Postić, SandraSarikas, SrdjanPfabe, JohannesPohorec, ViljemKrižančić Bombek, LidijaSluga, NastjaSkelin Klemen, MašaDolenšek, JurijKorošak, DeanStožer, AndražEvans-Molina, CarmellaJohnson, James D.Rupnik, Marjan Slak2024-01-112024-01-112023Postić S, Sarikas S, Pfabe J, et al. High-resolution analysis of the cytosolic Ca2+ events in β cell collectives in situ. Am J Physiol Endocrinol Metab. 2023;324(1):E42-E55. doi:10.1152/ajpendo.00165.2022https://hdl.handle.net/1805/37978The 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.en-USAttribution 4.0 InternationalAutomated analysisβ cellCalcium dynamicsCell collectivePancreas tissue slicesArticle