bluuebunny/crossref_metadata_2025 huggingface dataset:
Ethanol metabolite increases activity of CeA neurons and requires activation of local NMDA receptors (1125.5)
Authors: Chapp Andrew , Larson Robert , Huber Michael , Carter Jason , Chen Qing‐Hui
Published: June 2021

The central nucleus of amygdala (CeA) is involved in the sympathoexcitatory and pressor responses to ethanol intake. However, the underlying neural mechanisms have not been determined. Here we investigated the role of acetate, the ethanol metabolite, in regulating the in vitro excitability of CeA neurons with axon projecting to rostral ventrolateral medulla (CeA‐RVLM) under brain slice preparation. In current‐clamp recordings, graded current injections evoked graded increases in spike frequency. Maximum discharge was evoked by +250 pA injections and averaged 18 ± 1 Hz (n=7). Bath application of acetate (0, 7.5, 37.5 and 75 mM) increased the excitability of CeA‐RVLM neurons in a dose‐dependent manner. Maximum spike discharge in the presence of acetate (75 mM) was 53 ± 3 Hz (n=4), which was significantly (p<0.01) higher than vehicle control. Pre‐treatment with AP5 (60 µM, n=7), the NMDA receptor blocker, significantly prevented increase in excitability (37 ± 5 Hz, p<0.05) elicited by acetate. AP5 alone was without effect on baseline excitability. The contribution of acetate in regulating sympathetic nerve activity (SNA) was assessed by microinjecting acetate into the CeA of anesthetized rat. Acetate (0.2 µmol/100 nl) significantly (P<0.05) increased lumber SNA by 90 ± 1 %, splanchnic SNA by 76 ± 18% and MAP by 4 ± 1 mmHg (n=3). Immunohistochemistry study demonstrates expression of NMDA NR1 in CeA‐RVLM neurons. Our data indicate that activation of NMDA receptors by acetate contributes to the increased excitability of CeA‐RVLM neurons. The CeA acetate elicited sympathoexcitatory responses involve the activation of CeA‐RVLM neurons, which may underlie the mechanisms of increased SNA during the development of alcohol associated hypertension.

Grant Funding Source: Supported by AHA 10SDG2640130
α7 Nicotinic acetylcholine receptors induced an increase in intracellular calcium in supraoptic nucleus neurons (1126.20)
Authors: Song Zhilin
Published: June 2021

The SON neurons synthesize oxytocin (OT) and vasopressin (VP) and receive cholinergic innervations. AchRs are expressed in SON. Activation of AchRs has been implicated in the control of water loss via VP release and also regulates release of OT, an anorexic agent. Deficits in OT are associated with human obesity. Thus, the brain cholinergic system may regulate food intake via modulation of OT neurons. Previous studies have demonstrated that activation of nAchRs stimulated a rise in [Ca++]i in SON neurons. In our current studies, live‐cell calcium imaging was used to determine the subtypes of nAchRs responsible. Mouse hypothalamic explants that include SON were loaded with the calcium sensitive dye, Fura‐2AM. Changes in [Ca++]i were monitored by changes in the 340/380 nm emission. Ach (5 mM) induced a [Ca++]i rise in the absence or presence of atropine (10 μM, muscarinic AchR antagonist) in wild type (WT) mice. In the presence of atropine, the amplitude of the [Ca++]i rise was reduced in WT mice while in α7 nAchR knockout (KO) mice, Ach only generated a small increase in [Ca++]i. Furthermore, in the additional presence of DHPE, an antagonist for α4β2 nAchRs, the rise in [Ca++]i in KO mice was completely absent. These experiments demonstrated that in SON neurons, both α7 and α4β2 nAchRs are functionally present and upon activation, generate calcium signals. Future studies will identify the OT vs VP phenotype of the responsive neurons.

Grant Funding Source: Supported by a NORC pilot award from NIH DK048250.
Brain‐derived neurotrophic factor exerts depolarizing effects on hypothalamic paraventricular nucleus neurons (1126.6)
Authors: McIsaac William , Ferguson Alastair
Published: June 2021

Brain derived neurotrophic factor (BDNF) is a chemical messenger that has been implicated in the central control of food intake and regulation of the stress axis. Consistent with a role in feeding behavior, TrkB, the receptor through which BDNF exerts its effects, is expressed in the PVN along with its low affinity receptor p75. Considering the paraventricular nucleus (PVN) of the hypothalamus is well established as a critical homeostatic relay center involved in the regulation of both of these systems, we investigated BDNF’s effects on the excitability of PVN neurons. Whole cell current‐clamp experiments were implemented to record from PVN neurons (N=23) in a hypothalamic slice preparation. BDNF dimer was bath applied at a concentration of 2nM for 2 minutes and elicited almost exclusively depolarizing effects (N=15) with a mean depolarization of 10.5 ± 1.74 mV. Cells responding to BDNF typically showed a return to baseline membrane potential within 200 seconds, although occasionally recoveries were not observed until 4000 seconds after application of BDNF. The results presented further elucidate the putative role of BDNF in feeding and regulation of the stress axis through its actions on PVN neurons.

Grant Funding Source: Canadian Institutes of Health Research
Neuropeptide Y signaling from the hypothalamus inhibits sympathetic outflow to brown adipose tissue through GABA inhibition of the rostral medullary raphe (1126.8)
Authors: Nakamura Yoshiko , Nakamura Kazuhiro
Published: June 2021

The arcuate nucleus is a central area involved in the regulation of metabolic thermogenesis in peripheral organs including brown adipose tissue (BAT). However, the efferent pathways from this nucleus for metabolic regulation are unknown. Activation of neuropeptide Y (NPY) neurons in the arcuate nucleus results in lowered metabolism likely through their direct projection to the paraventricular hypothalamic nucleus (PVH). In this study, we tested the hypothesis that NPY‐triggered neural signaling from the PVH inhibits sympathetic premotor neurons in the rostral medullary raphe (rMR) that control BAT thermogenesis. Sympathetic nerve activity and temperature in interscapular BAT were recorded in anesthetized rats, whose trunk was covered with a water jacket to control the skin temperature. Skin cooling‐evoked increase in BAT thermogenesis was eliminated by injection of NPY into the lateral ventricle or PVH. NPY injection into the PVH also eliminated BAT thermogenesis evoked by glutamate receptor stimulation in the rMR with an NMDA nanoinjection. On the other hand, NPY injection into the PVH did not affect BAT thermogenesis evoked by antagonizing GABA receptors in the rMR with a bicuculline injection. These results suggest that NPY signaling from the PVH activates a GABAergic inhibitory drive to sympathetic premotor neurons in the rMR to attenuate BAT themogenesis, leading to reduced energy expenditure.

