Calcium-dependent increase in spike duration during repetitive firing of Aplysia axon in the presence of TEA

Repetitive stimulation was studied in the axon of the giant neuron, R2, of Aplysia in the presence of TEA. In 25 or 50 mM extracellular TEA, a plateau develops on the axon spike during repetitive stimulation at frequencies greater than 3/sec. The plateau in extracellular TEA is inhibited by 30 mM CoCl₂ or 1 mM CdCl₂, and is enhanced by raising the Ca concentration. Intracellular TEA induces a plateau on the axon spike at frequencies less than 1/30sec. This plateau increases in duration with repetitive stimulation at higher frequencies and is inhibited by 30 mM CoCl₂ or 1 mM CdCl₂. The increase in spike duration during repetitive firing in the presence of TEA is indicative of an increased entry of Ca during the spike train.     

Role of A-type K+ channels in spike broadening observed in soma and axon of Hermissenda type-B photoreceptors: A simulation study

In Hermissenda type-B photoreceptors, the spike is generated in the axon and back-propagated to the soma, resulting in smaller somatic spikes. Experimentally, blocking the A-type K⁺ current (I_K,A) results in broadening of somatic spikes. Similarly, in a compartmental model of the photoreceptor, reducing the maximum A-type K⁺ conductance (g_K,Amax) results in broadening of somatic spikes. However, simulations predict that little or no broadening of axonal spikes occurs when g_K,Amax is reduced. The results can be explained by the voltage-dependent properties of I_K,A and the different potential ranges that the somatic and axonal spike traverse. Because of the steeper I-V curve and faster activation of the K⁺ channels at higher potentials, the recruitment of additional K⁺ channels in the axon is able to compensate for the decrease in K⁺ conductance, yielding less spike broadening. These results also support the idea that spike duration in the axon may not be reliably inferred based upon recordings collected from the soma. Action Editor: Jonathan D. Victor     

Sodium- and Calcium-Dependent Spike Potentials in the Secretory Neuron Soma of the X-Organ of the Crayfish

Membrane characteristics of neuron somata in the medulla terminalis ganglionic X-organ of crayfish have been investigated with intracellular glass microelectrodes. The soma membrane developed action potentials with 10–20 mv of overshoot. Delayed rectification appeared at 10–20 mv above resting membrane potential. In 50% of the neuron somata examined, action potentials were observed in Na-free medium or TTX medium. The peak potential level of the spike in these media depended on the extracellular concentration of Ca ion. It increased with the Ca concentration. In low calcium media, the peak potential level of the spike varied with Na concentration. Action potentials of the X-organ-sinus gland tract disappeared after bathing in Na-free or TTX medium, suggesting that the conductive action potential was dependent on Na ions. From these results, it is concluded that there are two systems in the neuron soma, one of which responds to the Na ion and the other, to the Ca ion. Inhibitory innervation of the X-organ by the cerebral ganglion was manifested by IPSP's when the optic peduncle was stimulated. A postulated connection between the Ca-dependent spike and the release of hormone in X-organ neuron somata is discussed.     

The action potential in the smooth muscle of the guinea pig taenia coli and ureter studied by the double sucrose-gap method

The configuration of the electrotonic potential and the action potential observed by the double sucrose-gap method was similar to that observed with a microelectrode inserted into a cell in the center pool between the gaps. In the taenia and the ureter, the evoked spike was larger in low Na or in Na-free (sucrose substitute) solution than in normal solution. However, the plateau component in the ureter was suppressed in the absence of Na. In Ca-free solution containing Mg (3–5 mM) and Na (137 mM), the membrane potential and membrane resistance were normal, but no spike could be elicited in both the taenia and ureter. Replacement of Ca with Sr did not affect the spike in the taenia, nor the spike component of the ureter but prolonged the plateau component. The prolonged plateau disappeared on removal of Na, while repetitive spikes could still be evoked. It was concluded that the spike activity in the taenia and in the ureter of the guinea pig is due to Ca entry, that the plateau component in the ureter is due to an increase in the Na conductance of the membrane, and that both mechanisms, for the spike and for the plateau, are separately controlled by Ca bound in the membrane.     

