Rate constants associated with changes in sodium conductance in axons perfused with sodium fluoride

1. Membrane currents during step depolarizations were measured in axons which were perfused with 300 mM-NaF and placed in K-free artificial sea-water, -0·3-4° C. The Na conductance was fitted by the modified Hodgkin—Huxley model, g_Na = ḡ_Nam³(h₁ + h₂). Changes in h₁ and h₂ were assumed to follow [Formula: see text] where x represents the inactive state.2. The rate constants and steady-state values for m were in agreement with the Hodgkin—Huxley equations except that the experimental relationship of m_∞³ against V was shifted 10-15 mV in the negative direction. This discrepancy, which was not found in an experiment with choline sea-water, can be explained on the basis of a resistance in series with the membrane between the voltage measuring electrodes.3. At 0° C the rate constants (in msec^-1) associated with changes in h₁ and h₂ were fitted using the following equations: β_h1 = 0·5/{exp [- (V + 32)/10] + D₁exp (- V/V₁)}, α_h2 = pexp (V/V₂), β_h2 = pexp (V/V₂ - V/23·5) + pD₂, with the condition that at 0 mV, (α_h2 + β_h2) = p(D₂ + 2) = 0·55 msec^-1. The experiments gave average values D₁ = 3·6, V₁ = 240 mV, p = 0·08 msec^-1 and V₂ = 70 mV. The average value of ḡ_Na was 66 mmho/cm².4. At negative voltages where m_∞³ against V is steep, the points for β_h1 and α_h2/β_h2 from axons in Na sea-water were not fitted well by the above equations whereas data from an axon in choline sea-water were. These discrepancies can be explained on the basis of a series resistance.5. Measurements made at 16-17° C indicated that ḡ_Na has a Q₁₀ of 1·6, τ_m^-1 a Q₁₀ of 2·8 and β_h1 a Q₁₀ of 3·5. The ratio α_h2/β_h2 was decreased relative to the value at 0° C and could be fitted by using Q₁₀ = 0·6.6. Measurements made with 250 mM-NaF + 50 mM-KF inside gave rate constants which were very similar to those obtained with 300 mM-NaF. Perfusion with 300 mM-KF appeared to double the value of β_h1, relative to that obtained with 300 mM-NaF, and to reduce α_h2/β_h2 by about half.7. The voltage dependence of α_h2 makes it likely that following depolarization recovery from the inactive state x occurs via x → h₁ rather than x → h₂ → h₁.     

Evidence for two types of sodium conductance in axons perfused with sodium fluoride solution

1. Voltage clamp experiments were carried out on squid giant axons internally perfused with 300 mM-NaF + sucrose. K-free artificial sea-water, -0.3 to 3.5 degrees C, was used externally.2. Membrane currents were corrected for capacitative and leakage components, and the resulting Na current was converted to Na conductance, g(Na). An attempt was made to fit changes in g(Na) according to the Hodgkin-Huxley model, namely [Formula: see text].According to the model g(Na) is a constant, m(infinity) and h(infinity) are steady-state values which depend only on voltage, tau(m) (-1) and tau(h) (-1) are rate constants which also are functions only of voltage.3. Stepwise depolarizations from the holding potential (-67 to -83 mV) to a potential which varied from -10 to +63 mV resulted in an exponential decline of h from its initial level to a final, non-zero level. If the test depolarization was preceded by a positive prepulse (duration, 19-105 msec; voltage, -6 to 94 mV) the rate constant for h, tau(h) (-1), was increased roughly threefold with practically no change in the final level.4. The steady-state level of h was studied by using prepulses of varying amplitude followed by a test depolarization. In one such experiment a value of 0.34 was obtained for a 105 msec prepulse to -49 mV. The same value for the steady level of h was obtained from analysing a record taken at +52 mV. If the potential was switched from -49 to +52 mV there was a transient increase in g(Na) although h(infinity) had the same value at these two potentials.5. Recovery from depolarization was studied by repolarizing the fibre for varying lengths of time, then applying a test depolarization. If the first depolarization was strongly positive (for example, 70 mV), so that the steady level of h was large (0.39), the currents associated with the test pulse could not be fitted on the basis of an exponential increase in h during the recovery period. Rather, the results suggested that on repolarization h rapidly decreased initially, then slowly increased.6. These results can be explained by assuming that h is given by the sum of two components, h(1) and h(2). Changes are represented kinetically by h(1) right harpoon over left harpoon x right harpoon over left harpoon h(2), where x signifies the inactive state. The distribution is shifted to the left at negative potentials and to the right for positive ones. The resulting Na conductance is comprised of two types: the first type, g(Na)m(3)h(1), is similar to the Hodgkin-Huxley system and underlines the usual transient increase in g(Na) associated with depolarization; the second type, g(Na)m(3)h(2), is maintained with depolarization and gives rise to a steady level of g(Na).     

