Resistivity of axoplasm. I. Resistivity of extruded squid axoplasm

Six methods have given squid axoplasm resistivities of from 1.0 to 6.9 times seawater (X SW), so another was tried. A 100-mum platinized electrode was to be inserted from each end of an axion in iso-osmotic sucrose and impedance between them measured vs. separation. But observations that the resistance of axons in sucrose increased steadily ruled this out. Axoplasm from two or three axons was transferred to a glass capillary, 0.6 mm ID, and the 1-kHz series resistance and reactance were measured at electrode separations from 16 to 2 mm. The resistance was linear vs. distance, giving the resistivity, while the reactance was nearly constant, implying constant electrode contributions. Frequency runs from 10 Hz to 30 kHz at 10 mm gave electrode impedances of the form (jomega)-alpha, allowing 1-2% effects on the axoplasm resistivities. In nine experiments, one was discarded for cause, the range and average resistivities were, respectively, 1.2-1.6 and 1.4 times those of artificial seawater (19.7 omegacm at 24.4 degrees C). No single cause for the variability was apparent. These experiments essentially confirm the means and variations of two early experiments with intact axons and recent results with a single internal electrode to give overall resistivities of 1.4 +/- 0.2 X SW.     

Sodium efflux in Myxicola giant axons

Several properties of the Na pump in giant axons from the marine annelid Myxicola infundibulum have been determined in an attempt to characterize this preparation for membrane transport studies. Both NaO and KO activated the Na pump of normal microinjected Myxicola axons. In this preparation, the KO activation was less and the NaO activation much greater than that found in the squid giant axon. However, when the intracellular ATP:ADP ratio of the Myxicola axon was elevated by injection of an extraneous phosphagen system, the K sensitivity of Na efflux increased to the magnitude characteristic of squid axons and the activating effect of NaO disappeared. Several axons were injected with Na2SO4 in order to determine the effect of elevated Nai on the Na efflux. Increasing Nai enhanced a component of Na efflux which was insensitive to ouabain and dependent on [Ca] in Na-free (Li) seawater. After subtracting the CaO-dependent fraction, Na efflux was related linearly to [Na]i in all solutions except in K-free (Li) seawater, where it appeared to reach saturation at high [Na]i.     

Determination of the resistance in series with the membranes of giant axons.

Measurements of the resistance in series with the excitable membrane for giant axons of two different phylla (the squid Loligo pealii and the marine worm Myxicola infundibulum) were obtained. Efforts were made to take into account the errors introduced by the finite rise-time of the measuring apparatus. The series resistance value, obtained very quickly by the method described, may be used in setting the compensation potentiometer to offset this resistance in voltage-clamp measurements. Estimates of the resistance of the periaxonal tissue layer were made. Analyses were done on some of the problems involved in attempting to make an unambiguous determination of the series resistance.     

Survival of the K+ channel in axons externally and internally perfused with K+-free media.

In perfused squid giant axons, potassium channels irreversibly deteriorate when the internal K+ is removed and replaced by impermeant ions. Under the same conditions in perfused Myxicola giant axons, the K+ conductance is also irreversibly lost with a time constant of 10-15 min. In contrast, the K+ conductance in Myxicola giant axons dialyzed with impermeant ions and bathed in monovalent cation free solutions does not deteriorate, even over 5-6 h. Thus we suggest that washout of some internal component may be an important additional factor in the deterioration of K+ channels in perfused giant axons.     

Seasonal variation in conduction velocity of action potentials in squid giant axons

To determine whether the electrical properties of the squid giant axon are seasonally acclimated, action potentials, recorded at different temperatures, were compared between giant axons isolated from Loligo pealei caught in May, from relatively cold waters (approximately 10 degrees-12 degrees C), and in August, from relatively warm waters (approximately 20 degrees C). Parameters relating to the duration of the action potential (e.g., maximum rate of rise, maximum rate of fall, and duration at half-peak) did not change seasonally. The relationship between conduction velocity and temperature remained constant between seasons as well, in spite of the fact that May axons were significantly larger than August axons. When normalized to the fiber diameter, mean May conduction velocities were 83% of the August values at all temperatures tested, and analysis of the rise time of the action potential foot suggested that a change in the axoplasmic resistivity was responsible for this difference. Direct measurements of axoplasmic resistance further supported this hypothesis. Thus seasonal changes in the giant axon's size and resistivity are not consistent with compensatory thermal acclimation, but instead serve to maintain a constant relationship between conduction velocity and temperature.     

