuroanatomical technique to examine integrative interactions among two subpopulations of neurons located within the hypothalamic paraventricular nucleus. By combining the simultaneous intracellular and whole-cell patch- clamp recordings, intracellular dye injection and immunohistochemical, he will detect synaptic intercommunication between neurons that synthesize and release different hormones. These findings will provide new insights about the basic functioning of the hypothalamus. In addition, knowledge about the interdependence of neurosecretory functions and behavior will increase our understanding of how seemingly disparate physiological responses of the body influence one another. ***

Under conditions of increased hormone secretion (e.g., during reproductive functions), neurosecretory neurons in the hypothalamic paraventricular nucleus (PVN) develop characteristic patterns of electrical activity and neurohormone release. These patterns of activation are determined by the intrinsic electrical properties as well as by the synaptic organization of the hormone-secreting cells. Local synaptic circuits are crucial for the generation of patterned electrical activity, yet very little is known about the local synaptic regulation of neurosecretory neurons. The long-term objectives of this study are to characterize physiologically and anatomically the local synaptic organization of PVN magnocellular and parvocellular neurons, and to determine how local synaptic inputs regulate the electrical activity and hormonal/synaptic output of PVN neurons. The specific aims of this proposal are 1) to characterize pharmacologically and map topographically local synaptic inputs to PVN neurons from cells outside the PVN, 2) to determine whether PVN magnocellular and parvocellular neurons are synaptically coupled and the transmitters or hormones which mediate these interactions, and 3) to identify anatomically cell populations of the PVN which receive local synaptic inputs. During the course of these experiments, we will determine the effects of local synaptic inputs on the electrical activity of identified PVN cells. Experiments will be performed in rat hypothalamic slices using intracellular, whole-cell patch-clamp and extracellular recordings of PVN cells. Cells will be identified provisionally as magnocellular or parvocellular by their intracellular electrical characteristics. Glutamate microapplication will be used to stimulate selectively local neurons for topographic mapping of intra- and extra-PVN regions containing cells which provide inhibitory and/or excitatory synaptic inputs to recorded PVN neurons. Conclusive demonstration of synaptic coupling between PVN cells and local interneurons (PVN and non-PVN) will be accomplished with paired recordings and cross-correlation of action potentials and synaptic activity. Specific antagonists will be applied to determine the transmitters and transmitter receptors which mediate these local synaptic interactions. The effect of activation of local synaptic inputs on the patterned activity of PVN cells (e.g., phasic firing of vasopressin cells) will be analyzed. All intracellular/patch-clamp-recorded cells will be intracellularly labeled and immunohistochemically identified with antibodies directed against general neurophysin (to distinguish between magnocellular and parvocellular neurons), oxytocin, vasopressin or corticotropin-releasing hormone. These combined electrophysiological- anatomical studies will elucidate the local synaptic organization of the hypothalamic PVN, and will provide physiological data on the regulation of PVN cell output by local synaptic inputs.

The long-term goal of this study is to determine the role of glutamate modulation in the generation of patterned output by identified PVN neurons. Experiments will be performed in acute hypothalamic slices using a combination of electrophysiological, anatomical, and in situ hybridization methods. We will use whole-cell patch-camp recordings to monitor changes in resting, voltage-gated and synaptic currents in response to mGluR activation, and pharmacological/ionic manipulations will be performed to isolate the receptor subtypes, ionic currents and second messenger mechanisms involved. Recorded cells will be marked with an intracellular dye, and they will be identified using antisera specific for different hypothalamic neuropeptides and non-radioactive riboprobes to label specific mRNA. The specific bypotheses to be test are: 1) mGluR activation enhances PVN cell excitability by reducing postsynaptic leak and voltage-gated k+ currents; 2) specific mGluR subtypes and 2nd messengers are responsible for the different mGluR actions at pre- and postsynaptic sites; 3) mGluRs play a role in the synaptic regulation of PVN neurons; 4) magnocellular and parvocellular neurons, and presynaptic neurons innervating them, express similar mGluR subtypes. The patterned electrical behaviors of hypothalamic neurons are likely to be under neuromodulatory control, but neuromodulation in the hypothalamus remains largely unexplored. These studies will provide basic information on the glutamate modulation of identified subpopulations of hypothalmic neurons. Assigning modulatory actions to glutamate to pre- and postsynaptic sites of specific hypothalamic subpopulations will enhance our understanding of the synaptic mechanisms that shape the electrical activity of neurons that control endocrine and autonomic function, and may provide the basis for future clinical pharmaceutical applications.

