bluuebunny/crossref_metadata_2025_split huggingface dataset:
miR‐125a‐5p a Novel Regulator of SLC26A6 in Intestinal Epithelial Cells
Authors: Anbazhagan Arivarasu Natarajan , Priyamvada Shubha , Alrefai Waddah A. , Saksena Seema , Gill Ravinder K. , Dudeja Pradeep K. 
Published: November 2023

Putative Anion Transporter‐1 (PAT‐1 or SLC26A6) is expressed on apical membrane of the intestinal epithelial cells primarily in small intestine and functions as a Cl−/HCO3−, Cl−/formate or Cl−/oxalate exchanger. PAT‐1 has high affinity for oxalate and plays a crucial role in oxalate homeostasis. Mice deficient in Slc26a6 exhibit enhanced oxalate absorption resulting in nephrolithiasis. SLC26A6 has emerged as a potential target for reducing the risk of kidney stone formation. In this regard microRNAs (miRs) are small RNAs that play a key role in modulating gene expression via blocking the translation and/or promoting mRNA degradation of the target gene. In silico analysis of PAT‐1‐3¢UTR using Targetscan 7.1 revealed potential binding sites for several miRNAs. We hypothesized that PAT‐1 expression in intestinal epithelial cells is modulated by microRNAs. 3¢‐untranslated region (UTR) of PAT‐1 (263 bp) was cloned into pmirGLO vector upstream of luciferase. 3¢UTR PAT‐1 was transiently transfected in Caco‐2, T‐84 and HT‐29 cells and dual luciferase reporter activity was measured. Levels of microRNA, mRNA and protein expression were determined in transiently transfected Caco‐2 cells by RT‐PCR and western blotting, respectively. Significant decrease in the relative luciferase activity was observed in PAT‐1‐3¢UTR transfected Caco‐2 (53.87±3.02 p<0.0005) HT‐29 (50.51±3.05 p<0.0001) and T‐84 (44.93±4.47 p<0.0005) cells compared to empty vector. These data indicate the presence of intracellular factors in intestinal epithelial cells that target 3¢UTR of PAT‐1. In silico anaylsis showed highest context score for miR‐125a‐5p among the microRNAs targeting PAT‐1. Co‐transfection with microRNA mimic‐125a‐5p resulted in a further decrease in the relative luciferase activity of PAT‐1‐3¢UTR (~26% p<0.0001). In parallel, the decrease in luciferase activity by miR‐125a‐5p mimic concomitantly decreased PAT‐1 mRNA (~49% p<0.004) and protein expression (~44% p<0.006) in Caco‐2 cells. These data demonstrate a novel regulatory mechanism of intestinal PAT‐1 expression via miR‐125a‐5p and indicate that targeting this microRNA may serve to be a potential therapeutic strategy for counteracting defective SLC26A6‐mediated intestinal oxalate secretion leading to nephrolithiasis.Support or Funding InformationNIDDK and Dept of Veteran Affairs
Pharmacological but not genetically alteration of neural Epo modifies the CO2/H+central chemosensitivity in postnatal mice at birth
Authors: Laouafa Sofien , Perrin‐Terrin Anne‐Sophie , Jeton Florine , Elliot‐Portal Elizabeth , Tam Rose , Bodineau Laurence , Voituron Nicolas , Soliz Jorge 
Published: November 2023

The central chemosensitivity to CO2/H+ is considered essential for breathing during the maturation of the mammalian respiratory network. In this study we tested the hypothesis that at birth, the neural Epo is involved in the breathing stimulation induced by the activation of central CO2/H+ chemoreceptors. Measurements were performed on newborn Tg21 (overexpressing cerebral Epo), Epo‐TAgh (cerebral Epo deficient mice) and their control (WT‐Tg21 and WT‐Epo‐TAgh) mice aged of 4 day. Fictive breathing (in‐vitro) response to CO2‐induced acidosis or metabolic acidosis was measured by extracting the brainstem with a part of the spinal cord and placing it in a chamber where it was superfused