Fluorescence imaging study of extracellular zinc at the hippocampal mossy fiber synapse.
Although synaptically released, vesicular Zn(2+) has been proposed to play a neuromodulatory or neuronal signaling role at the mossy fiber-CA3 synapse, Zn(2+) release remains controversial, especially when detected using fluorescent imaging. In the present study, we investigated synaptically released Zn(2+) at the mossy fiber (MF) synapse in rat hippocampal slices using three chemically distinct, fluorescent Zn(2+) indicators. The indicators employed for this study were cell membrane impermeable (or extracellular) Newport Green [K(DZn2+) approximatelly 1 microM] , Zinpyr-4 K(DZn2+) approximately 1 nM and FluoZin-3 K(DZn2+) approximately 15 nM, chosen, in part, for their distinct dissociation constants. Among the three indicators, FluoZin-3 was also sensitive to Ca(2+) K(DCa2+) approximately 200-300 microM which was present in the extracellular medium ([Ca(2+)](o)>2mM). Hippocampal slices loaded with either Newport Green or FluoZin-3 showed increases in fluorescence after electrical stimulation of the mossy fiber pathway. These results are consistent with previous studies suggesting the presence of synaptically released Zn(2+) in the extracellular space during neuronal activities; however, the rise in FluoZin-3 fluorescence observed was complicated by the data that the addition of exogenous Zn(2+) onto FluoZin-3 loaded slices gave little change in fluorescence. In the slices loaded with the high-affinity indicator Zinpyr-4, there was little change in fluorescence after mossy fiber activation by electrical stimulation. Further study revealed that the sensitivity of Zinpyr-4 was mitigated by saturation with Zn(2+) contamination from the slice. These data suggest that the sensitivity and selectivity of a probe may affect individual outcomes in a given experimental system.
Bastian
Chinthasagar
C
Department of Biomedical Science, Program in Biological Sciences, Ohio University, Athens, OH 45701, USA.
Li
Yang V
YV
D000818:Animals; D002118:Calcium / Q000032:analysis / Q000378:metabolism; D005110:Extracellular Space / Q000378:metabolism; D005452:Fluoresceins / Q000592:standards; D005456:Fluorescent Dyes / Q000592:standards*; D006624:Hippocampus / Q000166:cytology / Q000378:metabolism*; D007202:Indicators and Reagents / Q000592:standards; D008297:Male; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D008856:Microscopy, Fluorescence / Q000379:methods*; D019599:Mossy Fibers, Hippocampal / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D009924:Organ Culture Techniques; D011083:Polycyclic Compounds / Q000592:standards; D011189:Potassium Chloride / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D013569:Synapses / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D009435:Synaptic Transmission / Q000502:physiology; D015032:Zinc / Q000032:analysis / Q000378:metabolism*
C451182:FluoZin-3; D005452:Fluoresceins; D005456:Fluorescent Dyes; D007202:Indicators and Reagents; D011083:Polycyclic Compounds; C501988:Zinpyr-4; D011189:Potassium Chloride; D015032:Zinc; D002118:Calcium