A re-excitation mechanism for strychnine-induced doublets in molluscan neurons

The effects of strychnine on Aplysia R2 neurons were evaluated using simultaneous intracellular recordings of the soma and axon potentials. 1 mM strychnine produced a slight enlargement of the somatic spike and a large increase of the axon spike duration. Following direct stimulation, the soma displayed depolarizing afterpotentials ( DAPs ) which might trigger extra-spikes, both produced electronically by long-lasting axon spikes. Cobalt suppressed both the axon spike lengthening and the somatic extra-spikes or DAPs , and induced large depolarizing shifts in the soma. The region of largest spike lengthening (proximal axon) had a large density of Ca channels. The different effects of strychnine on the soma and on the axon were assumed to result from a selective blockage of the V-dependent K channels which would predominate in the axon whereas Ca-activated K channels would predominate in the soma.     

Axonal contribution to subthreshold currents inaplysia bursting pacemaker neurons

The contribution of axonal activity to the ionic currents which generate bursting pacemaker activity was studied by using the two-electrode voltage-clamp technique in Aplysia bursting neuron somata in conjunction with intraaxonal voltage recordings. Depolarizing voltage-clamp pulses applied to bursting cell somata triggered axonal action potentials. The voltage-clamp current recording exhibited transient inward current "notches" corresponding to each of the axonal spikes. The addition of 50 microM tetrodotoxin (TTX) to the bathing medium blocked the fast axonal spikes and current notches, revealing a slower axonal spike which was blocked by the replacement of external Ca2+ with Co2+. The inward current evoked by applying a depolarizing voltage-clamp pulse in the soma is distorted by the occurrence of the axonal Ca2+ spike. Elimination of the axonal spike, by injecting hyperpolarizing current into the axon, changes both the time course and the magnitude of the inward current. The axonal Ca2+ spikes are followed by a series of Ca2+-dependent afterpotentials: a rapid postspike hyperpolarization, a depolarizing afterpotential (DAP) and, finally, a long-lasting postburst hyperpolarization. The long-lasting hyperpolarization is not blocked by 50 mM external tetraethyl ammonium, an effective blocker of Ca2+-activated K+ current [IK(Ca)], and does not appear to reverse at EK. Hence, the axonal long-lasting hyperpolarization may not be due to IK(Ca). Somatic voltage-clamp pulses in bursting neurons are followed by a slow inward tail current, which is sometimes coincident with a DAP in the axon. In some cells, the amplitude of the slow inward tail current is greatly reduced if axonal spikes and DAPs are prevented by hyperpolarization of the axon, while, in other cells, elimination of axonal activity has little effect. Therefore, the slow inward tail current is not necessarily an artifact of poor voltage-clamp control over the axonal membrane potential but probably results from the activation of an ionic conductance mechanism located partly in the axon and partly in the soma.     

Caffeine and excitation-contraction coupling in the guinea pig taenia coli

The effects of caffeine (0.2–10 mM) on the electrical and mechanical activities of guinea pig taenia coli were investigated with the double sucrose-gap method. Caffeine evoked a small tension with a latency of 20–30 sec, then phasic contraction developed and finally relaxation. The initial tension development also appeared in the Na-free solution without any marked changes in the membrane potential and membrane resistance. The phasic contraction disappeared in the Na-free solution. The relaxation in the presence of caffeine was accompanied by depolarization block of the spike generation. The minimum concentration of Ca ion needed to evoke the tension development by the caffeine was 10^-7 M. Caffeine also potentiated the twitch tension below a concentration of 5 mM either in the Na-free solution or at low temperature (5°C). NO₃ ^- and Br^- showed a similar response to caffeine on the potentiation of the twitch tension at low temperature.     

