Current-clamp Mode Research Articles

Electrophysiological properties of neurons in the visual sensory layers of the superior colliculus in early postnatal mice

Objective: To investigate the action potential firing patterns of neurons in the visual sensory layers of the superior colliculus in early postnatal mice and the electrophysiological characteristics of neurons with different firing patterns. Methods: This experimental study utilized whole-cell patch-clamp recordings performed on neurons in the visual sensory layers of the superior colliculus using brain slices from 57 healthy male C57BL/6J mice aged 14 to 20 days (weighing 5.0 to 8.9 g) using brain slices. In current-clamp mode, action potential characteristics were analyzed based on the first action potential generated by depolarizing current, and the firing patterns of neurons were recorded using step depolarizing currents. Neuronal firing patterns were analyzed using hierarchical clustering, and the active electrical properties of neurons with different firing patterns were compared. Results: A total of 135 neurons from the visual sensory layers of the superior colliculus were successfully recorded. Cluster analysis of the neuronal firing patterns identified three types of firing patterns: tonic firing (97, 72%), phasic firing (26, 19%), and single firing (12, 9%). The number of action potentials for each firing pattern was 13.30±7.38, 3.73±3.61, and 0.83±0.39, respectively, with significant differences (P<0.001). There was no significant difference in the membrane potential response to step currents among the three firing pattern types (P>0.05). The action potential amplitudes were (60.45±12.22), (53.67±13.20), and (44.04± 12.92) mV, and the afterhyperpolarization amplitudes were (13.45±13.79), (12.02±13.11), and (20.75±2.85) mV, respectively. The maximum rising slopes were (171.29±77.46), (130.14±61.83), and (78.89±37.08) V/s, and the maximum falling slopes were (-76.33±33.61), (-68.17±31.65), and (-47.97±13.92) V/s, respectively, with all differences being statistically significant (all P<0.05). There were no significant differences in the resting membrane potential, action potential threshold, half-width, and afterhyperpolarization duration among the three firing pattern types (all P>0.05). Conclusions: In the early postnatal mice, neurons in the visual sensory layers of the superior colliculus exhibit three distinct firing patterns: tonic, phasic, and single firing. These firing pattern types show significant differences in action potential amplitude, afterhyperpolarization amplitude, maximum rising slopes, and maximum falling slopes.

The evolution of patch-clamp electrophysiology: robotic, multiplex, and dynamic.

The patch-clamp technique has been the gold standard for analysis of excitable cells. Since its development in the 1980s it has contributed immensely to our understanding of neurons, muscle cells, and cardiomyocytes, and the ion channels and receptors that reside within them. This technique, predicated on Ohm's law, enables precise measurements of macroscopic excitability patterns, and ionic and gating conductances that can be assessed even down to the single channel level. Over the years, patch-clamp electrophysiology has undergone extensive modifications, with the introduction of new applications that have enhanced its power and reach. The most recent evolution of this technique occurred with the introduction of robotic high throughput automated platforms that enable high quality simultaneous recordings, in both voltage- and current-clamp modes, from 10s to 100s of cells, including cells freshly isolated from their native tissues. Combined with new dynamic-clamp applications, these new methods provide increasingly powerful tools for studying the contributions of ion channels and receptors to electrogenesis. In this brief review, we provide an overview of these enhanced patch-clamp techniques, followed by some of the applications presently being pursued, and a perspective into the potential future of the patch-clamp method. Significance Statement The patch-clamp technique, introduced in the 1980s, has revolutionized understanding of electrogenesis. Predicated on Ohm's law, this approach facilitates exploration of ionic conductances, gating mechanisms of ion channels and receptors, and their roles in neuronal, muscular, and cardiac excitability. Robotic platforms for high-throughput patch-clamp, and dynamic-clamp, have recently expanded its reach. Here, we outline new advances in patch-clamp including high throughput analysis of freshly-isolated neurons, and discuss the increasingly powerful trajectory of new patch-clamp techniques.

SIGNIFICANCE OF THE NAV 1.8. VOLTAGE-GATED, TETRODOTOXIN-RESISTANT SODIUM CHANNEL FOR THE RENAL SENSORY INNERVATION IN MICE AND RATS

