Publications by authors named "Javier E Stern"

64 Publications

Astrocytes mediate the effect of oxytocin in the central amygdala on neuronal activity and affective states in rodents.

Nat Neurosci 2021 Feb 15. Epub 2021 Feb 15.

Centre National de la Recherche Scientifique, University of Strasbourg, Institute of Cellular and Integrative Neurosciences, Strasbourg, France.

Oxytocin (OT) orchestrates social and emotional behaviors through modulation of neural circuits. In the central amygdala, the release of OT modulates inhibitory circuits and, thereby, suppresses fear responses and decreases anxiety levels. Using astrocyte-specific gain and loss of function and pharmacological approaches, we demonstrate that a morphologically distinct subpopulation of astrocytes expresses OT receptors and mediates anxiolytic and positive reinforcement effects of OT in the central amygdala of mice and rats. The involvement of astrocytes in OT signaling challenges the long-held dogma that OT acts exclusively on neurons and highlights astrocytes as essential components for modulation of emotional states under normal and chronic pain conditions.
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http://dx.doi.org/10.1038/s41593-021-00800-0DOI Listing
February 2021

Decreased parenchymal arteriolar tone uncouples vessel-to-neuronal communication in a mouse model of vascular cognitive impairment.

Geroscience 2021 Jan 7. Epub 2021 Jan 7.

Department of Physiology, Augusta University, Augusta, GA, 30912, USA.

Chronic hypoperfusion is a key contributor to cognitive decline and neurodegenerative conditions, but the cellular mechanisms remain ill-defined. Using a multidisciplinary approach, we sought to elucidate chronic hypoperfusion-evoked functional changes at the neurovascular unit. We used bilateral common carotid artery stenosis (BCAS), a well-established model of vascular cognitive impairment, combined with an ex vivo preparation that allows pressurization of parenchymal arterioles in a brain slice. Our results demonstrate that mild (~ 30%), chronic hypoperfusion significantly altered the functional integrity of the cortical neurovascular unit. Although pial cerebral perfusion recovered over time, parenchymal arterioles progressively lost tone, exhibiting significant reductions by day 28 post-surgery. We provide supportive evidence for reduced adenosine 1 receptor-mediated vasoconstriction as a potential mechanism in the adaptive response underlying the reduced baseline tone in parenchymal arterioles. In addition, we show that in response to the neuromodulator adenosine, the action potential frequency of cortical pyramidal neurons was significantly reduced in all groups. However, a significant decrease in adenosine-induced hyperpolarization was observed in BCAS 14 days. At the microvascular level, constriction-induced inhibition of pyramidal neurons was significantly compromised in BCAS mice. Collectively, these results suggest that BCAS uncouples vessel-to-neuron communication-vasculo-neuronal coupling-a potential early event in cognitive decline.
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http://dx.doi.org/10.1007/s11357-020-00305-xDOI Listing
January 2021

High Salt Intake Recruits Tonic Activation of NR2D Subunit-Containing Extrasynaptic NMDARs in Vasopressin Neurons.

J Neurosci 2021 Feb 10;41(6):1145-1156. Epub 2020 Dec 10.

Department of Physiology, Chungnam National University, Daejeon 35015, Republic of Korea

In addition to producing a classical excitatory postsynaptic current via activation of synaptic NMDA receptors (NMDARs), glutamate in the brain also induces a tonic NMDAR current () via activation of extrasynaptic NMDARs (eNMDARs). However, since Mg blocks NMDARs in nondepolarized neurons, the potential contribution of eNMDARs to the overall neuronal excitatory/inhibitory (E/I) balance remains unknown. Here, we demonstrate that chronic (7 d) salt loading (SL) recruited NR2D subunit-containing NMDARs to generate an Mg-resistant tonic in nondepolarized [ (holding potential) -70 mV] vasopressin (VP; but not oxytocin) supraoptic nucleus (SON) neurons in male rodents. Conversely, in euhydrated (EU) and 3 d SL mice, Mg-resistant tonic was not observed. Pharmacological and genetic intervention of NR2D subunits blocked the Mg-resistant tonic in VP neurons under SL conditions, while an NR2B antagonist unveiled Mg-sensitive tonic but not Mg-resistant tonic In the EU group VP neurons, an Mg-resistant tonic was not generated by increased ambient glutamate or treatment with coagonists (e.g., d-serine and glycine). Chronic SL significantly increased NR2D expression but not NR2B expression in the SON relative to the EU group or after 3 d under SL conditions. Finally, Mg-resistant tonic selectively upregulated neuronal excitability in VP neurons under SL conditions, independent of ionotropic GABAergic input. Our results indicate that the activation of NR2D-containing NMDARs constitutes a novel mechanism that generates an Mg-resistant tonic in nondepolarized VP neurons, thus causing an E/I balance shift in VP neurons to compensate for the hormonal demands imposed by a chronic osmotic challenge. The hypothalamic supraoptic nucleus (SON) consists of two different types of magnocellular neurosecretory cells (MNCs) that synthesize and release the following two peptide hormones: vasopressin (VP), which is necessary for regulation of fluid homeostasis; and oxytocin (OT), which plays a major role in lactation and parturition. NMDA receptors (NMDARs) play important roles in shaping neuronal firing patterns and hormone release from the SON MNCs in response to various physiological challenges. Our results show that prolonged (7 d) salt loading generated a Mg-resistant tonic NMDA current mediated by NR2D subunit-containing receptors, which efficiently activated nondepolarized VP (but not OT) neurons. Our findings support the hypothesis that NR2D subunit-containing NMDARs play an important adaptive role in adult brain in response to a sustained osmotic challenge.
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http://dx.doi.org/10.1523/JNEUROSCI.1742-20.2020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7888215PMC
February 2021

Correction to: Three-dimensional morphometric analysisreveals time-dependent structural changesin microglia and astrocytes in the centralamygdala and hypothalamicparaventricular nucleus of heart failure rats.

J Neuroinflammation 2020 Nov 22;17(1):348. Epub 2020 Nov 22.

Center for Neuroinflammation and Cardiometabolic Diseases, Georgia StateUniversity, Atlanta, USA.

An amendment to this paper has been published and can be accessed via the original article.
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http://dx.doi.org/10.1186/s12974-020-02035-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7680589PMC
November 2020

Social touch promotes interfemale communication via activation of parvocellular oxytocin neurons.

Nat Neurosci 2020 09 27;23(9):1125-1137. Epub 2020 Jul 27.

Department of Neuropeptide Research in Psychiatry, Central Institute of Mental Health, Medical Faculty Mannheim, University of Heidelberg, Mannheim, Germany.

Oxytocin (OT) is a great facilitator of social life but, although its effects on socially relevant brain regions have been extensively studied, OT neuron activity during actual social interactions remains unexplored. Most OT neurons are magnocellular neurons, which simultaneously project to the pituitary and forebrain regions involved in social behaviors. In the present study, we show that a much smaller population of OT neurons, parvocellular neurons that do not project to the pituitary but synapse onto magnocellular neurons, is preferentially activated by somatosensory stimuli. This activation is transmitted to the larger population of magnocellular neurons, which consequently show coordinated increases in their activity during social interactions between virgin female rats. Selectively activating these parvocellular neurons promotes social motivation, whereas inhibiting them reduces social interactions. Thus, parvocellular OT neurons receive particular inputs to control social behavior by coordinating the responses of the much larger population of magnocellular OT neurons.
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http://dx.doi.org/10.1038/s41593-020-0674-yDOI Listing
September 2020

Three-dimensional morphometric analysis reveals time-dependent structural changes in microglia and astrocytes in the central amygdala and hypothalamic paraventricular nucleus of heart failure rats.

J Neuroinflammation 2020 Jul 23;17(1):221. Epub 2020 Jul 23.

