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High signal-to-noise imaging of spontaneous and 5 ns electric pulse-evoked Ca.sup.2+ signals in GCaMP6f-expressing adrenal chromaffin cells isolated from transgenic mice
Ist Teil von
PloS one, 2023-03, Vol.18 (3), p.e0283736
Ort / Verlag
Public Library of Science
Erscheinungsjahr
2023
Link zum Volltext
Quelle
EZB Free E-Journals
Beschreibungen/Notizen
In studies exploring the potential for nanosecond duration electric pulses to serve as a novel modality for neuromodulation, we found that a 5 ns pulse triggers an immediate rise in [Ca.sup.2+ ].sub.i in isolated bovine adrenal chromaffin cells. To facilitate ongoing efforts to understand underlying mechanisms and to work toward carrying out investigations in cells in situ, we describe the suitability and advantages of using isolated murine adrenal chromaffin cells expressing, in a Cre-dependent manner, the genetically-encoded Ca.sup.2+ indicator GCaMP6f. Initial experiments confirmed that Ca.sup.2+ responses evoked by a 5 ns pulse were similar between fluorescent Ca.sup.2+ indicator-loaded murine and bovine chromaffin cells, thereby establishing that 5 ns-elicited excitation of chromaffin cells occurs reproducibly across species. In GCaMP6f-expressing murine chromaffin cells, spontaneous Ca.sup.2+ activity as well as nicotinic receptor agonist- and 5 ns evoked-Ca.sup.2+ responses consistently displayed similar kinetic characteristics as those in dye-loaded cells but with two-twentyfold greater amplitudes and without photobleaching. The high signal-to-noise ratio of evoked Ca.sup.2+ responses as well as spontaneous Ca.sup.2+ activity was observed in cells derived from Sox10-Cre, conditional GCaMP6f mice or TH-Cre, conditional GCaMP6f mice, although the number of cells expressing GCaMP6f at sufficiently high levels for achieving high signal-to-noise ratios was greater in Sox10-Cre mice. As in bovine cells, Ca.sup.2+ responses elicited in murine GCaMP6f-expressing cells by a 5 ns pulse were mediated by the activation of voltage-gated Ca.sup.2+ channels but not tetrodotoxin-sensitive voltage-gated Na.sup.+ channels. We conclude that genetically targeting GCaMP6f expression to murine chromaffin cells represents a sensitive and valuable approach to investigate spontaneous, receptor agonist- and nanosecond electric pulse-induced Ca.sup.2+ responses in vitro. This approach will also facilitate future studies investigating the effects of ultrashort electric pulses on cells in ex vivo slices of adrenal gland, which will lay the foundation for using nanosecond electric pulses to stimulate neurosecretion in vivo.