Voltage-clamp Mode Research Articles

Exploring the role of Cdk5 on striatal synaptic plasticity in a 3-NP-induced model of early stages of Huntington's disease.

Impaired mitochondrial function has been associated with the onset of neurodegenerative diseases. Specifically, certain mitochondrial toxins, such as 3-nitropropionic acid (3-NP), initiate cellular changes within the striatum that closely resemble the pathology observed in Huntington's disease (HD). Among the pivotal signaling molecules contributing to neurodegeneration, cyclin-dependent kinase 5 (Cdk5) stands out. In particular, Cdk5 has been implicated not only in cellular pathology but also in the modulation of synaptic plasticity. Given its widespread presence in the striatum, this study seeks to elucidate the potential role of Cdk5 in the induction of corticostriatal synaptic plasticity in murine striatal cells subjected to subchronic doses of 3-NP in vivo, aiming to mimic the early stages of HD. Immunostaining analyses revealed an increase in Cdk5 in tissues from animals treated with 3-NP, without a significant change in protein levels. Regarding striatal plasticity, long-term depression (LTD) was induced in both control and 3-NP cells when recorded in voltage clamp mode. The Cdk5 inhibitor roscovitine-reduced LTD in most cells. A minority subset of cells exhibited long-term potentiation (LTP) generation in the presence of roscovitine. The inhibitor of D1 receptors SCH23390 prevented LTP in three of nine cells, implying that MSN cells lacking D1/PKA activation were capable of LTP induction when Cdk5 was also blocked. Nevertheless, the co-administration of H89, a PKA inhibitor, along with roscovitine, prevented the generation of any type of plasticity in all recorded cells. These findings show the impact of 3-NP treatment on striatal plasticity and suggest that Cdk5 during early neurodegeneration may attenuate signaling pathways that lead neurons to increase their activity.

Selective KCNQ2/3 Potassium Channel Opener ICA069673 Inhibits Excitability in Mouse Vagal Sensory Neurons

The excitability of vagal sensory neurons is abnormally enhanced in inflammatory visceral diseases, contributing to the unproductive and diffcult-to-treat neuronally based symptoms such as non-cardiac chest pain and excessive coughing. Identification of targets and modulators capable of regulating the excitability of vagal sensory neurons may lead to novel therapeutic options. The KCNQ/KV7/M-channel is increasingly recognized as an intrinsic regulator of excitability in neurons, and several neuronal KCNQ2/3-selective M-channel openers have been developed. This study aimed to characterize the effects of one of these KCNQ2/3-selective activators, ICA-069673, on the M-current (IM) in mouse nodose neurons, and to determine its effects on excitability and action potential firings using perforated patch-clamp technique. The results showed that ICA-069673 enhanced IM density, accelerated the activation and delayed the deactivation of M-channels in a concentration-dependent manner. The EC50 of ICA-069673 in enhancing IM determined at -30 mV is 0.52 μM. ICA-069673 at 10 μM shifted the voltage-dependent activation of IM in hyperpolarizing direction by -25.5 ± 4.1 mV (p<0.001) and increased the maximal conductance from 8.1 ± 4.0 to 12.6±7.4 nS/pF (p=0.031). In a total of 48 nodose neurons studied on voltage-clamp mode, ICA-069673 enhanced IM in 41 neurons (~85%). Consistent with its effects on IM, ICA-069673 induced a marked hyperpolarization of resting potential and reduced the input resistance. The hyperpolarizing effect was more pronounced in partially depolarized neurons. Moreover, ICA-069673 caused a 3-fold increase in the minimal amount of depolarizing current needed to evoke an action potential, and significantly limited the action potential firings in response to sustained suprathreshold stimulations. These results indicate that opening KCNQ2/3-mediated M-channels is suffcient to suppress the excitability and enhance spike accommodation in vagal sensory neurons, and provide evidence supporting further investigation into the effcacy of ICA-069673 in normalizing the aberrant vagal visceral nociception. The study is supported by NIH Grant R35HL155671. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Substitution of bridge carbons for sulphur in calix[4]arene-bis-α-hydroxymethylphosphonic acid transformed mobile carrier into ionic channel accompanied with evoked muscle contraction and impaired neurotransmission powered by membrane action of resulting thiocalix[4]arene-bis-α-hydroxymethylphosphonic acid

