Activation of GCaMP6f-Expressing Chromaffin and Satellite Glial Cells in Murine Adrenal Slices by Nanosecond Electric Pulses

Abstract

Nanosecond electric pulses (NEP) are being explored as a novel bioelectric stimulus to modulate neurosecretion. Recently we reported that NEP trigger transient, voltage-gated Ca²⁺ channel-mediated Ca²⁺ influx in cultured murine adrenal chromaffin cells (ACC) expressing the genetically-encoded Ca²⁺ indicator GCaMP6f. The present study investigated ACC Ca²⁺ responses to NEP in acute adrenal slices from mice that expressed GCaMP6f in both ACC and satellite glial cells (SGC), as well as exclusively in SGC. Adrenal glands from male and female mice were sectioned into 100 μm slices that were placed in a chamber and perfused at a rate of 3–4 mL/min with a balanced salt solution maintained at 35°C. A custom-fabricated non-contact electrode delivered NEP ranging from 12 to 90 ns to a large area of the adrenal medulla. Our results show that a single 30 ns pulse elicited a rapid, transient rise in intracellular Ca²⁺ in ACC in situ. Like NEP-induced Ca²⁺ responses in cultured ACC, ACC Ca²⁺ responses in situ depended on extracellular Ca²⁺ and voltage-gated Ca²⁺ channel activation. The nicotinic receptor antagonist hexamethonium failed to inhibit these responses, indicating that NEP stimulation, in contrast to stimulation with conventional duration electrical pulses, activates ACC directly rather than indirectly through activation of splanchnic nerve terminals. Furthermore, NEP exposure of the adrenal medulla also resulted in Ca²⁺ increases in surrounding SGC that were slower in onset and longer in duration than those evoked by NEP in ACC. Finally, comparison of Ca²⁺ responses elicited by NEP trains in ACC in situ to those evoked by such trains in ACC in vitro revealed that the native tissue environment promotes more highly synchronized Ca²⁺ responses. Together, these results lay the foundation for future studies exploring the potential for NEP to modulate catecholamine release from ACC remotely and non-invasively in vivo.