Objective: Our previous work repeatedly showed that the sensory innervation of the kidney in rats has a peculiarity containing predominantly (more than 50%) highly active tonic neurons to electrical stimulation. In a previous publication demonstrated an increased mRNA expression of the TTX-resistant sodium channel Nav1.8 in renal sensory neurons. Hence, we tested the hypothesis that tonic firing pattern is related to the specific expression of Nav1.8 on the cell surface of neurons with renal sensoric axons in the dorsal root ganglia (DRG Th12-L2). Design and method: Harvested dorsal root ganglion neurons (DRG Th11-L2) from Sprague Dawley (SD) with renal afferents were investigated in neuronal cell culture using current clamp mode to assess action potential generation during current injection and to characterize neurons as tonic highly active and phasic less active neurons using a Nav1.8 blocker (A-803467) before and after stimulation. Further, renal DRG neurons from a Nav1.8 knock out mouse (C57BL6J-Scn10atm1Jwo) were investigated in a current clamp mode. C57BL6 mice were used as controls. Results: At a concentration of 0.3μM the maximum AP firing frequency of tonic neurons was blocked from 13+/- 1.1 APs/600ms to 7.6+/-1.4 APs/600ms under superfusion with a Nav1.8 blocker. No blocker effects were seen at a concentration of 0.1 μM and due to superfusion with the solvent methanol alone. The firing pattern of renal neurons in the C57BL6 mouse was similar to that in the SD rat with a dominance of the tonic highly active neurons. In a Nav1.8 knock out mouse (C57BL6J-Scn10atm1Jwo) in the population of neurons with dendrites from the kidney only a single cell out of 70 showed tonic firing behavior (control vs Nav1.8 KO mouse, z-test, p&lt;0.05). Conclusions: Under physiological conditions, renal sensory neurons exhibit predominantly a firing pattern associated with higher excitability. Our findings in this study support the significance of the TTX-resistant sodium channel Nav1.8 for the specific tonic firing pattern of neurons with renal projections. That might be of importance for pharmacological interventions to influence renal nerve activity, which likely plays a crucial role in the regulation of blood pressure and control of cardiovascular function.

Participation of calcium-permeable AMPA receptors in the regulation of epileptiform activity of hippocampal neurons.

Epileptiform activity is the most striking result of hyperexcitation of a group of neurons that can occur in different brain regions and then spread to other sites. Later it was shown that these rhythms have a cellular correlate in vitro called paroxysmal depolarization shift (PDS). In 13-15 DIV neuron-glial cell culture, inhibition of the GABA(A) receptors induces bursts of action potential in the form of clasters PDS and oscillations of intracellular Ca2+ concentration ([Ca2+]i). We demonstrate that GABAergic neurons expressing calcium-permeable AMPA receptors (CP-AMPARs) as well as Kv7-type potassium channels regulate hippocampal glutamatergic neurons' excitability during epileptiform activity in culture. A combination of whole-cell patch-clamp in current clamp mode and calcium imaging microscopy was used to simultaneously register membrane potential and [Ca2+]i level. To identify GABAergic cell cultures were fixed and stained with antibodies against glutamate decarboxylase GAD 65/67 and neuron-specific enolase (NSE) after vital [Ca2+]i imaging. It was shown that CP-AMPARs are involved in the regulation of the PDS clusters and [Ca2+]i pulses accompanied them. Activation of CP-AMPARs of GABAergic neurons is thought to cause the release of GABA, which activates the GABA(B) receptors of other GABAergic interneurons. It is assumed that activation of these GABA(B) receptors leads to the release of beta-gamma subunits of Gi protein, which activate potassium channels, resulting in hyperpolarization and inhibition of these interneurons. The latter causes disinhibition of glutamatergic neurons, the targets of these interneurons. In turn, the CP-AMPAR antagonist, NASPM, has the opposite effect. Measurement of membrane potential in GABAergic neurons by the patch-clamp method in whole-cell configuration demonstrated that NASPM suppresses hyperpolarization in clusters and individual PDSs. It is believed that Kv7-type potassium channels are involved in the control of hyperpolarization during epileptiform activity. The blocker of Kv7 channels, XE 991, mimicked the effect of the CP-AMPARs antagonist on PDS clusters. Both drugs increased the duration of the PDS cluster. In turn, the Kv7 activator, retigabine, decreased the duration of the PDS cluster and Ca2+ pulse. In addition, retigabine led to deep posthyperpolarization at the end of the PDS cluster. The Kv7 channel is believed to be involved in the formation of PDS, as the channel blocker reduced the rate of hyperpolarization in the PDS almost three times. Thus, GABAergic neurons expressing CP-AMPARs, regulate the membrane potential of innervated glutamatergic neurons by modulating the activity of postsynaptic potassium channels of other GABAergic neurons.

Effects of kappa-opioid agonist U-50488 and p38 MAPK inhibitor SB203580 on the spike activity of pyramidal neurons in the basolateral amygdala

Introduction: Kappa-opioid receptor (KOR) signaling in the basolateral amygdala (BLA) underlies KOR agonist-induced aversion. In this study, we aimed to understand the individual and combined effects of KOR agonist U-50488 and p38 MAPK inhibitor SB203580 on the spiking activity of pyramidal neurons in the BLA to shed light on the complex interplay between KORs, the p38 MAPK, and neuronal excitability. Materials and Methods: Electrophysiological experiments were performed using the patch-clamp technique in the whole-cell configuration. Rat brain slices containing the amygdala were prepared, and pyramidal neurons within the BLA were visually patched and recorded in the current clamp mode. The neurons were identified by their accommodation properties and neural activity signals were amplified and analyzed. Using local perfusion, we obtained three dose-response curves for: (a) U-50488 (0.001–10 μM); (b) U-50488 (0.001–10 μM) in the presence of SB203580 (1 μM); and (c) U-50488 (0.01–10 μM) in the presence of SB203580 (5 μM). Results: After the application of U-50488, pyramidal neurons had a higher action potential firing rate in response to a current injection than control neurons (p&lt;0.001). The dose-dependent curves we obtained indicate that the combination of U-50488 and SB203580 results in non-competitive antagonism. This conclusion is supported by the observed change in the curve’s slope with reduction in the maximum effect of U-50488. Thus, it can be assumed that the increase in spike activity of pyramidal neurons of the amygdala is mediated through the beta-arrestin pathway. When this pathway is blocked, the spike activity reverts to its baseline level. Conclusion: Our study found that the KOR agonist-induced spiking activity of the BLA pyramidal neurons is mediated by the beta-arrestin pathway and can be suppressed by the application of the p38 MAPK inhibitor SB203580.

