Opposing GPCR signaling programs protein intake setpoint in Drosophila.

Animals defend a target level for their fundamental needs, including food, water, and sleep. Deviation from the target range, or "setpoint," triggers motivated behaviors to eliminate that difference. Whether and how the setpoint itself is encoded remains enigmatic for all motivated behaviors. Employing a high-throughput feeding assay in Drosophila, we demonstrate that the protein intake setpoint is set to different values in male, virgin female, and mated female flies to meet their varying protein demands. Leveraging this setpoint variability, we found, remarkably, that the information on the intake setpoint is stored within the protein hunger neurons as the resting membrane potential. Two RFamide G protein-coupled receptor (GPCR) pathways, by tuning the resting membrane potential in opposite directions, coordinately program and adjust the protein intake setpoint. Together, our studies map the protein intake setpoint to a single trackable physiological parameter and elucidate the cellular and molecular mechanisms underlying setpoint determination and modulation.

New insights into the physiology and pathophysiology of the atypical sodium leak channel NALCN.

Cell excitability and its modulation by hormones and neurotransmitters involve the concerted action of a large repertoire of membrane proteins, especially ion channels. Unique complements of coexpressed ion channels are exquisitely balanced against each other in different excitable cell types, establishing distinct electrical properties that are tailored for diverse physiological contributions, and dysfunction of any component may induce a disease state. A crucial parameter controlling cell excitability is the resting membrane potential (RMP) set by extra- and intracellular concentrations of ions, mainly Na+, K+, and Cl-, and their passive permeation across the cell membrane through leak ion channels. Indeed, dysregulation of RMP causes significant effects on cellular excitability. This review describes the molecular and physiological properties of the Na+ leak channel NALCN, which associates with its accessory subunits UNC-79, UNC-80, and NLF-1/FAM155 to conduct depolarizing background Na+ currents in various excitable cell types, especially neurons. Studies of animal models clearly demonstrate that NALCN contributes to fundamental physiological processes in the nervous system including the control of respiratory rhythm, circadian rhythm, sleep, and locomotor behavior. Furthermore, dysfunction of NALCN and its subunits is associated with severe pathological states in humans. The critical involvement of NALCN in physiology is now well established, but its study has been hampered by the lack of specific drugs that can block or agonize NALCN currents in vitro and in vivo. Molecular tools and animal models are now available to accelerate our understanding of how NALCN contributes to key physiological functions and the development of novel therapies for NALCN channelopathies.

Increased expression of chondroitin sulfate proteoglycans in dentate gyrus and amygdala causes postinfectious seizures.

Alterations in the extracellular matrix are common in patients with epilepsy and animal models of epilepsy, yet whether they are the cause or consequence of seizures and epilepsy development is unknown. Using Theiler's murine encephalomyelitis virus (TMEV) infection-induced model of acquired epilepsy, we found de novo expression of chondroitin sulfate proteoglycans (CSPGs), a major extracellular matrix component, in dentate gyrus (DG) and amygdala exclusively in mice with acute seizures. Preventing the synthesis of CSPGs specifically in DG and amygdala by deletion of the major CSPG aggrecan reduced seizure burden. Patch-clamp recordings from dentate granule cells revealed enhanced intrinsic and synaptic excitability in seizing mice that was significantly ameliorated by aggrecan deletion. In situ experiments suggested that dentate granule cell hyperexcitability results from negatively charged CSPGs increasing stationary cations on the membrane, thereby depolarizing neurons, increasing their intrinsic and synaptic excitability. These results show increased expression of CSPGs in the DG and amygdala as one of the causal factors for TMEV-induced acute seizures. We also show identical changes in CSPGs in pilocarpine-induced epilepsy, suggesting that enhanced CSPGs in the DG and amygdala may be a common ictogenic factor and potential therapeutic target.

Impact of Matrix Gel Variations on Primary Culture of Microvascular Endothelial Cell Function.

