Jan 23, 2025
Hippocampal slice preparation for electrophysiology
- Quinn Pauli1,
- Robert Bonin1
- 1Leslie Dan Faculty of Pharmacy, University of Toronto, Toronto, Ontario, Canada
Protocol Citation: Quinn Pauli, Robert Bonin 2025. Hippocampal slice preparation for electrophysiology . protocols.io https://dx.doi.org/10.17504/protocols.io.eq2ly6j9egx9/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: December 05, 2024
Last Modified: January 23, 2025
Protocol Integer ID: 114347
Keywords: Electrophysiology, hippocampus, mouse, slice, brain dissection, hippocampus slices
Funders Acknowledgements:
Natural Sciences and Engineering Research Council of Canada
Grant ID: RGPIN-2024-06215
Abstract
This protocol describes the preparation and maintenance of acute hippocampal slices from mice for electrophysiology. We describe the dissection, slicing and incubation methods to produce healthy slices. Slice preparation can be followed by same-day electrophysiology experiments (e.g. to assess synaptic plasticity). Acute slices can be further processed for protein detection (e.g. immunofluorescence, immunoblotting) as shown by our lab.
Materials
Solution Recipes:
| A | B | C | D | E | |
| Reagent | Mass (g) for 1 L | Mass (g) for 500 mL | MW | mM | |
| NaCl | 7.2466 | 3.6233 | 58.44 | 124 | |
| Glucose | 1.8016 | 0.9008 | 180.156 | 10 | |
| NaHCO3 | 2.1843 | 1.0922 | 84.007 | 26 | |
| KCl | 0.2236 | 0.1118 | 74.55 | 3 | |
| NaH2PO4•H2O | 0.1932 | 0.0966 | 137.99 | 1.4 | |
| MgSO4 | 1 mL | 0.5 mL | 1M Solution | 1 | |
| CaCl2 | 2 mL | 1 mL | 1M Solution | 2 | |
| Osmolarity 300-310 mOsm |
ACSF Recipe
| A | B | C | D | E | |
| Reagent | Mass (g) for 1 L | Mass (g) for 500 mL | MW | mM | |
| Sucrose | 17.1145 | 8.5573 | 342.3 | 50 | |
| NaCl | 5.3765 | 2.6883 | 58.44 | 92 | |
| Glucose | 2.7024 | 1.3512 | 180.156 | 15 | |
| KCl | 0.3728 | 0.1864 | 74.55 | 5 | |
| NaH2PO4•H2O | 0.1987 | 0.0994 | 137.99 | 1.4 | |
| NaHCO3 | 2.1843 | 1.0922 | 84.007 | 26 | |
| CaCl2 | 0.5 mL | 0.25 mL | 1M Solution | 0.5 | |
| MgCl2•6H2O | 1.4231 | 0.7116 | 203.30 | 7 | |
| Kynurenic Acid | 0.1892 | 0.0946 | 189.17 | 1 | |
| Osmolarity 322-330 mOsm |
Sucrose Solution Recipe
Dissection tools:
- Fine scissors
- Angled fine scissors
- Surgical scissors
- Dumont Forceps
- Hemostat
- Dressing forceps
- Scalpel handle and blade (#21)
- Spatula
- Razor blades
- Glass petri dish
Other supplies:
- 25 or 50 mL syringe
- Tygon tubing
- 27g needle (for i.p. injection)
- 16g needle (for perfusion)
- Anesthetic (e.g. Avertin)
- Filter paper
- Tissue adhesive (e.g. 3M Vetbond)
- Ice
- 95% oxygen/5% carbon dioxide (carbogen tank and lines)
- Cell strainers, falcon tubes, super glue and a 250 mL beaker (for homemade brain slice keeper)
- Beakers (250-500 mL)
Equipment:
- Vibratome
- Brain slice keeper
Brain slice keeper
Safety warnings
If you choose to use this protocol, you do so at your own risk and must ensure that any local guidance is adhered to.