Estrogen-induced phenotype in hiPSC-derived cardiomyocytes of a woman affected by both Long QT Syndrome Type 2 and Polycystic Ovary Syndrome

Abstract Introduction Long QT Syndrome (LQTS) is an arrhythmogenic disorder caused either by genetic alteration or by exogenous factors. Polycystic Ovary Syndrome (PCOS) is a disease associated with high levels of testosterone and low levels of estrogen (E2). It has been suggested that hormones can influence the occurrence of malignant cardiac events in LQTS type 2 (LQT2) women, carriers of mutations on the potassium channel (hERG). However, the precise mechanisms governing this process still need to be clarified. Purpose We studied a patient affected by both LQT2 and PCOS who started experiencing cardiac symptoms after initiation of hormonal replacement therapy. We observed a correlation with the occurrence of arrhythmic events and the irregular assumption of the hormonal replacement therapy, thus we aim to dissect the underlying mechanism in patient-in-a-dish cardiac cellular model. Methods Induced Pluripotent Stem Cells (hiPSC) were generated from a woman affected by both LQT2 and PCOS and from a healthy donor, and then differentiated into cardiomyocytes (hiPSC-CMs). hiPSC-CMs were stimulated with E2 10nM for five time points (1, 5, 10, 15 and 30 minutes). The expression of potassium channel hERG, and of the G-coupled estrogen receptor (GPER) regulating the fast non-genomic pathway, were quantified after E2 stimulation at different time points by In Cell Western (ICW). The Unstimulated sample were used to normalize GPER and hERG expression to 100%. Electrophysiological experiments were performed using the whole-cell patch-clamp technique in current-clamp mode to record spontaneous action potentials (APs). AP duration at 30%, 50% and 90% repolarization (APD30, APD50 and APD90) was quantified. During spontaneous activity, AP duration values were rate-corrected with Bazett’s formula, thus obtaining cAPD. Results LQT2 hiPSC-CMs express higher levels of GPER (p&amp;lt;0.05) and hERG (p&amp;lt;0.01) than healthy hiPSC-CMs. A significant decrease in GPER expression was detected during 10nM E2 stimulation in healthy cardiomyocytes at the five time points (p&amp;lt;0.0001), while any difference was detected in hERG expression levels. E2 stimulation at different time points did not affect neither GPER nor hERG expression in LQT2 hiPSC-CMs. In LQT2 hiPSC-CMs, APDC values were significantly increased (p&amp;lt;0.0001) compared to healthy cells; pre-incubation with E2 for 10 minutes significantly shortened cAPD50 and increased spontaneous firing rate. In healthy hiPSC-CMs, E2 had no effect. Conclusions The hiPSC-CM model reproduces the LQT2 clinical phenotype. cAPD50 shortening by E2 was unexpected based on the proarrhythmic effect in the patient. The mechanism of LQT2-specific effects of E2 are under investigation.

Abstract 057: The Positive Effect Of Renal Denervation Persists Even Through Regrowth Of Afferent Axons Improving Renal Afferent Nerve Activity During High Sodium Intake In Rats

Introduction: Previous work of ours suggests that pathologically decreased sensitivity of renal afferent neurons (reduced number of highly active tonic neurons) due to high salt diet is normalized 1 wk after renal denervation (DNX). Now we tested the hypothesis that normalized sensitivity of renal neurons persists after DNX even through regrowth of afferent axons after 12 wk. This morphological regrowth of afferent nerves 12wk after renal DNX (postDNX) was shown by us previously. Material and methods: 6 male Sprague Dawley (SD) rats were put on high salt diet (HS; 8% NaCl) for 10 days. In another group of 12 rats on high salt diet (HS) left kidneys were denervated (postDNX) 12 weeks prior to examination. Rats on standard diet were used as controls. Harvested dorsal root ganglion neurons (DRG Th11-L2) with renal afferents were investigated in primary neuronal cell culture using current clamp mode to assess action potential generation during current injection and to characterize neurons as tonic highly active and phasic less active neurons. Results: In renal neurons from rats on HS the relation of tonic to phasic neurons shifted towards less active phasic units (62% tonic neurons in controls [n=72 out of 114] vs. 42% [n=37 out of 86] on HS, (p&lt;0.05, z-test)). Denervation of the left kidney in rats on high salt diet (HS-DNX) led to a recovery of afferent renal DRG neurons after 1 wk; (40% tonic neurons [n=32 out of 80] on HS vs. 72% tonic neurons [n=58 out of 81] on HS+DNX)(p&lt;0.05, z-test)). Even 12 weeks after renal denervation this regained electrophysiological property of tonic firing persisted (43% tonic neurons [n=35 out of 82] on HS vs. 69% tonic neurons [n=46 of 67]on HS-postDNX, p&lt;0.05, z-test)). Conclusion: The reduced proportion of highly active tonic neurons increased 1 wk after renal denervation to control levels. Even 12 weeks after renal denervation, this effect of renal denervation persisted, and the sensitivity of renal neurons to electrical stimuli did not change despite high-salt diet. Hence, the positive effects of renal denervation persisted even through the regrowth of renal afferent axons to the kidney.