OBJECTIVE: The endothelium regulates crucial aspects of vascular function, including hemostasis, vasomotor tone, proliferation, immune cell adhesion, and microvascular permeability. Endothelial cells (ECs), especially in arterioles, are pivotal for flow distribution and peripheral resistance regulation. Investigating vascular endothelium physiology, particularly in microvascular ECs, demands precise isolation and culturing techniques. METHODS: Freshly isolated ECs are vital for examining protein expression, ion channel behavior, and calcium dynamics. Establishing primary endothelial cell cultures is crucial for unraveling vascular functions and understanding intact microvessel endothelium roles. Despite the significance, detailed protocols and comparisons with intact vessels are scarce in microvascular research. We developed a reproducible method to isolate microvascular ECs, assessing substrate influence by cultivating cells on fibronectin and gelatin matrix gels. This comparative approach enhances our understanding of microvascular endothelial cell biology. RESULTS: Microvascular mesenteric ECs expressed key markers (VE-cadherin and eNOS) in both matrix gels, confirming cell culture purity. Under uncoated conditions, ECs were undetected, whereas proteins linked to smooth muscle cells and fibroblasts were evident. Examining endothelial cell (EC) physiological dynamics on distinct matrix substrates revealed comparable cell length, shape, and Ca2+ elevations in both male and female ECs on gelatin and fibronectin matrix gels. Gelatin-cultured ECs exhibited analogous membrane potential responses to acetylcholine (ACh) or adenosine triphosphate (ATP), contrasting with their fibronectin-cultured counterparts. In the absence of stimulation, fibronectin-cultured ECs displayed a more depolarized resting membrane potential than gelatin-cultured ECs. CONCLUSIONS: Gelatin-cultured ECs demonstrated electrical behaviors akin to intact endothelium from mouse mesenteric arteries, thus advancing our understanding of endothelial cell behavior within diverse microenvironments.

Modulating voltage-gated sodium channels to enhance differentiation and sensitize glioblastoma cells to chemotherapy.

BACKGROUND: Glioblastoma (GBM) stands as the most prevalent and aggressive form of adult gliomas. Despite the implementation of intensive therapeutic approaches involving surgery, radiation, and chemotherapy, Glioblastoma Stem Cells contribute to tumor recurrence and poor prognosis. The induction of Glioblastoma Stem Cells differentiation by manipulating the transcriptional machinery has emerged as a promising strategy for GBM treatment. Here, we explored an innovative approach by investigating the role of the depolarized resting membrane potential (RMP) observed in patient-derived GBM sphereforming cell (GSCs), which allows them to maintain a stemness profile when they reside in the G0 phase of the cell cycle. METHODS: We conducted molecular biology and electrophysiological experiments, both in vitro and in vivo, to examine the functional expression of the voltage-gated sodium channel (Nav) in GSCs, particularly focusing on its cell cycle-dependent functional expression. Nav activity was pharmacologically manipulated, and its effects on GSCs behavior were assessed by live imaging cell cycle analysis, self-renewal assays, and chemosensitivity assays. Mechanistic insights into the role of Nav in regulating GBM stemness were investigated through pathway analysis in vitro and through tumor proliferation assay in vivo. RESULTS: We demonstrated that Nav is functionally expressed by GSCs mainly during the G0 phase of the cell cycle, suggesting its pivotal role in modulating the RMP. The pharmacological blockade of Nav made GBM cells more susceptible to temozolomide (TMZ), a standard drug for this type of tumor, by inducing cell cycle re-entry from G0 phase to G1/S transition. Additionally, inhibition of Nav substantially influenced the self-renewal and multipotency features of GSCs, concomitantly enhancing their degree of differentiation. Finally, our data suggested that Nav positively regulates GBM stemness by depolarizing the RMP and suppressing the ERK signaling pathway. Of note, in vivo proliferation assessment confirmed the increased susceptibility to TMZ following pharmacological blockade of Nav. CONCLUSIONS: This insight positions Nav as a promising prognostic biomarker and therapeutic target for GBM patients, particularly in conjunction with temozolomide treatment.

Investigating Contributions of Canonical Transient Receptor Potential Channel 3 to Hippocampal Hyperexcitability and Seizure-Induced Neuronal Cell Death.

