bluuebunny/crossref_metadata_2025 huggingface dataset:
Acetylcholine combined with acetylcholinesterase blockade within the commissural nucleus of the solitary tract increases respiratory activity
Authors: Furuya Werner Issao , Bassi Mirian , Colombari Eduardo , Menani Jose Vanderlei , Zoccal Daniel Breseghello , de Almeida Colombari Débora Simões
Published: November 2023

Acetylcholine (ACh) microinjections in the commissural nucleus of the solitary tract (cNTS) increase phrenic nerve (PN) burst frequency. However, the effects of the activation of cholinergic mechanisms in the cNTS on the activity of other motor respiratory outputs are still unknown. In the present study, we recorded the hypoglossal nerve (HN), cervical vagus nerve (cVN) and PN activities using decorticated arterially‐perfused in situ preparations of juvenile male Holtzman rats (70–90 g, n=8), and evaluated the effects elicited by microinjections of ACh in the cNTS, combined with prior physostigmine administration (PHY, acetylcholinesterase inhibitor) in the same area. Microinjections of ACh (10 mM, 60 nL) were performed 5 min after PHY (1 mM, 40–60 nL, 0.3 and 0.6 mm caudal to calamus scriptorius) in the cNTS and the changes in the respiratory motor activities were analyzed during 1 min following microinjections. The combination of PHY and ACh within the cNTS induced an initial (6 s after ACh) slight increase in the PN burst frequency (4.8 ± 1.9 vs. vehicle 2.9 ± 1.0%, p > 0.05) and amplitude (9.0 ± 1.8 vs. vehicle 2.7 ± 0.5%, p < 0.05). The amplitude of post‐inspiratory (post‐I) activity in the cVN (18.7 ± 3.8 vs. vehicle 3.5 ± 0.9%, p < 0.05) and the pre‐inspiratory period (pre‐I) of the HN (425 ± 46 vs. vehicle 100 ± 43 ms, p < 0.05) also increased. Sixty seconds after ACh microinjections, PN frequency decreased (−27.1 ± 0.4 vs. vehicle 2.4 ± 0.6 %, p < 0.05). On the other hand, PN amplitude (11.2 ± 0.5 vs. vehicle 3.4 ± 0.3%, p < 0.05), cVN post‐I amplitude (16.5 ± 0.9 vs. vehicle 1.9 ± 0.5%, p < 0.05) and the HN pre‐I period (436 ± 26 vs. vehicle 149 ± 57 ms, p < 0.05) were maintained. The data show that stimulation of cholinergic receptors in the cNTS promotes significant changes in different respiratory motor nerves that resemble the respiratory responses elicited by peripheral chemoreflex activation (early tachypnea followed by bradypnea, increase in the PN and cVN amplitude and increase in the pre‐I period of HN), which might suggest an involvement of cholinergic mechanisms in the cNTS on chemoreflex pathway.

Support or Funding Information

FAPESP, CAPES and CNPq.
Cardiorespiratory Coupling is Dependent on a Conditionally Expressed Inspiratory Antecedent in the In Situ Rodent Preparation
Authors: Baekey David M
Published: November 2023

The in situ rodent model was previously used to describe respiratory modulation of cardiovascular activities expressed in heart rate (RSA), sympathetic nerves (SNA), and vascular pressure. While this previous study implicated both medullary and pontine involvement, the neural substrate for this coordination remains undefined. Our hypothesized model assumes some direct connectivity within the medulla, wherein the respiratory central pattern generating (rCPG) nuclei influence more caudal premotor respiratory populations (VRG) and adjacent autonomic nuclei (RVLM/CVLM), but recent observations challenge the straightforwardness of this proposed network. A retrospective analysis of 37 preparations in which the phrenic nerve was recorded prior to the first reanimated breath revealed features suggesting involvement of a unique inspiratory neural population. To observe this coupling, juvenile rats are anaesthetized, subdiaphragmatically bisected, decerebrated, and submerged in ice cold artificial cerebrospinal fluid (aCSF) during surgical exposure of neural recording sites. Aortic cannulation and perfusion with aCSF warms the preparations and provides dissolved O2 and CO2 (95/5) while electrodes are placed to monitor respiratory and autonomic nerves. A gradual increase in perfusion flow results in respiratory efforts as noted by both visible contraction of the chest musculature prior to application of paralytic and bursting activity of respiratory efferents thereafter. Phrenic nerve activity (PNA) has a relatively consistent expression at 12–15 breaths per minute with no initial coordination of heart rate, modulation of SNA, or oscillations of perfusion pressure. As phrenic bursting continues, one of two patterns occurs: 1) a sudden transition to ramp patterned PNA with reduced Ti and increased Te (N=30), or 2) high amplitude, short duration phrenic bursts are interspersed amongst the stable baseline rhythm, eventually entraining and merging with the baseline pattern to form the ramping phrenic with reduced Ti and increased Te (N=7). The significance of both pattern changes is that they occur simultaneously with expression of RSA and respiratory modulation of vascular pressure. In the interspersed expression, cardiorespiratory coupling was limited to the short duration, high amplitude bursts. In summary, this suggests that cardiorespiratory coupling requires activation of a conditionally expressed neural population that is active during late inspiration. Given that cardio‐respiratory coupling is reduced with aging and decreased RSA is proposed as a predictor of mortality, identification and modulation of this “missing link” might provide a powerful therapeutic target in prevention of cardiovascular disease.