Grant Funding Source: Supported by NEXT Program and by Grants‐in‐Aid for Scientific Research, MEXT, Japan
The influence of fetal cellular therapy on the functions’ restoration of central nervous system at rats after fatal hypobaric hypoxia (1127.1)
Authors: Aitbayeva Zh. , Tazhibaeva D. , Kabdualieva N. , Beglarova G. , Ermentaeva L. , Alipbekova A.
Published: June 2021

The aim of research is the influence study in the comparative aspect of fetal hepatocytes and «mediatory substances» of liver fetal tissues on the functions□ restoration of central nervous system at rats after fatal hypobaric hypoxia. Tasks of investigation: to study the influence of fetal cells and «mediatory substances» of liver fetal tissues on the survivalexponents of rats and general condition of organism after carried fatal form of hypobaric hypoxia. The experiments were carried out on 238 white rats of oth sexes. Fatal form of exogenic hypobaric hypoxia modeled in the Komovskyi apparatus (Tell L.Z.). Intra‐abdominal injection of fetal hepatocytes and «mediatory substances» were made in the postreanimation period. Results, received during this investigation, are allowed to establish more higher exponents of survival and improvement of animals□ general condition in the earliest postreanimational period in the group with use on the 30th minute after animation of «mediatory substances» in order to suspensions□injections of fetal cells of liver tissues.
Sustained hypoxia alters expression and function of excitatory amino acid transporters in the nucleus of the solitary tract (1127.2)
Authors: Matott Michael , Schramm Christine , Dantzler Heather , Kline David
Published: June 2021

Glutamate is the major excitatory neurotransmitter in the brain. Its removal occurs via excitatory amino acid transporters (EAATs) located on glia (EAAT1 & 2) and neurons (EAAT3 & 4). This removal is important in normal synaptic functioning as well as in pathological conditions. The goal of this study was to determine the molecular and functional changes in glial EAATs in the nTS due to sustained hypoxia (SH). Male Sprague‐Dawley rats (4‐7 weeks) were exposed to 1 or 3 days of SH (10% O2) and compared to normoxic (N) controls. The nTS was harvested for quantitative real‐time PCR, immunohistochemistry and immunoblots to evaluate expression of EAAT1 & 2. To examine EAAT function, nTS slices were made for whole‐cell electrophysiological recordings. EAAT1 and 2 in the nTS was confirmed by RT‐PCR, immunoblot and immunohistochemistry. EAAT2 mRNA was greater than EAAT1 in N, 1 and 3 d SH. One day SH did not alter EAAT 1 & 2 RT‐PCR, immunoblots and immunohistochemistry. 3 d SH did not alter EAAT1 RT‐PCR or immunoblots, but increased EAAT2 mRNA with a trend for increased protein. Functionally, blockade of EAAT2 with dihydrokainate (DHK, 300µM) produced a significant inward current and depolarization of membrane potential. These effects were reduced in slices from 3‐days SH but not 1‐day SH. These data suggest that SH alters the expression and function of EAAT2 which may have a nueroprotective effect.

Grant Funding Source: Supported by: RO1 HL098602
Nesfatin‐1 influences the excitability of subfornical organ neurons through actions on the Ik conductance (1129.2)
Authors: Kuksis Markus , Ferguson Alastair
Published: June 2021

Nesfatin‐1 is produced in several brain areas involved in metabolic processes and has been implicated in the control of ingestive behaviors and cardiovascular function. In this study, we confirm the presence of mRNA for the nucleobindin‐2 gene in the subfornical organ (SFO). We used whole‐cell patch clamp recordings in current clamp configuration to investigate the influence of nesfatin‐1 on the membrane potential of dissociated SFO neurons. We found that 80.3% (49 of 61) of neurons tested showed a response to nesfatin‐1 (100 nM, 10 nM and 1 nM) with 47.5% depolarising and 32.8% hyperpolarising. We then used voltage clamp configuration to investigate the conductances through which nesfatin‐1 may act to change the membrane potential of SFO neurons. Voltage clamp ramp and step protocols revealed a nesfatin‐1 induced enhancement of the delayed rectifier potassium conductance, IK. Pharmacological blockade of this conductance using tetraethyl ammonium chloride (TEA) greatly reduced the magnitude and occurrence of the observed hyperpolarisations, which implies that nesfatin‐1 induced hyperpolarisations are mediated through IK. This study has demonstrated that nesfatin‐1 has the ability to influence the membrane potential of SFO neurons by enhancing the IK conductance, and thus has identified the SFO as a potential site at which nesfatin‐1 may act to regulate ingestive behaviour and cardiovascular control.

