Pancreatic islet crosstalk is essential to maintain blood glucose homeostasis. Central to this crosstalk are the somatostatin (Sst)-secreting delta cells, which serve to regulate the activity of insulin-secreting beta and glucagon-secreting alpha cells. Crosstalk between beta and delta cells is of particular importance as it is often lost or dysregulated in diseases characterized by dysglycemia like diabetes and congenital hyperinsulinism (CHI). It is well established that insulin and Sst secretion are coordinated, but there is no consensus on the mechanism of this coordination. Published data demonstrating a 30 second lag between insulin and Sst secretion coupled with our previously published observation that the beta cell hormone Urocortin 3 stimulates delta cell Sst secretion illustrate the importance of paracrine coordination of beta-delta cell secretion. However, an alternative and potentially parallel mechanism is gap junction coupling. Preliminary data using the calcium reporter GCaMP6s simultaneously expressed in beta and delta cells demonstrates that delta cell response to high glucose is profoundly heterogenous compared to beta cells, with both beta cell coordinated and uncoordinated calcium oscillations observed in the majority of delta cells. Based on these data delta cells can be subdivided into 7 distinct subpopulations suggesting that a single mechanism may be insufficient to fully describe beta-delta cell coordination. This has informed the hypothesis that delta cells coordinate with beta cells predominantly via paracrine signaling with a minor subpopulation of highly synchronous delta cells showing calcium behaviors consistent with gap junction coupling. The mechanism(s) of crosstalk between beta and delta cells will be addressed in two aims. Aim 1 will focus on how gap junction coupling could contribute to coordinating a small subpopulation of highly synchronous delta cells. Aim 2 will focus on the role paracrine signaling plays in coordinating the majority of delta cells with beta cells during glucose stimulation. To assess the extent of gap junction coupling in Aim 1 the whole cell patch clamp technique will be used to deliver small gap junction permeable tracers into beta and delta cells within intact islets. To investigate the role of gap junction coupling between beta cells and this small subset of highly synchronous delta cells live cell Ca2+ imaging will be followed by post-hoc immunohistochemical staining of Cx36. To address the predominant role of paracrine signaling in coordinated delta and beta cell activity in Aim 2, inhibitors of secretion will be used while assessing changes in the level of coordination between beta and delta cells via Ca2+ imaging. These approaches are innovative by: 1) connecting heterogeneous delta cell behavior to underlying mechanisms of coordination in response to glucose stimulation in intact islets. 2) resolving the predominant mechanism(s) of coordination between beta and delta cells. Thes...