Identification of the subunit proteins of 10-nm neurofilaments isolated from axoplasm of squid and Myxicola giant axons

Neurofilaments were isolated from the axoplasm of the giant axons of Myxicola infundibulum and squid. The axoplasm was fractionated by discontinuous sucrose gradient centrifugation and gel filtration on Sepharose 4B. The fractions were monitored for neurofilaments by electron microscopy. When isolated in the presence of chelating agents, the neurofilaments of Myxicola are composed almost entirely of protein subunits with mol wt of 150,000 and 160,000. Squid neurofilaments contain two major proteins with mol wt of 200,000 and 60,000. These proteins are compared with other intermediate filament proteins which have been reported in the literature.     

The presence of transfer RNA in the axoplasm of the squid giant axon

Previous work has revealed that 4S RNA is the primary species of RNA in the axoplasm from the giant axons of the squid and Myxicola. This study shows that axoplasmic 4S RNA from the squid giant axon has the functional properties of tRNA. Axoplasmic RNA was charged with amino acids by aminoacyl-tRNA synthetases prepared from squid brain. The aminoacylation was prevented by incubating the RNA with RNase prior to running the reaction. The amino acid-RNA complex was labile at pH 9, which is characteristic of the acyl linkage between an amino acid and its tRNA. Aminoacyl-tRNA synthetase activity was also present in the axoplasm, primarily in the soluble fraction.     

K+ accumulation in the space between giant axon and Schwann cell in the squid Alloteuthis. Effects of changes in osmolarity.

In a train of impulses in squid giant axon, accumulation of extracellular potassium causes successive afterhyperpolarizations to be progressively less negative. In Loligo, Frankenhaeuser and Hodgkin had satisfactorily accounted for the characteristics of this effect with a model in which the axon is surrounded by a space, width theta, and a barrier of permeability P. In axons isolated from Alloteuthis, we found that the model fitted the observations quite well. Superfusing the axon with hypotonic artificial seawater (ASW) caused theta and P to decrease, and, conversely, hypertonic ASW caused them to increase: this would be the case if both the space and the pathway through the barrier were extracellular. In some cases, in normal ASW, the afterhyperpolarizations in a train decreased very little, less than 0.7 mV. In these extreme cases, theta was estimated to be 190 nm and P to be 7 x 10(-4) cm s-1, both several times the values of 30 nm and 6 x 10(-5) cm s-1 estimated by Frankenhaeuser and Hodgkin. We suggest that in vivo the periaxonal space may be considerably wider than that seen in conventionally fixed squid tissue.     

Determination of the resistance in series with the membranes of giant axons

Summary Measurements of the resistance in series with the excitable membrane for giant axons of two different phylla (the squidLoligo pealii and the marine wormMyxicola infundibulum) were obtained. Efforts were made to take into account the errors introduced by the finite rise-time of the measuring apparatus. The series resistance value, obtained very quickly by the method described, may be used in setting the compensation potentiometer to offset this resistance in voltage-clamp measurements. Estimates of the resistance of the periaxonal tissue layer were made. Analyses were done on some of the problems involved in attempting to make an unambiguous determination of the series resistance.     

Potassium homeostasis in the nervous system of cephalopods and crustacea

1. Previous work has shown that nerve activity is associated with a significant release of potassium in the vicinity of the axonal membrane. Several mechanisms are normally present which reduce K+ accumulation in the extra-axonal space. 2. In intact connectives of the crayfish, Procambarus clarkii, repetitive stimulation of the giant axons was associated with an apparent hyperpolarization measured by an interstitial microelectrode, which most probably corresponds to depolarization of the inner face of the perineurial cells by K+ ions leaving the axons. 3. In desheathed connectives of the crayfish, potassium accumulated during long depolarizing voltage-clamp pulses but cleared away very quickly at the end of the pulse. 4. In the small squid, Alloteuthis subulata, repetitive stimulation of giant axons in situ in fresh and well-perfused animals did not result in a large decrease in the positive after potential (undershoot), reflecting the absence of potassium accumulation. A similar absence of accumulation was observed in vitro for carefully and freshly dissected isolated axons from live squids. 5. In both cases, deterioration of the physiological state of the axon was accompanied by a significant potassium accumulation. Potassium accumulation could also be reversibly enhanced by decreasing the osmotic pressure of the bathing medium, whereas hyperosmotic solutions had the opposite effect. These results are compatible with the idea that Schwann cells around the axon play a key role in K+ homeostasis. 6. Experiments on giant axons of the large squid species, Loligo forbesi confirmed the observations made on Alloteuthis in that fresh preparations exhibited little potassium accumulation. Under voltage-clamp conditions, 10 ms depolarizing pulses to various potential levels did not induce any accumulation in these preparations as reflected by the outward tail current. Large accumulation was observed in older axons under similar experimental conditions. 7. A large peri-axonal space associated with healthy glial cells appears to be a prerequisite for efficient K+ homeostasis in both crayfish and squid. Other mechanisms involving specific transport mechanisms across axonal and glial membranes are also likely to be involved.     

