Outward Current Research Articles

Cannabinoid receptor-mediated modulation of inhibitory inputs to mitral cells in the main olfactory bulb.

The endocannabinoid (eCB) signaling system has been functionally implicated in many brain regions. Our understanding of the role of cannabinoid receptor type 1 (CB1) in olfactory processing remains limited. Cannabinoid signaling is involved in regulating glomerular activity in the main olfactory bulb (MOB). However, the cannabinoid-related circuitry of inputs to mitral cells in the MOB has not been fully determined. Using anatomical and functional approaches we have explored this question. CB1 was present in periglomerular processes of a GAD65-positive subpopulation of interneurons but not in mitral cells. We detected eCBs in the mouse MOB as well as the expression of CB1 and other genes associated with cannabinoid signaling in the MOB. Patch-clamp electrophysiology demonstrated that CB1 agonists activated mitral cells and evoked an inward current, while CB1 antagonists reduced firing and evoked an outward current. CB1 effects on mitral cells were absent in subglomerular slices in which the olfactory nerve layer and glomerular layer were removed, suggesting the glomerular layer as the site of CB1 action. We previously observed that GABAergic periglomerular cells show the inverse response pattern to CB1 activation compared with mitral cells, suggesting that CB1 indirectly regulates mitral cell activity as a result of cellular activation of glomerular GABAergic processes . This hypothesis was supported by the finding that cannabinoids modulated synaptic transmission to mitral cells. We conclude that CB1 directly regulates GABAergic processes in the glomerular layer to control GABA release and, in turn, regulates mitral cell activity with potential effects on olfactory threshold and behavior.NEW & NOTEWORTHY Cannabinoid signaling with cannabinoid receptor type 1 (CB1) is involved in the regulation of glomerular activity in the main olfactory bulb (MOB). We detected endocannabinoids in the mouse MOB. CB1 was present in periglomerular processes of a GAD65-positive subpopulation of interneurons. CB1 agonists activated mitral cells. CB1 directly regulates GABAergic processes to control GABA release and, in turn, regulates mitral cell activity with potential effects on olfactory threshold and behavior.

Inhibition of Kv2.1 Potassium Channels by MiDCA1, A Pre-Synaptically Active PLA2-Type Toxin from Micrurus dumerilii carinicauda Coral Snake Venom.

MiDCA1, a phospholipase A2 (PLA2) neurotoxin isolated from Micrurus dumerilii carinicauda coral snake venom, inhibited a major component of voltage-activated potassium (Kv) currents (41 ± 3% inhibition with 1 μM toxin) in mouse cultured dorsal root ganglion (DRG) neurons. In addition, the selective Kv2.1 channel blocker guangxitoxin (GxTx-1E) and MiDCA1 competitively inhibited the outward potassium current in DRG neurons. MiDCA1 (1 µM) reversibly inhibited the Kv2.1 current by 55 ± 8.9% in a Xenopus oocyte heterologous system. The toxin showed selectivity for Kv2.1 channels over all the other Kv channels tested in this study. We propose that Kv2.1 channel blockade by MiDCA1 underlies the toxin’s action on acetylcholine release at mammalian neuromuscular junctions.

A de novo gain-of-function KCND3 mutation in early repolarization syndrome

Early repolarization syndrome (ERS) is characterized by J-point elevation on electrocardiograms and ventricular fibrillation (VF). Early repolarization arises from augmentation of the transmural electrical gradient in the cardiac action potential; therefore, the transient outward potassium current (Ito) has been regarded as a key candidate current for elucidating the mechanism of ERS. KCND3 encoding Kv4.3, an α-subunit of the Ito channel, is considered as one of target genes. The purpose of this study was to search for novel KCND3 mutations associated with ERS and to clarify the pathogenesis. We performed genetic screening for 11 unrelated probands with ERS and analyzed the electrophysiological properties of detected mutations by patch-clamp methods. A novel de novo KCND3 heterozygous mutation, Gly306Ala (c.917g>c), was found in 1 proband. The proband was a 12-year-old boy, who suffered VF storm and showed significant J-point elevation in multiple leads. Intravenous isoproterenol and subsequent administration of quinidine were effective in preventing VF recurrence and restored the J-point elevation. In electrophysiological analysis, cultured cells expressing mutant Kv4.3 showed significantly increased current densities, slow inactivation, and slow recovery from inactivation compared to wild type. Extracellular application of quinidine significantly restored the inactivation time course in mutant Kv4.3. A simulation study confirmed the relationship between the novel KCND3 mutation and early repolarization on electrocardiograms. A novel KCND3 heterozygous mutation was found to be associated with ERS. The pathogenesis can be explained by the increased Ito. Genetic screening for KCND3 could be useful for understanding the pathogenesis and selecting effective treatment.

