EXTRACTION OF AN EXCITATORY SUBSTANCE 

 FROM DOG'S BRAIN 



TAKASHI HAYASHI.* KAZUO NAGAIJ AND KEISABURO MIYATAf 



* Department of Physiology, School of Medicine, Keio University, Tokyo, and 

 t Department of Physiology, School of Dental Surgery, Nihon University, Tokyo 



When an electrical stimulation was applied to the exposed surface of the 

 motor cortex, or an electric-shock current applied through the skull of the 

 dogs, generahzed seizures appeared. These continued 60-180 sec after cessation 

 of the stimulation. During these convulsions, an excitatory substance was 

 released from the motor cells which rapidly diffused into the cerebrospinal fluid 

 and which could be collected. For this purpose, a small metal syringe was 

 inserted into a lateral ventricle, the electric current applied through the skull 

 and 2-0 ml of the c.s.f. was taken during the lapse of the seizure of the donor 

 dog. The fluid taken was introduced into the c.s.f. of another dog. The experi- 

 ment showed that it produced clonic convulsions with a latent period of 

 5-18 sec in the receiver dog. We constructed a special dog holder so that the 

 procedure could be rapidly accomplished. There were, however, a few diffi- 

 culties with these experiments (Hayashi, 1959). One, the time relation of the 

 appearance of the exciting substance: the c.s.f. of the donor dogs did not 

 contain the effective substance 3-4 min after the cessation of the seizure, in 

 other words, the exciting factor was very unstable. Second, success of the 

 experiments was not confirmed in every case and out of twelve experiments 

 we could get the effective substance only in one or two cases. It was uncertain 

 whether this came from the instability of the substance or other reasons. 



Experiments were conducted in order to determine whether the reactive 

 substance is destroyed by some enzyme contained in the cerebrospinal fluid 

 or whether the substance released would re-combine and re-enter the brain. 



DEPROTEINIZATION 



If the cerebral spinal fluid was collected during the seizure into a syringe 

 filled whh 10% sublimate or with alcohol, the fluid, after centrifugation of 

 the precipitated proteins, had no convulsive action if introduced into a 

 receiver dog. Deproteinization of the cerebrospinal fluid by treatment in a 

 water bath at 100 'C for 5 min likewise resulted in inactivity. The effective 

 substance could, however, be extracted in the following way: the small metal 



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