Support or Funding Information

This research was supported by a UF CVM Faculty Award.
Improvement in Cerebral Autoregulation During Recovery Phase Following Concussion is associated with Reduced Postural Sway: A Pilot Study
Authors: Maffuid Kaitlyn , Ryan Larry , Murphy Megan N , Davis Peter F , Purkayastha Sushmita
Published: November 2023

Objective

Postural instability has been reported in patients with concussion and this symptom may be related to compromised cerebral blood flow regulation as a result of the injury. The purpose of the study was to examine whether changes in cerebral autoregulation from acute injury phase to the recovery phase following a concussion were associated with changes in postural sway in collegiate athletes participating in high‐contact sports.

Methods

Five NCAA Division I male athletes (21±2 years) with a confirmed sports‐related concussion were enrolled in the study. Data was collected on days 3 and 21 post‐concussion. Arterial blood pressure (ABP) was obtained with finger photoplethysmography. Middle cerebral artery blood velocity (MCAV) was collected using transcranial Doppler ultrasound for 12 minutes while subjects were seated upright. Dynamic cerebral autoregulation was estimated from transfer function analysis of beat‐to‐beat fluctuations in ABP and MCAV. Postural control was measured from the deviation in the center of pressure in both the mediolateral and anterioposterior directions during quiet standing on a force plate for 60 seconds. Four trials were randomized, two with eyes closed and two with eyes open. Multiple linear regression analyses were used to examine the association between changes in low frequency (LF, 0.07–0.2 Hz) transfer function phase and mediolateral range and variability of postural sway on days 3 and 21 post‐concussion.

Results

Improvement in LF transfer function phase from day 3 to day 21 post‐concussion was associated with reductions in mediolateral range (β estimate= −19, p=0.02) and variability (β estimate= −4.3, p=0.04) of postural sway with eyes closed.

Conclusion

Cerebral blood flow regulation is associated with alterations in postural control and may be useful as a potential vascular biomarker for tracking recovery following concussion.

Support or Funding Information

Supported by: University Research Council Grant at SMU, Dallas, Texas
Depletion of Brain Perivascular Macrophages Mitigates Depressive‐like Behavioral Consequences of Chronic Mild Stress
Authors: Grigoleit Jan‐Sebastian , Alvarez Elena , Farrokhi Catherine Borna , Sawchenko Paul E.
Published: November 2023

Despite intensive research, the etiology of depression remains poorly understood. Stress is implicated in the pathogenesis of most depressive disorders, as reflected by the preponderance of animal models that employ chronic stress to induce depressive‐like behavior. There is increasing evidence for involvement of immune/inflammatory mechanisms in the genesis of depression, in part by mediating the effects of stress, with microglia and also macrophage‐type immune cells in the brain being implicated. Recent work from our laboratory has identified brain perivascular macrophages (PVM), not only as crucial mediators of inflammatory signaling from the periphery to the CNS, but as also involved in mediating the consequences of acute emotional stress on central neuroendocrine and inflammatory responses. The objective of the current pilot experiment was to evaluate the potential involvement of brain PVM in the induction of depressive‐like behaviors caused by chronic stress exposure. Adult male rats either remained untreated (n=6) or received an intracerebroventricular injection of mannosylated liposomes containing the pro‐apoptotic drug, clodronate (n=6), to exploit the constitutive phagocytic activity of PVM, leading to depletion of this cell type, but not microglia, throughout the brain within 3–5 d. Both groups were then subjected to 5 weeks of chronic mild stress (CMS), a well‐established protocol for the induction of depression through repeated and unpredictable exposure to a variety of mild insults. Anhedonic behavior, a key component of depression, was assessed by monitoring sucrose intake (provided as a 2% solution twice weekly before and during the CMS phase). Additional behavioral tests (open field/OF, elevated plus maze/EPM, Porsolt forced swim test/PFST) were used to assess further key components of depressive‐like behavior. All animals preferred sucrose strongly over normal drinking water. However, there was a significant drop in sucrose intake after 1 week of CMS in the control group, which was sustained for the duration of the experiment. No such effect was observed in the PVM‐depleted group. In addition, stressed control animals displayed a significant decrease in exploratory behavior in the EPM, remaining almost exclusively in the closed arms of the maze, an effect that was not observed in PVM‐depleted rats. In the PFST, which is often used to assess the potency of anti‐depressant drugs, PVM‐depleted rats tried significantly longer to escape from a water‐filled jar than animals from the control group. No differences in general locomotor activity (OF) were observed among groups at any time point. These findings support the hypothesis that brain PVMs are involved in depressive behaviors seen in a chronic mild stress model.

Support or Funding Information

NIH grant NS21182, Clayton Medical Research Foundation, German Research Foundation (DFG) grant GR4370/1‐1, CONACyT Mexico
Differential Responses of Male and Female Microglia to LPS and Estrogen May Contribute to Sexually Dimorphic Effects of Inflammation on Phrenic Long‐term Facilitation
Authors: Gardner Alyssa N. , Dougherty Brendan J. , Watters Jyoti J
Published: November 2023