Voltage-activated lonic currents in differentiating rat cerebellar granule neurons cultured from the external germinal layer

The electrical properties of the precursor cells of the external germinal layer of rat cerebellum were assessed during their differentiation in control medium (Dulbecco's modified Eagle's medium) supplemented or not with either basic fibroblast growth factor (bFGF) or 25 mM potassium chloride (KCI). Resting potential was shown to be –10 mV in all three conditions 3 hours after plating [days in vitro (DIV)0]. By DIV 5, it reached -63 mV for cells cultured in 25 mM KCI but only –28 mV in control and bFGF media. The main voltage-sensitive ionic current measured at DIV 0 under all conditions was a composite I_k consisting in a sustained K⁺ current blocked by tetraethylammonium (I_k(TEA)), plus a rapidly activating and inactivating TEA-insensitive I_k(A). Both currents increased with time in all conditions, but after 5 days I_K(A) became dominant in terms of density. I_K(TEA) is likely an I_K(Ca), since it was blocked by 67% in 1 mM TEA. On DIV O, I_Na and I_Ca were absent or small in amplitude. By DIV 3, 80% of the cells had currents able to generate a spike. Interestingly, I_Ca mean amplitude and current density measured at –10 mV in control condition on DIV 1 was singnificantly larger than those recorded in bFGF and 25 mM KCI. The order of appearance of the ionic currents, I_K, I_Ca, and I_Na, leads directly to fast spike activity allowing for poor calcium entry. Firing rate likely depends on I_K(A), which increased during the first 6 days of development but could be differentially regulated by bFGF. © 1995 John Wiley & Sons, Inc.     

The Electrical Activity of Canine Cardiac Purkinje Fibers in Sodium-Free, Calcium-Rich Solutions

Propagated action potentials can be obtained in canine cardiac Purkinje fibers exposed to Na-free solutions containing no inorganic cation other than Ca and K. Essentially similar action potentials are obtained if Na is replaced by tetraethylammonium (TEA), tetramethylammonium (TMA), or choline. In a solution containing 128 mM TEA and 16.2 mM Ca the characteristics of these electrical responses were: maximum diastolic potential, -59 ± 3.3 mV; overshoot, 20 ± 6.8 mV; maximum upstroke velocity, 3.7 ± 2.3 V/s; conduction velocity, 0.1 m/s; and action potential duration, 360 ± 45 ms. The magnitude of the overshoot varied with log Ca_o with a slope of about 30 mV/10-fold concentration change. The upstroke velocity was an approximately linear function of Ca_o. The active response was greatly diminished or abolished by Mn and D-600 but was unaffected by tetrodotoxin. These Ca-dependent responses appeared in a region of transmembrane potential (about -50 mV) at which the rapid Na-dependent upstroke is abolished even when Na is present.     

Effect of sodium on cellular calcium transport in rat kidney

Summary The influence of extracellular Na (Na _o ) on cellular Ca transport and distribution was studied in rat kidney slices. Calcium efflux from prelabeled slices was depressed when Na _o was completely replaced by choline or tetraethylammonium (TEA) ions and it was markedly stimulated when Na was reintroduced in a Na-free medium. However, reducing Na _o (with choline or TEA as substituting ions) did not increase the total slice⁴⁰Ca, their total exchangeable Ca pool, or the⁴⁰Ca or⁴⁵Ca of mitochondria isolated from these slices. Kinetic analyses of steady-state⁴⁵Ca desaturation curves showed that reducing Na _o depressed the exchange of Ca across the plasma membrane, slightly decreased the cytosolic Ca pool, but did not significantly affect the mitochondrial Ca pool and Ca cycling. Ouabain (10^–3 m) which should reduce the Na gradient across the plasma membrane had no effect on calcium distribution and transport. These results suggest that in kidney cells low Na _o depresses Ca influx as well as Ca efflux; there may be an interaction between Na and Ca at a possible carrier located in the plasma membrane, but there is no Na/Ca exchange as described in several excitable tissues.     

Zinc modulates a-type potassium currents and neuronal excitability in snail neurons

Summary 1. Zinc-induced actions were studied on the A-current and neuronal activity in identified and unidentified nerve cells of the snail,Helix pomatia L., under voltage and current clamp conditions.2. Extracellularly applied Zn²⁺ attenuated the peak amplitude of the A-current in a potential- and dose-dependent way (K _i=1.8 mM at –30 mV,n _H=0.6).3. Attenuation of the A-currents was initiated as Zn²⁺ shifted the potential dependence of both activation and inactivation of the currents toward more positive potential values.4. Zinc concomitantly prolonged the time to peak and decay time constant of the A-currents (K _d=1.7 mM,n _H=1.4) as well.5. Zn²⁺ decreased the resting membrane potential and the spike amplitude and increased the action potential duration and the input resistance of the cells in current clamp experiments.6. A complex action of zinc increased the neuronal excitability, indicating spontaneous and synaptically evoked spike discharges.7. Common and specific zinc binding sites are supposed on vertebrate and invertebrate A-type potassium channel proteins, where binding Zn²⁺ can modulate the gating properties and kinetics of the fast outward potassium currents.     

