We examined the impact of intracellular dialysis on fluorescence detection of neuronal intracellular Zn²⁺ accumulation. Comparison between two dialysis conditions (standard; 20 min, brief; 2 min) by standard whole-cell clamp revealed a high vulnerability of intracellular Zn²⁺ buffers to intracellular dialysis. Thus, low concentrations of zinc-pyrithione generated robust responses in neurons with standard dialysis, but signals were smaller in neurons with short dialysis. Release from oxidation-sensitive Zn²⁺ pools was reduced by standard dialysis, when compared with responses in neurons with brief dialysis. The dialysis effects were partly reversed by inclusion of recombinant metallothionein-3 in the dialysis solution. These findings suggested that extensive dialysis could be exploited for selective detection of transmembrane Zn²⁺ influx. Different dialysis conditions were then used to probe responses to synaptic stimulation. Under standard dialysis conditions, synaptic stimuli generated significant FluoZin-3 signals in wild-type (WT) preparations, but responses were almost absent in preparations lacking vesicular Zn²⁺ (ZnT3-KO). In contrast, under brief dialysis conditions, intracellular Zn²⁺ transients were very similar in WT and ZnT3-KO preparations. This suggests that both intracellular release and transmembrane flux can contribute to intracellular Zn²⁺ accumulation after synaptic stimulation. These results demonstrate significant confounds and potential use of intracellular dialysis to investigate intracellular Zn²⁺ accumulation mechanisms.

Item Type:	Refereed Article
Keywords:	hippocampal slice, metallothionein, pyrithione, whole-cell, Zinc, ZnT3
Research Division:	Biomedical and Clinical Sciences
Research Group:	Neurosciences
Research Field:	Central nervous system
Objective Division:	Health
Objective Group:	Clinical health
Objective Field:	Clinical health not elsewhere classified
UTAS Author:	West, AK (Professor Adrian West)
ID Code:	85242
Year Published:	2013
Web of Science® Times Cited:	7
Deposited By:	Menzies Institute for Medical Research
Deposited On:	2013-06-20
Last Modified:	2017-11-06
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