Glenn M. Toney - US grants

DESCRIPTION: Autonomic neurons in the hypothalamic paraventricular nucleus (PVN) are considered to have a major influence on cardiovascular and body fluid regulatory functions through their dense innervation of the rostral ventrolateral medulla (RVLM), the nucleus tractus solitarius (NTS) and the intermediolateral cell column (IML). In particular, PVN neurons mediate sympathetic responses to both isotonic volume expansion and central osmoreceptor activation. How volume and osmotic afferent signal s are integrated at the level of single PVN neurons is not understood. The proposed experiments will test the hypothesis that neurons projecting to the IML are a primary PVN substrate for the cardiovascular and sympathetic responses to volume expansion and depletion. Neural pathways connecting the PVN to the RVLM, NTS and IML are hypothesized to each play a unique role in the response to central osmotic stimulation. Integrated responses to simultaneous activation of osmotic and volume-sensitive afferents is hypothesized to involve interactions among angiotensinergic and GABAergic inputs. Combining retrograde transport and Fos protein staining will be used to compare the relative degree to which each of these 3 PVN efferent pathways responds to volume depletion and central hyperosmolality. Inactivation studies will be used to determine how each pathway participates in hemorrhage- and hyperosmolality-induced responses. Responses to volume expansion, hemorrhage and central osmotic stimulation will be compared among individual PVN neurons with axonal projections to the RVLM, NTS and the IML. Iontophoretic application of antagonists will be used to determine the role of GABAergic and angiotensinergic neurotransmission in mediating these single unit responses. The results will provide new insight into the role of diverse autonomic pathways arising from the PVN in maintaining volume and osmotic homeostasis.

DESCRIPTION (provided by applicant): Exaggerated sympathetic nervous system activity is a major contributor to the pathogenesis of congestive heart failure (CHF). Two factors appear critical in producing the sympathoexcitatory state. First, an elevated level of plasma angiotensin II (ANG II) in CHF acts within the brain to increase sympathetic nerve activity (SNA). Second, chronic extracellular fluid accumulation reduces cardiopulmonary reflex function and thus inhibitory restraint of SNA. These findings are key to the present proposal because neurons in the hypothalamic paraventricular nucleus (PVN) are required for both the sympathoexcitatory actions of elevated ANG II as well as the sympathoinhibitory effects of volume expansion. Experiments will be performed in rats with CHF induced by ligating the left anterior descending coronary artery and in sham-operated control rats. We will test the hypothesis that sympathetic hyperactivity in CHF results from increased ANG II-mediated synaptic input to the PVN and that this has an enhanced excitatory effect due to a concurrent reduction in GABA-mediated cardiopulmonary reflex inhibition. In Aim 1, effects of acute GABA-A and ANG II AT1 receptor blockade in the PVN on the level of ongoing renal and lumbar SNA will be determined. Effects will be compared among rats with graded levels of CHF. Studies will also determine how SNA responses to physiological activation of ANG II and GABA inputs to the PVN are altered in CHF. In Aim 2, we will record the response of individual RVLM-, IML- and NTS-projecting PVN neurons to activation of volume-/mechanosensitive cardiopulmonary inputs and ANG II-sensitive inputs in vivo. We anticipate that PVN unit responses to cardiopulmonary input will be significantly attenuated in CHF. This, in turn, is postulated to leave sympathoexcitatory effects of central ANG II relatively unopposed. Therefore, we expect ANG II effects on SNA and PVN unit discharge to be enhanced. In Aim 3, brain slices will be prepared and whole cell patch-clamp recordings will be performed in vitro from PVN neurons retrogradely labelled from the RVLM, IML and NTS. We will determine how the amplitude and frequency of synaptic activity are altered in each cell group in CHF. We will determine interactions between ANG II and GABA inputs in regulating PVN neuronal excitability and how these are altered in CHF. Finally, we will also determine how postsynpatic depolarization, discharge and inward current responses to ANG II are altered in CHF. The proposed studies are the first to examine mechanisms of synaptic plasticity in CHF among identified sympathetic-regulatory neurons. This information will yeild new and potentially important insight into the processes that lead to sympathetic activation and progression of disease severity in CHF.