Grant Funding Source: Canadian Institutes for Health Research
An intestinal gap‐junction protein is needed for normal acetylcholine response in Caenorhabditis elegans (1129.8)
Authors: Moore Gabriel , Hammer Phoebe , Peters Maureen
Published: June 2021

The Caenorhabditis elegans (C. elegans) nervous system is understood in detail, yet predicting the behavior it controls is challenging. Dynamic regulation of the nervous system may be caused by neuromodulators released in response to calcium. In the C. elegans intestine, a calcium wave regulates peptide release that triggers muscle contractions associated with defecation. Intestinal calcium waves require gap junctions that include the innexin‐16 (inx‐16) subunit for normal propagation and patterning. Upon observing that inx‐16 mutants exhibit abnormal movement, we hypothesize that calcium‐triggered peptide release from the intestine regulates locomotory circuits. We determined the relationship between an intestinal calcium wave and locomotion signaling by assessing neurotransmission rates in the intestinal calcium wave mutant, inx‐16. Two assays of acetylcholine responsiveness, one using an acetylcholine agonist and the other an acetylcholinesterase inhibitor, were performed in the inx‐16 mutant. The results suggest a pre‐synaptic deficit in acetylcholine release. We propose that peptides released in response to intestinal calcium waves acts as neuromodulators of acetylcholine‐based neurotransmission. Our study suggests a connection between defecation‐related signaling and nervous system function.
Effect of liposome entrapped‐GABA on the expression of nNOS and GABAA receptors in neurons (1130.11)
Authors: Vaz Gisele , Sharma Neeru , Zheng Hong , Bahia Ana Paula , Zimmerman Matthew , Frezard Frederic , Fontes Marco , Patel Kaushik
Published: June 2021

Liposomes are concentric lipid vesicles that allow a sustained release of entrapped substances. GABA is the most prevalent inhibitory neurotransmitter in the central nervous system. Here we determined the effect of sustained release of GABA on the expression of neuronal nitric oxide synthase (nNOS) and GABAA receptor (GABAAR) in a neuronal culture (NG108‐15 cells). GABA entrapped‐liposomes particles (GEL) were prepared by the freeze‐thawing method. In vitro studies using NG108‐15 neurons treated with different doses of GEL for 12 hr showed an increase in nNOS (138, 157 and 165% at doses of 20, 50 and 100 ng respectively) and GABAAR (54 and 50% at the doses of 10 and 20 ng respectively) compared with control. Moreover, we found a decreased expression of protein inhibitor of nNOS (PIN) with GEL treatment (26, 66 and 57% at doses of 20, 50 and 100 ng, respectively). Time course experiments in cells treated with 50 ng of GEL, showed an increase in GABAAR (23%) after 1 hr followed by an increase in nNOS (55, 46 and 55%) and decrease in PIN (22, 28 and 53%) at 8, 12, 24 hr, respectively. Immunostaining also showed an increase in nNOS expression (134%) in cells after 12 hr of GEL treatment. These results suggest that the slow release of GABA can change the expression of nNOS in neuronal cultures, potentially by altering the expression of PIN. Supported by NIH grants HL62222 & DK82956 & CNPq fellowship to GCV.

Grant Funding Source: NIH grants HL62222 & DK82956 & CNPq fellowship to GCV
Blockade of mineralocorticoid receptors in the dorsal hindbrain inhibits stress‐induced hypertension in rats (1130.12)
Authors: Clifton Rebekah , Backes Iara , Scheuer Deborah
Published: June 2021

This study tested the hypothesis that chronic blockade of mineralocorticoid receptors (MR) in the dorsal hindbrain (DHB) attenuates cardiovascular responses to stress in male Sprague‐Dawley rats subjected to chronic variable stress (CVS) for 2 weeks. Blood pressure was measured using radiotelemetry. Baseline data were collected for 1 wk then all rats were given a continuous choice of water or 0.3M NaCl to drink, and pellets made of cholesterol (Chol) or the MR antagonist spironolactone (SPL) were implanted over the DHB. Rats were then subjected to CVS or remained unstressed (n=4‐6/group). Baseline blood pressure averaged 95±1 and 99±1 mmHg during the light (L) and dark (D) periods respectively. During the last week, CVS significantly (P<0.05) increased baseline blood pressure in the Chol + stressed (∆8±2 (L) and 9±3 (D) mmHg) but not in the SPL + stressed rats (∆1±1 and 1±1 mmHg). Blood pressure was not changed in the unstressed Chol (∆1±2 mmHg and ∆3±2) or SPL (∆‐1±1 and ∆1±1 mmHg) rats. SPL significantly attenuated the arterial pressure response to a novel cage stress, but not to other stressors. The results suggest that chronic blockade of DHB MR can eliminate stress‐induced hypertension.

Grant Funding Source: NIH #HL076807, AHA #GRNT4460047
Acute intermittent hypoxia induces sympathetic long‐term facilitation but does not alter synchronization between phrenic and sympathetic nerves (1130.15)
Authors: Yamamoto Kenta , Lalley Peter , Mifflin Steve
Published: June 2021

Acute intermittent hypoxia (AIH) can induce phrenic and sympathetic long‐term facilitation (LTF). Respiration modulates the sympathetic nervous system, leading to an idea that respiratory‐sympathetic coupling plays a role in the generation of sympathetic LTF. To determine if augmentation of synchronization between phrenic and sympathetic nerve activities is necessary to generate sympathetic LTF, we investigated the effect of AIH on phrenic nerve activity (PNA) and renal sympathetic nerve activity (RSNA) using spectral analysis in anesthetized, vagotomized and artificially ventilated Sprague‐Dawley rats (n=6). AIH was performed by ventilation with 10% oxygen for 1min every 6min for a total of 10 hypoxic exposures within an hour. PNA amplitude and RSNA increased by 110% and 100% respectively measured 60 min after AIH (P<0.05). We estimated spectral power of PNA and RSNA and coherence between PNA and RSNA at central respiratory drive frequency, defined as frequency of the main peak in PNA. Spectral power of PNA and RSNA increased by 640% and 270% respectively measured 60 min after AIH (P<0.05). However, coherence between PNA and RSNA did not change from baseline (0.62 +/‐ 0.34) to 60 min after AIH (0.71 +/‐ 0.25). These results suggest that AIH can induce sympathetic LTF in the absence of augmented synchronization between central respiratory drive and RSNA.

