bluuebunny/crossref_metadata_2025 huggingface dataset:
Role of orexin neurons in prostaglandin E2‐induced fever and the defense against environmental cooling
Authors: Kuwaki Tomoyuki , Takahashi Yoshiko , Zhang Wei , Sameshima Kohei , Maeda Chiharu , Sakurai Takeshi , Kanmura Yuichi
Published: June 2021

We have previously shown that handling stress‐induced thermogenesis depends on orexin‐containing neurons but not on orexin itself (J Physiol 588: 4117, 2010). In the present study, we examined whether the same is true or not with prostaglandin E2 (PGE2)‐induced fever and the defense against environmental cooling. We used prepro‐orexin knockout mice (ORX‐KO), orexin neuron‐ablated mice (ORX‐AB), and their controls. Increases in PGE2‐induced body temperature were significantly attenuated in ORX‐AB but not in ORX‐KO. PGE2‐induced shivering was also attenuated in ORX‐AB. In wild‐type mice, PGE2 induced fever was attenuated by pretreatment with glutamate receptor antagonists (AP5, CNQX) but not with orexin receptor antagonists (SB334867, OX2 29). ORX‐KO but not ORX‐AB tolerated a prolonged (>60 min, 5ºC) environmental cooling. Both mutants similarly responded to an environmental heating (39ºC). We conclude that orexin neurons play an important role in heat production during stress, fever, and cold exposure, while their contribution is minimal to an alleviation of body heat. Glutamate in orexin neurons probably mediates thermogenic responses.
Neuropeptide Y type 1 receptors are expressed in pre‐autonomic neurons in the hypothalamic paraventricular nucleus
Authors: Clute-Reinig Nicholas M. , Cassaglia Priscila A. , Brooks Virginia L.
Published: June 2021

We recently demonstrated that exogenous and endogenous Neuropeptide Y (NPY) decreases sympathetic nerve activity and baroreflex function via an action on Y1 receptors (Y1R) in the hypothalamic paraventricular nucleus (PVN). However, whether NPY acts directly on presympathetic neurons projecting to the rostral ventrolateral medulla (RVLM) is unknown. To test this hypothesis, we used immunofluorescence to determine if Y1R are present in PVN neurons that project to RVLM, identified by prior RVLM microinjection of the retrograde tracer cholera toxin subunit B (CTb, 0.25%; List Laboratories). NPY Y1R‐immunoreactivity (ir) (1:100; Santa Cruz; n=4) was found in scattered neurons in multiple PVN parvocellular subdivisions, including the dorsal, medial, lateral, and ventrolateral subnuclei. NPY Y1R‐ir was also prominent in the PVN posterior magnocellular subnucleus. In separate experiments, NPY Y1R‐ir colocalized with vasopressin‐ir (1:800; gift of H. Gainer) in neurons in this region. While no colocalization of NPY Y1R‐ir with CTb‐ir (1:1000; AbCam) was observed in the more rostral PVN (−1.6 mm from bregma), a significant fraction (16–38%) of NPY Y1R neurons in caudal PVN regions (−1.8 to −2.1 mm from bregma) co‐expressed CTb. In summary, NPY Y1R are expressed in PVN neurons projecting to the RVLM, suggesting that NPY acts directly on these neurons to inhibit sympathetic nerve activity and baroreflex regulation. Supported in part by HL088552.
Isolating a role for deep breathing in reducing activation of stress‐related limbic circuits
Authors: Noble Donald James , Sawchuk Michael , O'Neill Brannan , Hochman Shawn
Published: June 2021

Meditation and yoga techniques involving deep breathing (DB) have shown promise for treating a variety of cardiorespiratory and stress‐related disorders, but to date no studies have mechanistically investigated the role of DB in conferring these benefits. The brainstem nucleus of the solitary tract (NTS) is the major relay nucleus for all vagal afferent input including stretch‐sensitive pulmonary afferents. NTS projects densely to the central nucleus of the amygdala (CeA) and the paraventricular nucleus of the hypothalamus (PVN), both implicated in the response to threatening or stressful stimuli. As Fos immunolabeling can be used to assay stress‐evoked neuronal activation, we hypothesized that, via NTS projection pathways, DB would suppress Fos expression in these circuits. In anesthetized, mechanically ventilated rats, we compared the number of Fos‐positive neurons following a two hour period of DB vs. normal breathing. Preliminary results support reduced Fos expression following DB in both CeA (112 vs. 139 Fos+ cells per section) and PVN (69 vs. 108 Fos+ cells per section). Further studies are aimed at verifying these preliminary findings and identifying mechanisms that link DB to stress circuit deactivation. Funded by Emory University's Scholars Program in Interdisciplinary Neuroscience Research.
Microglial‐neuronal interactions in the paraventricular nucleus (PVN): a potential mechanism underlying neurogenic hypertension
Authors: Shi Peng , Desland Fiona A. , Zhou Guannan , Zhou Fuwen , Roper Steven N. , Dong Ying , Shan Zhiying , Grobe Justin L. , Raizada Mohan K. , Sumners Colin
Published: June 2021

Neuroinflammation has been suggested to contribute to neurogenic hypertension. Microglia, the resident immune cells in the CNS, are the major source of proinflammatory cytokines (PIC). We and others have shown microglial activation and elevated activity of the renin‐angiotensin system (RAS) in the hypothalamic PVN in hypertension. Therefore we hypothesized that RAS components (prorenin and angiotensin [Ang] II) activate microglia, and that microglial activation increases PVN neuronal firing. We found that Ang II and prorenin significantly increased the expression of PIC mRNA in microglia cultured from rat hypothalamus. These increases were respectively abolished by corresponding receptor blockers. Next, we activated microglia with LPS, and tested the effect on sympathetic PVN neuronal firing. Whole cell patch‐clamp recordings were made on identified PVN neurons projecting to the vasomotor neurons in the brain stem. LPS caused a significant increase in neuronal firing frequency. These increases were abolished by minocycline, an inhibitor of microglial activation. Moreover, LPS significantly increased PIC mRNA expression in microglia. PIC receptors were highly expressed on the PVN neurons. In summary we show that RAS components activate microglia, causing increased production of PIC, which leads to the increased firing frequency of PVN neurons. AHA11SDG6770006 to PS; HL098276 to JLG; HL076803 to CS.
ACE2 Shedding: A New Mechanism For Neurogenic Hypertension
Authors: Xia Huijing , Sriramula Srinivas , Scroggin Melissa , Chhabra Kavaljit , Lazartigues Eric
Published: June 2021