[unreadable] DESCRIPTION (provided by applicant): Sleep apnea (SA) affects up to 15% of the US population. Many who suffer from SA develop hypertension, which depends in large part on elevated sympathetic nerve activity (SNA). Although central neural mechanisms underlying SA-induced sympathetic activation are largely unknown, efforts to investigate these processes benefit from the fact that the hypertension can be modeled experimentally by exposing rats to chronic intermittent hypoxia (CIH). Studies using the CIH model have implicated two main factors in the development of hypertension - activation of the renin-angiotensin system and the arterial chemoreceptor reflex. Because pre-sympathetic PVN neurons are key mediators of increased SNA evoked by circulating angiotensin II (ANG II) and arterial chemoreceptor activation, co-activation of these inputs by CIH could induce cellular adaptations that increase PVN neuronal activity and excitability, thereby causing a persistent rise in SNA. Preliminary studies indicate that these actions can be modified by local tissue hypoxia, which is postulated to also contribute to the CIH-induced increase in PVN neuronal activity. Specific Aims are to: (1) Establish the contributions of arterial chemoreceptor and ANG II inputs to the PVN in CIH-induced sympathoexcitation and hypertension. (2) Record from pre-sympathetic PVN neurons in vivo and determine how CIH affects the rate and patterning of tonic discharge, basal and hypoxia-induced reductions of PVN pO2 as well as neuronal response to acute activation and silencing of chemoreceptor- and ANG II-sensitive inputs. (3) Perform whole-cell patch clamp recordings from pre-sympathetic PVN neurons in brain slices and determine effects of CIH on cell activity and excitability as well as membrane potential and current responses to glutamate and ANG II. Determine how tissue hypoxia modulates glutamatergic and persistent sodium currents in pre-sympathetic PVN neurons and how these are modulated by prior exposure to CIH. Record and manipulated pO2 in the PVN to mimic values observed in conscious rats during exposure to CIH and determine how tissue hypoxia modulates glutamatergic and ANG II-stimulated PVN neuronal activity. By determining how CIH modifies PVN neuronal responses to specific afferent inputs and how tissue hypoxia further modulates these effects, the proposed studies should provide new and potentially important insight into the neurogenic mechanisms of SA-induced hypertension. [unreadable] [unreadable] Project Narrative: Sleep apnea is a form of sleep disordered breathing that affects a large percentage of the adult population in the US. Sleep apnea is associated with many cardiovascular complications including high blood pressure. This project seeks to uncover fundamental cellular adaptations that occur among sympathetic- regulatory neurons in the hypothalamus upon exposure to episodes of hypoxia. Our goal is to identify new molecular targets for treatment of elevated sympathetic activity and high blood pressure that accompany sleep apnea. [unreadable] [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): This project will investigate neural mechanisms of elevated sympathetic nerve activity (SNA), which is now widely recognized to play a key role in many forms of human hypertension (HTN). We will use our angiotensin II-dependent salt-sensitive model of HTN (AngII-salt HTN) to explore a number of innovations in this project, the first of which is conceptual. We hypothesize that the neurogenic phase of AngII-salt HTN is supported by exaggerated discharge of vasomotor neurons in the rostral ventrolateral medulla (RVLM) in response to excitatory input from the central respiratory network. Thus exaggerated respiratory-vasomotor neuron coupling is postulated to support elevated SNA and ABP in AngII-salt HTN. Specifically, we propose that post-inspiratory burst amplitude in splanchnic SNA (SSNA) is particularly important. This concept is consistent with the fact that SSNA is strongly respiratory modulated and with published data showing that interruption of SSNA by celiac ganglionectomy prevents the neurogenic phase of AngII-salt HTN. A second major innovation is the concept that exaggerated respiratory-SSNA coupling is mediated by [1] activation of AngII AT1 receptors and [2] prostaglandin E2 (PGE2) EP3 receptors in the RVLM. We propose that AT1 receptor