Spontaneous Action Potential Generation Research Articles

Spontaneous synaptic drive in detrusor smooth muscle: computational investigation and implications for urinary bladder function.

The detrusor, a key component of the urinary bladder wall, is a densely innervated syncytial smooth muscle tissue. Random spontaneous release of neurotransmitter at neuromuscular junctions (NMJs) in the detrusor gives rise to spontaneous excitatory junction potentials (SEJPs). These sub-threshold passive signals not only offer insights into the syncytial nature of the tissue, their spatio-temporal integration is critical to the generation of spontaneous neurogenic action potentials which lead to focal contractions during the filling phase of the bladder. Given the structural complexity and the contractile nature of the tissue, electrophysiological investigations on spatio-temporal integration of SEJPs in the detrusor are technically challenging. Here we report a biophysically constrained computational model of a detrusor syncytium overlaid with spatially distributed innervation, using which we explored salient features of the integration of SEJPs in the tissue and the key factors that contribute to this integration. We validated our model against experimental data, ascertaining that observations were congruent with theoretical predictions. With the help of comparative studies, we propose that the amplitude of the spatio-temporally integrated SEJP is most sensitive to the inter-cellular coupling strength in the detrusor, while frequency of observed events depends more strongly on innervation density. An experimentally testable prediction arising from our study is that spontaneous release frequency of neurotransmitter may be implicated in the generation of detrusor overactivity. Set against histological observations, we also conjecture possible changes in the electrical activity of the detrusor during pathology involving patchy denervation. Our model thus provides a physiologically realistic, heuristic framework to investigate the spread and integration of passive potentials in an innervated syncytial tissue under normal conditions and in pathophysiology.

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

Abstract 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.

Electrophysiological and transcriptomic correlates of neuropathic pain in human dorsal root ganglion neurons.

Neuropathic pain encompasses a diverse array of clinical entities affecting 7-10% of the population, which is challenging to adequately treat. Several promising therapeutics derived from molecular discoveries in animal models of neuropathic pain have failed to translate following unsuccessful clinical trials suggesting the possibility of important cellular-level and molecular differences between animals and humans. Establishing the extent of potential differences between laboratory animals and humans, through direct study of human tissues and/or cells, is likely important in facilitating translation of preclinical discoveries to meaningful treatments. Patch-clamp electrophysiology and RNA-sequencing was performed on dorsal root ganglia taken from patients with variable presence of radicular/neuropathic pain. Findings establish that spontaneous action potential generation in dorsal root ganglion neurons is associated with radicular/neuropathic pain and radiographic nerve root compression. Transcriptome analysis suggests presence of sex-specific differences and reveals gene modules and signalling pathways in immune response and neuronal plasticity related to radicular/neuropathic pain that may suggest therapeutic avenues and that has the potential to predict neuropathic pain in future cohorts.

Theoretical predication of temperature effects at 20oC–42oC on adaptive processes in simulated amyotrophic lateral sclerosis

Strength-duration time constants, rheobase currents, and recovery cycles allow the nerve adaptive processes to single or pairs of depolarizing stimuli to be assessed. This study investigates the temperature dependence of these excitability indices of human motor nerve fibers with one of three increasingly-severe type of amyotrophic lateral sclerosis pathology, referred to as ALS1, ALS2, and ALS3. The temperature dependence of the excitability indices was investigated during hypothermia ( ≤ 25 o C), hyperthermia ( ≥ 40 o C), and in the physiological temperature range (30-37 o C). Numerical solutions were computed using a temperature-dependent multi-layered model. Results showed the following trends: (i) while the strength-duration time constants gradually decreased with a temperature increase from 20 o C to 42 o C, they were longer in the three ALS cases than those of the normal case; (ii) the reciprocally dependent strength-duration time constants and rheobase currents were more sensitive to hyperthermia, especially at 42 o C, than at temperatures across the physiological range of 30-37 o C; (iii) the shape of temperature-dependent recovery cycles was similar for both the normal and ALS1 cases; (iv) in the ALS2 case, each test stimulus applied at the end of 100 ms recovery cycle failed to initiate a second action potential during hypothermia at 20 o C; and (v) in the ALS3 case during hypothermia, hyperthermia and across the physiological temperature range, each test stimulus applied beyond a given conditioning-test interval was blocked. This blockage was a result of the spontaneous action potential generation caused by the conditioning (first) stimulus. The changes obtained for the temperature-dependent strength-duration time constants, rheobase currents, and recovery cycles reflect nodal and internodal ion channel dysfunctions in the three amyotrophic lateral sclerosis cases. It is proposed that these excitability indices can be applied clinically as specific indicators for amyotrophic lateral sclerosis motor neuron disease.

