title: Intracellular dialysis disrupts Zn2+ dynamics and enables selective detection of Zn2+ influx in brain slice preparations.
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.
authors.affiliation: Department of Neurosciences, University of New Mexico, Albuquerque, New Mexico 87131-0001, USA.
authors.forename: Isamu
authors.initials: I
authors.lastname: Aiba
authors.forename: Adrian K
authors.initials: AK
authors.lastname: West
authors.forename: Christian T
authors.initials: CT
authors.lastname: Sheline
authors.forename: C William
authors.initials: CW
authors.lastname: Shuttleworth
mesh_terms: D000818:Animals; D001921:Brain / Q000378:metabolism*; D002352:Carrier Proteins / Q000235:genetics; D027682:Cation Transport Proteins; D002413:Cations, Divalent; D005260:Female; D066298:In Vitro Techniques; D042541:Intracellular Space / Q000378:metabolism; D008297:Male; D008565:Membrane Proteins / Q000235:genetics; D026901:Membrane Transport Proteins; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D017551:Microdialysis; D009474:Neurons / Q000378:metabolism; D010084:Oxidation-Reduction; D018408:Patch-Clamp Techniques; D013569:Synapses / Q000378:metabolism; D015032:Zinc / Q000378:metabolism*
chemical_list: D002352:Carrier Proteins; D027682:Cation Transport Proteins; D002413:Cations, Divalent; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; C491177:Slc30a3 protein, mouse; D015032:Zinc