Electrical and morphological factors influencing the depolarizing after-potential in rat and lizard myelinated axons.

1. Intra-axonal recording and electron microscopy were applied to intramuscular myelinated axons in lizards and rats to investigate factors that influence the amplitude and time course of the depolarizing after-potential. 2. Depolarizing after-potentials in lizard axons had larger peak amplitudes and longer half-decay times than those recorded in rat axons (mean values 10 mV, 35 ms in lizard; 3 mV, 11 ms in rat). These differences were not due to differences in temperature, resting potential or action potential amplitude or duration. 3. For a given axon diameter, the myelin sheath in lizard fibres was thinner and had fewer wraps than in rat fibres. There was no significant difference in myelin periodicity. Calculations suggest that the thinner myelin sheath accounts for < 30% of the difference between depolarizing after-potential amplitudes recorded in lizard and rat axons. 4. Consistent with a passive charging model for the depolarizing after-potential, the half-time of the passive voltage transient following intra-axonal injection of current was shorter in rat than in lizard axons. 5. Aminopyridines prolonged the falling phase of the action potential and increased the amplitude of the depolarizing after-potential in both types of axon. 6. During repetitive stimulation the depolarizing after-potentials following successive action potentials exhibited little or no summation. Axonal input conductance in the interspike interval increased during the train. 7. These findings suggest that the amplitude and time course of the depolarizing after-potential are influenced not only by the passive properties of the axon and myelin sheath, but also by persisting activation of axolemmal K+ channels following action potentials.

Effects of tetraethylammonium on the depolarizing after-potential and passive properties of lizard myelinated axons.

1. Intra-axonal recordings were obtained from myelinated axons innervating a lizard skeletal muscle. 2. Bath application of tetraethylammonium (TEA, 1-10 mM) depolarized the resting potential, prolonged the action potential and increased the amplitude and duration of the ensuing passive depolarizing after-potential (DAP) in a dose-dependent and reversible manner. TEA increased the axonal input resistance and the slow time constant of the passive voltage response, not only in depolarized axons, but also in resting and hyperpolarized axons. The resting input resistance was tripled in 10 mM-TEA. 3. TEA's effects on the resting potential and action potential usually approached a steady state within 5 min, whereas TEA's effects on input resistance and on the amplitude and time course of the DAP increased progressively for 10-15 min or more, and persisted for 10-15 min after removal of TEA from the bath. 4. 4-Aminopyridine (4-AP, 1 mM), which prolonged the action potential by about the same extent as 10 mM-TEA, did not depolarize the resting potential or increase the resting input resistance, and produced a much smaller increase in DAP time course than 10 mM-TEA. Gallamine (1 mM) had effects more similar to those of TEA. 5. These results suggest that the resting input conductance and DAP time course in lizard motor axons are controlled in part by K+ channels that are blocked by TEA and gallamine, but not by 4-AP. The slow development of the TEA-induced increase in input resistance and DAP time course suggests that some of these channels are located in paranodal or internodal axolemma. 6. In TEA and gallamine additional depolarizing potentials were superimposed on the falling phase of the action potential and on the passive DAP. These superimposed potentials were abolished by 1 mM-Mn2+, and were probably caused by Ca2+ influx into motor terminals.

Effects of furosemide on the neuromuscular junction.

These studies investigated the direct effects of furosemide on neuromuscular transmission using the in vitro rat phrenic nerve diaphragm and the in vivo at soleus nerve muscle preparations. Furosemide (10(-6)-10(-4)m) reduced the concentration of d-tubocurarine required to achieve 50% twitch tension depression in the indirectly stimulated rat diaphragm. Intraarterial injection of furosemide had a biphasic effect on the cat neuromuscular junction. At low doses (0.1-10.0 micrograms/kg) the drug had a depressant effect, reduced the force of muscle contraction, prevented nerve and muscle responses to NaF and dibutryl cyclic AMP, and intensified the neuromuscular blockade produced by d-tubocurarine and succinylcholine. In contrast, in higher doses (1-4 mg/kg) furosemide produced stimulus-bound repetitive neural activity, initiated neural activity, increased the force of muscle contraction, enhanced nerve and muscle responses to NaF and dibutyrl cyclic AMP, and antagonized d-tubocurarine and succinylcholine blockades. Furosemide had no effect on denervated preparations. High doses of furosemide inhibit non-competitively both the high- and low-affinity forms of the enzyme cyclic AMP phosphodiesterase in both soluble and particulate fractions of cat sciatic nerve. Thus, furosemide has direct effects on neuromuscular transmission, but the direction of these effects is dose-dependent.

Effects of furosemide on neural mechanisms in Aplysia.

The effects of furosemide on action potentials and responses to several neurotransmitters have been studied in the neurons of Aplysia. Furosemide (10(-7) and 10(-3) M) does not visibly affect the normal action potential in R15 neurons. However, when TTX (30 microM) is used to block the sodium component in R15, the remaining spike (presumably the calcium component) is increased in amplitude in the presence of furosemide. Furosemide also alters transmitter-induced conductances. Furosemide greatly reduces the amplitude and shifts, in a depolarizing direction, the reversal potential of chloride-dependent responses to gamma-aminobutyric acid (GABA) and acetylcholine (ACh). This suggests that furosemide both blocks the chloride channel and inhibits a chloride pump. ACh-induced sodium responses were also reduced by furosemide but to a lesser extent than chloride responses. The potassium response to ACh and a voltage-dependent calcium response to serotonin were not altered. These results indicate that furosemide could alter synaptic responses both presynaptically by enhancement of calcium flux during the action potential and postsynaptically by blockade of chloride and sodium conductances.