Mathematical Modeling and Analyses of Interspike-Intervals of Spontaneous Activity in Afferent Neurons of the Zebrafish Lateral Line

Without stimuli, hair cells spontaneously release neurotransmitter leading to spontaneous generation of action potentials (spikes) in innervating afferent neurons. We analyzed spontaneous spike patterns recorded from the lateral line of zebrafish and found that distributions of interspike intervals (ISIs) either have an exponential shape or an “L” shape that is characterized by a sharp decay but wide tail. ISI data were fitted to renewal-process models that accounted for the neuron refractory periods and hair-cell synaptic release. Modeling the timing of synaptic release using a mixture of two exponential distributions yielded the best fit for our ISI data. Additionally, lateral line ISIs displayed positive serial correlation and appeared to exhibit switching between faster and slower modes of spike generation. This pattern contrasts with previous findings from the auditory system where ISIs tended to have negative serial correlation due to synaptic depletion. We propose that afferent neuron innervation with multiple and heterogenous hair-cells synapses, each influenced by changes in calcium domains, can serve as a mechanism for the random switching behavior. Overall, our analyses provide evidence of how physiological similarities and differences between synapses and innervation patterns in the auditory, vestibular, and lateral line systems can lead to variations in spontaneous activity.

New insights into the pathophysiology of primary hemifacial spasm

Primary hemifacial spasm (pHFS) is due to a benign compression of the facial motor nerve by an offending vessel, leading to increased nerve excitability. Facial nerve hyperexcitability presents two different aspects. First, there is a spontaneous and ectopic generation of action potentials on the incriminated nerve and then this ectopic impulse can propagate and spread “laterally” from one facial nerve branch to another. This results in spontaneous and synkinetic spasms affecting one hemiface. Although the increase in excitability certainly concerns the nucleus of the facial motor nerve in the brainstem, it seems unlikely that the primary origin of this hyperexcitability and the associated phenomenon of lateral spreading strictly originate at the nuclear level. In fact, the mechanisms causing facial nerve hyperexcitability per se remain unknown. The leading implication of a structural nerve lesion, such as segmental demyelination, induced by vessel compression, is also unconvincing. In contrast, a functional mechanical factor increasing nerve excitability is extremely probable, that it is either due to compression or stretch resulting from the neurovascular conflict. Axonal ion channel changes are obviously associated with this mechanism. Then the lateral spreading of nerve fibre hyperexcitability probably results from an ephaptic process, the “cross-talk” between axons being located in the region of the conflict or in the transition zone between central and peripheral myelin, at the end of the facial nerve root exit zone. In any event, pHFS is due to a functional increase in facial nerve excitability triggered by an offending vessel and this clearly explains the remarkable and rapid efficacy of surgical microvascular decompression.

