A novel and simple fluorescence method for the measurement of presynaptic vesicular zinc release in acute hippocampal slices with a fluorescence plate reader.
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.
Supramolecular and Nanostructured Materials Research Group, Hungarian Academy of Sciences, Szeged, Hungary. datkiz@mdche.szote.u-szeged.hu
Zsolt L
ZL
Datki
Akos
A
Hunya
Botond
B
Penke
D000367:Age Factors; D000704:Analysis of Variance; D000818:Animals; D006624:Hippocampus / Q000648:ultrastructure*; D066298:In Vitro Techniques; D008297:Male; D008856:Microscopy, Fluorescence / Q000295:instrumentation / Q000379:methods*; D015336:Molecular Probe Techniques; D011083:Polycyclic Compounds / Q000493:pharmacokinetics; D017729:Presynaptic Terminals / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar; D013572:Synaptic Vesicles / Q000378:metabolism*; D013997:Time Factors; D015032:Zinc / Q000378:metabolism*
C451182:FluoZin-3; D011083:Polycyclic Compounds; D015032:Zinc