Center for Neuroinflammation and Cardiometabolic Diseases, Georgia State University, Atlanta, USA.

Background: Cardiovascular diseases, including heart failure, are the most common cause of death globally. Recent studies support a high degree of comorbidity between heart failure and cognitive and mood disorders resulting in memory loss, depression, and anxiety. While neuroinflammation in the hypothalamic paraventricular nucleus contributes to autonomic and cardiovascular dysregulation in heart failure, mechanisms underlying cognitive and mood disorders in this disease remain elusive. The goal of this study was to quantitatively assess markers of neuroinflammation (glial morphology, cytokines, and A1 astrocyte markers) in the central amygdala, a critical forebrain region involved in emotion and cognition, and to determine its time course and correlation to disease severity during the progression of heart failure.

Methods: We developed and implemented a comprehensive microglial/astrocyte profiler for precise three-dimensional morphometric analysis of individual microglia and astrocytes in specific brain nuclei at different time points during the progression of heart failure. To this end, we used a well-established ischemic heart failure rat model. Morphometric studies were complemented with quantification of various pro-inflammatory cytokines and A1/A2 astrocyte markers via qPCR.

Results: We report structural remodeling of central amygdala microglia and astrocytes during heart failure that affected cell volume, surface area, filament length, and glial branches, resulting overall in somatic swelling and deramification, indicative of a change in glial state. These changes occurred in a time-dependent manner, correlated with the severity of heart failure, and were delayed compared to changes in the hypothalamic paraventricular nucleus. Morphometric changes correlated with elevated mRNA levels of pro-inflammatory cytokines and markers of reactive A1-type astrocytes in the paraventricular nucleus and central amygdala during heart failure.

Conclusion: We provide evidence that in addition to the previously described hypothalamic neuroinflammation implicated in sympathohumoral activation during heart failure, microglia, and astrocytes within the central amygdala also undergo structural remodeling indicative of glial shifts towards pro-inflammatory phenotypes. Thus, our studies suggest that neuroinflammation in the amygdala stands as a novel pathophysiological mechanism and potential therapeutic target that could be associated with emotional and cognitive deficits commonly observed at later stages during the course of heart failure.
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http://dx.doi.org/10.1186/s12974-020-01892-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7379770PMC
July 2020

Somato-dendritic vasopressin and oxytocin secretion in endocrine and autonomic regulation.

J Neuroendocrinol 2020 06 14;32(6):e12856. Epub 2020 May 14.

Neuroscience Institute, Georgia State University, Atlanta, GA, USA.

Somato-dendritic secretion was first demonstrated over 30 years ago. However, although its existence has become widely accepted, the function of somato-dendritic secretion is still not completely understood. Hypothalamic magnocellular neurosecretory cells were among the first neuronal phenotypes in which somato-dendritic secretion was demonstrated and are among the neurones for which the functions of somato-dendritic secretion are best characterised. These neurones secrete the neuropeptides, vasopressin and oxytocin, in an orthograde manner from their axons in the posterior pituitary gland into the blood circulation to regulate body fluid balance and reproductive physiology. Retrograde somato-dendritic secretion of vasopressin and oxytocin modulates the activity of the neurones from which they are secreted, as well as the activity of neighbouring populations of neurones, to provide intra- and inter-population signals that coordinate the endocrine and autonomic responses for the control of peripheral physiology. Somato-dendritic vasopressin and oxytocin have also been proposed to act as hormone-like signals in the brain. There is some evidence that somato-dendritic secretion from magnocellular neurosecretory cells modulates the activity of neurones beyond their local environment where there are no vasopressin- or oxytocin-containing axons but, to date, there is no conclusive evidence for, or against, hormone-like signalling throughout the brain, although it is difficult to imagine that the levels of vasopressin found throughout the brain could be underpinned by release from relatively sparse axon terminal fields. The generation of data to resolve this issue remains a priority for the field.
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http://dx.doi.org/10.1111/jne.12856DOI Listing
June 2020

Functional coupling between NMDA receptors and SK channels in rat hypothalamic magnocellular neurons: altered mechanisms during heart failure.

J Physiol 2021 Jan 4;599(2):507-520. Epub 2019 Dec 4.

Neuroscience Institute, Georgia State University, Atlanta, GA, USA.

Key Points: Glutamatergic NMDA receptors (NMDARs) and small conductance Ca -activated K (SK) channels are critical synaptic and intrinsic mechanisms, respectively, that regulate the activity of hypothalamic magnocellular neurosecretory neurons (MNNs). In this work, we investigated whether NMDARs and SK channels in MNNs are functionally coupled, and whether an altered coupling may contribute to exacerbated neuronal activity in this condition. We report that NMDARs and SK channels form a functional Ca -dependent negative feedback loop that restrains the excitatory effect on membrane potential and firing activity evoked by NMDAR activation. The negative feedback loop between NMDARs and SK channels was blunted or absent in MNNs of heart failure (HF) rats. These results help us better understand how synaptic and intrinsic mechanisms regulate hypothalamic neuronal activity, as well as how changes in the interaction among these disparate mechanisms contribute to altered neuronal activity during prevalent neurogenic cardiovascular diseases.

Abstract: Glutamatergic NMDA receptors (NMDARs) and small conductance Ca -activated K (SK) channels are critical synaptic and intrinsic mechanisms, respectively, that regulate the activity of hypothalamic magnocellular neurosecretory neurons (MNNs), both under physiological and pathological states, such as lactation and heart failure (HF). However, whether NMDARs and SK channels in MNNs are functionally coupled, and whether changes in this coupling contribute to exacerbated neuronal activity during HF is at present unknown. In the present study, we addressed these questions using patch-clamp electrophysiology and confocal Ca imaging in a rat model of ischaemic HF. We found that in MNNs of sham rats, blockade of SK channels with apamin (200 nM) significantly increased the magnitude of an NMDAR-evoked current (I ). We also observed that blockade of SK channels potentiated NMDAR-evoked firing, and abolished spike frequency adaptation in MNNs from sham, but not HF rats. Importantly, a larger I -ΔCa response was observed under basal conditions in HF compared to sham rats. Finally, we found that dialysing recorded cells with the Ca chelator BAPTA (10 mM) increased the magnitude of I in MNNs from both sham and HF rats, and occluded the effects of apamin in the former. Together our studies demonstrate that in MNNs, NMDARs and SK channels are functionally coupled, forming a local negative feedback loop that restrains the excitatory effect evoked by NMDAR activation. Moreover, our studies also support a blunted NMDAR-SK channel coupling in MNNs of HF rats, establishing it as a pathophysiological mechanism contributing to exacerbated hypothalamic neuronal activity during this prevalent neurogenic cardiovascular disease.
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http://dx.doi.org/10.1113/JP278910DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7392245PMC
January 2021

Role of endothelin receptor type A on catecholamine regulation in the olfactory bulb of DOCA-salt hypertensive rats: Hemodynamic implications.

Biochim Biophys Acta Mol Basis Dis 2019 11 6;1865(11):165527. Epub 2019 Aug 6.