Ethics statement
Experiments involving animals must be conducted according to internationally-accepted standards and should always have prior approval from an Institutional Animal Care and Use Committee (IACUC) or equivalent ethics committee(s). Animal care and experimental procedures were reviewed and approved by the
Animal Ethics and Compliance Program at the University of Toronto and conducted
in accordance with the Canadian Council on Animal Care (CACC) guidelines.
Set up
Set up
55m
55m
Prepare solutions (see Materials) 1 week to 1 day in advance (store at 4 °C )
Bubble 100 mL sucrose solution and 200 mL ACSF for 20 minutes in 95% oxygen/5% carbon dioxide for 00:20:00
20m
Freeze sucrose solution at -20 °C until a slurry begins to form (~00:15:00 )
15m
Prepare fresh ACSF (day of) that will be used for recording
20m
Once prepared, continue bubbling all solutions with 95% oxygen/5% carbon dioxide throughout the duration of slice preparation
Add ACSF to a brain slice keeper (e.g. 250 mL beaker with cell strainers affixed to cut falcon tubes to ensure continuous solution flow; see Materials) and heat in a water bath to 28 °C . Ensure that the cell holders are submerged and the solution is bubbled with 95% oxygen/5% carbon dioxide for the duration of the procedure.
Prepare dissection area with dissection tools, an absorbent pad, and bench protectors. Add ice to vibratome well (around buffer tray)
Perfusion and brain dissection
Perfusion and brain dissection
12m
12m
Weigh and anesthetize mouse with the appropriate dosage (i.p.) and place back into home cage.
5m
Assess depth of anesthesia using toe-pinch response (ensure response is gone before beginning procedure)
Add ice cold sucrose solution to 50mL syringe attached to tubing and needle for perfusion
Make a lateral incision through the abdomen at the xiphoid process using fine scissors
Pulling up on the xiphoid process, carefully cut the diaphragm and then cut along the base of the ribcage on both sides to expose the heart
Clamp the xiphoid process using a hemostat to expose the chest cavity
Securing the heart with dressing forceps, insert 18g needle attached to a 50mL syringe with tubing into left ventricle
Make a small incision in the right ventricle using fine scissors
Compress the syringe slowly to perfuse the mouse until the liver is visibly clear or approximately 30-40 mL of solution has been used
2m
Decapitate the mouse with large surgical scissors
Pull the skin back and make two lateral incisions at the base of the skull using fine scissors
Make a cut down the midline of the skull using angled fine scissors, taking care not to damage the brain
Gently peel away the two sections of the skull with Dumont forceps
Scoop the brain out of the skull using a spatula and immediately submerge in cold, bubbling ACSF for 00:05:00
5m
Slicing
Slicing
1h 45m
1h 45m
Wet filter paper in ice cold ACSF and place in a glass petri dish
Remove the brain from solution and place on the wet filter paper. Use a scalpel to remove the cerebellum and olfactory bulb, then make two thin cuts along the sagittal plane of the brain bilaterally to provide a flat surface for slicing
Make a cut down the midline of the brain and glue the two hemispheres of the brain to the vibratome platform, with the hippocampus facing up and cortex towards the blade
Fill the buffer chamber with ice cold ACSF and continuously bubble the solution. Attach the razor blade to the vibratome and position it right above the tissue as the starting position
15m
Slice 400 µm slices at 0.08 mm/s (on continuous slicing mode). Carefully remove the CA3 region of each slice using a scalpel. Remove any excess tissue around the dorsal hippocampal slices. Pause the vibratome when performing microdissections of slices.
Transfer each slice to 28 °C bubbling ACSF in the brain slice keeper to rest for a minimum of 01:30:00 before recording in a chamber maintained at 28 °C
1h 30m
If recording from multiple slices, turn off the water bath after 03:00:00 of incubation and allow slices to gradually come to room temperature.