Brain ACE2 expression is reduced during neurogenic hypertension and ACE2 gene therapy blunts high blood pressure (BP). ADAM17 stimulates ACE2 shedding, while calmodulin binding is protective. To assess the role of ACE2 shedding in the development of hypertension, somatic and soluble ACE2 activities (arbitrary units, AU) were measured in brain tissue (AU/mg/min) and microdialysed cerebrospinal fluid (CSF; AU/ml/min) of wildtype (NT) and transgenic mice (n=7/group) overexpressing hACE2 in the brain (hA), at baseline and during DOCA‐salt hypertension. Basal ACE2 activity was ~5‐fold higher in brain tissue (2335±713 vs. 482±57) and 2‐fold higher in the CSF of hA (38±3 vs. 17±1) vs. NT, while BP (telemetry) was similar (104±3 vs. 106±2 mmHg). In NT+DOCA, the BP increase (146±3 mmHg, P<0.01 vs. NT) paralleled a decrease in ACE2 activity in brain tissue (243±74) and an increase in the CSF (29±3). In hA+DOCA mice, with blunted hypertension (131±4 mmHg, P<0.05 vs. NT+DOCA), ACE2 activity remained unaltered in either brain homogenates (1469±991) or CSF (35±1). ADAM17 activity (AU) was increased in NT+DOCA (6.3±0.4 vs. 3.9±0.2) but not in hA+DOCA (2.7±0.01 vs. 4.5±0.08) mice, while calmodulin‐bound ACE2 was only reduced in NT+DOCA (−67%). Our data suggest that ACE2 shedding is involved in hypertension and that maintenance of somatic ACE2 activity levels in the brain is critical for the prevention of hypertension. (HL093178)
Low‐dose aspirin augments carotid‐cardiac baroreflex sensitivity during concurrent muscle mechanoreflex and metaboreflex activation in humans
Authors: Drew Rachel , Muller Matthew D. , Herr Michael , Blaha Cheryl , Mast Jessica , Nicklas Todd , Sinoway Lawrence I.
Published: June 2021

Metabolite sensitization of muscle mechanoreceptors alters carotid baroreflex (CBR) sensitivity but the role of thromboxane (TX) A2 is unknown. Therefore, we examined the effect of inhibiting TX A2 via low‐dose aspirin on carotid‐cardiac (CBR‐HR) and carotid‐vasomotor (CBR‐MAP) sensitivity during mechanoreflex and metaboreflex activation in humans. 9 subjects performed 1.5mins of 70% MVC isometric calf exercise, 3.5mins of post‐exercise circulatory occlusion, then 3mins of occlusion plus passive calf stretch. This occurred twice after 7 days of either low‐dose aspirin (Asp) or placebo (Pla). Asp reduced baseline TX B2 by ~90% (p<0.05). Maximal gains at rest were similar in all trials for CBR‐HR and CBR‐MAP (−0.44±0.1 vs. −0.37±0.1 b.min−1.mmHg−1 and − 0.23±0.0 vs. −0.20±0.0 mmHg.mmHg−1 for Asp and Pla, respectively). CBR‐HR maximal gain during stretch with Asp was significantly higher than with Pla (−0.64±0.1 vs. −0.33±0.1 b.min− 1.mmHg−1, respectively) (p<0.05). CBR‐MAP maximal gain during stretch with Asp was similar to with Pla (−0.29±0.0 vs. −0.33±0.1 mmHg.mmHg−1, respectively). These results suggest that low‐dose Asp augments CBR‐HR but not CBR‐MAP sensitivity during concurrent mechanoreflex and metaboreflex activation in humans. This could be due to increased cardiac vagal activity caused by reduced TX sensitization of mechanoreceptors. Supported by P01 HL096570 (LIS); UL1 RR033184 (LIS).
A single, acute bout of yogic breathing reduces arterial catecholamines and cortisol
Authors: Wehrwein Erica A , Johnson C P , Charkoudian N , Wallin B G , Joyner M J
Published: June 2021

Yoga is thought to reduce stress; however, few studies have quantified plasma levels of stress hormones. Yoga includes physical postures, meditation, and pranayama (controlled breathing) therefore it is difficult to determine which dose and combination of these components is most beneficial. We hypothesized that a single 15 min bout of 6 breaths per minute would be sufficient to cause a reduction in stress hormones. Ten young healthy males (age 18–35, n=10) were instrumented with brachial arterial catheter for blood draws and respiration was monitored continuously using a double pneumobelt. Blood draws were taken after 15 min of supine rest with baseline ventilation and again after 15 min of slow paced breathing. Average baseline supine respiratory rate was 12.1 ± 0.5 breaths/min and during slow breathing it was 5.5 ± 0.4 breaths/min (p<0.05). After 15 min of slow breathing in the supine position there was a significant reduction in arterial norepinephrine (173.9 ± 15.5 vs 135.6 ± 42.9, p=0.002), cortisol (8.2 ± 0.7 vs 7.3 ± 0.59, p=0.002), and adrenocorticotrophic hormone (ACTH, 15.5 ± 2.4 vs 13.4 ± 2.2, p=0.009). As well, there was a trend for reduction in arterial epinephrine (82 ±28.2 vs 42.7 ± 9.7, p=0.15). These changes are consistent with a reduction in sympathetic nerve activity and a blunting of cortisol. In summary, acute yogic breathing significantly reduces stress hormones.
Endogenous ATP and the Modulation of Sympathetic Vasoconstriction in Contracting Skeletal Muscle of Humans
Authors: Kirby Brett S , Richards Jennifer C , Crecelius Anne R , Garcia Leora J , Luckasen Gary J , Larson Dennis G , Dinenno Frank A
Published: June 2021

