Duration Of Single Action Potentials Research Articles

Electrophysiologic and cellular characteristics of cardiomyocytes after X-ray irradiation

The aim of this study was to investigate possible effects of ionizing irradiation on the electrophysiological functionality of cardiac myocytes in vitro. Primary chicken cardiomyocytes with spontaneous beating activity were irradiated with X-rays (dose range of 0.5–7Gy). Functional alterations of cardiac cell cultures were evaluated up to 7 days after irradiation using microelectrode arrays. As examined endpoints, cell proliferation, apoptosis, reactive oxygen species (ROS) and DNA damage were evaluated. The beat rate of the cardiac networks increased in a dose-dependent manner over one week. The duration of single action potentials was slightly shortened. Additionally, we observed lower numbers of mitotic and S-phase cells at certain time points after irradiation. Also, the number of cells with γH2AX foci increased as a function of the dose. No significant changes in the level of ROS were detected. Induction of apoptosis was generally negligibly low.This is the first report to directly show alterations in cardiac electrophysiology caused by ionizing radiation, which were detectable up to one week after irradiation.

Angiotensin II potentiates adrenergic and muscarinic modulation of guinea pig intracardiac neurons

The intrinsic cardiac plexus represents a major peripheral integration site for neuronal, hormonal, and locally produced neuromodulators controlling efferent neuronal output to the heart. This study examined the interdependence of norepinephrine, muscarinic agonists, and ANG II, to modulate intrinsic cardiac neuronal activity. Intracellular voltage recordings from whole-mount preparations of the guinea pig cardiac plexus were used to determine changes in active and passive electrical properties of individual intrinsic cardiac neurons. Application of either adrenergic or muscarinic agonists induced changes in neuronal resting membrane potentials, decreased afterhyperpolarization duration of single action potentials, and increased neuronal excitability. Adrenergic responses were inhibited by removal of extracellular calcium ions, while muscarinic responses were inhibited by application of TEA. The adrenergic responses were heterogeneous, responding to a variety of receptor-specific agonists (phenylephrine, clonidine, dobutamine, and terbutaline), although α-receptor agonists produced the most frequent responses. Application of ANG II alone produced a significant increase in excitability, while application of ANG II in combination with either adrenergic or muscarinic agonists produced a much larger potentiation of excitability. The ANG II-induced modulation of firing was blocked by the angiotensin type 2 (AT(2)) receptor inhibitor PD 123319 and was mimicked by the AT(2) receptor agonist CGP-42112A. AT(1) receptor blockade with telmasartin did not alter neuronal responses to ANG II. These data demonstrate that ANG II potentiates both muscarinically and adrenergically mediated activation of intrinsic cardiac neurons, doing so primarily via AT(2) receptor-dependent mechanisms. These neurohumoral interactions may be fundamental to regulation of neuronal excitability within the intrinsic cardiac nervous system.

Role of a Tetraethylammonium-Sensitive Component of Potassium Currents in High-Frequency Tonic Impulsation Generated by Rat Retinal Ganglion Cells

Using the voltage/current clamp technique in the whole-cell configuration, we studied the role of the highly tetraethylammonium (TEA) -sensitive component of integral potassium current in the generation of high-frequency tonic impulsation by rat retinal ganglion cells (RGCs). Application of 0.5 mM TEA led to a decrease in the frequency of evoked tonic impulsation by RGCs by 63% (from 55 ± 10 sec–1 in the control to 26 ± 5 sec–1 in the presence of the blocker; n = 11). In this case, the duration of single action potentials at the level of 50% their amplitude increased by 64% (from 1.1 ± 0.1 to 1.8 ± 0.1 msec; n = 11), the rate of repolarization decreased by 54% (from −101 ± 9 to −46 ± 5 mV/msec; n = 11), and the amplitude of afterhyperpolarization dropped by 62% (from −16 ± 2 to −6 ± 2 mV; n = 11). Upon the action of 0.5 mM TEA, the amplitude of the integral potassium current in RGCs decreased; the current component sensitive to the above blocker was equal to 0.41 ± 0.05 nA (n = 6), while the respective value in the control was 1.62 ± 0.14 nA (n = 12). Thus, a moderate (on average, by 25%) decrease in the amplitude of the above potassium current significantly influenced the characteristics of impulse activity generated by RGCs. The TEA-sensitive component of the current was similar to the Kv3.1/Kv3.2 potassium current described earlier. The obtained data are indicative of the key role of the highly TEA-sensitive component of the potassium current (passed probably via Kv3.1/Kv3 channels) in high-frequency tonic activity generated by RGCs.