The sorting receptor Rer1 controls Purkinje cell function via voltage gated sodium channels

Rer1 is a sorting receptor in the early secretory pathway that controls the assembly and the cell surface transport of selected multimeric membrane protein complexes. Mice with a Purkinje cell (PC) specific deletion of Rer1 showed normal polarization and differentiation of PCs and normal development of the cerebellum. However, PC-specific loss of Rer1 led to age-dependent motor deficits in beam walk, ladder climbing and gait. Analysis of brain sections revealed a specific degeneration of PCs in the anterior cerebellar lobe in old animals. Electrophysiological recordings demonstrated severe deficits in spontaneous action potential generation. Measurements of resurgent currents indicated decreased surface densities of voltage-gated sodium channels (Nav), but not changes in individual channels. Analysis of mice with a whole brain Rer1-deletion demonstrated a strong down-regulation of Nav1.6 and 1.1 in the absence of Rer1, whereas protein levels of the related Cav2.1 and of Kv3.3 and 7.2 channels were not affected. The data suggest that Rer1 controls the assembly and transport of Nav1.1 and 1.6, the principal sodium channels responsible for recurrent firing, in PCs.

Channel based generating function approach to the stochastic Hodgkin-Huxley neuronal system.

Internal and external fluctuations, such as channel noise and synaptic noise, contribute to the generation of spontaneous action potentials in neurons. Many different Langevin approaches have been proposed to speed up the computation but with waning accuracy especially at small channel numbers. We apply a generating function approach to the master equation for the ion channel dynamics and further propose two accelerating algorithms, with an accuracy close to the Gillespie algorithm but with much higher efficiency, opening the door for expedited simulation of noisy action potential propagating along axons or other types of noisy signal transduction.

Forced cell cycle exit and modulation of GABAA, CREB, and GSK3β signaling promote functional maturation of induced pluripotent stem cell-derived neurons.

Although numerous protocols have been developed for differentiation of neurons from a variety of pluripotent stem cells, most have concentrated on being able to specify effectively appropriate neuronal subtypes and few have been designed to enhance or accelerate functional maturity. Of those that have, most employ time courses of functional maturation that are rather protracted, and none have fully characterized all aspects of neuronal function, from spontaneous action potential generation through to postsynaptic receptor maturation. Here, we describe a simple protocol that employs the sequential addition of just two supplemented media that have been formulated to separate the two key phases of neural differentiation, the neurogenesis and synaptogenesis, each characterized by different signaling requirements. Employing these media, this new protocol synchronized neurogenesis and enhanced the rate of maturation of pluripotent stem cell-derived neural precursors. Neurons differentiated using this protocol exhibited large cell capacitance with relatively hyperpolarized resting membrane potentials; moreover, they exhibited augmented: 1) spontaneous electrical activity; 2) regenerative induced action potential train activity; 3) Na(+) current availability, and 4) synaptic currents. This was accomplished by rapid and uniform development of a mature, inhibitory GABAAreceptor phenotype that was demonstrated by Ca(2+) imaging and the ability of GABAAreceptor blockers to evoke seizurogenic network activity in multielectrode array recordings. Furthermore, since this protocol can exploit expanded and frozen prepatterned neural progenitors to deliver mature neurons within 21 days, it is both scalable and transferable to high-throughput platforms for the use in functional screens.

Real-time relationship between PKA biochemical signal network dynamics and increased action potential firing rate in heart pacemaker cells: Kinetics of PKA activation in heart pacemaker cells