Exogenous ATP administration in humans can blunt sympathetic vasoconstriction in a dose‐dependent manner, similar to what occurs within contracting skeletal muscle (sympatholysis). Here, we tested the hypothesis that endogenous ATP increases during exercise and is associated with the magnitude of sympatholysis in humans (N = 9). We measured deep venous plasma [ATP] and forearm blood flow (Doppler ultrasound), and calculated ATP effluent (FBF x [ATP]) at rest and during 5 and 15% MVC rhythmic handgrip exercise (EX), before and during superimposed lower body negative pressure (LBNP; −40 mmHg). LBNP‐induced vasoconstriction was similar during 5% EX vs rest (ΔFBF = −25 ± 2% vs −33 ± 5%), whereas this was blunted during 15% EX (−4 ± 2%). LBNP at rest did not alter [ATP] nor ATP effluent. During the 5% EX trial, [ATP] and ATP effluent were 44 ± 7, 70 ± 9, and 50 ± 10 nmol/L and 1 ± 1, 7 ± 1, and 4 ± 1 nmol/min at rest, EX, and EX + LBNP, respectively. During the 15% EX trial, [ATP] and ATP effluent were 47 ± 5, 79 ± 11, and 71 ± 8 nmol/L and 1 ± 1, 16 ± 3, and 14 ± 1 nmol/min at rest, EX, and EX + LBNP, respectively. We conclude that endogenous ATP release is increased during exercise and must remain elevated during sympathetic stimulation in order to blunt the vasoconstrictor response. Further, the contribution of intravascular ATP to vascular control may ultimately be dependent on an interaction between blood flow and [ATP].

Supported by NIH HL102720
Regulation of ventral surface chemoreceptors by purinergic signaling
Authors: Wenker Ian Christopher , Takakura Ana , Moreira Thiago , Mulkey Daniel Kent
Published: June 2021

A brainstem region called the retrotrapezoid nucleus (RTN) contains a population of CO2/H+‐sensitive neurons that appear to function as important central chemoreceptors (i.e. CO2/H+ sensors). Evidence also indicates that CO2/H+‐evoked ATP release contributes to RTN chemoreception. The goals of this study are to determine the extent to which ATP contributes to RTN chemoreception both in vivo and in vitro, and determine whether purinergic drive to chemoreceptors relies on extracellular Ca2+ or gap junction hemichannels. In vivo, phrenic nerve recordings of respiratory activity in adult rats show that bilateral injection of P2‐ receptor antagonists decreased the ventilatory response to CO2 by 30%. Likewise, in vitro, recordings from functionally identified RTN chemoreceptor neurons in brainstem slices from rat pups (p7–p12), we show P2‐receptor antagonists decreased the firing rate response of RTN neurons to CO2 also by 30%. In the slice, this purinergic drive to breathe did not increase with temperature and was retained in low [Ca2+]o. Conversely, gap junction blockers carbenoxolone (100 μM) and cobalt (500 μM) decreased neuronal CO2/H+‐sensitivity by an amount similar to P2‐receptor antagonists. These results support the possibility that astrocytes but not neurons mediate CO2/H+‐evoked ATP release possibly by a mechanism involving gap junction hemichannels.
Opioid μ‐receptors in the rostral medullary raphe modulate hypoxia‐induced hyperpnea in unanesthetized rats
Authors: Dias Mirela Barros , Nucci Tatiane B , Branco Luiz Guilherme Siqueira , Gargaglioni Luciane H
Published: June 2021

It has been suggested that the medullary raphe (MR) plays a key role in the physiological responses to hypoxia. As the rostral MR (rMR) expresses an extensive population of opioid μ‐receptors, we studied the putative role of opioid μ‐receptors in the rMR region on ventilation in normal and 7% hypoxic conditions. We measured pulmonary ventilation and the body temperatures of male Wistar rats before and after the selective opioid μ‐receptor antagonist CTAP (D‐Phe‐Cys‐Tyr‐D‐Trp‐Arg‐Thr‐Pen‐Thr‐NH2, cyclic, 0.1 μg/0.1 μL) was microinjected into the rMR during normoxia or after 60 minutes of hypoxia. The animals treated with intra‐rMR CTAP exhibited an attenuation of the ventilatory response to hypoxia (430 ± 86 mL kg−1 min−1) compared to the control group (790 ± 82 mL kg−1 min−1) (P<0.05) due to a reduced tidal volume (3.0 ± 0.41 versus 6.1 ± 0.6 ml kg−1; P<0.05). During hypoxic recovery, the ventilation remained reduced in the CTAP intra‐rRM group. No differences in the Tb were observed among groups during hypoxia. These data suggest that opioids acting on μ‐receptors in the rMR exert an excitatory modulation of hyperventilation induced by hypoxia.

Financial support: FAPESP, CNPq, PRONEX.
Functional expression of the extracellular Ca2+‐sensing receptor in pulmonary neuroepithelial bodies
Authors: Lembrechts Robrecht , Brouns Inge , Schnorbusch Kathy , Pintelon Isabel , Kemp Paul J , Timmermans Jean-Pierre , Riccardi Daniela , Adriaensen Dirk
Published: June 2021

The extracellular Ca2+‐sensing receptor (CaR) is a multimodal sensor that allows cells to detect and respond to changes in the extracellular Ca2+ concentration [Ca2+]o. Besides its role in Ca2+ homeostasis, the CaR has been shown in various specialized cells, suggesting its involvement in multiple cellular functions. Prenatal CaR expression was detected in mouse airway epithelium between E11,5 and E16,5 and was shown to play a role in airway branching morphogenesis. In postnatal lung tissue, the CaR so far has not been reported. Neuroepithelial bodies (NEBs) are densely innervated groups of neuroendocrine airway epithelial cells, believed to represent complex sensory receptors that are ideally placed to detect changes in the microenvironment. Immunostaining of postnatal mouse lungs showed a selective CaR expression on the basolateral membrane of NEB cells. Confocal Ca2+ imaging of NEBs in live mouse lung slices revealed that NEBs respond with a Ca2+ rise both to changes in [Ca2+]o and to various known CaR agonists (spermine, neomycin, calcimimetic R‐568). All responses were blocked by Calhex, a calcilytic CaR antagonist. The presented expression and proven functionality of the CaR in postnatal NEBs offer interesting perspectives for further unraveling NEB cell physiology.