DESCRIPTION (adapted from applicant's abstract): Hypothalamic neuroendocrine cells develop patterned electrical activity under certain physiological conditions, which leads to a pulsatility of hormone secretion into the blood. Glutamatergic and noradrenergic synaptic mechanisms appear to play an important interactive role in the generation of bursting electrical behavior in these cells. Preliminary evidence suggests that magnocellular neuroendocrine cells of the paraventricular (PVN) and supraoptic nuclei (SON) receive excitatory synaptic inputs from norepinephrine-sensitive glutamate interneurons located within the respective nuclei. This suggests that local glutamate neurons may relay excitatory signals from noradrenergic afferents to the magnocellular neuroendocrine cells, which provides a potential mechanism for the initiation and synchronization of bursting activity in these cells. This proposal will test the hypothesis that synchronous burst generation among hypothalamic neuroendocrine cells is the result of noradrenergic activation of local glutamatergic synaptic inputs. The specific aims are to determine whether 1) norepinephrine excites magnocellular neuroendocrine cells of the SON by activating intranuclear glutamate circuits; 2) magnocellular neurons of the SON and paraventricular nucleus (PVN) are synaptically coupled via internuclear glutamate circuits. Whole-cell patch-clamp recordings from magnocellular neuroendocrine cells will be performed in acute hypothalamic slices. Intra-and internuclear circuits will be activated using electrical and focal chemical stimulation techniques. Population responses will be studied with multiunit recordings and calcium imaging to detect synchronized synaptic inputs to magnocellular neurons from local glutamate circuits. Magnocellular neurons will be identified as oxytocin and vasopressin cells with intracellular dye injection and post-hoc immunohistochemical labeling. Determining the mechanisms responsible for the generation of specific bursting patterns among different neuroendocrine neuronal populations is one of the main objectives of the physiological study of neuroendocrine systems. Pulsatility, a hallmark of neuroendocrine systems, is caused by patterned electrical activity in the hormone-secreting cells. The development of therapeutic strategies for treating disrupted or abnormal hormonal cycles requires an understanding of the mechanisms responsible for the generation of patterned activity in these cells. This proposal should make a substantial contribution to understanding the mechanisms of burst generation in hypothalamic neuroendocrine cells.

DESCRIPTION (provided by applicant): Blood glucocorticoid levels rise in response to stress activation of the hypothalamic- pituitary-adrenal (HPA) axis. They feed back onto the brain, where they exert both rapid and delayed inhibitory effects on HPA axis activation, corresponding generally to non-genomic and genomic actions, respectively. Dysfunctional glucocorticoid negative feedback is associated with a wide variety of disorders, including stress disorders and metabolic syndrome. The general goal of this project is to determine the site and mechanisms of rapid glucocorticoid feedback actions. Our working hypothesis is that rapid glucocorticoid actions in the hypothalamus are important for the integration of neuroendocrine signaling during stress. During the first period of funding of this grant, we identified a novel rapid glucocorticoid action in hypothalamic paraventricular nucleus (PVN) neurons that involves the activation of a putative membrane G protein-coupled glucocorti in PVN parvocellular neurons and both retrograde endocannabinoid and NO release in magnocellular neurons. Endocannabinoids are also released and suppress excitatory inputs in response to depolarization of PVN neurons. Thus, glucocorticoids elicit multiple retrograde signals in PVN neurons, suggesting a divergence in membrane glucocorticoid receptor signaling pathways, and both glucocorticoids and electrical activity elicit endocannabinoid synthesis, suggesting a convergence in the signaling mechanisms activated by these stimuli. In this proposal, we will build on our previous findings with experiments that address the following aims: Aim 1 is to study the intracellular signaling mechanisms engaged by membrane glucocorticoid receptor actions and depolarization that lead to endocannabinoid and NO synthesis; Aim 2 is to investigate the role of glia in restricting the extracellular actions of endocannabinoids to glutamate synapses; Aim 3 is to determine the role of glucocorticoid-indus-related disorders, including depression, hypertension and obesity. Corticosteroids represent a critical endocrine signal activated during the stress response, and they play various roles throughout the body that increase the chance for survival during stress. Corticosteroids have different mechanisms of action, both rapid and delayed, which are mediated ostensibly by different membrane and intracellular receptors. A clear understanding of the differences in the mechanisms and properties of these receptors will allow the development of pharmacological therapies for stress-related disorders, such as anxiety, depression and feeding disorders, with distinct pharmacodynamics and reduced side effects.

