Release of neurotransmitters and depletion of synaptic vesicles in cerebral cortex slices by a-latrotoxin from black widow spider venom * ( ' y-aminobutyric acid / brain cortex / neurotoxin )

The effect of a-latrotoxin on cerebral cortex slices was studied by both biochemical and morphological methods. This toxin greatly stimulates the release of preloaded -y-amino[3H butyric acid from cortex slices. The response increases linearly with dose. The release is not dependent on the presence of extracellular Ca2+, and therefore it is not mediated by the release of other transmitters from other types of neurons. In contrast, no significant increase in the release of a nontransmitter substance a-amino[14Clisobutyric acid is observed. Since previously we have shown that a-latrotoxin stimulated the re ease of acetylcholine and norepinephrine from cortex slices, it appears that the toxin probably selectively releases all neurotransmitters. The toxin also profoundly depletes the synaptic vesicle population in boutons in the cortex slices. The results suggest that the release of neurotransmitter and the depletion of synaptic vesicle in boutons are manifestations of a single action of the toxin. Therefore, a-latrotoxin can be used as a good tool for the identification of neurotransmitters and in studies on the mechanism of neurotransmitter release. Previous work (1) has described the fractionation of an extract of the black widow spider venom gland (BWSV) into several toxic protein fractions. One of the fractions was purified to the degree of no detectable contaminating proteins and was recently named a-latrotoxin (2). This single toxin factor was demonstrated to be responsible for all the effects of BWSV on frog and mouse neuromuscular junctions, namely, the increase in the frequency of miniature end-plate potentials, the complete depletion of synaptic vesicles in the nerve terminals (boutons), and the ultimate blockage of neuromuscular transmission (1). Since BWSV has many actions on both vertebrates and invertebrates, particularly their nervous systems (reviewed in ref. 1), and since several toxic fractions have been found in the venom, it is of interest to investigate the spectrum of action of this highly purified a-latrotoxin in a single species. In a previous communication (2) we demonstrated that a-latrotoxin caused an increase in the release of both acetylcholine and norepinephrine (NE) from cerebral cortex slices of mouse. We report here: (i) that a-latrotoxin enhances the release of another putative neurotransmitter, -y-aminobutyric acid (GABA), from mouse cortical slices, but not the release of a non-transmitter substance, a-aminoisobutyric acid (AIB, an analogue of GABA); and (ii) that, as in the case of neuromuscular junction, it also causes depletion of synaptic vesicles in cerebral cortex slices. MATERIALS AND METHODS The purification and assay of purity of a-latrotoxin were performed as described (1); occasionally another step of Sephadex G-200 chromatography was carried out after the DEAESephadex step to ensure homogeneity of the toxin. For each experiment, one male mouse (25-30 g) was killed by decapitation and its brain quickly removed. One slice was taken with a blade and blade guide from the top layer of cerebral cortex of each of the two hemispheres. The two slices (30-40 mg) were first incubated for 30 min at 370 in 1.0 ml of a modified Krebs-Ringer solution with the following composition: 120 mM NaCl, 4 mM KC1, 1.8 mM CaCl2, 0.8 mM MgSO4, 4 mM Na2HPO4/HCl buffer (pH 7.4), 15 mM NaHCO&, and 10 mM glucose. The incubation solution was equilibrated with a gas mixture of 95% 02/5% CO2 throughout the experiment. In experiments assayingfor GABA release, 0.3 MAM [3H]GABA (35 Ci/mmol, New England Nuclear) was present in the incubation solution, and 0.5 mM (aminooxy)acetic acid (Eastman Co.) was used to inhibit the metabolism of GABA by GABA-glutamate transaminase (3). In the experiments with [14C]AIB, a greater concentration, 35 AM, was used because of the lower specific radioactivity (57 mCi/mmol, Amersham/Searle). After 30 min incubation, the slices were removed and washed once by immersion for 10 min in fresh solution. They were then transferred to a perfusion vessel containing 1.5 ml of solution. The vessel was immersed in a shaker maintained at 370. Fresh oxygenated modified KrebsRinger solution was perfused through the vessel at a rate of 0.5 ml/min by a peristaltic pump. The perfusate of the first 30 min was discarded, and thereafter 1-ml fractions were collected every 2 min into vials in a fraction collector. At a suitable time during the perfusion, a-latrotoxin was injected into the perfusion chamber. Radioactivity discharged into the perfusate was determined after the addition of Aquasol, and radioactivity remaining in the brain slices was determined after solubilization of the slices with Protosol. In some experiments, Ca2+ was omitted, 0.5 mM EGTA was present, and the MgSO4 concentration was increased to 3.0 mM. In order to take into account the variations among the various experiments in the uptake of the radioactive substances and to correct for the diminution of radioactivity in the tissue during the perfusion, we expressed the radioactivity in the perfusate at any given time as a percentage of the radioactivity still present in the tissue at that time (4). We also assayed, by a procedure modified from that of Nadler and Cooper (5), aliquots from some fractions to deterAbbreviations: AIB, a-aminoisobutyric acid; BWSV, black widow spider venom gland extract; NE, norepinephrine; GABA, 'y-aminobutyric acid. * This work was presented in abstract form by M. Tzeng and P. Siekovitz at the Seventh Annual Meeting of the Neuroscience Society, 1977. t Present address: Department of Anatomy, University of Illinois Medical School, Chicago, IL. 4016 The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact. Proc. Natl. Acad. Sci. USA 75 (1978) 4017 mine the degree of metabolism of [3H]GABA. The perfusates were first chromatographed through an AG 50W (BisRad) (Na+ form, pH 10) column. Neutral and acidic com s were eluted with three 1-ml H20 washes, and basic compounds were then washed out with 2 M NaOH. The H20 eluates were combined and acidified to pH <2, and then further fractionated on an AGSOW column (NH4+ form, pH 2.5). The column was eluted in sequence by two 1-ml washes of each of the following: H20, 0.3 M NH40H, 0.6 M NH40H, 1.0 M NH4OH, 1.5 M NH4OH, and 2.0 M NH40H (all solutions of NH40H having pH of 2.5). Al the GABA appeared in the 1.0 M and 1.5 M HN40H fractions. In some experiments the amount of 3H in the tissue that remained as GABA was also determined. To do this, the brain slices were homogenized in 10% (wt/vol) trichloroacetic acid and then centrifuged in a microcentrifuge for 10 min. The supernatant was treated twice with two volumes of ethyl ether, the water phase was neutralized with NaOH and then analyzed for GABA as described above. For studies by electron microscopy, brain slices were cut into small strips and then incubated for 1 hr at 370 in 1.0 ml of the modified Krebs-Ringer solution with 10 gg a-latrotoxin added at zero time and another 5 ,ug added at 30 min. Control samples were handled in the same manner except that no toxin was added. The strips were then fixed with 1% glutaraldehyde/1% paraformaldehyde in 0.12 M phosphate buffer, pH 7.2, and postfixed with 1% OS04 in 0.03 M barbital buffer, pH 7.4. The tissue was then stained en bloc with 0.5% uranyl acetate, dehydrated by standard procedures, and embedded in Epon, which was polymerized at 15.50 for 3 days. Thin sections were cut with a Porter-Blum MT2B microtome, stained sequentially with 8% uranyl acetate and 4% lead citrate, and examined with both Hitatchi HU-11B and Siemens-Elmiskop 101 electron microscopes. Only the outermost 20-,um layer of the strips was used for quantitative analysis, because the inner part of the tissue slices might not have been exposed to the toxin due to diffusion problems.