Grant Funding Source: HL088052
HIF‐1α increases sympathoexcitation via upregulation of NMDA receptors in the PVN during heart failure (1130.16)
Authors: Sharma Neeru , Cunningham Craig , Liu Xuefei , Zheng Hong , Patel Kaushik
Published: June 2021

Increased sympathetic outflow is a prominent feature of chronic heart failure (HF) associated with higher mortality. Multiple studies from our lab have reported that higher glutamatergic tone (increase in N‐methyl‐D‐aspartate (NMDA)‐type 1 (NR1) receptors expression) within the paraventricular nucleus (PVN) increase the sympathetic drive in HF. Further, HF (coronary artery ligation‐induced) augmented the expression of hypoxia‐inducible‐factor (HIF)‐1α both at mRNA (9 fold) and protein (2.5 fold) levels in the PVN which, led us to hypothesized that increased levels of HIF‐1α may contribute to exaggerated sympathetic drive via increasing NR1 expression in the PVN of rats with HF. Neuronal cell line (NG108‐15) exposed to hypoxia (2% O₂, 5% CO₂, 93% N₂) showed 2‐fold increase in HIF‐1α (12h) expression with concomitant increases in NR1 at transcription and protein levels after 4h (6.5 fold) and 12h (40%) respectively. Live cell imaging also showed marked increase in intracellular calcium in response to glutamate under hypoxic conditions. Mechanistically, hypoxia promotes the direct binding of HIF‐1α (3.2 fold) at NR1 promoter in neurons exposed to hypoxia as demonstrated by chromatin immunoprecipitation analysis. Furthermore, silencing of HIF‐1α by using HIF‐1α ‐targeted small interfering RNA in neurons led to 2.9 fold decrease in the expression of NR1. Validating our in vitro data, HIF‐1α knockdown by HIF‐1α‐siRNA in the PVN of rats with HF ameliorated the increased sympathoexcitation to NMDA (57±5% vs. 27±1% of basal value). Taken together, our study suggests a contribution of HIF‐1α in the regulation of NR1 expression within the PVN in HF, and provides a novel mechanism for the modulation of sympathoexcitation in HF. Supported by NIH HL62222.

Grant Funding Source: NIH HL62222.
RVLM sympathetic premotor neurons are not synaptically linked to each other (1130.17)
Authors: Bou Farah Lama , McMullan Simon
Published: June 2021

Sympathetic premotor neurons residing within the rostral ventrolateral medulla (RVLM) control the activity of sympathetic nerves and blood pressure. In vivo, the activities of sympathetic nerves and spinally projecting cells in the RVLM display rhythmic bursting that is independent of baroreceptor input. We have previously proposed that such synchronisation could result from functional coupling between spinally projecting neurons.

In this study we directly test that hypothesis by examining the prevalence of synaptic connections between pairs of RVLM sympathetic premotor neurons, identified by retrograde transport of tracer microinjected into the spinal cord.

We simultaneously recorded membrane voltage and currents in pairs (34 pairs from x neurons) of bulbospinal neurons recorded in whole cell mode in acute brainstem slices prepared from P8 ‐ P20 rats. We injected depolarizing currents to evoke trains of action potentials in the ‘presynaptic’ neuron and used ‘presynaptic’ action potentials to trigger averages of holding current recorded in the ‘post‐synaptic’ neuron.

No evidence of monosynaptic connections was observed in any case, despite long and stable recordings from neurons in close proximity to one another. We conclude that direct synaptic connections between bulbospinal neurons are sparse or non‐existent and that direct connections between these cells toes not play a significant functional role.
A nigro‐vagal pathway controls gastric motility (1130.18)
Authors: Toti Luca , Travagli R. Alberto
Published: June 2021

Degeneration of dopaminergic (DA) neurons of the substantia nigra pars compacta (SNpc) induces Parkinson’s disease (PD). Since almost all patients with PD show gastrointestinal (GI) dysfunctions, such as gastroparesis, and GI functions are controlled by neurons in the dorsal motor nucleus of the vagus (DMV), we aimed to determine whether manipulations of the SNpc induces a vagally‐mediated modulation of gastric motility.

Microinjection of the retrograde tracer CTB in the DMV labelled neurons in the SNpc, indicating a monosynaptic nigro‐vagal complex connection.

In an anesthetized rat preparation in which gastric motility was recorded using miniaturized strain gauges sewn on the anterior stomach wall, NMDA was microinjected in the left SNpc (210nL of a 25mM). In control rats, NMDA increased tone and motility in both the corpus and in the antrum (tone: +211±37, N=11 and +295±53 mg, N= 8; motility: +85±21, N=9 and +82±19%, N=9, of baseline, respectively). This effect was blocked by ipsilateral vagotomy (N=4) or by application to the fourth ventricle of DA‐1 (SCH‐23,390, 45nM) and DA‐2 (L‐741,626, 45nM) antagonists (N=4). In contrast, following microinjection of 6OH‐DA in the SNpc, application of NMDA had no effect on gastric motility (N=4).

Our data indicate a monosynaptic connection exists between the SNpc and DMV that modulates gastric tone and motility, suggesting a novel pathway in the regulation of GI functions which may be compromised in PD.

Grant Funding Source: DK55530
Blockade of mineralocorticoid receptors in the dorsal hindbrain enhances baroreflex sensitivity (1130.4)
Authors: Backes Iara , Clifton Rebekah , Scheuer Deborah , Adler Gail
Published: June 2021

This study tested the hypothesis that chronic blockade of mineralocorticoid receptors (MR) in the dorsal hindbrain (DBH) enhances spontaneous baroreflex sensitivity (sBRS) in male Sprague‐Dawley rats subjected to chronic variable stress (CVS) for 2 weeks. Rats were instrumented with radiotelemetry transmitters to obtain cardiovascular data, and pellets made of cholesterol (Chol) or the MR antagonist spironolactone (SPL) were implanted on the DBH (n=5‐6 per group). Two weeks later rats were subjected to CVS, which included insulin‐induced hypoglycemia on the first day of stress. SPL significantly (P<0.05) increased the sBRS prior to (2.1±0.1 vs. 1.5±0.2 ms/mmHg in SPL vs Chol rats) and after 2 weeks of CVS (2.1±0.1 vs. 1.6±0.2 ms/mmHg in SPL vs Chol rats). Neither CVS nor hypoglycemia altered sBRS. Blood glucose was measured at 20, 40, 60, 90, 120 and 180 min after insulin (7‐8 U/kg, i.p.) was administered to non‐fasted rats. Glucose reached a nadir at 2 hr, when levels were significantly higher in SPL compared to Chol rats, (37±3 vs.28±1 mg/dl, P<0.05). The results indicate that chronic blockade of DHB MR enhances sBRS and blunts the hypoglycemic effect of insulin.

