Intracellular dialysis disrupts Zn 2+ dynamics and enables selective detection of Zn 2+ influx in brain slice preparations

We examined the impact of intracellular dialysis on fluorescence detection of neuronal intracellular Zn 2+ accumulation. Comparison between two dialysis conditions (standard; 20 min, brief; 2 min) by standard whole‐cell clamp revealed a high vulnerability of intracellular Zn 2+ 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 Zn 2+ 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 Zn 2+ 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 Zn 2+ (ZnT3‐ KO ). In contrast, under brief dialysis conditions, intracellular Zn 2+ transients were very similar in WT and ZnT3‐ KO preparations. This suggests that both intracellular release and transmembrane flux can contribute to intracellular Zn 2+ accumulation after synaptic stimulation. These results demonstrate significant confounds and potential use of intracellular dialysis to investigate intracellular Zn 2+ accumulation mechanisms.