High-resolution analysis of the cytosolic Ca 2+ events in β cell collectives in situ.

The release of peptide hormones is predominantly regulated by a transient increase in cytosolic Ca concentration ([Ca]). To trigger exocytosis, Ca ions enter the cytosol from intracellular Ca stores or from the extracellular space. The molecular events of late stages of exocytosis, and their dependence on [Ca], 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] 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 [Ca] dynamics can be simultaneously assessed on both cellular and collective level. Here, we measured [Ca] transients across all relevant timescales, from a subsecond to a minute time range, using high-resolution imaging with a low-affinity Ca sensor. We quantified the recordings with a novel computational framework for automatic image segmentation and [Ca] event identification. Our results demonstrate that under physiological conditions the duration of [Ca] 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 Ca receptors is both sufficient and necessary for glucose-dependent [Ca] oscillations in β cell collectives, and that a subset of [Ca] events could be triggered even in the absence of Ca influx across the plasma membrane. In aggregate, our experimental and analytical platform was able to readily address the involvement of intracellular Ca receptors in shaping the heterogeneity of [Ca] responses in collectives of endocrine cells in situ. Physiological glucose or ryanodine stimulation of β cell collectives generates a large number of [Ca] events, which can be rapidly assessed with our newly developed automatic image segmentation and [Ca] 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 Ca receptors is both sufficient and necessary for glucose-dependent [Ca] oscillations in β cell collectives.