Project Summary Heart failure (HF) is the leading cause of death worldwide. A hallmark of HF is sympathetic hyperactivity and increased circulating levels of neurohormones such as vasopressin (VP). VP is produced by magnocellular neurosecretory cells (MNCs) in the hypothalamic supraoptic and paraventricular nuclei and is transported to axon terminals in the posterior pituitary, from where it is released into the bloodstream to act as a vasoconstrictor. VP release is activity-dependent and involves Ca2+-dependent exocytosis. Importantly, the volume of VP release is directly proportional to MNC spike activity. During HF, VP neurons become hyperexcitable, contributing to neurohumoral activation, morbidity, prognosis and mortality of HF patients. Thus, understanding the mechanisms leading to aberrant VP neuronal activity in HF is of critical importance. The slow afterhyperpolarization (sAHP) is recognized as a key mechanism that influences VP firing. This is a Ca2+-dependent outwardly rectifying K+ conductance that throttles spiking by hyperpolarizing the membrane potential, playing a critical role in shaping the stereotyped phasic bursting patterns in VP neurons. The sAHP is well characterized, but a comprehensive mechanistic understanding of the steps involved in activating the sAHP is lacking. Here we provide novel preliminary data showing that Ca2+-induced Ca2+ release (CICR) from the endoplasmic reticulum (ER) is necessary for sAHP activation. Additionally, we show that mitochondria (MITO) Ca2+ buffering modulates the sAHP time-course. Importantly, we also show that the sAHP in VP neurons of HF rats is blunted, and along with previous work, we have described ER and MITO structural/functional dysfunction in cardiovascular diseases. We therefore present the innovative hypothesis that sAHP activation and time-course involve a tight coordination of Ca2+ signals between CICR from ER and Ca2+ buffering by MITO, and that impairment of these mechanisms shuts off the sAHP, contributing to the hyperexcitability observed in VP neurons of HF rats. We propose to incorporate the novel virally encoded organelle-specific Ca2+ sensors, the CatchER+ and CatchMito+ to monitor luminal Ca2+ levels in ER and MITO, respectively, in VP neurons from GFP-VP transgenic control in a rat model of ischemic HF. In the first aim, we will patch clamp GFP-VP neurons expressing the red-shifted CatchER+/CatchMITO+ in acute brain slices and study the relationship between neuronal spiking (various frequencies and patterns), global and organelle Ca2+ levels, and sAHP time-course. We aim to identify the critical mechanisms for sAHP generation and modulation by organelle Ca2+. The experiments of Aim 1 attempt to isolate the most important aspects to generating sAHPs and thus, Aim 2 will focus on these mechanisms in the context of HF. For Aim 2, we will test the hypothesis that ER Ca2+ dynamics is greatly impaired in VP neurons of HF rats, in part due to ER Ca2+ depletion. We anticipate...