A novel and simple fluorescence method for the measurement of presynaptic vesicular zinc release in acute hippocampal slices with a fluorescence plate reader

Abstract

The synaptic vesicles in the hippocampal neuronal terminals are abundantly supplied with zinc ions (Zn2+), which can be released into the synaptic cleft. In the glutamatergic systems (e.g. the hippocampus and the amygdala), the vesicular Zn2+ is co-localized with glutamate (Glu). Glu functions as a neurotransmitter, and Zn2+ as a neuromodulator (effecting basic synaptic functions). Electrical stimulation or chemical treatment (e.g. KCl) of hippocampal slices evokes the release of presynaptic vesicular Zn2+ into the synapse, together with Glu. This paper reports on the development of a rapid and simple method with which to assess the vesicular Zn2+ release and the effects of Zn2+-binding chelators in rat acute hippocampal slices. This method uses a 96-well fluorescence plate reader and the well-known zinc-sensitive fluorescence dye, FluoZin-3, which is cell-impermeable. This dye forms a stable complex with Zn2+ (Kd = 15 nM at pH 7.4); the amount of Zn2+ can be measured by fluorometry (lambda ex. 480-485 nm, em. 520-535 nm). Using 96-well plates, we could measure the Zn2+ release with high sensitivity, in at most 10 slices with a 15-s cycle time. This novel method can readily be used for the ex vivo modelling of the stress-evoked neuronal presynaptic Zn2+ release characteristic of neurodegenerative processes (e.g. Alzheimer's disease), or for the testing of Zn2+ chelators including putative drug candidates. This novel fluorescence plate reader method offers a simple, rapid and cost-effective technique for the measurement of vesicular Zn2+ release. It permits the simultaneous measurement of all mechanically undamaged hippocampal slices, regardless of size, thereby reducing the number of rats required experimentally.