Lysosomal Storage and Albinism Due to Effects of a De Novo CLCN7 Variant on Lysosomal Acidification

Optimal lysosome function requires maintenance of an acidic pH maintained by proton pumps in combination with a counterion transporter such as the Cl-/H+ exchanger, CLCN7 (ClC-7), encoded by CLCN7. The role of ClC-7 in maintaining lysosomal pH has been controversial. In this paper, we performed clinical and genetic evaluations of two children of different ethnicities. Both children had delayed myelination and development, organomegaly, and hypopigmentation, but neither had osteopetrosis. Whole-exome and -genome sequencing revealed a de novo c.2144A>G variant in CLCN7 in both affected children. This p.Tyr715Cys variant, located in the C-terminal domain of ClC-7, resulted in increased outward currents when it was heterologously expressed in Xenopus oocytes. Fibroblasts from probands displayed a lysosomal pH approximately 0.2 units lower than that of control cells, and treatment with chloroquine normalized the pH. Primary fibroblasts from both probands also exhibited markedly enlarged intracellular vacuoles; this finding was recapitulated by the overexpression of human p.Tyr715Cys CLCN7 in control fibroblasts, reflecting the dominant, gain-of-function nature of the variant. A mouse harboring the knock-in Clcn7 variant exhibited hypopigmentation, hepatomegaly resulting from abnormal storage, and enlarged vacuoles in cultured fibroblasts. Our results show that p.Tyr715Cys is a gain-of-function CLCN7 variant associated with developmental delay, organomegaly, and hypopigmentation resulting from lysosomal hyperacidity, abnormal storage, and enlarged intracellular vacuoles. Our data supports the hypothesis that the ClC-7 antiporter plays a critical role in maintaining lysosomal pH.

The role of vacuolar ion channels in salt stress tolerance in the liverwort Conocephalum conicum

Liverworts are pioneer plants that colonized lands. They had to cope with frequent sea water flooding causing salt stress. The role of vacuoles and in particular slow-activating (SV) channels in the salt stress tolerance was addressed in the present study. A patch-clamp method was used to study sodium fluxes through the tonoplast of the liverwort Conocephalum conicum. The whole-vacuole measurements carried out in a symmetrical Na+ concentration allowed recording of slowly activated outward currents typical for SV channels. In a Na+ gradient promoting an efflux of Na+ from the vacuole, the outward rectifying properties of SV channels were reduced and inward Na+ currents with different inactivation rates were recorded. Single channel analysis proved that a decrease in cytoplasmic Na+ concentration evoked an increase in the open probability of the channels and shifted the activation voltages towards negative values. The number of SV channels recorded at negative voltages was dependent on the vacuolar calcium and decreased at the high concentration of this ion in the vacuole. In some of the tested patches, the channels exhibited a flickering type of activity and two different conductance levels. The role of SV channels in Na+ accumulation during salt stress and its removal after periods of flooding is discussed in the present paper.

Ultrasound Stimulation Modulates Voltage-Gated Potassium Currents Associated With Action Potential Shape in Hippocampal CA1 Pyramidal Neurons.

