Figure 1.
Simultaneous recording of membrane potential oscillations occurring in the patch-clamped cell and [Ca2+]i oscillations occurring in other cells of the same islet of Langerhans.
A [Ca2+]i oscillations were measured with a CCD camera at a temporal resolution of 2 Hz in cells in the tissue slice loaded with OGB-1. The patch-clamp pipette position is indicated with white lines. B During stimulation with 12 mM glucose, [Ca2+]i oscillations spread across the plane of the islet (from the lower right to the upper left corner in this figure). Responses of cells shown in A during a single [Ca2+]i oscillation are color-coded to represent time lags between the onset of [Ca2+]i increase in the cell where the visible part of the oscillation started and every other cell. Due to temporal resolution (2 Hz), cells were grouped into 4 groups only. The position of the patch-clamp pipette and the patch-clamped cell is indicated. The hatched pattern in the figure and legend bar indicates patch-clamp Data. C Two consecutive [Ca2+]i (upper panel) and membrane potential (lower panel) oscillations during stimulation with glucose. Each [Ca2+]i oscillation was accompanied by a membrane potential change consisting of a depolarization with superimposed spikes followed by a repolarization. Membrane potential was recorded at a temporal resolution of 1000 Hz. D A more detailed representation of the area indicated in C. The onset of high frequency spikes in the patched cell is temporally best correlated with the onset of [Ca2+]i oscillation in the group of cells indicated in orange. For this group of cells the distance to oscillation origin was roughly the same as for the patched cell. E Time delays between the first spike in the patch-clamped cell and the onset of [Ca2+]i oscillation in every other cell. Shortest time delays correspond to the orange group of cells for which the distance to wave origin was roughly the same as for the patch-clamped cell. Color coding as in B-D.
Figure 2.
[Ca2+]i oscillations during stimulation with 12 mM glucose.
A A high resolution image of an islet of Langerhans showing cells loaded with the Oregon Green 488 BAPTA-1 (OGB-1) Ca2+ indicator. B Time lags between the start50 of [Ca2+]i oscillation in the first and every other cell during stimulation with 12 mM glucose are color-coded. Note a wave-like spread of [Ca2+]i increase from left to right in this focal plane. C A single oscillation from three cells indicated in B during stimulation with 12 mM glucose. Gray rectangular areas indicate time intervals for increase and decrease of [Ca2+]i across different cells. Arrows indicate the duration of oscillations at their half-maximal amplitude. Y axis represents the normalized fraction of the difference between maximum and plateau baseline fluorescence. D A pseudo-colored representation of time lags between oscillation end50 in the cell in which [Ca2+]i decreased first and every other cell. Note a wave-like spread of [Ca2+]i decrease from left to right in this focal plane. E Trajectories describing the wave path of the oscillation start50 (blue, dashed line) and end50 (red, dashed line). The trajectories describing average directions of spreading are drawn with solid lines. Note that the wave of [Ca2+]i decrease roughly follows the wave of [Ca2+]i increase. F Average wave trajectories of oscillation ends50 (red) with respect to oscillation starts50 (blue) for a single oscillation from 6 different islets of Langerhans. Numbers indicate angles in degrees. Note that the wave-fronts of ends50 spread in roughly the same direction as the wave-fronts of starts50 in most cases. Sampling rate was 50 Hz at a spatial resolution of 256x64 pixels.
Figure 3.
Membrane potential oscillations during stimulation with 12 mM glucose.
A The VF dye labeled the majority of membranes in this islet of Langerhans from an acute mouse pancreas tissue slice. B During stimulation with 12 mM glucose, a wave of depolarization spread across the islet plane from left to right. Time lags between the start50 of membrane potential oscillation in the first cell and the rest of the cells are color-coded. C The oscillatory behavior during stimulation consisted of depolarizations and repolarizations. A single wave is shown; numbers denote cells´ spatial positions indicated in A. Gray rectangular areas indicate time intervals for depolarizations and repolarizations across different cells. Arrows indicate the duration of oscillations at their half-maximal amplitudes. Y axis represents the normalized fraction of the difference between maximum and plateau baseline fluorescence. D In general, the spreading of ends50 of oscillations did not take the same route as the starts50 of membrane potential oscillations. Time lags are color-coded. E Trajectories (dashed lines) and the average trajectory (solid line) for the start50 (blue) and end50 (red) of the membrane potential oscillation. D Repolarization did not follow any clearly predictable pattern in the 6 islets. Shown are angles of average trajectories of oscillation ends50 (red) relative to the respective starts50 trajectory (blue). Sampling rate was 5 Hz at a spatial resolution of 512x256 pixels.
Figure 4.
Durations of oscillations and velocities of waves during stimulation with 12 mM glucose are comparable for the [Ca2+]i and membrane potential signals.
A Durations of [Ca2+]i oscillations at their half-maximal amplitude in randomly picked cells from 7 islets assessed by OGB-1 (OGB-1, 1st quartile=1.4 s, median=1.9 s, 3rd quartile=2.2 s, n=7 islets), of membrane potential oscillations in randomly picked cells from 7 islets assessed by VF (VF, 1st quartile=1.8 s, median=2.4 s, 3rd quartile=3.2 s, n=7 islets) and of membrane potential oscillations assessed in 7 whole-cell patch-clamped cells (PATCH, 1st quartile=1.6 s, median=2.1 s, 3rd quartile=2.5 s, n=7 islets) are not statistically significantly different (Kruskal-Wallis test). B Median membrane potential wave velocities (VF: 1st quartile=55 µm/s, median=69 µm/s, 3rd quartile=97 µm/s, n=7) and [Ca2+]i wave velocities (OGB-1: 1st quartile=54 µm/s, median=89 µm/s, 3rd quartile=139 µm/s, n=6) were not statistically significantly different (Mann-Whitney test).