Chronic intermittent hypoxia (CIH) is a hallmark of obstructive sleep apnea (OSA) that causes neuroinflammation, and acutely (1 night) impairs one form of respiratory motor plasticity, phrenic long‐term facilitation (pLTF). The prevalence of OSA is sexually dimorphic (higher in males) although this difference greatly decreases after menopause, suggesting that estrogen may play a role in protecting the respiratory system in females. Preliminary data suggest that impairment of pLTF by neuroinflammation induced by challenge with gram‐negative bacterial lipopolysaccharide (LPS; 0.1 mg/kg) may also be sexually dimorphic; LPS treatment abolished pLTF in gonadally intact males but not in females treated with estrogen. Although estrogen is well‐known to be anti‐inflammatory, little is known about its ability to control neuroinflammation as it impacts respiratory neuroplasticity. We tested the hypothesis that LPS‐induced neuroinflammation is higher in males, and that estrogen exerts sex‐specific effects on neuroinflammatory gene expression. To begin to test this hypothesis, primary microglia from P4‐P6 male and female rat pups were cultured separately and treated with LPS (100 ng/ml) in the presence and absence of 17β‐estradiol (E2; 1nM). Total RNA was isolated and gene arrays (SA Biosciences) were performed; individual gene changes were confirmed by qRT‐PCR. Whereas LPS increased the expression of multiple pro‐inflammatory cytokines and chemokines in microglia from both sexes, the effects were stronger in male microglia. E2 treatment alone strongly reduced basal pro‐inflammatory gene expression in female microglia, but had little effect in male microglia. E2 had generally similar effects on LPS‐induced gene expression in microglia from both sexes. These data suggest that male microglia have stronger pro‐inflammatory responses to LPS, and that reductions in basal levels of inflammatory genes by estrogen are greater in female microglia. Together, these mechanisms may contribute to higher levels of neuroinflammation in the male CNS following LPS, and to the corresponding sex differences in inflammation‐mediated impairment of pLTF. Results from this work will provide significant insight into mechanisms that can be exploited to minimize destabilization of respiratory plasticity by inflammation in both sexes.

Support or Funding Information

Supported by R01 NS085226 and an OWHR supplement to R01 HL111598
Phrenic long‐term facilitation (pLTF) fluctuates over the estrous cycle and is abolished by ovariectomy in young female rats
Authors: Dougherty Brendan J. , Kopp Elizabeth S. , Watters Jyoti J.
Published: November 2023

Circulating sex steroids modulate spinal plasticity, including phrenic long‐term facilitation (pLTF), a persistent increase in phrenic nerve activity observed after acute exposure to intermittent hypoxia (AIH; three 5 min episodes of 10.5% O2, delivered at 5 minute intervals). Previous studies indicate that young male rats require the conversion of testosterone to 17β‐estradiol (E2) to express pLTF, and pLTF is not present in estrus or diestrus (when circulating estradiol levels are low) in young female rats, suggesting that estrogen availability and signaling may underlie sexually dimorphic pLTF expression. In this study, we tested the hypothesis that pLTF would be enhanced in young female rats during proestrus when levels of circulating estrogen are high, and blunted during estrus when circulating estrogen levels are low. In addition, we hypothesized that ovarian function is specifically necessary for pLTF expression in young female rats. Phrenic nerve activity was recorded in anesthetized, paralyzed and ventilated female Sprague‐Dawley rats (3–4 mos) that were separated into the following experimental groups: ovarian intact females in estrus or proestrus, and ovariectomized (OVX) females that received daily vehicle (sesame oil) or E2 (17β‐estradiol benzoate, 100 μg/kg, i.p.) supplementation for 7 days. Initial results confirmed that females in proestrus exhibited significantly higher pLTF compared to females in estrus, and that removal of the ovaries abolished pLTF in young female rats. These data suggest that pLTF expression varies over the estrous cycle in a manner proportional to circulating E2 levels. To test the sufficiency of E2 to restore pLTF to ovariectomized females, E2 replacement studies are currently underway. Together, these data suggest that ovarian hormones exert important effects on respiratory motor plasticity, and underscore the utility of this model to dissect the role of E2 signaling in the sexually dimorphic expression of spinal neuroplasticity.

Support or Funding Information

This work was supported by R01 NS085226 and an OWHR supplement to NIH R01 HL111598.
Increased Probability of Release in Huntington's Disease Neuromuscular Junctions
Authors: Khedraki Ahmad , Reed Eric Joshua , Rich Mark M. , Voss Andrew Alvin
Published: November 2023

Huntington's disease (HD) is a progressive and fatal degenerative disorder that results in debilitating cognitive and motor dysfunction. The majority of research related to HD has been focused on neurodegeneration in the central nervous system. Recently, we discovered defects in the membrane properties and mRNA processing of skeletal muscle from the R6/2 transgenic mouse line of HD. The decrease in the resting chloride and potassium conductance result in hyperexcitable skeletal muscle that is hypersensitive to neuronal stimulation. The objective of this study was to examine the neuromuscular events that trigger muscle excitation. We used a fully innervated ex vivo preparation of the levator auris longus muscle from late‐stage R6/2 mice and age‐matched wild type controls. Our voltage clamp records of spontaneous miniature endplate currents (mEPCs) and nerve‐evoked endplate currents (eEPCs) were independent of the changes in muscle membrane properties, unlike previous current clamp studies. Using the charge moved during the mEPCs and eEPCs, preliminary data indicates that the number of vesicles released per action potential (quantal content) was not significantly different between HD and WT muscle. This argues against denervation/renervation, which has been suggested in other studies. Furthermore, in response to trains of nerve stimulation (10 action potentials at 50 Hz), eEPCs in HD muscle reached a peak amplitude sooner and then depressed to a greater degree than in WT. This amplified depression suggests an increased probability of vesicle release in HD neuromuscular junctions. This hypothesis was supported by data obtained under low Ca2+ and high Mg2+ extracellular conditions, where quantal content in HD is higher than in WT. Because studies of central synapses face more experimental limitations, our voltage clamp examination of neuromuscular transmission is hitherto the most complete analysis of synaptic function in HD. The defects we discovered serve as a model to understand the contribution of synaptic defects to the devastating cognitive and motor symptoms in Huntington's disease.