Universidad de Buenos Aires, Facultad de Farmacia y Bioquímica, Cátedra de Fisiología, Buenos Aires, Argentina; CONICET-Universidad de Buenos Aires, Instituto de Química y Metabolismo del Fármaco (IQUIMEFA), Buenos Aires, Argentina. Electronic address:

Increasing evidence shows that the olfactory bulb is involved in blood pressure regulation in health and disease. Enhanced noradrenergic transmission in the olfactory bulb was reported in hypertension. Given that endothelins modulate catecholamines and are involved in the pathogenesis of hypertension, in the present study we sought to establish the role of the endothelin receptor type A on tyrosine hydroxylase, the rate limiting enzyme in catecholamine biosynthesis, in the olfactory bulb of DOCA-salt hypertensive rats. Sprague-Dawley male rats, randomly divided into Control and DOCA-Salt hypertensive groups, were used to assess endothelin receptors by Western blot and confocal microscopy, and their co-localization with tyrosine hydroxylase in the olfactory bulb. Blood pressure and heart rate as well as tyrosine hydroxylase expression and activity were assessed following BQ610 (ET antagonist) applied to the brain. DOCA-Salt hypertensive rats showed enhanced ET and decreased ET expression. ET co-localized with tyrosine hydroxylase positive neurons. Acute ET blockade reduced blood pressure and heart rate and decreased the expression of total tyrosine hydroxylase and its phosphorylated forms. Furthermore, it also diminished mRNA tyrosine hydroxylase expression and accelerated the enzyme degradation through the proteasome pathway as shown by pretreatment with MG132, (20s proteasome inhibitor) intracerebroventricularly applied. Present findings support that the brain endothelinergic system plays a major role through ET activation in the increase of catecholaminergic activity in the olfactory bulb of DOCA-Salt hypertensive rats. They provide rationale evidence that this telencephalic structure contributes in a direct or indirect way to the hemodynamic regulation in salt dependent hypertension.
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http://dx.doi.org/10.1016/j.bbadis.2019.08.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7392244PMC
November 2019

Exacerbated effects of prorenin on hypothalamic magnocellular neuronal activity and vasopressin plasma levels during salt-sensitive hypertension.

Am J Physiol Heart Circ Physiol 2019 09 5;317(3):H496-H504. Epub 2019 Jul 5.

Department of Physiology, Medical College of Georgia, Augusta University, Georgia.

Accumulating evidence supports that the brain renin-angiotensin system (RAS), including prorenin (PR) and its receptor (PRR), two newly discovered RAS players, contribute to sympathoexcitation in salt-sensitive hypertension. Still, whether PR also contributed to elevated circulating levels of neurohormones such as vasopressin (VP) during salt-sensitive hypertension, and if so, what are the precise underlying mechanisms, remains to be determined. To address these questions, we obtained patch-clamp recordings from hypothalamic magnocellular neurosecretory neurons (MNNs) that synthesize the neurohormones oxytocin and VP in acute hypothalamic slices obtained from sham and deoxycorticosterone acetate (DOCA)-salt-treated hypertensive rats. We found that focal application of PR markedly increased membrane excitability and firing responses in MNNs of DOCA-salt, compared with sham rats. This effect included a shorter latency to spike initiation and increased numbers of spikes in response to depolarizing stimuli and was mediated by a more robust inhibition of A-type K channels in DOCA-salt compared with sham rats. On the other hand, the afterhyperpolarizing potential mediated by the activation of Ca-dependent K channel was not affected by PR. mRNA expression of PRR, VP, and the Kv4.3 K channel subunit in the supraoptic nucleus of DOCA-salt hypertensive rats was increased compared with sham rats. Finally, we report a significant decrease of plasma VP levels in neuron-selective PRR knockdown mice treated with DOCA-salt, compared with wild-type DOCA-salt-treated mice. Together, these results support that activation of PRR contributes to increased excitability and firing discharge of MNNs and increased plasma levels of VP in DOCA-salt hypertension. Our studies support that prorenin (PR) and its receptor (PRR) within the hypothalamus contribute to elevated plasma vasopressin levels in deoxycorticosterone acetate-salt hypertension, in part because of an exacerbated effect of PR on magnocellular neurosecretory neuron excitability; Moreover, our study implicates A-type K+ channels as key underlying molecular targets mediating these effects. Thus, PR/PRR stands as a novel therapeutic target for the treatment of neurohumoral activation in salt-sensitive hypertension.
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http://dx.doi.org/10.1152/ajpheart.00063.2019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6766724PMC
September 2019

NMDA receptors potentiate activity-dependent dendritic release of neuropeptides from hypothalamic neurons.

J Physiol 2019 03 30;597(6):1735-1756. Epub 2019 Jan 30.

Medical College of Georgia, Augusta University, Augusta, GA, USA.

Key Points: Using 'sniffer' cell biosensors, we evaluated the effects of specific firing patterns and frequencies on activity-dependent somatodendritic release of vasopressin from paraventricular nucleus neurones. Somatodendritic release of vasopressin was rarely observed during continuous firing but was strengthened by clustered activity. Moreover, release evoked at any given frequency was robustly potentiated by NMDA receptor (NMDAR)-mediated firing. Differently from axonal release, NMDAR activation was necessary for somatodendritic release to occur at physiological firing frequencies, acting thus as a gating mechanism by which activity-dependent release from these two neuronal compartments could be independently regulated. The NMDAR-mediated potentiation was independent of a specific firing pattern and was not accompanied by increased spike broadening, but correlated with higher dendritic Ca levels. Our studies provide fundamental novel information regarding stimulus-secretion coupling at somatodendritic compartments, and shed light into mechanisms by which activity-dependent release of neuronal signals from axonal terminals and dendrites could be regulated in a spatially compartmentalized manner.

Abstract: Dendrites are now recognized to be active transmitting neuronal compartments subserving complex brain functions, including motor behaviours and homeostatic neurohumoral responses. Still, the precise mechanisms underlying activity-dependent release of dendritic signals, and how dendritic release is regulated independently from axonal release, remains largely unknown. We used 'sniffer' biosensor cells to enable the measurement and study of activity-dependent dendritic release of vasopressin (VP) from hypothalamic neurones in brain slices. Sniffer responses were dose-dependent, with a threshold detection level of 0.5 nM for VP, being thus a highly sensitive tool to detect endogenous physiological levels of the neuropeptide. Somatodendritic release of VP was rarely observed in response to a burst of action potentials fired in continuous mode, but was strengthened by clustered firing activity. Moreover, release evoked at any given frequency was robustly potentiated when firing was triggered by NMDA receptor (NMDAR) activation. Differently from axonal release, NMDAR activation was necessary for dendritic release to occur at physiological firing frequencies. Thus, we propose that NMDARs may act as a gating mechanism by which activity-dependent release from these two neuronal compartments can be independently regulated. The NMDAR-mediated potentiation of dendritic release was independent of a particular action potential waveform, firing pattern evoked, or a more pronounced spiked broadening, but correlated with higher dendritic Ca levels. Overall, our studies provide fundamental novel information regarding stimulus-secretion coupling at neuronal dendrites, and shed light into mechanisms by which activity-dependent release of neuronal signals from axonal terminals and dendrites can be regulated in a spatially compartmentalized manner.
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http://dx.doi.org/10.1113/JP277167DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6418761PMC
March 2019

Altered NMDA receptor-evoked intracellular Ca dynamics in magnocellular neurosecretory neurons of hypertensive rats.

J Physiol 2017 12 15;595(24):7399-7411. Epub 2017 Nov 15.

Department of Physiology, Medical College of Georgia, Augusta University, 1120 15th Street, Augusta, GA 30912, USA.

Key Points: NMDA receptor (NMDAR)-mediated Ca signalling plays a critical role in modulating hypothalamic neurosecretory function. However, whether an altered NMDAR-evoked changes in Ca (NMDAR-ΔCa ) signalling in magnocellular neurosecretory cells (MNCs) may contribute to neurohumoral activation during disease states is unknown. We show that activation of NMDARs evoked similar inward currents in MNCs of sham and renovascular hypertensive (RVH) rats. Despite this, a prolonged and larger NMDAR-ΔCa response was observed in the latter. The exacerbated NMDAR-ΔCa responses in MNCs of RVH rats affected both somatic and dendritic compartments. Inhibition of the endoplasmic reticulum sarcoendoplasmic reticulum calcium trasport ATPase (SERCA) pump prolonged NMDAR-ΔCa responses in sham rats, but not in RVH rats. Our study supports an altered spatiotemporal dynamic of NMDAR-ΔCa signalling in MNCs from RVH rats, partly due to blunted endoplasmic reticulum Ca buffering capacity.