Slow changes in potassium permeability in skeletal muscle

1. Voltage clamp experiments on sartorius muscle fibres at 3 degrees C showed that the potassium current is divisible into three components, namely:(a) Current in the delayed rectifier channel, which reached a maximum in about 0.1 sec at -30 mV, and declined with a time constant of about 4 msec when the fibre was repolarized to -100 mV; this component had an approximately linear instantaneous current-voltage relation and an equilibrium potential E(1) at 10-15 mV positive to the resting potential.(b) A slow component which reached a maximum in about 3 sec at -30 mV, and declined with a time constant of about 0.5 sec when the fibre was repolarized to -100 mV; this component had an approximately linear instantaneous current-voltage relation and a mean equilibrium potential E(2) at -83 mV in fibres where E(1) averaged -75 mV.(c) Current in the inward rectifier channel which decreased with a time constant of about 0.25 sec when the fibre was hyperpolarized to -150 mV. This component had an equilibrium potential close to the resting potential and an instantaneous current-voltage relation which was that of an inward rectifier.2. The general characteristics of the late after-potential in muscles in hypertonic solutions at 3 degrees C are consistent with those of the slow conductance change. The sign of the late after-potentials was reversed by depolarizing below -80 mV.3. The decline of current during a maintained hyperpolarization cannot be attributed solely to a decrease in tubular potassium concentration, since there may be a large decrease in current without much alteration of equilibrium potential. The negative slope conductance often seen at -150 mV is also difficult to reconcile with the tubular depletion hypothesis.4. Replacement of 10 mM-K by 10 mM-Rb abolished inward rectification but had less effect on the fast and slow components of the potassium conductance.     

Effect of stimulation and hyperpolarization on non-electrolyte and sodium permeability in perfused axons of squid

1. The permeability for micro-injected [(3)H]ethylene glycol was measured in resting state and during stimulation at 100/sec in squid giant axons. No detectable changes during electrical activity were observed.2. The influxes of urethane, tritiated water, ethylene glycol, urea and sodium were measured in internally perfused squid axons. Ethylene glycol and urea influxes were determined simultaneously with sodium influxes. The electrical stimulation of the fibre produced an increase in the influx of sodium but did not alter the influxes of the non-electrolytes listed above.3. Experiments were done with the combined voltage clamp-perfusion technique. The influxes of ethylene glycol and sodium were simultaneously measured in resting state and during maximum sodium current under stimulation at 10/sec. The influx of sodium increased in these conditions but the influx of ethylene glycol remained constant. In some experiments, the fibre was hyperpolarized to 10 or 20 mV, above the resting potential and the influxes of ethylene glycol and sodium were measured. The sodium influx decreased to 60% at 20 mV above the resting potential whereas the influx of ethylene glycol remained constant.4. These results indicate that in the giant axons of the squid Dosidicus gigas, sodium and non-electrolytes fluxes are not coupled.     