activation results from inputs to RVLM from the hypothalamic PVN. Preliminary data in the application support this view. We further propose that EP3 receptor activation in rats with AngII-salt HTN likely results from local production of PGE2 in the RVLM. Support for PGE2 in the RVLM playing a functional role in AngII-salt HTN comes from our microinjection studies in which PGE2 in the RVLM increases SSNA and ABP in hypertensive rats, but not in normotensive controls. Collectively, these data led us to formulate the following specific aims: (1) To test the hypothesis that PVN inputs and AT1R activation in the RVLM are important in the development and maintenance of AngII-salt HTN. (2) To test the hypothesis that PGE2 and activation of EP3R in the RVLM also contribute significantly to the HTN. (3) To test the hypothesis that activation of local AT1R and EP3R each contribute to exaggerated respiratory-rhythmic burst discharge of RVLM vasomotor neurons. In Aim 3 studies, we will also incorporate state of the art gene profiling methods to identify participating gene networks in the RVLM and to identify phenotypic markers of these neurons so that detailed cellular electrophysiology and imaging studies can be performed in the future to isolate favorable targets for anti-hypertensive treatment. PUBLIC HEALTH RELEVANCE: Hypertension (HTN) is a major risk factor for death due to cardiovascular disease, which accounted for 36% of US deaths in 2004 according to NIH NHLBI statistics. By the year 2020, the World Health Organization predicts that HTN will be the greatest single cause of death and disability worldwide. Because most (65-70%) hypertension is not effectively treated, there is an urgent need to understand the biological mechanisms of this disease so that more effective treatments can be developed.

DESCRIPTION (provided by applicant): Neural Mechanisms in Cardiovascular Regulation is a conference held on a recurring basis (every 3rd year) that has been sponsored by the Federation of American Societies for Experimental Biology (FASEB) since 1982. Our goal for this conference series is to bring together researchers from around the world with diverse interests in cardiovascular neural control. Our primary objectives are (1) to create a forum for in depth discussions of the latest research discoveries, and (2) to involve and engage young investigators, including students, post-doctoral fellows and junior researchers in an international high-quality scientific meeting with neural mechanisms of cardiovascular regulation as its central theme. As our previous conferences aptly show, we attract a diverse audience of world-class researchers by focusing equally on neural mechanisms that regulate normal cardiovascular responses to physical and environmental stressors and on neural mechanisms of cardiovascular-related diseases such as arterial hypertension/preeclampsia, heart failure, obesity/diabetes, sleep apnea, stroke, nephrotic syndrome, fever/sepsis, etc. Since our first conference in 1982, the field of cardiovascular neural control has grown tremendously, as has our understanding of the pivotal role CNS mechanisms play in the initiation, maintenance and progression of cardiovascular diseases. Our major goal for the 2013 meeting will be to provide a dynamic forum to help develop the careers of young investigators in the field. Accordingly, we plan to include ten recipients of NHLBI Travel Awards as featured speakers in a specially programmed symposium. In addition, all trainees will i) participate in a mentoring program during the meeting, ii) be provided numerous opportunities to interact with senior researchers, including leaders in the field, at many scheduled events (e.g. poster sessions, breaks, lunches, beach excursions and an always popular conference banquet). Selection of travel awardees will be based on the quality and relevance of their submitted abstracts to neural mechanisms in cardiovascular regulation/disease, as well as their overall academic credentials. The first meeting for which funds are sought will be held July 14-19, 2013 at the Salishan Lodge, Gleneden Beach, Oregon. The primary aim of this R13 application is to provide financial support to enable these young investigators to attend and participate in the 2013 meeting as well as to support future young investigators to participate in subsequent meetings in 2016 and 2019.