The action of calix[4]arenes C-424, C-425 and C-1193 has been investigated on suspended cholesterol/egg phosphatidylcholine lipid bilayer in a voltage-clamp mode. Comparative analysis with the membrane action by calix[4]arene-bis-α-hydroxymethylphosphonic acid (C-99) has shown that the substitution of bridge carbons for sulphur and addition of another methyl group to two alkyl tales in the lower rim of former dipropoxycalix[4]arene C-99 transformed mobile carrier that C-99 created in lipid bilayer (Shatursky et al., 2014) into a transmembrane pore as exposure of the bilayer membrane to sulphur-containing derivative dibutoxythiocalix[4]arene C-1193 resulted in microscopic transmembrane current patterns indicative of a channel-like mode of facilitated diffusion. Within all calix[4]arenes tested a net steady-state voltage-dependent transmembrane current was readily achieved only after addition of calix[4]-arene C-1193. In comparison with the membrane action of C-99 the current induced by calix[4]-arene C-1193 exhibited a much weakened anion selectivity passing slightly more current at positive potentials applied from the side of bilayer membrane to which the calix[4]-arene was added. Testing C-1193 for the membrane action against smooth muscle cells of rat uterus or swine myometrium and synaptosomes of rat brain nerve terminals revealed an increase in intracellular concentration of Ca2+ with reduction of the effective hydrodynamic diameter of the smooth muscle cells and enhanced basal extracellular level of neurotransmitters (glutamate and γ-aminobutyric acid) after C-1193-induced depolarization of the nerve terminals.

Voltage-Clamp Analysis of Synaptic Transmission at the Drosophila Larval Neuromuscular Junction.

Although it is particularly valuable in revealing membrane potential changes, intracellular recording has a number of limitations. Primarily, it does not offer information on the kinetics of membrane currents associated with ion channels or synaptic receptors responsible for the potential change. Furthermore, the resting potential of the Drosophila body wall muscle varies naturally such that the driving force also varies considerably, making it difficult to accurately compare the amplitude of miniature synaptic potentials (minis) or evoked excitatory junction potentials (EJPs). Finally, accurate determination of quantal content based on minis and EJPs is possible only under low-release conditions when nonlinear summation is not a major issue. As the EJP amplitude increases, it creates a "ceiling effect," because the same amount of transmitter will be less effective in depolarizing the membrane when the potential is approaching the reversal potential of glutamate receptors/channels. To overcome these limitations, the voltage-clamp technique can be used, which uses negative feedback mechanisms to keep the cell membrane potential steady at any reasonable set points. In voltage-clamp mode, the amplitude and kinetics of membrane currents can be determined. In the large larval muscle cells of Drosophila, the two-electrode voltage-clamp (TEVC) method is used, in which one electrode monitors the cell membrane potential while the other electrode passes electric currents. This protocol introduces the application of TEVC in analysis of synaptic currents using the larval neuromuscular junction preparation.

Outward potassium current in neurons of aestivated land snail Achatina fulica.

Aestivation and hibernation represent distinct forms of animal quiescence, characterized by physiological changes, including ion composition. Intracellular ion flows play a pivotal role in eliciting alterations in membrane potential and facilitating cellular communication, while outward K+ currents aid in the restitution and upkeep of the resting membrane potential. This study explores the relationship between inward and outward currents during aestivation in Achatina fulica snails. Specimens were collected near MSUBIT University in Shenzhen and divided into two groups. The first group was kept on a lattice diet, while the second one consisted of aestivating individuals, that were deprived of food and water until a cork-like structure sealed their shells. Recording of current from isolated neurons were conducted using the single-electrode voltage clamp mode with an AxoPatch 200B amplifier. Electrophysiological recordings on pedal ganglia neurons revealed significant differences in the inactivation processes of the Ia and Ikdr components. Alterations in the Ikdr component may inhibit pacemaker activity in pedal ganglion neurons, potentially contributing to locomotion cessation in aestivated animals. The KS current remains unaffected during aestivation. Changes in slow K+ current components could disrupt the resting membrane potential, possibly leading to cell depolarization and influx of Ca2+ and Na+ ions, impacting cell homeostasis. Thus, maintaining the constancy of outward K+ current is essential for cell stability.

