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.     

Propagation of action potentials in squid giant axons. Repetitive firing at regions of membrane inhomogeneities

Effects of reduction in potassium conductance on impulse conduction were studied in squid giant axons. Internal perfusion of axons with tetraethylammonium (TEA) ions reduces G K and causes the duration of action potential to be increased up to 300 ms. This prolongation of action potentials does not change their conduction velocity. The shape of these propagating action potentials is similar to membrane action potentials in TEA. Axons with regions of differing membrane potassium conductances are obtained by perfusing the axon trunk and one of its two main branches with TEA after the second branch has been filled with normal perfusing solution. Although the latter is initially free of TEA, this ion diffuses in slowly. Up until a large amount of TEA has diffused into the second branch, action potentials in the two branches have very different durations. During this period, membrane regions with prolonged action potentials are a source of depolarizing current for the other, and repetitive activity may be initiated at transitional regions. After a single stimulus in either axon region, interactions between action potentials of different durations usually led to rebound, or a short burst, of action potentials. Complex interactions between two axon regions whose action potentials have different durations resembles electric activity recorded during some cardiac arrhythmias.     

A study of the effects of externally applied sodium-ions and detection of spatial non-uniformity of the squid axon membrane under internal perfusion

Electrical properties of the axon membrane were examined under internal perfusion of squid giant axons with a dilute solution of NaF or CsF. The rate of propagation of the action potential was markedly enhanced when NaCl was added to the external CaCl₂ solution. The membrane conductance both at rest and during the action potential was increased with increasing Na-concentration in the external medium. In the perfusion zone of these axons, the action potentials in different parts of the membrane were found to terminate in a more-or-less spatially random and temporally irregular fashion. When the electric field outside the axon membrane was examined with hyperfine glass-pipette electrodes, small rectangular potential changes of uniform amplitude were observed. The small potential changes, which resemble those obtained by Bean in EIM-treated lipid bilayer, were interpreted as indicating spatial non-uniformity of the axon membrane during excitation. The importance of long-range electric interaction between different parts of the axon membrane is emphasized.     

A prolonged,voltage-dependent calcium permeability revealed by tetraethylammonium in the soma and axon of Aplysia giant neuron

The soma but not the axon of the giant neuron, R2, of Aplysia can generate an all-or-none Ca spike in Na-free or TTX-containing medium (Junge and Miller, 1974). Extracellular axonal recordings made at several distances from the soma provide evidence that the transition in ability to fire a spike in Na-free medium occurs within the first 250 μm of the axon. Application of 25 mM TEA-Br to the bathing medium causes a more than tenfold increase in the duration of the somatic action potential. The duration of the axonal action potential in TEA decreases with distance from the soma. At distances greater than 3 mm from the soma this concentration of TEA causes little or no increase in the duration of the axon spike. The effect of 25 mM TEA on both the soma and proximal axon is blocked reversibly by 30 mM CoCl₂ or 1 mM CdCl₂. The duration of the somatic action potential in TEA increases with an increase in Ca concentration of the bath. At a constant concentration of Na, the voltage level of the somatic plateau increases with Ca concentration in the manner predicted for a Ca electrode. In the presence of 11 mM Ca²⁺ the potential of the plateau is relatively insensitive to Na concentration. The TEA plateau in R2 reveals a prolonged voltage-dependent permeability to Ca. The duration of the plateau may indicate the degree of Ca activation during a spike.     

Effects of external ions on membrane potentials of a crayfish giant axon

Transmembrane potentials in the crayfish giant axon have been investigated as a function of the concentration of normally occurring external cations. Results have been compared with data already available for the lobster and squid giant axons. The magnitude of the action potential was shown to be a linear function of the log of the external sodium concentration, as would be predicted for an ideal sodium electrode. The resting potential is an inverse function of the external potassium concentration, but behaves as an ideal potassium electrode only at the higher external concentrations of potassium. Decrease in external calcium results in a decrease in both resting potential and action potential; an increase in external calcium above normal has no effect on magnitude of transmembrane potentials. Magnesium can partially substitute for calcium in the maintenance of normal action potential magnitude, but appears to have very little effect on resting potential. All ionic effects studied are completely reversible. The results are in generally good agreement with data presently available for the lobster giant axon and for the squid giant axon.     