Project Summary Sleep apnea (SA) affects ~15% of the US population. Most people with SA develop neurogenic hypertension (HTN) associated with elevated sympathetic nerve activity (SNA). During the previous funding cycle, we modeled SA HTN by exposing rats to chronic intermittent hypoxia (CIH). We determined that HTN induced by 7 days of CIH is maintained by pre-sympathetic PVN neuronal discharge driven by exaggerated NMDA receptor (NMDAR) tone. Importantly, we discovered that PVN activation by CIH involves local adaptive responses (i.e., plasticity) wherein expression of neuronal NMDAR subunits (GluN1 & GluN2B) is reduced and expression of the glial L-glutamate (L-Glu) transporter EEAT2 is increased. These responses reflect homeostatic adaptations to exaggerated glutamatergic input, which we postulate arises, at least in part, from the hindbrain NTS (Project 1) and the forebrain MnPO (Project 2). CIH also decreased PVN expression of the adaptor protein PSD95 that forms a ternary complex with the NMDAR GluN2B subunit and neuronal nitric oxide (NO) synthase (nNOS). This complex couples NMDAR Ca2+ influx with production of NO. We hypothesize that these adaptations to CIH have two offsetting actions. (1) Reduced NMDAR and increased EAAT2 expression blunt glutamatergic PVN activation and thereby buffer development of HTN. (2) Reduced PSD95 blunts NMDAR-driven NO production and lessens its tonic facilitation of GABA release. This disinhibits the PVN and thereby supports development of HTN. The net effect of these opposing adaptations is that HTN induced by CIH is less pronounced than it would be in their absence. In addition to HTN, SA increases the risk of ischemic stroke by ~4 fold. The same PVN adaptations to SA/CIH that participate in development of HTN are hypothesized to reduce ischemic injury by limiting the rise of extracellular L-Glu and reducing the production of neurotoxic NO. Studies in this revised proposal will expose rats to 7 and 28 days of CIH and CIH with hypercapnia (CIHHC) to more closely model human SA. Our specific aims will: (1) Determine effects of CIH & CIHHC on neurogenic HTN, PVN expression of synaptic/excitotoxic signaling proteins and PVN neuronal/tissue survival after local ischemia. (2) Determine effects of CIH/CIHHC adaptations on PVN control of SNA/MAP and mechanisms of neuronal vulnerability to local ischemia. (3) Use viral-mediated gene transfer/shRNA knockdown to mimic and rescue specific PVN adaptations and determine their contributions to hypertensive and neuroprotective effects of CIH & CIHHC.

Dysfunctional serotonergic neuromodulation in mood-regulating circuits underlies many psychiatric diseases, thus understanding regulation of serotonin (5-HT) transmission is of fundamental importance. The 5-HT transporter (SERT) clears 5-HT from extracellular fluid with high-affinity, and is considered a primary controller of the strength and duration of 5-HT signaling. Our studies have revealed that organic cation transporter 3 (OCT3), a low-affinity, but high-capacity transporter of monoamines, plays a critical role in 5-HT clearance as well. Though circuits modulating arousal and emotion are highly complex, processing within the basolateral amygdala (BLA) is considered essential, especially for fear conditioning. The BLA receives dense input from 5-HT neurons in dorsal raphe nucleus (DRN), and BLA principal neurons have numerous fear-regulatory outputs, including dense projections to medial entorhinal cortex (mEC), which serves as a gateway for fear memory information flow into and out of hippocampus. Like SERT, OCT3 is highly expressed in BLA, ideally positioning these transporters to powerfully control extracellular 5-HT and its local neuro-modulatory efficacy. Proposed studies test the hypothesis that 5-HT clearance by OCT3 and SERT in BLA facilitates acquisition and consolidation of fear memory by buffering the rise of 5-HT that normally restrains BLA-mEC neuronal activation by excitatory fear memory-promoting limbic inputs. We posit that fear conditioning stimuli, which lead to fear memory, co-activate limbic and DRN 5-HT inputs to BLA along with activating the hypothalamic-pituitary-adrenal stress axis. OCT3 is potently inhibited by corticosterone, indicating that diminished OCT3 clearance allows 5-HT to rise high enough during fear conditioning to activate 5-HT receptors and effectively buffer limbic excitation of BLA-mEC neurons, decreasing their output and reducing fear memory. We will use state-of-the-art conditional gene deletion strategies to separately and collectively deplete SERT and OCT3 from DRN neurons, combined with optogenetic activation and inhibition of DRN 5-HT neurons projecting directly to BLA. AAV shRNA will be used to knockdown SERT and/or OCT3 on all cell types in BLA. These approaches will be used to determine the relative contributions of SERT and OCT3 to 1) 5-HT clearance in BLA in vivo using high-speed chronoamperometry; 2) 5-HT modulation of BLA-mEC neuronal activity using in vivo single neuron and whole-cell patch clamp recording in brain slices; 3) fear conditioning behavior. Because of their important roles in fear conditioning and 5-HT signaling in BLA, we will interrogate the functional contributions of 5-HT2A and 5-HT1A receptors in this circuit. Serotonergic neurotransmission potently modulates behavior, and its dysregulation is strongly implicated in psychiatric diseases. Proposed, discovery driven, studies will provide unprecedented mechanistic insights into the role 5-HT, and its regulation by SERT and OCT3, play specifically within the DRN-BLA-mEC fear conditioning hub.

