P2559The mechanisms of spontaneous action potential generation in sinoauricular valve of the chick embryos

Background The changes in the density of ion channels and/or damage of ion channel proteins violates the homeostasis of the potential forming ions in myocardium cells and may contribute to arrhythmogenesis. The study of ion channel function in embryos is important in order to understand the mechanisms of arrhythmia. The purpose of the present study was to identify the key ion channels that form action potentials (APs) in pacemaker cells in the area of the sinoauricular (SA) chicken embryo valve. Material and methods The embryos were removed from fertilized White Leghorn breed chicken eggs on the 10±1 day of incubation (ED10). The experiments were performed on freshly prepared strips (3 mm × 2 mm) of the right atrium, which included the left SA valve itself as well as the surrounding tissue. After that the APs were recorded from the area of left SA valve using the microelectrode technique. The functional analysis of ion channels was performed using the specific ion channel blockers. Results The strips generated APs with slow diastolic depolarization (SDD) and frequency from 132 to 251 impulses per min (ipm) (average - 177±22 ipm). The APs amplitude and dV/dtmax were 102±9 mV and 98±25 V/s (nstrips=49). At the third minute of exposure to E-4031 (1 μmol), a blocker of rapid outward K-current channels, IKr, the increase in the duration of the AP repolarization phase by 30% and the shortening of SDD by 20–25% (p<0.05; n=6) were registered. The APs generation frequency increased by 10%. Subsequently we observed the monotonic decrease in the AP amplitude due to the reduced overshoot and depolarized Emax. The electrical activity of all strips was blocked on the 20±2 min (Fig.1.a-c). Lidocaine (500 μmol), a blocker of Nav1.5 channels, increased a duration of SDD and APD90 twice, but decreased dV/dtmax by 2.5 times. The APs generation frequency decreased by 30% in comparison to the control. After 8±2 min exposure to lidocaine, the APs generation was blocked in 56% of samples. It is important to mention that in these strips right before the APs generation was blocked dV/dtmax was 30±15 V/s. In the samples, where the APs were not blocked after the exposure to lidocaine this parameter was 14±10 V/s (Fig. 1d-e). Nifedipine (10 μmol), a blocker of L-type Ca2+-channels, decreased APD90 by 56% and SDD by 25%. The APs generation frequency increased twice in comparison to the control samples (fig.1f-h). The chronotropic effects of nifedipine were dose-dependent. Nifedipine did not induce the blocking of APs generation. Figure 1. Effects of E-4031 (a-c), lidocaine (d-e) and nifedipine (e-g) on APs. Conclusions The received data demonstrate that the key role in the APs formation in ED10 embryos belong to the outward IKr and inward INa, but not to ICa,L. Thus, the mechanisms of spontaneous action potential generation in ED10 chick embryos have more in common with that in human iPSC-derived cardiomyocytes rather than in SA area cells of the ED11 mice embryos. Acknowledgement/Funding Rus fund Basic Research No. 18-34-00654; Project RAS No. 0415-2019-0006 and No. AAAA-A17-117012310152-2

Mechanisms Underlying Spontaneous Action Potential Generation Induced by Catecholamine in Pulmonary Vein Cardiomyocytes: A Simulation Study

Cardiomyocytes and myocardial sleeves dissociated from pulmonary veins (PVs) potentially generate ectopic automaticity in response to noradrenaline (NA), and thereby trigger atrial fibrillation. We developed a mathematical model of rat PV cardiomyocytes (PVC) based on experimental data that incorporates the microscopic framework of the local control theory of Ca2+ release from the sarcoplasmic reticulum (SR), which can generate rhythmic Ca2+ release (limit cycle revealed by the bifurcation analysis) when total Ca2+ within the cell increased. Ca2+ overload in SR increased resting Ca2+ efflux through the type II inositol 1,4,5-trisphosphate (IP3) receptors (InsP3R) as well as ryanodine receptors (RyRs), which finally triggered massive Ca2+ release through activation of RyRs via local Ca2+ accumulation in the vicinity of RyRs. The new PVC model exhibited a resting potential of −68 mV. Under NA effects, repetitive Ca2+ release from SR triggered spontaneous action potentials (APs) by evoking transient depolarizations (TDs) through Na+/Ca2+ exchanger (APTDs). Marked and variable latencies initiating APTDs could be explained by the time courses of the α1- and β1-adrenergic influence on the regulation of intracellular Ca2+ content and random occurrences of spontaneous TD activating the first APTD. Positive and negative feedback relations were clarified under APTD generation.