Support or Funding Information

Wright State University Startup to AAV.
Tolerance to Alcohol‐Stimulated Glutamate Receptor Phosphorylation in the Central Amygdala and Prefrontal Cortex after Chronic Nicotine Exposure
Authors: McGinn Mary Adrienne , Itoga Christy A , Paulsen Rod I , Reppel Jonathan E , Farooq Muhammad A , Gilpin Nicholas W , Edwards Scott
Published: November 2023

Nicotine and alcohol are two of the most commonly abused substances. Nicotine use increases alcohol drinking, suggesting that the combination of these drugs may produce synergistic effects in activating reward circuitry. Alternatively, use of either of these drugs may facilitate the development of cross‐tolerance to the other to promote intake escalation. Mechanisms underlying these interactions are largely unknown, and there is little information on neuronal signaling events that occur in animals exposed to both drugs. In the present experiment, adult male Wistar rats were exposed to room air (non‐dependent group) or intermittent nicotine vapor (nicotine‐dependent group) for 12 hours per day (resulting in 12 hours of withdrawal per day) for three weeks. This treatment regimen has been shown to produce symptoms of nicotine dependence as evidenced by hyperalgesia and elevated nicotine self‐administration during withdrawal. On the final day of nicotine exposure, at 10 hours of withdrawal, animals were challenged with either saline or alcohol (1 g/kg, IP) and euthanized 15 minutes later. Brains were snap frozen and dissected for processing via Western analysis. We examined changes in the phosphorylation status of glutamate receptor subunits in stress‐ and reward‐related brain regions since excitatory neuroadaptations are heavily implicated in both alcohol and nicotine addiction. We discovered differential alterations in glutamate receptor subunit phosphorylation following nicotine withdrawal and alcohol challenge. For example, alcohol significantly increased PKA phosphorylation of GluR1 (a subunit of the AMPA receptor) across multiple brain regions, including the hippocampus, dorsomedial prefrontal cortex (dmPFC), and central amygdala (CeA). However, this neuroadaptation was largely absent in the dmPFC and CeA in animals exposed to chronic intermittent nicotine vapor (significant interaction between alcohol challenge and nicotine exposure). These data suggest a molecular tolerance to alcohol‐stimulated phosphorylation of GluR1 (a subunit of the AMPA receptor) in these regions in the context of nicotine dependence. As a consequence, more alcohol may be required to influence glutamatergic plasticity in the nicotine‐dependent state, and this may in part mediate increased drinking in heavy smokers.

Support or Funding Information

Our research is supported by the following grants from the National Institute on Alcohol Abuse and Alcoholism: R01AA023305 (NWG) R00AA020839 (SE) T32AA007577 (MAM)
Kv4.2/4.3 Channels in the Hypothalamic PVN Contribute to Sympathetic Activation in Angiotensin II‐Salt Hypertension
Authors: Toney Glenn M , Andrade MaryAnn
Published: November 2023

Recent studies in mice implicate increased NMDA receptor expression and function in the PVN in development of the slow pressor model of angiotensin II (AngII) hypertension. Here, we studied adult male rats with AngII‐salt hypertension. Rat were placed on a high salt (2% NaCl) diet for 6 weeks with systemic AngII treatment (150 ng/kg/min, s.c.) for the final 2 weeks. Consistent with studies in mice, we found that responsiveness to PVN NMDA was increased in hypertensive rats as was PVN NMDA receptor tonus. Previous studies of rats with reno‐vascular hypertension reported increased excitability of pre‐sympathetic PVN neurons accompanied by diminution of the transient potassium A‐current. In the PVN, Kv4.2/4.3 channels are major mediators of A‐current, but the present studies revealed that acute and selective blockade of these channels with phrixotoxin (5 pmol in 50 nl) had no effect on resting MAP, RSNA or SSNA in anesthetized normotensive or AngII‐salt hypertensive rats – suggesting that A‐current is not a significant determinant of in vivo spike probability among PVN pre‐sympathetic neurons of either group. However, phrixotoxin did enhance pressor and sympatho‐excitatory response to PVN injections of NMDA (200 nmol in 50 nl) in normotensive rats (P<0.05 – 0.01), while failing to do so in hypertensive rats. These findings suggest several alternative explanation for A‐current function in the PVN. Most parsimoniously, Vm of pre‐sympathetic neurons could be sufficiently hyperpolarized and discharge sufficiently sparse under normal conditions that A‐current is maintained at a relatively low level of steady state inactivation, allowing it to actively attenuate spiking induced by activation of glutamatergic input (NMDA). By contrast, Vm depolarization and more frequent spiking of neurons in rats with AngII‐salt hypertension could prevent hyperpolarization‐induced de‐inactivation of A‐current and thereby increase steady‐state inactivation such that A‐current mediated by Kv4.2/4.3 channels cannot effectively oppose spiking. The latter is consistent with lack of enhancement of responses to PVN NMDA by phrixotoxin in hypertensive rats. The above model of diminished PVN A‐current contribution to AngII‐salt hypertension requires further study to assess whether diminished Kv4.2/4.3 channel expression and ongoing covalent modulation of channel gating might also contribute to PVN activation and heightened PVN support of sympathetic activity in AngII‐salt hypertension.

Support or Funding Information

HL102310 & HL088052 (GMT)
Neuronal specific Caveolin‐1 overexpression enhances dendritic growth and arborization in differentiated human neuronal stem cells derived from induced pluripotent stem cells
Authors: Zhang Zheng , Almenar‐Queralt Angels , Roth David M. , Patel Hemal H. , Head Brian P.
Published: November 2023

Introduction

Neuroplasticity is evoked by extracellular signals that bind receptors in the plasma membrane to induce signal transduction that results in polarized actions in the neuronal plasma membrane. Membrane/lipid rafts (MLR) and cholesterol binding protein caveolin‐1 (Cav‐1) provide a signaling membrane platform that tethers and regulates cytoskeletal components necessary for proper dendritic growth, maintenance and neuroplasticity. Our lab has demonstrated in rodents primary neurons that overexpression of neuronal cav‐1 enhances pro‐growth/pro‐survival signaling, and improves dendritic growth and arborization. We utilized neurons derived from human induced pluripotent stem cells (iPSCs) to ask whether Cav‐1 controls similar signaling and functions in human neurons as it does in rodent neurons. We overexpressed Cav‐1 driven by the neuronal specific synapsin promoter (SynCav1) and evaluated changes in dendritic morphology after the differentiation process.