with an artificial cerebrospinal fluid (aCSF). The output signal from the respiratory network was recorded by a suction electrode placed on the fourth ventral root (C4) representing the inspiratory signal sent to the diaphragm via the phrenic nerve allowing to determine fictive breathing frequency, amplitude and neural ventilation (Frequency × amplitude). Our results did not show differences between preparations from control and transgenic animals Tg21 and Epo‐TAgh for the fictive breathing frequency and amplitude. However, when Tg21 brainstem preparations were incubated for 1h with sEpoR (the antagosnist of Epo), or with inhibitors of ERK/Akt (thus blocking the activation of the Epo molecular pathway), the fictive breathing response to CO2‐induced acidosis was blunted and showed a significant decrease of the neural ventilation (Tg21‐sEpoR: from 111.7±41.1 to 100.0±20.6; & Tg21‐ERK/Akt inhibitors: from 112.3±20.3 to 100.8±23.8; P<0.05) due to a significant decrease of the amplitude (Tg21‐sEpoR: from 100.0±17.1 to 47.4±11.0; and Tg21‐ERK/Akt inhibitors: from 104.6±10.6 to 54.9±8.4; P<0.05). Our data suggest that the variation of the Epo/sEpoR ratio is central in the breathing modulation, and open up clinical perspectives based on the use of Epo drugs at birth in hypoventilation cases.Support or Funding InformationFounded by FRQS, Région Rhône‐Alpes and consulat de France à Québec
Cellular localization of spinal A2A receptors giving rise to phrenic motor facilitation in rats
Authors: Seven Yasin Baris , Perim Raphael R , Hobson Orinda R , Simon Alec , Mitchell Gordon S 
Published: November 2023

Cervical spinal adenosine 2A (A2A) receptor activation elicits plasticity in the phrenic motor system. For example, spinal A2A receptor‐dependent phrenic motor facilitation (pMF) is observed following severe acute intermittent hypoxia (Nichols et al., 2012), and can be elicited by intrathecal A2A receptor agonist administration (Golder et al., 2008). Since the specific cells expressing the relevant A2A receptors for this form of phrenic motor plasticity are not known, we tested the hypothesis that the relevant A2A receptors are expressed in phrenic motor neurons per se. To test this hypothesis, we combined neurophysiology, neuropharmacology, immunofluorescence and RNA interference. Intrapleural injections of cholera toxin B subunit (CtB) were used to label phrenic motor neurons. Intrapleural injections of siRNAs (3 consecutive days) targeting A2A receptor mRNA (siA2A), or non‐targeting siRNAs (siNT) were used to knock‐down A2A expression within phrenic motor neurons. Successful knock‐down of A2A receptors was confirmed by ~45% decrease in A2A receptor immunolabeling within CtB identified phrenic motor neurons (p<0.05). Minimal A2A receptor expression could be detected in adjacent astrocytes or microglia; further, there was no evidence for knockdown in C4 interneurons or non‐phrenic motor neurons. Selective phrenic motor neuron knock‐down using this method was similar to prior reports using siRNAs targeting either TrkB or PKCθ. In anesthetized, paralyzed and ventilated rats, pMF induced by intrathecal injections of the A2A receptor agonist CGS21680 was significantly attenuated in the siA2A (37%) versus the siNT group (147%; p<0.05). There were no significant effects of siA2A on phrenic burst frequency. Our results support the hypothesis that the A2A receptors giving rise to A2A receptor‐induced PMF are expressed within phrenic motor neurons versus glia, non‐phrenic motor neurons or segmental interneurons.Support or Funding InformationSupported by NIH HL69064 and the McKnight Brain Institute.
