Obtaining Horizontal Mouse Hippocampal Brain Slices

프로토콜

All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.

1. Preparation of high-sucrose slice solution and artificial cerebrospinal fluid (ACSF)

1. Prior to experimental day
   1. Prepare 1 L of 10x slice pre-solution with laboratory grade Type 1 water containing (in mM): 25 KCl, 20 CaCl₂, 10 MgSO₄, 12.5 KH₂PO₄ (Table 1). In order to prevent calcium phosphate precipitation, slowly mix the chemicals in a beaker pre-filled with 800 mL H₂O while constantly stirring with a magnetic stirrer. Store the solution at 4 °C or room temperature (RT).
2. On the experimental day
   1. Prepare 1 L of 1x ACSF with laboratory grade Type 1 water containing (in mM): 125 NaCl, 2.5 KCl, 2 CaCl₂, 1 MgSO₄, 1.25 NaH₂PO₄, 26 NaHCO₃, 25 Glucose (Table 2). Use a vapor pressure osmometer to validate the osmolarity between 305–315 mOsm (pH ~ 7.55–7.6).
      NOTE: In order to prevent calcium phosphate precipitation, slowly mix all solid chemicals in a beaker pre-filled with 800 mL of H₂O while constantly stirring with a magnetic stirrer. Add MgSO₄ and CaCl₂ at the very end, slowly dripping in the necessary amount from 1 M stock solutions.
   2. Continuously bubble 1x ACSF solution at RT with carbogen to set pH between 7.3–7.4.
      NOTE: If the pH is slightly too high or too low, small adjustments in the carbogenation strength would be sufficient. If the pH is higher than 7.45 with carbogenation, adjust it by adding few drops of 1 M NaH₂PO₄ solution.
   3. Prepare 250 mL (per brain) of 1x high-sucrose slice solution in a beaker containing 25 mL of 10x slice pre-solution and (in mM): 252 Sucrose, 26 NaHCO₃, and 10 Glucose (Table 3). Verify that the osmolarity is between 320–325 mOsm (pH ~ 7.55–7.6).
   4. Bubble the high-sucrose slice solution for 10–15 min with carbogen to control the pH between 7.3–7.4.
      NOTE: If the pH is higher than 7.45 with carbogenation, adjust it by adding a few drops of 1 M KH₂PO₄ solution.
   5. Store the high-sucrose slice solution for 20–30 min in the ultra freezer (-80 °C) until it is partially frozen.

2. Dissection and positioning of the murine brain

1. Transfer the brain to the chilled 35 mm culture dish and remove all hair or blood particles from the tissue by gently washing the brain with an ACSF-filled Pasteur pipette (blood has cytotoxic effects on brain tissue).
2. Transfer the brain with the help of the spatula on the soaked filter paper on top of the chilled 90 mm culture dish (Figure 1A).
3. Use a blade to cut the brain in half, separating the two hemispheres, and place both hemispheres on the freshly cut medial side.
4. Use a blade to remove the dorsal part (5%–10%) of the brain from each hemisphere with a parallel cut to the dorsal top (Figure 1C)and place both hemispheres on the freshly cut side with the ventral part of the brain facing upwards.
5. Position a drop of super glue on the specimen plate and spread out properly with a pipette tip to accommodate both hemispheres.
6. Use a filter paper strap to pick up one hemisphere with capillary forces by touching the ventral side with the filter paper strap, thereby not damaging the tissue.
7. Use another filter paper strap to carefully semi-dry the dorsal side of the brain before positioning the hemisphere, dorsal side down, on top of the glue on the specimen plate. Repeat the same procedure with the second hemisphere.
   NOTE: The hemispheres should be positioned in horizontal alignment in a mirrored fashion on the vibratome plate, with the rostral sides pointing toward the outside and the caudal sides facing (but not touching) each other in the middle. The medial sides of both hemispheres should point toward the vibratome blade and the lateral sides toward the experimenter (Figure 1D).
8. Place the specimen plate in the slicing chamber and quickly, but carefully, cover it with ice-cold high-sucrose slice solution slush. As soon as the solution touches the glue, it will solidify and properly glue the hemispheres to the specimen plate.
9. Assure that the hemispheres are properly covered with high-sucrose slice solution and confirm that the solution is bubbled with carbogen.
   NOTE: The entire dissection procedure should be performed as fast as possible. Please ensure that the brain does not stay without the supply of oxygen for a very long time. It should take only around 1–1.5 min from decapitation to brain submersion in the high-sucrose slice solution slush. This is the most critical requirement for acute brain slice preparations in order to warrant high quality of the slice.

3. Horizontal slicing of the brain

1. Position the vibratome blade in front of the medial side of the hemispheres and lower it to the same height as the ventral sides of the hemispheres that are now facing upwards. Lower the blade with the help of the vibratome control to 600 µm further in the dorsal direction and start slicing. The blade should hit the tissue (if not, reverse the blade and lower it a bit more). Slice until the first two slices are completely separated from the two hemispheres.
2. Reverse the blade and lower another 300 µm and slice again.
3. When the hippocampus becomes visible (use the mouse brain atlas for help, if necessary) (Figure 1E) collect the slices with the widened plastic Pasteur pipette. Collect the slices until the caudate putamen becomes visible next to the hippocampus. Typically, between 8–12 slices of 300 µm (4–6 per hemisphere) can be collected for the mouse brain.
4. Use the plastic Pasteur pipette to collect the slices and transfer them to the recovery chamber in the water bath (Figure 1F) (collect slices after each round of slicing to prevent them from floating around in the slice chamber).
   NOTE: Work as fast as possible and ensure that the high-sucrose slice solution is ice-cold and carbogenated during the entire procedure. If necessary, refill the ice surrounding the slice chamber.