The influence of external cations and membrane potential on Ca- activated Na efflux in Myxicola giant axons

In microinjected Myxicola giant axons with elevated [Na]i, Na efflux was sensitive to Cao under some conditions. In Li seawater, sensitivity to Cao was high whereas in Na seawater, sensitivity to Cao was observed only upon elevation of [Ca]o above the normal value. In choline seawater, the sensitivity of Na efflux to Cao was less than that observed in Li seawater whereas Mg seawater failed to support any detectable Cao-sensitive Na efflux. Addition of Na to Li seawater was inhibitory to Cao-sensitive Na efflux, the extent of inhibition increasing with rising values of [Na]o. The presence of 20 mM K in Li seawater resulted in about a threefold increase in the Cao-activated Na efflux. Experiments in which the membrane potential, Vm, was varied or held constant when [K]o was changed showed that the augmentation of Ca- activated Na efflux by Ko was not due to changes in Vm but resulted from a direct action of K on activation by Ca. The same experimental conditions that favored a large component of Cao-activated Na efflux also caused a large increase in Ca influx. Measurements of Ca influx in the presence of 20 mM K and comparison with values of Ca-activated Na efflux suggest that the Na:Ca coupling ratio may be altered by increasing external [K]o. Overall, the results suggest that the Cao- activated Na efflux in Myxicola giant axons requires the presence of an external monovalent cation and that the order of effectiveness at a total monovalent cation concentration of 430 mM is K + Li greater than Li greater than Choline greater than Na.     

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.     

An optical determination of the series resistance in Loligo

The resistance in series with the membrane capacitance in the giant axon of the squid Loligo pealei was measured using potentiometric probes that exhibit absorbance changes proportional to the voltage across the plasma membrane proper. The method relies upon the fact that a voltage drop across the series resistance produces a deviation in the true transmembrane voltage from that imposed by a voltage clamp. Optical measurement of the true transmembrane potential, together with electrical measurement of the ionic current, permits the immediate determination of the series resistance by Ohm's law. An alternative method monitored the amount of electronic series resistance compensation required to force the optical signal to match the shape of the reference potential. The value of the series resistance measured in artificial seawater was 3.78 +/- 0.95 omega X cm2. The estimated value of the contribution of the Schwann cell layer to the series resistance was 2.57 +/- 0.89 omega X cm2.     

Potassium current kinetics in Myxicola axons. Effects of conditioning prepulses

In Myxicola giant axons the time constants for activation of the potassium conductance (GK) after prepulses less depolarized than a test pulse are comparable to the time constants for turn off of GK after prepulses more depolarized than the same test pulse. The absolute magnitude of the steady-state level of GK is also independent of prepulse amplitude in Myxicola. The results are contrasted with recent observations on voltage-clamped frog nodes.     

Inhibition of potassium conductance with external tetraethylammonium ion in Myxicola giant axons.

In voltage clamp experiments, externally applied tetraethylammonium ion (TEA) was found to have minimal effects on transient sodium currents and to suppress steady-state potassium currents of Myxicola giant axons by causing a specific decrease in the maximum potassium conductance gK. The dose-response curve suggests a one-to-one stoichiometry for TEA-receptor binding with an apparent dissociation constant on 24 mM. The suppression of IK is essentially reversible. Experiments performed on high external potassium ion concentrations indicate that both outward and inward IK were blocked by external TEA. The results thus suggest the presence of TEA receptors on the outer surface of Myxicola axonal membrane similar to those reported in the frog node.     

