Jan 29, 2025

Public workspaceWide-field imaging of voltage sensors expressed in ex vivo mouse brain slices

DOI
dx.doi.org/10.17504/protocols.io.yxmvm3q46l3p/v1
  • 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;
  • 3Oxford Parkinson’s Disease Centre, University of Oxford, Oxford, United Kingdom;
  • 4Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford OX1 3PT, UK
Cláudia C. Mendes
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DOI: dx.doi.org/10.17504/protocols.io.yxmvm3q46l3p/v1
Protocol Citation: Yan-Feng Zhang, Stephanie J Cragg 2025. Wide-field imaging of voltage sensors expressed in ex vivo mouse brain slices. protocols.io https://dx.doi.org/10.17504/protocols.io.yxmvm3q46l3p/v1
Manuscript citation:
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 27, 2023
Last Modified: January 29, 2025
Protocol Integer ID: 91484
Keywords: voltage, ex vivo, mouse, dopamine
Funders Acknowledgements:
Aligning Science Across Parkinson’s
Grant ID: ASAP-020370
Disclaimer
This protocol uses the ASAP3 voltage sensor (AAV5-EF1α-DIO-ASAP3WPRE) kindly donated by the Lin lab.
Abstract
This protocol describes how to perform wide-field imaging of voltage sensors using high frame rates (660 Hz minimum every 2.5 minutes) in mouse midbrain using ex vivo brain slices.
Materials
Equipment:
  • Olympus BX51Wl microscope equipped with a OptoLED Lite system (CAIRN Research);
  • iXon EMCCD Camera (ANDOR);
  • x40/0.8 NA water-objective (Olympus UK)

Virus:
  • AAV5-EF1α-DIO-ASAP3WPRE (ASAP3) from Stanford Gene Vector and Virus Core.

Software:
  • Micro-Manager v1.4
  • PClamp
  • Matlab vR2019b
  • Fiji v1.5
Before start
We injected the voltage sensor ASAP3 without a soma-targeting signal (AAV5-EF1α-DIO-ASAP3WPRE) following the steps described in Protocol: Intracranial injections of viral vectors in mouse midbrain and striatum. We injected the virus diluted to 2.4E+12 vg/ml in the midbrain (1 μL per site) of heterozygous DAT-IRES-Cre mice.

The coordinates used by us for targeting the midbrain were as follows:

Ventral tegmental area (VTA) (AP = -3.1 mm, ML = ± 0.5 mm, DV = -4.4 mm)
Substancia nigra pars compacta (SNc) (AP = -3.5 mm, ML = ± 1.2 mm , DV = -4.0 mm)

Animals were maintained for at least three weeks following surgery to allow virus expression in the midbrain. We then prepare ex vivo 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
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 600 Hz every 2.5 min using Micro-Manager v1.4.
Apply electrical stimulus pulses singly and in trains (4 pulses, 50 Hz) using PClamp.


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.

Observations were time-locked to the deflection.

Record changes in fluorescence intensity using PClamp.
Image Analysis
Image Analysis
The following steps were performed in MATLAB vR2019b and Fiji v1.5.

Extract fluorescence intensity from the region of interest (~5 µm * 5 µm).
Bleach-correct the ASAP3 transients by fitting an exponential curve function.
Expressed data as ΔF/F where F is the fitted curve.