The release of peptide hormones is predominantly regulated by a transient increase in cytosolic Ca²⁺ concentration ([Ca²⁺]_c). To trigger exocytosis, Ca²⁺ ions enter the cytosol from intracellular Ca²⁺ stores or from the extracellular space. The molecular events of late stages of exocytosis, and their dependence on [Ca²⁺]_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 [Ca²⁺]_c events contributing to a collective functional response of a gland. For this, β cell collectives in a pancreatic islet are particularly well suited as they are the smallest, experimentally manageable functional unit, where [Ca²⁺]_c dynamics can be simultaneously assessed on both cellular and collective level. Here, we measured [Ca²⁺]_c transients across all relevant timescales, from a subsecond to a minute time range, using high-resolution imaging with a low-affinity Ca²⁺ sensor. We quantified the recordings with a novel computational framework for automatic image segmentation and [Ca²⁺]_c event identification. Our results demonstrate that under physiological conditions the duration of [Ca²⁺]_c events is variable, and segregated into three reproducible modes, subsecond, 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 Ca²⁺ receptors is both sufficient and necessary for glucose-dependent [Ca²⁺]_c oscillations in β cell collectives, and that a subset of [Ca²⁺]_c events could be triggered even in the absence of Ca²⁺ influx across the plasma membrane. In aggregate, our experimental and analytical platform was able to readily address the involvement of intracellular Ca²⁺ receptors in shaping the heterogeneity of [Ca²⁺]_c responses in collectives of endocrine cells in situ.NEW & NOTEWORTHY Physiological glucose or ryanodine stimulation of β cell collectives generates a large number of [Ca²⁺]_c events, which can be rapidly assessed with our newly developed automatic image segmentation and [Ca²⁺]_c event identification pipeline. The event durations segregate into three reproducible modes produced by a progressive temporal summation. Using pharmacological tools, we show that activation of ryanodine intracellular Ca²⁺ receptors is both sufficient and necessary for glucose-dependent [Ca²⁺]_c oscillations in β cell collectives.

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