The release of peptide hormones is predominantly regulated by a transient increase in cytosolic Ca 2+ concentration ([Ca 2+ ] c ). To trigger exocytosis, Ca 2+ ions enter the cytosol from intracellular Ca 2+ stores or from the extracellular space. The molecular events of late stages of exocytosis, and their dependence on [Ca 2+ ] c , were extensively described in isolated single cells from various endocrine glands. Notably less work has been done on endocrine cells in situ to address the heterogeneity of [Ca 2+ ] c events contributing to a collective functional response of a gland. For this beta cell collectives in a pancreatic islet are particularly well suited as they are the smallest, experimentally manageable functional unit, where [Ca 2+ ] c dynamics can be simultaneously assessed on both cellular and collective level. Here we measured [Ca 2+ ] c transients across all relevant timescales, from a sub-second to a minute time range, using high-resolution imaging with low-affinity Ca 2+ sensor. We quantified the recordings with a novel computational framework for semi-automatic image segmentation and [Ca 2+ ] c event identification. Our results demonstrate that under physiological conditions the duration of [Ca 2+ ] c events is variable, and segregated into 3 reproducible modes, sub-second, second and tens of seconds time range, and are a result of a progressive temporal summation of the shortest events. Using pharmacological tools we show that activation of intracellular Ca 2+ receptors is both sufficient and necessary for glucose-dependent [Ca 2+ ] c oscillations in beta cell collectives, and that a subset of [Ca 2+ ] c events could be triggered even in the absence of Ca 2+ influx across the plasma membrane. In aggregate, our experimental and analytical platform was able to readily address the involvement of intracellular Ca 2+ receptors in shaping the heterogeneity of [Ca 2+ ] c responses in collectives of endocrine cells in situ.

High resolution analysis of the cytosolic Ca2+events in beta cell collectives in situ