Potassium channels (K+) play an important role in the regulation of cellular signaling. Dysfunction of potassium channels is associated with several severe ion channels diseases, such as long QT syndrome, episodic ataxia and epilepsy. Ultrasound stimulation has proven to be an effective non-invasive tool for the modulation of ion channels and neural activity. In this study, we demonstrate that ultrasound stimulation enables to modulate the potassium currents and has an impact on the shape modulation of action potentials (AP) in the hippocampal pyramidal neurons using whole-cell patch-clamp recordings in vitro. The results show that outward potassium currents in neurons increase significantly, approximately 13%, in response to 30 s ultrasound stimulation. Simultaneously, the increasing outward potassium currents directly decrease the resting membrane potential (RMP) from −64.67 ± 1.10 mV to −67.51 ± 1.35 mV. Moreover, the threshold current and AP fall rate increase while the reduction of AP half-width and after-hyperpolarization peak time is detected. During ultrasound stimulation, reduction of the membrane input resistance of pyramidal neurons can be found and shorter membrane time constant is achieved. Additionally, we verify that the regulation of potassium currents and shape of action potential is mainly due to the mechanical effects induced by ultrasound. Therefore, ultrasound stimulation may offer an alternative tool to treat some ion channels diseases related to potassium channels.

Glibenclamide and HMR1098 normalize Cantú syndrome-associated gain-of-function currents.

Cantú syndrome (CS) is caused by dominant gain‐of‐function mutation in ATP‐dependent potassium channels. Cellular ATP concentrations regulate potassium current thereby coupling energy status with membrane excitability. No specific pharmacotherapeutic options are available to treat CS but IKATP channels are pharmaceutical targets in type II diabetes or cardiac arrhythmia treatment. We have been suggested that IKATP inhibitors, glibenclamide and HMR1098, normalize CS channels. IKATP in response to Mg‐ATP, glibenclamide and HMR1098 were measured by inside‐out patch‐clamp electrophysiology. Results were interpreted in view of cryo‐EM IKATP channel structures. Mg‐ATP IC50 values of outward current were increased for D207E (0.71 ± 0.14 mmol/L), S1020P (1.83 ± 0.10), S1054Y (0.95 ± 0.06) and R1154Q (0.75 ± 0.13) channels compared to H60Y (0.14 ± 0.01) and wild‐type (0.15 ± 0.01). HMR1098 dose‐dependently inhibited S1020P and S1054Y channels in the presence of 0.15 mmol/L Mg‐ATP, reaching, at 30 μmol/L, current levels displayed by wild‐type and H60Y channels in the presence of 0.15 mmol/L Mg‐ATP. Glibenclamide (10 μmol/L) induced similar normalization. S1054Y sensitivity to glibenclamide increases strongly at 0.5 mmol/L Mg‐ATP compared to 0.15 mmol/L, in contrast to D207E and S1020P channels. Experimental findings agree with structural considerations. We conclude that CS channel activity can be normalized by existing drugs; however, complete normalization can be achieved at supraclinical concentrations only.

SCN5A variant R222Q generated abnormal changes in cardiac sodium current and action potentials in murine myocytes and Purkinje cells

The cardiac sodium channel (SCN5A) mutation R222Q neutralizes a positive charge in the domain I voltage sensor. Mutation carriers display very frequent ectopy and dilated cardiomyopathy. To describe the effect of SCN5A R222Q on murine myocyte and Purkinje fiber electrophysiology, and identify underlying mechanisms. We generated mice carrying humanized wild-type (H) and mutant (RQ) SCN5A channels. We characterized whole-heart and isolated ventricular and Purkinje myocyte properties. RQ/RQ mice were not viable. INa from RQ/H ventricular myocytes displayed increased "window current" and hyperpolarizing shifts in both inactivation and activation compared to H/H, as previously reported in heterologous expression systems. Surprisingly, action potentials were markedly abbreviated in RQ/H myocytes (action potential durations at 90% repolarization: 12.6± 1.3 ms vs 29.1 ± 1.0 ms in H/H, P < .01, n = 10 each). We identified a large [K+]o-dependent outward gating pore current in RQ/H but not H/H myocytes, and decreasing [K+]o elicited early afterdepolarizations (EADs) and triggered activity in isolated myocytes and ectopic beats in whole hearts. Further, RQ/H Purkinje cells displayed striking, consistent low-voltage EADs. Invivo, however, RQ/H mice displayed little ectopy and contractile function was normal. While SCN5A R222Q increases plateau inward sodium current, action potentials were unexpectedly shortened, likely reflecting an outward gating-pore current. Low extracellular potassium increased this pore current, and was arrhythmogenic invitro and exvivo.