Abstract: A growing body of evidence supports an elevated NMDA receptor (NMDAR)-mediated glutamate excitatory function in the supraoptic nucleus and paraventricular nucleus of hypertensive rats that contributes to neurohumoral activation in this disease. However, the precise mechanisms underlying altered NMDAR signalling in hypertension remain to be elucidated. In this study, we performed simultaneous electrophysiology and fast confocal Ca imaging to determine whether altered NMDAR-mediated changes in intracellular Ca levels (NMDAR-ΔCa ) occurred in hypothalamic magnocellular neurosecretory cells (MNCs) in renovascular hypertensive (RVH) rats. We found that despite evoking a similar excitatory inward current, activation of NMDARs resulted in a larger and prolonged ΔCa in MNCs from RVH rats. Changes in NMDAR-ΔCa dynamics were observed both in somatic and dendritic compartments. Inhibition of the sarcoendoplasmic reticulum calcium trasport ATPase (SERCA) pump activity with thapsigargin prolonged NMDAR-ΔCa responses in MNCs of sham rats, but this effect was occluded in RVH rats, thus equalizing the magnitude and time course of the NMDA-ΔCa responses between the two experimental groups. Taken together, our results support (1) an exacerbated NMDAR-ΔCa response in somatodendritic compartments of MNCs of RVH rats, and (2) that a blunted ER Ca buffering capacity contributes to the altered NMDAR-ΔCa dynamics in this condition. Thus, altered spatiotemporal dynamics of the NMDAR-ΔCa response stands as an underlying mechanism contributing to neurohumoral activation in neurogenic hypertension.
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http://dx.doi.org/10.1113/JP275169DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5730840PMC
December 2017

A reduction in SK channels contributes to increased activity of hypothalamic magnocellular neurons during heart failure.

J Physiol 2017 10 2;595(20):6429-6442. Epub 2017 Aug 2.

Department of Physiology, Augusta University, Augusta, GA, USA.

Key Points: Small conductance Ca -activated K (SK) channels play an important role in regulating the excitability of magnocellular neurosecretory cells (MNCs). Although an increased SK channel function contributes to adaptive physiological responses, it remains unknown whether changes in SK channel function/expression contribute to exacerbated MNC activity under disease conditions. We show that the input-output function of MNCs in heart failure (HF) rats is enhanced. Moreover, the SK channel blocker apamin enhanced the input-output function in sham, although not in HF rats. We found that both the after-hyperpolarizing potential magnitude and the underlying apamin-sensitive I are blunted in MNCs from HF rats. The magnitude of spike-induced increases in intracellular Ca levels was not affected in MNCs of HF rats. We found a diminished expression of SK2/SK3 channel subunit mRNA expression in the supraoptic nucleus of HF rats. Our studies suggest that a reduction in SK channel expression, but not changes in Ca -mediated activation of SK channels, contributes to exacerbated MNC activity in HF rats.

Abstract: Small conductance Ca -activated K channels (SK) play an important role in regulating the activity of magnocellular neurosecretory cells (MNCs) and hormone release from the posterior pituitary. Moreover, enhanced SK activity contributes to the adaptive responses of MNCs to physiological challenge, such as lactation. Nevertheless, whether changes in SK function/expression contribute to exacerbated MNC activity during diseases such as heart failure (HF) remains unknown. In the present study, we used a combination of patch clamp electrophysiology, confocal Ca imaging and molecular biology in a rat model of ischaemic HF. We found that the input-output function of MNCs was enhanced in HF compared to sham rats. Moreover, although the SK blocker apamin (200 nm) strengthened the input-output function in sham rats, it failed to have an effect in HF rats. The magnitude of the after-hyperpolarizing potential (AHP) following a train of spikes and the underlying apamin-sensitive I were blunted in MNCs from HF rats. However, spike-induced increases in intracellular Ca were not affected in the MNCs of HF rats. Real-time PCR measurements of SK channel subunits mRNA in supraoptic nucleus punches revealed a diminished expression of SK2/SK3 subunits in HF compared to sham rats. Together, our studies demonstrate that MNCs from HF rats exhibit increased membrane excitability and an enhanced input-output function, and also that a reduction in SK channel-mediated, apamin-sensitive AHP is a critical contributing mechanism. Moreover, our results suggest that the reduced AHP is related to a down-regulation of SK2/SK3 channel subunit expression but not the result of a blunted activity-dependent intracellular Ca increase following a burst of action potentials.
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http://dx.doi.org/10.1113/JP274730DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5638886PMC
October 2017

A-type K channels contribute to the prorenin increase of firing activity in hypothalamic vasopressin neurosecretory neurons.

Am J Physiol Heart Circ Physiol 2017 Sep 16;313(3):H548-H557. Epub 2017 Jun 16.

Department of Physiology, Medical College of Georgia, Augusta University, Augusta, Georgia

Recent studies have supported an important contribution of prorenin (PR) and its receptor (PRR) to the regulation of hypothalamic, sympathetic, and neurosecretory outflows to the cardiovascular system, including systemic release of vasopressin (VP), both under physiological and cardiovascular disease conditions. Still, the identification of precise cellular mechanisms and neuronal/molecular targets remain unknown. We have recently shown that PRR is expressed in VP neurons and that their activation increases neuronal activity. However, the underlying ionic channel mechanisms are undefined. Here, we performed patch-clamp electrophysiology from identified VP neurons in acute hypothalamic slices obtained from enhanced green fluorescent protein-VP transgenic rats. Voltage-clamp recordings showed that PR inhibited the magnitude of A-type K current (; ~50% at -25 mV), a subthreshold voltage-dependent current that restrains VP firing activity. PR also increased the inactivation rate of and shifted the steady-state voltage-dependent inactivation function toward more hyperpolarized membrane potential (~7 mV shift), thus resulting in less channel availability to be activated at any given membrane potential. PR also inhibited a sustained component of ("window" current). PR-mediated changes in action potential waveform and increased firing activity were occluded when was blocked by 4-aminopyridine. Finally, PR failed to increase superoxide production within the supraoptic nucleus/paraventricular nucleus, and PR excitatory effects persisted in slices treated with the SOD mimetic tempol. Taken together, these experiments indicated that PR excitatory effects on vasopressin neurons involve inhibition of , due, in part, to increases in its voltage-dependent inactivation properties. Moreover, our results indicate that PR effects did not involve an increase in oxidative stress. Here, we demonstrate that prorenin/the prorenin receptor is an important signaling unit for the regulation of vasopressin firing activity and, thus, systemic hormonal release. We identified A-type K channels as key molecular targets mediating prorenin stimulation of vasopressin neuronal activity, thus standing as a potential therapeutic target for neurohumoral activation in cardiovascular disease.
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http://dx.doi.org/10.1152/ajpheart.00216.2017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5625174PMC
September 2017

TNFα drives mitochondrial stress in POMC neurons in obesity.

Nat Commun 2017 05 10;8:15143. Epub 2017 May 10.

Institute for Diabetes and Obesity, Helmholtz Diabetes Center at Helmholtz Zentrum München, Division of Metabolic Diseases, Department of Medicine, Technische Universität München, German Center for Diabetes Research (DZD), 85764 München-Neuherberg, Germany.