4. Recovery of brain slices for electrophysiological recordings

1. Leave the slices in the ACSF-filled recovery chamber in the 32 °C water bath for 1 h.
   NOTE: Recovery chambers are also commercially available.
2. Take the recovery chamber out of the water bath.
3. Place the slices at RT for at least another 30 min for recovery before starting any further application.

Buffer and Media Recipes.

Table 1: 10 x slice pre-solution (1 L).

Compound	Concentration (mM)	Molecular weight (g/mol)	Amount (g)
KCl	25	74.55	1.86
CaCl2 * 2H2O	20	147.01	2.94
MgSO4 * 7H2O	10	246.48	2.46
KH2PO4	12.5	136.08	1.7

Table 2: 1x ACSF (1 L) (osmolarity between 305–315 mOsm).

Compound	Concentration (mM)	Molecular weight (g/mol)	Amount (g)
NaCl	125	58.44	7.3
KCl	2.5	74.55	0.19
CaCl2 * 2H2O	2	from 1 M CaCl2 solution	2 mL
MgSO4 * 7H2O	1	from 1 M MgSO4 solution	1 mL
NaH2PO4 * 2H2O	1.25	156.02	0.2
NaHCO3	26	84.01	2.18
Glucose * H2O	25	198.17	4.95

Table 3: 1x high-sucrose slice solution (250 mL) (osmolarity between 320–325 mOsm).

Compound	Concentration (mM)	Molecular weight (g/mol)	Amount (g)
10x slice presolution	N/A	N/A	25 mL
Sucrose	252	342.3	21.57
NaHCO3	26	84.01	0.55
Glucose * H2O	10	198.17	0.49

결과

Figure 1: Detailed information on the preparation of horizontal hippocampal brain slices. (A) Image of tools required for dissection and slicing of the rodent brain: (a) ±2 cm long and ±0.5 cm wide straps of filter paper (e.g., grade 413); (b) blade; (c) super glue; (d) pipette tip; (e) specimen plate (comes with vibratome); (f) 35 mm culture dish; (g) fine brush; (h) spatula; (i) curve...

자료

Name	Company	Catalog Number	Comments
Carbogen tank	Air Liquide	Alphagaz mix B50	Gasmixture CO2/O2: 5/95, purity 5
Culture dish (35 mm)	Corning Life Sciences	353001	https://ecatalog.corning.com/life-sciences/b2c/US/en/Cell-Culture/Cell-Culture-Vessels/Dishes%2C-Culture/Falcon®-Cell-Culture-Dishes/p/353001
Culture dish (90 mm)	Thermo Fisher Scientific	101VR20	https://www.thermofisher.com/order/catalog/product/101R20#/101R20
Curved forceps	Fine Science tools	11270-20	https://www.finescience.de/de-DE/Products/Forceps-Hemostats/Dumont-Forceps/Dumont-7b-Forceps/11270-20
D-(+)-Glucose monohydrate	Sigma Aldrich	16301	https://www.sigmaaldrich.com/catalog/product/sial/16301?lang=en&region=BE
Filter paper	VWR	516-0818	grade 413
Fine brush	Raphael Kaerell	8204	Size #1
Loctite 406	Henkel Adhesive technologies	Loctite 406	Super glue
Potassium chlorid	Chem-lab	CL00.1133	https://www.chem-lab.be/#/en-gb/prod/1393528
Potassium dihydrogen phosphate	Merck	104873	https://www.merckmillipore.com/BE/en/product/Potassium-dihydrogen-phosphate,MDA_CHEM-104873?ReferrerURL=https%3A%2F%2Fwww.google.com%2F
Razor blade to prepare hemispheres	SPI supplies	Safety Cartridge Dispenser - Pkg/10	GEM Scientific Single Edge Razor Blades
Razor blade for vibratome	Ted Pella Inc	121-6	double edge breakable style razor blades (PTFE-coated stainless steel)
Recovery chamber	home made - Generic	N/A	to collect and store brain slices in (see details in manuscript)
Scissors	Any company	N/A	Blade should be well sharpened and at least 15 cm long for easy decapitation
Sodium dihydrogen phosphate dihydrate	Merck	106342	https://www.merckmillipore.com/BE/en/product/Sodium-dihydrogen-phosphate-dihydrate,MDA_CHEM-106342?ReferrerURL=https%3A%2F%2Fwww.google.com%2F
Sodium hydrogen carbonate	Alfa Aesar	14707	https://www.alfa.com/en/catalog/014707/
Sodium chlorid	Fisher Scientific	S/3160/60	https://www.fishersci.co.uk/shop/products/sodium-chloride-certified-ar-analysis-meets-analytical-specification-ph-eur/10428420
Spatula	Sigma Aldrich	S9147-12EA	https://www.sigmaaldrich.com/catalog/product/sigma/s9147?lang=en&region=BE
Sucrose	VWR International Ltd.	102745C	https://es.vwr-cmd.com/ex/downloads/magazine/lupc_userguide_uk.pdf
Tubing for carbogen, perfusion and suction lines 1	Warner Instruments	64-0167	Tygon tubing (TY-50) for standard valve systems
Tubing for carbogen, perfusion and suction lines 2	Fisher Scientific	800/100/200 & 800/100/280	Smiths Medical Portex Fine Bore LDPE Tubing
Vibratome	Leica	14912000001	Semi-automatic vibrating blade microomei VT1200
Water bath	Memmert	WNB 7	https://www.memmert.be/wp-content/uploads/2019/09/Memmert-Waterbath-WNB-7.en_.pdf
Water purification system	Merck	Synergy millipore	to obtain highly purified water