Phrenic long‐term facilitation (pLTF) following acute intermittent hypoxia (AIH) is TrkB and PI3K/Akt dependent in rats with motor neuron death from intrapleural CTB‐saporin injections
Authors: Nichols Nicole L. , Tanner Miles 
Published: November 2023

Intrapleural injections of cholera toxin B fragment conjugated to saporin (CTB‐SAP) selectively kills respiratory motor neurons, and mimics motor neuron death observed in rats with amyotrophic lateral sclerosis (ALS) (Nichols et al., 2015). This CTB‐SAP model allows us to study the impact of respiratory motor neuron death on breathing without many complications attendant to ALS, and is sufficient to enhance phrenic long‐term facilitation (pLTF, a form of phrenic motor facilitation; Nichols and Mitchell, ibid, 2014) after 7 days of CTB‐SAP (CTB‐SAP 145±22%; control 54±5%). However, the mechanism responsible for this enhancement is unknown. pLTF in naïve rats predominately requires 5HT2 receptors, the new synthesis of BDNF, and MEK/ERK signaling; however, there is an alternative mechanism for pLTF that requires A2A receptors, the new synthesis of TrkB, and PI3K/Akt signaling. Preliminary data suggest that A2A receptors are increased in the phrenic motor nucleus of CTB‐SAP treated rats (Craig et al., ibid) suggesting that CTB‐SAP rats utilize the alternative mechanism for pLTF. Here, we tested the hypothesis that pLTF is TrkB and PI3K/Akt, not BDNF and MEK/ERK, dependent in anesthetized, paralyzed and ventilated adult Sprague Dawley rats. Specifically, AIH (3, 5 min bouts of 10.5% O2) induced pLTF was studied 7 days following bilateral, intrapleural injections of: 1) CTB‐SAP (25μg), or 2) un‐conjugated CTB and SAP (control). Intrathecal C4 delivery included either: 1) small interfering RNA that targeted BDNF or TrkB mRNA; 2) U0126 (MEK/ERK inhibitor); or 3) PI828 (PI3K/Akt inhibitor). Our preliminary data suggest that pLTF in CTB‐SAP treated rats is TrkB dependent (siTrkB: 1%, n = 1; siBDNF: 127%, n=1), whereas pLTF in controls is BDNF dependent (siTrkB: 35%, n = 1; siBDNF: 11%, n=1). U0126, not PI828, nearly abolished pLTF in controls (U0126: 13%, n=2; PI828: 52%, n=2); in contrast, pLTF was attenuated in CTB‐SAP treated rats following U0126 and PI828 (U0126: 71%, n=2; PI828: 85%, n=2). This suggests that CTB‐SAP treated rats express pLTF primarily via an alternative mechanism that requires the new synthesis of TrkB and both MEK/ERK and PI3K/Akt signaling. Future studies will be focused on completing this analysis and testing whether pLTF in CTB‐SAP treated rats is abolished following C4 intrathecal, A2A receptor antagonist treatment. This project increases our understanding of respiratory plasticity and its implications for breathing following motor neuron death.Support or Funding InformationSupported by NIH K99/R00 HL119606
Mechanisms of cross‐talk inhibition between phrenic motor facilitation elicited by 5‐HT2B and 5‐HT7 receptors
Authors: Perim Raphael R , Fields Daryl P , Mitchell Gordon S 
Published: November 2023

Intermittent spinal serotonin receptor activation elicits long‐lasting phrenic motor facilitation (pMF), a model of respiratory motor plasticity. When activated alone, spinal Gq protein‐coupled serotonin type‐2 receptors (5‐HT2) initiate pMF by a mechanism that requires ERK MAP kinase signaling and new synthesis of BDNF (i.e. the Q pathway to pMF). Activation of spinal Gs protein‐coupled serotonin type‐7 receptors (5‐HT7) also elicits pMF, but by a distinct mechanism that requires Akt (not ERK) signaling and new TrkB (not BDNF) synthesis. When both receptor subtypes are activated simultaneously, pMF is attenuated or even abolished, demonstrating powerful cross‐talk inhibition between these competing mechanisms. However, we know little concerning the mechanisms of cross‐talk inhibition between these competing pathways to pMF. Here, we tested the hypotheses that: 1) concurrent 5‐HT2 subtype‐B (5‐HT2B) and 5‐HT7 receptors undermine pMF; 2) 5‐HT7 receptors inhibit the 5‐HT2B receptor induced Q pathway to pMF via a protein kinase A dependent mechanism; and 3) 5‐HT2B receptors inhibit the 5‐HT7 receptor induced S pathway to pMF via a NADPH oxidase dependent mechanism. Selective agonists for 5‐HT2B (BW 723C86 hydrochloride) and 5HT7 receptors (AS‐19) were administered intrathecally at C4 (3×, 5‐min injections) in anesthetized, vagotomized and ventilated rats. After spinal 5‐HT2B or 5‐HT7 receptor activation alone, integrated phrenic nerve burst amplitude increased progressively (i.e. pMF). In contrast, concurrent 5‐HT2B and 5‐HT7 activation failed to elicit pMF. With concurrent receptor activation, pMF was restored by inhibiting NADPH oxidase activity (apocynin) or protein kinase A activity (Rp‐8‐Br‐cAMPS), consistent with our hypotheses. We suggest an elaborated model accounting for inter‐receptor cross‐talk interactions regulating serotonin‐induced phrenic motor facilitation.Support or Funding InformationSupported by NIH HL69064 and the McKnight Brain Institute.
