Intracellular dialysis disrupts Zn2+ dynamics and enables selective detection of Zn2+ influx in brain slice preparations

J Neurochem. 2013 Jun;125(6):822-31. doi: 10.1111/jnc.12246. Epub 2013 Apr 22.

Abstract

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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Brain / metabolism*
  • Carrier Proteins / genetics
  • Cation Transport Proteins
  • Cations, Divalent
  • Female
  • In Vitro Techniques
  • Intracellular Space / metabolism
  • Male
  • Membrane Proteins / genetics
  • Membrane Transport Proteins
  • Mice
  • Mice, Inbred C57BL
  • Mice, Knockout
  • Microdialysis
  • Neurons / metabolism
  • Oxidation-Reduction
  • Patch-Clamp Techniques
  • Synapses / metabolism
  • Zinc / metabolism*

Substances

  • Carrier Proteins
  • Cation Transport Proteins
  • Cations, Divalent
  • Membrane Proteins
  • Membrane Transport Proteins
  • Slc30a3 protein, mouse
  • Zinc