Cx43 Hemichannel and Panx1 Channel Modulation by Gap19 and 10Panx1 Peptides.

Cx43 hemichannels (HCs) and Panx1 channels are two genetically distant protein families. Despite the lack of sequence homology, Cx43 and Panx1 channels have been the subject of debate due to their overlapping expression and the fact that both channels present similarities in terms of their membrane topology and electrical properties. Using the mimetic peptides Gap19 and 10Panx1, this study aimed to investigate the cross-effects of these peptides on Cx43 HCs and Panx1 channels. The single-channel current activity from stably expressing HeLa-Cx43 and C6-Panx1 cells was recorded using patch-clamp experiments in whole-cell voltage-clamp mode, demonstrating 214 pS and 68 pS average unitary conductances for the respective channels. Gap19 was applied intracellularly while 10Panx1 was applied extracellularly at different concentrations (100, 200 and 500 μM) and the average nominal open probability (NPo) was determined for each testing condition. A concentration of 100 µM Gap19 more than halved the NPo of Cx43 HCs, while 200 µM 10Panx1 was necessary to obtain a half-maximal NPo reduction in the Panx1 channels. Gap19 started to significantly inhibit the Panx1 channels at 500 µM, reducing the NPo by 26% while reducing the NPo of the Cx43 HCs by 84%. In contrast 10Panx1 significantly reduced the NPo of the Cx43 HCs by 37% at 100 µM and by 83% at 200 µM, a concentration that caused the half-maximal inhibition of the Panx1 channels. These results demonstrate that 10Panx1 inhibits Cx43 HCs over the 100-500 µM concentration range while 500 µM intracellular Gap19 is necessary to observe some inhibition of Panx1 channels.

Inhibition of high-voltage-activated calcium currents by acute hypoxia in cultured retinal ganglion cells.

Hypoxia is a common factor of numerous ocular diseases that lead to dysfunctions and loss of retinal ganglion cells (RGCs) with subsequent vision loss. High-voltage-activated calcium channels are the main source of calcium entry into neurons. Their activity plays a central role in different signaling processes in health and diseases, such as enzyme activation, gene transcription, synaptic transmission, or the onset of cell death. This study aims to establish and evaluate the initial effect of the early stage of acute hypoxia on somatic HVA calcium currents in cultured RGCs. HVA calcium currents were recorded in RGCs using the whole-cell patch-clamp technique in the voltage-clamp mode. The fast local superfusion was used for a brief (up to 270 s) application of the hypoxic solution (pO2 < 5 mmHg). The switch from normoxic to hypoxic solutions and vice versa was less than 1 s. The HVA calcium channel activity was inhibited by acute hypoxia in 79% of RGCs (30 of 38 RGCs) in a strong voltage-dependent manner. The level of inhibition was independent of the duration of hypoxia or repeated applications. The hypoxia-induced inhibition of calcium currents had a strong correlation with the duration of hypoxia and showed the transition from reversible to irreversible at 75 s of hypoxia and longer. The results obtained are the first demonstration of the phenomena of HVA calcium current inhibition by acute hypoxia in RGCs and provide a conceptual framework for further research.

Group I metabotropic glutamate receptor-triggered temporally patterned action potential-dependent spontaneous synaptic transmission in mouse MNTB neurons

Rhythmic action potentials (AP) are generated via intrinsic ionic mechanisms in pacemaking neurons, producing synaptic responses of regular inter-event intervals (IEIs) in their targets. In auditory processing, evoked temporally patterned activities are induced when neural responses timely lock to a certain phase of the sound stimuli. Spontaneous spike activity, however, is a stochastic process, rendering the prediction of the exact timing of the next event completely based on probability. Furthermore, neuromodulation mediated by metabotropic glutamate receptors (mGluRs) is not commonly associated with patterned neural activities. Here, we report an intriguing phenomenon. In a subpopulation of medial nucleus of the trapezoid body (MNTB) neurons recorded under whole-cell voltage-clamp mode in acute mouse brain slices, temporally patterned AP-dependent glycinergic sIPSCs and glutamatergic sEPSCs were elicited by activation of group I mGluRs with 3,5-DHPG (200 µM). Auto-correlation analyses revealed rhythmogenesis in these synaptic responses. Knockout of mGluR5 largely eliminated the effects of 3,5-DHPG. Cell-attached recordings showed temporally patterned spikes evoked by 3,5-DHPG in potential presynaptic VNTB cells for synaptic inhibition onto MNTB. The amplitudes of sEPSCs enhanced by 3,5-DHPG were larger than quantal size but smaller than spike-driven calyceal inputs, suggesting that non-calyceal inputs to MNTB might be responsible for the temporally patterned sEPSCs. Finally, immunocytochemical studies identified expression and localization of mGluR5 and mGluR1 in the VNTB-MNTB inhibitory pathway. Our results imply a potential central mechanism underlying the generation of patterned spontaneous spike activity in the brainstem sound localization circuit.

