Spatiotemporal characterization of [Ca²⁺]_i oscillations superimposed on the sustained plateau. A

<p>High resolution image (1024×256 pixels) that served as a reference to select regions of interest indicated in B and to assess possible motion artefacts. Scale bar indicates 20 micrometers. <b>B</b> Color-coded time delays for every cell demonstrating the average direction of spreading of [Ca²⁺]_i waves for 6 consecutive [Ca²⁺]_i oscillations. <b>C</b> 45 individual cells whose signals were included in the analyses. Temporal traces of highlighted cells indicated with 1–4 are plotted in D. <b>D</b> 3 consecutive [Ca²⁺]_i oscillations in 4 cells indicated in C. Scale bar indicates 2 seconds. On Y-axes, values represent normalized fraction of the difference between maximum and plateau baseline fluorescence. <b>E</b> Time delays between the beginning of a [Ca²⁺]_i oscillation in the cell in which the wave originated and the beginning of the [Ca²⁺]_i oscillation in any given cell as a function of the Euclidean distance between the cell of wave origin and the respective cell, for a single [Ca²⁺]_i oscillation in 45 cells from the islet shown in A-C. The regression line gives an average velocity of 92 µm s⁻¹ (R² = 0.76, p<0.001). <b>F</b> After taking into account 6 consecutive spikes in the same set of cells, the same average speed was obtained (R² = 0.73, p<0.001). <b>G</b> For 4 different islets, with 6 consecutive [Ca²⁺]_i oscillations in 10 cells from each islet, the average speed was 80 µm s⁻¹ (R² = 0.62, p<0.001). The respective values obtained in 4 islets were 98 µm s⁻¹ (R² = 0.83, p<0.001), 88 µm s⁻¹ (R² = 0.34, p<0.001), 80 µm s⁻¹ (R² = 0.40, p<0.001), and 74 µm s⁻¹ (R² = 0.46, p<0.001). In Figures E-G, x and y axes are chosen as to enable representation of speed by the slope of the regression lines. However, velocities were calculated with distances representing the independent and time lags being the dependent variable.</p

Spatiotemporal [Ca²⁺]_i patterns in a representative islet of Langerhans upon stimulation with 12 mM glucose. A

<p>A high resolution image, used as a reference to choose regions of interest corresponding to individual cells and to assess motion artefacts, showing that Oregon Green® 488 BAPTA-1 effectively labeled most of the cells within the focal plane. Scale bar indicates 50 micrometers. <b>B</b> A schematic color-coded representation of the position of cells in A that responded to stimulation with 12 mM glucose as shown in C–E (N = 177 cells). The grey area indicates unlabelled or unresponsive cells. We detected six different types of responses to glucose. The types of responses presented in C and D were predominant and are characteristic of beta cells. <b>C</b> Slow transient response followed by oscillations superimposed on a sustained plateau (type 1, N = 63 cells). Note the difference in time required for activation between the upper and lower trace, the synchronicity of Ca²⁺ oscillations superimposed on the sustained plateau and of deactivation, as well as the presence of a transient increase in Ca²⁺ in the lower trace after the sustained plateau has subsided. <b>D</b> Response as in C but without a clear transient phase (type 2, N = 61 cells). <b>E–H</b> The responses representative of non typical beta cells and non beta cells (see text for further details). <b>I</b> The average time course of fluorescence over the whole islet. Due to synchronicity of Ca²⁺ oscillations in individual beta cells, the oscillations are clearly distinguishable in the average signal. Scale bar indicates 200 seconds. On Y-axes, values represent normalized fraction of the difference between maximum and basal fluorescence.</p

Glucose-Stimulated Calcium Dynamics in Islets of Langerhans in Acute Mouse Pancreas Tissue Slices

<div><p>In endocrine cells within islets of Langerhans calcium ions couple cell stimulation to hormone secretion. Since the advent of modern fluorimetry, numerous <em>in vitro</em> studies employing primarily isolated mouse islets have investigated the effects of various secretagogues on cytoplasmic calcium, predominantly in insulin-secreting beta cells. Due to technical limitations, insights of these studies are inherently limited to a rather small subpopulation of outermost cells. The results also seem to depend on various factors, like culture conditions and duration, and are not always easily reconcilable with findings <em>in vivo</em>. The main controversies regard the types of calcium oscillations, presence of calcium waves, and the level of synchronized activity. Here, we set out to combine the <em>in situ</em> acute mouse pancreas tissue slice preparation with noninvasive fluorescent calcium labeling and subsequent confocal laser scanning microscopy to shed new light on the existing controversies utilizing an innovative approach enabling the characterization of responses in many cells from all layers of islets. Our experiments reproducibly showed stable fast calcium oscillations on a sustained plateau rather than slow oscillations as the predominant type of response in acute tissue slices, and that calcium waves are the mechanistic substrate for synchronization of oscillations. We also found indirect evidence that even a large amplitude calcium signal was not sufficient and that metabolic activation was necessary to ensure cell synchronization upon stimulation with glucose. Our novel method helped resolve existing controversies and showed the potential to help answer important physiological questions, making it one of the methods of choice for the foreseeable future.</p> </div

Summary of types of responses to stimulation with 12 mM glucose.