Analysis of K Inactivation and TEA Action in the Supramedullary Cells of Puffer

Under the voltage clamp condition, the K inactivation was analyzed in cells bathed in the isosmotic KCl Lophius-Ringer solution. After conditioning hyperpolarization, the cells respond to depolarizations with increased K permeability, which in turn is decreased during maintained depolarizations. The steady-state levels of the K inactivation as a function of the membrane potential are related by an S-shaped curve similar to that which describes the steady-state Na inactivation in the squid giant axon. TEA reduced the K conductance by a factor which is independent of the potential, and without a shift of the inactivation curve along the voltage axis. The rapid phase of the K activation is less susceptible to TEA than the slow phase of the K activation. Hyperpolarizing steps remove the K inactivation, the rate of the removal being faster the larger the hyperpolarization from the standard potential of about -60 mv.     

Tetracycline fluorescence as calcium-probe for nerve membrane with some model studies using erythrocyte ghosts

Summary The tetracycline dyes, particularly chlorotetracycline, have been employed as probes of membrane-associated calcium during the excitation process of nerve. Both squid giant axons, stained internally, and lobster nerves, stained externally, show a small increase in fluorescent light during the action potential. Increasing the calcium concentration bathing a lobster nerve leads to a larger optical signal. Adding fluoride ion to the inside of a squid axon, which might be expected to influence the internal calcium-ion concentration, also leads to a larger optical signal. Squid axons have been studied under conditions of voltage clamp and the hyperpolarizing response. Model studies were done with erythrocyte ghosts to clarify the influence of membranes and calcium on the fluores-cence of the tetracyclines. Chlorotetracycline may be monitoring calcium concentration associated with the inner surface of the nerve membrane.     

Electrical Changes in Pre- and Postsynaptic Axons of the Giant Synapse of Loligo

Potential changes both in pre- and postsynaptic axons were recorded from the giant synapse of squid with intracellular electrodes. Synaptic current was also recorded by a voltage clamp method. Facilitation of postsynaptic potential caused by applying two stimuli several milliseconds apart was accompanied by an increase in the amplitude of the presynaptic action potential. Depression of the postsynaptic potential occurred without changes in the presynaptic action potential. Increase in the concentration of Ca in sea water caused an increase in amplitude of the synaptic current. On the other hand increase in Mg concentration decreased the amplitude of the synaptic current. In these cases no appreciable change in the presynaptic action potential was observed. Extracellularly recorded potential changes of the presynaptic axon showed mainly a positive deflexion at the synaptic region and a negative deflexion in the more proximal part of the presynaptic axon. Mechanism of synaptic transmission is discussed.     

Reversible electrical breakdown of squid giant axon membrane

Charge pulse relaxation experiments were performed on squid giant axon. In the low voltage range, the initial voltage across squid axon membrane was a linear function of the injected charge. For voltages of the order of 1 V this relationship between injected charge and voltage across the membrane changes abruptly. Because of a high conductance state caused by these large electric fields the voltage across the membrane cannot be made large enough to exceed a critical value, Vc, defined as the breakdown voltage, Vc has for squid axon membrane a value of 1.1 V at 12 degrees C. During breakdown the specific membrane conductance exceeds 1 S. cm-2. Electrical breakdown produced by charge pulses of few microseconds duration have no influence on the excitability of the squid axon membrane. The resealing process of the membrane is so fast that a depolarizing breakdown is followed by the falling phase of a normal action potential. Thus, membrane voltages close to Vc open the sodium channels in few microseconds, but do not produce a decrease of the time constant of potassium activation large enough to cause the opening of a significant percentage of channels in a time of about 10 mus. It is probable that the reversible electrical breakdown is mainly caused by mechanical instability produced by electrostriction of the membrane (electrochemical model), but the decrease in the Born energy for ion injection into the membrane, accompanying the decrease in membrane thickness, may play also an important role. Because of the high conductance of the membrane during breakdown it seems very likely that this results in pore formation.     

Excitability, instability and phase transitions in squid axon membrane under internal perfusion with dilute salt solutions

Using giant axons of squid, Doryteuthis, available in Hokkaido, Japan, it was shown that axons internally perfused with a dilute sodium salt solution undergo an abrupt transition from a resting to a depolarized state on addition of KCl to an external medium containing CaCl₂. Under internal perfusion with a dilute solution of sodium or cesium salt, it was possible to induce abrupt transitions between the two (i.e., resting and depolarized) states of the membrane by changing the temperature. “Giant fluctuations” in the state of the axon membrane were demonstrated at and near the critical points of the axon membrane. These findings are interpreted as supporting the view that an abrupt change in the membrane potential and conductance is an electrochemical manifestation of a phase transition of the membrane macromolecules.     

