bluuebunny/crossref_metadata_2025 huggingface dataset:
Increased Cerebral Endothelial Transcytosis and Blood‐Brain Barrier Permeability in “Slow Pressor” Angiotensin‐II Hypertension
Authors: Iadecola Costantino , Faraco Giuseppe , Lane Diane , Anrather Josef
Published: June 2021

Chronic hypertension (HTN) has deleterious cerebrovascular effects, leading to increased risk of stroke and dementia (AJP 304:H398, 2013). In a the sustained HTN induced by “slow‐pressor” doses of angiotensin‐II (AngII), neurovascular coupling and endothelium‐dependent responses are suppressed. However, the effects of “slow pressor” AngII on the structure and function of the blood‐brain barrier (BBB) remain unclear. Therefore, we examined BBB permeability and ultrastructure in this model of HTN. C57Bl6 male mice were implanted with minipumps delivering saline or AngII (600ng/kg/min). Two weeks later, TRITC‐dextran (4kDa) was administered i.v and the brain conc. of dextran was determined after clearing the intravascular tracer. AngII increased blood pressure (+43±4mmHg; p<0.05; n=8/group) and brain dextran conc. (vehicle: 26±3; AngII: 56±13ng/g; p<0.05). By electron microscopy, slow pressor AngII enhanced endothelial vesicular transcytosis (+80±2%; p<0.05) and reduced by 41±5% the cerebrovascular expression of msfd2a, a BBB‐associated endothelial transmembrane protein that suppresses vesicular transport (Nature 509:507, 2014). Biotinylated AngII, administered i.v. after 2 weeks of AngII, was found in endothelial vesicles, vascular and perivascular cells, and astrocytes. Thus, slow pressor AngII increases BBB permeability. The effect may be due to suppression of msfd2a, leading to enhanced endothelial transcytosis. Circulating AngII crosses the BBB entering perivascular space and brain. Such increase in BBB permeability to circulating agents, including AngII, may contribute to the cerebrovascular and cognitive dysfunction associated with HTN.
Placental Ischemia Impairs Cerebral Blood Flow Autoregulation and Increases Blood‐Brain Barrier Permeability in Pregnant Rats
Authors: Warrington Junie , Fan Fan , Murphy Sydney , Roman Richard , Drummond Heather , Granger Joey , Ryan Michael
Published: June 2021

Cerebrovascular events contribute to ~40% of preeclampsia/eclampsia related deaths, and neurological symptoms are common among preeclamptic patients. We previously reported that placental ischemia, induced by reducing utero‐placental perfusion pressure, leads to impaired cerebrovascular myogenic reactivity and cerebral edema in the pregnant rat. Whether the impaired myogenic reactivity is associated with altered cerebral blood flow (CBF) autoregulation and the edema is due to altered blood‐brain barrier (BBB) permeability is unclear. Therefore, we tested the hypothesis that placental ischemia leads to impaired CBF autoregulation and a disruption of the BBB. CBF autoregulation, measured in vivo by laser Doppler flowmetry, was significantly impaired in placental ischemic rats compared to normal pregnant rats. Brain water content was increased in the anterior cerebrum of placental ischemic rats and BBB permeability, assayed using the Evans blue extravasation method, was also increased in the anterior cerebrum. The expression of the tight junction proteins: claudin‐1 was increased in the posterior cerebrum while zonula occludens‐1, and occludin, were not significantly altered in either the anterior or posterior cerebrum. These results are consistent with the hypothesis that placental ischemia mediates anterior cerebral edema through impaired CBF autoregulation and the associated increased transmission of pressure to microvessels, resulting in increased BBB permeability and cerebral edema.
Aortic Depressor Response is Blunted Following Ovariectomy: Evidence for Involvement of Central but not Peripheral Afferent Pathways in Rat
Authors: Santa Cruz Chavez Grace , Schild John
Published: June 2021

Sex differences in aortic baroreflex (BRx) function are well documented. In female rats, selective electrical stimulation of myelinated aortic baroreceptor fibers evokes a much greater reduction in blood pressure and heart rate than in age‐matched males. Our lab has a longstanding interest in the electrophysiological properties of vagal neurons that give rise to these sexually dimorphic barosensory fibers. We have previously shown that myelinated “Ah‐type” afferent neurons lose their capacity for sustained repetitive discharge after ovariectomy (OVX). Furthermore, 4 or more weeks after OVX the enhanced BRx response of female rats is blunted. This longitudinal study aims to quantify the transition in BRx dynamics over the first 4 weeks after OVX. Age‐matched cohorts of normal (OVI, n = 11) and OVX rats (n = 26) were prepared for bipolar electrical stimulation of aortic depressor nerve. Rats at 1 week post OVX were not significantly different than OVI controls. However, rats that were 2 to 4 weeks post OVX exhibited blunted depressor responses that were 3 – 4 times smaller than OVI. To determine whether Ah‐type afferent fiber conduction was being compromised, we measured electrically evoked compound action potentials (CAP) from additional cohorts of OVI (n = 10) and OVX rats (n = 15) at 2, 3 and 4 weeks after OVX. These studies revealed no significant changes in the recruitment profile of Ah myelinated fibers nor the integrated electrical energy of the CAP. Interestingly, at 4 or more weeks following OVX the CAP showed evidence of temporal dispersion. Collectively, our results suggest that the onset of early changes in BRx function after OVX are likely driven by central mechanisms as the evoked conduction profile of Ah barosensory fibers appears unaltered.
Non‐targeted Effects of a Targeted Toxin
Authors: Talman William , Nitschke Dragon Deidre , Lin Li‐Hsien
Published: June 2021