DESCRIPTION (provided by applicant): Magnocellular neuroendocrine cells in the hypothalamus are responsible for the synthesis and release of vasopressin and oxytocin, neurohormones involved in fluid balance, blood pressure regulation, parturition and lactation. Much of the synaptic regulation of these neurons is under the control of the neurotransmitters glutamate, GABA and norepinephrine. Anatomical studies have shown that the magnocellular neurosecretory systems undergo dramatic neuronal-glial and synaptic reorganization under conditions of dehydration, representing a unique model of physiologically linked structural plasticity in the adult brain. This includes extensive retraction of glial processes from around the magnocellular neurons and the formation of new glutamatergic, GABAergic and noradrenergic synapses. We postulate that these structural changes lead to an increase in the glutamate, GABA and noradrenergic synaptic inputs to the magnocellular neurons as well as to a decrease in their transporter-mediated clearance, resulting in an increase in the ambient extracellular levels of these neurotransmitters. We will test this first hypothesis by comparing in untreated and dehydrated rats the levels of glutamatergic and GABAergic synaptic inputs to magnocellular neurons of the supraoptic nucleus, as well as the modulation of these inputs by activation of presynaptic metabotropic receptors by ambient neurotransmitter levels. We posit that changes in the expression of glutamate may be responsible for the induction of the structural plasticity caused by dehydration, as these receptors have been implicated in the formation and stabilization of synaptic contacts associated with structural plasticity in developing and adult brain. We will test this hypothesis by assessing dehydration-induced changes in the expression of specific glutamate receptor subunits, and by altering subunit expression in normal animals through viral delivery of specific receptor subunit genes in vivo. These studies are designed to accomplish two goals: 1) to determine whether the neuronal-glial structural changes induced by dehydration lead to changes in the synaptic innervation and in the excitability of magnocellular neurons, and 2) to determine whether changes in glutamate receptor expression are causal in the induction of the structural changes associated with dehydration. The successful completion of these studies will reveal the physiological significance of anatomical changes observed under conditions of dehydration, and will provide insight into the molecular mechanisms responsible for these changes.