Grant Funding Source: Supported by NIH #HL076807 and AHA #GRNT4460047
Enhanced leptin‐TRPC signaling in the hypothalamus contributes to the exaggerated sympatho‐excitation in type 2 diabetic rats (1130.9)
Authors: Zheng Hong , Liu Xuefei , Li Yulong , Patel Kaushik
Published: June 2021

Accumulated evidence indicates that type 2 diabetes (T2D) is associated with enhanced sympathetic activation. Transient receptor potential canonical channels (TRPC) have been found in pro‐opiomelanocortin neurons and activated by leptin. The present study was conducted to investigate the role for leptin‐TRPC signaling in centrally mediated sympathetic response in rats with T2D. In high‐fat diet and low‐dose strepzotocin (30mg/kg)‐induced T2D rats, mRNA levels of TRPC subtype including TRPC1, 4, 5 and 6 were significantly increased in the paraventricular nucleus (PVN) and arcuate nucleus (ARCN) compared to control rats (n=8/group). Whole‐cell patch‐clamp was conducted in primary cultured hypothalamic neurons to record TPPC currents. Neurons from T2D rats had significant increased TRPC currents (about 2 folds to the control). In anaesthetized condition, microinjections of leptin (5ng~100ng) into the ARCN and PVN induced increases in renal sympathetic nerve activity and blood pressure. These effects were significantly blocked by pre‐microinjections of TRPC inhibitors (2APB and SKF96365). In cultured neuronal cell NG108, pre‐incubation with leptin (0.625~2.5μM) for 24hrs induced significant increases of TRPC mRNA expressions (TRPC1, 4 and 5). Taken together, these data suggest that within the hypothalamic nuclei, leptin‐induced sympatho‐excitation in T2D maybe mediated through an activation of TRPC. Supported by NIH grant DK82956.

Grant Funding Source: NIH DK82956
A neurophysiological study of the medullary sympathetic pathway to the adrenal gland (1131.7)
Authors: Verberne Anthony , Korim Willian , Sabetghadam Azadeh
Published: June 2021

Neurons in the rostral ventrolateral medulla (RVLM) control adrenaline secretion from the adrenal gland and participate in glucose homeostasis. Here, we examined sympathetic evoked responses recorded from the adrenal sympathetic nerve in response to intermittent stimulation of the RVLM. All experiments were performed using isoflurane/urethane‐anaesthetised, artificially‐ventilated, paralysed, male Sprague‐Dawley rats. The RVLM was first identified by extracellular single unit recording combined with field potential mapping of the facial nucleus. We compared the latencies of the evoked adrenal sympathetic nerve (ASNA) response to the lumbar sympathetic nerve (LSNA) response upon electrical stimulation of RVLM (0.5 Hz, 1 ms pulse width, 50‐300 μA) after conversion to urethane (1.4 g/kg, i.v.). RVLM stimulation elicited an evoked ASNA response that depended on whether the recording was primarily pre‐ or post‐ganglionic activity as judged by the effect of the ganglion blocker hexamethonium (Hex; 20‐40 mg/kg, i.v.). Pre‐ganglionic ASNA consisted of an early peak (latency ‐ 79±9 ms, n=4) and a late peak (latency ‐ 149±6 ms). Lumbar sympathetic nerve activity exhibited a prominent early peak (latency ‐ 87±7 ms; n = 5) that was eliminated by Hex. Pre‐ganglionic ASNA was reduced by 62 % after intrathecal kynurenate (0.5 μmole/10 μl; P<0.01, late peak, n=3). The early peak may represent sympathetic vasomotor activity in both the adrenal and lumbar sympathetic nerves. However, the late sympathetic response noted in the adrenal nerve is mediated by a glutamatergic input and may be involved in the control of catecholamine secretion from adrenal chromaffin cells.

Grant Funding Source: National Health and Medical Research Council of Australia
Effects of digitoxin treatment on reflex cardiovascular control in rats with heart failure (1132.2)
Authors: Fardin Núbia , Antonio Ednei , Montemor Jairo , Tucci Paulo , Campos Ruy
Published: June 2021

There is evidence that digitalis promote changes in the autonomic and reflex control of the cardiovascular system and enhances survival in rats with Heart Failure (HF). The major aim of the present study was to evaluate the effects of digitoxin treatment on basal renal sympathetic nerve activity (rSNA) and on the reflex control of the cardiovascular system by the arterial baroreceptors and cardiopulmonary reflex in HF rats. Male Wistar rats (±200g) were divided into two experimental groups: HF and HF treated with digitoxin in the diet for 60 days (1mg/Kg/day) (HF+DIG). A significant increase in rSNA were found in HF (190 ± 29pps; N= 5) compared to control rats (98 ± 14pps, N=5). Digitoxin treatment leads to a significant decrease and normalizes rSNA in HF+DIG rats (98 ± 14pps, N= 7). The baroreflex sensitivity for the control of rSNA was significantly decreased by treatment (slope: ‐0.69 ± 0.10pps/mmHg, N= 7) when compared to HF group (slope: ‐2.66 ± 0.12pps/mmHg, N= 5). A similar result was found for the cardiopulmonary reflex, digitoxin reduced the fall in heart rate (HF: 180 ± 7bpm, N=8; HF+DIG: ‐126 ± 14bpm, N=9) in response to acute phenylbiguanide administration. Altogether the results demonstrate that the fall in rSNA in response to digitoxin in HF rats is not related to improvement in the reflex control of the cardiovascular system. Central actions of digitoxin may explain the sympathoinhibitory effects of digitalis.