Increasing NaCl and CaCl<Subscript>2</Subscript> concentrations in the growth medium of quince leaves: I. Effects on somatic embryo and root regeneration

Summary The effects of increasing concentrations of NaCl and CaCl₂ on quince (Cydonia oblonga Mill. BA 29 clone) somatic embryogenesis and adventitious root regeneration were investigated. Leaves collected from in vitro-grown shoots were used as explants and induced for 2d in liquid Murashige and Skoog medium containing 11.3 μM 2,4-dichlorophenoxyacetic acid. Explants were then cultured on semisolid Murashige and Skoog medium enriched with 4.7 μM kinetin and 0.5 μM naphthaleneacetic acid under red light for 25 d and under white light for another 25 d. Two experiments were performed: in the first, NaCl was used at 0,25, 50, 100, and 200 mM in factorial combination with CaCl₂ at 3, 9, and 27 mM; in the second, NaCl was applied at 0, 5, 10, 20, 40, and 80 mM in combination with CaCl₂ at 0.3, 1.0, and 3.0 mM. Quince leaves revealed the capacity to regenerate somatic embryos and/or adventitious roots. Quantitative and qualitative regeneration from leaves was affected by NaCl treatments: increasing NaCl concentrations, in combination with CaCl₂ at 1 mM, led to an increase in the proportion of leaves producing somatic embryos only, and to a decrease of both leaves regenerating roots only and leaves simultaneously producing somatic embryos and adventitious roots. This suggests a beneficial effect of salt stress on the embryogenic process. The regeneration response decreased with increasing salt concentrations and was almost totally inhibited above 50 mM NaCl and 9 mM CaCl₂. The presence of CaCl₂ in the culture medium apparently mitigated the effects of salt stress, but only when NaCl was applied at 40 mM. NaCl at 5 mM, in the presence of 0.3 or 1 mM CaCl₂, was favorable both to somatic embryo and root production. No value of the ratio Na⁺/Ca²⁺ was found to be optimal for the regeneration processes.     

三叉神经中脑核神经元膜的电学特性

用大鼠脑干脑片,给三叉神经中脑核79个神经元作了细胞内记录,测算了20个神经元膜的电学特性:静息电位-60.3±5.6mV;输入阻抗为10.5±5.4MΩ;时间常数1.3±0.5ms。电刺激可诱发动作电位,测算32个神经元的有关参数:阈电位-50—-55mV;波幅69.5±6.1mV;超射11.9±3.6mV;波宽0.8±0.2ms。TTX(0.3μmol/L)或无钠使之消失。通以长时程矩形波电流可引起200—250Hz的2—15个重复放电,但在通电停止前终止,TEA或4-AP可延长放电。膜电位-60—-55mV时在动作电位之后可看到阈下电位波动,它不受TTX的影响,无钙时消失,TEA或4-AP使波幅增大。静息电位去极化可使45个神经元中的40个发生外向整流作用,并被TEA,4-AP或无钙抑制,超极化则发生内向整流作用,Cs或无钠抑制之。灌流液中加入各种钾通道阻断药时神经元的稳态I-V曲线发生相应变化,提示I_(DR),l_A,I_(K(Ca))及I_Q可能都与静息时的膜电导有关。     

Axonal spike generation near the giant neuron soma computed by the Hodgkin-Huxley equation

The behavior of the antidromic spike and the origin of the axonal spike evoked by direct stimulation of the soma were studied with the aid of the Hodgkin-Hexley equation. It is suggested that the mechanisms responsible for electrical excitation of the axon are qualitatively and quantitatively similar to those described by Hodgkin and Huxley for the squid axon. The amplitude of the antidromic spike diminishes rapidly close to the soma. In the example studied, only subthreshod changes of membrane potential take place in the soma. During direct stimulation of the soma the site of primary origin of the axonal spike depends on the strength of the stimulating current. With an increase in its strength the site of primary generation of the spike moves closer to the soma.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 7, No. 4, pp. 422–427, July–August, 1975.     