cAMP-PKA protein kinase is a key nodal signaling pathway that regulates a wide range of heart pacemaker cell functions. These functions are predicted to be involved in regulation of spontaneous action potential (AP) generation of these cells. Here we investigate if the kinetics and stoichiometry of increase in PKA activity match the increase in AP firing rate in response to β-adrenergic receptor (β-AR) stimulation or phosphodiesterase (PDE) inhibition, that alters the AP firing rate of heart sinoatrial pacemaker cells. In cultured adult rabbit pacemaker cells infected with an adenovirus expressing the FRET sensor AKAR3, the EC50 in response to graded increases in the intensity of β-AR stimulation (by Isoproterenol) the magnitude of the increases in PKA activity and the spontaneous AP firing rate were similar (0.4±0.1nM vs. 0.6±0.15nM, respectively). Moreover, the kinetics (t1/2) of the increases in PKA activity and spontaneous AP firing rate in response to β-AR stimulation or PDE inhibition were tightly linked. We characterized the system rate-limiting biochemical reactions by integrating these experimentally derived data into a mechanistic–computational model. Model simulations predicted that phospholamban phosphorylation is a potent target of the increase in PKA activity that links to increase in spontaneous AP firing rate. In summary, the kinetics and stoichiometry of increases in PKA activity in response to a physiological (β-AR stimulation) or pharmacological (PDE inhibitor) stimuli match those of changes in the AP firing rate. Thus Ca2+-cAMP/PKA-dependent phosphorylation limits the rate and magnitude of increase in spontaneous AP firing rate.

Abstract 16515: Differential Functions of HCN1 and HCN4 Pacemaker Channels Within the Sinoatrial Node for the Stable and Precise Beating of the Heart

The heart beat is initiated by the generation of spontaneous action potentials in pacemaker cells of the sinoatrial node (SAN) region. The maintenance of a stable heart beat requires mechanisms which protect the SAN pacemaker cells from potential perturbing influences which arise from inside and outside the sinoatrial network. Our previous work suggests that the hyperpolarization-activated cyclic nucleotide gated channel subtype 1 (HCN1) protects against such perturbations and thereby increases the stability, the precision and the safety of the sinoatrial network. Here, we investigate the role of HCN4 channels within this context. Using genetic mouse models deficient for HCN channels as well as mouse models expressing engineered HCN channels, we performed a detailed functional characterization of pacemaker mechanisms in single isolated sinoatrial node cells, explanted beating sinoatrial node preparations, with telemetric in vivo electrocardiography, echocardiography, and in vivo electrophysiology. We provide evidence that HCN4 has a critical role in counteracting and balancing potentially destabilizing effects of the autonomic nervous system on the regulation of the heart rate. Specifically, HCN4 channels smooth the transition of the heart rate to a new equilibrium. Furthermore, we provide evidence that the cAMP- dependent fine tuning of HCN4 channel activity could provide the exact dosage of current to balance and counteract overshooting responses of the heart rate to autonomic regulation. In the absence of such a protecting effect, mice display a brady- tachy syndrome.

Parametric inference of neuronal response latency in presence of a background signal

Neurons are commonly characterized by spontaneous generation of action potentials (spikes), which appear without any apparent or controlled stimulation. When a stimulus is applied, the spontaneous firing may prevail and hamper identification of the effect of the stimulus. Therefore, for any rigorous analysis of evoked neuronal activity, the presence of spontaneous firing has to be taken into account. If the background signal is ignored, however small it is compared to the response activity, and however large is the delay, estimation of the response latency will be wrong, and the error will persist even when sample size is increasing. The first question is: what is the response latency to the stimulus? Answering this question becomes even more difficult if the latency is of a complex nature, for example composed of a physically implied deterministic part and a stochastic part. This scenario is considered here, where the response time is a sum of two components; the delay and the relative latency. Parametric estimators for the time delay and the response latency are derived. These estimators are evaluated on simulated data and their properties are discussed. Finally, we show that the mean of the response latency is always satisfactorily estimated, even assuming a wrong distribution for the response latency.

