Alternating current threshold excitation of space-clamped squid giant axons was measured as a function of frequency, external calcium concentration, temperature (from 10° to 35°C), and hyper- and depolarizing steps. In normal axons there is usually an optimum frequency at about 120 Hz, at which the threshold is a minimum. The threshold rises at both lower and higher frequencies to give a resonance curve. Low calcium causes an increase in optimum frequency, a decrease in current threshold, and an increase in sharpness of tuning in both real axons and axons computed according to the Hodgkin-Huxley formulation; high calcium causes opposite effects. An increase in temperature causes an increase of optimum frequency, an increase in sharpness of tuning, and an increase in threshold current in both real and computed axons. The Q 10 for the effect of temperature upon optimum frequency is 1.8 in real and computed axons at moderate temperatures. Hyperpolarization causes ( a ) a decrease in optimum frequency, ( b ) a decrease in sharpness of tuning, and ( c ) an increase in threshold. Depolarization causes opposite effects.

Space-clamped squid axons treated with low calcium and computed Hodgkin-Huxley (HH) axons were stimulated by steps of superthreshold current from 101 to 400% of the rheobasic value over a temperature range of 5–27°C. The natural frequency of sustained repetitive firing of real and computed axons depended weakly upon stimulus intensity and strongly upon temperature, with a Q 10 of 2.7 (experimental) and 2.6 (computed). For real axons, but not the computed axon, the intervals between the first two spikes were shorter than between subsequent spikes. Constant spike frequencies from 75 Hz at low intensities and temperatures to 330 Hz at high intensities and temperatures were soon achieved. Subthreshold and superthreshold responses were sometimes intermixed in a train of responses from a real axon responding to a constant step of current, but not predicted by HH. The time interval following a spike was always longer than that following a subthreshold oscillation in slightly decalcified real axons, as Huxley and FitzHugh also found for computed axons. There was a bias toward spikes at the beginning of the train and toward subthreshold responses later on. Some repeated patterns were found, every second, third, or fourth response being a spike. Neither the HH equations nor the computed or experimental threshold behaviors show a critical temperature to support a membrane phase transition.

Accommodation and excitation in space-clamped squid axons were studied with the double sucrose gap technique, using linear current ramps, short (50 µsec) square wave pulses, and rheobasic square wave pulses as stimuli. The temperature was varied from 5° to 35°C. Experimental results showed a Q 10 for accommodation which was 44% higher than that for excitation. Yet calculations on the basis of the Hodgkin-Huxley equations predict equal Q 10 's for excitation and accommodation. Although the Hodgkin-Huxley equations are spectacularly successful for so many nerve phenomena, the differences between calculations of accommodation and these experiments, which were designed to test the equations, show that the equations need modification in this area.

The lowering of external sodium raised both the constant quantity threshold, Q o , and the rheobase, I o , in both real space-clamped squid axons and the theoretical axon as computed on the basis of the standard Hodgkin-Huxley equations. In both real and theoretical axons the minimum intensity for excitability for short pulses, which occurs at about 15°C, was still present when low sodium replaced seawater. Low sodium did not affect the temperature dependence of the strength-duration relationship in the range, 5° to 25°C. The excitability of tetrodotoxin-treated real axons was found to be more temperature-dependent than that of normal real axons. Also the data on dosage-response to TTX of real axons fit the dose-response relationship of a hypothetical system in which one TTX ion binds reversibly to its receptor to produce a fraction of the inhibitory effect, the curve being identical to a simple adsorption isotherm. The Hodgkin-Huxley equations describe the broad outline of events occurring during excitation quite well.

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 Q 10 of 1.15. The average threshold current density at rheobase was 12 µa/cm 2 at 20°C with a Q 10 of 2.35 compared to 2.3 obtained previously. At short times the threshold charge was 1.5·10 -8 coul/cm 2 . 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.

The effect of temperature on the potential and current thresholds of the squid giant axon membrane was measured with gross external electrodes. A central segment of the axon, 0.8 mm long and in sea water, was isolated by flowing low conductance, isoosmotic sucrose solution on each side; both ends were depolarized in isoosmotic KCl. Measured biphasic square wave currents at five cycles per second were applied between one end of the nerve and the membrane of the central segment. The membrane potential was recorded between the central sea water and the other depolarized end. The recorded potentials are developed only across the membrane impedance. Threshold current values ranged from 3.2 µa at 267deg;C to 1 µa at 7.5°C. Threshold potential values ranged from 50 mv at 26°C to 6 mv at 7.5°C. The mean Q 10 of threshold current was 2.3 (SD = 0.2), while the Q 10 for threshold potentials was 2.0 (SD = 0.1).