Canonical transient receptor potential channel 3 (TRPC3) is the most abundant TRPC channel in the brain and is highly expressed in all subfields of the hippocampus. Previous studies have suggested that TRPC3 channels may be involved in the hyperexcitability of hippocampal pyramidal neurons and seizures. Genetic ablation of TRPC3 channel expression reduced the intensity of pilocarpine-induced status epilepticus (SE). However, the underlying cellular mechanisms remain unexplored and the contribution of TRPC3 channels to SE-induced neurodegeneration is not determined. In this study, we investigated the contribution of TRPC3 channels to the electrophysiological properties of hippocampal pyramidal neurons and hippocampal synaptic plasticity, and the contribution of TRPC3 channels to seizure-induced neuronal cell death. We found that genetic ablation of TRPC3 expression did not alter basic electrophysiological properties of hippocampal pyramidal neurons and had a complex impact on epileptiform bursting in CA3. However, TRPC3 channels contribute significantly to long-term potentiation in CA1 and SE-induced neurodegeneration. Our results provided further support for therapeutic potential of TRPC3 inhibitors and raised new questions that need to be answered by future studies.

Resting membrane potential is less negative in trabeculae from right atrial appendages of women, but action potential duration does not shorten with age.

AIMS: The incidence of atrial fibrillation (AF) increases with age. Women have a lower risk. Little is known on the impact of age, sex and clinical variables on action potentials (AP) recorded in right atrial tissue obtained during open heart surgery from patients in sinus rhythm (SR) and in longstanding AF. We here investigated whether age or sex have an impact on the shape of AP recorded in vitro from right atrial tissue. METHODS: We performed multivariable analysis of individual AP data from trabeculae obtained during heart surgery of patients in SR (n = 320) or in longstanding AF (n = 201). AP were recorded by sharp microelectrodes at 37 °C at 1 Hz. Impact of clinical variables were modeled using a multivariable mixed model regression. RESULTS: In SR, AP duration at 90% repolarization (APD90) increased with age. Lower ejection fraction and higher body mass index were associated with smaller action potential amplitude (APA) and maximum upstroke velocity (Vmax). The use of beta-blockers was associated with larger APD90. In tissues from women, resting membrane potential was less negative and APA as well as Vmax were smaller. Besides shorter APD20 in elderly patients, effects of age and sex on atrial AP were lost in AF. CONCLUSION: The higher probability to develop AF at advanced age cannot be explained by a shortening in APD90. Less negative RMP and lower upstroke velocity might contribute to lower incidence of AF in women, which may be of clinical relevance.

Sodium background currents in endocrine/neuroendocrine cells: Towards unraveling channel identity and contribution in hormone secretion.

In endocrine/neuroendocrine tissues, excitability of secretory cells is patterned by the repertoire of ion channels and there is clear evidence that extracellular sodium (Na+) ions contribute to hormone secretion. While voltage-gated channels involved in action potential generation are well-described, the background 'leak' channels operating near the resting membrane potential are much less known, and in particular the channels supporting a background entry of Na+ ions. These background Na+ currents (called here 'INab') have the ability to modulate the resting membrane potential and subsequently affect action potential firing. Here we compile and analyze the data collected from three endocrine/neuroendocrine tissues: the anterior pituitary gland, the adrenal medulla and the endocrine pancreas. We also model how INab can be functionally involved in cellular excitability. Finally, towards deciphering the physiological role of INab in endocrine/neuroendocrine cells, its implication in hormone release is also discussed.

The sodium leak channel NALCN regulates cell excitability of pituitary endocrine cells.

Anterior pituitary endocrine cells that release hormones such as growth hormone and prolactin are excitable and fire action potentials. In these cells, several studies previously showed that extracellular sodium (Na+ ) removal resulted in a negative shift of the resting membrane potential (RMP) and a subsequent inhibition of the spontaneous firing of action potentials, suggesting the contribution of a Na+ background conductance. Here, we show that the Na+ leak channel NALCN conducts a Ca2+ - Gd3+ -sensitive and TTX-resistant Na+ background conductance in the GH3 cell line, a cell model of pituitary endocrine cells. NALCN knockdown hyperpolarized the RMP, altered GH3 cell electrical properties and inhibited prolactin secretion. Conversely, the overexpression of NALCN depolarized the RMP, also reshaping the electrical properties of GH3 cells. Overall, our results indicate that NALCN is functional in GH3 cells and involved in endocrine cell excitability as well as in hormone secretion. Indeed, the GH3 cell line suitably models native pituitary cells that display a similar Na+ background conductance and appears as a proper cellular model to study the role of NALCN in cellular excitability.

Chronic Ouabain Prevents Radiation-Induced Reduction in the α2 Na,K-ATPase Function in the Rat Diaphragm Muscle.