<p>(+) and (−) indicate presence and absence of the specified phenomenon. (++) indicates an increase in frequency.</p

Spatiotemporal characterization of deactivation of beta cells. A

<p>Deactivation of beta cells in the islet from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054638#pone-0054638-g002" target="_blank">Figure 2</a>. Images are presented as F/F₀ and pseudocolored with blue and white representing low and high intensity signals, respectively. Scale bar indicates 50 µm. <b>B</b> Regions that deactivated at a certain time are indicated as red areas that are not present in subsequent images. Differences between individual cells inside these regions were not resolvable at the recording rate of 0.5 Hz. No regular spatial pattern of deactivation is visible. <b>C</b> Time traces showing delays in deactivation for the regions indicated in B. Scale bar indicates 20 seconds. On Y-axes, values represent normalized fraction of the difference between maximum and basal fluorescence. <b>D</b> Time delays from the end of stimulation to deactivation for 43 cells from this islet (median = 274 s, 1^st quartile = 270 s, and 3^rd quartile = 274 s). <b>E</b> Cumulative distribution of time delays from the end of stimulation to deactivation for 43 cells from this islet. First regions deactivated 256 seconds after lowering glucose to 6 mM. Within the following 36 seconds all other groups of cells deactivated. A half of cells deactivated within 17 seconds after deactivation of the first cell. <b>F</b> Distribution of time delays from the end of stimulation to deactivation of the first cell in each islet for 10 different islets (median = 195 s, 1^st quartile = 151 s, and 3^rd quartile = 261 s). <b>G</b> Distribution of time delays from deactivation of the first cell in each islet to deactivation of any given cell from the same islet for 366 cells from 11 different islets (median = 18 s, 1^st quartile = 9 s, and 3^rd quartile = 24 s). <b>H</b> Cumulative distribution of time delays in G. A half of cells deactivated within 22 seconds after the first. All cells deactivated within 45 seconds after the first.</p

Spatiotemporal characterization of activation of beta cells. A

<p>Activation of beta cells. Images are presented as F/F₀ and pseudocolored with blue and white representing low and high intensity signals, respectively. Scale bar indicates 50 µm. Numbers indicate time after beginning of stimulation in seconds. <b>B</b> Regions that activated at a certain time are indicated as black-bordered red areas and regions already activated with red only. Differences between individual cells inside these regions were not resolvable at the recording rate of 0.5 Hz. No regular spatial pattern of activation is visible. <b>C</b> Time traces for the regions indicated in B. Scale bar indicates 20 seconds. On Y-axes, values represent normalized fraction of the difference between maximum and basal fluorescence. <b>D</b> Distribution of time delays from the beginning of stimulation to activation for 78 cells from this islet (median = 128 s, 1^st quartile = 115 s, 3^rd quartile = 138 s). <b>E</b> Cumulative distribution of time delays from the beginning of stimulation to activation in this islet. First regions responded 80 seconds after the rise in glucose. Within the following 76 seconds all other groups of cells activated. A half of cells activated within 42 seconds after the activation of the first cell. <b>F</b> Distribution of time delays from the beginning of stimulation to activation of the first cell in each islet for 17 different islets (median = 93 s, 1^st quartile = 72 s, 3^rd quartile = 135 s). <b>G</b> Distribution of time delays from activation of the first cell in each islet to activation of any given cell from the same islet for 700 cells from 17 different islets (median = 41 s, 1^st quartile = 24 s, 3^rd quartile = 62 s). <b>H</b> Cumulative distribution of time delays shown in G. A half of cells activated within 40 seconds after the activation of the first cell. All cells activated within 312 seconds after the first.</p

Characterization of durations of [Ca²⁺]_i oscillations superimposed on the sustained plateau. A