Chloroquine inhibits tumor-related Kv10.1 channel and decreases migration of MDA-MB-231 breast cancer cells in vitro

Chloroquine (CQ) is an old antimalarial drug currently being investigated for its anti-tumor properties. As chloroquine has been shown to inhibits several potassium channels, we decided to study its effect on the tumor-related Kv10.1 channel by using patch-clamp electrophysiology and cell migration assays. We found that chloroquine inhibited Kv10.1 channels transiently expressed in HEK-293 cells in a concentration- and voltage-dependent manner acting from the cytoplasmic side of the plasma membrane. Chloroquine also inhibited the outward potassium currents from MDA-MB-231 cells, which are mainly carried through Kv10.1 channels as was confirmed using astemizole. Additionally, chloroquine decreased MDA-MB-231 cell migration in the in vitro scratch wound healing assay. In conclusion, our data suggest that chloroquine decreases MDA-MB-231 cell migration by inhibiting Kv10.1 channels. The inhibition of Kv10.1 channels could represent another mechanism of the antitumoral action of chloroquine, besides autophagy inhibition and tumor vessel normalization.

Average firing rate rather than temporal pattern determines metabolic cost of activity in thalamocortical relay neurons

Thalamocortical (TC) relay cells exhibit different temporal patterns of activity, including tonic mode and burst mode, to transmit sensory information to the cortex. Our aim was to quantify the metabolic cost of different temporal patterns of neural activity across a range of average firing rates. We used a biophysically-realistic model of a TC relay neuron to simulate tonic and burst patterns of firing. We calculated the metabolic cost by converting the calculated ion fluxes into the demand for ATP to maintain homeostasis of intracellular ion concentrations. Most energy was expended on reversing Na+ entry during action potentials and pumping Ca2+ out of the cell. Average firing rate determined the ATP cost across firing patterns by controlling the overall number of spikes. Varying intraburst frequency or spike number in each burst influenced the metabolic cost by altering the interactions of inward and outward currents on multiple timescales, but temporal pattern contributed substantially less to the metabolic demand of neural activity as compared to average firing rate. These predictions should be considered when interpreting findings of functional imaging studies that rely of estimates of neuronal metabolic demand, e.g., functional magnetic resonance imaging.

Mediating Retinal Ganglion Cell Spike Rates Using High-Frequency Electrical Stimulation.

Recent retinal studies have directed more attention to sophisticated stimulation strategies based on high-frequency (>1.0 kHz) electrical stimulation (HFS). In these studies, each retinal ganglion cell (RGC) type demonstrated a characteristic stimulus-strength-dependent response to HFS, offering the intriguing possibility of focally targeting retinal neurons to provide useful visual information by retinal prosthetics. Ionic mechanisms are known to affect the responses of electrogenic cells during electrical stimulation. However, how these mechanisms affect RGC responses is not well understood at present, particularly when applying HFS. Here, we investigate this issue via an in silico model of the RGC. We calibrate and validate the model using an in vitro retinal preparation. An RGC model based on accurate biophysics and realistic representation of cell morphology, was used to investigate how RGCs respond to HFS. The model was able to closely replicate the stimulus-strength-dependent suppression of RGC action potentials observed experimentally. Our results suggest that spike inhibition during HFS is due to local membrane hyperpolarization caused by outward membrane currents near the stimulus electrode. In addition, the extent of HFS-induced inhibition can be largely altered by the intrinsic properties of the inward sodium current. Finally, stimulus-strength-dependent suppression can be modulated by a wide range of stimulation frequencies, under generalized electrode placement conditions. In vitro experiments verified the computational modeling data. This modeling and experimental approach can be extended to further our understanding on the effects of novel stimulus strategies by simulating RGC stimulus-response profiles over a wider range of stimulation frequencies and electrode locations than have previously been explored.

The Problem of Outward Cellular Electric Current

An outward electrical current from a living cell during repolarisation implies a flow of electrons in the opposite direction according to the definition of electricity (electrons moving). As there are no free electrons in the interstitial fluid that bathes the living cell membrane, an outward current is an impossibility unless an ATP driven exchanger is utilized.

