Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2024 Jan 23:12:1320672.
doi: 10.3389/fcell.2024.1320672. eCollection 2024.

Dynamics of astrocytes Ca2+ signaling: a low-cost fluorescence customized system for 2D cultures

Rosa Musotto  1 , Ulderico Wanderlingh  2 , Angela D'Ascola  3 , Michela Spatuzza  4 , Maria Vincenza Catania  4 , Maurizio De Pittà  5   6   7   8 , Giovanni Pioggia  1
Affiliations

Dynamics of astrocytes Ca2+ signaling: a low-cost fluorescence customized system for 2D cultures

Rosa Musotto et al. Front Cell Dev Biol. .

Abstract

In an effort to help reduce the costs of fluorescence microscopy and expand the use of this valuable technique, we developed a low-cost platform capable of visualising and analysing the spatio-temporal dynamics of intracellular Ca2+ signalling in astrocytes. The created platform, consisting of a specially adapted fluorescence microscope and a data analysis procedure performed with Imagej Fiji software and custom scripts, allowed us to detect relative changes of intracellular Ca2+ ions in astrocytes. To demonstrate the usefulness of the workflow, we applied the methodology to several in vitro astrocyte preparations, specifically immortalised human astrocyte cells and wild-type mouse cells. To demonstrate the reliability of the procedure, analyses were conducted by stimulating astrocyte activity with the agonist dihydroxyphenylglycine (DHPG), alone or in the presence of the antagonist 2-methyl-6-phenylethyl-pyridine (MPEP).

Keywords: analysis; astrocytes; calcium waves; customized system; fluorescence.

PubMed Disclaimer

Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

FIGURE 1
FIGURE 1
Specially adapted inverted fluorescence microscope (A) Picture of the adapted microscope and its supplements. (B) Detail of the positioning of the dichroic mirror (C) Housing of the adapted microscope inside the incubator.
FIGURE 2
FIGURE 2
Fluorescence of intracellular Ca2+ signal of astrocytes detected by time-lapse methodology. (A) Fluorescence of a sample of mouse astroglial cells detected in time-lapse of one frame every 5 s (PCLWDCD20XPL NA 0,40 W.D.5,4 mm). (B) Fluorescence of a sample of human astrocyte cells detected in time-lapse of one frame every 5 s (PCD10XPL NA 0,25 W.D. 7,2 mm). (C) Example of some time-lapse frames of the cell circled in B. Using time-lapse technology, it is possible to observe the increase in intracellular calcium and its diffusion through astrocytes in real time. (D) Representative trace of Ca2+ in the cell underlined in red in B showing temporal changes in the signal.
FIGURE 3
FIGURE 3
Fluorescence image of intracellular Ca2+ of human astrocytes. To increase the visibility of low-contrast features (A) and help the human eye to compare different images by enhancing the differences in intensity of the samples, a look-up table was applied (B).
FIGURE 4
FIGURE 4
Sum of the individual images of a time series. (A) Construction of a “slice sum”; all pixels with the same xy coordinates are summed up. The time dimension is used as the z-direction (B) Original individual stack (C) Sum of the individual images of a time series.
FIGURE 5
FIGURE 5
The average intensities of the selected regions of interest (ROI). ROI#1: drawn on the entire astrocytic cell; ROI#3 and ROI#4 on the sides of the astrocytic cell; ROI#2 in the centre of the astrocytic cell. The graphs of the fluorescence versus time of the individual 4 ROIs, shown in the figure, highlighted that whatever is measured within the astrocytic cell, the Ca2+ signal is both evident and comparable.
FIGURE 6
FIGURE 6
The TP calculation algorithm. We apply the TP method to the raw fluorescence intensity data of intracellular Ca2+ waves of astrocyte cells, however it is possible to apply the method to any type of raw data. (A) Example of a curve on which to apply the TP algorithm (B) Identification of the maximum points of the distribution. (C) Calculation of the lowest contour that encircles each maximum but not any higher peaks. (D) Calculation of TP as the relative height of each maximum above each baseline. (E) Example of raw recording of a Ca2+ signal in immortalized human astrocytes (F) Example of Ca2+ peak determination in immortalized human astrocytes after application of the Topographic Prominence algorithm.
FIGURE 7
FIGURE 7
Fluorescence images of immortalised human astrocytes labelled with Fluo-8. (A) Identification of astrocytic cells and tracing of regions of interest (ROI). (B) Representative fluorescence intensity profiles showing spontaneous intracellular Ca2+ activity in immortalised human astrocytes under basal conditions. (C) 3D analysis of intensity intracellular Ca2+ in astrocytes (D) Intracellular Ca2+ peaks determined by prominence calculation by means of a Gnuplot script.
FIGURE 8
FIGURE 8
Histogram showing the number of spikes per second vs. time of intracellular Ca2+ events in: (A) immortalized human astrocytes under basal conditions. (B) WT mouse astrocytes under basal conditions.
FIGURE 9
FIGURE 9
Sequence of fluorescence intensity profiles of intracellular Ca2+ in Wild Type (WT) mouse astrocytes (A) WT mouse astrocytes under basal condition (B) WT mouse astrocytes treated with 10 μM the selective metabotropic glutamate receptor agonist 3,5-dihydroxyphenylglycine (DHPG) mGluR5. (C) WT mouse astrocytes were pre-treated with 50 μM 2-methyl-6-(phenylethynyl)pyridine (MPEP), a selective antagonist of the metabotropic glutamate receptor subtype mGluR5, for 15 min and then exposed to the DHPG agonist.
See this image and copyright information in PMC

Similar articles

See all similar articles

References

    1. Allaman I., Bélanger M., Magistretti P. J. (2011). Astrocyte–neuron metabolic relationships: for better and for worse. Trends Neurosci. 34 (2), 76–87. 10.1016/j.tins.2010.12.001 - DOI - PubMed
    1. Armbruster M., Naskar S., Garcia J. P., Sommer M., Kim E., Adam Y., et al. (2022). Neuronal activity drives pathway-specific depolarization of peripheral astrocyte processes. Nat. Neurosci. 25 (5), 607–616. 10.1038/s41593-022-01049-x - DOI - PMC - PubMed
    1. Barres B. A. (2008). The mystery and magic of glia: a perspective on their roles in health and disease. Neuron 60 (3), 430–440. 10.1016/j.neuron.2008.10.013 - DOI - PubMed
    1. Basarsky T. A., Parpura V., Haydon P. G. (1994). Hippocampal synaptogenesis in cell culture: developmental time course of synapse formation, calcium influx, and synaptic protein distribution. J. Neurosci. 14 (11), 6402–6411. 10.1523/JNEUROSCI.14-11-06402.1994 - DOI - PMC - PubMed
    1. Berridge M. J., Bootman M. D., Roderick H. L. (2003). Calcium signalling: dynamics, homeostasis and remodelling. Nat. Rev. Mol. Cell Biol. 4 (7), 517–529. 10.1038/nrm1155 - DOI - PubMed

Grants and funding

The author(s) declare financial support was received for the research, authorship, and/or publication of this article. This work was funded by the NUTR-AGE—“Nutrizione, Alimentazione and Invecchiamento Attivo” project of the National Research Council of Italy (CNR).