Consuming a calorically dense diet stimulates microglial reactivity in the mediobasal hypothalamus (MBH) in association with decreased number of appetite-curbing pro-opiomelanocortin (POMC) neurons; whether the reduction in POMC neuronal function is secondary to the microglial activation is unclear. Here we show that in hypercaloric diet-induced obese mice, persistently activated microglia in the MBH hypersecrete TNFα that in turn stimulate mitochondrial ATP production in POMC neurons, promoting mitochondrial fusion in their neurites, and increasing POMC neuronal firing rates and excitability. Specific disruption of the gene expressions of TNFα downstream signals TNFSF11A or NDUFAB1 in the MBH of diet-induced obese mice reverses mitochondrial elongation and reduces obesity. These data imply that in a hypercaloric environment, persistent elevation of microglial reactivity and consequent TNFα secretion induces mitochondrial stress in POMC neurons that contributes to the development of obesity.
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http://dx.doi.org/10.1038/ncomms15143DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5436136PMC
May 2017

Vasopressin casts light on the suprachiasmatic nucleus.

J Physiol 2017 06 14;595(11):3497-3514. Epub 2017 May 14.

Centre for Integrative Physiology, University of Edinburgh, Edinburgh, UK.

Key Points: A subpopulation of retinal ganglion cells expresses the neuropeptide vasopressin. These retinal ganglion cells project predominately to our biological clock, the suprachiasmatic nucleus (SCN). Light-induced vasopressin release enhances the responses of SCN neurons to light. It also enhances expression of genes involved in photo-entrainment of biological rhythms.

Abstract: In all animals, the transition between night and day engages a host of physiological and behavioural rhythms. These rhythms depend not on the rods and cones of the retina, but on retinal ganglion cells (RGCs) that detect the ambient light level in the environment. These project to the suprachiasmatic nucleus (SCN) of the hypothalamus to entrain circadian rhythms that are generated within the SCN. The neuropeptide vasopressin has an important role in this entrainment. Many SCN neurons express vasopressin, and it has been assumed that the role of vasopressin in the SCN reflects the activity of these cells. Here we show that vasopressin is also expressed in many retinal cells that project to the SCN. Light-evoked vasopressin release contributes to the responses of SCN neurons to light, and enhances expression of the immediate early gene c-fos in the SCN, which is involved in photic entrainment of circadian rhythms.
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http://dx.doi.org/10.1113/JP274025DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5451709PMC
June 2017

An increased extrasynaptic NMDA tone inhibits A-type K current and increases excitability of hypothalamic neurosecretory neurons in hypertensive rats.

J Physiol 2017 07 23;595(14):4647-4661. Epub 2017 May 23.

Department of Physiology, Medical College of Georgia, Augusta University, 1120 15th Street, Augusta, GA, 30912, USA.

Key Points: A functional coupling between extrasynaptic NMDA receptors (eNMDARs) and the A-type K current (I ) influences homeostatic firing responses of magnocellular neurosecretory cells (MNCs) to a physiological challenge. However, whether an altered eNMDAR-I coupling also contributes to exacerbated MNC activity and neurohumoral activation during disease states is unknown. We show that activation of eNMDARs by exogenously applied NMDA inhibited I in MNCs obtained from sham, but not in MNCs from renovascular hypertensive (RVH) rats. Neither the magnitude of the exogenously evoked NMDA current nor the expression of NMDAR subunits were altered in RVH rats. Conversely, we found that a larger endogenous glutamate tone, which was not due to blunted glutamate transport activity, led to the sustained activation of eNMDARs that tonically inhibited I , contributing in turn to higher firing activity in RVH rats. Our studies show that exacerbated activation of eNMDARs by endogenous glutamate contributes to tonic inhibition of I and enhanced MNC excitability in RVH rats.

Abstract: We recently showed that a functional coupling between extrasynaptic NMDA receptors (eNMDARs) and the A-type K current (I ) influences the firing activity of hypothalamic magnocellular neurosecretory neurons (MNCs), as well as homeostatic adaptive responses to a physiological challenge. Here, we aimed to determine whether changes in the eNMDAR-I coupling also contributed to exacerbated MNC activity during disease states. We used a combination of patch-clamp electrophysiology and real-time PCR in MNCs in sham and renovascular hypertensive (RVH) rats. Activation of eNMDARs by exogenously applied NMDA inhibited I in sham rats, but this effect was largely blunted in RVH rats. The blunted response was not due to changes in eNMDAR expression and/or function, since neither NMDA current magnitude or reversal potential, nor the levels of NR1-NR2A-D subunit expression were altered in RVH rats. Conversely, we found a larger endogenous glutamate tone, resulting in the sustained activation of eNMDARs that tonically inhibited I and contributed also to higher ongoing firing activity in RVH rats. The enhanced endogenous glutamate tone in RVH rats was not due to blunted glutamate transporter activity. Rather, a higher transporter activity was observed, which possibly acted as a compensatory mechanism in the face of the elevated endogenous tone. In summary, our studies indicate that an elevated endogenous glutamate tone results in an exacerbated activation of eNMDARs, which in turn contributes to diminished I magnitude and increased firing activity of MNCs from hypertensive rats.
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http://dx.doi.org/10.1113/JP274327DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5509869PMC
July 2017

A Unique "Angiotensin-Sensitive" Neuronal Population Coordinates Neuroendocrine, Cardiovascular, and Behavioral Responses to Stress.

J Neurosci 2017 03 20;37(13):3478-3490. Epub 2017 Feb 20.

Department of Pharmacodynamics, College of Pharmacy, University of Florida, Gainesville, Florida 32611, and

Stress elicits neuroendocrine, autonomic, and behavioral responses that mitigate homeostatic imbalance and ensure survival. However, chronic engagement of such responses promotes psychological, cardiovascular, and metabolic impairments. In recent years, the renin-angiotensin system has emerged as a key mediator of stress responding and its related pathologies, but the neuronal circuits that orchestrate these interactions are not known. These studies combine the use of the Cre-recombinase/loxP system in mice with optogenetics to structurally and functionally characterize angiotensin type-1a receptor-containing neurons of the paraventricular nucleus of the hypothalamus, the goal being to determine the extent of their involvement in the regulation of stress responses. Initial studies use neuroanatomical techniques to reveal that angiotensin type-1a receptors are localized predominantly to the parvocellular neurosecretory neurons of the paraventricular nucleus of the hypothalamus. These neurons are almost exclusively glutamatergic and send dense projections to the exterior portion of the median eminence. Furthermore, these neurons largely express corticotrophin-releasing hormone or thyrotropin-releasing hormone and do not express arginine vasopressin or oxytocin. Functionally, optogenetic stimulation of these neurons promotes the activation of the hypothalamic-pituitary-adrenal and hypothalamic-pituitary-thyroid axes, as well as a rise in systolic blood pressure. When these neurons are optogenetically inhibited, the activity of these neuroendocrine axes are suppressed and anxiety-like behavior in the elevated plus maze is dampened. Collectively, these studies implicate this neuronal population in the integration and coordination of the physiological responses to stress and may therefore serve as a potential target for therapeutic intervention for stress-related pathology. Chronic stress leads to an array of physiological responses that ultimately rouse psychological, cardiovascular, and metabolic impairments. As a consequence, there is an urgent need for the development of novel therapeutic approaches to prevent or dampen deleterious aspects of "stress." While the renin-angiotensin system has received some attention in this regard, the neural mechanisms by which this endocrine system may impact stress-related pathologies and consequently serve as targets for therapeutic intervention are not clear. The present studies provide substantial insight in this regard. That is, they reveal that a distinct population of angiotensin-sensitive neurons is integral to the coordination of stress responses. The implication is that this neuronal phenotype may serve as a target for stress-related disease.
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http://dx.doi.org/10.1523/JNEUROSCI.3674-16.2017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5373130PMC
March 2017

Astrocytes Contribute to Angiotensin II Stimulation of Hypothalamic Neuronal Activity and Sympathetic Outflow.

Hypertension 2016 12 3;68(6):1483-1493. Epub 2016 Oct 3.