Resistivity of axoplasm. II. Internal resistivity of giant axons of squid and Myxicola

The specific resistivity of the axoplasm of giant axons of squid and Myxicola was measured utilizing a single metal microelectrode subjected to alternating current in a circuit in which the voltage output varies with the conductivity of the thin layer of fluid at the exposed electrode tip. The average specific resistivity of stellar axons of Loligo pealei was 31 omegacm (1.55 times seawater [X SW]) while for Loligo opalescens it was 32 omegacm (1.30 X SW). Smaller giant axons had a higher average resistivity. Myxicola giant axons had a resistivity of 68 omegacm (2.7 X SW) in normal seawater, and 53 omegacm (2.1 X SW) in a hypertonic high-Mg++ seawater. The temperature dependence of squid axon resistivity does not differ from that of an equally conductive dilution of seawater.     

Current-Voltage Relations in the Lobster Giant Axon Membrane Under Voltage Clamp Conditions

The sucrose-gap method introduced by Stämpfli provides a means for the application of a voltage clamp to the lobster giant axon, which responds to a variety of different experimental procedures in ways quite similar to those reported for the squid axon and frog node. This is particularly true for the behavior of the peak initial current. However, the steady state current shows some differences. It has a variable slope conductance less than that of the peak initial current. The magnitude of the steady state slope conductance is related to the length of the repolarization phase of the action potential, which does not have an undershoot in the lobster. The steady state outward current is maintained for as long as 100 msec.; this is in contrast to a decline of about 50 per cent in the squid axon. Lowering the external calcium concentration produces shifts in the current-voltage relations qualitatively similar to those obtained from the squid axon. On the basis of the data available, there is no reason to doubt that the Hodgkin and Huxley analysis for the squid giant axon in sea water can be applied to the lobster giant axon.     

Surface Charge of Giant Axons of Squid and Lobster

A method is described for the determination of the electrophoretic mobility of single, isolated, intact, giant axons of squid and lobster. In normal physiological solutions, the surface of hydrodynamic shear of these axons is negatively charged. The lower limit of the estimated surface charge density is -1.9 × 10^-8 coul cm^-2 for squid axons, -4.2 × 10^-8 coul cm^-2 for lobster axons. The electrophoretic mobility of squid axons decreases greatly when the applied transaxial electric field is made sufficiently intense; action potential propagation is blocked irreversibly by transaxial electric fields of the same intensity. The squid axon recovers its mobility hours later and is then less affected by transaxial fields. Eventually, a state is reached in which the transaxial field irreversibly reverses the sign of the surface charge. In contrast, there is no obvious effect of electric field on the mobility of lobster axons. The mobility of lobster axons becomes undetectable in the presence of Th⁴⁺ at a concentration which blocks the action potential, and in the presence of La³⁺ at a concentration which does not affect propagation. Quinine does not alter lobster axon mobility at a concentration which blocks action potential conduction. Replacement of extracellular Na⁺ by K⁺ is without effect upon lobster axon mobility. The electrophysiological implications of the results are discussed.     

Effects of narcotics on the giant axon of the squid

—Levorphanol (10^-3 M) reversibly blocked conduction in the giant axon of the squid and axons from the walking legs of spider crab and lobster. Similar concentrations of levallorphan and dextrorphan blocked conduction in the squid giant axon. Under the same experimental condition morphine caused an approximately 40 per cent decrease in spike height. Levorphanol did not affect the resting potential or resistance of the squid axon. Spermidine, spermine and dinitrophenol had little or no direct effect on the action potential nor did they alter the potency of levorphanol. Concentrations of levorphanol as low as 5 × 10^-5 M blocked repetitive or spontaneous activity in the squid axon induced by decreasing the divalent cations in the medium. After exposure to tritiated levorphanol, the axoplasm and envelope of the squid axon accumulated up to 500 per cent of the concentration of tritium found in the external medium, dependent on time of exposure, and other variables. At pH 6 the levels of penetration were 33-50% of those found at pH 8, which correlates with our observation that levorphanol is about 33 % as potent in blocking the action potential at pH 6. The penetrability of levorphanol was not affected by spermidine, dinitrophenol or cottonmouth moccasin venom. Levorphanol did not alter the penetration of [C¹⁴]acetylcholine nor did it render the squid axon sensitive to it. The block of axonal conduction by compounds of the morphine series is discussed both as to possible mechanisms and significance.     

Methylmercury: Effects on electrical properties of squid axon membranes

At low concentrations (25–100 μM) methylmercury chloride caused a steady increase in the threshold for excitation and on eventual block of action potentials without changing the resting membrane potential in squid giant axons. In the axons exposed to 25 μM methylmercury chloride, peak transient and steady-state conductances were decreased by 58.8 ± 5.1% and 35.9 ± 4.3% (mean ± SEM, 4 axons), respectively and leakage conductance increased to about five times of the control value. Higher concentrations of methylmercury chloride decreased the resting membrane potential. A concentration of 0.5 mM depolarizing the nerve membrane by 16 ± 2 mV (mean ± SEM, 3 axons) in 40 minutes. These changes in ionic conductances and membrane potential were irreversible on washing the axon with drug-free sea water.     

