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.     

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.     

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.     

Osmometrically determined characteristics of the cell membrane of squid and lobster giant axons

Lobster and squid giant nerve fibers respond differently when subjected to osmotic challenges. The axons proper, as distinct from the total (fiber) complex formed by the axon and connective sheath, both behave as "fast" osmometers for changes in the concentration of NaCl, but the maximum degree of swelling in hyposmotic media is by about 60% in lobster and only by 20% in squid. The relative volume intercepts of the van't Hoff relation are about 0.2 for lobster and 0.4 for squid. The sheaths of both axons undergo only small, apparently passive changes in volume. Lobster axons are permeable to Cl, but squid axons are impermeable to this anion. Lobster axons are also permeable to glycerol. The implications of the data as to the nature of volume regulation of cells are discussed.     

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.     

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.     

Cholinesterase activity per unit surface area of conducting membranes

According to theory, the action of acetylcholine (ACh) and ACh-esterase is essential for the permeability changes of excitable membranes during activity. It is, therefore, pertinent to know the activity of ACh-esterase per unit axonal surface area instead of per gram nerve, as it has been measured in the past. Such information has now been obtained with the newly developed microgasometric technique using a magnetic diver. (1) The cholinesterase (Ch-esterase) activity per mm² surface of sensory axons of the walking leg of lobster is 1.2 x 10^-3 µM/hr. (σ = ± 0.3 x 10^-3; SE = 0.17 x 10^-3); the corresponding value for the motor axons isslightly higher: 1.93 x 10^-3 µM/hr. (σ = ± 0.41 x 10^-3; SE = ± 0.14 x 10^-3). Referred to gram nerve, the Ch-esterase activity of the sensory axons is much higher than that of the motor axons: 741 µM/hr. (σ = ± 73.5; SE = ± 32.6) versus 111.6 µM/hr. (σ = ± 28.3; SE = ± 10). (2) The enzyme activity in the small fibers of the stellar nerve of squid is 3.2 x 10^-4 µM/mm²/hr. (σ = ± 0.96 x 10^-4; SE = ± 0.4 x 10^-4). (3) The Ch-esterase activity per mm² surface of squid giant axon is 9.5 x 10^-5 µM/hr. (σ = ± 1.55 x 10^-5; SE = ± 0.38 x 10^-5). The value was obtained with small pieces of carefully cleaned axons after removal of the axoplasm and exposure to sonic disintegration. Without the latter treatment the figurewas 3.85 x 10^-5 µM/mm²/hr. (σ = ± 3.24 x 10^-5; SE = ± 0.93 x 10^-5). The experiments indicate the existence of permeability barriers in the cell wall surrounding part of the enzyme, since the substrate cannot reach all the enzyme even when small fragments of the cell wall are used without disintegration. (4) On the basis of the data obtained, some tentative approximations are made of the ratio of ACh released to Na ions entering the squid giant axon per cm² per impulse.     

Effects of calcium lack on action potential of motor axons of the lobster limb

The effects of external calcium deprivation on certain characteristics of the action potential of the lobster motor axon have been studied. Upon exposure to calcium-free solution the spike amplitude is rapidly decreased within a few minutes and is followed by a slow linear decline. The rates of spike rise and fall are proportionally reduced more than the spike but follow similar time courses during calcium lack. Associated with these phenomena are the loss in the normal slow spike repolarization process, the development of a large and lengthy undershoot, and the appearance of a high degree of refractoriness. The mean increase in the refractory period is 525 per cent upon 10 minutes' exposure to calcium-free solution. These effects are completely reversible upon returning the axons to normal solution. These results are compared to similar effects of calcium deprivation on frog myelinated axons and squid and lobster giant axons recently observed by other workers.     

Temperature Characteristics of Excitation in Space-Clamped Squid Axons

Temperature characteristics of excitability in the squid giant axon were measured for the space-clamped axon with the double sucrose gap technique. Threshold strength-duration curves were obtained for square wave current pulses from 10 µsec to 10 msec and at temperatures from 5°C to 35°C. The threshold change of potential, at which an action potential separated from a subthreshold response, averaged 17 mv at 20°C with a Q¹⁰ of 1.15. The average threshold current density at rheobase was 12 µa/cm² at 20°C with a Q¹⁰ of 2.35 compared to 2.3 obtained previously. At short times the threshold charge was 1.5·10^-8 coul/cm². This was relatively independent of temperature and occasionally showed a minimum in the temperature range. At intermediate times and all temperatures the threshold currents were less than for both the single time constant model and the two factor excitation process as developed by Hill. FitzHugh has made computer investigations of the effect of temperature on the excitation of the squid axon membrane as represented by the Hodgkin-Huxley equations. These are in general in good agreement with our experimental results.     

