Calcium indicator loading of neurons using single-cell electroporation.
Studies of subcellular Ca(2+) signaling rely on methods for labeling cells with fluorescent Ca(2+) indicator dyes. In this study, we demonstrate the use of single-cell electroporation for Ca(2+) indicator loading of individual neurons and small neuronal networks in rat neocortex in vitro and in vivo. Brief voltage pulses were delivered through glass pipettes positioned close to target cells. This approach resulted in reliable and rapid (within seconds) loading of somata and subsequent complete labeling of dendritic and axonal arborizations. By using simultaneous whole-cell recordings in brain slices, we directly addressed the effect of electroporation on neurons. Cell viability was high (about 85%) with recovery from the membrane permeabilization occurring within a minute. Electrical properties of recovered cells were indistinguishable before and after electroporation. In addition, Ca(2+) transients with normal appearance could be evoked in dendrites, spines, and axonal boutons of electroporated cells. Using negative-stains of somata, targeted single-cell electroporation was equally applicable in vivo. We conclude that electroporation is a simple approach that permits Ca(2+) indicator loading of multiple cells with low background staining within a short amount of time, which makes it especially well suited for functional imaging of subcellular Ca(2+) dynamics in small neuronal networks.
Nevian
Thomas
T
Abteilung Zellphysiologie, Max-Planck-Institut für medizinische Forschung, Jahnstrasse 29, 69120 Heidelberg, Germany. nevian@pyl.unibe.ch
Helmchen
Fritjof
F
D000818:Animals; D001921:Brain / Q000166:cytology / Q000378:metabolism; D002118:Calcium / Q000378:metabolism*; D002470:Cell Survival / Q000502:physiology; D018274:Electroporation / Q000379:methods*; D005456:Fluorescent Dyes / Q000378:metabolism; D008564:Membrane Potentials / Q000502:physiology; D009474:Neurons / Q000166:cytology / Q000378:metabolism*; D018408:Patch-Clamp Techniques; D051381:Rats; D017208:Rats, Wistar; D009435:Synaptic Transmission / Q000502:physiology
D005456:Fluorescent Dyes; D002118:Calcium