Whole-cell patch-clamp recording and parameters.

The patch-clamp technique represents an electrophysiology type of method. This is one of several insightful approaches with five major configurations, namely a loose patch, cell-attached (also known as on-cell), whole-cell, inside-out, and outside-out modes. The patch-clamp method is more advanced compared to classical electrophysiology since it elucidates single-channel activation in a tiny portion of the membrane in addition to action potential (AP), junction potential (JP), endplate potential (EP), electrical coupling between two adjacent cells via Gap junction hemi-channels, excitatory/inhibitory postsynaptic potentials, and resting membrane potential (RMP). In fact, a malfunction of only one channel or even one component will alter AP amplitude or duration in vitro. If parameters are inferred appropriately and recordings are performed properly, the patch-clamp trace readouts and results are robust. The main hallmarks of currents via voltage-dependent calcium (Cav), hyperpolarization-activated cyclic nucleotide gated non-selective cation (HCN), inwardly rectifying potassium (Kir), voltage-dependent potassium (Kv), and voltage-dependent sodium (Nav) channels are similar and tractable among cells even when they are derived from evolutionary distinct organs and species. However, the size of the membrane area, where the functional subunits reside, and current magnitudes vary among cells of the same type. Therefore, dividing current magnitudes by cell capacitance- current density enables the estimate of functional and active channels relative to recorded cytoplasmic membrane area. Since the patch-clamp recordings can be performed in both current- and voltage-clamp modes, the action potential or spike durations can be adequately elucidated. Sometimes, optical methods are preferred to patch-clamp electrophysiology, but the obtained signals and traces are not robust. Finally, not only an alternans of AP durations, but also that of 'action potential shape' is observed with electrophysiology.

VPAC2 receptor mediates VIP-potentiated insulin secretion via ion channels in rat pancreatic β cells

Vasoactive intestinal peptide (VIP), a small neuropeptide composing of 28 amino acids, functions as a neuromodulator with insulinotropic effect on pancreatic β cells, in which it is of vital importance in regulating the levels of blood glucose. VIP potently agonizes VPAC2 receptor (VPAC2-R). Agonists of VPAC2-R stimulate glucose-dependent insulin secretion. The purpose of this study was to further investigate the possible ion channel mechanisms in VPAC2-R-mediated VIP-potentiated insulin secretion. The results of insulin secretion experiments showed that VIP augmented insulin secretion in a glucose-dependent manner. The insulinotropic effect was mediated by VPAC2-R rather than VPAC1 receptor (VPAC1-R), through the adenylyl cyclase (AC)/protein kinase A (PKA) signalling pathway. The calcium imaging analysis demonstrated that VIP increased intracellular Ca2+ concentration ([Ca2+]i). In addition, in the whole-cell voltage-clamp mode, we found that VIP blocked the voltage-dependent potassium (Kv) channel currents, while this effect was reversed by inhibiting the VPAC2-R, AC or PKA respectively. Taken together, these findings suggest that VIP stimulates insulin secretion by inhibiting the Kv channels, activating the Ca2+ channels, and increasing [Ca2+]i through the VPAC2-R and AC/PKA signalling pathway. These findings provide theoretical basis for the research of VPAC2-R as a novel therapeutic target.

Effect of high-frequency membrane potential alternation between depolarization and hyperpolarization on dorsal root ganglion neurons of rats.