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

The effects of varying external concentrations of normally occurring cations on membrane potentials in the lobster giant axon have been studied and compared with data presently available from the squid giant axon. A decrease in the external concentration of sodium ions causes a reversible reduction in the amplitude of the action potential and its rate of rise. No effect on the resting potential was detected. The changes are of the same order of magnitude, but greater than would be predicted for an ideal sodium electrode. Increase in external potassium causes a decrease in resting potential, and a decrease in potassium causes an increase in potential. The data so obtained are similar to those which have been reported for the squid giant axon, and cannot be exactly fitted to the Goldman constant field equation. Lowering external calcium below 25 mM causes a reduction in resting and action potentials, and the occasional occurrence of repetitive activity. The decrease in action potential is not solely attributable to a decrease in resting potential. Increase of external calcium from 25 to 50 mM causes no change in transmembrane potentials. Variations of external magnesium concentration between zero and 50 mM had no measurable effect on membrane potentials. These studies on membrane potentials do not indicate a clear choice between the use of sea water and Cole's perfusion solution as the better external medium for studies on lobster nerve.     

Electric field-induced lateral mobility of photosystem I in the photosynthetic membrane: A study by electrophotoluminescence

Electrophoretic movement of photosystem I (PS I) along the photosynthetic membrane of hypotonically swollen thylakoid vesicles was studied by analyzing the electric field-stimulated delayed luminescence (electrophotoluminescence) emitted from PS I. The electrophoretic mobility was inferred from the differences in electrophotoluminescence (EPL) of the photosynthetic vesicles in presence and absence of trains of low amplitude (<80 V/cm) prepulses of 1 ms duration at 4 ms spacing. The average apparent electric mobility, determined from the time course of EPL increase on one hemisphere or its decrease on the other one, as function of prepulse length and intensity was of the order of 3 · 10^-5 cm²V^-1s^-1. The assymetric distribution of the PS I reached a steady state when the diffusional, electrostatic, and elastic forces balanced the electrophoretic driving force. A lateral diffusion coefficient of ~5 · 10^-9 cm²s^-1 was found for the PS I complex from the diffusional relaxation after cessation of the electric field pulse train. Experimental conditions such as concentration, temperature, and viscosity of the aqueous solution were not critical for the effect. Between 23 and 150 electron charges per moving particle were estimated from the measured electrophoretic mobility.     

Nonelectrolyte Permeability, Sodium Influx, Electrical Potentials, and Axolemma Ultrastructure in Squid Axons of Various Diameters

The penetration of ¹⁴C-labeled ethylene glycol, erythritol, mannitol, and sucrose was measured in giant axons of various diameters isolated from the hindmost stellar nerves of Doryteuthis plei squid. Axon diameter depends mainly on the age of the squid. The influx of ²²Na, some electrical properties, and the ultrastructure of the axolemma were also studied. The results confirm our previous observation that in medium sized axons of D. plei stimulation causes an increase in the permeability to the penetration of erythritol, mannitol, and sucrose. They also demonstrate that the magnitude of the increase in the penetration of these probing molecules diminishes progressively as the axon diameter increases. The diminution in permeability may be due to a reduction in size of the pathways used by nonelectrolytes to enter the axon. No effect of stimulation on the ethylene glycol permeability is observed. The sodium influx and electrical properties are independent of axon size. The ultrastructural study shows that the axolemma thickness increases with axon diameter. The present experiments indicate that the nonelectrolyte permeability of stimulated axons depends on nerve fiber properties related to axon diameter and on the size of the hydrophilic nonelectrolyte probe.     

Magnesium influx in dialyzed squid axons

Summary The influx of magnesium from seawater into squid giant axons has been measured under conditions where internal solute control in the axon was maintained by dialysis. Mg influx is smallest (1 pmol/cm² sec) when both Na and ATP have been removed from the axoplasm by dialysis. The addition of 3mm ATP to the dialysis fluid gives a Mg influx of 2.5 pmol/cm² sec while the addition of [Na] _i and [ATP] _i gives 3 pmol/cm² sec as a value for Mg influx; this corresponds well with fluxes measured in intact squid giant axons.The Mg content of squid axons is 6 mmol/kg axoplasm; this is unaffected by soaking axons in Li or Na seawater for periods of up to 100 min.     

Tetrodotoxin Blockage of Sodium Conductance Increase in Lobster Giant Axons

Previous studies suggested that tetrodotoxin, a poison from the puffer fish, blocks conduction of nerve and muscle through its rather selective inhibition of the sodium-carrying mechanism. In order to verify this hypothesis, observations have been made of sodium and potassium currents in the lobster giant axons treated with tetrodotoxin by means of the sucrose-gap voltage-clamp technique. Tetrodotoxin at concentrations of 1 x 10^-7 to 5 x 10^-9 gm/ml blocked the action potential but had no effect on the resting potential. Partial or complete recovery might have occurred on washing with normal medium. The increase in sodium conductance normally occurring upon depolarization was very effectively suppressed when the action potential was blocked after tetrodotoxin, while the delayed increase in potassium conductance underwent no change. It is concluded that tetrodotoxin, at very low concentrations, blocks the action potential production through its selective inhibition of the sodium-carrying mechanism while keeping the potassium-carrying mechanism intact.     