Cardiac electrical and contractile disorders promoted by anabolic steroid overdose are associated with late autonomic imbalance and impaired Ca2+ handling

AimInvestigate cardiac electrical and mechanical dysfunctions elicited by chronic anabolic steroid (AS) overdose. MethodsMale Wistar rats were treated with nandrolone decanoate (DECA) or vehicle (CTL) for 8 weeks. Electrocardiography and heart rate variability were assessed at weeks 2, 4, and 8. Cardiac reactivity to isoproterenol was investigated in isolated rat hearts. Action potential duration (APD) was measured from left ventricular (LV) muscle strips. L-type Ca2+ current (ICaL), and transient outward potassium current (Ito) were recorded by whole-cell patch-clamp in LV cardiomyocytes. Sarcoplasmic reticulum (SR) Ca2+ mobilization and Ca2+-induced contractile response sensitivity were evaluated in skinned cardiac fibers. Muscarinic type 2 receptor (M2R), β1-adrenergic receptor (β1AR), sarcoplasmic Ca2+ ATPase (SERCA-2a), type 2 ryanodine receptor (RyR2), L-type Ca2+ channel (CACNA1), Kv4.2 (KCND2), and Kv4.3 (KCND3) mRNA expression levels were measured by quantitative RT-PCR. ResultsCompared with CTL group, DECA group exhibited decreased high frequency band power density (HF) and increased low frequency power density (LF), Cardiac M2R mRNA level was decreased. QTc interval at 2nd, 4th, and 8th week as well as APD30 and APD90 were increased by DECA. Ito density was decreased, while ICaL density was increased by DECA. SR Ca2+ loading and release were decreased by DECA, while contractile sensitivity to Ca2+ was increased versus CTL group. ConclusionDECA overdose induced cardiac rhythmic and mechanical abnormalities that can be associated with autonomic imbalance, up-regulated ICaL and down-regulated Ito, abnormal SR Ca2+ mobilization, and increased contractile sensitivity to Ca2+.

Optogenetic Hyperpolarization of Cardiomyocytes Terminates Ventricular Arrhythmia.

Cardiac defibrillation to terminate lethal ventricular arrhythmia (VA) is currently performed by applying high energy electrical shocks. In cardiac tissue, electrical shocks induce simultaneously de- and hyperpolarized areas and only depolarized areas are considered to be responsible for VA termination. Because electrical shocks do not allow proper control over spatial extent and level of membrane potential changes, the effects of hyperpolarization have not been explored in the intact heart. In contrast, optogenetic methods allow cell type-selective induction of de- and hyperpolarization with unprecedented temporal and spatial control. To investigate effects of cardiomyocyte hyperpolarization on VA termination, we generated a mouse line with cardiomyocyte-specific expression of the light-driven proton pump ArchT. Isolated cardiomyocytes showed light-induced outward currents and hyperpolarization. Free-running VA were evoked by electrical stimulation of explanted hearts perfused with low K+ and the KATP channel opener Pinacidil. Optogenetic hyperpolarization was induced by epicardial illumination, which terminated VA with an average efficacy of ∼55%. This value was significantly higher compared to control hearts without illumination or ArchT expression (p = 0.0007). Intracellular recordings with sharp electrodes within the intact heart revealed hyperpolarization and faster action potential upstroke upon illumination, which should fasten conduction. However, conduction speed was lower during illumination suggesting enhanced electrical sink by hyperpolarization underlying VA termination. Thus, selective hyperpolarization in cardiomyocytes is able to terminate VA with a completely new mechanism of increased electrical sink. These novel insights could improve our mechanistic understanding and treatment strategies of VA termination.

JZTX-V Targets the Voltage Sensor in Kv4.2 to Inhibit Ito Potassium Channels in Cardiomyocytes.

