Brain Research, 305 (1984) 259-270 259 Elsevier BRE 10170 Characterization of Transmitter Release as a Response of Vertebrate Neural Tissue to Erythrosin B PATRICIA D. WADE 1,2,EVE MARDER 2and PHILIP SIEKEVITZ l 1Rockefeller University, 1230 York A venue, New York, NY 10021 and 2Brandeis University, Biology Department, Waltham, MA 02254 (U.S.A.) (Accepted December 12th, 1983) Key words: transmitter release - - food dyes - - brain slice - - neuromuscular junction A rat cerebral cortical slice preparation was used to study the response of transmitter release to the application of the food dye, Erythrosin B, a tetraiodinated derivative of fluorescein. Erythrosin B (100~M) stimulated net release of previously taken up [3H]no- repinephrine and [3H]7-aminobutyric acid (GABA). The Erythrosin-induced release of GABA (the only transmitter studied ) oc- curred in the absence of added Ca2+, and in the presence of tetrodotoxin (TTX). Ultrastructural analysis of the vesicle content of frog neuromuscular junctions treated with Erythrosin B revealed a diminution in the number of synaptic vesicles present in the nerve termi- nal. By using fluorescein and some halogen-substituted derivatives including Erythrosin B, it was found that incubation with the unha- Iogenated compound caused no net release, whereas incubation with the iodine-, chlorine- or bromine-substituted compound did cause release. It was also found that somewhat greater release induced by Erythrosin B (at 100/~M) occurred in the light than in the dark. That Erythrosin B inhibits the Na+,K ÷,Mg2+-ATPasewas confirmed in this preparation; it did so in both light and dark. The dis- crepancy between release and Na +,K÷,Mg2+-ATPase blockade in the dark suggests that release either occurs by some other mecha- nism than by Na ÷,K÷,Mg2+-ATPase blockade, or that an additional light-dependent process contributes to the release. We conclude that Erythrosin B can presumably induce net release of transmitters generally, that release does not occur via the TTX-sensitive Na ÷ channel, that release via vesicles does occur, and that light somewhat enhances the release. INTRODUCTION One approach to elucidating the molecular steps leading to neurotransmitter release is to perturb the release process with an agent and characterize the re- sponse to the agent. A chemical agent of possible use in provoking transmitter release is the tetraiodinated derivative of fluorescein, Erythrosin B. Among oth- er effects, it had been shown to block accumulation of transmitters into a preparation of rat brain homo- genate 30, and into synaptosomes 26, and to cause a dramatic increase in miniature end-plate potential (MEPP) frequency at the frog neuromuscular junc- tion 2.4. Because Erythrosin B can affect a number of processes 41, various possibilities exist for its mecha- nism in causing transmitter release. Release could occur by axonal excitability changes or by some direct effect on the nerve terminal. Inso- far as axonal excitability occurs by activating a tetro- dotoxin (TTX)-sensitive channel, release by means of an action potential would also be dependent on such a channel. Blocking the channel with TTX would test the dependence of the response on it. A further property of action potential-evoked release is that it requires Ca 2+ in the external medium 24. O n the other hand, at the neuromuscular junction Eryth- rosin B 2.4 is one of a number of agents which causes transmitter release in the absence of added Ca 2+ 5-7, 31,36,37. The Ca2+-dependence of the response to Erythrosin in a central nervous system preparation would determine if this release parallels release me- diated by action potentials. Erythrosin inhibits the Na ÷,K+-ATPase activity in a rat cerebral cortical membrane 43,44 and similar47 Correspondence: P. D. Wade, Rockefeller University, 1230 York Avenue, New York, NY 10021, U.S.A. 0006-8993/84/$03.00 © 1984 Elsevier Science Publishers B.V.