Presynaptic Na/Ca action potentials in unmyelinated axons of olfactory cortex

(1) Pial surface slices of guinea-pig olfactory cortex were cut to have a thickness of 150 m. Action potentials were recorded from the sectioned ends of the unmyelinated afferent axons originating from the lateral olfactory tract (LOT). These potentials were prolonged by the K-channel blocker 3,4-diaminopyridine (0.1 mmol/l) and further lengthened by tetraethylammonium (10 mmol/l). The action potential was also greatly prolonged by partly replacing the K⁺ in the bathing solution by Cs⁺. (2) These prolonged action potentials were shortened by Cd²⁺; Gd³⁺ (gadolinium); Ni²⁺; Mn²⁺; Co²⁺, in order of potency. The residual early component of the action potential was tetrodotoxi (TTX) sensitive. In contrast, the LOT action potential wal little affected by Ca-channel blockade. (3) Organic Ca-channel blockers either had no effect (0.05 mmol/l nifedipine), or depressed the early and later phases of the prolonged action potential equally (0.05–0.5 mmol/l verapamil or 0.05–0.2 mmol/l diltiazem). (4) a propagated action potential was also obtained in solution containing TTX and low Na⁺. This potential was supported by Ca²⁺, Sr²⁺ or Ba²⁺ and completely suppressed by Cd²⁺. (5) The later parts of the action potential, after K-channel blockade, had a pharmacological sensitivity towards Ca-channel blockers matching that of synaptic transmission. This suggests the falling phase of the action potential is caused by charge carrier (mainly Ca²⁺) passing through Ca-channels that have similar properties to, or are the same as those which open prior to transmitter release.     

Slow integration leads to persistent action potential firing in distal axons of coupled interneurons

The conventional view of neurons is that synaptic inputs are integrated on a timescale of milliseconds to seconds in the dendrites, with action potential initiation occurring in the axon initial segment. We found a much slower form of integration that leads to action potential initiation in the distal axon, well beyond the initial segment. In a subset of rodent hippocampal and neocortical interneurons, hundreds of spikes, evoked over minutes, resulted in persistent firing that lasted for a similar duration. Although axonal action potential firing was required to trigger persistent firing, somatic depolarization was not. In paired recordings, persistent firing was not restricted to the stimulated neuron; it could also be produced in the unstimulated cell. Thus, these interneurons can slowly integrate spiking, share the output across a coupled network of axons and respond with persistent firing even in the absence of input to the soma or dendrites.     

Isoproterenol-induced myocardial necrosis and membrane permeability alterations in the isolated perfused rabbit heart

The quantitative effects of isoproterenol on myocardial necrosis and its effect on membrane permeability using the tracer horseradish peroxidase were studied in isolated, Langendorff-perfused rabbit hearts. The hearts were perfused at a column height of 70 cm with a modified Krebs — Henseleit bicarbonate buffer gassed with 95% O₂: 5% CO₂. Isoproterenol was infused at constant rates with separate molar concentrations of 10^?6, 10^?5, 10^?4, and 10^?3M. Horseradish peroxidase was infused manually for 3 min at the end of the experiment and reacted with Hanker — Yates reagent. Two predominant forms of “contraction band” lesions were observed: small “paradiscal” lesions involving clumping of a few sarcomeres adjacent to the intercalated disc and “holocytic” contraction band lesions involving clumping of myofilaments throughout the myocardial cell. Each dose of isoproterenol produced significantly increased numbers of both types of lesions. Incubation of tissue for horseradish peroxidase produced significant accumulation within myocardial cells damaged with “contraction band” lesions. This accumulation was occasionally seen in “normal” appearing cells with isoproterenol, but not in control hearts. The results support the use of quantitation of lesions as an index of severity of isoproterenol cardiotoxicity and documented the early involvement of membrane permeability alterations in the pathogenesis of these “contraction band” lesions.     

Dorsal horn potentials and current source densities evoked by single action potentials in single slowly adapting type I axons