Demonstration of two stable potential states in the squid giant axon under tetraethylammonium chloride

1. Intracellular injection of tetraethylammonium chloride (TEA) into a giant axon of the squid prolongs the duration of the action potential without changing the resting potential (Fig. 3). The prolongation is sometimes 100-fold or more. 2. The action potential of a giant axon treated with TEA has an initial peak followed by a plateau (Fig. 3). The membrane resistance during the plateau is practically normal (Fig. 4). Near the end of the action potential, there is an apparent increase in the membrane resistance (Fig. 5D and Fig. 6, right). 3. The phenomenon of abolition of action potentials was demonstrated in the squid giant axon treated with TEA (Fig. 7). Following an action potential abolished in its early phase, there is no refractoriness (Fig. 8). 4. By the method of voltage clamp, the voltage-current relation was investigated on normal squid axons as well as on axons treated with TEA (Figs. 9 and 10). 5. The presence of stable states of the membrane was demonstrated by clamping the membrane potential with two voltage steps (Fig. 11). Experimental evidence was presented showing that, in an "unstable" state, the membrane conductance is not uniquely determined by the membrane potential. 6. The effect of low sodium water was investigated in the axon treated with TEA (Fig. 12). 7. The similarity between the action potential of a squid axon under TEA and that of the vertebrate cardiac muscle was stressed. The experimental results were interpreted as supporting the view that there are two stable states in the membrane. Initiation and abolition of an action potential were explained as transitions between the two states.     

Internal perfusion of crayfish, giant axons: action of tannic acid, DDT, and TEA

Crayfish giant axons remain viable following internal perfusion with a mixture of fluoride and citrate salts. The relative favorability of various internal anions, and the dependence of resting and action potentials on internal cations are both similar to results on internally perfused squid axons. TEA widens the falling phase of the spike only from inside the axon, while DDT is active from either side of the membrane. Records of impedance changes show that effects of TEA and DDT on components of ionic conductances are similar to those found in other axons by voltage clamp measurements. Tannic acid perfused internally at a concentration of the order of 10 μM produces spontaneous activity, and a progressive increase in spike width. After 30 minutes, action potentials are “cardiac” type and are up to several minutes in duration. Records of impedance changes, and data from rapid changes in external ionic concentrations, suggest that the plateau phase of the spike is due to a maintained increase in sodium conductance. Since tannic acid is capable of crosslinking proteins and “rigidifying” protein monolayers, it is suggested that its effects on the axon may be the result of an interference with a conformational change in a membrane protein or protein-phospholipid complex during excitation.     

On the mechanism of cesium-induced voltage and current tails in single ventricular myocytes

The mechanisms by which different concentrations of cesium modify membrane potentials and currents were investigated in guinea pig single ventricular myocytes. In a dose-dependent manner, cesium reversibly decreases the resting potential and action potential amplitude and duration, and induces a diastolic decaying voltage tail (V_ex), which increases at more negative and reverses at less negative potentials. In voltage-clamped myocytes, Cs⁺ increases the holding current, increases the outward current at plateau levels while decreasing it at potentials closer to resting potential, induces an inward tail current (I_ex) on return to resting potential and causes a negative shift of the threshold for the inward current. During depolarizing ramps, Cs⁺ decreases the outward current negative to inward rectification range, whereas it increases the current past that range. During repolarizing ramps, Cs⁺ shifts the threshold for removal of inward rectification negative slope to less negative values. Cs⁺-induced voltage and current tails are increased by repetitive activity, caffeine (5 mM) and high [Ca²⁺]_o (8.1 mM), and are reduced by low Ca²⁺ (0.45 mM), Cd²⁺ (0.2 mM) and Ni²⁺ (2 mM). Ni²⁺ also abolishes the tail current that follows steps more positive than E_Ca. We conclude that Cs⁺ (1) decreases the resting potential by decreasing the outward current at more negative potentials, (2) shortens the action potential by increasing the outward current at potentials positive to the negative slope of inward rectification, and (3) induces diastolic tails through a Ca²⁺-dependent mechanism, which apparently is an enhanced electrogenic Na-Ca exchange.     

