Journal zyxwvutsrqponm of Neurochemistry, zyxwvutsrqponmlkji 1977. Vol. 29. pp. 335-343. Pergarnon Press. Printed zyxwvut in Great Britain. ON THE SITE OF ORIGIN OF TRANSMITTER AMINO ACIDS RELEASED BY DEPOLARIZATION OF NERVE TERMINALS zyxwv IN zyxwv VITRO J. S. DE BELLEROCHE' and H. F. BRADFORD Biochemistry Department, Imperial College, London S.W.7. U.K. (Received 17 November 1976. Revised 18 February 1977. Accepted 25 February 1977) Abstract-The site of origin of transmitter amino acids released by depolarizing agents from nerve endings was studied. The model used was the incubated and depolarized synaptosome preparation from which the component soluble, synaptic vesicle, membrane and mitochondria1 sub-fractions were obtained. Synaptosomal amino acids were radioactively labelled from ~-[U-'~C]glucose in viuo by intraventricular injection and in uitro during subsequent incubation. The specific radioactivities of amino acids released in response to K' (56 mM) or veratrine (75 p ~ ) were found to closely resemble those of the soluble cytoplasmic fraction, in most cases differing significantly from those of the other fractions. The specific radioactivity of the GABA and aspartate released by K+ stimulation and the GABA and glutamate released by veratrine were significantly different from that of the vesicles in each case. The specific radioactivities of glutamate released by both agents, and also GABA with K' stimulation, were approximately double that of the amino acid released in control conditions. Depletion of the soluble cytoplasmic pools of glutamate, GABA and aspartate occurred following stimulation, corre- sponding to the induced-release of these compounds. Turnover of the amino acids in the other subfrac- tions was too low to account for their participation in the release process in addition to the soluble cytoplasmic pool. A cytoplasmic origin of release of neurotransmitter amino acids from nerve endings is proposed. IN SPITE of clear evidence for quanta1 release of trans- mitter at certain CNS synapses (KATZ zyxwvut & MILEDI, 1963; KUNO, 1964; WEAKLY, 1969) direct evidence im- plicating synaptic vesicles in this process has not been forthcoming and the purpose of the present investiga- tion was to localise the subcellular origin of released transmitter. A synaptosome preparation from cerebral cortex was the in vitro system chosen for the study. It is now well established that a selective release of endogenous and preloaded transmitter amino acids is induced by treatment of synaptosomes with such depolarizing stimuli as electrical pulses, potassium and veratrine. Thus, glycine is released from spinal cord synaptosomes and glutamate, GABA and aspar- tate from cortical synaptosomes by calcium-depen- dent mechanisms (BRADFORD, 1970; DE BELLEROCHE & BRADFORD, 1972; OSBORNE zyxwvuts et a!., 1973; REDBURN & COTMAN, 1974). This in vitro stimulus-coupled release is blocked by agents shown to prevent trans- mitter release in vivo. Thus tetanus toxin blocks gly- cine and GABA release (OSBORNE et al., 1973), tetro- dotoxin prevents noradrenaline release (BLAUSTEIN et al., 1972) and botulinum toxin prevents acetylcholine release (WONNACOTT & MARCHBANKS, 1976). These and other properties together form a strong case for considering synaptosomes a valid in vitro model for studying neurotransmitter release mechanisms. In the experiments reported here different degrees of radio- active labelling of transmitter amino acids in vesicles, cytoplasm and other synaptosome compartments were established, and the specific radioactivities of amino acids released by depolarizing agents was used as an index of their site of origin. METHODS Intrauentricular injection of D-[U-'4C]ghcoSe Eight female Sprague-Dawley rats (22W250 g) were used per experiment. These were anaesthetized with diethyl ether-air and the junction of the coronal and sagittal sutures of the skull was exposed to use as a reference point. A molded Perspex guide was used for injection into the lateral ventricles. The injection was made at a point 1 mm posterior to the coronal suture and 2mm lateral to the sagittal suture and on one side only. A microlitre syringe and needle with a nylon stop were used to inject to a depth of 3.5-4mm. Injection of 12.5pCi of aqueous ~-[U-'~C]glucose (283 mCi/mmol) containing 3% ethanol was made in a volume of 50 pl. Rats recovered from anaes- thesia 3 4 min after injection and were killed 30 min later. The brains were removed and the cerebral cortices placed in 0.32 M-sucrose. Although injection was into one hemi- sphere only, it was shown that both ventricles became filled with injection fluid by the use of aqueous methylene blue. Preparation and incubation of synaptosomes Synaptosomes were prepared by the method of GRAY & WHITTAKER (1962) as modified by BRADFORD et al. (1973). Synaptosomes (40 mg) were suspended in Krebs bicarbonate medium (7.5 ml) of composition: (mM); NaC1, 124; KCI, 5: Na2HP04, 1.2; MgSO,, 1.3; CaCI,, 0.75; NaHCO,, 26; pH 7.5 containing 10.3mM-glucose (0.934pCi/pmol) and gassed with 95% 0,/5% CO,. Incu- bation was at 37°C for periods of up to 20 min as indicated 335