Jan 29, 2025
Imaging axonal calcium dynamics in ex vivo mouse brain slices
- Yan-Feng Zhang1,2,
- Stephanie J Cragg3,4
- 1Department of Clinical and Biomedical Sciences, University of Exeter, Exeter, United Kingdom;
- 2Aligning Science Across Parkinson's (ASAP) Collaborative Research Network, Chevy Chase, MD, USA;
- 3Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford OX1 3PT, UKAligning Science Across Parkinson's (ASAP) Collaborative Research Network, Chevy Chase, MD, USA;
- 4Oxford Parkinson’s Disease Centre, University of Oxford, Oxford, United Kingdom
Protocol Citation: Yan-Feng Zhang, Stephanie J Cragg 2025. Imaging axonal calcium dynamics in ex vivo mouse brain slices. protocols.io https://dx.doi.org/10.17504/protocols.io.81wgbx9eolpk/v1
Manuscript citation:
Zhang et al. (2024) An axonal brake on striatal dopamine output by cholinergic interneurons, bioRxiv
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: November 28, 2023
Last Modified: January 29, 2025
Protocol Integer ID: 91481
Keywords: calcium, ex vivo, calcium, mouse, dopamine, striatum, calcium dynamics in striatal dopaminergic axon, imaging axonal calcium dynamic, encoded calcium indicator gcamp6f, striatal dopaminergic axon, calcium indicator gcamp6f, ex vivo mouse brain slice, calcium transients in response, ex vivo mouse brain slices this protocol, calcium transient, calcium dynamic, mouse brain, electrical stimulus pulse, calcium, ai95d mice, imaging, pulse, vivo
Funders Acknowledgements:
Aligning Science Across Parkinson’s
Grant ID: ASAP-020370
Abstract
This protocol describes how to image calcium dynamics in striatal dopaminergic axons in ex vivo mouse brain slices. We imaged calcium transients in response to single and trains (4 pulses, 100 Hz) of electrical stimulus pulses using genetically encoded calcium indicator GCaMP6f expressed in DAT-Cre:Ai95D mice.
Materials
Equipment:
- Olympus BX51Wl microscope equipped with a OptoLED Lite system (CAIRN Research);
- Prime Scientific CMOS (sCMOS) Camera (Teledyne Photometrics);
- x40/0.8 NA water-objective (Olympus UK)
- ITC-18 A/D board (Instrutech)
Software:
- Micro-Manager v1.4
- Matlab vR2019b
- Fiji v1.5
- Igor Pro 6 (WaveMetrics)
Troubleshooting
Before start
This protocol was performed in heterozygous DAT-Cre:Ai95D (4-7 weeks) mice. These mice were bred from homozygous DAT-Cre mice (B6.SJL-Slc6a3tm1.1(cre)Bkmn/J, JAX stock number 006660) crossed with homozygous Ai95D mice (B6;129S-Gt(ROSA)26Sortm95.1(CAG-GCaMP6f)Hze/J, JAX stock number 028865).
Note
Ai95(RCL-GCaMP6f)-D (also called Ai95D; RRID:IMSR_JAX:028865) mice are a Cre-dependent, fluorescent, calcium-indicator tool strain. Ai95D has a floxed-STOP cassette preventing transcription of the GCaMP6 fast variant calcium indicator (GCaMP6f; a detector of single neuronal action potentials with fast response kinetics). After Cre exposure, bright EGFP fluorescence is observed following calcium binding (such as neuronal activation).
This Ai95D allele is on a C57BL/6J genetic background.
We prepare ex vivo mouse brain slices by performing steps 1 to 11 from Protocol: Fast-scan cyclic voltammetry to assess dopamine release in ex vivo mouse brain slices.
Image Acquisition
Using a x40/0.8 NA water-objective (Olympus UK), position the stimulating electrode on the surface of the brain slice and centre it in the field of view.
Change the exposure time to reach a frame rate of around 16.6 Hz every 2.5 min using Micro-Manager v1.4. 16.6 Hz frame rate every 2.5 min using Micro-Manager 1.4.
Apply electrical stimulus pulses singly and in trains (4 pulses, 100 Hz) using custom-written procedures in Igor Pro 6 (WaveMetrics) and an ITC-18 A/D board (Instrutech).
Note
The order of single and train stimulations was alternated and equally distributed and data were collected in duplicate before and after a change in extracellular experimental condition.
Record changes in fluorescence intensity using custom-written procedures in Igor Pro 6 (WaveMetrics) and an ITC-18 A/D board (Instrutech).
Image Analysis
The following steps were performed in MATLAB vR2019b and Fiji v1.5.
Extract fluorescence intensity from the region of interest 25 µm * 25 µm which was 50 µm away from the electrical stimulating electrode tip.
After background subtraction, bleach-correct the Ca2+ transients by fitting an exponential curve function through both the baseline (2 s prior to stimulation) and the last 1 s in a 7.2 s recording window.
Expressed data as ΔF/F where F is the fitted curve.