Saporin (SAP) alone or conjugated either with stabilized substance P (SSP‐SAP) or with an antibody to dopamine β‐hydroxylase (anti‐DBH‐SAP) leads to attenuation of arterial baroreflexes when injected bilaterally into the nucleus tractus solitarii (NTS) of rats. Effects on cardiovascular reflex control could be due to an impact of the toxin on cells that express not only the target protein but also other receptors. To test that hypothesis we assessed effects of glutamate, N‐methyl‐D‐aspartate (NMDA), or α‐amino‐3‐hydroxy‐5‐methylisoxazole‐4‐proprionic acid (AMPA) injected into the NTS at the same site as a prior injection of SAP, anti‐DBH‐SAP, SSP‐SAP, 6‐hydroxydopamine (6‐OHDA) or phosphate buffered saline (PBS). 6‐OHDA was included because our previous studies showed that its injection into the NTS led to loss of noradrenergic neurons while not affecting other neuronal types. Also, 6‐OHDA did not lead to loss of astrocytes as seen after injection of anti‐DBH‐SAP, SSP‐SAP, or SAP into NTS. In control rats that received PBS, glutamate agonists produced dose‐related hypotensive, bradycardic effects, which were significantly reduced in animals treated with SAP containing toxins. Prior injection of 6‐OHDA did not affect bradycardic or hypotensive responses elicited by glutamate receptor agonists in the NTS. This study provides a cautionary note in interpreting physiological responses resulting from use of saporin conjugates in that, while selectively damaging neurons with a specific target, the toxins will also disrupt other neuronal functions medicated by the same neuron but through different mechanisms. Further, the study supports the concept that astrocytes in NTS play a role in mediating central cardiovascular control. Support: VA Merit Review and NIH R01 HL59593
Baroreflex impairment in a rat model of Parkinson's disease
Authors: Cavalleri Marina , Barna Barbara , Moreira Thiago , Takakura Ana
Published: June 2021

Parkinson's disease (PD) exhibit motor and non‐motor symptoms. Among non‐motor symptoms, the cardiovascular autonomic dysfunction is frequently. The aim of this work was to evaluate whether in a model of PD induced by injection of the toxin 6‐hydroxy‐dopamine (6‐OHDA) into the striatum was able to produce changes in the baroreflex as well as in neurons in brainstem areas enrolled in cardiovascular control. Wistar rats (300‐320 g, n = 5) received bilateral injections of 6‐OHDA or vehicle into the striatum. After 60 days,baroreflex was assessed by determining the heart rate (HR) responses to ramped infusions of phenylephrine (PE) and sodium nitroprusside (SNP)induced mean arterial pressure (MAP) changes. After, the rats were anesthetized for brain perfusion and processed for tyrosine hydroxylase (TH) and the transcription factor Phox2b immunoreactive. PE produced a similar increased in MAP in 6‐OHDA and control groups. However, the bradycardia was blunted in 6‐OHDA (D = ‐7.5±0.5 to ‐62±6 vs. vehicle: ‐29.5±1.2 to ‐148.5±9.8 bpm). In 6‐OHDA, the SNP produced a smaller decrease in MAP (Δ = ‐18±2.6 to ‐24.5±0.8 vs. vehicle: ‐60.3±2 to ‐96.5±1.9 mmHg) and a smaller increase in HR (Δ = 34.75±3.4 to 59.5±7.8 vs. vehicle: 61±7.8 to 101.3±6 bpm). Ocurred a reduction in TH neurons in the substantia nigra (85%), in the C1 regions (70%) and in the Phox2b+ neurons in the NTS (48%). We observed an impairment of baroreflex sensitivity in the 6‐OHDA model of PD, which could be correlated with a reduction in the neuronal cytoarchitecture of brainstem regions involved in cardiovascular control

Financial support: FAPESP, CNPq, CAPES/PROEX
Role of Alpha 2‐adrenoceptors in Hemodynamic Instability in Rats Under Cooling Stress
Authors: Tung Che‐Se , Liu Yia‐Ping , Lee Po‐Lei , Tseng Ching‐Jiunn
Published: June 2021

Cooling causes hemodynamic instability as changes in blood pressure variability and heart rate variability (BPV, HRV). We observed that increased sympathomyogenic responses (SMR) as changes in very‐low frequency of BPV (VLFBPV) and low frequency of BPV (LFBPV) which were dependent on the evoked pressor to cooling; the present study was designed to examine whether the α2‐adrenoceptor (AR) is involved in such cooling effects of rats. Hemodynamic changes were telemetry monitored throughout the experiments. Vehicle or yohimbine (YOH, 2.5 or 5.0 mg/kg) was administered with a latency (5 min, 30 min) before a cooling impact (CI, 4±2oC). Plasma nitric oxide (NO) was measured in presence or absence of the added hexamethonium (HEX). Spectral powers of high frequency (HF, 0.6‐3.0 Hz), low frequency (LF, 0.2‐0.6 Hz), and very‐low frequency (VLF, 0.02‐0.2 Hz) in BPV bands were compared with those in HRV bands. Results generally showed 1) cooling‐evoked pressor was significantly attenuated after YOH treatment, 2) Compared with vehicle prior to the CI start, YOH produced tachycardia and increased most frequency powers but reduced the normalized HFHRV (nHFHRV), 3) In contrast, compared with control vehicle throughout the CI trial, YOH produced less pressor, more tachycardia, and increased nHFHRV but reduced most the other frequency powers. Besides, the dicrotic notch was undetectable and NO was increased in CI trial after YOH treatment, in which the increase in NO was diminished after HEX addition. This study confirmed a functional role of α2‐AR which is imperative for the immediate SMR to the cooling‐induced hemodynamic instability.
Assessment of Guanethidine‐induced Neuronal Loss on Basal and Reflex Sympathetic Support of Blood Pressure and Heart rate in the Anesthetized Rat
Authors: Zahner Matthew , Liu Chang‐Ning , Okerberg Carlin , Somps Chris
Published: June 2021