Phrenic Motor Neuron Expression of Receptors Known to Elicit Phrenic Motor Facilitation After Daily Acute Intermittent Hypoxia Preconditioning
Authors: Allen Latoya Lynn , Satriotomo Irawan , Simon Alec K. , Mitchell Gordon S. 
Published: November 2023

Spinal serotonin receptor activation elicits a form of respiratory motor plasticity known as phrenic motor facilitation (pMF). One specific form of pMF is phrenic long‐term facilitation (pLTF) following acute intermittent hypoxia (AIH) exposure. pMF exhibits metaplasticity, defined as a future change in the capacity to express plasticity based on experience. For example, repetitive AIH preconditioning enhances AIH‐induced pLTF.Distinct cellular mechanisms give rise to pMF, including the Q and S pathways, which are induced by 5HT2A/5HT2B and 5HT7 or adenosine 2A signaling, respectively. Although Q and S pathways each elicit pMF when acting alone, they are mutually inhibitory; thus, pLTF following moderate AIH results predominantly via the Q pathway, and is partially restrained by concurrent S pathway activation. Our working hypothesis is that repetitive AIH preconditioning causes pLTF metaplasticity by: 1) reducing inhibitory interactions between the S and Q pathways, and 2) increasing the relative contribution of the S pathway to pLTF. To begin exploring this global hypothesis, we began by investigating the impact of repetitive AIH on the phrenic motor neuron expression of receptors initiating the Q (5HT2A,5HT2B) versus S (5HT7, A2A) pathways to pMF. Four days after intrapleural injections of Cholera toxin B fragment (CTB) to identify phrenic motor neurons, rats were exposed to normoxia or daily AIH (10, 5 min episodes per day, 5 min intervals; 14 days). The rats were perfused and tissue sections were analyzed for 5HT2A, 5HT2B, 5HT7 and A2A receptors, and a downstream mediator of the S pathway (EPAC2) via immunofluorescence. Following dAIH: 1) 5HT2A, 5HT2B and A2A receptor expression were unchanged; but 2) 5HT7 and EPAC2 expression were increased versus controls. Increased 5HT7 and EPAC2 protein expression may support enhanced contributions of the S pathway to pMF, but do not provide information concerning the possibility that cross‐talk inhibition between the Q and S pathways was reduced. Priming a system with dAIH may increase the capacity for respiratory plasticity, and may be a useful therapeutic intervention to treat disorders that cause respiratory motor deficits, including spinal injury, sleep apnea or motor neuron disease.Support or Funding InformationSupported by: T32 Neuromuscular Plasticity Training Program NICHD: T32‐HD043730, NIH HL69064, and the McKnight Brain Institute. We acknowledge Dr. Mark Brownfield for providing us with the serotonin receptor antibodies.
LPS‐induced systemic inflammation impairs phrenic long‐term facilitation via okadaic acid‐sensitive protein phosphatase activity
Authors: Tadjalli Arash , Perim Raphael , Satriotomo Irawan , Santiago‐Moreno Juan , Seven Yasin , Mitchell Gordon S. 
