Oct 09, 2025
CALCIUM IMAGING PROTOCOL
- Carmen Perez De Nanclares1
- 1University of Minnesota
- Team Lee
Protocol Citation: Carmen Perez De Nanclares 2025. CALCIUM IMAGING PROTOCOL. protocols.io https://dx.doi.org/10.17504/protocols.io.e6nvw4k22lmk/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: July 21, 2025
Last Modified: October 10, 2025
Protocol Integer ID: 223838
Keywords: ASAPCRN, electrophysiology, calcium imaging protocol, calcium imaging protocol this protocol detail, activity in astrocyte, astrocyte, ca1 region, stratum radiatum of the ca1 region, mouse hippocampus, imaging
Abstract
This protocol details Ca2+ imaging to analyze activity in astrocytes of the stratum radiatum of the CA1 region of the mouse hippocampus.
Attachments
Troubleshooting
Locate Ca2+ levels in astrocytes in the stratum radiatum of the CA1 region of the hippocampus are monitored using the Ca2+ indicator fluo-4.
Load Astrocytes with the dye by incubating the slices with fluo-4-AM (2 micromolar (µM) and 0.01% of pluronic) for 00:45:00 -01:00:00 at Room temperature (Araque et al. 2002; Kang et al. 1998; Nemani et al. 2010; Nett et al. 2002; Navarrete and Araque 2008).
1h 45m
This protocol has been proven to be suitable for measuring the calcium activity of astrocytes (Nanclares et al. 2023).
Briefly, fluo-4-positive cells present typical electrophysiological properties for astrocytes, this is, low input resistance, a quasi-linear voltage–current relationship under voltageclamped conditions, and absence of action potentials.
On the contrary, recordings from fluo-4-negative cells in the stratum radiatum exhibit typical neuronal characteristics, such as higher input resistances, a non-linear voltage– current curve, and firing of action potentials.
Additionally, incubate the slices with fluo-4 and sulforhodamine 101 (SR101), a widely used astrocyte marker known to label astrocytes in the hippocampus (Schnell, Hagos, and Hülsmann 2012), provide good co-localization between SR101 and fluo-4-positive cells (Nanclares et al. 2023).
Perform the recordings in the presence of a cocktail of neurotransmitter receptor antagonists to minimize neuronal contribution to astrocyte Ca2+ activity (CNQX 20 micromolar (µM) , AP5 50 micromolar (µM) , MPEP 50 micromolar (µM) , LY367385 100 micromolar (µM) , picrotoxin 50 micromolar (µM) , CGP54626 1 micromolar (µM) , atropine 50 micromolar (µM) , CPT 2 micromolar (µM) , suramin 100 micromolar (µM) , flupenthixol 30 micromolar (µM) , AM251 2 micromolar (µM) and TTX 1 micromolar (µM) to block glutamatergic, GABAergic, cholinergic, purinergic, endocannabinoid and dopaminergic transmission).
Image Astrocytes using a custom-made confocal microscope (Thorlabs).
Obtain the videos at 512 × 512 resolution with a sampling interval of 00:00:01 .
1s
Use ImageJ software (NIH) for quantitative fluorescence measurements.
Estimate Ca2+ variations as changes in the fluorescence signal over baseline (DF/F0), and cells are considered to show a Ca2+ event when DF/F0 increases two times the standard deviation of the baseline.
Quantify the astrocyte Ca2+ signal as the number of Ca2+ events per min within 00:10:00 of recording.
10m
Obtain Astrocyte Ca2+ events from 5 to 24 astrocytes in the field of view during the recording.
Protocol references
Araque, Alfonso, Eduardo D. Martı́n, Gertrudis Perea, Jon I. Arellano, and Washington Buño. 2002. “Synaptically Released Acetylcholine Evokes Ca 2+ Elevations in Astrocytes in Hippocampal Slices.” The Journal of Neuroscience 22 (7): 2443–50. https://doi.org/10.1523/JNEUROSCI.22-07-02443.2002.
Kang, Jian, Li Jiang, Steven A. Goldman, and Maiken Nedergaard. 1998. “Astrocyte-Mediated Potentiation of Inhibitory Synaptic Transmission.” Nature Neuroscience 1 (8): 683–92. https://doi.org/10.1038/3684.
Nanclares, Carmen, Jonah Poynter, Hector A. Martell-Martinez, et al. 2023. “Dysregulation of Astrocytic Ca2+ Signaling and Gliotransmitter Release in Mouse Models of α-Synucleinopathies.” Acta Neuropathologica 145 (5): 597–610. https://doi.org/10.1007/s00401-023-02547-3.
Navarrete, Marta, and Alfonso Araque. 2008. “Endocannabinoids Mediate Neuron-Astrocyte Communication.” Neuron 57 (6): 883–93. https://doi.org/10.1016/j.neuron.2008.01.029.
Nemani, Venu M., Wei Lu, Victoria Berge, et al. 2010. “Increased Expression of α-Synuclein Reduces Neurotransmitter Release by Inhibiting Synaptic Vesicle Reclustering after Endocytosis.” Neuron 65 (1): 66–79. https://doi.org/10.1016/j.neuron.2009.12.023.
Nett, Wolfgang J., Scott H. Oloff, and Ken D. McCarthy. 2002. “Hippocampal Astrocytes In Situ Exhibit Calcium Oscillations That Occur Independent of Neuronal Activity.” Journal of Neurophysiology 87 (1): 528–37. https://doi.org/10.1152/jn.00268.2001.