Submillisecond optical reporting of membrane potential in situ using a neuronal tracer dye.

A major goal in neuroscience is the development of optical reporters of membrane potential that are easy to use, have limited phototoxicity, and achieve the speed and sensitivity necessary for detection of individual action potentials in single neurons. Here we present a novel, two-component optical approach that attains these goals. By combining DiO, a fluorescent neuronal tracer dye, with dipicrylamine (DPA), a molecule whose membrane partitioning is voltage-sensitive, optical signals related to changes in membrane potential based on FRET (Förster resonance energy transfer) are reported. Using DiO/DPA in HEK-293 cells with diffraction-limited laser spot illumination, depolarization-induced fluorescence changes of 56% per 100 mV (tau approximately 0.1 ms) were obtained, while in neuronal cultures and brain slices, action potentials (APs) generated a Delta F/F per 100 mV of >25%. The high sensitivity provided by DiO/DPA enabled the detection of subthreshold activity and high-frequency APs in single trials from somatic, axonal, or dendritic membrane compartments. Recognizing that DPA can depress excitability, we assayed the amplitude and duration of single APs, burst properties, and spontaneous firing in neurons of primary cultures and brain slices and found that they are undetectably altered by up to 2 microm DPA and only slightly perturbed by 5 microm DPA. These findings substantiate a simple, noninvasive method that relies on a neuronal tracer dye for monitoring electrical signal flow, and offers unique flexibility for the study of signaling within intact neuronal circuits.

Abbreviated action potential kinetics in a mouse model of potassium channel overexpression during hippocampal development.

Loss or "gain" of function mutations in voltage-gated ion channels often results in an adverse neurological phenotype. We have examined the electrical characteristics of hippocampal pyramidal cells in a transgenic mouse model to determine how overexpression of a Shaker-type potassium channel subunit during early postnatal development might alter excitability properties of developing neurons. We found that in CA3 neurons potassium channel overexpression led to a transient shortening in duration of single action potentials during the first two postnatal weeks. There was an increase in maximal repolarization rate, without significant effect on the rate of rise. Transgenic CA3 neurons also showed a decrease of firing frequency in response to sustained depolarizing current injection. In contrast, repolarization of action potentials in CA1 neurons was not significantly altered by trangene expression. Western Blot Analysis of membrane-associated transgene protein indicated that transgene protein levels decreased during development, in agreement with functional measures of spike width. Our data indicate that the functional consequences of potassium channel transgene expression are dependent on cellular environment and developmental stage. A transient period of hypoexcitability during a critical period of development for CA3 neurons may contribute to the hyperexcitable phenotype observed in adult animals.

The role of calcium-activated potassium channels in respiratory control.

The introduction of pharmacological and genetic tools has helped to understand the functional role of specific ion channel subtypes in the neural control of breathing. This review outlines the role one family of potassium channels, those, which are regulated by intracellular calcium. Calcium-activated potassium channels (K(+)ca) play important roles in determining the duration of single action potentials and in the frequency of action potentials in excitable cells. They have been classified based on their unitary conductances combined with their inhibition by a variety of pharmacologic substances. In this review the role of K(+)ca channels in respiratory control is outlined. The predictions of their contributions from modeling the respiratory system and the results of experiments in which they are individually blocked are discussed. Recently mice have been generated in which the expression of a single member of the K(+)ca channel family can be conditionally regulated. The repression of this small-conductance K(+)ca channel (SK3) as well as its overexpression affect baseline respiratory rhythm and the ventilatory response to hypoxia.

Plant bioelectrochemistry: effects of CCCP on electrical signaling in soybean

Carbonyl cyanide 3-chlorophenylhydrazone (CCCP) induces fast action potentials and decreases the variation potential in soybean plants. The propagation speed of the action potentials in a soybean plant produced by CCCP reaches up to 25 m/s. The duration of single action potentials after treatment by CCCP is 0.6 ms. The addition of CCCP to soil reduces variation or streaming potential to zero. The mechanism by which CCCP decreases plant maturation most likely includes depolarization of the plasma membrane, retardation of photosynthetic water oxidation, and respiratory electron transfer.

Imaging stochastic spatial variability of active channel clusters during excitation of single neurons.