Methods

iPSC derived neuronal stem cells (NSCs) were cultured in DMEM/F12+Gluamax media with N‐2 supplement, B‐27 supplement, Pen/Strep, and basic Fibroblast Growth Factor (bFGF). Neuronal differentiation was started by bFGF withdrawal from media. NSCs were transfected with either SynCav1 or SynGFP as control when differentiation was started. Cells were fixed 3 weeks after differentiation and stained for Cav‐1, the dendritic marker microtubule‐associated protein 2 (MAP2), and the axonal marker neurofilament (SMI‐31). Dendritic length, branching (arborization), area, and volume were measured by Autoneuron (MBF Bioscience), a tracing algorithm that measures 3D image volume stacks. Cells were also collected and homogenized to measure Cav‐1 levels via western blots.

Results

Western blots showed that Cav‐1 was increased in the cells transfected with SynCav‐1 compared to SynGFP 3 weeks after differentiation. Compared with SynGFP, SynCav1 transfection significantly enhanced dendritic length (p=0.002), branching points/arborization (p=0.0004), dendrite area (p=0.003), and dendrite volume (p=0.009)

Conclusion

Neuron‐targeted expression of Cav‐1 significantly enhanced dendritic growth of human iPSC‐derived neurons suggesting the presence of efficient Cav‐1 dependent signaling in these cells.
Gender Differences in the Association of Neutrophil Gelatinase‐Associated Lipocalin with Cognitive Impairment and Depression in Heart Failure
Authors: Schoemaker Regien G , Gouweleeuw Leonie , Hovens Iris B , Liu Hui , Naude Pieter JW , Eisel Uli ML
Published: November 2023

Introduction

Heart failure increases the risk to develop depression, which worsens prognosis. Depression is more common in women than in men. Neutrophil Gelatinase‐Associated Lipocalin (NGAL), a regulator of inflammation and indicator of prognosis in heart failure patients, is also associated with depression. The aim of this study was to investigate the association of NGAL with behavior in both male and female rats with heart failure.

Methods

Male and female Wistar rats were subjected to coronary artery ligation (MI) or sham surgery. Three weeks later, depressive symptoms were obtained from the open field test (OF) and cognitive functioning from the novel object and novel location test. Plasma levels of NGAL were measured with Elisa, and NGAL expression in the brain was obtained using immunohistochemistry.

Results

In males, but not in females, plasma NGAL appeared correlated to infarct size. We found both a gender (F=6.53, p=0.005) and an MI (F=6.63, p=0.013) effect in plasma NGAL levels, with higher levels in male compared to female rats and MI compared to sham rats. An MI effect (F=4.19, p=0.049) and interaction effect (F=7.87, p=0.09) were found in the number of NGAL positive cells in the paraventricular nucleus of the hypothalamus (PVN), post‐hoc analysis showed that NGAL was elevated only in male rats upon MI. Spatial object recognition was impaired in male MI compared to sham rats (discrimination index 54.3% vs 78.3%, p<0.01). No significant effect of MI was seen in the OF. In male rats, plasma NGAL showed a negative correlation with distance in the OF (r=−0.384, p=0.044) and discrimination index of the moved object in the novel location test (r=−0.570, p=0.033). Additionally, PVN NGAL negatively correlated to distance spent in the OF in male rats (r=0.464, p=0.039). In female rats no behavioral changes were seen after MI and NGAL did not correlate with behavioral outcomes.

Discussion

Data indicate gender specific effects of MI on the expression of NGAL and on behavior. This suggests that post‐MI inflammatory processes and their potential effects on depressive‐like behavior and cognition, may be differently regulated in male and female rats.
Altered Microglia Morphology in The Hypothalamus after Myocardial Infarction in Mice
Authors: Schoemaker Regien G , Gouweleeuw Leonie , Wajant Harald , Maier Olaf , Eisel Uli ML
Published: November 2023

Background

Myocardial infarction (MI) evokes an inflammatory response that may present itself in the brain as neuroinflammation in the paraventricular nucleus of the hypothalamus (PVN), which may contribute to the elevated sympathetic nerve activity found in heart failure. Since increased expression of TNF‐alpha has been implicated in neuroinflammation, we investigated whether interference with TNF‐alpha signaling could affect neuroinflammation after MI by affecting the brain immune cells; microglia.

Methods

Male 12‐week old C57bl/6 mice underwent coronary artery ligation or sham surgery. MI mice were treated with either saline, the TNF‐inhibitor etanercept, a TNF‐R1 antagonist or a TNF‐R2 agonist, starting 60 minutes after MI. Sham mice received saline injections. Mice were sacrificed 18 days later and the dorsal hippocampus and PVN were stained with an anti‐Iba1 antibody to visualize microglia. Changes in microglia were obtained from cell density, total coverage, and activity. The latter is based on morphology and calculated as the ratio of cell body/total cell size.

Results

Whereas in the hippocampus no differences in the number, cell size or cell body/cell size ratio were observed between groups, in the PVN a significant decrease in the number of microglia in the MI+TNF‐R2 agonist group was found compared to sham (16.1 ± 1.6 vs 27.4 ± 7.5, p<0.05). The cell body/cell size ratio was significantly decreased in all MI groups compared to sham (4.3 ± 1.5 – 5.5 ± 0.8 vs 8.0 ± 0.7%, p<0.05), which could be attributed to an increase in dendrite size, since cell body size did not differ between groups.