The damaging effect of ionizing radiation (IR) on skeletal muscle Na,K-ATPase is an open field of research. Considering a therapeutic potential of ouabain, a specific ligand of the Na,K-ATPase, we tested its ability to protect against the IR-induced disturbances of Na,K-ATPase function in rat diaphragm muscle that co-expresses the α1 and α2 isozymes of this protein. Male Wistar rats (n = 26) were subjected to 6-day injections of vehicle (0.9% NaCl) or ouabain (1 µg/kg/day). On the fourth day of injections, rats were exposed to one-time total-body X-ray irradiation (10 Gy), or a sham irradiation. The isolated muscles were studied 72 h post-irradiation. IR decreased the electrogenic contribution of the α2 Na,K-ATPase without affecting its protein content, thereby causing sarcolemma depolarization. IR increased serum concentrations of ouabain, IL-6, and corticosterone, decreased lipid peroxidation, and changed cellular redox status. Chronic ouabain administration prevented IR-induced depolarization and loss of the α2 Na,K-ATPase electrogenic contribution without changing its protein content. This was accompanied with an elevation of ouabain concentration in circulation and with the lack of IR-induced suppression of lipid peroxidation. Given the crucial role of Na,K-ATPase in skeletal muscle performance, these findings may have therapeutic implications as countermeasures for IR-induced muscle pathology.

Short-Term Mild Hypoxia Modulates Na,K-ATPase to Maintain Membrane Electrogenesis in Rat Skeletal Muscle.

The Na,K-ATPase plays an important role in adaptation to hypoxia. Prolonged hypoxia results in loss of skeletal muscle mass, structure, and performance. However, hypoxic preconditioning is known to protect against a variety of functional impairments. In this study, we tested the possibility of mild hypoxia to modulate the Na,K-ATPase and to improve skeletal muscle electrogenesis. The rats were subjected to simulated high-altitude (3000 m above sea level) hypobaric hypoxia (HH) for 3 h using a hypobaric chamber. Isolated diaphragm and soleus muscles were tested. In the diaphragm muscle, HH increased the α2 Na,K-ATPase isozyme electrogenic activity and stably hyperpolarized the extrajunctional membrane for 24 h. These changes were accompanied by a steady increase in the production of thiobarbituric acid reactive substances as well as a decrease in the serum level of endogenous ouabain, a specific ligand of the Na,K-ATPase. HH also increased the α2 Na,K-ATPase membrane abundance without changing its total protein content; the plasma membrane lipid-ordered phase did not change. In the soleus muscle, HH protected against disuse (hindlimb suspension) induced sarcolemmal depolarization. Considering that the Na,K-ATPase is critical for maintaining skeletal muscle electrogenesis and performance, these findings may have implications for countermeasures in disuse-induced pathology and hypoxic therapy.

Depolarization-Dependent C-Raf Signaling Promotes Hyperexcitability and Reduces Opioid Sensitivity of Isolated Nociceptors after Spinal Cord Injury.

Chronic pain caused by spinal cord injury (SCI) is notoriously resistant to treatment, particularly by opioids. After SCI, DRG neurons show hyperactivity and chronic depolarization of resting membrane potential (RMP) that is maintained by cAMP signaling through PKA and EPAC. Importantly, SCI also reduces the negative regulation by Gαi of adenylyl cyclase and its production of cAMP, independent of alterations in G protein-coupled receptors and/or G proteins. Opioid reduction of pain depends on coupling of opioid receptors to Gαi/o family members. Combining high-content imaging and cluster analysis, we show that in male rats SCI decreases opioid responsiveness in vitro within a specific subset of small-diameter nociceptors that bind isolectin B4. This SCI effect is mimicked in nociceptors from naive animals by a modest 5 min depolarization of RMP (15 mm K+; -45 mV), reducing inhibition of cAMP signaling by µ-opioid receptor agonists DAMGO and morphine. Disinhibition and activation of C-Raf by depolarization-dependent phosphorylation are central to these effects. Expression of an activated C-Raf reduces sensitivity of adenylyl cyclase to opioids in nonexcitable HEK293 cells, whereas inhibition of C-Raf or treatment with the hyperpolarizing drug retigabine restores opioid responsiveness and blocks spontaneous activity of nociceptors after SCI. Inhibition of ERK downstream of C-Raf also blocks SCI-induced hyperexcitability and depolarization, without direct effects on opioid responsiveness. Thus, depolarization-dependent C-Raf and downstream ERK activity maintain a depolarized RMP and nociceptor hyperactivity after SCI, providing a self-reinforcing mechanism to persistently promote nociceptor hyperexcitability and limit the therapeutic effectiveness of opioids.SIGNIFICANCE STATEMENT Chronic pain induced by spinal cord injury (SCI) is often permanent and debilitating, and usually refractory to treatment with analgesics, including opioids. SCI-induced pain in a rat model has been shown to depend on persistent hyperactivity in primary nociceptors (injury-detecting sensory neurons), associated with a decrease in the sensitivity of adenylyl cyclase production of cAMP to inhibitory Gαi proteins in DRGs. This study shows that SCI and one consequence of SCI (chronic depolarization of resting membrane potential) decrease sensitivity to opioid-mediated inhibition of cAMP and promote hyperactivity of nociceptors by enhancing C-Raf activity. ERK activation downstream of C-Raf is necessary for maintaining ongoing depolarization and hyperactivity, demonstrating an unexpected positive feedback loop to persistently promote pain.