HIGH SODIUM INTAKE - EFFECTS ON AFFERENT RENAL NERVE ACTIVITY

Objective: Previous work of ours suggests that the sensitivity of renal afferent neurons is decreased under pathological conditions. Here we tested the hypothesis that pathologically decreased sensitivity of renal afferent neurons due to high salt diet is normalized after renal denervation. Design and method: Six male Sprague Dawley (SD) rats were put on high salt diet (HS; 8% NaCl) for 10 days. In another group of 18 rats on high salt diet (HS) left kidneys were denervated (DNX) 7 days prior to examination. 17 rats on standard diet with and without DNX were used as controls. Dorsal root ganglion neurons with renal afferents were investigated in primary neuronal cell culture using current clamp mode to assess action potential generation during current injection. Neurons were characterized as tonic highly active (less than 5 action potentials, AP) and phasic less active neurons (more than 5 AP upon stimulation). Results: In renal neurons from rats on HS the relation of tonic to phasic neurons shifted towards less active phasic units (62% tonic neurons in control vs. 42% on HS, p &lt; 0.05, z-test). Denervation (DNX) of the left kidney in rats on high salt diet (HS-DNX) led to a recovery of afferent renal DRG neurons remaining vital after denervation. They regained their electrophysiological property of mainly tonic firing (42% tonic neurons in HS vs. 71% HS-DNX, p &lt; 0.05, z-test). Conclusions: In rats on high salt diet the proportion of highly active tonic neurons with renal afferents decreased at the expense of less active phasic neurons. This HS effect could be abolished by DNX. Hence the input of afferent renal pathways to the central nervous system was normalized form the first neuron onward to the brain.

EMPAGLIFLOZIN IMPROVES IMPAIRED SENSITIVITY OF AFFERENT NEURONS WITH RENAL AXONS

Objective: Afferent renal nerve pathways likely play a role in salt sensitive hypertension. We recently reported that high salt diet (HS) impairs these afferent renal pathways in rats. Now we tested the hypothesis that during HS a decrease in sensitivity of renal afferent neurons is prevented by the SGLT2 inhibitor empagiflozin. Design and method: Respective groups of rats were put on HS containing 8% NaCl or a normal diet. Two groups (HS, controls) received empagiflozin 20 mg/kg BW/day orally. Renal neurons were retrogradely labeled with DiI. In culture, labeled dorsal root ganglion neurons (DRG Th11-L2) with renal afferents were investigated electrophysiologically using current clamp mode to assess action potential generation during current injection. Neurons were characterized as tonic highly active ( less than 5 action potentials, AP) and phasic less active neurons (more than 5 AP upon stimulation). Results: In neurons from rats on HS, the relation of tonic highly active neurons to less active phasic neurons shifted consistently towards phasic units (63,8% tonic neurons in controls vs. 42%* on HS, *p &lt; 0.05, z-test). However, continuous treatment with empagiflozin preserved the proportion of tonic neurons as in controls (67,9% on HS with concomitant administration of empagiflozin). In controls, empagiflozin did not affect the proportion of tonic to phasic neurons (63,8% tonic neurons in controls vs. 67,9% on HS &amp; empagliflozin, p = 0.7, z-test). Blood pressure and heart rate were not altered by HS and/or treatment with any chosen dose of empagiflozin. Conclusions: In rats, chronically elevated sodium intake (8% NaCl) reduced the sensitivity and stimulability of renal afferent DRG neurons. Under these circumstances, concomitant treatment with the SGLT2 inhibitor empagiflozin preserved the function of renal afferent DRG neurons. SGLT 2 inhibitors may help to treat dysfunction of renal innervation in cardiovascular disease.