The purpose of this study was to determine how sensory neurons respond to high-frequency membrane potential alternation between depolarization and hyperpolarization. Membrane currents were recorded from dissociated dorsal root ganglion (DRG) neurons of adult rats using the whole cell patch clamp technique in voltage clamp mode. Stepwise depolarization of the membrane was applied first to determine the threshold membrane potential for inducing an action potential (AP) current. Then, membrane potential alternation between depolarization (to +20 mV) and hyperpolarization (to -110 mV) was applied to the neuron for 10s at different frequencies (10 Hz to 1 kHz). The tested DRG neurons had APs of either a long duration (>10ms) or a short duration (<10ms). Membrane potential alternation at ≥500 Hz completely disrupted the AP generation, disabled the ion channel gating function, and produced membrane current alternating symmetrically across zero. Replacing extracellular sodium with potassium increased the amplitude of the membrane current response and caused the membrane current to be larger during hyperpolarization than during depolarization. These results support the hypothesis that high-frequency biphasic stimulation blocks axonal conduction by driving the potassium channel open constantly. Understanding neural membrane response to high-frequency membrane potential alternation is important to reveal the possible mechanisms underlying axonal conduction block induced by high-frequency biphasic stimulation.

Furosemide blocks the anion channel caused by Staphylococcus aureus α-hemolysin

To investigate the effect of furosemide on Staphylococcus aureus α-hemolysin (α-HL) channel in planar lipid bilayers by electrophysiological characterization and molecular docking studies. Planar lipid bilayer membranes were prepared and α-HL (0.07 mg/mL) was added to the standard solution in cis compartment of the experimental chamber. All experiments were performed at room temperature using an Axopatch 200A amplifier in the voltage clamp mode. At pH 7.5, α-HL channels were usually in a high conductance ~4 nS and rarely switch to low conductance states. After the ion channel was incorporated in bilayer membrane, the furosemide was also added to the standard solution to the cis compartment. To docking studies, atomics coordinates for the α-HL heptameric channel was retrieved from PDB ID (7AHL) and the structure of furosemide was removed from the PubChem, their coordinates were built and minimized with Avogadro software. The molecular docking experiments were performed using the Dockthor online. The furosemide inhibited (P&lt;0.05) conductance α-HL channel and it was a voltage-dependent manner. The two best docking solutions and the α-HL channel were evaluated, it was observed the connection mode with the highest affinity of interaction has a greater number of hydrogen bonding. The residues were 113 and 147 that form the remainders of the constriction α-HL channel. In conclusion, furosemide blocks ion currents in the constriction of channel caused by Staphylococcus aureus α-hemolysin.

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.

GABAA Receptor Autoantibodies Decrease GABAergic Synaptic Transmission in the Hippocampal CA3 Network.

Autoimmune encephalitis associated with antibodies (Abs) against α1, β3, and γ2 subunits of γ-aminobutyric acid receptor A (GABAAR) represents a severe form of encephalitis with refractory seizures and status epilepticus. Reduction in inhibitory GABAergic synaptic activity is linked to dysfunction of neuronal networks, hyperexcitability, and seizures. The aim in this study was to investigate the direct pathogenic effect of a recombinant GABAAR autoantibody (rAb-IP2), derived from the cerebrospinal fluid (CSF) of a patient with autoimmune GABAAR encephalitis, on hippocampal CA1 and CA3 networks. Acute brain slices from C57BL/6 mice were incubated with rAb-IP2. The spontaneous synaptic GABAergic transmission was measured using electrophysiological recordings in voltage-clamp mode. The GABAAR autoantibody rAb-IP2 reduced inhibitory postsynaptic signaling in the hippocampal CA1 pyramidal neurons with regard to the number of spontaneous inhibitory postsynaptic currents (sIPSCs) but did not affect their amplitude. In the hippocampal CA3 network, decreased number and amplitude of sIPSCs were detected, leading to decreased GABAergic synaptic transmission. Immunohistochemical staining confirmed the rAb-IP2 bound to hippocampal tissue. These findings suggest that GABAAR autoantibodies exert direct functional effects on both hippocampal CA1 and CA3 pyramidal neurons and play a crucial role in seizure generation in GABAAR autoimmune encephalitis.