Published: November 2023

Moderate acute intermittent hypoxia (mAIH; 3, 5‐min hypoxic episodes, 5‐min normoxic intervals) elicits a form of respiratory motor plasticity known as phrenic long‐term facilitation (pLTF). Although mild systemic inflammation elicited by a low dose of lipopolysaccharide (LPS; 100 ug/kg ip) undermines the capacity for mAIH‐induced pLTF 24 hours post‐injection (Huxtable et al., 2013), cellular mechanisms of this impairment are unknown. We previously demonstrated that inflammation undermines pLTF by a p38 MAP kinase dependent mechanism. In our working model, p38 MAP kinase activates serine/threonine protein phosphatases that dephosphorylate and inactivate kinases critical for mAIH‐induced pLTF expression, particularly the ERK MAP kinases. Here, we tested the hypothesis that LPS impairs pLTF by a mechanism that requires spinal okadaic acid‐sensitive serine/threonine protein phosphatase activity (eg. PP1 and/or PP2A). In anesthetized, paralyzed and ventilated adult male Sprague Dawley rats, we confirmed that mAIH‐induced pLTF is abolished 24 hours post LPS (LPS: 1 ± 4% baseline at 90‐min post hypoxia; vs. vehicle: 49 ± 12%; p = 0.003). We extend prior reports with the observation that other forms of mAIH‐induced respiratory motor plasticity are also impaired; specifically, hypoglossal LTF was also abolished 24 hours post‐LPS (LPS: −12 ± 5% baseline 90‐min post‐AIH; vs. vehicle controls: 30 ± 11% baseline; p = 0.02). In support of our hypothesis, spinal intrathecal (C3) pre‐treatment with okadaic acid, a PP1/2A inhibitor (25 nM, 30‐min before mAIH) restored pLTF in LPS treated rats (33 ± 8% baseline; p = 0.01). Thus, okadaic acid‐sensitive protein phosphatases constrain pLTF expression with systemic inflammation. As expected, hypoglossal LTF was not restored since okadaic acid distribution was limited to the spinal cord (1 ± 5% baseline; p = 0.79). Preliminary data from separate rats demonstrate that: 1) LPS increased phosphorylated p38 MAP kinase expression within phrenic and hypoglossal motor neurons (immunofluorescence) consistent with a role in LPS impairment of phrenic and hypoglossal LTF; and 2) diminished ERK MAP kinase phosphorylation/activation following mAIH in phrenic motor neurons. Thus, a likely target for the relevant okadaic acid‐sensitive phosphatase is ERK, a key enzyme in the cellular mechanism of mAIH‐induced pLTF. Since moderate AIH has demonstrated therapeutic potential to restore lost respiratory and non‐respiratory motor function in neuromuscular disorders that compromise movement, and the high prevalence of systemic and neuroinflammation in those same disorders, greater understanding of mechanisms whereby inflammation undermines mAIH‐induced motor plasticity may guide combinatorial therapies to enhance the therapeutic potential of this promising new treatment.Support or Funding InformationNIH HL111598 and the McKnight Brain Institute
The effect of urethane anesthesia on central coordination of cardiorespiratory coupling
Authors: Howard Courtney , Nall Andrea C , Zhu Vivian , Denson Heather B , Baekey David M 
Published: November 2023

Rodent models are commonly used to study respiratory and cardiovascular control systems and this research is often confounded by the unknown effects of commonly used anesthetics. A primary example of this challenge is seen in in vivo urethane anesthetized rats which, when surgically anesthetized, demonstrate appropriate respiratory rate, blood pressure, and heart rate. Less apparent is the attenuated coordination of respiratory and cardiovascular rhythms expressed as respiratory modulation of heart rate (respiratory sinus arrhythmia ‐ RSA) and blood pressure (Traube‐Hering waves). In contrast, in situ decerebrate rodent preparations (unanesthetized) have well‐coordinated cardiorespiratory coupling with a robust RSA and respiratory modulation of perfusion pressure. This influence of the respiratory rhythm on cardiovascular parameters can be measured after both removal of the lungs and neuromuscular blockade indicating that sufficient coupling occurs at the neural level to express the coordination. Based on these observations, we hypothesized that