Project Summary Pre-sympathetic neurons (PSNs) of the hypothalamic paraventricular nucleus (PVN) are essential drivers of physiological and pathological increases of sympathetic nerve activity (SNA). Perhaps their most robust property is their resting state of discharge quiescence. Early studies linked quiescence to the dominance of synaptic inhibition, but mechanisms that establish and defend GABAergic inhibitory tonus in the PVN are understood only on a rudimentary level. This is an important knowledge gap because pathogenic factors that increase PVN-driven SNA must ultimately subvert or overwhelm mechanisms that regulate the quiescent resting state of PSNs. In preliminary studies, we uncovered a presynaptic mechanism that is novel to the PVN, referred to as ?Glutamate-GABA strengthening (GGS)?, that increases GABAergic inhibition in pace with synaptic glutamate (Glu) spillover. To do so, GGS regulates the amplitude of GABA-A receptor-mediated inhibitory postsynaptic currents (IPSCs) through uptake of synaptically released Glu, ostensibly into local GABA terminals, by the neuronal excitatory amino acid transporter 3 (EAAT3). Once internalized, Glu is converted to GABA and GABA molecules are packaged into synaptic vesicles at greater than normal density. Stressors that acutely increase PVN-driven SNA are hypothesized to increase synaptic Glu release without changing extrinsic GABAergic input. As a result, ?over-filled? GABA vesicles are released that dampen excitation and aid restoration of PSN quiescence. During chronic sympathoexcitation challenges accompanied by reduced GABA input, GGS is subverted (due to low GABA release) and can therefore provide little opposition to synaptic excitation. Proposed studies will use state-of-the-art transgenic mouse models, optogenetics and virus-mediated gene over-expression and CRISPR-Cas9 knockdown to assess mechanisms and functional outcomes of GGS. Kinetics, sensitivity and efficacy of GGS will be established at the single PSN level using a novel horizontal brain slice preparation that preserves Glu input from the forebrain median preoptic nucleus (MnPO) as well as GABA input from the PVN peri-nuclear zone (PNZ). Retrogradely transported AAV will be injected into the PVN of vGlut2-Cre mice to express channelrhodopsin (ChR2) in glutamatergic MnPO-PVN neurons. Optogenetic activation will determine the capacity of MnPO inputs to drive GGS amongst RVLM-projecting PVN PSNs. Using vGlut2fl/fl mice, we will determine functional effects of GGS on GABA-A receptor inhibitory tone and SNA responses to forebrain angiotensin II (AngII) and hyperosmolality when glutamatergic MnPO neurons have normal (vGlut2 intact) or diminished (vGlut2 knockdown) capacity to release Glu from PVN synapses. To further illuminate in vivo mechanisms and efficacy of GGS, EAAT3 on PNZ GABA inputs to the PVN will be increased and decreased to grade PVN GABAergic tonus and the magnitude of PVN-driven SNA responses to (1) acute forebrain AngII and hyperosmolality as well as (2) sub-acute water deprivation and high salt intake. Proposed studies will provide unprecedented mechanistic insight into the physiological role GGS plays in generating and defending PVN PSN quiescence, and are essential for advancing the goal of preventing and reversing disease-promoting sympathoexcitation.