Kv4 potassium channels are responsible for transient outward K+ currents in the cardiac action potential (AP). Previous experiments by our group demonstrated that Jingzhaotoxin-V (JZTX-V) selectively inhibits A-type potassium channels. However, the specific effects of JZTX-V on the transient outward (Ito) current of cardiomyocytes and underlying mechanism of action remain unclear. In the current study, 100 nM JZTX-V effectively inhibited the Ito current and extended the action potential duration (APD) of neonatal rat ventricular myocytes (NRVM). We further analyzed the effects of JZTX-V on Kv4.2, a cloned channel believed to underlie the Ito current in rat cardiomyocytes. JZTX-V inhibited the Kv4.2 current with a half-maximal inhibitory concentration (IC50) of 13 ± 1.7 nM. To establish the molecular mechanism underlying the inhibitory action of JZTX-V on Kv4.2, we performed alanine scanning mutagenesis of Kv4.2 and JZTX-V and assessed the effects of the mutations on binding activities of the proteins. Interestingly, the Kv4.2 mutations V285A, F289A, and V290A reduced the affinity for JZTX-V while I275A and L277A increased the affinity for JZTX-V. Moreover, mutation of positively charged residues (R20 and K22) of JZTX-V and the hydrophobic patch (formed by W5, M6, and W7) led to a significant reduction in toxin sensitivity, indicating that the hydrophobic patch and electrostatic interactions played key roles in the binding of JZTX-V with Kv4.2. Data from our study have shed light on the specific roles and molecular mechanisms of JZTX-V in the regulation of Ito potassium channels and supported its utility as a potential novel antiarrhythmic drug.

Early repolarisation syndrome – where do we stand now?

Early repolarization syndrome is associated with high risk of lethal arrhythmias. There is still a huge lack of evidence in the area of arrhythmogenesis, genetics and molecular mechanisms of J wave syndromes. Early repolarization is characterized by so called J waves which are seen in infero-lateral leads (II, III, aVF, V4, V5, V6). The early repolarization is dependent on rapid outward potassium current. One of the latest thesis on ventricular arrhythmias induction are various refraction times of Ito currents in epicardium and endocardium forming electrical current, which induces phase 2 re-entry arrhythmias. The treatment after cardiac arrest survival is implantation of cardioverter-defibrillator. There are few pharmacological methods of repolarization pattern restoration. Reviews of premature ventricular contractions inducing ventricular fibrillation, mainly in Brugada syndrome, ablation have been presented. The main prophylactic issue is to identify the most endangered patients.

Action of Norepinephrine on Lamina X of the Spinal Cord

Lamina X is localized in the spinal cord within the region surrounding the central canal and receives descending projections from the supraspinal nuclei. Norepinephrine (NE) is a neurotransmitter in descending pathways emanating from the brain stem; NE-containing fibers terminate in the spinal dorsal cord, particularly in the substantia gelatinosa (SG). NE enhances inhibitory synaptic transmission in SG neurons by activating presynaptic α1-receptors and hyperpolarizes the membranes of SG neurons by acting on α2-receptors; NE may thus act directly on SG neurons of the dorsal spinal cord and inhibit nociceptive transmission at the spinal level. NE-containing fibers also reportedly terminate in lamina X, suggesting that NE also modulates synaptic transmission in lamina X. However, the cellular mechanisms underlying such action have not been investigated. We hypothesized that NE might directly act on lamina X and enhance inhibitory synaptic transmission therein. Using rat spinal cord slices and in vitro whole-cell patch-clamps, we found that the bath-application of NE to lamina X does not affect the excitatory interneurons but enhances GABAergic and glycinergic miniature inhibitory postsynaptic currents (mIPSCs) and induces an outward current. NE-induced enhancement of mIPSCs was blocked by α1A-receptor antagonists, and NE-induced outward current was blocked by α2-receptor antagonists. NE did not affect GABA- or glycine- induced outward currents. These findings are similar to those obtained from SG neurons: NE may act at presynaptic terminals of GABAergic and glycinergic interneurons on lamina X to facilitate inhibitory-transmitter release through α1A-receptor activation and directly induce inhibitory interneuron membrane hyperpolarization through α2-receptors activation.