[unreadable] DESCRIPTION (provided by applicant): Chronic stress and depression lead to a sustained increase in the activation of the hypothalamic-pituitary adrenal (HPA) axis and tonically elevated levels of circulating HPA hormones, including glucocorticoids. Sequelae of chronic stress in experimental models and in humans include hypersensitivity of the HPA axis to stressful stimuli, and reduced sensitivity of the HPA axis to negative feedback regulation by circulating glucocorticoids. Likely causes of the chronic stress-induced hypersensitivity of the HPA axis and sustained hypersecretion of HPA hormones is an increased excitatory synaptic drive to and a reduced sensitivity to glucocorticoids of the cells that trigger HPA axis activation, the corticotropin releasing hormone (CRH) neurons of the paraventricular nucleus (PVN). Synaptic activation of the CRH neurons appears to involve an interaction between glutamatergic, GABAergic and noradrenergic systems, suggesting that structural changes in these systems may be responsible for the altered responsiveness of the HPA axis during chronic stress and depression. Rapid feedback inhibitory actions of glucocorticoids appear to be mediated, in part, by activation of endocannabinoid release within the PVN and a resulting retrograde suppression of glutamate release onto the PVN CRH neurons. Through a collaborative network of investigators studying the HPA axis, we have acquired preliminary anatomical and molecular data to suggest that the synaptic innervation of PVN CRH neurons is structurally altered by exposure to chronic stress. This proposal is the cellular physiology component of an IRPG application designed to address the overarching hypothesis that chronic stress leads to long-term molecular, anatomical and functional changes in the synaptic circuitry and glucocorticoid feedback that regulate PVN CRH neurons and the hypothalamic response to stress. We will use whole-cell patch-clamp recordings and genomic analyses to determine whether exposure to chronic stress causes an increase in the excitability of PVN CRH neurons 1) by altering glutamatergic, GABAergic and/or noradrenergic synaptic inputs, and/or 2) by reducing glucocorticoid inhibitory feedback regulation. These studies will provide important insight into the functional changes that occur in the brain during chronic stress, and will offer potential targets for the clinical treatment of certain stress-related affective disorders, such as severe depression. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Post-traumatic stress disorder (PTSD) is triggered by a traumatic life event and is characterized by the recurrent retrieval of the traumatic memory in the form of context-induced flashbacks and recurrent nightmares. The amygdala is a critical brain structure involved in both the formation and the extinction of emotional memories. Glucocorticoids, steroid hormones secreted as part of the general stress response, and endocannabinoids, lipid molecules that bind to CB1 receptors in the brain, have been shown to be important for the consolidation and extinction of fear conditioning. Both glucocorticoids and endocannabinoids enhance fear memory formation and extinction via actions within the basolateral amygdaloid complex (BLA). Patients suffering from PTSD typically show low circulating levels of corticosteroids, particularly at the nadir of the diurnal cortisol secretory rhythm, and corticosteroid treatment causes improvement in subjective measures of PTSD symptoms. A recent study showed that the glucocorticoid facilitation of conditioned fear extinction is dependent on CB1 receptor activation in the BLA, linking the actions of glucocorticoids and endocannabinoids in the BLA in conditioned fear extinction. The current study is designed to determine the cellular mechanisms that link glucocorticoid and endocannabinoid effects on fear conditioning in the BLA. We will test the hypothesis that glucocorticoids trigger endocannabinoid synthesis and retrograde release at GABA synapses in the BLA, leading to the suppression of synaptic inhibitory input to BLA neurons. The specific aims of the proposal are: 1) to test biochemically for a rapid glucocorticoid-induced increase in endocannabinoid synthesis in the BLA and CeA using a liquid chromatography-mass spectrometry approach;and 2) to determine electrophysiologically whether glucocorticoids induce a rapid suppression of GABA synaptic inputs to BLA neurons via activation of a membrane receptor and the retrograde release of endocannabinoids using whole-cell patch clamp recordings in acute in vitro slices of amygdala. Pharmacological and genetic manipulations of glucocorticoid, mineralocorticoid and cannabinoid receptors and intracellular signaling pathways will be employed to characterize the novel molecular interactions between glucocorticoids and endocannabinoids in the BLA. The importance of endocannabinoids and glucocorticoids in the BLA in the consolidation and extinction of fear conditioning, and the relevance of fear conditioning to memory processing in PTSD, suggests that the outcome of this study will provide important insight into, and possible targets for, pharmacological treatment of stress-related disorders such as PTSD. PUBLIC HEALTH RELEVANCE: The proposed research on glucocorticoid-endocannabinoid interactions in the amygdala will provide critical insight into the basic biological mechanisms responsible for emotional memory formation and retention/extinction. The better understanding of emotional memory mechanisms gained from these studies will enhance our ability and improve the tools available to address increasingly prevalent and devastating mental illnesses brought on by stress and trauma, including posttraumatic stress disorder, anxiety disorders and phobias. The link between corticosteroids and endogenous cannabinoids is relevant not only to the basic biology of emotional memory formation, but also to the interaction between illicit drug use and anxiety disorders, as increased drug abuse in certain emotionally disturbed populations may be the result of compensation for a deficit in endogenous psychoactive chemicals involved in the generation of positive emotions.