The slow-wave response of cat dorsal horn, elicited by single action potentials in single slowly adapting type I (SAI) axons, was mapped by averaging the slow wave recorded from each locus in a rectangular array of recording loci in the transverse plane. Current source-density (CSD) waveforms were computed from these averages. Evoked potentials always included N-waves, with mean latency = 4.9 ms, rise time (base line to peak) = 2.3 ms, and duration (base line-peak-base line) = 13.1 ms. In some planes, the N-wave was followed by a longer P-wave. The N-wave timing corresponded to previously described excitatory postsynaptic potentials (EPSPs) recorded intracellularly and excitatory discharges recorded extracellularly from single units evoked by single SAI spikes. The P-wave timing corresponded to a previously described postexcitatory suppression of SAI spike-evoked EPSPs and discharges following single conditioning SAI action potentials. The current sink during the N-wave had the following properties: It occurred in a column of tissue, perpendicular to the laminar borders, less than 600 microns wide; this is similar to the terminal domain of a single SAI collateral. It remains stationary during the N-wave, indicating that the excited population of dorsal horn elements does not spread or shift within the transverse plane during the response. It is somatotopically organized in the mediolateral dimension, in a manner similar to the somatotopic organization of primary afferent terminations and of dorsal horn cell receptive fields.     

Potentiometric measurement of membrane action potentials in frog muscle fibres

1. A potentiometric method for recording membrane action potentials from small segments of isolated skeletal muscle fibres has been described.2. The value of the method in muscle physiology has been appraised by recording from fast and slow skeletal muscle fibres of the frog under a variety of conditions.3. Artifacts associated with this method have been described and their sources investigated.     

Simultaneous measurements of magnesium, calcium and sodium influxes in perfused squid giant axons under membrane potential control.

1. Giant axons from the squids Dosidicus gigas, Loligo forbesi and Loligo vulgaris were internally perfused with 550 or 275 mM KF plus sucrose and bathed in artificial sea water containing 45Ca, 28Mg or mixtures of 45Ca-28Mg or 45Ca-22Na. Resting influxes and extra influxes during voltage-clamp pulses were measured by collecting and counting the internal perfusate. 2. For Dosidicus axons in 10 mM-CaCl2 the resting influx of calcium was 0-016 +/- 0-007 p-mole/cm2 sec and a linear function of external concentration. For two experiments in 10 and 84-7 mM-CaCl2, 100 nM tetrodotoxin had no effect. Resting calcium influx in 10 mM-CaCl2 was 0-017 +/- 0-013 p-mole/cm2 sec for Loligo axons. 3. With 55 mM-MgCl2 outside the average resting magnesium influx was 0-124 +/- 0-080 p-mole/cm2 sec for Loligo axons. Discarding one aberrant point the value is 0-105 +/- 0-046 which is not significantly different from the resting calcium influx for Dosidicus fibres in 55 mM-CaCl2, given as 0-094 p-mole/cm2 sec by the regression line shown in Fig. 1. In two experiments 150 nM tetrodotoxin had no effect. 4. With 430 mM-NaCl outside 100 nM tetrodotoxin reduced the average resting influx of sodium in Dosidicus axon from 27-7 +/- 4-5 to 25-1 +/- 6-2 p-mole/cm2 sec and for Loligo fibres in 460 mM-NaCl from 50-5 +/- 4 to 20 +/- 8 p-mole/cm2 sec. 5. Using depolarizing pulses of various durations, the extra calcium influx occurred in two phases. The early phase was eliminated by external application of tetrodotoxin. The results of analysis are consistent with, but do not rigorously demonstrate, the conclusion that the tetrodotoxin sensitive calcium entry is flowing through the normal sodium channels (cf. Baker, Hodgkin & Ridgway, 1971). 6. Measurements of extra influxes using 22Na and 45Ca simultaneously indicate that the time courses of tetrodotoxin sensitive calcium and sodium entry are similar but not necessarily identical. It is very doubtful that any significant calcium entry occurs before the sodium or is involved in the activation of the sodium system. 7. These measurements confirm for Loligo, as previously shown for Dosidicus axons, that the magnitude and time course of the sodium entry during a depolarizing pulse deduced from electrical measurements is the same as that measured with 22Na. 8. Using 28Mg, or mixtures of 45Ca and 28Mg, we observed a single phase of magnesium entry which was insensitive to external tetrodotoxin or internal tetraethyl ammonium. The magnitude of the magnesium influx was considerably greater than the calcium extra entry and large enough to have been detected in the experiments of Meves & Vogel (1973) if it represented current. 9. We suggest the possibility that the calcium and magnesium extra influxes, after external treatment with tetrodotoxin, during a depolarizing pulse, do not contribute to the measured current.