The goal of this study was to determine the degree of sympathetic postganglionic neuronal loss required to impair cardiovascular related sympathetic activity. Rats were treated daily with vehicle (saline, n=10) or guanethidine (100 mg/kg). To produce variable degrees of neuronal loss two separate groups were treated, one for 5 days (n=9) and another for 11 days (n=10), followed by recovery to allow acute effects of guanethidine to dissipate. Sympathetic reflex activity was measured by renal sympathetic nerve activity (RSNA) during baroreflex and chemoreflex testing. Prior to testing, basal RSNA, blood pressure (BP), and heart rate (HR) were recorded. Thoracic ganglia (T13) were examined to determine the extent of postganglionic neuronal loss. Despite some neuronal loss in both treated groups there was no significant effect on resting BP or HR in either group. Rats treated for 5 days had a modest decrease in basal RSNA (63 ± 13%) whereas rats treated for 11 days had significantly lower basal RSNA (37 ± 8%, P < 0.05) compared with vehicle‐treated rats (100 ± 5%). Baroreflex testing in vehicle‐treated rats elicited a 183 ± 20% increase in RSNA. Compared with vehicle‐treated rats baroreflex mediated increases in RSNA were significantly attenuated in 11‐day (59 ± 11%, P < 0.05) but not 5‐day treated rats (122 ± 23%). Chemoreflex testing in vehicle‐treated rats elicited a 270 ± 23% increase in RSNA. Compared with vehicle‐treated rats chemoreflex‐mediated increases in RSNA were significantly attenuated in both the 5‐day (158 ± 32%, P < 0.05) and the 11‐day treated rats (76 ± 15%, P < 0.05). These data provide important insight on the degree of neuronal loss required to impair sympathetic responsiveness and support of BP and HR.
Nitrous Oxide Inhalation Attenuates an Increase in Muscle Sympathetic Nerve Activity during Cold Stress
Authors: Isono Kazushige , Okada Yoshiyuki , Mitsui Tatsuhisa , Masuda Yuji , Ogasawara Tadashi
Published: June 2021

Nitrous oxide inhalation was reported to suppress pressor response to pain stimulation, while an increase in heart rate (HR) remained unchanged. We evaluated whether nitrous oxide inhalation attenuates an increase in muscle sympathetic nerve activity (MSNA) during cold stress. MSNA (microneurography), arm cuff and beat‐to‐beat blood pressure (BP), and HR were measured during 5‐min baseline, 2‐min cold pressor test (CPT) in 12 young men [30±1 (SE) yrs] after they receive room air, 100% oxygen, 30%, and 40% nitrous oxide for 20 min through a mask, respectively. BP increased during CPT in all conditions, while the increase of systolic BP tended to be smaller during inhalation of 40% nitrous oxide than room air (26±5 vs 15±4 mmHg, P=0.081). HR also increased during CPT without any difference the increase between the conditions. MSNA burst frequency increased during CPT, while the increase was smaller during the inhalation of 40% nitrous oxide than room air (21±4 vs 12±3 burst/min, P=0.045). Change (Δ) in systolic BP during CPT was correlated with ΔMSNA burst frequency (r=0.40), but not ΔHR (r=0.06) in all conditions. These results suggest that suppressed pressor response to pain stimuli by inhalation of nitrous oxide is attributable to the attenuation of increase in muscle sympathetic nerve activity.

Supported by JSPSKAKENHI70566661.
Afferent Activity is Greatly Increased by Spontaneous Phasic Contractions in Mouse Ex Vivo Urinary Bladder
Authors: Heppner Thomas , Tykocki Nathan , Nelson Mark
Published: June 2021

Spontaneous phasic contractions (SPCs) are transient, non‐voiding contractions during urinary bladder filling that hallmark many bladder pathologies. However, the origin and physiological importance of SPCs are largely unknown. Using an ex vivo mouse urinary bladder preparation, we examined the relationship between SPCs and afferent nerve activity. Continuous bladder filling (30 µl/min) alone increased baseline afferent activity (~100 Hz per 4 mmHg pressure). Strikingly, over the same pressure range, SPCs evoked bursts of afferent activity that were more than 3‐fold greater than baseline afferent activity (>350 Hz) and often contained large‐amplitude action potentials not normally observed in the absence of SPCs. The frequency of afferent bursts correlated with the SPC rate of rise, and peaked prior to the associated SPC. Inhibition of neuronal Na+ channels blocked afferent nerve activity without altering SPC amplitude, duration or rate of rise. In contrast, inhibition of smooth muscle voltage‐dependent Ca2+ channels (VDCCs) abolished SPCs as well as all associated afferent bursts, indicating afferent bursts coupled directly to the smooth muscle contractile machinery. However, In the presence of VDCC blockers, mechanical deformation of the bladder evoked SPC‐like events and afferent bursts with an amplitude and SPC/afferent ratio typical of “natural” SPCs. This supports the concept that mechanical distention of the bladder by SPCs generates bursts of sensory nerve activity that dominate bladder afferent outflow. These findings suggest that SPC‐dependent bursts of afferent activity may convey sensory information about bladder fullness to the CNS, and thus contribute to the regulation of micturition frequency. Supported by NIH R37DK053832.
Self‐Paced Slow Deep Breathing: Persistence of Effects on Vascular Function
Authors: Kelly K , Dick T , Lin J , Moser J , Wehrwein E
Published: June 2021

Slow deep breathing (SDB) exercises have been practiced for centuries for their health benefits. In order to have a long‐term therapeutic benefit the effects of SDB must persist. We tested the hypothesis that physiological effects persist after an acute SDB bout. Healthy subjects (n=14, 10M/4F, age 18‐35) rested supine for 20 minutes (baseline), followed by 20 minutes of self‐paced SDB without any device‐guided cues, then completed 20 minutes of endogenous breathing (recovery). Venous occlusion plethysmography was used to measure forearm blood flow (FBF: ml/min/100 dl tissue) to assess vasodilation. Respiratory rate (RR), monitored by a double pneumobelt, was 13.4 ± 0.74 breaths/min during baseline, 6.03 ± 0.51 during SDB (p<0.05), and 12.75 ± 0.95 during recovery. FBF increased immediately during the first minute of SDB (SDB: 2.3 ± 0.2 vs Baseline: 1.7 ± 0.2, p<0.05) and persisted to the end of recovery. Participants were categorized into four bins: high (n=3), medium (n=7), low (n=4), and negative (n=2) responders, which had mean percent changes in FBF during SDB of +49.1%, +27.6%, +8.4%, and ‐5.6%, respectively, where positive changes indicate vasodilation. SDB resulted in vasodilation in 86% of subjects and 43% had sustained effects lasting at least 20 min. One potential factor in responses to SDB is the level of mindfulness achieved by the subjects, which may account for the varied responses. We are now assessing state and trait anxiety scores along with psychological assessments of mindfulness to see if psychological aspects are predictive of response magnitude. The persistence of responses is promising for long‐term efficacy of a regular SDB practice and it is vital to better understand the variability in responses to ensure that SDB practitioners yield maximum benefit.
Dissociating the Metabolic and Cardiovascular Effects of Leptin Through mTORC1 Signaling
Authors: Rouabhi Mohamed , Bell Balyssa , Morgan Donald , Rahmouni Kamal
Published: June 2021