Conclusion

In conclusion, interference with TNF‐alpha signaling after MI in mice resulted in area‐specific changes in microglia, with TNF‐R2 agonist treatment resulting in a lower number of microglia in the PVN. Morphological analysis suggests hyperramification of microglia in the PVN after MI. This might indicate a polarization towards type M2 microglia, associated with protective functions; though to be confirmed with specific markers.
Association of Impaired Hypoxia‐Inducible Factor‐1 Transcriptional Activity with Severer Brain Injury in Aged Rats in Comparison with Young Rats after Acute Ischemic Stroke
Authors: Guo Yingjia , Li Xianglong , Han Pengfei , Xiao Ying , Zhu Ping , Gou Zhaoyu , Kang Y. James
Published: November 2023

Aged mammals in comparison with young suffer severer damage post ischemic stroke, and hypoxia‐inducible factor‐1 (HIF‐1) activation is neuroprotective in ischemic stroke. The present study was undertaken to investigate whether there is a difference in HIF‐1 between aged and young rats and its association with the degree in brain damage post ischemic stroke. Aged (18–20 months) and young (3–4 months) male Sprague‐Dawley rats were subjected to transient middle cerebral artery occlusion (MCAO) for 60‐min, followed by reperfusion for 3, 7, 14, or 21 days. There was no significant difference in the infarct volume in the brain between aged and young rats 3 days after MCAO, but the infarct volume in the aged was significantly larger than in the young animals 21 days after MCAO. The overall survival rate in the aged was significantly lower than in the young rats. In addition, delayed body weight gain, retarded neurobehavioral recovery, and depressed angiogenesis were observed in the aged rats compared to the young at 3, 7, and 21 days after MCAO. There was no significant difference in the protein levels of HIF‐1 accumulated in the ischemic area between the aged and the young rats at 3 days after MCAO, however, the HIF‐1 transcriptional activity was significantly suppressed, along with increased apoptotic cell death, in the aged rats. Furthermore, the levels of mRNAs involved in modulating HIF‐1α stabilization and HIF‐1 target genes were dramatically different between aged and young rats. These findings thus revealed the association of impaired HIF‐1 transcriptional activity with the delayed neurobehavioral recovery after focal ischemic stroke in aged rats.

Support or Funding Information

The National Basic Research Program of China (973 Program Grant no. 2013CB530702).
Persistent Memory Deficits and Histone Modification after a Systemic Inflammatory Event
Authors: Collette Katie , Donzis Elissa , Tronson Natalie C
Published: November 2023

Inflammatory events, including myocardial infarction (MI), illness, or major surgery, commonly lead to cognitive deficits that persist for months or years, far beyond the duration of inflammation. The cognitive deficits correlate with poorer outcomes as well as increased morbidity and mortality. These clinical observations are paralleled in animal models of MI and inflammation. However, the mechanisms mediating such persistent effects remain unknown. Epigenetic modifications induced by cytokine signaling are one potential mechanism. In particular, dysregulation of histone acetylation can be triggered by inflammation and also induce memory deficits. We hypothesized that, although the initial cytokine signaling would be resolved within 8 weeks after MI, changes in histone modifications would persist and mediate lasting cognitive impairments.

Methods

We used a surgical model of MI in male and female mice, with sham‐operated (sham) and non‐operated (non‐op) control groups. Eight weeks after the MI group's surgery, memory was assessed using context fear conditioning (3 min exposure to context followed by a single 0.8mA, 2sec foot shock). In separate animals, hippocampus and blood serum were collected 8 weeks after MI. We used multiplex cytokine analysis to determine levels in the hippocampus. In addition, we conducted western blotting to determine persistent alterations in histone acetylation, phospho‐acetylation and methylation. Finally, we analyzed hearts from MI and sham animals to determine infarct size.

Results and Discussion

Eight weeks after MI surgery, both male and female mice exhibited impairments in fear conditioning compared with non‐op control animals. In addition, female ‐ but not male ‐ sham mice exhibited deficits in context fear conditioning. This suggests that female mice are more susceptible to persistent memory deficits even after a lower level inflammatory event. There were no differences in peripheral or hippocampal cytokine levels 8 wks post‐MI; therefore, the cognitive deficits were not due to ongoing inflammatory signaling. Future work will determine a causal role for dysregulated histone acetylation in the persistent memory deficits following an inflammatory event. Identifying the mechanisms underlying this process will provide novel targets for prevention and treatment of persistent cognitive deficits after MI, illness or major surgery.

Support or Funding Information

This research was supported by a National Institutes of Health grant (R00 MH093459, NCT)
Possible Respiratory Modulation of Sympathetic Activity in Rats After Sino‐aortic Denervation
Authors: Amorim Mateus R , Bonagamba Leni G. H. , Souza George M. P. R. , Moraes Davi J. A. , Machado Benedito H.
Published: November 2023