The slow tonic responses of the anterior byssus retractor of Mytilus edulis to rapid cooling were investigated by simultaneously recording tension and resting potential changes after soaking the muscle in banthine, a powerful neuromuscular blocking agent. The quantitative relations between the amount of cooling and the amount of associated depolarization necessary for contraction at various concentrations of potentiating potassium can be expressed in a family of curves. The plateaus of the curves for sea water and for potassium-free sea water were beneath the depolarization value necessary for contraction, so that it is clear that no amount of cooling with sea water alone or with potassium-free sea water would ever be effective. When the muscle is treated with subthreshold amounts of potassium and rapidly cooled in various concentrations of sodium ion and calcium ion, respectively, the sodium and calcium do not affect the amount of depolarization. Acetylcholine, in subthreshold amounts, has a potentiating effect, but, unlike potassium and cooling, acts through the nervous apparatus. Mytilus muscle will respond to cooling with tonic contraction whenever a critical threshold amount of depolarization is achieved. Cooling alone cannot trigger the contraction since it cannot bring about sufficient depolarization. Cooling can result in contraction, however, if used in conjunction with some other subthreshold depolarizing agent. Cooling affects the contractile mechanism by first causing membrane breakdown and depolarization.

Electrical rectification was demonstrated in whole sartorius muscle and sciatic nerve of Rana pipiens and also in the single giant nerve fiber of the northern squid, Ommastrephes illecibrossus . It is probably a property of the plasma membrane. Rectification decreases reversibly under the influence of increased concentrations of the potassium ion and with chloroform, veratrine sulfate and isoamyl carbamate. No effect was found with lack of calcium, excess calcium, or barium chloride. Decrease in rectification is invariably accompanied by simultaneous decrease in resting potential. A proposed explanation of the mechanism of rectification is discussed. Rectification in a living membrane, viz . a change in resistance with change in direction of current flow, may possibly be explained in terms of a change in the concentration of potassium ions in the membrane.

1. The alkaline earths, Ba, Sr, Ca, and Mg, in isotonic solutions of their chlorides, have, in general, no effect upon the resting potential of non-medullated spider crab nerve. 2. Ba, Sr, and Ca can, however, prevent the depressing action of K upon the resting potential. The order of effectiveness of these ions in this regard is the following: Ba > Sr > Ca. 3. Ba, Sr, Ca, and Mg oppose the depressing action of veratrine sulfate upon the resting potential. The order of effectiveness is Ba > Sr > Ca > Mg. The relation between drop in potential caused by veratrine sulfate and the logarithm of the veratrine sulfate concentration is a linear one. 4. The action of various other organic ions and molecules which depress the resting potential: saponin, amyl urethane, chloral hydrate, and Na salicylate is neutralized by Ba. 5. Hypertonic sea water solutions do not affect the resting potential. Also, preliminary experiments indicate that the nerves do not shrink in hypertonic solutions although they swell in hypotonic sea water. 6. The alkaline earths depress excitability reversibly. The various organic agents which depress the resting potential also depress excitability, in most cases, reversibly, but the concentrations necessary to depress excitability are much smaller than those necessary to depress the resting potential. 7. The relation of these findings to theories put forward as possible explanations of resting potential phenomena is considered.

1. The effect of certain inorganic cations upon the electrical impedance of the sartorius muscle of the frog was investigated. While Na, K, and Mg have little effect upon the resistance of muscle, Ba and Ca cause it to fall. The use of physiologically "unbalanced" salt solution does not in itself seem to affect muscle impedance. 2. The time course of the effect upon muscle impedance of the penetration of substances into the intercellular spaces was studied by treating the muscle with sugar solutions. Half of the effect is over in three-quarters of a minute when the sugar solution is permitted to circulate past both sides of the muscle. This sets an upper limit for the time necessary for inorganic cations and organic narcotics to reach the cell surfaces. The action of inorganic cations and organic narcotics upon muscle is slow compared to the time necessary for them to reach the scene of action. The penetration of the sugar solutions into the intercellular spaces of muscle was found to follow the well known diffusion law, the amount diffusing in being proportional to the square root of the time. Average values of 77.7 per cent for ρ, the volume concentration of fibers; 231 ohms specific resistance for r 2 , the resistance of the interior of the fibers; and 71.0° for θ, the phase angle of the impedance locus, were obtained for the muscle in Ringer's solution. How these values change when the muscle is placed in various concentrations of sugar was also studied. 3. The action of a number of organic narcotics upon muscle was studied. All decrease 1000 cycle resistance if the concentration is sufficiently high. A detailed analysis of the action of the narcotic, iso-amyl carbamate, was made, and it was noted that low concentrations increase resistance while higher concentrations decrease it. By investigating the effect of narcotics upon muscle impedance over a wide frequency range, it was found that during narcosis the resistance of the fiber membranes first increases and then decreases, and, if the drug is present in sufficiently great concentration, membrane resistance may completely disappear. Membrane capacity is only very slightly affected.