Leptin is an adipocyte‐derived hormone which acts in the brain to regulate energy homeostasis by suppressing appetite and increasing energy expenditure. Leptin also promotes arterial pressure elevation by increasing sympathetic nerve activity (SNA). Recent evidences indicate that the mechanistic target of rapamycin complex 1 (mTORC1) plays a key role in mediating the metabolic and sympathetic effects of leptin. To test the consequence of disrupting mTORC1 on leptin actions, we conditionally deleted Raptor, a critical subunit of mTORC1, in leptin receptor (LRb) expressing cells using genetically modified mice in which the Raptor gene is flanked by Lox‐P sites (Raptorfl/fl) and Cre recombinase driven by the LRb promoter (LRbCre). We measured body weight weekly from 4 weeks of age and observed no differences between LRbCre/Raptorfl/fl and control littermates (29.6±0.8g vs 31.0±0.8g at 14 weeks). Additionally, food intake and body weight were recorded daily at baseline and after treatment with vehicle or leptin (1mg/g bw, intraperitoneally, twice daily) for 4 days. We found no significant difference in the cumulative decrease in food intake (‐1.6±0.8g vs ‐1.1±1.7g) or body weight (‐5.9±0.8% vs ‐5.7±0.7%) caused by leptin between LRbCre/Raptorfl/fl mice and WT littermates. Finally, we performed multifiber nerve recordings of renal SNA to determine the contribution of mTORC1 to the cardiovascular sympathetic action of leptin. Interestingly, intracerebroventricular injection of leptin (2 mg) increased renal SNA in control mice (106±20%), but not in LRbCrexRaptorfl/fl mice (‐28±11%, P<0.05 vs controls). We concluded that mTORC1 is necessary for the renal SNA effect of leptin, but not critical for the metabolic actions of leptin.
Assessment of Neuronal Activity in the Rostral Ventrolateral Medulla (RVLM) of Conscious Rats
Authors: Huereca D , Wong C , Ingles J , Ghoddoussi F , Berkowitz B , Holt A , Mueller P
Published: June 2021

Hyperactive bulbospinal neurons in the RVLM and elevated sympathetic activity likely contribute to cardiovascular disease (CVD). Measuring RVLM neuronal activity in conscious rats would represent a major advance in understanding brain‐related CVD. The present study addressed this by developing manganese (Mn2+) enhanced magnetic resonance imaging (MEMRI). Mn2+, a paramagnetic ion, enters active neurons via Ca2+ channels and with MRI, allows visualization and quantification of neuronal activity. The purposes of this study were to use MEMRI to: 1) identify regions in RVLM with tonically active neurons, 2) characterize a time course of Mn2+ in RVLM after systemic injection, 3) evaluate the sensitivity of MEMRI by varying dose, and 4) compare Mn2+ in rostral vs caudal RVLM. We hypothesized that Mn2+ increases in RVLM 1 d after i.p. injection; is dose‐dependent; and exhibits rostrocaudal variation. Tract‐tracing and direct RVLM injection anatomically and functionally confirmed RVLM on MRI images. Ten male SD rats were administered MnCl2 (33 or 66 mg/kg i.p., n=5 ea). MRI scans on a 7T ClinScan demonstrated increased Mn2+ in RVLM at 1 d (p=0.002) and was higher after 66 mg/kg vs 33mg/kg (p=0.019). Mn2+ returned to baseline by 15 and 22 days (33 and 66 mg/kg, respectively). After 66 mg/kg, increased Mn2+ occurred in more rostral vs caudal regions of RVLM (p=0.004), consistent with our previous dendritic branching study. Results highlight MEMRI as a viable and powerful new method for examining neuronal activity in the RVLM of conscious rats and understanding mechanisms related to brain‐related CVD.
Chemical Stimulation of the Arterial Chemoreflex Activates Neuroendocrine and Pre‐sympathetic Corticotropin Releasing Hormone (CRH) Neurons in the Paraventricular Nucleus of the Hypothalamus (PVN)
Authors: Coldren K. , Kline David , Hasser Eileen , Heesch Cheryl
Published: June 2021

Acute hypoxia (AH) increases corticosteroid secretion through activation of the arterial chemoreflex central pathway including the PVN. Previously we found that CRH was present in pre‐sympathetic neurons, but, although AH (10% O2, 2 hrs) activated CRH‐IR cells (~14%), pre‐sympathetic neurons in the PVN were not activated. We hypothesized that, compared to neuroendocrine cells, a more potent chemoreflex stimulus might be required to activate pre‐sympathetic PVN neurons. Conscious rats received repeated i.v. sodium cyanide (NaCN) or saline (Sal); 10 injections once every 3 minutes, followed by immunohistochemical evaluation of Fos‐ (index of activation) and CRH‐IR. Pre‐sympathetic cells in the PVN were identified by prior retrograde tracer injections into the RVLM & IML. More CRH cells were activated by NaCN (CRH+Fos; 34±5%) compared to Sal treated rats (9±2%), with a substantial portion located in the medial parvocellular region (presumed neuroendocrine). NaCN also activated RVLM (11±2 vs 6±1%) and IML projecting cells (13±2 vs 6±1%). Of the activated pre‐sympathetic cells in NaCN treated rats, 83±4% of RVLM and 80±4% of IML projecting cells were CRH‐IR. Thus, activation of the chemoreflex with NaCN excites both neuroendocrine (medial parvocellular) and pre‐sympathetic CRH neurons in the PVN. Compared to AH, NaCN treatment activated more CRH (34±5 vs 15±3%) and IML projecting cells (13±2 vs 5±0.1%). Future experiments will evaluate differential regulation of CRH‐neuroendocrine vs CRH‐pre‐sympathetic cells in the PVN. [NIH HL 98602]
Acute Hypoxia (AH) Potentiates Cardiorespiratory Responses to Blockade of the Anti‐oxidant Enzyme, Glutathione Peroxidase (GPX), in the Nucleus Tractus Solitarius (nTS)
Authors: Heesch Cheryl , Phaup J. , Kline David , Hasser Eileen
Published: June 2021