Determining the opioid sensitivity of projections from the Kölliker‐Fuse to respiratory rhythm generators

Opioid‐induced respiratory suppression is the primary cause of death in individuals who overdose on opioids, but the mechanism remains elusive. Mu opioid receptor agonists have been shown to hyperpolarize a population of neurons in the Kölliker‐Fuse nucleus (KF) due to activation of G protein‐mediated inwardly rectifying (GIRK) conductance (Levitt et al., J Physiol, 2015). The KF, required for normal eupneic breathing, adjusts respiratory rhythm and rate by modulating respiratory rhythm generators. Given the interconnectivity of the KF and respiratory rhythm generators, we hypothesize that KF neurons projecting to respiratory rhythm generators are opioid‐sensitive and thus a possible mechanism by which opioids reduce respiratory rate. The goal of these experiments was to determine if opioid‐sensitive KF neurons project to respiratory rhythm generators. Fluorescent beads (FluoSpheres 580/605, ThermoFisher) were injected into respiratory rhythm generators, including pre‐Bötzinger complex (preBötC) and post‐inspiratory complex (PiCo), of mice. Whole‐cell voltage‐clamp recordings were made from retrograde‐labeled neurons in the KF. Mu opioid receptor‐mediated outward currents were identified in KF neurons that project to the preBötC. Therefore, opioid‐sensitive connections between the KF and respiratory rhythm generators may be involved in opioid‐induced respiratory suppression.Support or Funding InformationSupported by NIH grant DA038069.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Microglia regulate RVLM‐projecting PVN neuronal activity

Microglia, the primary resident immune cells in the CNS, communicate with neurons very dynamically. Our previous studies show that activated microglia are involved in neurogenic hypertension. In the present study, we are investigating the mechanism by which microglia regulate RVLM‐projecting PVN neuronal activity in resting state. Adult male transgenic CD11b‐DTR mice were randomly assigned into two groups: sham control and diphtheria toxin (DT) groups, which received ICV infusion of saline or DT (depletion of microglia) respectively. Sympathetic neurons in the hypothalamic PVN are retrograde labeled by fluorescent microsphere from the brain stem RVLM. The electronic profile of RVLM‐PVN neurons are recorded in voltage clamp mode. Our results indicate that depletion of naïve microglia did not alter resting membrane potential nor membrane resistance. However, loss of microglia significantly inhibited outward potassium currents in RVLM‐PVN neurons. The frequency of mEPSP were also decreased in the PVN neurons in the absence of microglia. By confocal microscopy analysis, we found that there was a significantly decrease of spine density in the PVN neurons without microglia compared to the ones from the sham controls. Our results suggest that microglia directly regulate both intrinsic and extrinsic activities of the PVN sympathetic neurons.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

S‐nitroso‐L‐cysteine increases intracellular calcium in prenatal rat carotid body glomus cells

Carotid body (CB) glomus (Type I) cells detect decreases in arterial oxygen (O2) tension (hypoxia) by depolarizing, opening voltage‐gated calcium channels (VGCCs) and releasing neurotransmitters. These neurotransmitters activate chemoafferent terminals within the carotid sinus nerve (CSN), which signal respiratory regions of the brainstem to increase ventilation and restore blood O2 levels. Low‐molecular mass S‐nitrosothiols (SNOs), such as S‐nitroso‐Lcysteine (LSNC), are recently being used as chemical agents for studying the biochemistry underlying the ventilatory response to hypoxia. Unpublished work from our lab shows that LSNC alters macroscopic outward potassium currents in carotid body glomus cells under both normoxic and hypoxic conditions. However, the effect of LSNC on intracellular calcium concentration ([Ca2+]i) is unknown. Our objective was to determine whether LSNC elicits changes in [Ca2+]i in CB glomus cells. We hypothesized that LSNC increases [Ca2+]i via influx through VGCCs. To test this we isolated CB glomus cells from postnatal (P) P11–P15 Sprague Dawley male rat pups, and used Ca2+ imaging to investigate for the first time the effect of LSNC on CB glomus cell [Ca2+]i. Our preliminary data reveals that 50μM LSNC causes a rise in [Ca2+]i under normoxic conditions (n=2). This data suggests that LSNC increases CB glomus cell [Ca2+]i, and this rise may trigger Ca2+‐dependent release of neurotransmitters and activation of chemoafferent fibers within the CSN.Support or Funding InformationNIH SPARC 1OT2OD023860‐02 SJLewisThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.