Eugenosedin-A improves obesity-related hyperglycemia by regulating ATP-sensitive K+ channels and insulin secretion in pancreatic β cells

Eugenosedin-A (Eu-A) has been shown to protect against hyperglycemia- and hyperlipidemia-induced metabolic syndrome. We investigated the relationship of KATP channel activities and insulin secretion by Eu-A in vitro in pancreatic β-cells, and examined the effect of Eu-A on streptozotocin (STZ)/nicotinamide (NA)-induced type 2 diabetes mellitus (T2DM) in vivo. We isolated pancreatic islets from adult male Wistar rats (250-350g) and identified pancreatic β-cells by the cell size, capacitance and membrane potential. Perforated patch-clamp and inside-out recordings were used to monitor the membrane potential (current-clamp mode) and channel activity (voltage-clamp mode) of β-cells. The membrane potential of β-cells was raised by Eu-A and reversed by the KATP channel activator diazoxide. Eu-A inhibited the KATP channel activity measured at -60mV and increased the intracellular calcium concentration ([Ca2+]i), resulting in enhanced insulin secretion. Eu-A also reduced Kir6.2 protein on the cell membrane and scattered in the cytosol under normal glucose conditions (5.6mM). In our animal study, rats were divided into normal and STZ/NA-induced T2DM groups. Normal rats fed with regular chow were divided into control and control+Eu-A (5mg/kg/day, i.p.) groups. The STZ/NA-induced diabetic rats fed with a high-fat diet (HFD) were divided into three groups: T2DM, T2DM+Eu-A (5mg/kg/day, i.p.), and T2DM+glibenclamide (0.5mg/kg/day, i.p.; a KATP channel inhibitor). Both Eu-A and glibenclamide decreased the rats' blood glucose, prevented weight gain, and enhanced insulin secretion. We found that Eu-A blocked pancreatic β-cell KATP channels, caused membrane potential depolarization, and stimulated Ca2+ influx, thus increasing insulin secretion. Furthermore, Eu-A decreased blood glucose and increased insulin levels in T2DM rats. These results suggested that Eu-A might have clinical benefits for the control of T2DM and its complications.

Presynaptic mechanisms underlying GABAB-receptor-mediated inhibition of spontaneous neurotransmitter release.

SUMMARYInhibition of neurotransmitter release by neurotransmitter substances constitutes a fundamental means of neuromodulation. In contrast to well-delineated mechanisms that underlie inhibition of evoked release via suppression of voltage-gated Ca2+ channels, processes that underlie neuromodulatory inhibition of spontaneous release remain unclear. Here, we interrogated inhibition of spontaneous glutamate and GABA release by presynaptic metabotropic GABAB receptors. Our findings show that this inhibition relies on Gβγ subunit action at the membrane, and it is largely independent of presynaptic Ca2+ signaling for both forms of release. In the case of spontaneous glutamate release, inhibition requires Gβγ interaction with the C terminus of the key fusion machinery component SNAP25, and it is modulated by synaptotagmin-1. Inhibition of spontaneous GABA release, on the other hand, is independent of these pathways and likely requires alternative Gβγ targets at the presynaptic terminal.

Subanesthetic Dose of Propofol Activates the Reward System in Rats.

