23° 



HANDBOOK OF PHYSIOLOGY 



NEUROPHYSIOLOGY 



THE CHOLINERGIC NERVE TRANSMITTER 



Identification 



Dixon & Hamill (36) pointed out as early as 1909 

 that there was very little inherent difference between 

 the action of muscarine on the heart and electrical 

 excitation of the vagus. They continue: "If it is per- 

 missible to argue from analogy there is reason in the 

 suggestion that excitation of a nerve induces the local 

 liberation of a hormone which causes specific activity 

 by combination with some constituent by the end 

 organ muscle or gland." Only a few years later 

 Dale (25) and Dale & Ewins (30) related the phe- 

 nomena observed on stimulation of parasympathetic 

 nerves to some earlier research by Hunt & Taveau 

 (71). Among a large series of choline esters prepared 

 by them, acetylcholine was shown to be the most bio- 

 logically active, on an average about 1000 times more 

 active than choline. During studies on ergot extracts. 

 Dale (26) found a substance which produced actions 

 similar to muscarine and identified this substance 

 with acetylcholine. In his further workJDale was able 

 to state that the actions of vagus stimulation and also 

 other actions of the cranial and sacral divisions of the 

 autonomic system were mimicked very faithfully by 

 ac_etylcholine. The effects were remarkably evanescent 

 and were always abolished by a small dose of atro- 

 pine. On the basis of these observations by Dale it 

 became extremely likely that acetylcholine actually 

 was the substance which causes the effect of para- 

 sympathetic nerve impulses on the target cells. 

 Further support for the idea that the substance re- 

 leased at the parasympathetic nerve endings was 

 acetylcholine was supplied by Dale & Dudley (29) 

 who showed in 1929 that it was present in the spleen 

 of the horse and the ox. They prepared the substance 

 and isolated it as chloroplatinate. 



The identification of the parasympathetic cholin- 

 ergic nerve transmitter is based upon biological tests. 

 The amounts of acetylcholine which are liberated 

 and occur in the organism are generally too small to 

 be determined by chemical methods. Some of the bio- 

 logical methods are very sensitive, but on the other 

 hand the specificity is not always above doubt. The 

 methods most widely used are the negative inotropic 

 action of acetylcholine on the heart of the frog, the 

 hypotensive effect in the cat and the contracting; effect 

 on the intestine of the guinea pig or other animals. 

 Other preparations which may yield more specific 

 results are the leech muscle, the rectus abdominis 

 muscle and the isolated lung of the frog. The isolated 



heart of the clam Venus mercenaria has also been used. 

 For the identification of acetylcholine, the finding of 

 Fiihner (49) that the dorsal muscle of the leech was 

 greatly .sensitized to acetylcholine by addition of 

 physostigmine was one of the more important. The 

 preparation was introduced as a specific and quanti- 

 tative biologic test for acetylcholine in 1932 by Minz 

 (95). After preparation the muscle is suspended in 

 Ringer's solution from one-half to several hours to 

 relax it, and physostigmine is added to the solution in 

 a concentration of 1-200,000 to 1-2,000,000. After 

 about 20 min., the muscle is highly sensitized to 

 acetylcholine so as to detect and measure acetylcho- 

 line in concentrations as low as io~'. The frog rectus 

 is less .sensitive but fairly specific for acetylcholine. 

 The isolated frog lung has also been used and may 

 have an even higher sensitivity than the leech muscle; 

 it is claimed to contract in an acetylcholine solution 

 of io~'* (34)- The heart of Venus mercenaria has also 

 been reported to have high sensitivity to acetylcholine, 

 up to io~^-, although it varies at different times of the 

 year. 



In order to allow the conclusion that the actions 

 observed on these test preparations actually have 

 been due to acetylcholine, certain other conditions 

 must be fulfilled. The action has to be increased by 

 drugs inhibiting the acetylcholine esterase such as_ 

 physostigmine, the activity should disappear after 

 incubation with blood and the active principle should 

 be inactivated when exposed to in alkali for 10 min. 

 at room temperature, which is typical of choline 

 esters. As a general rule different kinds of tests have 

 to be consistent, i.e. when compared with a standard 

 of acetylcholine the unknown extracts should elicit 

 the same quantitative action in relation to acetyl- 

 choline (fig. 12). 



One of the chief difficulties in demonstrating the 

 neurochemical transmission from cholinergic nerves 

 arises from the fact that in most cases the para- 

 sympathetic nerves have their autonomic synapses 

 very close to the target organ. Therefore, stimulation 

 of the nerves also releases acetylcholine from the 

 preganglionic nerve. The acetylcholine released by 

 stimulation of vagus nerve in the frog's heart may 

 actually be due partly to the release of the substance 

 from the synapses. 



The introduction of physostigmine in experimental 

 work made it possible to demonstrate the mediated 

 effect with greater certainty since the substance was 

 not immediately destroyed. Loewi's original experi- 

 ments were later confirmed by many others. Among 

 the sources of transmitter which have been tried mav 