Mechanical changes in squid giant axons associated with production of action potentials

The action potential of squid giant axons is accompanied by a quick and small swelling, about 0.5 nm in displacement of the surface, and about 1 dyne/cm² in pressure increase.     

Surface charges on membranes

Summary The equations of membrane potential developed by Kobatake and coworkers have been applied to the literature data on the resting membrane potential of the crayfish andMyxicola axons to derive values for the surface charge density present on the axon membranes. Some shortcomings of the method are briefly discussed. The value for the surface charge density derived for the squid axon membrane agreed with a similar value derived from measurements of shifts in Na and/or potassium conductance-voltage relations following changes in the concentration of calcium in the solutions bathing the axons.     

Temporally coherent organization and instabilities in squid giant axons

Instabilities and dynamic structure of the modified Hodgkin-Huxley equations (Adelman & FitzHugh, 1975) for sensitized axons were studied as a function of the sodium concentration in the external medium surrounding the axon. At the same time electrophysiological activities in squid giant axons were experimentally observed to confirm the results of the numerical calculation. It was found that the resting state of the axon was thermodynamically equivalent to a thermodynamic structure of an asymptotically stable equilibrium point. The state of spontaneous repetitive firing of action potentials corresponds to the dissipative structure with a stable limit cycle. The temporally coherent organization is realized through instability of the equilibrium point.     

The incorporation of microinjected 14C-amino acids into TCA insoluble fractions of the giant axon of the squid

Giant squid axons were microinjected with serine, valine and leucine-C¹⁴ under controlled electrophysiological conditions. These amino acids are incorporated into TCA insoluble fraction in the isolated axon. This incorporation is higher in the stimulated axons as compared to non-stimulated ones. By processing separately the axoplasm and axon sheath, it was found that the last one is responsible almost entirely for the observed incorporation. Through differential centrifugation of homogenates of microinjected axons was shown that the highest incorporation occurred in the 1500 × g sediment, which probably corresponds to membranes. The incorporation of amino acids in stimulated axons, is strongly inhibited by chloramphenicol and actinomycin D.     

Protein release from the internal surface of the squid giant axon membrane during excitation and potassium depolarization

The proteins in the perfusate collected from intracellularly perfused squid giant axons were analyzed after being labeled with radioactive ¹²⁵I-labeled Bolton-Hunter reagent. The rate of protein release into the perfusate was found to be increased by the following electrophysiological manipulations of the axons: (1) repetitive electrical stimulation at 60 Hz in axons perfused with normal potassium fluoride-containing solution or at 0.125 Hz in axons perfused with tetraethylammonium containing solution, (2) perfusion with 4-amino-pyridine solution which induces spontaneous electrical activity in the axon, and (3) depolarization of the axon induced by raising the external potassium concentration. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of the proteins released under these conditions yielded molecular weight profiles different from those of the extruded axoplasmic proteins. These observations indicate that there exists, in close association with the axonal membrane, a particular group of proteins, the solubility of which is readily affected by changes in the state of the membrane.     

Electrophoretic mobility of lambda phage HIND III and HAE III DNA fragments in agarose gels: a detailed study

The electrophoretic mobility (μ) of DNA fragments from λ phage and ΦX 174, split by restriction enzyme to molecular lengths from 3 × 10² to 2.36 × 10⁴ base pairs, has been investigated in 0.6–4% agarose gels at various field strengths, ionic strengths, and temperatures. As already observed, μ is seen to be very sensitive to the field, increasing with field strength. The sensitivity increases with the molecular length of the DNA and decreases at high gel concentration. Our data are in qualitative agreement with recent theoretical predictions that concern the influence of the electric field on electrophoretic mobility. Mobility data have been extrapolated to zero field. This enables a comparison of our experimental results with theoretical predictions on the dependence of μ on the molecular weight of the DNA fragments. Our data fit, quite closely, a reptation model, where the tube path is described as a semiflexible entity with a persistence length equal to the pore diameter. The influence of the agarose concentration and the ionic strength of the buffer on the two parameters of the model—intrinsic electrophoretic mobility (μ₀) and the number of base pairs per element of the tube (g)—are well described by the model. The temperature dependence of the electrophoretic mobility, together with the influence of the agarose concentration on μ₀, indicate that the hydrodynamic drag is the leading frictional force on the DNA molecules in the gel.