From the Department of Physiology, Augusta University, GA (J.E.S., S.S., V.C.B.); and Department of Cellular and Integrative Physiology, University of Nebraska Medical Center, Omaha (H.Z., N.S., K.P.P.).

Angiotensin II (AngII) is a key neuropeptide that acting within the brain hypothalamic paraventricular nucleus regulates neurohumoral outflow to the circulation. Moreover, an exacerbated AngII action within the paraventricular nucleus contributes to neurohumoral activation in hypertension. Although AngII effects involve changes in paraventricular nucleus neuronal activity, the precise underlying mechanisms, cellular targets, and distribution of AngII receptors within the paraventricular nucleus remain largely unknown. Thus, whether AngII effects involve direct actions on paraventricular neurons, or whether it acts via intermediary cells, such as astrocytes, is still controversial. To address this important gap in our knowledge, we used a multidisciplinary approach combining patch-clamp electrophysiology in presympathetic paraventricular neurons and astrocytes, along with in vivo sympathetic nerve recordings and astrocyte-targeted gene manipulations. We present evidence for a novel mechanism underlying central AngII actions, which involves astrocytes as major intermediary cellular targets. We found that AngII type 1 receptor mRNA is expressed in paraventricular astrocytes. Moreover, we report that AngII inhibited glutamate transporter function, increasing in turn extracellular glutamate levels. This resulted in the activation of neuronal extrasynaptic NMDA (N-methyl-d-aspartate) receptors, increased presympathetic neuronal activity, enhanced sympathoexcitatory outflow, and increased blood pressure. Together, our studies support astrocytes as critical intermediary cell types mediating brain AngII regulation of the circulation and indicate that AngII-mediated neuronal and sympathoexcitatory effects are dependent on a unique neuroglial signaling modality involving nonsynaptic glutamate transmission.
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http://dx.doi.org/10.1161/HYPERTENSIONAHA.116.07747DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5159229PMC
December 2016

Mechanisms underlying prorenin actions on hypothalamic neurons implicated in cardiometabolic control.

Mol Metab 2016 Oct 4;5(10):858-868. Epub 2016 Aug 4.

Department of Physiology, Medical College of Georgia, Augusta University, United States. Electronic address:

Background: Hypertension and obesity are highly interrelated diseases, being critical components of the metabolic syndrome. Despite the growing prevalence of this syndrome in the world population, efficient therapies are still missing. Thus, identification of novel targets and therapies are warranted. An enhanced activity of the hypothalamic renin-angiotensin system (RAS), including the recently discovered prorenin (PR) and its receptor (PRR), has been implicated as a common mechanism underlying aberrant sympatho-humoral activation that contributes to both metabolic and cardiovascular dysregulation in the metabolic syndrome. Still, the identification of precise neuronal targets, cellular mechanisms and signaling pathways underlying PR/PRR actions in cardiovascular- and metabolic related hypothalamic nuclei remain unknown.

Methods And Results: Using a multidisciplinary approach including patch-clamp electrophysiology, live calcium imaging and immunohistochemistry, we aimed to elucidate cellular mechanisms underlying PR/PRR actions within the hypothalamic supraoptic (SON) and paraventricular nucleus (PVN), key brain areas previously involved in cardiometabolic regulation. We show for the first time that PRR is expressed in magnocellular neurosecretory cells (MNCs), and to a lesser extent, in presympathetic PVN neurons (PVNPS). Moreover, we show that while PRR activation efficiently stimulates the firing activity of both MNCs and PVNPS neurons, these effects involved AngII-independent and AngII-dependent mechanisms, respectively. In both cases however, PR excitatory effects involved an increase in intracellular Ca(2+) levels and a Ca(2+)-dependent inhibition of a voltage-gated K(+) current.

Conclusions: We identified novel neuronal targets and cellular mechanisms underlying PR/PRR actions in critical hypothalamic neurons involved in cardiometabolic regulation. This fundamental mechanistic information regarding central PR/PRR actions is essential for the development of novel RAS-based therapeutic targets for the treatment of cardiometabolic disorders in obesity and hypertension.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5034613PMC
http://dx.doi.org/10.1016/j.molmet.2016.07.010DOI Listing
October 2016

Angiotensin Type-2 Receptors Influence the Activity of Vasopressin Neurons in the Paraventricular Nucleus of the Hypothalamus in Male Mice.

Endocrinology 2016 08 6;157(8):3167-80. Epub 2016 Jun 6.

Department of Physiology and Functional Genomics (A.D.d.K., D.J.P., C.S.), College of Medicine, University of Florida, Gainesville, Florida 32610; Physiology (S.P., J.E.S.), Medical College of Georgia, Augusta University, Augusta, Georgia 30912; and Department of Pharmacodynamics (L.W., H.H., J.A.S., E.G.K.)., College of Pharmacy, University of Florida, Gainesville, Florida 32610.

It is known that angiotensin-II acts at its type-1 receptor to stimulate vasopressin (AVP) secretion, which may contribute to angiotensin-II-induced hypertension. Less well known is the impact of angiotensin type-2 receptor (AT2R) activation on these processes. Studies conducted in a transgenic AT2R enhanced green fluorescent protein reporter mouse revealed that although AT2R are not themselves localized to AVP neurons within the paraventricular nucleus of the hypothalamus (PVN), they are localized to neurons that extend processes into the PVN. In the present set of studies, we set out to characterize the origin, phenotype, and function of nerve terminals within the PVN that arise from AT2R-enhanced green fluorescent protein-positive neurons and synapse onto AVP neurons. Initial experiments combined genetic and neuroanatomical techniques to determine that γ-aminobutyric acid (GABA)ergic neurons derived from the peri-PVN area containing AT2R make appositions onto AVP neurons within the PVN, thereby positioning AT2R to negatively regulate neuroendocrine secretion. Subsequent patch-clamp electrophysiological experiments revealed that selective activation of AT2R in the peri-PVN area using compound 21 facilitates inhibitory (ie, GABAergic) neurotransmission and leads to reduced activity of AVP neurons within the PVN. Final experiments determined the functional impact of AT2R activation by testing the effects of compound 21 on plasma AVP levels. Collectively, these experiments revealed that AT2R expressing neurons make GABAergic synapses onto AVP neurons that inhibit AVP neuronal activity and suppress baseline systemic AVP levels. These findings have direct implications in the targeting of AT2R for disorders of AVP secretion and also for the alleviation of high blood pressure.
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http://dx.doi.org/10.1210/en.2016-1131DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4967126PMC
August 2016

A Functional Coupling Between Carbon Monoxide and Nitric Oxide Contributes to Increased Vasopressin Neuronal Activity in Heart Failure rats.

Endocrinology 2016 05 16;157(5):2052-66. Epub 2016 Mar 16.

Department of Physiology, Medical College of Georgia, Georgia Regents University, Augusta, Georgia 30912.

Despite the pathophysiological importance of neurohumoral activation in patients with heart failure (HF), the precise underlying mechanisms contributing to elevated vasopressin (VP) activation in HF remains unknown. Carbon monoxide (CO) is a gaseous neurotransmitter in the central nervous system that stimulates VP neuronal firing activity. Recently, we showed that the excitatory effect of CO on VP neurons in the hypothalamic paraventricular nucleus (PVN) was mediated by inhibition of nitric oxide (NO). Given that previous studies showed that VP neuronal activity is enhanced, whereas NO inhibitory signaling is blunted in HF rats, we tested whether an enhanced endogenous CO availability within the PVN contributes to elevated VP neuronal activity and blunted NO signaling in HF rats. We found that both haeme-oxygenase 1 (the CO-synthesizing enzyme) protein and mRNA expression levels were enhanced in the PVN of HF compared with sham rats (∼18% and ∼38%, respectively). We report that in sham rats, bath application of a CO donor (tricarbonyldichlororuthenium dimer) increased the firing activity of identified PVN VP neurons (P < .05), whereas inhibition of endogenous CO production (Tin-protoporphyrin IX [SnPP]) failed to affect neuronal activity. In HF rats, however, SnPP decreased VP activity (P < .05), an effect that was occluded by previous NO synathase blockade NG-nitro-larginine methyl ester. Finally, we found that SnPP increased the mean frequency of γ-aminobutyric acid inhibitory postsynaptic currents in VP neurons in HF (P < .05) but not sham rats. Our results support an enhanced endogenous CO excitatory signaling in VP neurons, which likely contributes to blunted NO and γ-aminobutyric acid inhibitory function in HF rats.
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http://dx.doi.org/10.1210/en.2015-1958DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4870874PMC
May 2016

Functional topography of cardiovascular regulation along the rostrocaudal axis of the rat posterior insular cortex.