It is well known that several days after sino‐aortic denervation (SAD) rats present normal levels of sympathetic activity and mean arterial pressure (MAP). However, the mechanisms involved in the modulation of the sympathetic activity after SAD remain unclear. Here we hypothesized that the respiratory network may contribute to the modulation of the pre‐sympathetic neurons preventing sustained increase in MAP in SAD rats. To study this mechanism we recorded the activities of phrenic, thoracic sympathetic (tSNA) and cervical vagus nerves (cVN) in male juvenile Wistar rats in the working heart‐brainstem preparation, at high or low respiratory drive induced by hypercapnia or hypocapnia, respectively (SAD, n = 26 and Sham, n = 33). The duration of inspiration significantly increased in SAD rats (1.46 ± 0.05 vs 1.14 ± 0.07 s, P < 0.001). It was also observed that SAD increases cVN inspiratory activity (25 ± 1 vs 32 ± 2%, P < 0.05) and decreases in its post‐inspiratory component (76 ± 1 vs 68 ± 2%, P < 0.05), suggesting an increased upper airway resistance during inspiration in SAD rats. Regarding the baseline tSNA, no changes were observed in the baseline tSNA in all phases of the respiratory cycle in SAD rats, which presented significant reduction in the Traube‐Hering waves magnitude (2.57 ± 0.04 vs 1.19 ± 0.20 mmHg, P = 0.0048). In order to reveal the influence of the respiratory network on the modulation of sympathetic activity rats were exposed to hypercapnia (from 5 to 10 % of CO2) or hypocapnia (from 5 to 3 and 1 % of CO2). High respiratory drive induced by hypercapnia increases tSNA in all phases of respiration in Sham rats (n=13), but in SAD rats (n=14) a significant increase in tSNA was observed only during inspiration (43 ± 3% vs 55 ± 3%, P < 0.01). On the other hand, hypocapnia reduced the respiratory drive and induced sympatho‐inhibition in the SAD (n=8) and in Sham rats (n=10). The data show that in spite of longer inspiratory activity, the baseline sympathetic activity is normal in SAD rats. Whether or not the longer inspiratory activity is driving the sympathetic activity in SAD rats is still matter for further investigation.

Support or Funding Information

FAPESP, CAPES and CNPq
Neural circuit mechanism for hypothalamic neuropeptide Y‐induced inhibition of brown adipose tissue thermogenesis
Authors: Nakamura Yoshiko , Yanagawa Yuchio , Morrison Shaun F , Nakamura Kazuhiro
Published: November 2023

Hunger stimulates neuropeptide Y (NPY)‐containing neurons in the arcuate nucleus of the hypothalamus, which then release NPY from their nerve endings in the paraventricular hypothalamic nucleus (PVH) to inhibit metabolic activities including brown adipose tissue (BAT) thermogenesis. However, the neural circuit mechanism by which the NPY‐triggered signaling from the PVH inhibits BAT thermogenesis remains unknown. We found that NPY injection into the PVH in rats inhibits BAT thermogenesis through GABAergic inhibitory inputs to BAT‐controlling sympathetic premotor neurons in the rostral medullary raphe (rMR). Our anterograde and retrograde tract tracing revealed that GABAergic neurons in the intermediate and parvicellular reticular nuclei (IRt/PCRt) of the medulla oblongata directly project to sympathetic premotor neurons in the rMR. In vivo stimulation of neurons in the IRt/PCRt with a nanoinjection of bicuculline, a GABAA receptor antagonist, strongly inhibited either skin cooling‐evoked or pyrogen‐evoked BAT thermogenesis. Furthermore, inactivation of IRt/PCRt neurons with bilateral nanoinjections of muscimol, a GABAA receptor agonist, reversed the inhibition of BAT thermogenesis by hypothalamic NPY. These data indicate that the hunger‐driven hypothalamic NPY signaling inhibits BAT thermogenesis through the GABAergic innervation from IRt/PCRt neurons to sympathetic premotor neurons in the rMR.

Support or Funding Information

Funding Program for Next Generation World‐Leading Researchers from the Japan Society for the Promotion of Science (LS070 to K.N.); Grants‐in‐Aids for Scientific Research from the MEXT of Japan (15H05932, 26118508 and 26713009 to K.N., 26290002 and 15H01415 to Y.Y., and 26860159 to Y.N.); Grant‐in‐Aid for Scientific Research on Innovative Areas (Comprehensive Brain Science Network) from the MEXT of Japan (to Y.Y.); NIH grant (R01NS40987 to S.F.M.); Takeda Science Foundation (to K.N. and Y.Y.); Nakajima Foundation (to K.N.); Brain Science Foundation (to K.N.); Kowa Life Science Foundation (to K.N.).
Cerebrovascular Reactivity Is Reduced in Postmenopausal Women with a History of Hypertensive Pregnancy Disorders
Authors: Harvey Ronee E , Johnson Maja C , Johnson Christopher P , Joyner Michael J , Miller Virginia M , Barnes Jill N
Published: November 2023

Women who have experienced hypertensive pregnancy disorders (HPD) are at an increased risk for developing cardiovascular disease later in life, including fatal and non‐fatal stroke. Cerebrovascular reactivity is related to stroke in individuals with carotid artery disease, and it is possible that this measure may be altered in women with a history of HPD prior to overt cerebrovascular disease. It is unknown if cerebrovascular reactivity is altered in postmenopausal women decades after experiencing a hypertensive pregnancy. Therefore, the goal of this study was to determine if cerebral blood flow velocity and cerebrovascular reactivity are altered in women with a history of HPD in comparison to women with no history of HPD. We studied a cohort of postmenopausal women approximately 35 years after they experienced an HPD (n=27, age at testing: 58±1) and a group of age‐ and parity‐matched women with no history of HPD (n=30, age at testing: 59±1). We measured middle cerebral artery velocity (MCAv, via transcranial Doppler), continuous blood pressure (finger photoplethysmography), and end‐tidal CO2 (nasal cannula) in these women at baseline (room air) and during stepped hypercapnia at 2, 4, and 6% inhaled CO2 for 3 minutes at each condition. Women with a history of HPD had a higher body mass index than women without a history of HPD (29±1 vs. 26±1 kg/m2, respectively; p<0.05). At baseline and during 6% CO2, MCAv was lower in women with a history of HPD (baseline: 60±4 vs. 71±3 cm/s, respectively; 6% CO2: 82±6 vs. 99±5 cm/s, respectively; p<0.05 for both conditions) compared to women without a history of HPD. Cerebrovascular reactivity, the slope defining the association between MCAv and end‐tidal CO2 across the entire breathing trial, was also lower in women with a history of HPD (2.0±0.2 vs. 2.7±0.2 cm/s/mmHg, respectively; p<0.05). Body mass index was not correlated with MCAv or cerebrovascular reactivity in either group (p>0.05). In conclusion, resting MCAv and cerebrovascular reactivity to CO2 is lower in women with a history of HPD in comparison with women who have never experienced HPD. Our findings suggest that HPD may have long‐term effects on cerebrovascular regulation in women who have suffered from these conditions.