Support: IWT fellowship SB/81162 (RL); FWO grants G.0081.08 (DA) and G.0589.11N (DA, JPT); UA grants GOA BOF 2007 (DA) and KP BOF 2006 (IB)
Gastric CO2 output via the esophagus increases during systemic hypercapnia in anesthetized cat
Authors: Held Heather E. , Dean Jay B. , Lindsey Bruce G. , Pitts Teresa C. , Rose Melanie J. , Mortensen Ashley , Nicholas Justine N. , Baekey David M. , Davenport Paul W. , Bolser Donald C.
Published: June 2021

The stomach extracts arterial CO2 during respiratory acidosis to produce HCl & HCO3−, which react in the lumen to re‐form CO2. This study tested the hypothesis that hypercapnia stimulates gastric CO2 removal via the esophagus. Tracheal and esophageal CO2 (PEsCO2) and flows were measured in 13 anesthetized, pyloric occluded cats. Tracheal and gastric CO2 output (V̇ GCO2) and gastric minute ventilation (V̇ G) were calculated during tracheal air breathing for 30min (Control) and CO2 breathing: 10%CO2 in air (1hr), recovery in air (30min), followed by 6×10min alternating paired bouts of 10%CO2 and air (10CO2, 10air). CO2 breathing increased PEsCO2 above control 77% after 1hrCO2 and 123% during 10CO2. Coughing revealed more CO2 was present in the stomach: PEsCO2 increased 134% (in air) and 115% (after 1hrCO2) compared to pre‐cough PEsCO2. V̇ GCO2 ranged from 0–15% (X̄=2.5%) of total CO2 output during 1hrCO2. V̇ G was 27% higher during 10CO2 versus 10air. Relative to control, V̇ G increased 95% after 1 hrCO2 and 148% after the first bout of 10minCO2. The results show a dynamic, supplemental CO2‐eliminating response by the gastroesophageal system during systemic hypercapnia (NIH HL89104, HL103415 & ONR).
Identification of lung priming area in frog brainstem, adjacent to the lung oscillator, distinct from the buccal oscillator: homologous to RTN/pFRG?
Authors: Baghdadwala Mufaddal , Duchcherer Maryana , Wilson Richard J.A.
Published: June 2021

Data from mammals and frogs suggest tetrapods have an anatomically‐discrete inspiratory oscillator, necessary and sufficient for expression of the powerstroke phase of lung inflation (preBötzinger Complex in mammals; Lung Area in frogs). In mammals a second Pre‐Inspiratory / late‐phase expiratory oscillator has been hypothesized; this oscillator likely resides in the retrotrapezoid/parafacial respiratory group (RTN/pFRG) and is “conditional”, recruited during high respiratory demand. In isolated frog brainstems, lung bursts often have a powerstroke and priming phase. Though the priming phase is always observed with a subsequent powerstroke, the priming phase is “conditional”: powerstrokes can occur without a priming phase.

Here we identify a region of the frog brainstem necessary and sufficient for the priming phase. This priming area is adjacent to the area that produces the powerstroke (between CNVII‐IX) and distinct from the buccal area (caudal of CN X). Using AMPA microinjections to locally excite the priming area, we demonstrate ectopic priming bursts uncoupled from powerstroke bursts. We propose the priming and powerstroke areas are homologous to the PreI and PreBötC, respectively. We further postulate that rhombomeric differences may be explained by a “gene cassette” for air breathing rhythmogenesis, turned on in different rhombomeres in different clades. Work funded by NSERC.
Stimulus‐specific cerebrovascular dysfunction in humans with metabolic syndrome
Authors: Harrell John W , McKenna Edward J , Linstroth Lee A , Morgan Barbara J , Schrage William G
Published: June 2021

Metabolic syndrome (MetS) and reduced cerebrovascular reactivity are each associated with increased risk of stroke in humans. Animal models of MetS demonstrate reduced cerebral artery dilation, but cerebrovascular function in humans with MetS remains untested. We tested the hypothesis MetS humans would have reduced cerebrovascular reactivity to hypoxia and hypercapnia. Blood pressure (BP), arterial oxygen saturation (SpO2), expired carbon dioxide (PETCO2), and middle cerebral artery blood velocity (CBV, transcranial Doppler ultrasound) were measured in MetS adults (n=9, 37 ± 3 yr) and healthy controls (n=10, 32 ± 2 yr). Cerebral conductance index (CVCI = CBV / BP) was calculated to normalize CBV for higher BP in MetS adults. Cerebral vasodilation (ΔCVCI) to hypoxia (SpO2 = 90% and 80%) and hypercapnia (+10 mmHg PETCO2) were assessed. MetS adults exhibited markedly lower ΔCVCI to 90% SpO2 (1 ± 2 vs 6 ± 2 cm s−1 mmHg−1, p<0.05) and 80% SpO2 (5 ± 2 vs 15 ± 3 cm s−1 mmHg−1, p<0.05). In contrast, MetS adults displayed preserved cerebrovascular reactivity to hypercapnia (ΔCVCI = 18 ± 2 vs 20 ± 2 cm s−1 mmHg−1). We conclude younger MetS adults exhibit markedly reduced stimulus‐specific cerebrovascular reactivity during hypoxia. Poor hypoxia‐mediated cerebral dilation may contribute to the increased risk and severity of cerebrovascular disease in MetS humans. Support: NIH HL091397 & AHA 11PRE7390038
Activation of TASK‐2 by hypoxia: is it via ROS, AMPK or H2S?
Authors: Martin Elizabeth A , Papreck Justin R , Kim Donghee
Published: June 2021