Effects of batrachotoxin on membrane potential and conductance of squid giant axons

The effects of batrachotoxin (BTX) on the membrane potential and conductances of squid giant axons have been studied by means of intracellular microelectrode recording, internal perfusion, and voltage clamp techniques. BTX (550–1100 nM) caused a marked and irreversible depolarization of the nerve membrane, the membrane potential being eventually reversed in polarity by as much as 15 mv. The depolarization progressed more rapidly with internal application than with external application of BTX to the axon. External application of tetrodotoxin (1000 nM) completely restored the BTX depolarization. Removal or drastic reduction of external sodium caused a hyperpolarization of the BTX-poisoned membrane. However, no change in the resting membrane potential occurred when BTX was applied in the absence of sodium ions in both external and internal phases. These observations demonstrate that BTX specifically increases the resting sodium permeability of the squid axon membrane. Despite such an increase in resting sodium permeability, the BTX-poisoned membrane was still capable of undergoing a large sodium permeability increase of normal magnitude upon depolarizing stimulation provided that the membrane potential was brought back to the original or higher level. The possibility that a single sodium channel is operative for both the resting sodium, permeability and the sodium permeability increase upon stimulation is discussed.     

Optical Coherence Tomography Phase Measurement of Transient Changes in Squid Giant Axons During Activity

Noncontact optical measurements reveal that transient changes in squid giant axons are associated with action potential propagation and altered under different environmental (i.e., temperature) and physiological (i.e., ionic concentrations) conditions. Using a spectral-domain optical coherence tomography system, which produces real-time cross-sectional images of the axon in a nerve chamber, axonal surfaces along a depth profile are monitored. Differential phase analyses show transient changes around the membrane on a millisecond timescale, and the response is coincident with the arrival of the action potential at the optical measurement area. Cooling the axon slows the electrical and optical responses and increases the magnitude of the transient signals. Increasing the NaCl concentration bathing the axon, whose diameter is decreased in the hypertonic solution, results in significantly larger transient signals during action potential propagation. While monophasic and biphasic behaviors are observed, biphasic behavior dominates the results. The initial phase detected was constant for a single location but alternated for different locations; therefore, these transient signals acquired around the membrane appear to have local characteristics.     

FINE STRUCTURAL ALTERATIONS ASSOCIATED WITH VENOM ACTION ON SQUID GIANT NERVE FIBERS

(1) Block of conduction and marked increase in permeability of the squid giant axon, when surrounded by adhering small nerve fibers, is caused by the venoms of cottonmouth, ringhals, and cobra snakes and by phospholipase A (PhA). This phenomenon is associated with a marked breakdown of the substructure of the Schwann sheath into masses of cytoplasmic globules. Low concentrations of these agents which render the axons sensitive to curare cause less marked changes in the structure of the sheath. (2) Rattlesnake venom, the direct lytic factor obtained from ringhals venom, and hyaluronidase caused few observable changes in structure, correlating with the inability of these agents to increase permeability. (3) Cottonmouth venom did not alter the structure of giant axons freed of all adhering small nerve fibers. This is in agreement with previous evidence that the venom effects are due to an action of lysophosphatides liberated as a result of PhA action. Cetyltrimethylammonium chloride, a cationic detergent, produces effects that resemble those of venom and PhA. (4) The results provide evidence that PhA is the component of the venoms that is responsible for their effects. It also appears that the Schwann cell and possibly the axonal membrane are the major permeability barriers in the squid giant axon.     

Interaction of DDT with the Components of Lobster Nerve Membrane Conductance

The falling phase of action potential of lobster giant axons is markedly prolonged by treatment with DDT, and a plateau phase appears as in cardiac action potentials. Repetitive afterdischarge is very often superimposed on the plateau. Voltage-clamp experiments with the axons treated with DDT and with DDT plus tetrodotoxin or saxitoxin have revealed the following: DDT markedly slows the turning-off process of peak transient current and suppresses the steady-state current. The falling phase of the peak transient current in the DDT-poisoned axon is no longer expressed by a single exponential function as in normal axons, but by two or more exponential functions with much longer time constants. The maximum peak transient conductance is not significantly affected by DDT. DDT did not induce a shift of the curve relating the peak transient conductance to membrane potential along the potential axis. The time to peak transient current and the time for the steady-state current to reach its half-maximum are prolonged by DDT to a small extent. The finding that, under the influence of DDT, the steady-state current starts flowing while the peak transient current is partially maintained supports the hypothesis of two operationally separate ion channels in the nerve membrane.