Clin Exp Pharmacol Physiol 2016 Apr;43(4):484-93

Department of Physiology and Biophysics, INCT, Institute of Biological Sciences, Federal University of Minas Gerais, Belo Horizonte, Brazil.

Cardiovascular (CV) representation has been identified within the insular cortex (IC) and a lateralization of function previously suggested. In order to further understand the role of IC on cardiovascular control, the present study compared the CV responses evoked by stimulation of N-metil-D-aspartate (NMDA) receptors in the right and left posterior IC at different rostrocaudal levels. Intracortical microinjections of NMDA were performed into the IC of male Wistar rats anaesthetized with urethane (1.4 g/kg) prepared for blood pressure, heart rate and renal sympathetic nerve activity. Gene expression of NMDA receptor subunits NR2A and NR2B in the IC was confirmed by RT-PCR. Immunofluorescence for the NMDA receptor NR1 subunit was demonstrated in the IC (coordinates anteroposterior (AP) +1.5, 0.0 and -1.5 mm). A cardiac sympathoinhibitory site was identified, more rostrally located than identified in previous studies. A site of sympathoexcitatory cardiac control was identified more caudal to this region in agreement with earlier work. Under the experimental conditions, no lateralization of cardiovascular function was identified with chemical stimulation eliciting the same responses from either left or right insular cortices. No tonic role of the insula on cardiovascular control was identified with the use of the NMDA antagonist, AP-5. Peri-insular microinjection of NMDA was without cardiovascular effect indicating the specificity of the insula as a cardiovascular regulatory site. The current study reveals a functional topography for autonomic cardiovascular control along the rostrocaudal axis of the posterior IC.
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http://dx.doi.org/10.1111/1440-1681.12542DOI Listing
April 2016

Cross talk between AT1 receptors and Toll-like receptor 4 in microglia contributes to angiotensin II-derived ROS production in the hypothalamic paraventricular nucleus.

Am J Physiol Heart Circ Physiol 2016 Feb 4;310(3):H404-15. Epub 2015 Dec 4.

Department of Physiology, Medical College of Georgia, Georgia Regents University, Augusta, Georgia;

ANG II is thought to increase sympathetic outflow by increasing oxidative stress and promoting local inflammation in the paraventricular nucleus (PVN) of the hypothalamus. However, the relative contributions of inflammation and oxidative stress to sympathetic drive remain poorly understood, and the underlying cellular and molecular targets have yet to be examined. ANG II has been shown to enhance Toll-like receptor (TLR)4-mediated signaling on microglia. Thus, in the present study, we aimed to determine whether ANG II-mediated activation of microglial TLR4 signaling is a key molecular target initiating local oxidative stress in the PVN. We found TLR4 and ANG II type 1 (AT1) receptor mRNA expression in hypothalamic microglia, providing molecular evidence for the potential interaction between these two receptors. In hypothalamic slices, ANG II induced microglial activation within the PVN (∼65% increase, P < 0.001), an effect that was blunted in the absence of functional TLR4. ANG II increased ROS production, as indicated by dihydroethidium fluorescence, within the PVN of rats and mice (P < 0.0001 in both cases), effects that were also dependent on the presence of functional TLR4. The microglial inhibitor minocycline attenuated ANG II-mediated ROS production, yet ANG II effects persisted in PVN single-minded 1-AT1a knockout mice, supporting the contribution of a non-neuronal source (likely microglia) to ANG II-driven ROS production in the PVN. Taken together, these results support functional interactions between AT1 receptors and TLR4 in mediating ANG II-dependent microglial activation and oxidative stress within the PVN. More broadly, our results support a functional interaction between the central renin-angiotensin system and innate immunity in the regulation of neurohumoral outflows from the PVN.
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http://dx.doi.org/10.1152/ajpheart.00247.2015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4796625PMC
February 2016

Endocrine-Autonomic Linkages.

Compr Physiol 2015 Jul;5(3):1281-323

Department of Physiology and Biophysics, Rosalind Franklin University of Medicine & Science, North Chicago, IL, USA.

Interaction between the autonomic nervous system and the neuroendocrine system is critical for maintenance of homeostasis in a wide variety of physiological parameters such as body temperature, fluid and electrolyte balance, and blood pressure and volume. The anatomical and physiological mechanisms underlying integration of the neuroendocrine and autonomic mechanisms responsible for eliciting integrated autonomic and neuroendocrine actions are the focus of this article. This includes a focus on the hypothalamic paraventricular nucleus, because it includes both neuroendocrine neurons and preganglionic autonomic neurons that regulate sympathetic and parasympathetic outflow. The "wired" and "nonwired" mechanisms within PVN that facilitate communication between these neuronal populations are described. The impact of peripheral hormones, specifically the adrenal and gonadal steroids, on the neuroendocrine and autonomic systems is discussed, and exercise is used as a specific example of a physiological challenge/stress that requires precise integration of neuroendocrine and autonomic responses to maintain cardiovascular, fluid, and energy homeostasis.
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http://dx.doi.org/10.1002/cphy.c140028DOI Listing
July 2015

Astrocyte contributions to flow/pressure-evoked parenchymal arteriole vasoconstriction.

J Neurosci 2015 May;35(21):8245-57

Georgia Regents University, Augusta, Georgia 30912, and

Basal and activity-dependent cerebral blood flow changes are coordinated by the action of critical processes, including cerebral autoregulation, endothelial-mediated signaling, and neurovascular coupling. The goal of our study was to determine whether astrocytes contribute to the regulation of parenchymal arteriole (PA) tone in response to hemodynamic stimuli (pressure/flow). Cortical PA vascular responses and astrocytic Ca(2+) dynamics were measured using an in vitro rat/mouse brain slice model of perfused/pressurized PAs; studies were supplemented with in vivo astrocytic Ca(2+) imaging. In vitro, astrocytes responded to PA flow/pressure increases with an increase in intracellular Ca(2+). Astrocytic Ca(2+) responses were corroborated in vivo, where acute systemic phenylephrine-induced increases in blood pressure evoked a significant increase in astrocytic Ca(2+). In vitro, flow/pressure-evoked vasoconstriction was blunted when the astrocytic syncytium was loaded with BAPTA (chelating intracellular Ca(2+)) and enhanced when high Ca(2+) or ATP were introduced to the astrocytic syncytium. Bath application of either the TRPV4 channel blocker HC067047 or purinergic receptor antagonist suramin blunted flow/pressure-evoked vasoconstriction, whereas K(+) and 20-HETE signaling blockade showed no effect. Importantly, we found TRPV4 channel expression to be restricted to astrocytes and not the endothelium of PA. We present evidence for a novel role of astrocytes in PA flow/pressure-evoked vasoconstriction. Our data suggest that astrocytic TRPV4 channels are key molecular sensors of hemodynamic stimuli and that a purinergic, glial-derived signal contributes to flow/pressure-induced adjustments in PA tone. Together our results support bidirectional signaling within the neurovascular unit and astrocytes as key modulators of PA tone.
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http://dx.doi.org/10.1523/JNEUROSCI.4486-14.2015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4444545PMC
May 2015

ATP stimulates rat hypothalamic sympathetic neurons by enhancing AMPA receptor-mediated currents.