The respiratory depression produced by hypoxia has been reported to involve TASK‐2 in chemoreceptor neurons of the brainstem. We tested the effect of hypoxia on TASK‐2 expressed in HeLa cells. In cell‐attached patches, hypoxia reversibly increased TASK‐2 activity. Sodium cyanide and FCCP augmented TASK‐2 activity that was not increased further by hypoxia. In inside‐out patches, hypoxia failed to activate TASK‐2. The roles of reactive oxygen species, AMP kinase and H2S as hypoxic signals were investigated. In inside‐out patches, H2O2 (10 μM‐326 mM) and xanthine/xanthine oxidase (X/XO) mixture showed no significant effect on TASK‐2 activity. H2O2 (>30 mM), however, activated TASK‐1 and TASK‐3. In outside‐out patches, extracellular H2O2 showed no effect but X/XO mixture elicited a stong activation of TASK‐2. Perfusion of cell‐attached patches with 1 mM AICAR or 100 μM A792662 (activators of AMP kinase) did not cause activation of TASK‐2. NaHS (100–200 μM), a H2S donor, produced an increase in TASK‐2 activity in some cell‐attached patches but not in excised patches. Hypoxia‐induced activation of TASK‐2 was not present when cells were pretreated with DL‐propargylglycine (inhibitor of cystathionine‐γ‐lyase) and aminooxyacetic acid (inhibitor of cystathionine‐β‐synthase). These findings suggest that in HeLa cells, TASK‐2 is a hypoxia‐sensitive K+ channel that is regulated via H2S and possibly via ROS as well.
Endogenous Hydrogen Sulfide in Carotid Bodies Correlates with the Initiation of Hypertension in Spontaneously Hypertensive Rats (SHR)
Authors: Lu Yongjun , Whiteis Carol A , Chapleau Mark W , Abboud Francois M
Published: June 2021

Hydrogen sulfide (H2S) is a new gaseous mediator of carotid chemoreceptor responses to hypoxia. Its production is determined by cystathionine γ lyase (CSE), cystathionine β synthetase (CBS), and 3‐mercaptopyruvate sulfurtransferase (3MST). Since CSE deletion severely impaired mouse carotid body response to hypoxia, we hypothesized that genetic overexpression of the H2S enzymes occurs in carotid bodies of SHR and explains their chemoreceptor hypersensitivity that contributes to hypertension. Carotid bodies were removed from anesthetized male SHR (and their normotensive control WKY rats) when they were young and prehypertensive at 4 weeks, when hypertension was established (at 12 weeks), and when hypertension had been long standing (at 38 weeks). The mRNA expression of CSE, CBS and 3MST was over 100 fold higher (p<0.01) in SHR than in WKY at 4 weeks of age. The expression decreased in SHR to levels that were only slightly higher than WKY at 12 and 38 weeks. The enzyme expression in WKY remained relatively low and stable over time. Expressions in brains of SHR and WKY were comparable. The results indicate a marked overexpression of H2S in carotid bodies of SHR during the critical prehypertensive stage. We suggest that this important molecular determinant of chemoreceptor hypersensitivity causes an increase in sympathetic nerve activity, and induces the initiation and development of hypertension. (HL14388)
Altered urinary excretion of allantoin and purine catabolites in neonates with necrotizing enterocolitis
Authors: Plank Megan , Slater Laurel , Asmerom Yayesh , Angeles Katherine R , Mannoia Kristyn , Boskovic Danilo , Gollin Gerald , Angeles Danilyn
Published: June 2021

Necrotizing enterocolitis (NEC) is a complex gastrointestinal disease that affects premature infants. We hypothesize that biochemical markers of hypoxia and oxidative stress may be predictive of the different stages of NEC. Urine specimens were collected from premature infants over day 2 and 3 of life. The concentrations of purines and allantoin were determined using HPLC and GCMS. All concentrations were taken as a ratio to creatinine. The presence and clinical severity of NEC was determined at week 2 of life using the Bell staging system. Compared to control subjects (n=16), we found allantoin and purine concentrations to be significantly higher in neonates who developed NEC. Our data revealed that neonates who developed NEC (n=8), had NEC scare (n=4), or died (n=6) had significantly elevated uric acid levels. Allantoin levels were found to be highest in neonates who died, increased over time in neonates who developed NEC, began high and decreased over time in neonates with NEC scare, and were lowest in control neonates. These preliminary data indicate that uric acid may be useful in identifying acutely ill neonates while allantoin appears to be most predictive of NEC. These data suggest that oxidative stress may play a significant role in the pathophysiology of NEC.
Corticosterone (CS) modulation of miniature excitatory postsynaptic currents is altered following exposure to CIH
Authors: Fan Yun , Mifflin Steve
Published: June 2021

CS is released after stress and can bind to intracellular receptors in the brain, which in activated form function as transcription factors. We tested the effect of CS on AMPA‐receptor–mediated transmission in NTS neurons from normoxic rats and in rats exposed to CIH ( 9% oxygen for 3min every 10 min, 8 h per day for 7 days). Whole‐cell recordings of second‐order NTS neurons receiving arterial chemoreceptor afferent inputs, labeled with DiA on the carotid body, were obtained in a brain slice. To focus on delayed, gene‐mediated effects of the hormone miniature postsynaptic excitatory currents (mEPSCs) were measured at least 1 h after 20min superfusion of the slice with 100 nM CS. In normoxic rats, CS increased the amplitude (control 16.8±1.0pA, n=10, CS 22.1±1.3pA, n=12, p<0.01) but not the interval (control 0.92±0.15s, n=11, CS 0.77±0.09s, n=11, p>0.05) of mEPSCs. CS had a much different effect in rats exposed to CIH. CS did not increase the amplitude of mEPSCs in the CIH exposed rats from 19.1±1.9pA to 20.1±1.7pA (n=10, p>0.05). Following exposure to CIH, CS reduced mEPSC interval from 1.6±0.4s to 0.75±0.1s (n=10, p<0.05). The present results show that in normoxic rats CS induces a delayed post‐synaptic enhancement of AMPA‐receptor–mediated responses. In rats exposed to CIH, CS does not induce any further post‐synaptic increase in AMPA current but rather increases the probability of glutamate release.
Intracellular recordings of respiratory and pre‐sympathetic neurons in the ventrolateral medulla of rats submitted to chronic intermittent hypoxia
Authors: Moraes Davi JA , Zoccal Daniel B , Machado Benedito H
Published: June 2021