<p>Representative traces of 3 subsequent [Ca²⁺]_i oscillations in 15 cells analyzed in B and C. Scale bar represents 5 seconds. On Y-axes, values represent normalized fraction of the difference between maximum and plateau baseline fluorescence. <b>B</b> Distribution of durations of Ca²⁺ oscillations for 15 subsequent [Ca²⁺]_i oscillations in 15 cells from a single islet presented for individual cells. Note that in every cell, roughly the same range of interval durations is present. The box plot indicated with “All cells” shows the distribution of interval durations pooled for 15 subsequent [Ca²⁺]_i oscillations in 15 cells from the same islet (median = 3.5 s, 1^st quartile = 2.9 s, 3^rd quartile = 4.1 s). The box plot indicated with “All islets” shows the distribution of durations for all [Ca²⁺]_i oscillations in 6 different islets (median = 2.2 s, 1^st quartile = 1.8 s, 3^rd quartile = 3.4 s). <b>C</b> Distribution of durations of [Ca²⁺]_i oscillations for 15 subsequent [Ca²⁺]_i oscillations in 15 cells from a single islet, presented for every individual [Ca²⁺]_i oscillation. Note that the durations vary from one oscillation to another in roughly the same way in all analyzed cells. <b>D</b> Time-variability of the duration of [Ca²⁺]_i oscillations in 6 islets of Langerhans over a period of 120 seconds, shown as the distribution of durations of [Ca²⁺]_i oscillations in four subsequent 30-seconds-long time intervals. For each of the 6 different islets median durations of [Ca²⁺]_i oscillations were calculated for each of the 30-seconds-long time intervals and compared with each other by employing nonparametric Friedmańs analysis of variance. The duration of [Ca²⁺]_i oscillations did not significantly change over the four time intervals.</p

Characterization of frequencies of [Ca²⁺]_i oscillations superimposed on the sustained plateau. A

<p>Representative traces of 3 subsequent [Ca²⁺]_i oscillations in 15 cells analyzed in B and C. Scale bar represents 5 seconds. On Y-axes, values represent normalized fraction of the difference between maximum and plateau baseline fluorescence. <b>B</b> Distribution of durations of intervals between consecutive [Ca²⁺]_i oscillations for 14 subsequent pairs of [Ca²⁺]_i oscillations in 15 cells from a single islet, presented for individual cells. In every cell, roughly the same range of interval durations is present. The box plot indicated with “All cells” shows the distribution of interval durations pooled for 14 subsequent pairs of [Ca²⁺]_i oscillations in 15 cells from the same islet (median = 13 s, 1^st quartile = 10 s, 3^rd quartile = 14 s). The box plot indicated with “All islets” shows the distribution of intervals for all pairs of [Ca²⁺]_i oscillations in 12 different islets (median = 12 s, 1^st quartile = 10 s, 3^rd quartile = 16 s). <b>C</b> Distribution of interval durations for 14 subsequent pairs of [Ca²⁺]_i oscillations in 15 cells from a single islet, presented for every pair of consecutive [Ca²⁺]_i oscillations. Note that intervals change from oscillation to oscillation in roughly the same way in every analyzed cell. <b>D</b> Time-variability of the interval durations in a single islet of Langerhans over a period of 500 seconds. Note the absence of any clear trend towards higher or lower frequencies with time. <b>E</b> Time-variability of the interval in 12 islets of Langerhans over a period of 500 seconds, shown as the distribution of intervals between [Ca²⁺]_i oscillations for five subsequent 100-seconds-long time intervals. For each of the 12 different islets median interval durations were calculated for each of the 100-seconds-long time intervals and compared with each other by employing nonparametric Friedmańs analysis of variance. The duration of intervals between two consecutive [Ca²⁺]_i oscillations did not significantly change over the five time intervals.</p

Alpha-MSH and pro-opiomelanocortin in Rab3a KO melanotrophs.

<p>Representative confocal microscopy micrographs from immunocytochemistry of γ-MSH/ pro-opiomelanocortin (POMC) (top) and α-MSH (bottom) in WT and Rab3a KO pituitary slices. Arrow indicates the intermediate lobe (PI). AP=anterior part, PP=posterior part. Rab3a KO melanotrophs contained POMC, but lacked α-MSH positive signal. </p

Initial high Ca²⁺ sensitive phase in Rab3a KO melanotrophs.

<p><i>A</i>-<i>C</i>, slow photo-release of caged Ca²⁺ triggered a multiphasic ΔC_m. The marked area is magnified in panel C. <i>D</i>, [Ca²⁺]_i threshold. <i>E</i>, cumulative ΔC_m. <i>F</i>, time derivative of the ΔC_m presented in B as a function of [Ca²⁺]_i during the first 2 s of the slow photo-release of caged Ca²⁺. Note that high Ca²⁺ sensitive phase (vesicle pool) was greatly reduced in Rab3a KO cells (arrow), and that Ca²⁺ triggered ΔC_m at significantly higher [Ca²⁺]_i in Rab3a KO melanotrophs compared to WT cells (inset); the inset shows normalized C_m rate fitted by the Hill function and is displayed as a function of [Ca²⁺]_i. <i>G</i>, half effective [Ca²⁺]_i (EC₅₀). <i>H</i>, amplitude of high Ca²⁺ sensitive pool (HCSP) quantified at 1.5 µM [Ca²⁺]_i . Numbers on bars indicate the number of tested cells. ^∗<i>P</i><0.05 versus WT.</p