Functional connectivity in islets of Langerhans from mouse pancreas tissue slices.

Ethics statement

The study was carried out in accordance with all national and European recommendations pertaining to work with experimental animals, and all efforts were made to minimize suffering of animals. The protocol was approved by the Veterinary Administration of the Republic of Slovenia (permit number: 34401-61-2009/2).

Tissue slice preparation

Tissue slices (140 µm) were prepared from pancreata of 10–20 week old NMRI mice killed by cervical dislocation as described previously [43] . Throughout preparation and during slicing, the tissue was held in an ice-cold extracellular solution (ECS, consisting of (in mM) 125 NaCl, 26 NaHCO 3 , 6 glucose, 6 lactic acid, 3 myo-inositol, 2.5 KCl, 2 Na-pyruvate, 2 CaCl 2 , 1.25 NaH 2 PO 4 , 1 MgCl 2 , 0.5 ascorbic acid; ph 7.4) continuously bubbled with a gas mixture containing 5% CO 2 and 95% O 2 . Slices were collected in 30 ml of HEPES-buffered saline at room temperature (HBS, consisting of (in mM) 150 NaCl, 10 HEPES, 6 glucose, 5 KCl, 2 CaCl 2 , 1 MgCl 2 ; pH 7.4) before they were transferred to the dye-loading solution. All chemicals were obtained from Sigma-Aldrich (St. Louis, Missouri, USA) unless otherwise specified. 4–10 slices were simultaneously loaded with the dye in a Petri dish, exposed to ambient air, protected from light, and filled with 3.333 ml of HBS containing 6 µM Oregon Green 488 BAPTA-1 AM calcium dye (OGB-1, Invitrogen, Eugene, Oregon, USA), 0.03% Pluronic F-127 (w/v), and 0.12% dimethylsulphoxide (v/v) for 1 hour on an orbital shaker (40 turns/min) at room temperature. Uptake of OGB-1 was limited to the first two or three most superficial cell layers as described previously for pituitary slices [30] , [44] and isolated islets [35] , [45] . Several-fold differences in fluorescence intensity were observed between cells, most probably due to differences in viability, enzyme activity, loading, and variable extrusion of the dye. Different loading did not influence fluorescence time profiles. After staining and before measurements, the slices were kept protected from ambient light in 30 ml of fresh HBS for up to 12 hours at room temperature. HBS was exchanged every 2 hours. Individual slices were transferred to a temperature-controlled bath chamber (37°C, Luigs & Neumann, Ratingen, Germany) continuously perifused with bubbled (5% CO 2 , 95% O 2 ) ECS and used in imaging experiments.

Calcium imaging

Imaging was performed on a Leica TCS SP5 AOBS Tandem II upright confocal system using a Leica HCX APO L water immersion objective (20×, NA 1.0). OGB-1 was excited by an argon 488 nm laser and fluorescence detected by Leica HyD hybrid detector in the range of 500–650 nm (all from Leica Microsystems GmbH, Wetzlar, Germany). 8-bit 512×512 pixels images were acquired at a frequency of 0.5 Hz. To avoid recording from cells at the potentially damaged cut surface, cells at 15 µm depth or more were imaged. Optical section thickness was 4 µm which gave a reasonable trade-off between satisfactory signal strength at lowest acceptable laser power (to avoid photobleaching and prolong the maximum time of recording) and the need to keep the section thickness as thin as possible to assure recording from a single cell only. Before and after recording each time series, a higher quality fluorescence image (1024×1024 pixels) was taken and used as a reference to assess motion artefacts and regions of interest (ROIs) during analysis. Calcium kinetics was measured off-line from ROIs and exported employing Leica Application Suite Advanced Fluorescence software (Leica Microsystems GmbH, Wetzlar, Germany). Further analysis was performed using custom-made scripts in MATLAB program (The MathWorks, Inc., Massachusetts, USA). Photobleaching was accounted for by a combination of linear and exponential fit. Traces were rejected if extensive motion artefacts were observed. The fluorescence signals of OGB-1 were expressed as F / F 0 ratios, F 0 representing the initial level of fluorescence and F the fluorescence signal recorded at individual time points during the experiment, respectively. Network construction Beta cells were distinguished from other cells on the basis of previous reports showing that cells within islets of Langerhans can reliably be identified by their type-specific calcium responses to stimulation with high glucose [46] – [48] . In cells identified as beta cells, intracellular concentration of calcium ([Ca 2+ ] i ) was low and rather stable under basal (6 mM) glucose. Upon stimulation with 12 mM glucose cells responded with a rapid increase in [Ca 2+ ] i , followed by a sustained plateau of elevated [Ca 2+ ] i with superimposed oscillations ( Figure 1B ). In different cells these oscillations had practically identical frequencies, but were slightly out of phase ( Figure 1C ). After the glucose was lowered back to 6 mM, [Ca 2+ ] i rapidly returned to the prestimulatory level. In further analyses, five different regimes will be considered: low glucose prior to stimulation (LG1), activation of beta cells (ON), high glucose regime (HG), deactivation of beta cells (OFF), and the low glucose after stimulation (LG2) ( Figure 1B ). Video S1 features responses of all cells in the islet. 10.1371/journal.pcbi.1002923.g001 Figure 1 Methodology used to extract functional connectivity patterns from cytosolic Ca 2+ traces. A Image of an islet of Langerhans showing the relative intensity of fluorescence signal during sustained activity (HG). Red circles indicate two cells, i and j , which we regard in continuation. B Temporal evolution of global calcium activity characterized by the mean-field of all beta cells in the islet. In the intervals 0≤ t ≤150 and 754≤ t ≤1960 cells were exposed to 6 mM glucose, whereas for 150≤ t ≤754 a stimulating concentration of glucose (12 mM) was applied. Arrows above the temporal trace denote five different dynamical regimes considered in this study: low glucose prior to stimulation (LG1) – 0≤ t