urethane anesthesia interferes with brainstem circuitry linking respiratory and cardiovascular control networks.To test our hypothesis, we performed experiments in decerebrate artificially perfused rodents (n=12) expressing baseline RSA and Traube‐Hering waves while monitoring phrenic nerve activity (PNA), thoracic sympathetic nerve activity (tSNA), ECG, and perfusion pressure. All preparations received increasing concentration of urethane anesthetic (1.5μg/ml: 0.1– 4.7ml/200ml perfusate) added to the perfusion bath and we subsequently characterized its influence on the monitored respiratory and cardiovascular signals. Atropine and hexamethonium were added to the perfusion bath in a subset of experiments (n=3) to confirm parasympathetic and sympathetic modulation was responsible for transmission of the respiratory pattern on the heart and vasculature. Additionally, in an overlapping subset (n=7), a multielectrode array was used to record ensembles of respiratory modulated neurons in the ventrolateral brainstem during application of the aforementioned drugs.Urethane anesthesia attenuated cardiorespiratory coupling in a dose dependent manner which corresponded with a substantial reduction in the number of active respiratory modulated neurons in the ventrolateral brainstem. In concert with the uncoupling, the overall phrenic amplitude was reduced and showed a loss of its ramp patterned bursting. As respiratory rate remained relatively stable prior and subsequent to complete uncoupling, we conclude that urethane acts on a respiratory population downstream of the respiratory central pattern generator which relays the respiratory rhythm to the autonomic efferents responsible for RSA and Traube Hering waves.Support or Funding InformationWe thank the University of Florida College of Veterinary Medicine and the Veterinary Scholars Program for their generous support.
Distributed Networks of Medullary Tonic Expiratory Neurons Regulate Cough Motor Drive
Authors: Shen Tabitha Y. , Morris Kendall F. , Baekey David M. , Musselwhite M. Nicholas , Segers Lauren S. , Nuding Sarah , Rose Melanie J. , Lindsey Bruce G. , Bolser Donald C. 
Published: November 2023

Recently, we showed that ventral medullary tonic E neurons participate in regulatory networks that control respiratory network excitability in response to alterations in chemical drive. The objective of this study was to determine the response patterns during cough of medullary neurons with continuous discharge during the breathing cycle that increased in the expiratory phase (tonic E). We hypothesized that dorsal and ventral medullary tonic E neurons would have activity patterns during coughing consistent with participation in the neurogenesis of this behavior. Further, we speculated that these neurons would participate in functionally‐identified neuronal circuits that regulate the excitability of cranial and spinal motor pathways to respiratory muscles. Using multiple electrode arrays, we recorded from many neurons simultaneously in the regions of the nucleus of the tractus solitarius (dorsal medullary), raphe nuclei, and ventral respiratory column (VRC) in 26 decerebrated, paralyzed, and artificially ventilated cats. A total of 106 tonic E neurons were recorded. During fictive cough, the discharge rates of 42 neurons increased, 21 decreased, and 43 did not change. Cross correlation analysis of simultaneously recorded dorsal and ventral medullary neurons revealed evidence for: a) circuits composed of tonic E neurons that excite one another and provide excitatory drive to expiratory laryngeal premotor and motoneurons, b) evidence for shaping of laryngeal motor discharge by suppression of tonic E neurons by phasic E decrementing neurons, and c) networks of cough‐responsive tonic E and non‐breathing modulated neurons that modulated the activities of recruited cough inspiratory neurons in the dorsal medulla and recruited cough expiratory neurons in the caudal VRC. The results support an important role of dorsal and ventral medullary tonic E neurons in controlling the excitability of cranial and spinal motoneurons that produce cough.Support or Funding InformationNIH HL 103415 and 1OT20D001983