Essential oil of Croton zehntneri prevents electrophysiological alterations in dorsal root ganglia of streptozotocin-induced diabetes mellitus in rats

Croton zehntneri is an aromatic shrub, native to Northeast Brazil, where it is used in folk medicine. The essential oil of C. zehntneri (EOCz) has shown potent antioxidant and anti-inflammatory activities. Our aim was to investigate the beneficial effect of EOCz on electrophysiological changes in dorsal root ganglia (DRG) of rats with streptozotocin-induced diabetes mellitus (DM). DRG excitability and passive and active membrane properties were studied in control and DM-induced rats. Intracellular recording in current-clamp mode with sharp microelectrode was used. For Na+ channel investigation, patch-clamp recording in whole-cell mode was used. DM caused depolarizations (from -60.6 to -55.0 mV) of resting membrane potential without alteration of cell membrane input resistance. Also, it showed a tendency towards a decrease in rheobase and a tendency for increase in excitability, which was confirmed by increase in Na+ current (from -113.6 to -178.3 pF/pA). EOCz did not reverse the hyperglycemia of DM. Regardless of that, upon oral treatment, EOCz prevented neuronal alterations. EOCz was demonstrated to have low toxicity. The preventive effects of EOCz on neuronal changes coupled with its low toxicity make EOCz a potential candidate for a type of treatment of diabetic neuropathy complementary to therapy with hypoglycemic agents.

Sinoatrial node cells and tissue operate via stochastic resonance.

Sinoatrial node (SAN), the primary heart pacemaker, is a highly heterogeneous, complex cellular network. Recent imaging data indicate that synchronized action potentials (APs) that exit the SAN emerge from heterogeneous signals, including subthreshold, irregular signals. We tested the hypothesis that SAN cells and tissue operate via stochastic resonance, i.e. biological noise within and among SAN cells plays an important role in generation of cardiac impulses. We performed patch-clamp experiments in current-clamp mode in isolated single rabbit SAN cells. When uniformly random current noise of a few pA amplitude with a zero average was applied via the patch pipette, cells that had been firing APs rarely and irregularly began firing more frequently and rhythmically. Further, similar noise application to non-firing cells (dormant cells or artificially hyperpolarized cells) initiated AP firing. These results were supported by simulations of Maltsev-Lakatta numerical model of SAN cells. Further, simulations using a numerical model of heterogeneous SAN cellular network, in which the rate of Ca pumping and ICaL conductance varied, revealed that random current noise applied within cells also improved the tissue function. In scenarios at the edge of criticality, in which the cell network could not operate (99.84% of non-firing cells), almost all cells (97.12%) fired APs when noise was applied to cells within the network. Although artificial noise was applied in our experiments and simulations, similar noise is likely to be generated under physiological conditions by stochastic openings of ion channels (RyRs and L-type Ca-channels) within cells and by subthreshold signals from neighboring cells in SAN tissue. Our results support the idea that the SAN operates via stochastic resonance, thus shifting the paradigm of SAN operation from dictatorship and democracy theories, to a complex neuronal-type modus operandi, i.e. the SAN operates as “the heart's brain”.

Characterization of atrial fibrillation linked mutations using the Nav1.5 knock-out of atrial cardiomyocytes derived from iPSCs.

Atrial fibrillation (AF) is the most common cardiac arrhythmia diagnosed and affects more than 33 million on people word wide. AF increases the risk of heart failure and myocardial infarction. Some studies carried out on HEK293 cells and murine models have shown that SCN5A mutations are linked to AF. However, the mechanism by which NaV1.5 mutations generate cardiac arrhythmias is still to be elucidated. Human induced pluripotent stem cells (hiPSCs) hold great promise to model NaV1.5-linked AF mutations and recapitulate the functional changes. Previously, our laboratory established an NaV1.5 knock-out (KO) iPSC line with CRISPR/Cas9 technology. In this study, atrial cardiomyocytes (iPSC-aCMs) were differentiated from the Control and NaV1.5 KO iPSC lines. We aim to characterise the atrial statue of the iPSC-aCMs, and then, use the model to express two AF Nav1.5-linked mutations (K1493R and M1875T). To verify that NaV1.5 protein expression was suppressed, immunocytofluorescence and Western blot analysis were performed. Action potentials (APs) recordings in current-clamp mode showed that APs from the NaV1.5 KO iPSC-aCMs exhibited a significant decrease of the AP overshoot and the upstroke velocity. Voltage-clamp analysis in NaV1.5/K1493R-transfected KO iPSC-aCMs showed a shift of the steady-state activation toward hyperpolarized voltage while the NaV1.5/M1875T expression provoked an increase of the Na+ current density and a shift of the steady-state inactivation toward depolarized voltage. These alterations increase the window current which indicate a channel gain-of-function. The AP recordings showed a decrease of the depolarization threshold corresponding to a cellular excitability increase. 61 and 80 % of NaV1.5/K1493R and /M1875T-transfected iPSC-aCMs respectively, exhibited arrhythmic events such as early and delayed afterdepolarizations (EADs and DADs). Our results confirm that the gain-of-function by shifting of the activation and inactivation properties is the cause of AF.

Nicotine but not saline self-administering or yoked control conditions produces sustained neuroadaptations in the accumbens shell.