AH in conscious rats activates the arterial chemoreflex and increases reactive oxygen species (ROS) in the nTS. We previously showed that mRNA for the anti‐oxidant enzyme GPX in the nTS is increased with AH (10% O2, 2 hr), possibly as an early protective response to AH. Current experiments evaluated effects of AH on cardiorespiratory responses to GPX blockade in the nTS with mercaptosuccinic acid (MCS). Mean arterial pressure (MAP), heart rate (HR), and splanchnic sympathetic & phrenic nerve activities (SSNA; PhNA) were measured in anesthetized (inactin, 100 mg/kg), paralyzed (gallamine, 25 mg/kg/hr), artificially ventilated, vagotomized male rats. Increasing concentrations of MCS (0.05, 0.1, and 0.5M) were microinjected (30 nl) into the nTS before (MCS‐1) and after (MCS‐2) 4 evenly spaced bouts (5 min) of hypoxia (O2 saturation ~50%) over a 2 hr period. SSNA and PhNA were expressed as % baseline before MCS‐1. In rats exposed to AH, compared to time controls (no hypoxic stimuli), baseline values increased between MCS‐1 and MCS‐2: HR (+29± 5 vs +1± 3 bpm), SSNA (+43± 8 vs +5± 8%), overall PhNA (+47± 24 vs ‐3± 2 %) and Ph amplitude (+112± 27 vs +26± 32%). Increasing concentrations of MCS progressively decreased MAP, HR, SSNA, and PhNA (rate and amplitude) in all rats. However, responses to GPX blockade in the nTS were augmented following AH (ΔMCS‐2 > ΔMCS‐1): Maximum ΔMAP (‐39± 2 > ‐33± 2 mm Hg); ΔSSNA (‐113± 9 > ‐80± 2 %); ΔPhNA (‐94± 19 > ‐ 38± 8 %). Thus, consistent with increased mRNA for GPX, cardiorespiratory effects of GPX in the nTS appear to be augmented following AH. [NIH HL 98602]
Glycemic State Regulates Brain Derived Neurotrophic Factor Responsiveness of Neurons in the Paraventrucular Nucleus
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 paraventricular nucleus (PVN). The PVN is a complex integrative autonomic centre with critical roles in the coordinated control of food intake. Recently, studies have shown that glucose‐sensing neurons in areas of the hypothalamus, including PVN, exhibit altered excitability in response to changes in extracellular glucose. Here, whole cell current‐clamp recordings were employed on rat PVN neurons in slice preparation to evaluate the ability of these cells to integrate multiple metabolically relevant signals. BDNF (2nM) was bath applied in both high (10mM) and low (0.2mM) extracellular glucose concentrations. BDNF applied to PVN neurons (N=48) in 10mM glucose elicited varying responses with 50% depolarizing (mean change in membrane potential ± standard error: 9.0 ± 1.2 mV), 23% hyperpolarizing (mean: ‐6.7 ± 1.4 mV), and 27% showing no response to BDNF treatment. However, BDNF's effects were profoundly altered in hypoglycemic conditions. BDNF application to PVN neurons in 0.2mM glucose (N=15) elicited primarily hyperpolarizing effects (73%, mean: ‐6.4 ± 0.9 mV) apart from one cell that depolarized (7%, mean: 4.8 mV). Our findings demonstrate the potential role of BDNF in feeding through its impact on the excitability of PVN neurons and how these responses are altered by glycemic state.
Cervical Epidural Stimulation Elicits Evoked Potentials and Modulates Diaphragm EMG
Authors: Dale Erica , Gad Parag , Zhong Hui , Roy Roland , Edgerton V Reggie
Published: June 2021

Approximately 2/3 of traumatic spinal injuries occur at the cervical level leading to severe respiratory dysfunction. There are currently few clinical interventions for recovery from complete ventilator dependence after spinal cord injury. Our lab has shown that the spinal circuitry involved in locomotion can be stimulated via epidural stimulation by modulating the excitability of the lumbar spinal circuitry and enabling complex motor tasks after complete paralysis. We hypothesized that cervical epidural stimulation could modulate excitability of phrenic motor circuitry (and, thus, breathing) in much the same way. In this study, adult, female, Sprague‐Dawley rats (n=2) were implanted with bilateral cervical (C4, an area within the phrenic motor pool) epidural stimulating electrodes and bilateral diaphragm recording electrodes. Under isoflurane anesthesia, one rat was stimulated at C4 with varying intensities, frequencies, and polarities. With increasing voltage (1‐8V), amplitude of spinal evoked potentials (SEP) increased accordingly. Ten SEPs were averaged in each voltage range from 1‐8V. Right to left bilateral stimulation elicited SEPs from 0.41± 0.002mV to 2.3±0.001mV (LDia) and 0±0.0001mV‐2.0±0.001mV (RDia); left to right: 1.50±0.001mV‐4.32±0.003mV (LDia) and 0.70±0.0001mV‐2.12±0.002mV (RDia); right to ground: 0.45±0.0002mV‐2.0±0.001mV (LDia) and 1.0±0.001mV‐3.9±0.002mV (RDia); left to ground: 0.82±0.001mV‐2.4±0.002mV (LDia) and 0.18±0.0002mV‐3.0±0.003mV (RDia). Latencies ranged from 2.9‐3.1ms. In other experiments, an unanesthetized rat was given the same epidural cervical stimulation but via current‐based stimulation parameters (400‐1000uA). Right to left stimulation elicited SEPs from 2.1±0.003mV‐5.8±0.004mV (LDia); left to right: 4.0±0.003mV‐6.8±0.004mV (LDia); right to ground: 1.9±0.002mV‐6.7±0.004mV (LDia); left to ground: 1.9±0.002mV‐2.8±0.002mV (LDia). Latencies ranged from 2.9‐3.3ms. LDia EMG amplitude increased by 23.37±3.2% of baseline value (vs ‐2.0±1.3% by RDia) at 8V, right to left stimulation, 1Hz indicating at least a short term modulation of phrenic motor networks. These data demonstrate that cervical epidural stimulation at the phrenic motor pool elicits activation of diaphragmatic motor units. This short‐term modulation of the phrenic motor network offers great therapeutic potential for patients who are ventilator‐dependent after spinal cord injury. Further study is required to determine if long‐term stimulation can lead to plasticity of these highly plastic motor networks. Supported by U01EB015521 and T32 NS058280.
Alaskan Botanicals Influence Neuronal Aging
Authors: Scerbak Courtney , Vayndorf Elena , McGill Colin , Taylor Barbara
Published: June 2021