DESCRIPTION (provided by applicant): A single, low dose of the n-methyl d-aspartate receptor (NMDAR) antagonist ketamine produces rapid anti-depressant actions in treatment-resistant depressed patients. This observation strongly supports a role for cortical NMDAR function in depression, which could lead to the generation of new disease models and novel therapeutic strategies. While it is clear that NMDAR antagonism causes a rapid increase in protein translation in cortical neurons, the exact mechanisms underlying these incredible effects remain unclear. One critical unanswered question is how does suppression of NMDAR signaling promote protein translation? NMDARs are heteromultimeric complexes containing two GluN1 subunits and two GluN2 subunits, the latter of which are encoded by four genes (GluN2A-D). Cortical NMDARs are dominated by GluN2A and GluN2B subunits. Recent data have shown that GluN2B-containing NMDARs can act to directly suppress mammalian/mechanistic target of rapamycin (mTOR)-mediated protein translation in cortical neurons, through a cellular signaling mechanism that is uniquely associated with this subunit. Based upon these data, an exciting hypothesis is that relief of GluN2B-mediated suppression of mTOR signaling is responsible for producing the rapid anti- depressant effects observed in response to low dose ketamine treatment. The experiments in this proposal will test this hypothesis with the goal of improving our understanding of the cellular signaling pathways associated with cortical GluN2B-containing NMDARs and determining their involvement in depression.

Ghrelin and vasopressin are two critical hormones that control physiological balance, or homeostasis, in animals including humans. Ghrelin is secreted by the gut and provides a hunger signal to the brain during fasting. Vasopressin, a key regulator of fluid balance (the antidiuretic hormone), is secreted from the brain under conditions of dehydration. Feeding and drinking are tightly coordinated behaviors that are largely controlled, respectively, by circulating ghrelin and vasopressin hormone levels in the blood. Understanding how the two systems that control eating and drinking interact at the cellular level in the brain is critical for understanding how they are coupled and how the two basic behaviors are coordinated. This research will determine the brain cell mechanisms of ghrelin control of vasopressin secretion, and how ghrelin influences blood fluid levels and drinking behavior. These studies will reveal a novel interaction in the brain between the nutritional signal ghrelin and the vasopressin fluid balance system, providing a physiological basis for the coordination of feeding and drinking behaviors. The project will involve undergraduate students from a neighboring historically black university in intensive research experiences over two months during the summers.

Ghrelin is a key regulator of energy balance and vasopressin provides the primary control of fluid balance. Ghrelin stimulates vasopressin secretion into the blood via a central excitatory effect on vasopressin neurons. The overarching hypothesis of this research effort is that ghrelin serves as a homeostatic regulatory signal in the brain that integrates energy balance with fluid balance. This study will investigate the cellular mechanisms responsible for the ghrelin stimulation of vasopressin neurons. Pilot studies suggest that, under fasting conditions, ghrelin activates a non-canonical excitatory GABAergic synaptic input to vasopressin neurons in the hypothalamus by stimulating a novel retrograde signaling cascade that recruits neighboring astrocytes via the dendritic release of vasopressin and gliotransmitter activation of presynaptic GABA neurons. The ghrelin-induced activation of astrocytes and presynaptic GABA neurons will be studied using in-vitro electrophysiological and imaging methods in brain slices from rats and wild-type and transgenic mouse models. These studies will reveal a novel interaction in the central nervous system between the nutritional signal ghrelin and the osmoregulatory control of vasopressin, and will provide a physiological substrate for the integration of energy and fluid homeostasis.

