IT HAS BEEN CONSIDERED that the end-plate potential (e.p.p.) is generated by the brief ionic flux across the end-plate membrane and the later slowly declining phase of the e.p.p. is due to the dissipation of the charge along and across the muscle membrane. This consideration was supported by some authors. Kuffler (21) observed with a single nerve-muscle preparation that the later slowly decaying part of the e.p.p. was destroyed by a propagated muscle impulse and obtained a duration of transmitter action (3-4 msec. at 20°C.) by observing the size of the e.p.p. that was built after the invasion of a propagated muscle impulse. Katz (18) demonstrated that the neuromuscular transmitter produced a brief phase of impedance loss at the end-plate region. Recently Fatt and Katz (10) observed by measuring the displacement of the total charge along and across the muscle membrane during the e.p.p. that the active depolarization process at the end-plate had ceased within 2 msec. On the other hand the time course of the actively depolarizing phase of the e.p.p. was estimated by an analysis of the time course of the e.p.p., it being assumed that the exponentially decaying phase was attributable to the passive repolarization of the muscle membrane (7,19). The purpose of the present experiment was to determine directly the time course of the active phase of the e.p.p. by using the voltage clamp method which was originally described by Hodgkin et al. (14) and was also applied to the squid giant synapse by Tasaki and Hagiwara (29). When the membrane potential is clamped at the resting membrane potential with negative feedback during the neuromuscular transmission, the electrotonic spread of the charge along the muscle fibre membrane can be eliminated. The feedback current which flows through the muscle membrane to hold the membrane potential at the resting value is due to the brief electric change at the end-plate, i.e., it will show the active phase of the e.p.p. To simplify the expression, the feedback current during neuromuscular transmission will be called provisionally the "end-plate current." A preliminary report of the present experiment appeared in 1958 (27). Materials and solutions M. sartorius with sciatic nerve was dissected from well-fed winter frogs of species Rana nigromacuZata. The neuromuscular transmission was usually blocked by adding d

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SUMMARY1. The effect of picrotoxin on the neuromuscular junction of the crayfish (Cambaru8 clarkii) was investigated. The potential changes were recorded intracellularly and extracellularly with micro-electrodes. The membrane conductance of the muscle fibre was also measured.2. Picrotoxin depressed the amplitudes of the inhibitory junctional potentials and the potential changes produced by iontophoretically applied y-aminobutyric acid (GABA), but had no appreciable effect on the excitatory junctional potentials and the potential changes produced by L-glutamate.3. The presynaptic action of GABA and the neural transmitter was depressed by picrotoxin. The presynaptic action of /J-guanidinopropionic acid was also depressed by picrotoxin.4. The increase in the membrane conductance produced by the addition of GABA in the bath fluid was depressed by picrotoxin. The dose-response relation showed that picrotoxin depressed the conductance increase produced by GABA in a non-competitive manner. The action of picrotoxin on the conductance increase produced by GABA was more effective in low Cl-solution.5. The analysis of the dose-response curves showed that the action of picrotoxin was well expressed by the Michaelis-Menten equation, but the slope of the dose-response curve of GABA was steeper than this relation. It is proposed that the conductance of the junctional membrane was increased by the combination of two molecules of GABA with a receptor, and the attachment of one molecule of picrotoxin to a specific site depressed the conductance increase.

Potential changes both in pre-and postsynapfic axons were recorded from the giant synapse of squid with intracellular electrodes. Synaptic current was also recorded by a voltage clamp method. Facilitation of postsynaptic potential caused by applying two stimuli several milliseconds apart was accompanied by an increase in the amplitude of the presynaptic action potential. Depression of the postsynaptic potential occurred without changes in the presynaptic action potential. Increase in the concentration of Ca in sea water caused an increase in amplitude of the synaptic current. On the other hand increase in Mg concentration decreased the amplitude of the synaptic current. In these cases no appreciable change in the presynaptic action potential was observed. Extracellularly recorded potential changes of the presynaptic axon showed mainly a positive deflexion at the synaptic region and a negative deflexion in the more proximal part of the presynaptic axon. Mechanism of synaptic transmission is discussed.At synapses where chemical transmission occurs, the arrival of a nerve impulse at the presynaptic terminal is considered to cause release of the transmitter which, in turn, produces a depolarization of the postsynaptic membrane. Several investigators have attempted to correlate electrical changes in the presynaptic structures with those occurring in the postsynaptic structures during transmission. Thus, del Castillo and Katz (1954 d) observed that hyperpolarizing current applied to the presynaptic nerve fiber at the neuromuscular junction augmented the amplitude of the end-plate potential. Also, during post-tetanic potentiation of the monosynaptic reflex of spinal motoneurons, hyperpolarization of primary afferent fibers was observed (Eccles and Krnjevid, 1959). In both these synapses, the postsy-naptic structure is relatively large and its electrical changes may be easily measured but the

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The resonance Raman spectra of ferric and ferrous forms of the heme-heme oxygenase (HO) complex (isoform 1) clarify several structural features of the catalytic active site. Isotopic substitution studies of the central iron atom of the heme demonstrate that the line at 218 cm-1 in the ferrous ligand-free form of the complex originates from the iron-histidine stretching mode. The presence of a Raman line at this frequency confirms that the fifth ligand coordinating to the heme is a neutral imidazole from a histidine residue. The modes associated with CO in the carboxy derivative of the ferrous enzyme complex have typical frequencies of histidine-bound heme proteins such as myoglobin. In the ferric form of the complex, at alkaline pH, hydroxide is identified as the bound exogenous ligand, and at neutral pH we infer that water is bound. Thus, the coordination of the heme-HO complex is the same as that in myoglobin. However, in a comparison of the low-frequency vibrational modes in the resonance Raman spectrum of the heme-HO complex to those of myoglobin, the spectra are found to be very different, indicating that the interactions between the heme and its amino acid pocket in these two proteins are quite different. The neutral imidazole may play several important roles in the physiological function of the heme-HO complex.

SUMMARY1. The membrane conductance of the crayfish muscle fibre was calculated and the increase in the membrane conductance brought about by the application of y-aminobutyric acid (GABA) was investigated under various conditions. The permeability of the activated inhibitory postsynaptic membrane was estimated from the conductance change.2. When the concentration of Cl-was changed by substituting CO-with methylsulphate, a linear relation was obtained between the membrane conductance change produced by GABA and the concentration of C1-. Cations have no appreciable effect on the GABA induced conductance change (less than 0-4 %).3. The conductance change produced by GABA on the inhibitory postsynaptic membrane was measured in various solutions in which the Clwas replaced by various anions. The degree of permeability depended on the anion species in the following order:Br-> Cl-> SON-> I-> N03-> HCOO-> C103-> C104-> BrO3-4. At high pH the conductance change produced by GABA was decreased, while at low pH it increased.5. These results show that the activated inhibitory post-synaptic membrane of crayfish muscle fibres has the characteristics of an anion selective membrane, suggesting the fixed positive charges in the inhibitory membrane.