Some Effects of Calcium Ions on the Action Potential of Single Nodes of Ranvier

Action potentials of single frog nerve fibers were recorded with the air-gap method in "low Ca" (0.26 mM) and "high Ca" (4.2 mM) solutions and compared to spikes in normal Ringer''s (1.05 mM Ca). On increasing (Ca)_o the action potentials became shorter, the "knee" during the falling phase as well as the threshold for abolition moved to internal potentials more positive, and the spike recovery during the relative refractory period was faster. Outward current pulses applied during an action potential affected its configuration more in low Ca than in high Ca. The onset of the delayed rectification (in the absence of Na) was found faster in high Ga. After-potentials during anelectrotonus declined more rapidly in high Ca than in low Ca. The results are compared primarily with the voltage-clamp analysis of Ca effects on squid axons and satisfactory qualitative agreement is reached.     

Electrical Properties of the Pacemaker Neurons in the Heart Ganglion of a Stomatopod, Squilla oratoria

In the Squilla heart ganglion, the pacemaker is located in the rostral group of cells. After spontaneous firing ceased, the electrophysiological properties of these cells were examined with intracellular electrodes. Cells respond to electrical stimuli with all-or-none action potentials. Direct stimulation by strong currents decreases the size of action potentials. Comparison with action potentials caused by axonal stimulation and analysis of time relations indicate that with stronger currents the soma membrane is directly stimulated whereas with weaker currents the impulse first arises in the axon and then invades the soma. Spikes evoked in a neuron spread into all other neurons. Adjacent cells are interconnected by electrotonic connections. Histologically axons are tied with the side-junction. B spikes of adjacent cells are blocked simultaneously by hyperpolarization or by repetitive stimulation. Experiments show that under such circumstances the B spike is not directly elicited from the A spike but is evoked by invasion of an impulse or electrotonic potential from adjacent cells. On rostral stimulation a small prepotential precedes the main spike. It is interpreted as an action potential from dendrites.     

Muscarinic activation causes biphasic inotropic response and decreases cellular Na+ activity in canine cardiac purkinje fibers

In this study, the effects of carbachol (CCh) on twitch tension, intracellular Na⁺ activity (a _Na ⁱ ), and action potential were simultaneously measured in canine cardiac Purkinje fibers in order to examine the regulation of inotropy through muscarinic receptors and its relation to a _Na ⁱ . In fibers driven at 1 Hz, CCh (10 µM) initially and transiently decreased and then increased the twitch tension by 36±8%. The action potential showed a significant elevation of the plateau and a significant shortening of the duration at 90% repolarization (APD₉₀), from 403±7 to 389±7 ms. The a _Na ⁱ decreased from 7.4±0.4 to 6.7±0.3 mM (n=23, p<0.05). Atropine (1 µM) decreased the twitch tension by 21±6% (n=7, p<0.05) without significant effects on the action potential and a _Na ⁱ , and inhibited the effects of CCh. Cs⁺ (20 mM) increased the plateau height and APD₉₀, enhanced the twitch tension by 66±24%, but decreased a _Na ⁱ from 7.3±0.3 to 6.3±0.4 mM (n=6, p<0.05). In the presence of 20 mM Cs⁺, some fibers generated slow responses. The addition of 10 µM CCh further increased the twitch tension and APD₉₀, and decreased a _Na ⁱ from 6.3±0.4 to 5.3±0.3 mM. Ouabain (0.3 µM) increased the twitch tension and a _Na ⁱ , and inhibited the CCh-induced decrease of a _Na ⁱ . In the presence of ouabain, 20 mM Cs⁺ depolarized the fiber and generated slow responses with a decreased a _Na ⁱ . The addition of 10 µM CCh enhanced the slow action potential, and increased a _Na ⁱ although there was a transient decrease during early exposure. These results suggest that activation of muscarinic receptors in canine Purkinje fibers results in an enhancement of the Na⁺-K⁺ pump activity and a biphasic inotropic response, probably via different receptor subtypes. The inhibitory effect, most likely through M₂ receptors, is associated with the activation of K⁺ channels. The stimulatory effect, on the other hand, is probably due to the action on the M₁ receptors, resulting in increases in Ca²⁺ currents.