Sympathetic activity is increased during late‐expiratory (late‐E) phase in CIH rats. Electrophysiological properties of medullary respiratory and pre‐sympathetic neurons were evaluated in CIH rats. At 11th day, using working heart brainstem preparation, we recorded respiratory and sympathetic nerves simultaneously with intracellular recordings of medullary respiratory and pre‐sympathetic neurons. In CIH rats the decrease in the frequency discharge of Bötzinger post‐inspiratory [post‐I, (n=28)] neurons reduced the post‐I activity of central vagus nerve (n=10). The overactivities of augmenting expiratory [aug‐E, (n=55)] and pre‐Bötzinger pre‐inspiratory [pre‐I, (n=30)] neurons increased the late‐E abdominal (n=28) and pre‐I hypoglossal (n=20) nerves activities. Due to these changes, the frequency discharge of pre‐sympathetic neurons (n=27) and thoracic sympathetic activity (n=30) were enhanced time‐locked with late‐E. However, CIH produced changes in the input resistance and excitability, independent of synaptic transmission only in the pre‐I (p<0.05; n=15) and post‐I (p<0.05; n=17) neurons. These data support the concept that CIH leads to changes in the intrinsic properties of pre‐I and post‐I respiratory neurons, which may contribute to the observed increase in the sympathetic activity in this experimental model. Supported by FAPESP and CNPQ.
Real‐Time Dynamics of Translation
Authors: Puglisi Joseph Daniel , Chen Jin , Kornberg Guy , O'Leary Sean , Petrov Alexey , Tsai Albert
Published: June 2021

Translation of proteins by the ribosome represents the endpoint of gene expression and is highly regulated. The structures of ribosomal complexes represent a triumph of structural biology. Yet these structures lack a time axis: dynamic measurements are needed to understand the mysteries of translation. Here we present our broad efforts to develop and use single‐molecule fluorescence methods to track translation in real time. Using fluorescent labels on transfer RNAs, factors, mRNAs and ribosomal subunits, we have mapped conformational and compositional dynamics of the translational machinery during protein synthesis. Our focus has recently been on the dynamics of initiation, by which translational reading frame is established.
Sexually dimorphic expression of S1PR2 as candidate for susceptibility to CNS autoimmunity
Authors: Cruz-Orengo Lillian , Basak Sarah Alison , Dorsey Denise , Crosby Seth Daniel , Klein Robyn Sue
Published: June 2021

Multiple Sclerosis (MS) is characterized by blood brain barrier (BBB) dysfunction with consequent autoimmune attack of oligodendrocytes. MS exhibits both a high sex bias and a sexually dimorphic progression, affecting more women than men whom exhibiting the relapsing‐remitting (RR) form. Specimens from MS patients and mice with EAE, exhibit relocation of CXCL12 to the lumenal surfaces of CNS vessels. This alteration in CXCL12 expression impacted on the infiltration of immune cells enhancing EAE severity. Here we show that naïve female SJL mice, which develop RR–EAE, exhibit lumenal CXCL12 expression within white matter microvasculature, while naïve male SJL mice, which do not develop RR‐EAE, exhibit ablumenal CXCL12 expression. Other experiments showed significantly increased expression of the Sphingosine 1‐Phosphate Receptor 2 (S1PR2), in the cerebella of female SJL mice compared with male SJL cerebella and cortices for both genders. Antagonists of S1P receptors have previously been shown to be effective targets for the treatment of EAE and MS, mainly through immunosuppressive mechanisms, inducing lymphopenia in vivo. Our preliminary data, however, suggests that an alternative receptor, S1PR2, may be a specific target for manipulation of BBB biology during CNS autoimmunity. In addition, our data reveals that white matter expression of this receptor exhibits sexual dimorphism, which suggests that it might be relevant for RR‐MS neuropathogenesis.

Research support: National Institute of Neurological Disorders and Stroke, 1F32NS074824‐01 (LCO), National Multiple Sclerosis Society, RG3982A‐2 (RSK) and Dana Foundation (RSK).
Does estrogen have immunomodulatory effects on pregnant women diagnosed with Multiple Sclerosis
Authors: Brown Amber , McNair Kimberly , Rawson Reneé , Sibley Turner , Raynes Edilberto
Published: June 2021

Multiple sclerosis is an autoimmune disease affecting the central nervous system, specifically demyelination of the axon sheaths. It is commonly diagnosed in women in their ideal reproductive ages. Current evidence has proposed a decrease in relapse rates in women diagnosed with multiple sclerosis during their third trimester of pregnancy with an increase in relapse rates post‐partum. We hypothesize that interventions using hormonal therapy could provide decreased exacerbations of the clinical manifestations of relapse‐remitting multiple sclerosis. This would lead to an increased quality of life and help increase the efficacy of therapeutic intervention. Using the Oxford Centre of Evidence‐Based Medicine, a systematic review of literature was completed. Keywords for the literature included multiple sclerosis, pregnancy, hormones, estrogen, progesterone, relapse rates, post‐partum, and experimental autoimmune encephalomyelitis. Based on the evidence gathered, combination hormonal therapy, including forms of estrogen, produces a decrease in multiple sclerosis disease activity, resulting in benefits for the women diagnosed with relapse‐remitting multiple sclerosis. We concluded that hormonal therapy would benefit significantly in the reduction of exacerbations in women diagnosed with relapse‐remitting multiple sclerosis leading to an increased quality of life.
Mechanisms of Water Deprivation‐Induced Enhancement of Glutamatergic Activity in the Hypothalamic Paraventricular Nucleus (PVN)
Authors: Bardgett Megan , Baumgartner Anne D. , Calderon Alfredo S. , Toney Glenn M.
Published: June 2021