Tonic mGluR5/CB1‐dependent suppression of inhibition as a pathophysiological hallmark in the striatum of mice carrying a mutant form of huntingtin

Changes in the activity of striatal output neurons (SONs) have been implicated in the pathogenesis of Huntington's disease (HD). In this inherited polyglutamine disorder, accumulation of intracellular toxins causes a variety of deficits, including synaptic dysfunction, but it is still unclear to what extent striatal GABA release is afflicted as well. Two murine HD models were used, a recently created knock-in mouse (Z_Q175_KI) and an established model of HD (R6/2). In sagittal slices with relatively well-preserved glutamatergic connections throughout the basal ganglia, we have characterized the following: (i) the excitability of SONs; (ii) their spontaneous action potential-dependent GABAergic synaptic activity; (iii) the capacity of exogenous GABA to inhibit spontaneous action potential generation; and (iv) the properties of GABAergic unitary evoked responses (eIPSCs) in response to intrastriatal minimal stimulation at low and high frequency. The HD SONs exhibited enhanced intrisic excitability and higher levels of GABAergic spontaneous activity without presenting evidence for homeostatic upregulation of endogenous or exogenous GABA actions. Unitary eIPSC amplitudes were reduced, with a clear deficit in the probability of release, as indicated by a higher paired-pulse ratio, failure rate and coefficient of variation. In conditions of high-frequency activation, GABAergic connections of HD SONs were prone to asynchronous release and delayed IPSC generation at the expense of synchronized release. Both in wild-type and in HD SONs, GABA was inhibitory. Our results support the conclusion that the enhanced spontaneous synaptic activity in the HD striatum reflects disinhibition. Pharmacological tests identified the HD-related tonic suppression of synaptic inhibition as a glutamate- and endocannabinoid-dependent process.

Reduced Excitability and Ca2+ Current Density in Sinoatrial Myocytes from Aged Mice

Cardiac pacemaking results from the generation of spontaneous action potentials (APs) by specialized myocytes in the sinoatrial node (sinoatrial myocytes, SAMs). These APs are produced by the coordinated activity of a number of different ion channels, including hyperpolarization-activated cyclic nucleotide-sensitive (HCN) channels and voltage-gated Ca2+ channels. Across all mammalian species, the maximum heart rate that can be elicited by stress or exercise decreases with age. However, little is known about age-related changes in the intrinsic excitability of SAMs. In this study, we examined differences in heart rate in young (<3 mo) versus old (>32 mo) mice, and the corresponding differences in spontaneous AP firing rate and ionic currents in isolated SAMs from young versus old animals. ECG measurements in vivo confirmed previous observations of age-related reductions in maximum heart rate and intrinsic heart rate (i.e., in the absence of autonomic input). Current clamp recordings from acutely isolated SAMs revealed age-related deficits in spontaneous AP firing rate that mirrored the age-related changes in heart rate; the basal AP firing rate was significantly slower in SAMs from elderly animals, and maximal stimulation with 1 μM isoproterenol (Iso, a β adrenergic agonist) resulted in a slower average maximum firing rate in SAMs from old mice. To explore the mechanistic bases of these age-related deficiencies in sinoatrial AP generation, we have recently begun voltage-clamp recordings of hyperpolarization-activated currents and voltage-gated calcium currents from isolated SAMs from young and old mice. To date, we have found significant reductions in Ca2+ current density in cells from older animals. These studies may provide mechanistic information about fundamental processes that limit the physical capability of the elderly.

Nociceptors as chronic drivers of pain and hyperreflexia after spinal cord injury: an adaptive-maladaptive hyperfunctional state hypothesis