Using yoked animals as the control when monitoring operant drug-self-administration is considered the golden standard. However, instrumental learning per se recruits several neurocircuits that may produce distinct or overlapping neuroadaptations with drugs of abuse. The aim of this project was to assess if contingent responding for nicotine or saline in the presence of a light stimulus as a conditioned reinforcer is associated with sustained neurophysiological adaptations in the nucleus accumbens shell (nAcS), a brain region repeatedly associated with reward related behaviors. To this end, nicotine-or saline-administrating rats and yoked-saline stimulus-unpaired training conditions were assessed in operant boxes over four consecutive weeks. After four additional weeks of home cage forced abstinence and subsequent cue reinforced responding under extinction conditions, ex vivo electrophysiology was performed in the nAcS medium spiny neurons (MSNs). Whole cell recordings conducted in voltage and current-clamp mode showed that excitatory synapses in the nAcS were altered after prolonged forced abstinence from nicotine self-administration. We observed an increase in sEPSC amplitude in animals with a history of contingent nicotine SA potentially indicating higher excitability of accumbal MSNs, which was further supported by current clamp recordings. Interestingly no sustained neuroadaptations were elicited in saline exposed rats from nicotine associated visual cues compared to the yoked controls. The data presented here indicate that nicotine self-administration produces sustained neuroadaptations in the nAcS while operant responding driven by nicotine visual stimuli has no long-term effects on MSNs in nAcS.

Ca2+ -activated Cl- channels (TMEM16A) underlie spontaneous electrical activity in isolated mouse corpus cavernosum smooth muscle cells.

Penile detumescence is maintained by tonic contraction of corpus cavernosum smooth muscle cells (CCSMC), but the underlying mechanisms have not been fully elucidated. The purpose of this study was to characterize the mechanisms underlying activation of TMEM16A Ca2+ -activated Cl- channels in freshly isolated murine CCSMC. Male C57BL/6 mice aged 10-18 weeks were euthanized via intraperitoneal injection of sodium pentobarbital (100 mg.kg-1 ). Whole-cell patch clamp, pharmacological, and immunocytochemical experiments were performed on isolated CCSM. Tension measurements were performed in whole tissue. TMEM16A expression in murine corpus cavernosum was confirmed using immunocytochemistry. Isolated CCSMC developed spontaneous transient inward currents (STICs) under voltage clamp and spontaneous transient depolarizations (STDs) in current clamp mode of the whole cell, perforated patch clamp technique. STICs reversed close to the predicted Cl- equilibrium potential and both STICs and STDs were blocked by the TMEM16A channel blockers, Ani9 and CaCC(inh)-A01. These events were also blocked by GSK7975A (ORAI inhibitor), cyclopiazonic acid (CPA, sarcoplasmic reticulum [SR] Ca2+- ATPase blocker), tetracaine (RyR blocker), and 2APB (IP3 R blocker), suggesting that they were dependent on Ca2+ release from intracellular Ca2+ stores. Nifedipine (L-type Ca2+ channel blocker) did not affect STICs, but reduced the duration of STDs. Phenylephrine induced transient depolarizations and transient inward currents which were blocked by Ani9. Similarly, phenylephrine induced phasic contractions of intact corpus cavernosum muscle strips and these events were also inhibited by Ani9. This study suggests that contraction of CCSM is regulated by activation of TMEM16A channels and therefore inhibition of these channels could lead to penile erection.

Artificial sleep-like up/down-states induce synaptic plasticity in cortical neurons from mouse brain slices.

During non-rapid eye movement (NREM) sleep, cortical neuron activity alternates between a depolarized (firing, up-state) and a hyperpolarized state (down-state) coinciding with delta electroencephalogram (EEG) slow-wave oscillation (SWO, 0. 5–4 Hz) in vivo. Recently, we have found that artificial sleep-like up/down-states can potentiate synaptic strength in layer V cortical neurons ex vivo. Using mouse coronal brain slices, whole cell voltage-clamp recordings were made from layer V cortical pyramidal neurons to record spontaneous excitatory synaptic currents (sEPSCs) and inhibitory synaptic currents (sIPSCs). Artificial sleep-like up/down-states (as SWOs, 0.5 Hz, 10 min, current clamp mode) were induced by injecting sinusoidal currents into layer V cortical neurons. Baseline pre-SWO recordings were recorded for 5 min and post-SWO recordings for at least 25–30 min. Compared to pre-SWO sEPSCs or sIPSCs, post-SWO sEPSCs or sIPSCs in layer V cortical neurons exhibited significantly larger amplitudes and a higher frequency for 30 min. This finding suggests that both sEPSCs and sIPSCs could be potentiated in layer V cortical neurons by the low-level activity of SWOs, and sEPSCs and sIPSCs maintained a balance in layer V cortical neurons during pre- and post-SWO periods. Overall, this study presents an ex vivo method to show SWO's ability to induce synaptic plasticity in layer V cortical neurons, which may underlie sleep-related synaptic potentiation for sleep-related memory consolidation in vivo.