Figure 5.
[Ca2+]i and membrane potential oscillations during stimulation with 12 mM glucose plus 10 mM tetraethylammonium (TEA).
A A high resolution image showing loading of cells with the calcium-sensitive dye OGB-1. B A color-coded representation of responsive cells from A with respect to time lags between the start50 of [Ca2+]i oscillation in the first cell and every other cell. C Time trace of a [Ca2+]i oscillation during stimulation with 12 mM glucose plus 10 mM TEA in the cell indicated in B. D A high resolution image showing labeling of cells with the voltage-sensitive dye VF. Note that almost exclusively plasma membranes are labeled. E A color-coded representation of responsive cells from D with respect to time lags between the start50 of membrane potential oscillation in the first cell and every other cell. F Time trace of a membrane potential oscillation during stimulation with 12 mM glucose plus 10 mM TEA in the cell indicated in D. The time scale is the same for C and F. Y axis represents the normalized fraction of the difference between maximum and plateau baseline fluorescence. G Durations of [Ca2+]i oscillations (OGB-1: 1st quartile=135 ms, median=166 ms, 3rd quartile=248 ms, n=57 cells from three islets) and membrane potential oscillations (VF: 1st quartile=81 ms, median=101 ms, 3rd quartile= 162 ms, n=107 cells from five islets) at their half-maximal amplitude. Asterisks indicate a statistically significant difference (p<0.001, Mann-Whitney rank sum test). H Velocities of [Ca2+]i waves (OGB-1: 1st quartile=372 µm/s, median=623 µm/s, 3rd quartile=1058 µm/s, n=8 values from three islets) and membrane potential waves (VF: 1st quartile=457 µm/s, median=577 µm/s, 3rd quartile=857 µm/s, n=12 values from five islets) were not statistically significantly different. Resolution was 256 x 64 pixels at 97 Hz for OGB-1 and 256x128 at 99Hz for VF.
Figure 6.
Simultaneous recording of [Ca2+]i and membrane potential oscillations during stimulation with 12 mM glucose.
A VF (left panel) stained mostly cell membranes while Rhod-2 (middle panel) stained cell cytoplasms, without any significant co-localization of the two dyes in the active regions (right panel; see also Figure S4). B A color-coded representation of selected responsive cells with respect to the time lag between the oscillation start50 of the first cell and every other cell for the VF signal (left panel), Rhod-2 signal (middle panel) and merge of the two (right panel). The outline stands for the VF signal and the interior for Rhod-2. Both membrane potential and [Ca2+]i wave roughly spread from top to bottom of the focal plane. Note that in any given cell, the membrane signal precedes (colder color) the [Ca2+]i signal, indicating that depolarization precedes the increase in [Ca2+]i. C Time traces of the VF and Rhod-2 signals from three cells indicated in the right panel of B show a wave-like spreading of both signals. In all cells, the VF signal clearly preceded the Rhod-2 signal. Arrows indicate the widths of oscillations at their half-maximal amplitude (see Methods). Y axis represents the normalized fraction of the difference between maximum and plateau baseline fluorescence. D Average trajectory for [Ca2+]i oscillation starts50 (solid line) and ends50 (dashed line). E Average trajectory for the membrane potential oscillation starts50 (solid line) and ends50 (dashed line). In D and E, numbers indicate angles in degrees. F Time lag between ends50 (1st quartile=284 ms, median= 360 ms, 3rd quartile=473 ms) was significantly larger than the time lag between starts50 (1st quartile=133 ms, median=170 ms, 3rd quartile=189 ms, n=21 cells) of the Rhod-2 and VF signals. Asterisks indicate p<0.001 (Mann-Whitney test). Resolution was 256x128 pixels at 52 Hz.
Figure 7.
Simultaneous recording of [Ca2+]i and membrane potential oscillations during stimulation with 12 mM glucose plus 10 mM tetraethylammonium (TEA).
A The voltage sensitive dye VF (upper panel) stained mostly cell membranes, whereas the calcium indicator Rhod-2 (middle panel) stained cytoplasms. For the active cells no significant co-localization was observed (lower panel). B Oscillation in the cell indicated with red asterisk in A during stimulation with 12 mM glucose plus 10 mM TEA. Arrows indicate the widths of oscillations at their half-maximal amplitude. Y axis represents the normalized fraction of the difference between maximum and plateau baseline fluorescence. C The delay between start50 of the Rhod-2 and VF signals (1st quartile=13 ms, median=20 ms, 3rd quartile=36 ms, n=11 cells from 4 islets) was statistically significantly shorter than the delay between end50 of the Rhod-2 and VF signals (1st quartile=150 ms, median=212 ms, 3rd quartile=239 ms, n=11 cells from 4 islets). Asterisks indicate p<0.001 (Mann-Whitney test). Resolution was 128x64 pixels at 170 Hz.