Spinal cord injury (SCI) causes chronic peripheral sensitization of nociceptors and persistent generation of spontaneous action potentials (SA) in peripheral branches and the somata of hyperexcitable nociceptors within dorsal root ganglia (DRG). Here it is proposed that SCI triggers in numerous nociceptors a persistent hyperfunctional state (peripheral, synaptic, and somal) that originally evolved as an adaptive response to compensate for loss of sensory terminals after severe but survivable peripheral injury. In this hypothesis, nociceptor somata monitor the status of their own receptive field and the rest of the body by integrating signals received by their peripheral and central branches and the soma itself. A nociceptor switches into a potentially permanent hyperfunctional state when central neural, glial, and inflammatory signal combinations are detected that indicate extensive peripheral injury. Similar signal combinations are produced by SCI and disseminated widely to uninjured as well as injured nociceptors. This paper focuses on the uninjured nociceptors that are altered by SCI. Enhanced activity generated in below-level nociceptors promotes below-level central sensitization, somatic and autonomic hyperreflexia, and visceral dysfunction. If sufficient ascending fibers survive, enhanced activity in below-level nociceptors contributes to below-level pain. Nociceptor activity generated above the injury level contributes to at- and above-level sensitization and pain (evoked and spontaneous). Thus, SCI triggers a potent nociceptor state that may have been adaptive (from an evolutionary perspective) after severe peripheral injury but is maladaptive after SCI. Evidence that hyperfunctional nociceptors make large contributions to behavioral hypersensitivity after SCI suggests that nociceptor-specific ion channels required for nociceptor SA and hypersensitivity offer promising targets for treating chronic pain and hyperreflexia after SCI.

The Negative Chronotropic Effect of Cs+ Ions on Generation of Transmembrane Potentials in Mouse Sinoatrial Node Cells

Experiments on spontaneously contracting strips from sinoatrial region of the hearts of 2-month-old albino mice showed that cesium (Cs(+)), a blocker of hyperpolarization-activated I(f) current, in a concentration of 1 mM produced the greatest negative chronotropic effect on the duration of diastolic depolarization phase (75%), its rate (59%), and action potential duration (29%). The threshold concentration of Cs(+) was approximately 0.15 mM. In a concentration of about 8.5 mM, spontaneous generation of action potentials stopped. The effect was reversible. Thus, blockade of I(f) current by Cs(+) reduced the rate of action potential generation in cells of mouse sinoatrial node by app. 42% in comparison with controls.

Rhythmic beating of stem cell-derived cardiac cells requires dynamic coupling of electrophysiology and Ca cycling

There is an intense interest in differentiating embryonic stem cells to engineer biological pacemakers as an alternative to electronic pacemakers for patients with cardiac pacemaker function deficiency. Embryonic stem cell-derived cardiocytes (ESCs), however, often exhibit dysrhythmic excitations. Using Ca2+ imaging and patch-clamp techniques, we studied requirements for generation of spontaneous rhythmic action potentials (APs) in late-stage mouse ESCs. Sarcoplasmic reticulum (SR) of ESCs generates spontaneous, rhythmic, wavelet-like Local Ca2+Releases (LCRs) (inhibited by ryanodine, tetracaine, or thapsigargin). L-type Ca2+current (ICaL) induces a global Ca2+ release (CICR), depleting the Ca2+ content SR which resets the phases of LCR oscillators. Following a delay, SR then generates a highly synchronized spontaneous Ca2+release of multiple LCRs throughout the cell. The LCRs generate an inward Na+/Ca2+exchanger (NCX) current (absent in Na+-free solution) that ignites the next AP. Interfering with SR Ca2+ cycling (ryanodine, caffeine, thapsigargin, cyclopiazonic acid, BAPTA-AM), NCX (Na+-free solution), or ICaL (nifedipine) results in dysrhythmic excitations or cessation of automaticity. Inhibition of cAMP/PKA signaling by a specific PKA inhibitor, PKI, decreases SR Ca2+ loading, substantially reducing both spontaneous LCRs (number, size, and amplitude) and rhythmic AP firing. In contrast, enhancing PKA signaling by cAMP increases the LCRs (number, size, duration) and converts irregularly beating ESCs to rhythmic “pacemaker-like” cells. SR Ca2+ loading and LCR activity could be also increased with a selective activation of SR Ca2+ pumping by a phospholamban antibody. We conclude that SR Ca2+ loading and spontaneous rhythmic LCRs are driven by inherent cAMP/PKA activity. ICaL synchronizes multiple LCR oscillators resulting in strong, partially synchronized diastolic Ca2+ release and NCX current. Rhythmic ESC automaticity can be achieved by boosting “coupling” factors, such as cAMP/PKA signaling, that enhance interactions between SR and sarcolemma.