Aging is a risk factor for many life‐threatening disorders, including neurodegenerative diseases. Many genetic mutations and environmental factors that increase lifespan may also postpone these age‐related diseases. The overall goal of this project is to examine the effect of Alaskan botanicals on the aging nervous system, using the nematode model system, Caenorhabditis elegans. We tested the impact of crude extracts from three edible, medicinally beneficial Interior Alaskan botanicals (Vaccinium uliginosum [bog blueberry], Empetrum nigrum [crowberry], Vaccinium vitis‐idaea [lowbush cranberry], Inonotus obliquus [chaga]) on whole animal lifespan and markers of neuronal aging. Specifically, we evaluated age‐ and botanical‐related changes in neuronal morphology and function of mechanosensory and GABAergic motor neurons using fluorescently labeled C. elegans strains. We find that these botanicals are able to extend wildtype C. elegans lifespan by up to 35%. Preliminary results suggest that mechanosensory neuronal morphology and function are also affected, but not GABA‐ergic neurons. We explored the mechanism of action of these botanicals through determining the extracts' phenolic, flavonoid, and anthocyanin composition and in vivo DAF‐16, SKN‐1, and HSF‐1 transcription factor activation following treatments. Through this botanical screen, we will scientifically validate and underscore the value of ecological resources endemic to the Arctic, elucidate the mechanism of changing neuronal morphology and functionality with age, and, perhaps, establish precedent for novel therapies for neurodegenerative diseases.
Osmotically‐induced Vasopressin Secretion is Not Altered in Novel Knock‐Out Rats Lacking Transient Receptor Potential Vanilloid (TRPV1) Channel
Authors: Tucker Andrew , Stocker Sean
Published: June 2021

Changes in osmolality are detected by specialized neurons in the hypothalamus to stimulate vasopressin (VP) secretion and thirst. Recent in vitro evidence suggests this is a mechanical process mediated by the TRPV1 channel. Using novel TRPV1‐/‐ rats produced by Zn‐finger nuclease deletion of Exon 13, we tested the hypothesis that VP secretion and thirst stimulated by hyperosmolality would be attenuated in conscious TRPV1‐/‐ (n=10) vs control rats (n=10) chronically instrumented with femoral arterial and venous catheters. There were no differences between TRPV1‐/‐ vs control rats in baseline plasma osmolality (295±1 vs 295±1 mOsm/L, P>0.7) and VP levels (2±1 vs 1±1 pg/mL, P>0.6). Infusion of 2M NaCl (1mL/h, IV) in TRPV1‐/‐ vs control rats produced similar increases in osmolality at 30 min (302±1 vs 301±2 mOsm/L; P>0.6) and 60 min (303±2 vs 305±3 mOsm/L, P>0.4). Surprisingly, plasma VP levels were not different between TRPV1‐/‐ vs control rats at 30 min (10±1 vs 8±1 pg/mL, P>0.5) and 60 min (17±2 vs 15±2 pg/mL, P>0.5). There was a significant correlation between plasma VP levels and osmolality (P<0.01) but no differences between TRPV1‐/‐ vs control rats (P>0.8). When water bottles were returned to cages after the vasopressin experiments, TRPV1‐/‐ vs control rats ingested similar amounts of water (6.6±1.1 vs 5.0±0.6 mL, P>0.2). Blood pressure, heart rate, and urinary sodium excretion were not different between groups. Deletion of TRPV1 was confirmed by PCR, western blot, and the depressor response to the TRPV1 agonist capsaicin (0.5µg, IV) (control: ‐47±7 vs TRPV1‐/‐: 3±1 mmHg, P<0.001). These findings suggest that a mechanism other than TRPV1 channels mediate osmosensory processes in the central nervous system.
Fructose Driven Vasopressin (VP) Secretion May Contribute to Chronic Kidney Diseases (CKD) under Chronic Dehydration
Authors: Song Zhilin , Garcia Miguel , Roncal Carlos
Published: June 2021

Chronic recurrent dehydration (WAN) can cause CKD, through a fructokinase (KHK) dependent way. VP is a peptide hormone that is synthesized in the hypothalamic supraoptic nucleus (SON) and paraventricular nucleus (PVN), and is subsequently transported to and stored in posterior pituitary (PP), from where it is released into systemic circulation. VP may mediate some types of CKD through currently unclear mechanisms. Here we propose a novel mechanism for CKD resulted from chronic dehydration, that chronic dehydration may activate a local polyol pathway: aldose reductase (AR)‐fructose‐KHK, leading to elevated VP and thus worsening CKD. Current studies have shown existence of KHK mRNA in hypothalamic area and expression of KHK protein in both SON and PVN. The rise of copeptin, a serum marker of VP, was observed in wild type (WT)/WAN mice, but not in KHK‐KO/WAN mice. Further analysis revealed that VP storage in PP was elevated in WT/WAN mice compared with control WT mice, but was greatly reduced to about control level in KHK‐KO/WAN mice. KHK‐KO did not significantly affect VP storage in PP or serum copeptin levels in control mice. These results demonstrate that chronic dehydration may lead to CKD, at least partially, through elevated VP synthesis and secretion driven by activation of the AR‐fructose‐KHK polyol pathway. Future studies will further clarify the underlying mechanisms.
5HT7 Receptor Induced Phrenic Motor Facilitation Requires EPAC and mTORC1, But Not PKA Activity
Authors: Fields Daryl , Mitchell Gordon
Published: June 2021