Evidence indicates that NMDA receptor “tone” in the PVN of 48 h water deprived (WD) rats is increased compared to euhydrated (EU) controls. Consistent with this, acute blockade of PVN NMDAR reduces sympathetic nerve activity (SNA) & mean arterial pressure (MAP) in WD rats, but has little effect in EU rats. Wet weight & water content of brain were determined to be nearly identical in EU & WD rats (n=5, P>0.9), suggesting that increased PVN NMDAR tone in WD rats is unlikely to result from increased L‐glutamate concentration arising from brain dehydration and ECF volume contraction. Next, we determined in anesthetized rats that bilateral PVN injection of the glutamate transporter blocker TBOA (250 pmol in 50 nl) evoked greater increases of renal SNA & MAP in EU than WD rats (RSNA: 26±4 % vs 15±2 %, P<0.05; MAP: 15±1 mmHg vs 7±1 mmHg, P<0.001). In whole cell voltage‐clamp recordings (Vhold: −60 mV) from PVN‐RVLM neurons in brains slices, TBOA (100 μM) likewise elicited greater inward current (P<0.05) in the EU (−144±21 pA, n=3) than WD (−40±10 pA, n=5) group. Both in vivo & in vitro TBOA evoked responses were abrogated by NMDAR blockade with AP5. We conclude that greater functional NMDAR tone in the PVN of WD rats may not simply reflect increased glutamatergic synaptic input, but instead involve increased PVN neuronal responsiveness to L‐glutamate or reduced ongoing L‐glutamate uptake, or both. Support: HL102310 & HL 088052 (GMT)
Upregulation of NMDA receptors in Spinal Cord Dorsal Horn Contributes to Diabetic Neuropathic Pain
Authors: Wang Xiu-Li , Zhang Ying-Ze , Liu Fei-Fei , Cao Qian-Qian , Guo Yue-Xian
Published: June 2021

Diabetic neuropathic pain is a common complication in patients with diabetes. Spinal N‐methyl‐D‐aspartate (NMDA) receptors play critical role in neuropathic pain. In this study, we determined that role of enhanced NMDA receptors function in diabetic neurophathic pain. Diabetic neuropathy was induced by a single injection of streptozotocin (STZ, 50 mg/kg, intraperitoneal). Blood glucose level and paw withdraw threshold (PWT) were measured 3 weeks after STZ treatment to verify diabetic neuropathy. Evoked NMDA excitatory postsynaptic currents (NMDAR‐EPSCs) were recorded in lamina II neurons by using whole‐cell voltage‐clamp patch in rats treated with STZ and vehicle. The blood glucose level was significantly higher in STZ‐treated rats than in vehicle‐treated rats. The PWT in STZ‐treated rats was significantly lower than that in vehicle group. Immunohistochemical staining revealed that the number of NR1 and NR2B‐positive neurons were significantly higher in STZ‐treated rats than in vehicle‐treated rats. Furthermore, the amplitude of evoked NMDA‐EPSCs in spinal neurons was significantly higher in STZ‐treated rats than vehicle‐treated rats. Application of 50 μM AP5 completely blocked the evoked NMDAR‐EPSCs. Taken together, these data suggest that upregulation of NMDA receptors in the spinal cord dorsal horn contributes to the diabetic neuropathic pain
Proneurotrophins mediate peri‐infarct sympathetic denervation following myocardial infarction
Authors: Parrish Diana C , Lorentz Christina U , Habecker Beth A
Published: June 2021

Altered sympathetic innervation of the heart is a major contributor to arrythmogenesis following myocardial infarction (MI). Changes to the innervation include denervation of peri‐infarct myocardium. Activation of the p75 neurotrophin receptor leads to axon degeneration, and we asked if the denervation of peri‐infarct myocardium was caused by p75 activation. Sympathetic innervation density was quantified by tyrosine hydroxylase immunohistochemistry in 12–18 week old p75 −/− and wild‐type control mice (C57Bl6/J) that were subjected to sham or ischemia‐reperfusion surgery. By 24 hours post‐MI, the peri‐infarct zone was largely denervated in wild‐type mice, and this denervation persisted up to 3 days. In contrast, peri‐infarct denervation was absent in p75 −/− mice. Both pro‐ and mature neurotrophins bind the p75 neurotrophin receptor. We quantified the levels of mature and proneurotrophins in the left ventricle of wild‐type mice 24 hours following MI by western blotting. Pro‐brain derived neurotrophic factor (proBDNF) was elevated in the infarcted left ventricle compared to sham‐operated controls, while mature BDNF was not detected. Both pro‐ and mature nerve growth factor (NGF) were detected in the infarcted left ventricle, with proNGF being the more abundant species. Application of proBDNF‐conditioned media to sympathetic ganglia explants resulted in reduced axon length. These data suggest a role for proneurotrophins in acute peri‐infarct sympathetic denervation of the left ventricle.
Mechanisms regulating norepinephrine‐mediated α2‐IPSCs in the rat locus coeruleus
Authors: Barclay Samantha Lee , Ford Christopher
Published: June 2021

Dopamine (DA) in the ventral tegmental area (VTA) and norepinephrine (NE) in the locus coeruleus (LC) provide feedback inhibition that allows these cells to auto‐regulate their excitability. Previously, we characterized DA D2‐receptor‐mediated inhibitory postsynaptic currents (D2‐IPSCs) in the VTA. Here, we characterized NE α2‐receptor‐mediated IPSCs (α2‐IPSCs) in the LC. Both D2 and α2 receptors activate potassium channels. Despite analogous signaling, the duration of the D2‐IPSC was about one‐fourth that of the α2‐IPSC (D2‐IPSC tau: 347 ms, n=8; α2‐IPSC tau: 1346 ms, n=17). To characterize transmission, we evoked IPSCs with varying‐intensity stimulations. In the VTA, increased stimulus intensity increased the amplitude, but not the time course, of the D2‐IPSCs (tau = 92 + 4%, n =12). In contrast, in the LC, increased stimulation intensity increased the amplitude and duration of the α2‐IPSCs (tau = 142 + 13%, n = 13). Increased stimulus intensity may evoke spillover of NE. To examine glutamate input, we applied the AMPA receptor antagonist DNQX. DNQX had no effect on the amplitude or duration of the D2‐IPSCs, but reduced the amplitude and duration of the α2‐IPSC (control tau: 1282 ms, DNQX tau: 660 ms, n=9). Thus, stimulation of the LC may drive glutamate release that potentiates NE release. These data indicate that different mechanisms may regulate catecholamine transmission. Support: DA026417.
Stress‐induced activation of distinct subpopulations of neurons in the basolateral amygdala of the rat
Authors: Bolanos Juan M. , Hamdan Jameel N. , Rosales Victor M. , Gosselink Kristin L.
Published: June 2021