Grant Funding Source: CAPES, CNPq and FAPESP (2009/54225‐8)
Device‐guided slow deep breathing in essential hypertension: is cardiac or sympathetic baroreflex sensitivity altered? (1132.7)
Authors: Shantsila Alena , Adlan Ahmed , Lip Gregory , Pickering Anthony , Paton Julian , Fisher James
Published: June 2021

The influence of device‐guided slow deep breathing (SDB; RESPeRATE□) on arterial baroreflex control of the heart and muscle sympathetic nerve activity (MSNA) in essential hypertension patients remains unclear. To address this, respiratory rate, partial pressure of end‐tidal carbon dioxide (PETCO2), MSNA (microneurography), heart rate (HR) and blood pressure (BP: Finometer) were monitored in 8 hypertensive patients (55±14 years, BMI 28±3 kg/m2, 152±26/71±11 mmHg [systolic/diastolic BP], duration of hypertension 6.4±6.7 years; mean±SD) during 10 min of normal breathing and 10 min of SDB. Cardiac baroreflex sensitivity (sequence technique) and spontaneous arterial baroreflex control of MSNA burst incidence were calculated offline. As expected, SDB decreased respiratory rate (13±4 to 6±1 breath/min, P=0.001), whereas PETCO2 (P=0.22), and mean BP (98±14 to 97±13 mmHg, P=0.11) were unchanged. MSNA burst incidence tended to fall during SDB (median with interquartile range from 46 [34‐56] to 40 [31‐48] bursts per 100 heart beats, P=0.12), however neither cardiac nor sympathetic baroreflex sensitivity were altered (P>0.05). In conclusion, acute device‐guided SDB does not appear to modify cardiac and sympathetic baroreflex sensitivity in patients with essential hypertension.

Grant Funding Source: Supported by British Heart Foundation
Activities in a single sensory fiber of the aortic depressor nerve were derived from multiple encoders (1132.8)
Authors: Liu Jun , Song Nana , Tian Shifu , Guardiola Juan , Proctor Mary , Yu Jerry
Published: June 2021

Arterial baroreceptors, resembling airway slowly adapting receptors (SARs) that regulate breathing, play a very important role in the regulation of blood pressure. However, the sensory processing mechanisms at the cellular level remain unclear. Recently, a SAR unit was found to contain multiple encoders. Therefore, we hypothesize that a barosensory unit is the same. We used immunofluorescent approach to examine the baroreceptor structure in rabbit aortic arches morphologically. A single myelinated axon may connect with multiple structures, clustered in the aortic arch near the bifurcation or origin of the common carotid and subclavian arteries, forming a network. Physiologically, we recorded single‐unit activity in the depressor nerve in anesthetized rabbits and examined their response to phenylephrine. The unit activity increased progressively as blood pressure (BP) increased and decreased abruptly (deactivation) after the BP attained a plateau for about 2 min. Their activity became slow first (indicating multiple encoders) and subsequently ceased. The unit could be reactivated by lowering BP and deactivated again by raising BP. Since baroreceptors resemble SARs morphologically and electrophysiologically, we conclude that a single barosensory unit contains multiple encoders. Thus, the sensory unit is not only a transducer but also a processor that integrates a significant amount of information.

Grant Funding Source: VA Merit Review PULM‐029‐10S
Hypertension impairs spontaneous baroreflex heart rate control during exercise and muscle metaboreflex activation (1132.9)
Authors: Augustyniak Robert , Spranger Marty , Abu‐Hamdah Rania , Kaur Jasdeep , Hammond Robert , Sala‐Mercado Javier , Ichinose Masashi , O'Leary Donal
Published: June 2021

Spontaneous baroreflex sensitivity (SBRS) in the control of heart rate (HR) is reduced in proportion to exercise intensity and magnitude of MMA. In hypertension (HTN), while SBRS is reduced at rest, the effect of exercise and MMA are unknown. In eight conscious dogs we measured SBRS in the control of HR (sequence method) at rest, during mild dynamic exercise, and during MMA (via imposed reductions of hindlimb blood flow), in the same animals before and 38±7 days after induction of HTN (via unilateral partial renal artery occlusion). HTN increased resting mean arterial blood pressure from 94±3 to 142±14 mmHg. In control, with the transition from rest to exercise, SBRS decreased from ‐4.01±0.77 to ‐2.38±0.61 bpm/mmHg and reduced further to ‐1.78±0.43 with MMA. In HTN, SBRS was significantly reduced at rest (‐2.38±0.34), during exercise (‐1.05±0.12) and MMA (‐0.90±0.14).We conclude that in HTN SBRS in the control of HR is impaired to similar degrees at rest, during exercise and MMA.

Grant Funding Source: HL‐55743 and HL‐095819
The proton channel HV1 is expressed in the medullary thick ascending limb of the kidney (1134.1)
Authors: Stilphen Carly , Sun Jingping , Lambert Nevin , O’Connor Paul
Published: June 2021

Oxidative stress in the renal outer‐medulla contributes to the development of hypertension and renal disease in Dahl salt‐sensitive (SS) rats. We have previously demonstrated that an unknown proton channel is responsible for enhanced superoxide production in medullary thick ascending limb (mTAL) of SS rats. We hypothesized that HV1 is responsible for superoxide producing H+ currents and is expressed in mTAL of SS rats. In order to test our hypothesis we quantitated HV1 mRNA (RT‐PCR) and protein (immunohistochemistry) specifically within mTAL of wild‐type SS rats and in a HV1‐/‐ strain of Dahl SS rats in which we mutated the HV1 gene using a Zn‐finger nuclease. We confirmed complete loss of Hv1 protein in HV1‐/‐ rats both by whole‐cell voltage clamp of peritoneal macrophages and by Western blot/anti‐HV1 staining in spleen (positive control tissue). Using the same antibody, HV1 protein expression was found to be significantly greater in mTAL of wild‐type rats compared to presumed background in HV1‐/‐ rats (4382 ± 921 vs 1459 ± 708AU respectively, n=5/p=0.036). In addition, mRNA for HV1 was expressed 6.4 fold higher in mTAL of wild‐type rats (n=4/p=.04). We conclude that HV1 is expressed in the mTAL of Dahl SS rats. Our data indicates that HV1 could potentially be responsible for superoxide producing H+ currents in mTAL of Dahl SS rats, which are thought to contribute to the development of hypertension and renal disease.
Changes in mitochondrial metabolism and morphology contribute to sepsis‐associated acute kidney injury (1134.10)
Authors: Thomas Joanna , Ayoyama Koji , Nourbakhsh Noureddin , Singh Prabhleen
Published: June 2021