Serotonin type‐7 (5HT7) receptors in the cervical spinal cord elicit complex effects on respiratory motor activity. For example, intermittent 5HT7 receptor activation gives rise to long‐lasting phrenic motor facilitation (pMF); on the other hand, 5HT7 receptor activation constrains competing serotonin type‐2 induced pMF via protein kinase A (PKA)‐dependent “cross‐talk inhibition.” We hypothesized that divergent signaling pathways give rise to these distinct 5HT7 receptor actions. Specifically, whereas PKA regulates cross‐talk inhibition of 5HT2‐dependent pMF, a different cyclic AMP signaling pathway that requires exchange protein activated by cyclic AMP (EPAC) underlies 5HT7‐dependent pMF. Anesthetized, paralyzed and ventilated rats receiving intermittent 5HT7 receptor agonist injections (3x5uL; AS‐19) into the C4 intrathecal space expressed pMF for at least 90 minutes. This 5HT7‐induced pMF was abolished by pretreatment with a selective EPAC inhibitor (ESI‐05), but not a selective PKA inhibitor (KT‐5720). Further, an intrathecal EPAC activator (8‐pCPT‐2′‐O‐Me‐cAMP) is sufficient to elicit pMF. mTORC1 inhibition (rapamycin) abolished both 5HT7‐ and EPAC‐induced pMF. Thus, spinal 5HT7 receptors elicit pMF by a non‐canonical EPAC‐AKT‐mTORC1 pathway, and constrain alternate pathways to spinal respiratory motor plasticity via PKA signaling.
How Respiratory Muscle Training Affects Breathing in Individuals with Spinal Cord Injury
Authors: Aslan Sinan , Brown Eddie , Chopra Manpreet , Legg Bonnie , Singh Goutam , Aslan Sevda , Ovechkin Alexander
Published: June 2021

There are an estimated 10,000 to 12,000 spinal cord injuries (SCI) every year in the United States. Besides motor and sensory functional deficits, respiratory insufficiency is also common in individuals with SCI due to paralysis, muscle weakness and/or spastic contractions of the muscles involved in respiration. Pulmonary diseases such as pneumonia and restrictive lung disease are the number one reason for death in individuals with SCI. The aim of the study was to see how respiratory muscle training (RMT) affected the pulmonary function measured with spirometry tests. The outcomes were the forced vital capacity (FVC) and forced expiratory volume in one second (FEV1) and surface electromyography (sEMG). The results showed that both the FVC and FEV1 improved significantly after RMT. Individuals with SCI showed significantly lower sEMG activities recorded from intercostal, diaphragm, rectus abdominis and oblique during expiratory part of spirometry test. The training increased sEMG activity during both inspiration and expiration parts of the test, and this improvement was higher in thoracic SCI compared to cervical SCI.
Mid‐Cervical Spinal Neuron Activity during Intermittent Hypoxia
Authors: Streeter Kristi , Patel Shreya , Gonzalez‐Rothi Elisa , Reier Paul , Baekey David , Fuller David
Published: June 2021

The cervical spinal cord contains a complex propriospinal network, and cervical spinal interneurons show both inspiratory‐ and expiratory‐related discharge patterns. However, the role of cervical interneurons in spinal respiratory plasticity is unknown. We hypothesize that a stimulus that induces plasticity in phrenic motor output – intermittent hypoxia (IH) ‐ also triggers plasticity of cervical interneuron activity. Bilateral phrenic motor output was recorded in anesthetized, ventilated adult rats and a multi‐electrode array used to record mid‐cervical spinal neuron activity during baseline, 3, 5‐min hypoxic episodes (11% O2) separated by 5‐min of baseline oxygen (50% O2), and for 60‐min after hypoxia. During baseline (i.e. hyperoxia, normocapnia), four basic interneuron discharge patterns have been observed: 1) phasic inspiratory, 2) phasic expiratory, 3) tonic, or 4) silent. IH altered the firing rate of most (>90%) phasic and tonic neurons, and some tonic neurons shifted to an inspiratory firing pattern. IH also triggered recruitment of previously “silent” neurons. Evaluation of the eta squared (h2) statistic, which estimates the strength of respiratory‐related discharge, indicated increased respiratory modulation in most neurons after IH. These preliminary data suggest that mid‐cervical spinal interneurons express IH‐induced plasticity consistent with the hypothesis that IH promotes synaptic coupling between phrenic motor neurons and interneurons. Support: NIH 1R01NS080180‐01A1 (DDF), State of Florida Brain and Spinal Cord Injury Research Program (DDF, PJR).
Anatomical Characterization of Phrenic Afferent Projections Following Cervical Spinal Cord Injury
Authors: Nair Jayakrishnan , Gonzalez‐Rothi Elisa , Armstrong Gregory , Reier Paul , Fuller David
Published: June 2021

