Alexander Mauro

Curarized cutaneous pectoris nerve-muscle preparations from frogs were stimulated at 10/s or at 2/s for periods ranging from 20 min to 4 h. End plate potential were recorded intracellularly and used to estimate the quantity of transmitter secreted during the period of stimulation. At the ends of the periods of stimulation the preparations were either fixed for electron microscopy or treated with black widow spider venom to determine the quantities of transmitter remainind in the terminal. Horseradish peroxidase or dextran was added to the bathing solution and used as a tracer to detect the formation of vesicles from the axolemma. During 4 h of stimulation at 2/s many new vesicles were formed from the axolemma and the quantity of transmitter secreted was several times greater than the quantity in the initial store. After this period of stimulation, the terminals were severely depleted of transmitter, but not of vesicles, and their general morphological organization was normal. During 20 min of stimulation at 10/s the nerve terminals swelled and were severely depleted both of vesicles and of transmitter. During a subsequent hour of rest the changes in morphology were largely reversed, many new vesicles were formed from the axolemma and the stores of transmitter were partially replenished. These results suggest (a) that synaptic vesicles fuse with, and re-form from, the membrane of the nerve terminal during and after stimulation and (b), that the re-formed vesicles can store and release transmitter.

The oscillatory behavior of the cephalopod giant axons in response to an applied current has been established by previous investigators. In the study reported here the relationship between the familiar "RC" electrotonic response and the oscillatory behavior is examined experimentally and shown to be dependent on the membrane potential. Computations based on the three-current system which was inferred from electrical measurements by Hodgkin and Huxley yield subthreshold responses in good agreement with experimental data. The point which is developed explicitly is that since the three currents, in general, have nonzero resting values and two currents, the "Na" system and the "K" system, are controlled by voltage-dependent time-variant conductances, the subthreshold behavior of the squid axon in the small-signal range can be looked upon as arising from phenomenological inductance or capacitance. The total phenomenological impedance as a function of membrane potential is derived by linearizing the empirically fitted equations which describe the time-variant conductances. At the resting potential the impedance consists of three structures in parallel, namely, two series RL elements and one series RC element. The true membrane capacitance acts in parallel with the phenomenological elements, to give a total impedance which is, in effect, a parallel R, L, C system with a "natural frequency" of oscillation. At relatively hyperpolarized levels the impedance "degenerates" to an RC system.

Curarized cutaneous pectoris nerve muscle preparations from frogs were subjected to prolonged indirect stimulation at 2/sec while recording from end plate regions. At the ends of the periods of stimulation, the curare was removed and the preparations were fixed for electron microscopy or treated with black widow spider venom to determine the degree to which their stores of transmitter had been depleted. After 6-8 hr of stimulation the nerve terminals were almost completely depleted of their stores of transmitter and of their population of vesicles. Most of the transmitter release occurred during the first 4 hr of stimulation, and after this time most (about 80%) of the fibers were depleted of about 80% of their transmitter. The organization of the nerve terminals in 4-hr preparations appeared normal and the terminals still contained many vesicles. When peroxidase was present in the bathing medium, terminals from stimulated preparations showed many vesicles that contained peroxidase, whereas the rested control preparations showed few such vesicles The fact that after 4 hr the total number of vesicles is not markedly changed while a large fraction (up to 45%) contained peroxidase suggests that in our experiments vesicles were continuously fusing with and reforming from the axolemma.