A sodium background conductance controls the spiking pattern of mouse adrenal chromaffin cells in situ.

KEY POINTS: Mouse chromaffin cells in acute adrenal slices exhibit two distinct spiking patterns, a repetitive mode and a bursting mode. A sodium background conductance operates at rest as demonstrated by the membrane hyperpolarization evoked by a low Na+ -containing extracellular saline. This sodium background current is insensitive to TTX, is not blocked by Cs+ ions and displays a linear I-V relationship at potentials close to chromaffin cell resting potential. Its properties are reminiscent of those of the sodium leak channel NALCN. In the adrenal gland, Nalcn mRNA is selectively expressed in chromaffin cells. The study fosters our understanding of how the spiking pattern of chromaffin cells is regulated and adds a sodium background conductance to the list of players involved in the stimulus-secretion coupling of the adrenomedullary tissue. ABSTRACT: Chromaffin cells (CCs) are the master neuroendocrine units for the secretory function of the adrenal medulla and a finely-tuned regulation of their electrical activity is required for appropriate catecholamine secretion in response to the organismal demand. Here, we aim at deciphering how the spiking pattern of mouse CCs is regulated by the ion conductances operating near the resting membrane potential (RMP). At RMP, mouse CCs display a composite firing pattern, alternating between active periods composed of action potentials spiking with a regular or a bursting mode, and silent periods. RMP is sensitive to changes in extracellular sodium concentration, and a low Na+ -containing saline hyperpolarizes the membrane, regardless of the discharge pattern. This RMP drive reflects the contribution of a depolarizing conductance, which is (i) not blocked by tetrodotoxin or caesium, (ii) displays a linear I-V relationship between -110 and -40 mV, and (iii) is carried by cations with a conductance sequence gNa  > gK  > gCs . These biophysical attributes, together with the expression of the sodium-leak channel Nalcn transcript in CCs, state credible the contribution of NALCN. This inaugural report opens new research routes in the field of CC stimulus-secretion coupling, and extends the inventory of tissues in which NALCN is expressed to neuroendocrine glands.

Giant Y79 retinoblastoma cells contain functionally active T-type calcium channels.

Retinoblastoma is the most common malignant intraocular tumor in children. Y79 human retinoblastoma cells are in vitro models of retinal tumors used for drug screening. Undifferentiated Y79 cells originate from a primitive multi-potential neuroectodermal cell and express neuronal and glial properties. However, the nature of cellular heterogeneity in Y79 cells is unclear because functional methods to characterize neurons or glial cells have not been employed to Y79 cells. Here, we perform patch-clamp recordings to characterize electrophysiological properties in retinoblastoma cells. We identified a population of large-sized Y79 cells (i.e., giant cells, ~ 40-µm diameter), hyperpolarized resting membrane potential (-54 mV), and low input resistance (~ 600 MΩ), indicating electrically mature cells. We also found that giant Y79 cells contain increased density of T-type calcium channels. Finally, we found that T-type calcium channels are active only in giant cells suggesting that cancer treatments aimed to prevent calcium influx in retinoblastomas should be tested in giant cells.