The function of the proximal synapses of the squid stellate ganglion

In the oxygenated excised squid (Loligo pealii) stellate ganglion preparation one can produce excitation of the stellar giant axons by stimulating the second largest (accessory fiber, Young, 1939) or other smaller preganglionic giant axons. Impulse transmission is believed to occur at the proximal synapses of the stellar giant axons rather than the distal (giant) synapses which are excited by the largest giant preaxon. Proximal synaptic transmission is more readily depressed by hypoxia and can be fatigued independently of, and with fewer impulses than, the giant synapses. Intracellular recording from the last stellar axon at its inflection in the ganglion reveals both proximal and distal excitatory postsynaptic potentials EPSP's). The synaptic delay, temporal form of the EPSP, and depolarization for spike initiation were similar for both synapses. If the proximal EPSP occurs shortly after excitation by the giant synapse it reduces the undershoot and adds to the falling phase of the spike. If it occurs later it can produce a second spike. Parallel results were obtained when the proximal EPSP's arrived earlier than the EPSP of the giant synapse. In fatigued preparations it was possible to sum distal and proximal or two proximal EPSP's and achieve spike excitation.     

Significant potassium ion accumulation at the external surface of Myxicola giant axons

Potassium accumulation associated with outward membrane potassium current was investigated experimentally in Myxicola giant axon. During prolonged voltage-clamp pulses to positive transmembrane potentials, the K+ equilibrium potential may approach zero mV, suggesting massive K+ accumulation outside the axonal membrane to concentrations many-fold higher than those in the bathing medium. The potassium accumulation can be satisfactorily described by a three-compartment model, consisting of the nerve fiber, a restricted physiological periaxonal space and the bulk solution. The average thickness, theta, of the periaxonal space is calculated as 177 +/- 59 A, i.e., comparable to that in the squid, while the permeability coefficient of the external barrier, PKs, was calculated to be (1.4 +/- 0.4) X 10(-4) cm/s. These conclusions are well supported by morphological study.     

Potassium flux ratio in voltage-clamped squid giant axons

The potassium flux ratio across the axolemma of internally perfused, voltage-clamped giant axons of Loligo pealei has been evaluated at various membrane potentials and internal potassium concentrations ([K]i). Four different methods were used: (a) independent measurement of one-way influx and efflux of 42K; (b) simultaneous measurement of net K current (IK) and 42K influx; (c) simultaneous measurement of IK and 42K efflux; and (d) measurement of potassium conductance and 42K influx at the potassium equilibrium potential. The reliability of each of these methods is discussed. The average value of the exponent n' in the Hodgkin-Keynes equation ranged from 1.5 at -4mV and 200 mM [K]i to 3.3 at -38 mV and 350 mM [K]i and appeared to be a function of membrane potential and possibly of [K]i. It is concluded that the potassium channel of squid giant axon is a multi-ion, single-file pore with three or more sites.     

Binding of tetrodotoxin to squid nerve fibers. Two kinds of receptors?

The effect of tetrodotoxin on the sodium currents of the squid (Doryteuthis plei and Sepioteuthis sepiodea) giant axons was studied under potential control conditions. The axons were immersed in artificial seawater at 21 degrees C and pH 7.5. When the effect of the toxin is studied in concentrations ranging from 0.1 to 50 nM the Eadie- Haldane plot is not a straight line and indicates that there are two populations of sodium channels open during activity. 19.0 +/- 4.7% of the channels are accociated to receptors with an apparent dissociation constant of 0.11 +/- 0.05 nM and 84.0 +/- 4.1% of the channels are related to receptors having an affinity constant of 4.90 +/- 0.49 nM (nine nerves).     

Binding of radioactively labeled saxitoxin to the squid giant axon

Summary The binding of saxitoxin, a specific inhibitor of the sodium conductance in excitable membranes, has been measured in giant axons from the squid,Loligo pealei. Binding was studied by labeling saxitoxin with tritium, using a solvent-exchange technique, and measuring the toxin uptake by liquid scintillation counting. Total toxin binding is the sum of a saturable, hyperbolic binding component, with a dissociation constant at 2–4°C of 4.3±1.7nm (meanse), and a linear, nonsaturable component. The density of saturable binding sites is 166±20.4 m^–2. From this density and published values of the maximum sodium conductance, the conductance per toxin site is estimated to be about 7 pS, assuming sequential activation and inactivation processes (F. Bezanilla & C.M. Armstrong, 1977,J. Gen. Physiol. 70: 549). This single site conductance value of 7 pS is in close agreement with estimates of the conductance of one open sodium channel from measurements of gating currents and of noise on squid giant axons, and is consistent with the hypothesis that one saxitoxin molecule binds to one sodium channel.