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1. The third cerebral ventricle of cats treated with nialamide and anaesthetized with chloralose was perfused, and the effluent was tested for 5-hydroxytryptamine (5-HT) and also for acetylcholine (ACh) when the perfusion fluid contained neostigmine.

2. Under `resting' conditions a 25 min sample of effluent contained from < 1 to 6 ng 5-HT; the release remained steady during many hours of perfusion. It was necessary to watch out for traces of blood which might contribute to the 5-HT content and which were only visible after centrifugation.

3. A number of regions in the ventral mid-brain and hind-brain were stimulated, including the two most anterior nuclei of the raphe, nucleus linearis rostralis and intermedius. Release of 5-HT (rarely more than 2 ng) was only obtained on stimulation of these two nuclei, whereas ACh was released by stimulating many points, such as the reticular formation or the decussation of the superior cerebellar peduncles, but not the two raphe nuclei.

4. Low frequencies of stimulation were more effective at releasing 5-HT, and high frequencies at releasing ACh.

5. Since the amount of 5-HT released on stimulation was rarely more than 2 ng, a powerful re-uptake process was suspected and confirmed by the use of chlorimipramine. Intravenous, intraperitoneal and intraventricular use of this drug temporarily increased the basal release to values ranging from 20 to 50 ng in 25 min samples, and about trebled the release on stimulation of either of the linear nuclei.

6. Intravenous administration of chlorimipramine (10 mg/kg) caused the disappearance of electrical responses evoked in the brain stem by afferent sensory stimuli.