SKELETAL NEUROMUSCULAR TRANSMISSION 



various stages by changes of temperature. The most 

 conspicuous result of lowering it is a prolongation of 

 the phase of transmitter action. This appears to be 

 due largely to a reduction in the activit) of cholin- 

 esterase since at low temperatures treatment with an 

 anticholinesterase produces little additional change 

 (4, 31). It is found, however, that, while the time 

 course of the curarized endplate potential is length- 

 ened, the peak amplitude is not significantly in- 

 creased as it should be if the early phase of transmitter 

 action were unaltered. In the mammalian muscle this 

 appears to be the result of curare competing more 

 effectively with acetylcholine at the reduced tempera- 

 ture and thus ofTsetting the effect of the reduction in 

 cholinesterase activity on the peak potential change. 

 An experiment, highly relevant to the conclusion 

 that the alteration of the properties of the muscle 

 fiber produced by a nerve impulse is consistent with 

 the operation of a chemical mediator, is the demon- 

 stration that the depolarization elicited by acetyl- 

 choline has its origin in the same conductance change 

 that has been shown to occur during transmission 

 (26). For this purpose the muscle has first been 

 nearly completely depolarized by immersing it in a 

 solution with a high concentration of potassium ions. 

 In this condition the application of acetylcholine pro- 

 duces no discernible change in inembrane potential. 

 When the membrane is now polarized in either direc- 

 tion by the passage of current across it, acetylcholine 

 produces a potential change that partly compensates 

 for the displacement from the unpolarized state, and 

 this is attributable to an increase in membrane con- 

 ductance similar to that observed for the preparation 

 initially in its normal environment. 



CONCLUSION : MECHANISM OF TR.ANSMISSION 



From the rate at which acetylcholine appears in the 

 effluent from a perfused muscle during repetitive 

 stimulation of the motor nerve fibers, it has been es- 

 timated that the quantity released from the nerve 

 endings at a single junction in response to a single 

 nerve impulse is about io~'- moles (i, 35). Although 

 the value obtained in this way is liable to be too small 

 because of losses in the collection procedure and be- 

 cause of a depression in the release mechanism by 

 previous activity, it is not likely to be in error in its 

 order of magnitude. It may be compared with the 

 minimum quantity of about 5 X lo""' moles of acetyl- 

 choline which is required to evoke a muscle action 

 potential when applied to the junctional region by a 



micropipette (25, 70). The factor of 200 between these 

 two quantities can be satisfactorily accounted for by 

 the geometry of the junction. The nerve endings from 

 which the acetylcholine is released are probably every- 

 where in very close proximity to the receptive region 

 of the postjunctional surface with a consequent high 

 efficiency for its reaching the receptor. On the other 

 hand, when acetylcholine is applied by a micro- 

 pipette, it would have to diffuse over a greater distance 

 and be considerably dispersed before reacting with 

 the receptor, and a larger quantity' would therefore 

 be required to produce a comparable effect. Even if 

 the micropipette were placed directly on a sensitive 

 region, the application of a moderate amount of 

 acetylcholine would no doubt lead to a rapid satura- 

 tion and inactivation of the receptor there because of 

 its high local concentration, and the initiation of an 

 action potential would require the action of acetyl- 

 choline over a greater part of the receptive area. 



From the concentration of acetylcholine required to 

 produce an action potential when applied uniformly 

 to the preparation and from the quantity that is re- 

 leased by a nerve impulse, it is possible to calculate 

 the volume in which the acetylcholine released from 

 the nerve terminals would be present when reacting 

 with the receptor (37). The result shows that the 

 acetylcholine must exert its maximum effect before 

 diffusing more than i //, a distance which is consistent 

 with morphological findings on the minute separation 

 of the pre- and postjunctional surfaces. Furthermore, 

 assuming that diffusion occurs away from the im- 

 mediate neighborhood of the junction, the time dur- 

 ing which the acetylcholine will remain in an effective 

 concentration is shown to be less than i msec. The 

 brief duration of transmitter action may reflect the 

 operation of this diffusion, though the possibility re- 

 mains that the reaction between the receptor and 

 acetylcholine does not reach an equilibrium in such a 

 short period of time and the kinetics of this reaction 

 may then influence the time course of transmitter 

 action. At least it is clear that the enzymatic destruc- 

 tion of acetylcholine is not involved in the early, high 

 intensity phase of transmitter action, as it is not 

 affected by the presence of an anticholinesterase. The 

 failure of the destruction of acetylcholine adds a 

 later low level phase of transmitter action which 

 probably occurs after the acetylcholine has diffused 

 away from the immediate neighborhood of the 

 terminals where it is released and is dispersed over 

 the entire junctional region. 



The high degree of chemical specificity of the 

 receptor and the competition for it of different sub- 