Support or Funding Information

NIH HL083947, NIA 1P50AG044170‐01, and CTSA UL1TR000135
Stimulation of beta adrenergic receptor type‐3 (β3‐AR) in the kidney triggers antidiuresis: physiological implications and possible therapeutic target to treat NDI
Authors: Procino Giuseppe , Carmosino Monica , Milano Serena , Dal Monte Massimo , Schena Giorgia , Mastrodonato Maria , Torretta Silvia , Gerbino Andrea , Bagnoli Paola , Svelto Maria
Published: November 2023

Background

Beta‐adrenergic receptors β1‐AR and β2‐AR regulate important renal functions. However, the presence, localization and possible physiological role of β3‐AR in the kidney is still debated.

Methods

Immunolocalization of β3‐AR was performed in mouse kidney. Live mouse kidney slices were treated with the β3‐AR selective agonist BRL37344, in the presence or in the absence of the PKA inhibitor H89 or β3‐AR antagonists.

To unmask the possible antidiuretic effect of β3‐AR stimulation, we tested the urine concentrating ability of β3‐AR KO mice and injected BRL37344 in wt and vasopressin V2 receptor (V2R) KO mice.

Results

β3‐AR is expressed at the basolateral membrane of most of the vasopressin‐sensitive nephron segments: thick ascending limb (TAL), distal convolute tubule (DCT) and the cortical/outer medullary collecting duct (CD).

Interestingly, in live kidney slices, BRL37344 elicited a vasopressin‐like effect on cAMP production, AQP2 apical expression and KNCC2 phosphorylation/activity. These effects were prevented by β3‐AR antagonists or H89. β3‐AR KO mice showed reduced AQP2 plasma membrane expression, increased diuresis, reduced urine osmolality and increased urine excretion of Na, K and Cl compared to control mice. Moreover, in both wt and V2R‐KO mice, BRL37344 increased Na, K, Cl and water reabsorption and promoted a significant antidiuretic effect.

Conclusions

Taken together, these findings are of great physiological importance as they uncover an antidiuretic effect of β3‐AR stimulation in the kidney and suggest that β3‐AR agonists might be useful to bypass V2R inactivating mutations.

Support or Funding Information

Telethon foundation grants n. GGP15083 and n. GGP12040
Anatomical substract of the masseter muscle myofascial trigger points
Authors: Akamatsu Flávia , Gaubeur Matheus Acquesta , Itezerote Ana Maria , Saleh Samir , Hojaij Flávio , Figueredo Mauro , Jacomo Alfredo
Published: November 2023

Myofascial pain syndrome (MPS) is a common cause of chronic musculoskeletal pain and is characterized by myofascial trigger points (MTP), limited range of motion in joints, referred pain and local twitch response during mechanical stimulation of MTP. MTP are clinically identified by palpation of a taut band of muscle or fascia. Physiopathology of MPS remains unclear and it has been suggested that MTP occur when a nociceptor and a muscular motor endplate coincide. Eight different MTP are clinically described for the masseter muscle, but anatomical correlation is still lacking. Masseter muscles from ten cadavers were carefully dissected from their origins in order to observe the exact point where nerve fibers penetrated the muscle belly.

As muscle size varies among individuals, we calculated the relative entry point of the nerve into the muscle by defining six different areas in the muscle belly: three superior (I, II and III) and three inferior (IV, V and VI), beginning at the posterior margin of the muscle. Areas II (36.6%), I (22.2%) and V (16.6%) concentrated the masseteric nerve entry points and statistical analyzis using Chi square fit quality test showed a significant higher in these areas (p <0.001). These locations correspond to the clinically described MTP. Anatomical correlation of the MTP may be useful for a better understanding of the physiopathology of these disorders and provide basis for their treatment.
Synthesis of a photolabile inhibitor for light‐dependent control of a kinase
Authors: Yogodzinski Christopher , Hughes Taylor , Brandt Gabriel
Published: November 2023

A photolabile inhibitor of Src kinase was synthesized and tested for light‐dependent inhibition of recombinant human Src. Typical photo triggered or ‘caged’ compounds acquire inhibitory activity in response to light. Light‐initiated removal of a photo removable protecting group reveals a bioactive functional group. Here, a less common strategy for establishing photo control over a biochemical pathway is presented, namely introduction of a photolabile inhibitor that loses potency upon irradiation with light. A typical photo triggered compound functions as a light‐induced knockout. Here, the goal is to revive an inhibited pathway, in order to mimic functional knock‐in of a particular protein. The lavendustin C scaffold was used as a platform for the development both photo activated and photolabile analogs. The synthesis and photochemical characterization of a photolabile nitrolavendustin analog are reported. The compound responds to light in the predicted fashion, undergoing photo cleavage in response to near‐UV light. Preliminary testing in vitro suggests that the effect of the compound on Src activity is light‐dependent. The compound is water‐soluble, suggesting the potential for use in cell culture.