? DESCRIPTION (provided by applicant): Stress plays a critical role in emotional memory formation. The emotional content of an experience often dictates which elements of the experience are remembered, and an important site in the brain for the formation of these emotional memories is the amygdala, particularly the basal lateral complex of the amygdala (BLA). The formation of emotional memories is caused by changes in the neural signaling in the BLA, and fear memory formation is characterized by the potentiation of excitatory synaptic circuits impacting the principal output neurons of the BLA. One means by which excitatory synaptic circuits in the BLA can be potentiated is by depressing inhibitory synaptic circuits, which leads to an increase in the excitability of the BLA neurons and a lower threshold for the long-term potentiation (LTP) of BLA excitatory circuits. Thus, the long-term depression of synaptic inhibition (LTDi) in the BLA promotes the LTP of excitatory circuits, which should increase anxiogenic output from the BLA to downstream target structures, including the hypothalamic-pituitary-adrenal neuroendocrine stress axis. Stress and the stress-induced increase in circulating glucocorticoids facilitate the anxiogenic output from the BLA, and this is mediated by intra-BLA endocannabinoid- and norepinephrine- dependent mechanisms. We have compelling preliminary evidence from patch-clamp recordings in brain slices for the induction by acute restraint stress of a form of LTDi that is mediated by rapid glucocorticoid-induced endocannabinoid depression of inhibitory transmission in the BLA. This represents, therefore, a form of stress and glucocorticoid-induced LTDi in the BLA. In this proposed project, we will distinguish the glucocorticoid and endocannabinoid mechanisms responsible for this stress-induced LTDi using pharmacological and genetic approaches, and we will determine whether stress-induced LTDi elicits anxiogenic behavior and facilitates fear memory formation by promoting LTP at excitatory synaptic circuits in the BLA. We will determine the noradrenergic mechanisms involved in the stress and glucocorticoid facilitation of anxiogenesis and fear memory formation. These studies will culminate in a fundamental understanding at the cellular level of how stress facilitates anxiogenesis and fear memory formation by inducing synaptic plasticity of inhibitory circuits in the BLA, and will provide possible cellular and molecular targts for future therapeutic intervention for the prevention and/or treatment of anxiety disorders. A potentially invaluable finding that may emerge from these studies is the identification of cannabinoid and/or noradrenergic pharmaceutical targets for the prevention of fear memory formation following exposure to trauma, when therapeutic intervention is feasible.

Summary Dysregulation of the hypothalamic-pituitary-adrenal (HPA) axis and altered circulating glucocorticoid levels are closely associated with mental health disorders. Corticotropin-releasing hormone (CRH) neurons of the hypothalamic paraventricular nucleus (PVN) control activation of the HPA axis, and direct stress- and anxiety- associated behaviors. Alterations of the CRH neuron excitatory-inhibitory balance caused by plastic changes in synaptic circuits result in altered HPA activity and shifting circulating glucocorticoids, which can lead to changes in physiological homeostasis and behavioral outputs. Afferent noradrenergic circuits are critical for controlling CRH neuron activity and HPA activation, yet little is known about the mechanism by which norepinephrine (NE) regulates the CRH neurons or its plasticity with stress exposure. Our preliminary findings reveal a novel mechanism of dendritic volume transmission in PVN CRH neurons that is activated by NE and mediated by an astrocytic retrograde relay and gliotransmission to stimulate local excitatory synaptic circuits. We have also found that stress-induced glucocorticoids cause a rapid suppression of the NE activation of the PVN CRH neurons that is mediated by ?1 adrenoreceptor desensitization. This rapid glucocorticoid effect is likely to contribute to the feedback inhibition of the HPA axis, but its specificity to physiological vs. psychological stress inputs and its role in stress-associated behaviors are not known. Here, we will use a combination of patch clamp recordings, live-cell imaging, biochemical analysis, and behavioral testing to address three specific aims. Aim 1 will focus on the pre- and postsynaptic mechanisms in CRH neurons in brain slices that are responsible for ?1 adrenoreceptor-induced neuronal-glial retrograde signaling that activates upstream glutamate circuits. Aim 2 will determine the cellular mechanisms of the stress-induced plasticity of the NE regulation of CRH neuron activity by probing the rapid glucocorticoid regulation of ?1 adrenoreceptor trafficking and signaling. Aim 3 will take an in vivo approach to examine the role of the NE afferents in HPA activation by physiological and psychological inputs, and to study the impact of the stress plasticity of NE regulation of CRH neurons on a core stress behavioral phenotype, anxiety. Together, these studies will fill an important gap in our understanding of the noradrenergic mechanisms of HPA regulation and the stress plasticity of central circuits controlling the CRH neurons and their physiological and behavioral outputs.