IDENTIFICATION OF MAMMALIAN INHIBITORY TRANSMITTERS 345 



spinal neurons (Curtis et al., 1960b) whilst lower concentrations effectively 

 excite these cells by producing a membrane depolarization. It is probably 

 that the depolarization produced by the high concentrations of these acids is 

 suflficient to inactivate the processes responsible for the rising phase of the 

 spike potential. Procaine and atropine also depress spinal neurons, probably 

 in the absence of a change in membrane conductance. These substances 

 stabihze the electrically excitable component of the neuronal membrane and 

 so reduce the effectiveness with which depolarization initiates a spike potential 

 (Curtis and Philhs, 1960). 



B. DEMONSTRATION OF THE PRESENCE OF THE 



SUBSTANCE WITHIN THE APPROPRIATE 



PRESYNAPTIC TERMINALS 



Although the identification of a substance, which has satisfied criterion (A), 

 within extracts of nervous tissue strengthens further its possible function as a 

 transmitter, mere presence is insufficient evidence in the absence of precise 

 anatomical locaHzation. If the substance had been tested upon a variety of 

 neurons and was found to be present in extracts only of those areas of the 

 nervous system in which cells were inhibited by it or in those areas containing 

 cell bodies of fibers having inhibitory synaptic connections with these 

 neurons, the inference that the tested agent was indeed the inhibitory trans- 

 mitter would be reasonable. The possibility that transmitters are stored in 

 synaptic vesicles of the presynaptic terminals has been proposed (Robertson, 

 1956; del Castillo and Katz, 1956; Eccles, 1957; Eccles and Jaeger, 1957 

 Palay, 1958; de Robertis, 1958) and attempts are being made to identify 

 such vesicles in homogenates of brain tissue (Hebb and Whittaker, 1958 

 Whittaker, 1959). It has been shown that, in extracts of nervous tissue 

 transmitters exist not only in "bound" form, perhaps contained in vesicles 

 but also in the free state, the proportions of the two forms depending upon 

 the manner in which the extracts are prepared. Thus procedures aimed at des- 

 troying the membrane of the vesicles would be expected to increase the 

 amount of free transmitter extracted (cf. Whittaker, 1959). 



However, the identification of particles in brain homogenates is exceedingly 

 difficult and in spite of any variations that may be observed in the amounts 

 of a particular substance so extracted from particular anatomical regions, 

 such methods do not necessarily indicate that the agent was originally present 

 in inhibitory neurons and their terminals. The anatomical locahzation 

 within these sites can only be determined by histochemical methods which, 

 to the present time, have not been refined sufficiently to deal with particles 

 of the vesicle type. In addition to the chemical problems involved in these 

 techniques, a difficulty arises in determining whether a particular synapse is 

 inhibitory in nature. Some attempts have been made in this direction 



