Apr 21, 2023

Public workspaceVisualisation and quantification of dendritic spines in cultured human Medium Spiny Neurons (MSNs) V.1

DOI
dx.doi.org/10.17504/protocols.io.261ge397jl47/v1
  • Quyen Do1,2,3
  • 1Oxford Parkinson's Disease Centre and Department of Physiology, Anatomy and Genetics, University of Oxford, South Park Road, Oxford OX1 3QU, United Kingdom;
  • 2Kavli Institute for Neuroscience Discovery, University of Oxford, Dorothy Crowfoot Hodgkin Building, South Park Road, Oxford OX1 3QU, United Kingdom;
  • 3Aligning Science Across Parkinson's (ASAP) Collaborative Research Network, Chevy Chase, MD, 20815, USA
Cláudia C. Mendes
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DOI: dx.doi.org/10.17504/protocols.io.261ge397jl47/v1
Protocol Citation: Quyen Do 2023. Visualisation and quantification of dendritic spines in cultured human Medium Spiny Neurons (MSNs). protocols.io https://dx.doi.org/10.17504/protocols.io.261ge397jl47/v1Version created by Cláudia C. Mendes
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: April 12, 2023
Last Modified: April 21, 2023
Protocol Integer ID: 80391
Keywords: Dendritic spine, Imaris, fluorescent, immunocytochemistry
Funders Acknowledgement:
Aligning Science Across Parkinson's (ASAP)
Grant ID: ASAP-020370
Abstract
This protocol describes the visualisation of dendritic spines of human neurons cultured on coverslips in vitro and subsequent quantification using the software Imaris.

Materials
Reagents:

Equipment:
  • Olympus FluoViewTM FV1000 Confocal Microscope

Software:
  • Imaris v9.6.0 (Bitplane, South Windsor, CT, USA)
Visualisation of fluorescent dendritic spines in MSNs
Visualisation of fluorescent dendritic spines in MSNs
Mount a coverslip of Medium Spiny Neurons (MSNs) immunolabelled for DARP32 and neurobiotin onto SlowFade™ Diamond Antifade mountant (follow Protocol: Immunocytochemistry of cultured human Medium Spiny Neurons (MSNs)).

Place slide under under Olympus FluoView FV1000 confocal microscope with argon and solid-state laser with 488 nm and 559 nm excitation, respectively.
Use the 60x oil-immersion objective (NA = 1.40) to image and capture MSNs coexpressing Darpp32 and Neurobiotin as Z-stacks, sampling sequentially at resolution of 1024 * 1024 pixels and at 1.05 µm steps, as optimised by Nyquist sampling theorem.
Use the zoom function (3x) in the Olympus Dendritic FluoView FV1000 software to capture dendritic branches of biotinylated neurons.


Note
Approximately 2-4 images are captured per neurons, depending on image quality.


Quantification of dendritic spines
Quantification of dendritic spines
Use the 'Surface' module to threshold and segment the confocal images.
Use the 'Filament' module to automatically render the dendrites from images obtained after step 5.
Choose the spine detection function in the 'Filament' module to detect spines as protrusions from the previously-identified dendritic filament.
Manually exclude putative spines at branch points or disconnected dots.