Previous studies showed that repeated exposure to 6h restraint stress causes hippocampal plasticity and shifts the profile of activated basolateral amygdala (BLA) neurons, suggesting alterations in mechanisms that affect learning and memory in repeatedly stressed rats. The purpose of this study was to determine whether a lesser stress exposure can induce similar consequences, and whether the effects are sex‐specific. Adult male and female rats were acutely (A; 30min x 1d) or repeatedly (R; 30min x 14d) restraint stressed and compared to unstressed controls (C). Stress occurred near the beginning of the light cycle (0900h). Series of perfused brain sections (30μm) were stained immunohistochemically for Fos, and the number of Fos‐expressing cells was quantified in the BLA. The number of Fos‐positive BLA cells tended to be higher in females than males in all groups, and varied by group: C/Male=47±19, A/Male=251±57, R/Male=152±44; C/Female=169±79, A/Female=151±83, R/Female=235±41. One significant difference was seen between C and A males (p<0.05). The data suggest that BLA responses to short‐term emotional stress vary according to sex and stress exposure. Further analyses will be done using fluorescent immunohistochemistry to quantify the specific types of stress‐activated BLA neurons in both sexes.

Supported by: UTEP's COURI Program, NIH/NCRR 5G12RR008124, and the Egbert Endowment (to KLG).
A computational model of neural activation, force generation and intracellular bioenergetics during muscle fatigue
Authors: Callahan Damien , Umberger Brian , Kent-Braun Jane
Published: June 2021

Muscle fatigue, an acute decrease in force‐generating capacity in response to repeated or prolonged activation, is a process whose mechanisms are difficult to discern due to the complicated nature of in vivo voluntary muscle force development. Computational modeling has been used to gain insight into the mechanisms of similarly complicated systems. The neuromuscular response to repeated voluntary activation is an attractive target for this approach. Here, we present a comprehensive model of neuromuscular activation, torque generation, bioenergetics and fatigue that provides a theoretical framework by which hypotheses related to the mechanisms of fatigue might be tested. A series of modular components were formulated, each representing a component of neuromuscular function. Multiple simulations were run to confirm accurate contractile and metabolic predictions when compared with in vivo data. A simulation that emulated a contraction protocol in the literature was run to evaluate the ability of the model to predict fatigue. Overall, the model predicted intracellular metabolic responses similar to in vivo data. Fatigue predicted by the model was within 6.6% of the reference data‐set from the literature, validating this model as a powerful tool for investigating the mechanisms of fatigue in voluntary isometric contractions.
Tracer washout from an organ is predicted from the tracer center of mass
Authors: Bassingthwaighte James B. , Raymond Gary M. , Chan J. I. S.
Published: June 2021

A tracer bolus injected into the arterial inflow of a tissue distributes by branching flows, permeation, and diffusion throughout a volume, Vd. Initially, concentrations are highest at the upstream end of the capillary‐tissue units but at larger times are nearer the downstream ends of the tissue and also more radially distant from the capillary. A useful way to comprehend the complex interrelating influences is to follow the trajectory of the “center of mass” of retained tracer as a function of time. This gives a measure of the fractional escape rate for the tracer and is predictive of the shape and slope of the washout curve measured by external monitoring of tracer content. When the capillary permeability‐surface area, PS, is high and diffusion distance short, washout rate provides estimates of flow. When PS is low, the washout rate approaches PS/Vd. Within a single capillary‐tissue unit the balance of downstream convection is balanced by upstream‐directed net diffusion leading to a stabilization of the center of mass and a mono‐exponential tail on the washout curve even though there are standing gradients in concentrations within the tissue. (Supported by NIH grants T15‐HL088516 and R01‐EB08407)
Production of a biodegradable electrospun biomaterial with tensile strength and elasticity
Authors: Wagner William D , Argenta Louis C , Morykwas Michael J , Levi-Polyachenko Nicole
Published: June 2021

Biodegradable biomaterials are essential to provide a temporary matrix for replacement or repair of tissues to resume function. The aim of the present study was 1) to develop a new biomaterial containing a blend of Collagen (80%) and poly (octanediol citrate), (POC) (20%), a soft material capable of providing an elastomeric cross‐linked polyester network and 2) to evaluate the physical, mechanical and functional properties of the biomaterial. Following chemical synthesis of POC, prepolymer was dissolved with type I Collagen and nanofibers were electrospun. Fibers within mats were thermally and chemically cross‐linked using heat and glutaraldehyde vapor respectively. Electrospun mats were hydrated in physiologic saline and physically were soft and elastic. Mechanical testing according to ASTM standards showed an elastic modulus of 0.46 ± 0.02 N/mm2 (mean ± SEM) and an elongation at break of 16.4 ± 0.9mm (mean ± SEM). Using a radial pressure expansion and relaxation system relaxation of the material was observed for over 10 cycles. Based on findings of the study and low tissue inflammatory responses of POC and Collagen the material described is versatile in biomedical applications especially in those such as skin repair where repetitive deformation can occur during healing.
Sphingosine 1‐phosphate Potentiated Endothelial Cell Attachment on De‐cellularized Human Umbilical Vein as a Scaffold for Vascular Tissue Engineering
Authors: Huang Yi-Long , Chen See-Yuan , Lu Jenher , Lee Hsinyu
Published: June 2021

Cardiovascular disease (CVD), disorders of the heart and blood vessels, is the leading cause of mortality and morbidity. Arterial bypass graft is a primary therapy for the CVD patients. However, choosing a proper material for arterial bypass grafting is still a critical issue. Human umbilical vein (HUV) is a natural vessel and has been suggested as a suitable scaffold for vascular tissue engineering. In this study, we de‐cellularized HUV by using sodium dodecyl sulfate (SDS) and further incubated with medium contains 12% FBS to remove the residual nucleic acid. After the de‐cellularization process, HUV was examined by histological analysis as well as DNA quantification to conform the elimination of residual cellular composition. Human umbilical vein endothelial cells (HUVECs) were then seeded back on de‐cellularized HUV with the treatment of 1uM sphingosine 1‐phosphate (S1P). S1P is a lysophospholipid (LPL) which binds to G protein‐coupled receptors (GPCRs) and enhance endothelial cell proliferation, migration, and adhesion. Our results showed that S1P enhances HUVECs attachment on de‐cellularized HUV and reduce the time consuming for vessel grafts. The finding potentially provides a new strategy to develop vascular tissues.