Chronic Ouabain Prevents Na,K-ATPase Dysfunction and Targets AMPK and IL-6 in Disused Rat Soleus Muscle.

Sustained sarcolemma depolarization due to loss of the Na,K-ATPase function is characteristic for skeletal muscle motor dysfunction. Ouabain, a specific ligand of the Na,K-ATPase, has a circulating endogenous analogue. We hypothesized that the Na,K-ATPase targeted by the elevated level of circulating ouabain modulates skeletal muscle electrogenesis and prevents its disuse-induced disturbances. Isolated soleus muscles from rats intraperitoneally injected with ouabain alone or subsequently exposed to muscle disuse by 6-h hindlimb suspension (HS) were studied. Conventional electrophysiology, Western blotting, and confocal microscopy with cytochemistry were used. Acutely applied 10 nM ouabain hyperpolarized the membrane. However, a single injection of ouabain (1 µg/kg) prior HS was unable to prevent the HS-induced membrane depolarization. Chronic administration of ouabain for four days did not change the α1 and α2 Na,K-ATPase protein content, however it partially prevented the HS-induced loss of the Na,K-ATPase electrogenic activity and sarcolemma depolarization. These changes were associated with increased phosphorylation levels of AMP-activated protein kinase (AMPK), its substrate acetyl-CoA carboxylase and p70 protein, accompanied with increased mRNA expression of interleikin-6 (IL-6) and IL-6 receptor. Considering the role of AMPK in regulation of the Na,K-ATPase, we suggest an IL-6/AMPK contribution to prevent the effects of chronic ouabain under skeletal muscle disuse.

Effects of extremely low-frequency electromagnetic field exposure on the skeletal muscle functions in rats.

The aim of the present study was to systematically investigate the effects of chronic exposure to extremely low-frequency electromagnetic field (ELF-EMF) on electrophysiological, histological and biochemical properties of the diaphragm muscle in rats. Twenty-nine newly weaned (24 days old, 23-80 g) female (n = 15) and male (n = 14) Wistar Albino rats were used in this study. The animals were randomly divided into two groups: the control group and the electromagnetic field (EMF) group. The control group was also randomly divided into two groups: the control female group and the control male group. The EMF exposure group was also randomly divided into two groups: the ELF-EMF female group and the ELF-EMF male group. The rats in the ELF-EMF groups were exposed for 4 h daily for up to 7 months to 50 Hz frequency, 1.5 mT magnetic flux density. Under these experimental conditions, electrophysiological parameters (muscle bioelectrical activity parameters: intracellular action potential and resting membrane potential and muscle mechanical activity parameter: force-frequency relationship), biochemical parameters (Na+, K+, Cl- and Ca+2 levels in the blood serum of rats; Na+-K+ ATPase enzyme-specific activities in muscle tissue; and free radical metabolism in both muscle tissue and serum) and transmission electron microscopic morphometric parameters of the diaphragm muscle were determined. We found that chronic exposure to ELF-EMF had no significant effect on the histological structure and mechanical activity of the muscle and on the majority of muscle bioelectrical activity parameters, with the exception of some parameters of muscle bioelectrical activity. However, the changes in some bioelectrical activity parameters were relatively small and unlikely to be clinically relevant.

Skeletal Muscle Na,K-ATPase as a Target for Circulating Ouabain.

While the role of circulating ouabain-like compounds in the cardiovascular and central nervous systems, kidney and other tissues in health and disease is well documented, little is known about its effects in skeletal muscle. In this study, rats were intraperitoneally injected with ouabain (0.1-10 µg/kg for 4 days) alone or with subsequent injections of lipopolysaccharide (1 mg/kg). Some rats were also subjected to disuse for 6 h by hindlimb suspension. In the diaphragm muscle, chronic ouabain (1 µg/kg) hyperpolarized resting potential of extrajunctional membrane due to specific increase in electrogenic transport activity of the 2 Na,K-ATPase isozyme and without changes in 1 and 2 Na,K-ATPase protein content. Ouabain (10-20 nM), acutely applied to isolated intact diaphragm muscle from not injected rats, hyperpolarized the membrane to a similar extent. Chronic ouabain administration prevented lipopolysaccharide-induced (diaphragm muscle) or disuse-induced (soleus muscle) depolarization of the extrajunctional membrane. No stimulation of the 1 Na,K-ATPase activity in human red blood cells, purified lamb kidney and Torpedo membrane preparations by low ouabain concentrations was observed. Our results suggest that skeletal muscle electrogenesis is subjected to regulation by circulating ouabain via the 2 Na,K-ATPase isozyme that could be important for adaptation of this tissue to functional impairment.

