Dec 16, 2024

Public workspaceEpifluorescence Time-Lapse Imaging of Hippocampal Neurons

DOI
dx.doi.org/10.17504/protocols.io.n92ldrmqog5b/v1
  • Mukesh Kumar1,
  • Timothy A. Ryan1,2
  • 1Department of Biochemistry, Weill Cornell Medicine, New York, NY 10065;
  • 2Aligning Science Across Parkinson's (ASAP) Collaborative Research Network, Chevy Chase, Maryland 20815, USA
Mukesh Kumar
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DOI: dx.doi.org/10.17504/protocols.io.n92ldrmqog5b/v1
Protocol Citation: Mukesh Kumar, Timothy A. Ryan 2024. Epifluorescence Time-Lapse Imaging of Hippocampal Neurons. protocols.io https://dx.doi.org/10.17504/protocols.io.n92ldrmqog5b/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: November 26, 2024
Last Modified: December 16, 2024
Protocol Integer ID: 114383
Keywords: ASAPCRN
Funders Acknowledgements:
NIH
Grant ID: NS036942
NIH
Grant ID: NS11739
ASAP
Grant ID: ASAP-024404
Abstract
This protocol outlines the detailed steps for epifluorescence time-lapse imaging of hippocampal neurons, enabling the study of neuronal responses to various pharmacological and metabolic conditions.
Guidelines
The protocol needs prior approval by the users' Institutional Animal Care and Use Committee (IACUC) or equivalent ethics committee.
Materials
Materials:

  • Tyrode’s solution: 119 mM NaCl, 2.5 mM KCl, 2 mM CaCl2, 2 mM MgCl2, 50 mM HEPES (pH 7.4), 0-5 mM glucose, 10 mM CNQX (Reagent6-Cyano-7-nitroquinoxaline-2,3-dioneMerck MilliporeSigma (Sigma-Aldrich)Catalog #C127 ), 50 mM APV (ReagentDL-2-Amino-5-phosphonopentanoic acidMerck MilliporeSigma (Sigma-Aldrich)Catalog #A5282 )

  • BSA-complexed palmitate: 150 μM for feeding media and 100 μM for perfusion (prepared as previously described in Fatty acid induction protocol)

  • Pharmacological inhibitors:
  1. 5 μM ReagentKLH45Merck MilliporeSigma (Sigma-Aldrich)Catalog #SML1998
  2. 40 μM ReagentAtglistatinMerck MilliporeSigma (Sigma-Aldrich)Catalog #SML1075
  3. 2 μM ReagentOligomycin from Streptomyces diastatochromogenesMerck MilliporeSigma (Sigma-Aldrich)Catalog #O4876
  4. 20 μM ReagentEtomoxirMerck MilliporeSigma (Sigma-Aldrich)Catalog #E1905
  5. Tetrodotoxin: 2 μM (Abcam, ab120055)


Equipments:

  • Microscope: Custom-modified Zeiss Axiovert 200
  • Camera: Andor iXon DU-897U-CS0-BVF
  • Objective: Zeiss 40X 1.3 NA Fluar with temperature control (37°C)
  • Lasers: OBIS solid-state 488 nm, 561nm and 635 nm
  • Perfusion system: Laminar flow with temperature control (37°C) at objective
  • Platinum-iridium electrodes
  • Field stimulation setup capable of generating ~10 V/cm





Preparation of Neurons
Preparation of Neurons
Culture hippocampal neurons on glass coverslips under standard conditions following previously described protocol.

Transfect neurons with desired constructs using calcium phosphate method previously described, and allow expression for 7 days.

Preparation of Perfusion Solution
Preparation of Perfusion Solution
Prepare Tyrode’s solution as described in materials.
Add Concentration100 micromolar (µM) BSA-complexed palmitate to the Tyrode’s solution if required for fatty acid studies.

For pharmacological experiments, add desired amount of inhibitors as described in material section.

Feeding with Fatty Acids
Feeding with Fatty Acids
4h
4h
Supplement neuronal feeding media with Concentration150 micromolar (µM) BSA-complexed palmitate.

Incubate neurons with the supplemented media for Duration04:00:00 .

4h
Incubation
JF dye labelling
JF dye labelling
30m
30m
For imaging of Halo tagged proteins, add 100 nM JF635 or JF585 dyes to feeding media and return to incubator for Duration00:30:00 .

30m
Incubation
Transfer the coverslip to Tyrode’s solution to wash unbound dyes.

Mounting Neurons for Imaging
Mounting Neurons for Imaging
Mount the coverslip with neurons onto the laminar flow perfusion system.

Ensure the perfusion system and objective are maintained at Temperature37 °C .

Perfusion and Inhibitor Treatment
Perfusion and Inhibitor Treatment
5m
5m
Perfuse neurons with the appropriate Tyrode’s solution forDuration00:05:00 before imaging or stimulation.

5m
Ensure inhibitors are present during perfusion if required.

Electrical Stimulation
Electrical Stimulation
Connect platinum-iridium electrodes with the stimulator.

Generate action potentials by applying brief 1 ms electrical pulses.

Ensure the field potential is maintained at ~10 V/cm.

Image Acquisition
Image Acquisition
Acquire time-lapse images using the Andor iXon camera.

Record images at the desired time intervals for the duration of the experiment.

Monitor temperature and flow conditions throughout the imaging session.

Note
  • Ensure all inhibitors are freshly prepared.
  • Use hippocampal neurons between 7 to 14 days of transfection.
  • Maintain consistent temperature (Temperature37 °C ) during all imaging and perfusion steps.
  • Protect light-sensitive compounds (BSA-complexed palmitate) from prolonged light exposure.
  • Ensure that the neurons are healthy and responsive during electrical stimulation.