The phrenic nerve has a rich component of sensory afferents, and there is some evidence that activation of these afferents can affect neuroplasticity in the phrenic motor system after cervical spinal cord injury (SCI). We are presently examining the neuroanatomical substrate that could enable phrenic afferent modulation of the spinal phrenic circuitry following high cervical SCI. Cholera toxin‐β (CT‐β) was applied to the cut end of the phrenic nerve to label spinal phrenic afferent projections in spinal‐intact adult Sprague‐Dawley rats and at 2‐8 wks‐ or 1 yr after lateralized C2 spinal hemisection (C2Hx). Cervical segments (C3‐C5) were also stained for C‐fiber projections using calcitonin gene related peptide (CGRP) and isolectin‐IB4. In spinal‐intact animals, CT‐β ‐labeled phrenic afferent projections terminated primarily in the ipsilateral mid‐cervical dorsal horn (laminae II‐III) and the intermediate grey matter (laminae IV‐V). CGRP and IB4 showed positive labeling in laminae I‐II. Coupling CT‐β labeling with transynaptic labeling of the phrenic motor circuit (via pseudorabies virus) demonstrated co‐labeling of afferent projections and spinal interneurons that are connected to the phrenic circuit. Contralaterally projecting phrenic afferents were not observed in spinal intact rats; however, axonal extensions across the midline were observed one year after C2HX. These early findings demonstrate primary afferent relationships with the spinal phrenic circuit that may play a role in post‐SCI respiratory neuroplasticity.Funding: NIH 1R01NS080180 (DDF), State of Florida Brain and Spinal Cord Injury Research Program
Serotonergic Innervation of Spinal Interneurons Synaptically Coupled with Phrenic and Intercostal Motor Pools
Authors: Gonzalez‐Rothi Elisa , Ross Heather , Armstrong Gregory , Streeter Kristi , Cerreta Anthony , Reier Paul , Fuller David
Published: June 2021

Spinal serotonin (5‐HT) receptor activation is both necessary and sufficient to trigger persistent increases in respiratory output. While there is a prevalence of interneurons synaptically integrated into the phrenic and intercostal circuits, it is unknown whether these cells are innervated by 5‐HT and/or express relevant receptors. Accordingly, we investigated 5‐HT innervation and receptor expression of respiratory interneurons in the cervical and thoracic spinal cord. Pseudorabies virus, a retrograde transynaptic tracer, was applied to the diaphragm of adult female Sprague‐Dawley rats to identify phrenic and rostral intercostal motoneurons and synaptically antecedent interneurons. Using immunohistochemical dual‐labeling techniques we confirmed extensive 5‐HT immunoreactivity within the immediate vicinity of phrenic and intercostal motoneurons, as well as robust immunoreactivity near pre‐motor interneurons of both motor pools. Additionally, robust 5‐HT2a, 5‐HT2c, and 5‐HT7 receptor immunoreactivity was also observed for both phrenic and intercostal pre‐motor interneurons in laminae VII and X. Protein and mRNA analyses confirmed receptor expression, and indicated regional differences in 5‐HT innervation and receptor expression in cervical vs. thoracic spinal cords. We conclude that respiratory pre‐motor interneurons in the cervical and thoracic spinal cords are innervated by 5‐HT and express receptors that have been previously implicated in triggering spinal respiratory neuroplasticity.
Differential Inhibition of Bilateral Phrenic Bursting following Pulmonary Chemoreflex in Rats with Chronic Cervical Spinal Hemisection
Authors: Lee Kun‐Ze
Published: June 2021

High cervical spinal injury usually interrupts bulbospinal pathways and results in inactivation of phrenic bursting ipsilateral to the lesion. The ipsilateral phrenic activity can partially recover over weeks to months post‐injury due to activation of the latent crossed phrenic pathway. Ipsilateral phrenic nerve exhibits a greater capacity to increase activity during respiratory challenges than the contralateral phrenic nerve. However, whether bilateral phrenic nerves show a differential response to respiratory inhibitory inputs is unclear. Accordingly, present study was designed to examine bilateral phrenic bursting in response to capsaicin‐induced pulmonary chemoreflex, a robust respiratory inhibitory stimulus. Bilateral phrenic nerve activity were recorded in anesthetized and mechanically‐ventilated adult rats at 8‐9 weeks post‐C2 hemisection (C2Hx) or C2 laminectomy. Intra‐jugular capsaicin (1.5 µg/kg) injection was used to activate pulmonary C‐fibers to evoke the pulmonary chemoreflex. Present results showed capsaicin‐induced prolongation of expiratory duration (i.e., apnea) was significantly attenuated in C2Hx animals. However, ipsilateral phrenic activity was robustly inhibited after capsaicin treatment compared to contralateral phrenic nerve of C2Hx animals or ipsilateral phrenic nerve of uninjured animals. In addition, discharge onset of ipsilateral phrenic bursting of C2Hx animals is significantly delayed after capsaicin delivery. These results suggest that phrenic motor output is more susceptible to inhibitory inputs after cervical hemisection. Moreover, central (i.e., apnea) and spinal (i.e., inhibition of phrenic bursting) component of the pulmonary chemoreflex were differentially modulated during chronic injury phase.
Disordered Breathing In An Aged Model of Ischemic Stroke
Authors: Mulkey Daniel , Patrizz Anthony , Chen Xinnian , Kolb Adam , McCullough Louise
Published: June 2021

Respiratory dysfunction is a common complication of stroke associated with increased mortality. Stroke‐disorder breathing typically presents as Cheyne‐Stokes respiration which is thought to result from hyper‐activation of central chemoreceptors. We test this hypothesis by characterizing the CO2 ventilatory response before and after ischemic stroke. Further, since neurons in the retrotrapezoid nucleus are important chemoreceptors and their activity is regulated by KCNQ channels, we also test whether KCNQ channels can be targeted for the treatment of stroke related breathing problems. We used aged mice for these experiments because stroke mainly affects the elderly. Stroke was induced by middle cerebral artery occlusion (MCAO) and breathing was measured by plethysmography. Sham animals are subjected to the same surgery but with no occlusion. All MCAO‐treated mice exhibited hypoventilation with increased apneas (both frequency and duration) that were often preceded by short bursts of respiratory activity. Stroked mice also showed decreased respiratory stability as evidenced by a drop in approximate entropy. However, the ventilatory response to CO2 as similar between stroke and sham and systemic injection of retigabine further suppressed basal respiratory activity in MCAO/sham mice. These results show that MCAO‐treated mice exhibit a phenotype reminiscent of periodic breathing in stroke patients and they suggest that increased chemoreceptor gain is not the underlying cause of stroke‐disorder breathing.