Topographical maps of membrane voltages were obtained during action potentials by imaging, at 1 microm resolution, live dissociated neurons stained with the voltage sensitive dye RH237. We demonstrate with a theoretical approach that the spatial patterns in the images result from the distribution of net positive charges condensed in the inner sites of the membrane where clusters of open ionic channels are located. We observed that, in our biological images, this spatial distribution of open channels varies randomly from trial to trial while the action potentials recorded by the microelectrode display similar amplitudes and time-courses. The random differences in size and intensity of the spatial patterns in the images are best evidenced when the time of observation coincides with the duration of single action potentials. This spatial variability is explained by the fact that only part of the channel population generates an action potential and that different channels open in turn in different trials due to their stochastic operation. Such spatial flicker modifies the direction of lateral current along the neuronal membrane and may have important consequences on the intrinsic processing capabilities of the neuron.

Green plants: electrochemical interfaces

Abstract The most rapid methods of long distance communication between plant tissues and organs are bioelectrochemical signals. The action potential propagates rapidly throughout the plant along electrified interfaces. A potential pathway for transmission of this electrical signal might be the phloem sieve-tube system since it represents a continuum of plasma membranes. A phloem is an electrical conductor of bioelectrochemical impulses over long distances. At the cellular level in plants, electrical potentials exist across membranes, and thus between cellular compartments, as well as within specific compartments. Electrochemical phenomena are primary, and then deep cytophysiological reactions occur. Possible effects of the electric double layer of the Earth on the electrical activity of plants are discussed. Effects of uncouplers and acid rains on green plants are studied. Uncoupler carbonyl cyanide-p-trifluoromethoxyphenyl hydrazone (FCCP) induces ultra fast action potentials and decreases the resting potential in a soybean. The speed of the propagation of action potentials in a soybean induced by FCCP reaches up to 40 m s−1. The duration of single action potentials after treatment by FCCP was 0.0003 s. Adding FCCP to soil decreases the resting potential to zero level. The automatic measurements of the electrical potential difference can be effectively used in bioelectrochemistry, environmental plant electrophysiology, for the study of molecular mechanisms of transport processes and the influence of external stimuli on plants.

The Relation of Exocytosis and Rapid Endocytosis to Calcium Entry Evoked by Short Repetitive Depolarizing Pulses in Rat Melanotropic Cells

Melanotropic cells release predocked, large, dense-cored vesicles containing alpha-melanocyte stimulating hormone in response to calcium entry through voltage-gated calcium channels. Our first objective was to study the relationship between exocytosis, rapid endocytosis, and calcium entry evoked by short step depolarizations in the order of duration of single action potentials (APs). Exocytosis and rapid endocytosis were monitored by capacitance measurements. We show that short step depolarizations (40 msec) evoke the fast release of only approximately 3% of the predocked release-ready vesicle pool. Second, we asked what the distance is between voltage-gated calcium channels and predocked vesicles in these cells by modulating the intracellular buffer capacity. Exocytosis and rapid endocytosis were differentially affected by low concentrations of the calcium chelator EGTA. EGTA slightly attenuated exocytosis at 100 microM relative to 50 microM, but exocytosis was strongly depressed at 400 microM, showing that calcium ions have to travel a large distance to stimulate exocytosis. Nevertheless, the efficacy of calcium ions to stimulate exocytosis was constant for pulse durations between 2 and 40 msec, indicating that in melanotropes, exocytosis is related linearly to the amount and duration of calcium entry during a single AP. Rapid endocytosis was already strongly depressed at 100 microM EGTA, which shows that the process of endocytosis itself is calcium dependent in melanotropic cells. Furthermore, rapid endocytosis proceeded with a time constant of approximately 116 msec at 33 degrees C, which is three times faster than at room temperature. There was a strong correlation between the amplitude of endocytosis and the amplitude of exocytosis immediately preceding endocytosis. Both this correlation and the fast time constant of endocytosis suggest that the exocytotic vesicle is retrieved rapidly.

Characterization of a transient outward current in a rapidly adapting insect mechanosensory neuron.