Excitatory Input Onto Hilar Somatostatin Interneurons Is Increased in a Chronic Model of Epilepsy

The density of somatostatin (SOM)-containing GABAergic interneurons in the hilus of the dentate gyrus is significantly decreased in both human and experimental temporal lobe epilepsy. We used the pilocarpine model of status epilepticus and temporal lobe epilepsy in mice to study anatomical and electrophysiological properties of surviving somatostatin interneurons and determine whether compensatory functional changes occur that might offset loss of other inhibitory neurons. Using standard patch-clamp techniques and pipettes containing biocytin, whole cell recordings were obtained in hippocampal slices maintained in vitro. Hilar SOM cells containing enhanced green fluorescent protein (EGFP) were identified with fluorescent and infrared differential interference contrast video microscopy in epileptic and control GIN (EGFP-expressing Inhibitory Neurons) mice. Results showed that SOM cells from epileptic mice had 1) significant increases in somatic area and dendritic length; 2) changes in membrane properties, including a small but significant decrease in resting membrane potential, and increases in time constant and whole cell capacitance; 3) increased frequency of slowly rising spontaneous excitatory postsynaptic currents (sEPSCs) due primarily to increased mEPSC frequency, without changes in the probability of release; 4) increased evoked EPSC amplitude; and 5) increased spontaneous action potential generation in cell-attached recordings. Results suggest an increase in excitatory innervation, perhaps on distal dendrites, considering the slower rising EPSCs and increased output of hilar SOM cells in this model of epilepsy. In sum, these changes would be expected to increase the inhibitory output of surviving SOM interneurons and in part compensate for interneuronal loss in the epileptogenic hippocampus.

Automaticity and conduction properties of bio-artificial pacemakers assessed in an in vitro monolayer model of neonatal rat ventricular myocytes

A better understanding of the ionic mechanisms for cardiac automaticity can lead to better strategies for engineering bio-artificial pacemakers. Here, we attempted to better define the relative contribution of I(f) and I(K1) in the generation of spontaneous action potentials (SAPs) in cardiomyocytes (CMs). Monolayers of neonatal rat ventricular myocytes (NRVMs) were transduced with a recombinant adenovirus (Ad) to express a gating-engineered HCN1 construct (HCN1-DeltaDeltaDelta) for patch-clamp and multielectrode array (MEA) recordings. Single NRVMs exhibited a bi-phasic response in the generation of SAPs (62.6 +/- 17.4 b.p.m., Days 1-2; 194.3 +/- 12.3 b.p.m., Days 3-4; 73% quiescent, Days 9-10). Although automaticity time-dependently decreased and subsequently ceased, I(f) remained fairly stable (-5.2 +/- 1.1 pA/pF, Days 1-2; -5.1 +/- 1.4 pA/pF, Days 7-8; -4.3 +/- 1.3 pA/pF, Days 13-14). In contrast, I(K1) declined rapidly (from -16.9 +/- 2.7 pA/pF on Days 1-2 to -4.4 +/- 1.6 pA/pF on Days 5-6). Maximum diastolic potential/resting membrane potential (r = 0.89) and action potential duration at 50% (APD(50), r = 0.73) and 90% (APD(90), r = 0.75) but not the firing rate (r = -0.3) were positively correlated to the I(K1). Similarly, monolayer NRVMs ceased to spontaneously fire after long-term culture. Ad-HCN1-DeltaDeltaDelta transduction restored pacing in silenced individual and monolayer NRVMs but with reduced conduction velocity and field potential amplitude. We conclude that the combination of I(K1) and I(f) primes CMs for bio-artificial pacing by determining the threshold. However, I(f) functions as a membrane potential oscillator to determine the basal firing frequency. Future engineering of automaticity in the multicellular setting needs to have conduction taken into consideration.