Acute Kidney Injury (AKI) is commonly seen in septic patients, but there are no effective treatments to prevent or treat it. New perspectives on the pathogenesis of AKI in sepsis are needed to improve the current standard of treatment. We examined renal mitochondrial function and expression of proteins with known roles in mitochondrial function and dynamics in the cecal ligation and puncture (CLP) model of sepsis in rats. At 24 hrs post CLP or sham surgery, kidney tissue and mitochondria were isolated for protein expression analyses and mitochondrial functional assays including mitochondrial oxygen consumption (QO2), ATP generation and reactive oxygen species (ROS) generation. Data represents mean ± SEM.

In isolated mitochondria, QO2 rates were higher in CLP compared to sham; with the largest difference observed during state 3 (199.9 ± 31.6 pMol/min vs. 163.2 ± 28.81 pMol/min, p<0.05). However, ATP generation was significantly lower in the mitochondria from CLP kidneys. With western blot expression, we found increased expression of mitochondrial fission proteins DRP1 (1.9‐fold higher in CLP, P<.0004) and Fis1 (trend for higher expression) in CLP kidneys. We also observed decreased expression of PGC‐1α, a key transcription factor in mitochondrial biogenesis. Analysis of the expression of the expression of the ETC complexes in the mitochondrial fraction showed a 25% reduction (P 蠄 .001) in Complex Va and Complex III in CLP mitochondria. In conclusion, our data indicate significant increase in renal oxidative metabolism but without the associated ATP generation and reduction in the expression of ETC complexes. Mitochondrial analyses reveal increased mitochondrial fission but with reduced biogenesis, indicating increased mitochondrial stress. Additional morphological and functional analyses are being conducted. We are also evaluating mechanisms underlying the altered mitochondrial function to identify potential therapeutic targets for the treatment of kidney injury during sepsis.

Grant Funding Source: NIH
Tumor necrosis factor‐α supression rescues glutathione peroxidase activity in kidney of insulin resistant rats (1134.12)
Authors: Carter Matthew , Lee Andrew , Rodriguez Ruben , Thorwald Max , Ortiz Rudy , Nishiyama Akira
Published: June 2021

The interactions between reactive oxygen species (ROS) production and kidney nephropathy in metabolic syndrome are not well described. This study assessed the effects of blocking 1) renin‐angiotensin system (RAS), 2) tumor necrosis factor‐alpha (TNFα)‐mediated inflammation, 3) and non‐RAS‐mediated hypertension on the activity of the antioxidant enzymes, glutathione peroxidase (GPx), catalase (CAT), and superoxide dismutase (SOD) in the kidney of insulin resistant (IR), Otsuka‐Long‐Evans‐Tokushima‐Fatty (OLETF) rats. Lean and obese rats (n = 5‐7/group) were randomly assigned to the following groups: 1) untreated Long‐Evans‐Tokushima‐Otsuka (LETO) (lean, strain control ), 2) untreated OLETF, 3) OLETF + angiotensin receptor blocker (ARB) (10 mg olmesartan/kg/d x 10 wk), 4) OLETF + TNFα blocker (ETAN) (1.25 mg entanercept/kg/d x 10 wk), 5) OLETF + calcium channel blocker (CCB) (3 mg amlodipine/kg/d x 10 wk), and 6) OLETF + ARB+ ETAN (COMBO) (same doses x 10 wk). OLETF exhibited a 23% decrease (41 ± 4 vs. 32 ± 3 nmol/min/ml) in mean GPx activity. TNFα blockade completely recovered the IR‐associated decrease in GPx activity (45 ± 4 nmol/min/ml) demonstrating that TNFα contributes to the dysregulation of antioxidant enzymes in the IR kidney. Catalase and SOD activities, while elevated, were not different among groups suggesting that the dysregulation of antioxidant enzymes is selective during IR conditions.

Grant Funding Source: APS Teacher Researcher
Disruption of cyclooxygenase type 2 exacerbates the progression of oxidative stress during obstructive nephropathy (1134.2)
Authors: Nilsson Line , Madsen Kirsten , Jensen Boye , Norregaard Rikke
Published: June 2021

Renal oxidative stress is increased in response to ureteral obstruction. In vitro, COX‐2 activity contributes to antioxidative protection. Here, we test the hypothesis that COX‐2 activity counters oxidative stress in an in vivo setting of obstructive nephropathy. The progression of renal oxidative stress was investigated in COX‐2‐/‐ and WT mice subjected to 3 days of unilateral ureteral obstruction (UUO).

Following UUO, kidney weight and functional parameters were analysed. Using immunoblot analysis, we examined the oxidative stress marker, heme oxygenase‐1 (HO‐1), antioxidant enzymes, superoxide dismutase 1 and 2 (SOD1 and ‐2), and caspase 3 in renal cortex and inner medulla (IM).

Plasma creatinine was increased in response to UUO, however, COX‐2‐/‐ had more increased plasma creatinine compared to WT. COX‐2 mRNA level was increased in WT mice subjected to UUO and undetectable in COX‐2‐/‐ mice. HO‐1 protein abundance increased in response to UUO and was further increased in COX‐2‐/‐ mice. Both SOD1 and SOD2 protein levels were downregulated in IM and cortex in WT mice after UUO. COX‐2 knockout prevented downregulation of SOD2 in both cortex and IM whereas SOD1 reduction was only attenuated in cortex. Cortical caspase 3 was increased in response to UUO, and further increased in COX‐2‐/‐ mice.

