Two-photon imaging of Zn2+ dynamics in mossy fiber boutons of adult hippocampal slices.
Mossy fiber termini in the hippocampus accumulate Zn(2+), which is released with glutamate from synaptic vesicles upon neural excitation. Understanding the spatiotemporal regulation of mobile Zn(2+) at the synaptic level is challenging owing to the difficulty of visualizing Zn(2+) at individual synapses. Here we describe the use of zinc-responsive fluorescent probes together with two-photon microscopy to image Zn(2+) dynamics mediated by NMDA receptor-dependent long-term potentiation induction at single mossy fiber termini of dentate gyrus neurons in adult mouse hippocampal slices. The membrane-impermeant fluorescent Zn(2+) probe, 6-CO2H-ZAP4, was loaded into presynaptic vesicles in hippocampal mossy fiber termini upon KCl-induced depolarization, which triggers subsequent endocytosis and vesicular restoration. Local tetanic stimulation decreased the Zn(2+) signal observed at individual presynaptic sites, indicating release of the Zn(2+) from vesicles in synaptic potentiation. This synapse-level two-photon Zn(2+) imaging method enables monitoring of presynaptic Zn(2+) dynamics for improving the understanding of physiological roles of mobile Zn(2+) in regular and aberrant neurologic function.
Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, ON, Canada M5G 1X5.
Mustafa
M
Khan
Christian R
CR
Goldsmith
Zhen
Z
Huang
John
J
Georgiou
Thomas T
TT
Luyben
John C
JC
Roder
Stephen J
SJ
Lippard
Kenichi
K
Okamoto
D000818:Animals; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism; D027682:Cation Transport Proteins; D005452:Fluoresceins / Q000138:chemical synthesis / Q000737:chemistry; D005456:Fluorescent Dyes / Q000138:chemical synthesis / Q000737:chemistry; D059907:Functional Neuroimaging / Q000379:methods*; D006367:HeLa Cells; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008565:Membrane Proteins / Q000172:deficiency / Q000235:genetics / Q000378:metabolism; D026901:Membrane Transport Proteins; D051379:Mice; D018345:Mice, Knockout; D036641:Microscopy, Fluorescence, Multiphoton / Q000379:methods*; D015394:Molecular Structure; D019599:Mossy Fibers, Hippocampal / Q000378:metabolism*; D055668:Photochemical Processes; D015032:Zinc / Q000378:metabolism*
D002352:Carrier Proteins; D027682:Cation Transport Proteins; D005452:Fluoresceins; D005456:Fluorescent Dyes; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; C491177:Slc30a3 protein, mouse; D015032:Zinc
metalloneurochemistry; zinc ion