This paper describes the first voltage-clamp recordings from an arthropod cuticular sensory neuron. In the femoral tactile spine neuron of the cockroach Periplaneta americana, a rapidly activating and inactivating outward current, IA, appeared when the neuron was hyperpolarized for a short period before a depolarizing test pulse. IA could be separated from the other outward currents using 5 mM 4-aminopyridine (4-AP), which specifically blocked it. Tetraethylammonium (TEA), (50 mM) did not remove IA, but decreased the steady-state outward current by about 50%. The threshold for IA activation was about -75 mV. The minimum activation and inactivation time constants were approximately 0.2 ms and 15 ms, respectively. The voltage dependencies of activation and inactivation were well fit-ted by Boltzmann distributions, giving values of membrane potential at half-maximal activation (V50) equal to -56.5 mV and an equivalent gating charge of n = 3.9 for activation and V50 = -86.7 mV and n = 3.4 for inactivation. In current-clamp recordings, 4-AP reversibly reduced the cell's normal adaptation by lowering the threshold for action potentials, but did not affect the amplitude or duration of single action potentials. These results indicate that IA plays a role in short-term adaptation by opposing membrane depolarization and reducing the spike frequency during maintained stimulation.

A [Na+]o-independent, pHo-dependent mechanism for reduction of intracellular [Ca2+] after influx through Ca2+ channels in mouse pituitary cells.

The effect of extracellular pH (pHo) on the duration of calcium-dependent chloride currents (ICl(Ca] was studied in voltage clamped AtT-20 pituitary cells. ICl(Ca) was activated by Ca2+ influx through plasma membrane Ca2+ channels, which were opened by step depolarization to voltages between -20 and +60 mV. Increasing pHo from 7.3 to 8.0 reversibly prolonged ICl(Ca) tail currents in perforated patch recordings from cells bathed in both Na(+)-containing and Na(+)-free solutions. This prolongation was prevented in standard whole cell recordings when the pipette solution contained 0.5 mM EGTA. The effects of raised pHo were not due to alteration of intracellular pH, since tail current prolongation still occurred when intracellular pH was buffered at 7.3 with 80 mM HEPES. The prolongation of ICl(Ca) at pHo 8 could not be accounted for by a direct action on Ca2+ channels, since tail currents were prolonged when pHo was changed rapidly during the tail current, after all Ca2+ channels were closed. The effects of increasing pHo on ICl(Ca) also could not be explained by a direct action on Cl- channels, since changing to pHo 8 did not prolong Cl- tail currents when intracellular Ca2+ concentration [( Ca2+]i) was fixed by EGTA in whole cell recordings. Raising pHo did, however, prolong depolarization-evoked [Ca2+]i transients, measured directly with the Ca2+ indicator dye, fura-2. Taken together, these data demonstrate the presence of a Na(+)-independent, pHo-sensitive mechanism for reduction of [Ca2+]i after influx through Ca2+ channels. This mechanism is associated with the plasma membrane, and is active on a time scale that is relevant to the duration of single action potentials in these cells. We suggest that this mechanism is the plasma membrane Ca2+ ATPase.

Differential sensitivity of cerebellar purkinje neurons to ethanol in selectivity outbred lines of mice: maintenance in vitro independent of synaptic transmission

The effects of ethanol on spontaneous firing of cerebellar Purkinje neurons were examined in outbred lines of mice (short-sleep, SS; and long-sleep, LS) which exhibit differential behavioral sensitivity to ethanol. In order to determine whether the differences in Purkinje cell ethanol sensitivity which are observed in situ reflect differences in intrinsic properties of Purkinje neurons, we developed an isolated in vitro preparation of mouse cerebellum. Even when synaptic transmission was largely inhibited by elevating Mg 2+ and decreasing Ca 2+ concentrations, Purkinje cells demonstrated stable long-term firing rates quite similar to those observed in vivo. Purkinje cells responded to superfusion of ethanol with both increases and decreases in firing rate. Inhibition of rate was more commonly observed, and was the only response which was demonstrably dose-dependent. The differential sensitivity to ethanol which we have previously reported in vivo was maintained even under these condtions, with the LS mice being approximately 5 times more sensitive to the depressant effects of ethanol. In addition, it was shown that ethanol, at the concentrations used in these experiments, decreased the amplitude and increased the duration of single action potentials. Thus, taken together, these results suggest that the differential sensitivity of outbred lines to the soporific effects of ethanol are paralleled by differences in the sensitivity of Purkinje neurons in vitro to superfusion with ethanol. Because these differences can be observed even when synaptic transmission is largely suppressed, it would appear that these differences are intrinsic to the Purkinje neurons themselves.