J Neurophysiol 2015 Jul 22;114(1):159-69. Epub 2015 Apr 22.

Department of Physiology, Georgia Regents University, Augusta, Georgia

We have previously shown that ATP within the paraventricular nucleus (PVN) induces an increase in sympathetic activity, an effect attenuated by the antagonism of P2 and/or glutamatergic receptors. Here, we evaluated precise cellular mechanisms underlying the ATP-glutamate interaction in the PVN and assessed whether this receptor coupling contributed to osmotically driven sympathetic PVN neuronal activity. Whole-cell patch-clamp recordings obtained from PVN-rostral ventrolateral medulla neurons showed that ATP (100 μM, 1 min, bath applied) induced an increase in firing rate (89%), an effect blocked by kynurenic acid (1 mM) or 4-[[4-Formyl-5-hydroxy-6-methyl-3-[(phosphonooxy)methyl]-2-pyridinyl]azo]-1,3-benzenedisulfonic acid tetrasodium salt (PPADS) (10 μM). Whereas ATP did not affect glutamate synaptic function, α-amino-3-hydroxy-5-methylisoxazole propionic acid (AMPA) receptor-mediated currents evoked by focal application of AMPA (50 μM, n = 13) were increased in magnitude by ATP (AMPA amplitude: 33%, AMPA area: 52%). ATP potentiation of AMPA currents was blocked by PPADS (n = 12) and by chelation of intracellular Ca(2+) (BAPTA, n = 10). Finally, a hyperosmotic stimulus (mannitol 1%, +55 mosM, n = 8) potentiated evoked AMPA currents (53%), an effect blocked by PPADS (n = 6). Taken together, our data support a functional stimulatory coupling between P2 and AMPA receptors (likely of extrasynaptic location) in PVN sympathetic neurons, which is engaged in response to an acute hyperosmotic stimulus, which might contribute in turn to osmotically driven sympathoexcitatory responses by the PVN.
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http://dx.doi.org/10.1152/jn.01011.2014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4507951PMC
July 2015

Neuronal-derived nitric oxide and somatodendritically released vasopressin regulate neurovascular coupling in the rat hypothalamic supraoptic nucleus.

J Neurosci 2015 Apr;35(13):5330-41

Department of Physiology, Georgia Regents University, Augusta, Georgia 30912

The classical model of neurovascular coupling (NVC) implies that activity-dependent axonal glutamate release at synapses evokes the production and release of vasoactive signals from both neurons and astrocytes, which dilate arterioles, increasing in turn cerebral blood flow (CBF) to areas with increased metabolic needs. However, whether this model is applicable to brain areas that also use less conventional neurotransmitters, such as neuropeptides, is currently unknown. To this end, we studied NVC in the rat hypothalamic magnocellular neurosecretory system (MNS) of the supraoptic nucleus (SON), in which dendritic release of neuropeptides, including vasopressin (VP), constitutes a key signaling modality influencing neuronal and network activity. Using a multidisciplinary approach, we investigated vasopressin-mediated vascular responses in SON arterioles of hypothalamic brain slices of Wistar or VP-eGFP Wistar rats. Bath-applied VP significantly constricted SON arterioles (Δ-41 ± 7%) via activation of the V1a receptor subtype. Vasoconstrictions were also observed in response to single VP neuronal stimulation (Δ-18 ± 2%), an effect prevented by V1a receptor blockade (V2255), supporting local dendritic VP release as the key signal mediating activity-dependent vasoconstrictions. Conversely, osmotically driven magnocellular neurosecretory neuronal population activity leads to a predominant nitric oxide-mediated vasodilation (Δ19 ± 2%). Activity-dependent vasodilations were followed by a VP-mediated vasoconstriction, which acted to limit the magnitude of the vasodilation and served to reset vascular tone following activity-dependent vasodilation. Together, our results unveiled a unique and complex form of NVC in the MNS, supporting a competitive balance between nitric oxide and activity-dependent dendritic released VP, in the generation of proper NVC responses.
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http://dx.doi.org/10.1523/JNEUROSCI.3674-14.2015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4381004PMC
April 2015

Brain innate immunity regulates hypothalamic arcuate neuronal activity and feeding behavior.

Endocrinology 2015 Apr 3;156(4):1303-15. Epub 2015 Feb 3.

Department of Physiology (W.L.R., J.E.S.), Medical College of Georgia, Georgia Regents University, Augusta, Georgia 30912; and Helmholtz Diabetes Center (C.-X.Y., Y.G., M.H.T.), Helmholtz Zentrum München and Technische Universität München, Munich 85764, Germany.

Hypothalamic inflammation, involving microglia activation in the arcuate nucleus (ARC), is proposed as a novel underlying mechanism in obesity, insulin and leptin resistance. However, whether activated microglia affects ARC neuronal activity, and consequently basal and hormonal-induced food intake, is unknown. We show that lipopolysaccharide, an agonist of the toll-like receptor-4 (TLR4), which we found to be expressed in ARC microglia, inhibited the firing activity of the majority of orexigenic agouti gene-related protein/neuropeptide Y neurons, whereas it increased the activity of the majority of anorexigenic proopiomelanocortin neurons. Lipopolysaccharide effects in agouti gene-related protein/neuropeptide Y (but not in proopiomelanocortin) neurons were occluded by inhibiting microglia function or by blocking TLR4 receptors. Finally, we report that inhibition of hypothalamic microglia altered basal food intake, also preventing central orexigenic responses to ghrelin. Our studies support a major role for a TLR4-mediated microglia signaling pathway in the control of ARC neuronal activity and feeding behavior.
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http://dx.doi.org/10.1210/en.2014-1849DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4399317PMC
April 2015

A functional coupling between extrasynaptic NMDA receptors and A-type K+ channels under astrocyte control regulates hypothalamic neurosecretory neuronal activity.

J Physiol 2014 Jul 16;592(13):2813-27. Epub 2014 May 16.

Department of Physiology, Medical College of Georgia, Georgia Regents University, Augusta, GA, 30912, USA

Neuronal activity is controlled by a fine-tuned balance between intrinsic properties and extrinsic synaptic inputs. Moreover, neighbouring astrocytes are now recognized to influence a wide spectrum of neuronal functions. Yet, how these three key factors act in concert to modulate and fine-tune neuronal output is not well understood. Here, we show that in rat hypothalamic magnocellular neurosecretory cells (MNCs), glutamate NMDA receptors (NMDARs) are negatively coupled to the transient, voltage-gated A-type K(+) current (IA). We found that activation of NMDARs by extracellular glutamate levels influenced by astrocyte glutamate transporters resulted in a significant inhibition of IA. The NMDAR-IA functional coupling resulted from activation of extrasynaptic NMDARs, was calcium- and protein kinase C-dependent, and involved enhanced steady-state, voltage-dependent inactivation of IA. The NMDAR-IA coupling diminished the latency to the first evoked spike in response to membrane depolarization and increased the total number of evoked action potentials, thus strengthening the neuronal input/output function. Finally, we found a blunted NMDA-mediated inhibition of IA in dehydrated rats. Together, our findings support a novel signalling mechanism that involves a functional coupling between extrasynaptic NMDARs and A-type K(+) channels, which is influenced by local astrocytes. We show this signalling complex to play an important role in modulating hypothalamic neuronal excitability, which may contribute to adaptive responses during a sustained osmotic challenge such as dehydration.
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http://dx.doi.org/10.1113/jphysiol.2014.270793DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4221822PMC
July 2014