Dapagliflozin exhibits class I antiarrhythmic effects which suppress action potential formation in human atrial cardiomyocytes

Abstract Background Recently, inhibitors of sodium-glucose transporter 2 (SGLT2i) were shown to have tremendous cardioprotective effects in patients with type 2 diabetes mellitus (T2DM) as well as heart failure (HF) patients, regardless of their glycemic status. Among other beneficial effects on cardiovascular outcome an antiarrhythmic effect of SGLT2i was indicated. In patients with T2DM therapy with SGLT2i was associated with a significantly reduced risk of atrial arrhythmias and sudden cardiac death. As sodium-glucose transporter 2 (SGLT2) is not expressed within the heart underlying molecular modes of action remain unclear. Therefore, investigating possible antiarrhythmic mechanisms is crucial to determine whether SGLT2i might be beneficial for all patients suffering from arrhythmias, regardless of other comorbidities. Purpose To assess antiarrhythmic effects of SGLT2i on a molecular and cellular level. Therefore, direct electrophysiological effects of dapagliflozin – the most advanced SGLT2i in clinical trials – on action potential (AP) formation in atrial cardiomyocytes (CM) was investigated. Methods Effects of dapagliflozin on human NaV1.5, heterologously expressed in Chinese hamster ovary (CHO) cells, was investigated using the patch-clamp method. Further, effects on voltage-gated sodium channels (NaV) were also measured in human atrial CM. Consequences of dapagliflozin treatment on AP formation were studied on isolated human and porcine atrial CM using the patch-clamp technique. APs were elicited in current-clamp mode by injection of brief current pulses. Results Dapagliflozin (100 μM) significantly decreased peak sodium currents in human atrial CM as well as CHO cells, expressing human NaV1.5 channel subunits. Additionally, half-activation potential of voltage-gated sodium channels was significantly increased after administration of dapagliflozin. Furthermore, dapagliflozin (100 μM) suppressed AP formation in CM isolated from human and porcine left and right atrial tissue. In human CM action potential amplitude (APA) was significantly reduced by 34.3%, while APA inhibition by dapagliflozin averaged out at 30.7% for porcine CM. In porcine CM action potential duration at 50% (APD50) and 90% repolarization (APD90) were also significantly reduced. Conclusion Dapagliflozin suppresses AP formation in human and porcine atrial CM by inhibiting voltage-gated sodium currents. Therefore, we suppose that dapagliflozin exhibits class I antiarrhythmic effects. Funding Acknowledgement Type of funding sources: None.

Radiation-induced electrophysiological remodelling in neonatal rat ventricular cardiomyocytes

Abstract Background Ventricular tachycardia (VT) carries a relatively high risk of recurrence and significantly impacts patients' quality of life, despite state of the art treatment including antiarrhythmic drug therapy and catheter ablation. Cardiac stereotactic body radiotherapy (SBRT) has emerged as a promising non-invasive treatment option for refractory VT. A marked reduction in VT episodes is observed within days of treating high-risk patients with a single fraction SBRT. The cellular mechanisms underlying the clinically demonstrated antiarrhythmic effect are still poorly understood. Radiation-induced fibrosis is observed several months after irradiation, suggesting other mechanisms that explain the immediate benefit of radiotherapy on the VT substrate. Purpose We hypothesized that early antiarrhythmic effects of single-fraction radiation are produced by direct molecular and functional electrophysiological changes, independent of fibrosis. Our aim was to investigate the effects of 20 Gy ionizing photon radiation on the electrophysiology of ventricular neonatal rat cardiomyocytes at time periods from 24 hours to 96 hours after irradiation. Methods Ventricular neonatal rat cardiomyocytes were isolated, cultivated for 24 to 96 hours and then exposed to photon beam with a single dose of 20 Gy. Expression changes of ion channels, calcium handling proteins and structural proteins affecting cellular electrophysiology were assessed via RT-qPCR and Western blotting. Beat frequency was evaluated by video analysis and action potentials (APD) were recorded using the whole-cell voltage clamp technique in current-clamp mode. Intracellular calcium transients in Fura-2 AM loaded cardiomyocytes were recorded with an IonOptix recording system. Results Irradiation lead to significant changes in the mRNA and protein level of ion channels, with most distinct changes being observed 96h after radiation. Increased mRNA and protein expression of (Cav1.2) and of the potassium channels Kcnd3 (Kv4.3), Kcnh2 (Kv11.1), Kcnq1 (Kv7.1), Kcnk2 (K2P2.1) and Kcnj2 (Kir2.1) was observed. Additionally, Connexin 43 (Cx43) was upregulated. Irradiated cardiomyocytes showed a significant increase in beat rate over time. APD was significantly shortened, with a significantly increased upstroke velocity. Regarding calcium handling, significant upregulations of Ryr2 (RYR2) and Slc8a1 (NCX) transcript and protein level was detected. Additionally, calcium handling properties were altered. Conclusion Our results suggest that acute irradiation effects are associated with electrophysiological remodelling in cardiomyocytes. Changes of cellular ion channel expression and calcium homeostasis lead to normalization and shortening of heart-failure associated APD prolongation. These results reveal new insights into acute antiarrhythmic effects on cardiomyocytes after SBRT-therapy. Funding Acknowledgement Type of funding sources: None.