Propofol has addictive properties, even with a single administration, and facilitates dopamine secretion in the nucleus accumbens (NAc). Activation of the dopaminergic circuits of the midbrain reward system, including the ventral tegmental area (VTA) and NAc, plays a crucial role in addiction. However, the effects of propofol on synaptic transmission and biochemical changes in the VTA-NAc circuit remain unclear. We investigated the effects of subanesthetic doses of propofol on rat VTA neurons and excitatory synaptic transmission in the NAc using slice patch-clamp experiments. Using immunohistochemistry and western blot analyses, we evaluated the effects of intraperitoneal propofol administration on the expression of addiction-associated transcription factor ΔFosB (truncated form of the FBJ murine osteosarcoma viral oncogene homolog B protein) in the NAcs in 5-week-old rats. In the current-clamp mode, a subanesthetic dose (0.5-5 µmol/L) of propofol increased the action potential frequency in about half the VTA neurons (excited neurons: control: 9.4 ± 3.0 Hz, propofol 0.5 µmol/L: 21.5 ± 6.0 Hz, propofol 5 µmol/L: 14.6 ± 5.3 Hz, wash: 2.0 ± 0.7 Hz, n = 14/27 cells; unchanged/suppressed neurons: control: 1.68 ± 0.94 Hz, propofol 0.5 µmol/L: 1.0 ± 0.67 Hz, propofol 5 µmol/L: 0.89 ± 0.87 Hz, wash: 0.16 ± 0.11 Hz, n = 13/27 cells). In the voltage-clamp mode, about half the VTA principal neurons showed inward currents with 5 µmol/L of propofol (inward current neurons: control: -20.5 ± 10.0 pA, propofol 0.5 µmol/L: -62.6 ± 14.4 pA, propofol 5 µmol/L: -85.2 ± 18.3 pA, propofol 50 µmol/L: -17.1 ± 39.2 pA, washout: +30.5 ± 33.9 pA, n = 6/11 cells; outward current neurons: control: -33.9 ± 14.6 pA, propofol 0.5 µmol/L: -29.5 ± 16.0 pA, propofol 5 µmol/L: -0.5 ± 20.9 pA, propofol 50 µmol/L: +38.9 ± 18.5 pA, washout: +40.8 ± 32.1 pA, n = 5/11 cells). Moreover, 0.5 µmol/L propofol increased the amplitudes of evoked excitatory synaptic currents in the NAc, whereas >5 µmol/L propofol decreased them (control: 100.0 ± 2.0%, propofol 0.5 µmol/L: 118.4 ± 4.3%, propofol 5 µmol/L: 98.3 ± 3.3%, wash [within 10 min]: 70.7 ± 3.3%, wash [30 minutes later]: 89.9 ± 2.5%, n = 13 cells, P < .001, Dunnett's test comparing control and propofol 0.5 µmol/L). Intraperitoneally administered subanesthetic dose of propofol increased ΔFosB expression in the NAc, but not in VTA, 2 and 24 hours after administration, compared with the Intralipid control group (propofol 2 hours: 0.94 ± 0.15, 24 hours: 0.68 ± 0.07; Intralipid 2 hours: 0.40 ± 0.03, 24 hours: 0.37 ± 0.06, P = .0002 for drug in the 2-way analysis of variance). Even a single administration of a subanesthetic dose of propofol may cause rewarding change in the central nervous system. Thus, there is a potential propofol rewarding effect among patients receiving anesthesia or sedation with propofol, as well as among health care providers exposed to propofol.

Development an ovine myocardial infarction- induced heart failure model and characterisation of altered calcium homeostasis mechanisms

Abstract Introduction Ventricular arrhythmias (VA) and heart failure (HF) are the major complications following myocardial infarction (MI). In both conditions, there is a key role for perturbed calcium homeostasis of which the underlying mechanisms remain unclear. A preclinical model that faithfully presents most of the features of MI-induced HF has been lacking. The complexity of this syndrome means that animal modelling is difficult. As the hearts of large animals share many electrophysiological similarities to humans, ovine modelling of cardiac diseases could better reflect human pathologies than in small mammals. Question Is it possible to develop a clinically relevant ovine model with moderate cardiac dysfunction following myocardial infarction? Methods MI was induced in sheep by inflating an angioplasty balloon distal to the second diagonal branch of the left anterior descending artery for 90 min. Cardiac function was monitored for 20 weeks using electrocardiography (ECG), echocardiography, blood biochemical analysis, and subjective signs of cardiac deterioration (lethargy, dyspnoea, and cough). 20 weeks post-MI, the animals were humanely killed and single left ventricular myocytes were isolated from the infarct border zone. Changes in cellular electrophysiology and intracellular calcium concentration were monitored using whole-cell patch technique in voltage-clamp mode and the calcium sensitive fluorescent indicator Fura-2 (K5 salt). Results By using minimally invasive procedures, we obtained a survival rate of 80% (n=15). During surgery, our data show clinical features of ischaemia, including changes in the ECG features (elevation of the ST and T segment, left bundle branch block and/or pathological Q waves) and elevation of the cardiac biomarker such as troponin I. Following MI, we observed a decline in ejection fraction (−25±3%, p&amp;lt;0.0001) and an increase in whole animal arrhythmias (incidence of VA 72 hours post-MI, ∼70%). On cardiac removal apico-septal transmural necrosis / scarring was evident. Importantly, the L-type calcium current (ICaL) was decreased in MI cells compared to healthy cells (−1.87±0.73 pA/pF, p&amp;lt;0.05), but isoprenaline had no effet on ICaL (0.48±1.2 pA/pF, p=0.70) in MI cells. Moreover, the amplitude of the systolic calcium transient (−0.33±0.1 F/F0, p&amp;lt;0.05) and the sarcoplasmic reticulum calcium content (−23±7 μmol/L, p&amp;lt;0.01) were also decreased. The shortening velocity of the sarcomere was also decreased in MI cells (−0.55±0.18 μm/s, p&amp;lt;0.01). Conclusion We successfully established an ovine MI model using minimally invasive procedure which displays a moderately impaired cardiac function, reduced contractility, and pro-arrhythmic electrophysiological remodelling. Future analysis will examine the role of the L-type calcium channel with respect to the excitation-contraction coupling process and myocyte contractility and how we can improve therapeutic strategies towards VA and HF. Funding Acknowledgement Type of funding sources: Foundation. Main funding source(s): British Heart Foundation