Novel Ca2+-dependent mechanisms regulate spontaneous release at excitatory synapses onto CA1 pyramidal cells

Although long thought to simply be a source of synaptic noise, spontaneous, action potential-independent release of neurotransmitter from presynaptic terminals has multiple roles in synaptic function. We explored whether and to what extent the two predominantly proposed mechanisms for explaining spontaneous release, stochastic activation of voltage-gated Ca2+ channels (VGCCs) or activation of Ca2+-sensing receptors (CaSRs) by extracellular Ca2+, played a role in the sensitivity of spontaneous release to the level of extracellular Ca2+ concentration at excitatory synapses at CA1 pyramidal cells of the adult male mouse hippocampus. Blocking VGCCs with Cd2+ had no effect on spontaneous release, ruling out stochastic activation of VGCCs. Although divalent cation agonists of CaSRs, Co2+ and Mg2+, dramatically enhanced miniature excitatory postsynaptic current (mEPSC) frequency, potent positive and negative allosteric modulators of CaSRs had no effect. Moreover, immunoblot analysis of hippocampal lysates failed to detect CaSR expression, ruling out the CaSR. Instead, the increase in mEPSC frequency induced by Co2+ and Mg2+ was mimicked by lowering postsynaptic Ca2+ levels with BAPTA. Together, our results suggest that a reduction in intracellular Ca2+ may trigger a homeostatic-like compensatory response that upregulates spontaneous transmission at excitatory synapses onto CA1 pyramidal cells in the adult hippocampus. NEW & NOTEWORTHY We show that the predominant theories for explaining the regulation of spontaneous, action potential-independent synaptic release do not explain the sensitivity of this type of synaptic transmission to external Ca2+ concentration at excitatory synapses onto hippocampal CA1 pyramidal cells. In addition, our data indicate that intracellular Ca2+ levels in CA1 pyramidal cells regulate spontaneous release, suggesting that excitatory synapses onto CA1 pyramidal cells may express a novel, rapid form of homeostatic plasticity.

Castration modulates singing patterns and electrophysiological properties of ra projection neurons in adult male zebra finches

Castration can change levels of plasma testosterone. Androgens such as testosterone play an important role in stabilizing birdsong. The robust nucleus of the arcopallium (RA) is an important premotor nucleus critical for singing. In this study, we investigated the effect of castration on singing patterns and electrophysiological properties of projection neurons (PNs) in the RA of adult male zebra finches.Adult male zebra finches were castrated and the changes in bird song assessed. We also recorded the electrophysiological changes from RA PNs using patch clamp recording. We found that the plasma levels of testosterone were significantly decreased, song syllable’s entropy was increased and the similarity of motif was decreased after castration. Spontaneous and evoked firing rates, membrane time constants, and membrane capacitance of RA PNs in the castration group were lower than those of the control and the sham groups. Afterhyperpolarization（AHP）time to peak of spontaneous action potential (AP) was prolonged after castration.These findings suggest that castration decreases song stereotypy and excitability of RA PNs in male zebra finches.

Castration modulates singing patterns and electrophysiological properties of ra projection neurons in adult male zebra finches

Castration can change levels of plasma testosterone. Androgens such as testosterone play an important role in stabilizing birdsong. The robust nucleus of the arcopallium (RA) is an important premotor nucleus critical for singing. In this study, we investigated the effect of castration on singing patterns and electrophysiological properties of projection neurons (PNs) in the RA of adult male zebra finches.Adult male zebra finches were castrated and the changes in bird song assessed. We also recorded the electrophysiological changes from RA PNs using patch clamp recording. We found that the plasma levels of testosterone were significantly decreased, song syllable’s entropy was increased and the similarity of motif was decreased after castration. Spontaneous and evoked firing rates, membrane time constants, and membrane capacitance of RA PNs in the castration group were lower than those of the control and the sham groups. Afterhyperpolarization（AHP）time to peak of spontaneous action potential (AP) was prolonged after castration.These findings suggest that castration decreases song stereotypy and excitability of RA PNs in male zebra finches.

Castration modulates singing patterns and electrophysiological properties of RA projection neurons in adult male zebra finches

Castration can change levels of plasma testosterone. Androgens such as testosterone play an important role in stabilizing birdsong. The robust nucleus of the arcopallium (RA) is an important premotor nucleus critical for singing. In this study, we investigated the effect of castration on singing patterns and electrophysiological properties of projection neurons (PNs) in the RA of adult male zebra finches.Adult male zebra finches were castrated and the changes in bird song assessed. We also recorded the electrophysiological changes from RA PNs using patch clamp recording. We found that the plasma levels of testosterone were significantly decreased, song syllable’s entropy was increased and the similarity of motif was decreased after castration. Spontaneous and evoked firing rates, membrane time constants, and membrane capacitance of RA PNs in the castration group were lower than those of the control and the sham groups. Afterhyperpolarization（AHP）time to peak of spontaneous action potential (AP) was prolonged after castration.These findings suggest that castration decreases song stereotypy and excitability of RA PNs in male zebra finches.