Coupling the Cardiac Voltage-Gated Sodium Channel to Channelrhodopsin-2 Generates Novel Optical Switches for Action Potential Studies.

Voltage-gated sodium (Na+) channels respond to short membrane depolarization with conformational changes leading to pore opening, Na+ influx, and action potential (AP) upstroke. In the present study, we coupled channelrhodopsin-2 (ChR2), the key ion channel in optogenetics, directly to the cardiac voltage-gated Na+ channel (Nav1.5). Fusion constructs were expressed in Xenopus laevis oocytes, and electrophysiological recordings were performed by the two-microelectrode technique. Heteromeric channels retained both typical Nav1.5 kinetics and light-sensitive ChR2 properties. Switching to the current-clamp mode and applying short blue-light pulses resulted either in subthreshold depolarization or in a rapid change of membrane polarity typically seen in APs of excitable cells. To study the effect of individual K+ channels on the AP shape, we co-expressed either Kv1.2 or hERG with one of the Nav1.5-ChR2 fusions. As expected, both delayed rectifier K+ channels shortened AP duration significantly. Kv1.2 currents remarkably accelerated initial repolarization, whereas hERG channel activity efficiently restored the resting membrane potential. Finally, we investigated the effect of the LQT3 deletion mutant ΔKPQ on the AP shape and noticed an extremely prolonged AP duration that was directly correlated to the size of the non-inactivating Na+ current fraction. In conclusion, coupling of ChR2 to a voltage-gated Na+ channel generates optical switches that are useful for studying the effect of individual ion channels on the AP shape. Moreover, our novel optogenetic approach provides the potential for an application in pharmacology and optogenetic tissue-engineering.

Effects of chronic intermittent hypoxia on atrial electrical remodeling in rats

The aim of this study was to investigate the effects of chronic intermittent hypoxia (CIH) on atrial electrical remodeling in Sprague-Dawley (SD) rats, which provide the explication for the mechanisms of CIH promoting atrial fibrillation (AF). Eighty SD rats were randomly divided into 2 groups: control group and CIH group (n=40). CIH rats were subjected to CIH 8 h/d for 30 days. After the echocardiography and hemodynamics examination, cardiac electrophysiological experiments, histological experiments, and molecular biological experiments were executed. AF susceptibility was measured by isolated heart electrophysiological experiments. Masson's trichrome stain was used to assess the degree of atrial fibrosis. The protein expression levels of sodium voltage-gated channel alpha subunit 5 (SCN5A/Nav1.5), calcium voltage-gated channel subunit alpha1 C (CACNA1C/Cav1.2) and potassium voltage-gated channel subfamily D member 3 (KCND3/Kv4.3) were measured by Western blot. In whole-cell patch clamp experiments, current clamp mode was used to record AP, and APD90 and APD50 were analyzed and compared between the two groups. In voltage clamp mode, INa, ICa-L, Ito and their kinetic parameters were recorded and compared between the two groups. Compared to the control rats, atrial interstitial collagen deposition (P＜0.01) and AF inducibility (P＜0.05) were increased in CIH rats, whereas the expression levels of Nav1.5, Cav1.2 and Kv4.3 were decreased (P＜0.05). APD90 and APD50 in CIH rats' atrial myocytes were longer than those of control rats, and CIH rats showed decreased current density of INa, ICa-L(P＜0.01) and Ito(P＜0.01). CIH-induced changes in the protein expression levels of ion channel subunits, current intensity, APD, and AF susceptibility, which may be the mechanisms of CIH promoting AF.

Abstract P338: High Sodium Intake - Effects On Afferent Renal Nerve Activity

Previous work of ours suggests that the sensitivity of renal afferent neurons is decreased under pathological conditions. Here we tested the hypothesis that pathologically decreased sensitivity of renal afferent neurons due to high salt diet is normalized after renal denervation. 6 male Sprague Dawley (SD) rats were put on high salt diet (HS; 8% NaCl) for 10 days. In another group of 18 rats on high salt diet (HS) left kidneys were denervated (DNX) 7 days prior to examination. 17 rats on standard diet with and without DNX were used as controls. Dorsal root ganglion neurons with renal afferents were investigated in primary neuronal cell culture using current clamp mode to assess action potential generation during current injection. Neurons were characterized as tonic highly active (&gt; 5 action potentials, AP) and phasic less active neurons (≤ 5 AP upon stimulation)In renal neurons from rats on HS the relation of tonic to phasic neurons shifted towards less active phasic units (62% tonic neurons in control vs. 42% on HS, p&lt;0.05, z-test). Denervation (DNX) of the left kidney in rats on high salt diet (HS-DNX) led to a recovery of afferent renal DRG neurons remaining vital after denervation. They regained their electrophysiological property of mainly tonic firing (42% tonic neurons in HS vs. 71% HS-DNX, p&lt;0.05, z-test).In rats on high salt diet the proportion of highly active tonic neurons with renal afferents decreased at the expense of less active phasic neurons. This HS effect could be abolished by DNX.