Contribution of KCNQ and TREK Channels to the Resting Membrane Potential in Sympathetic Neurons at Physiological Temperature.

The ionic mechanisms controlling the resting membrane potential (RMP) in superior cervical ganglion (SCG) neurons have been widely studied and the M-current (IM, KCNQ) is one of the key players. Recently, with the discovery of the presence of functional TREK-2 (TWIK-related K+ channel 2) channels in SCG neurons, another potential main contributor for setting the value of the resting membrane potential has appeared. In the present work, we quantified the contribution of TREK-2 channels to the resting membrane potential at physiological temperature and studied its role in excitability using patch-clamp techniques. In the process we have discovered that TREK-2 channels are sensitive to the classic M-current blockers linopirdine and XE991 (IC50 = 0.310 ± 0.06 µM and 0.044 ± 0.013 µM, respectively). An increase from room temperature (23 °C) to physiological temperature (37 °C) enhanced both IM and TREK-2 currents. Likewise, inhibition of IM by tetraethylammonium (TEA) and TREK-2 current by XE991 depolarized the RMP at room and physiological temperatures. Temperature rise also enhanced adaptation in SCG neurons which was reduced due to TREK-2 and IM inhibition by XE991 application. In summary, TREK-2 and M currents contribute to the resting membrane potential and excitability at room and physiological temperature in the primary culture of mouse SCG neurons.

The effects of repetitive transcranial magnetic stimulation on the cognition and neuronal excitability of mice.

This study aimed to investigate the effects of repetitive transcranial magnetic stimulation (rTMS) on the cognition and neuronal excitability of Kunming mice during the natural aging of the brain. Twenty young (2-3-month-old) female mice, 20 adult (9-10-month-old) female mice and 12 aged (14-15-month-old) female mice were divided into two groups (control and rTMS treatment). rTMS-treated groups were subjected to high-frequency (20 Hz) rTMS treatment for 15 days. Novel object recognition (NOR) and step-down tests were performed to examine cognition of learning and memory. The whole-cell patch clamp technique was used to record the resting membrane potential (RMP) and action potential (AP), and the intrinsic properties of the AP were analyzed (the frequence of AP, the after hyperpolarizing potential (AHP), the AP peak amplitude, the time to AP amplitude, the average rise/down slope). Results showed that the cognition and neuronal excitability of hippocampal dentate gyrus (DG) granule cells were significantly declined only in aged animals while no statistic differences were found between young and adult animals. Chronic high-frequency rTMS could significantly improve the age-related cognitive impairment in parallel with enhancing the DG granule cells' neuronal excitability.

[Repetitive transcranial magnetic stimulation significantly improves cognitive impairment and neuronal excitability during aging in mice].

Repetitive transcranial magnetic stimulation (rTMS) is a noninvasive brain stimulation technique that has been paid attention to with increasing interests as a therapeutic neural rehabilitative tool. Studies confirmed that high-frequency rTMS could improve the cognitive performance in behavioral test as well as the excitability of the neuron in animals. This study aimes to investigate the effects of rTMS on the cognition and neuronal excitability of Kunming mice during the natural aging. Twelve young mice, 12 adult mice, and 12 aged mice were used, and each age group were randomly divided into rTMS group and control group. rTMS-treated groups were subjected to high-frequency rTMS treatment for 15 days, and control groups were treated with sham stimulation for 15 days. Then, novel object recognition and step-down tests were performed to examine cognition of learning and memory. Whole-cell patch clamp technique was used to record and analyze resting membrane potential, action potential (AP), and related electrical properties of AP of hippocampal dentate gyrus (DG) granule neurons. Data analysis showed that cognition of mice and neuronal excitability of DG granule neurons were degenerated significantly as the age increased. Cognitive damage and degeneration of some electrical properties were alleviated under the condition of high-frequency rTMS. It may be one of the mechanisms of rTMS to alleviate cognitive damage and improve cognitive ability by changing the electrophysiological properties of DG granule neurons and increasing neuronal excitability.