The suitability of high throughput automated patch clamp for physiological applications.

Although automated patch clamp (APC) devices have been around for many years and have become an integral part of many aspects of drug discovery, high throughput instruments with gigaohm seal data quality are relatively new. Experiments where a large number of compounds are screened against ion channels are ideally suited to high throughput APC, particularly when the amount of compound available is low. Here we evaluate different APC approaches using a variety of ion channels and screening settings. We have performed a screen of 1920 compounds on GluN1/GluN2A NMDA receptors for negative allosteric modulation using both the SyncroPatch 384 and FLIPR. Additionally, we tested the effect of 36 arthropod venoms on NaV 1.9 using a single 384-well plate on the SyncroPatch 384. As an example for mutant screening, a range of acid-sensing ion channel variants were tested and the success rate increased through fluorescence-activated cell sorting (FACS) prior to APC experiments. Gigaohm seal data quality makes the 384-format accessible to recording of primary and stem cell-derived cells on the SyncroPatch 384. We show recordings in voltage and current clamp modes of stem cell-derived cardiomyocytes. In addition, the option of intracellular solution exchange enabled investigations into the effects of intracellular Ca2+ and cAMP on TRPC5 and HCN2 currents, respectively. Together, these data highlight the broad applicability and versatility of APC platforms and also outlines some limitations of the approach. KEY POINTS: High throughput automated patch clamp (APC) can be used for a variety of applications involving ion channels. Lower false positive rates were achieved using automated patch clamp versus a fluorometric imaging plate reader (FLIPR) in a high throughput compound screen against NMDA receptors. Genetic variants and mutations can be screened on a single 384-well plate to reduce variability of experimental parameters. Intracellular solution can be perfused to investigate effects of ions and second messenger systems without the need for excised patches. Primary cells and stem cell-derived cells can be used on high throughput APC with reasonable success rates for cell capture, voltage clamp measurements and action potential recordings in current clamp mode.

HCN4 current during human sinoatrial node-like action potentials

BackgroundDespite the many studies carried out over the past 40 years, the contribution of the HCN4 encoded hyperpolarization-activated ‘funny’ current (If) to pacemaker activity in the mammalian sinoatrial node (SAN), and the human SAN in particular, is still controversial and not fully established. ObjectiveTo study the contribution of If to diastolic depolarization of human SAN cells and its dependence on heart rate, cAMP levels, and atrial load. MethodsHCN4 channels were expressed in human cardiac myocyte progenitor cells (CMPCs) and HCN4 currents assessed using perforated patch-clamp in traditional voltage clamp mode and during action potential clamp with human SAN-like action potential waveforms with 500–1500 ms cycle length, in absence or presence of forskolin to mimic β-adrenergic stimulation and a −15 mV command potential offset to mimic atrial load. ResultsForskolin significantly increased the fully-activated HCN4 current density at −140 mV by 14% and shifted the steady-state activation curve by +7.4 mV without affecting its slope. In addition, forskolin significantly accelerated current activation but slowed deactivation. The HCN4 current did not completely deactivate before the subsequent diastolic depolarization during action potential clamp. The amplitude of HCN4 current increased with increasing cycle length, was significantly larger in the presence of forskolin at all cycle lengths, and was significantly increased upon the negative offset to the command potential. ConclusionsIf is active during a human SAN action potential waveform and its amplitude is modulated by heart rate, β-adrenergic stimulation, and diastolic voltage range, such that If is under delicate control.