Activation and fine‐tuning of Ca2+‐dependent slow afterhyperpolarizations by Ca2+‐handling organelles in vasopressin magnocellular neurosecretory neurons

Abstract

Vasopressin (VP) magnocellular neurons of the supraoptic nucleus become hyperexcitable in cardiovascular diseases such as heart failure. This hyperexcitability is caused by changes in both synaptic and intrinsic mechanisms, including the slow afterhyperpolarization (sAHP), a phenomenon underlain by a calcium‐dependent K+ current. While the general features of the sAHP and its effects on firing are well characterized, the precise spatiotemporal mechanisms of Ca2+ activation and buffering of the sAHP in VP neurons remains unknown. Combining patch clamp electrophysiology, live Ca2+ imaging, and photolytic Ca2+ uncaging in transgenic eGFP‐VP rats, we investigated the role of endoplasmic reticulum (ER) and mitochondria (MITO) in shaping sAHPs. We generated and measured sAHPs in current clamp via current injection or rapid targeted intracellular Ca2+ uncaging. Pre‐incubation of slices with thapsigargin (3μM) to deplete ER Ca2+ stores inhibited sAHPs significantly while leaving activity‐evoked Ca2+ transients unaffected. When sAHPs were evoked via Ca2+ uncaging, some sAHP remained, but was still significantly inhibited by thapsigargin. Additionally, acute caffeine mobilization of ER stores enhanced sAHP magnitude. These results demonstrate that ER Ca2+ release is necessary for sAHP activation in VP neurons. Conversely, blockade of MITO Ca2+ release via blocking MITO Ca2+ exchangers with TPP (20μM) failed to inhibit the sAHP. Conversely, blockade of mitochondrial Ca2+ buffering with bath application of the MITO uncoupler CCCP (1μM) or the MITO Ca2+ uniporter (MCU) blocker Ru360 (20μM) in the patch pipette significantly prolonged the decay time constant of both the sAHP and the underlying Ca2+ transient. These results demonstrate that the MCU regulates spatiotemporal dynamics of the sAHP. Combining thapsigargin with Ru360 showed an initial block of the sAHP, followed by an enhancement over 15 minutes as Ru360 dialyzed into the cell. Finally, targeted Ca2+ uncaging in the dendrites failed to generate an sAHP, providing insight into the morphological compartmentalization of sAHP channels. These results support the ER (but not MITO) as a major source of Ca2+ activating the sAHP, as well as a role for the MCU in regulating the magnitude and time course of the sAHP via Ca2+ buffering. Taken together, our studies support a fine‐tuned interaction between VGCCs, ER, and MITO in modulating the magnitude and time course of the sAHP in VP neurons. Future studies warrant investigation into whether changes in these interactions contribute to altered VP neuronal activity in cardiovascular disease states.