In summary, disruption of COX‐2 exacerbates oxidative stress and increases apoptosis markers in response to obstruction.
Effects of 17β‐estradiol replacement in a model of renal ischemia in the ovariectomized female apolipoprotein E knockout mouse (1135.2)
Authors: Li Lijun , Ji Hong , Holloway Chelsee , Sandberg Kathryn , Ecelbarger Carolyn
Published: June 2021

Cardiovascular disease is a major cause of morbidity and mortality in postmenopausal women. 17ß‐estradiol (E2) replacement in ovarian hormone deficient rodents inhibits renal and vascular damage in models of ischemia and attenuates inflammatory responses in models of atherosclerosis. We hypothesized that E2 replacement in ovariectomized (OVX) female atherosclerotic mice would protect against vasculature and kidney damage in renal ischemia by attenuating inflammation. Left renal artery incomplete ligation and ovariectomies were performed on 12‐week‐old, female apolipoprotein E (ApoE) knockout (KO) mice and either E2 (2 μg/day x 60 days) or placebo (P) pellets (n = 6/group) were inserted subcutaneously. After 60 days, animals were euthanized and kidneys, blood, and aortic and carotid vessels were obtained. E2 replacement attenuated the ischemia‐induced reduction in left kidney weight [g: OVX + E2, 0.13 ± 0.017 vs. OVX + P, 0.058 ± 0.016; p < 0.02]. E2 treatment had no effect on the contralateral right kidney weight or on body weight. E2 replacement lowered plasma insulin by 18% [µg/L: OVX + E2, 0.33 ± 0.009 vs. OVX + P, 0.40 ± 0.023; p<0.02] and plasma triglycerides by 46% [µg/dL: OVX + E2, 30.3 ± 3.5 vs. OVX + P, 56.6 ± 8.4; p<0.02]. Plasma interleukin‐6 (IL‐6) was increased by E2 [pg/ml: OVX + E2, 156 ± 67 vs. OVX + P, 16.7 ± 7.0; p<0.05]. E2 treatment attenuated the ischemia‐induced glomerular injury, tubular atrophy, interstitial fibrosis and inflammation and neointima proliferation in the thoracic and abdominal aorta and carotid artery. These findings suggest E2 markedly protects the ovarian‐hormone‐deficient atherosclerotic female from the renal and vascular damage induced by renal ischemia. Possible protective means include improvement of the metabolic profile, and a near 10X increase in mean circulating levels of the potentially anti‐inflammatory cytokine, IL‐6.

Grant Funding Source: Supported by NIH DK082507 and Marriott Fellowship
T cell‐dependent hypertension is attenuated in female mice during angiotensin II infusion (1136.10)
Authors: Pollow Dennis , Romero‐Aleshire Mellisa , Uhrlaub Jennifer , Nikolich‐Zugich Janko , Hay Meredith , Brooks Heddwen
Published: June 2021

Previous studies have shown that the adaptive immune system is required for the development of Ang II‐induced hypertension in male mice. Here we determine if there are sex differences in the ability of the adaptive immune system to induce hypertension and alter T cell infiltration and renal dysfunction in response to Ang II infusion. Male and female RAG‐1‐/‐ mice, which lack both T and B cells, received adoptive transfer of male CD3+ T cells 3 weeks prior to Ang II infusion (490ng/kg/min 14d) (n=8). Controls received T cells or Ang II. Blood pressure was monitored via tail cuff. In the absence of T cells, Ang II induced a small increase in systolic blood pressure (SBP) in males & females (Δ10.9 vs. Δ14.1mmHg). After adoptive transfer, Ang II induced a significant increase in SBP in males (Δ36.8mmHg, p< 0.01), while females were no different from Ang II‐only controls (Δ16.9mmHg). PAS staining identified that Ang II infusion significantly increased glomerular hypertrophy in both males and females, independent of T cells; glomerular area was significantly greater in males vs females, at baseline. Renal CD3+, CD4+, CD8+, and Foxp3+ lymphocyte infiltration was significantly greater in males compared to females, independent of Ang II. These results suggest that females are protected from Ang II‐induced T cell‐dependent hypertension, and that this protection may involve sex differences in T cell‐infiltration of the kidney.

Grant Funding Source: Supported by Training Grant HL007249 (J. Burt), UA Sarver Heart Center, ARCS Foundation
High salt intake aggravates aortic dysfunction in rats with adenine‐induced chronic renal failure (1136.13)
Authors: Nguy Lisa , Nilsson Holger , Guron Gregor
Published: June 2021

Rats with adenine‐induced chronic renal failure (ACRF) develop a reduced rate of relaxation, and increased stiffness of the aorta. The aim of this study was to investigate the effects of 2 weeks of high NaCl intake on vascular function in rats with ACRF.

Male Sprague‐Dawley rats received either chow containing adenine (ACRF) or were pair‐fed a control diet (Controls, C). Two weeks prior to sacrifice animals were randomized to diet containing either normal salt (0.6% NaCl, NS) or high salt (4% NaCl, HS), resulting in four groups: C‐NS, C‐HS, ACRF‐NS and ACRF‐HS (n=11‐15 per group). Animals were sacrificed at 7‐10 weeks after study start and mesenteric arteries and thoracic aorta were analyzed with wire myograph.

Animals with ACRF showed significant increases in serum creatinine and systolic blood pressure that was aggravated by high NaCl intake (138±14, 164±10, 152±7 and 202±14 mmHg in groups C‐NS, ACRF‐NS, C‐HS and ACRF‐HS, respectively). A decreased sensitivity to norepinephrine was found in mesenteric arteries of ACRF‐HS rats (P<0.05). In the thoracic aorta, the sensitivity to both sodium nitroprusside (SNP) and acetylcholine (ACh) were reduced only in ACRF‐HS animals (P<0.05). Furthermore, the ACRF‐HS group exhibited a marked decrease in aortic relaxation rate compared to ACRF‐NS animals (P<0.01).

In rats with ACRF two weeks of high NaCl intake reduced the sensitivity to ACh and SNP in the thoracic aorta and aggravated the impairment in relaxation rate whereas vasodilator responses were unaffected in resistance arteries. These results indicate an abnormality in vascular smooth muscle function specifically in the thoracic aorta of ACRF animals that is augmented by high NaCl intake.