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Imaging Calcium Dynamics in Subpopulations of Mouse Pancreatic Islet Cells

Hamilton, Alexander LU ; Vergari, Elisa ; Miranda, Caroline and Tarasov, Andrei I. (2019) In Journal of visualized experiments : JoVE
Abstract

Pancreatic islet hormones regulate blood glucose homeostasis. Changes in blood glucose induce oscillations of cytosolic calcium in pancreatic islet cells that trigger secretion of three main hormones: insulin (from β-cells), glucagon (α-cells) and somatostatin (δ-cells). β-Cells, which make up the majority of islet cells and are electrically coupled to each other, respond to the glucose stimulus as one single entity. The excitability of the minor subpopulations, α-cells and δ-cells (making up around 20% (30%) and 4% (10%) of the total rodent1 (human2) islet cell numbers, respectively) is less predictable and is therefore of special interest. Calcium sensors are delivered into the peripheral layer of cells within the isolated islet. The... (More)

Pancreatic islet hormones regulate blood glucose homeostasis. Changes in blood glucose induce oscillations of cytosolic calcium in pancreatic islet cells that trigger secretion of three main hormones: insulin (from β-cells), glucagon (α-cells) and somatostatin (δ-cells). β-Cells, which make up the majority of islet cells and are electrically coupled to each other, respond to the glucose stimulus as one single entity. The excitability of the minor subpopulations, α-cells and δ-cells (making up around 20% (30%) and 4% (10%) of the total rodent1 (human2) islet cell numbers, respectively) is less predictable and is therefore of special interest. Calcium sensors are delivered into the peripheral layer of cells within the isolated islet. The islet or a group of islets is then immobilized and imaged using a fluorescence microscope. The choice of the imaging mode is between higher throughput (wide-field) and better spatial resolution (confocal). Conventionally, laser scanning confocal microscopy is used for imaging tissue, as it provides the best separation of the signal between the neighboring cells. A wide-field system can be utilized too, if the contaminating signal from the dominating population of β-cells is minimized. Once calcium dynamics in response to specific stimuli have been recorded, data are expressed in numerical form as fluorescence intensity vs. time, normalized to the initial fluorescence and baseline-corrected, to remove the effects linked to bleaching of the fluorophore. Changes in the spike frequency or partial area under the curve (pAUC) are computed vs. time, to quantify the observed effects. pAUC is more sensitive and quite robust whereas spiking frequency provides more information on the mechanism of calcium increase. Minor cell subpopulations can be identified using functional responses to marker compounds, such as adrenaline and ghrelin, that induce changes in cytosolic calcium in a specific populations of islet cells.

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organization
publishing date
type
Contribution to journal
publication status
published
subject
in
Journal of visualized experiments : JoVE
issue
153
article number
e59491
publisher
JoVE
external identifiers
  • pmid:31840667
  • scopus:85076523399
ISSN
1940-087X
DOI
10.3791/59491
language
English
LU publication?
yes
id
60c3445c-f9b7-4dd9-a146-1c4c46e92a04
date added to LUP
2020-01-13 12:23:40
date last changed
2020-01-14 01:57:00
@article{60c3445c-f9b7-4dd9-a146-1c4c46e92a04,
  abstract     = {<p>Pancreatic islet hormones regulate blood glucose homeostasis. Changes in blood glucose induce oscillations of cytosolic calcium in pancreatic islet cells that trigger secretion of three main hormones: insulin (from β-cells), glucagon (α-cells) and somatostatin (δ-cells). β-Cells, which make up the majority of islet cells and are electrically coupled to each other, respond to the glucose stimulus as one single entity. The excitability of the minor subpopulations, α-cells and δ-cells (making up around 20% (30%) and 4% (10%) of the total rodent1 (human2) islet cell numbers, respectively) is less predictable and is therefore of special interest. Calcium sensors are delivered into the peripheral layer of cells within the isolated islet. The islet or a group of islets is then immobilized and imaged using a fluorescence microscope. The choice of the imaging mode is between higher throughput (wide-field) and better spatial resolution (confocal). Conventionally, laser scanning confocal microscopy is used for imaging tissue, as it provides the best separation of the signal between the neighboring cells. A wide-field system can be utilized too, if the contaminating signal from the dominating population of β-cells is minimized. Once calcium dynamics in response to specific stimuli have been recorded, data are expressed in numerical form as fluorescence intensity vs. time, normalized to the initial fluorescence and baseline-corrected, to remove the effects linked to bleaching of the fluorophore. Changes in the spike frequency or partial area under the curve (pAUC) are computed vs. time, to quantify the observed effects. pAUC is more sensitive and quite robust whereas spiking frequency provides more information on the mechanism of calcium increase. Minor cell subpopulations can be identified using functional responses to marker compounds, such as adrenaline and ghrelin, that induce changes in cytosolic calcium in a specific populations of islet cells.</p>},
  author       = {Hamilton, Alexander and Vergari, Elisa and Miranda, Caroline and Tarasov, Andrei I.},
  issn         = {1940-087X},
  language     = {eng},
  month        = {11},
  number       = {153},
  publisher    = {JoVE},
  series       = {Journal of visualized experiments : JoVE},
  title        = {Imaging Calcium Dynamics in Subpopulations of Mouse Pancreatic Islet Cells},
  url          = {http://dx.doi.org/10.3791/59491},
  doi          = {10.3791/59491},
  year         = {2019},
}