Generation of Spontaneous and On-Demand Ictal Events in Mouse Brain Cortical Slices

Protocolo

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

1. Preparation of dissection solutions and artificial cerebrospinal fluid

1. Carbogenate ultrapure water (which is water filtered with a resistivity of 18.2 MΩ·cm) with carbogen (95% O₂/5% CO₂) for 5 min using air stone bubble diffusers (aerators for aquariums).
   Note: Air stones can be connected to the carbogen tank's regulator via standard silicone tubing. If air stones are not available, seal shut the end of the silicone tubing and poke small holes in the seal to allow the carbogen gas to bubble out and carbogenate the solution.
2. Prepare a dissection solution by dissolving the solutes from Table 1 in ultrapure water. Add CaCl₂ to a solution that has been carbogenated for at least 5 min to help it dissolve.
   Note: Alternatively, add CaCl₂ first, so that it can dissolve easily in an unsaturated solution. The solution, in liquid form, should be 300 - 320 mOsm/L and have a pH of 7.4 after being saturated with carbogen.
   1. Chill the dissection solution to 0 - 4 °C. Leave the solution in the fridge overnight or place the solution in the freezer for 1 h and continually monitor the temperature.
      Note: The dissection solution can be stored for up to 3 nights; discard afterward.
   2. Carbogenate the dissection solution for 5 - 10 min before use.
      Note: Ideally, the solution should either appear as slush or be transitioning to slush prior to use.
3. Prepare rodent artificial cerebrospinal fluid (ACSF) by dissolving the solutes from Table 2 in ultrapure water. Carbogenate the ACSF solution while it is in a water bath heated to 35 °C until use. Add CaCl₂ to a solution that has been carbogenated for at least 5 min to help it dissolve.
   Note: Alternatively, add CaCl₂ first so that it can dissolve easier in an unsaturated solution. The solution, in liquid form, should be 290 - 320 mOsm/L and have a pH of 7.4 after being saturated with carbogen. Solutions should be made in 1, 2, or 4 L volumes depending on the duration of the experiments. Make 4 L for a full day (8 h) of experiments. The ACSF should be made fresh each day; do not store it for longer than 1 d.

2. Mouse dissection to collect brain tissue

1. Collect all the tools and materials necessary for animal dissections. Set up the workspace to prepare for animal dissections.
   1. Check the carbogen tank (95%O₂/5%CO₂) to ensure it is not empty. Replace the gas cylinder prior to experiments if there is less than 500 psi of pressure remaining.
   2. Calibrate the vibratome (vibrating blade tissue slicer) according to the manufacturer's instruction manual. Adjust the vibratome setting to have a cutting amplitude of 1 mm and a cutting speed of 0.12 mm/s.
      Note: The optimal settings may differ for each individual vibrating tissue blade cutter; however, in general, the amplitude should be high and the cutting speed should be low.
   3. Fill the brain slice incubation chamber (i.e., the brain slice keeper) with ACSF and bubble it with carbogen. Then place the incubation chamber in a water bath set at 35 °C.
2. Obtain a juvenile mouse of either sex, between the age of 13 d (p13) to 21 d (p21, where p0 is the date of birth).
3. Anesthetize the mouse with an intraperitoneal injection of sodium pentobarbital (55 mg/kg of body weight). Once the mouse is deeply anesthetized, as indicated by the absence of the toe pinch reflex, promptly use a vet-approved guillotine instrument to decapitate the mouse in one swift movement.
   Note: Prepare the sodium pentobarbital injection according to the procedures recommended by institutional guidelines.
4. Hold the mouse's nose to stabilize the decapitated head and gently make a midline incision starting between the mouse's eyes and along the entire length of the scalp to expose the skull. Ensure the skull is not compressed by the pressure applied by the razor. Use the corner of the razor's edge to increase cut precision.
5. Spread apart the flaps of the mouse's scalp using fingers to expose the skull. Then, use the fresh corner of a razor's edge to make an incision along the midline of the skull; pay careful attention to avoid any contact with the cerebral cortex.
6. Insert splinter forceps into the mouse's eye sockets to stabilize the decapitated head and place it in a Petri dish filled with cold (0 - 4 °C) dissection solution. Ensure that the head is fully submerged in the dissection solution.
7. Use a second set of splinter forceps to gently peel away the mouse's scalp, remove the nose bone by peeling it, and remove the back (caudal side) of the skull.
   Note: The nose bone of juvenile mice is very soft and easily broken.
8. Use a micro spatula to cut the optic nerves, trigeminal nerves, and spinal cord. Then, use the micro spatula to gently separate the brain from the cranium. Leave the brain fully submerged in the Petri dish filled with dissection solution.

3. Preparation of cortical slices from the somatosensory-motor cortex

1. Fill the vibratome's buffer tray with ~150 mL of cold (0 - 4°C) dissection solution.
   Note: Optionally, keep the buffer tray in the freezer until needed to maintain a low temperature during the slicing procedure.
2. Glue the brain tissue from mouse onto the vibratome stage (specimen holder/tray) using instant adhesive glue. For mice, cut off a small caudal portion of the brain (i.e., the cerebellum) so that it can be easily glued flat onto the specimen holder (allowing the rostral side of the brain to face the ceiling).
   Note: Alternatively, horizontal mouse brain slices can be prepared by gluing the dorsal side of the brain onto the specimen holder (the ventral side of the brain should face the ceiling).
3. Gently place the specimen holder (with brain tissue) into the buffer tray. Ensure that the dorsal portion of the brain is facing the vibratome's blade.
   Note: It is critical to minimize the amount of time the brain is exposed to air.

4. Brain tissue sectioning and collection

1. Slice the brain into 450 μm thick slices using the vibratome in the dorsal to ventral direction. Ensure that the brain remains completely anchored to the specimen tray while slicing it.
   1. Make the first cut in the mouse brain to remove the olfactory bulb. Then, make subsequent cuts until the somatosensory-motor area is observed (located approximately halfway between the olfactory bulb and bregma).
      Note: Optionally, slice the brain in thinner (200 μm) slices until the corpus callosum appears in the coronal view of the brain, which indicates that the somatosensory-motor area is close.
2. Use a wide-bore transfer pipette to collect coronal slices (450 μm) that contain the somatosensory-motor area and submerge them in a Petri dish containing cold (0 - 4 °C) dissection solution.
3. Use a new razor blade and cut off any excess tissue from the slices. For coronal slices from mice, perform a transverse cut just below the neocortical commissure (i.e., corpus callosum). Do not cut in a sawing motion; simply apply pressure on the blade into the tissue and use a detailing brush to gently separate the tissue. Make sure to minimize the movement of the coronal slice.
4. Use a wide-bore transfer pipette to transfer the dorsal portion of the coronal slices that contain the neocortex (layer 1 - 6) to a second Petri dish filled with warm (35 °C) ACSF for a moment (~1 s). Then, promptly transfer the slices to an incubation chamber containing warm (35 °C) carbogenated ACSF.
   Note: The purpose of the transfer into the Petri dish with ACSF is to minimize the transfer of dissection solution to the incubation chamber while transferring brain slices with the wide-bore pipette. Utilize the incubation chamber's multiple wells to keep different brain slices organized.
5. Discard the rest of the brain and animal carcass according to the institutional guidelines.

5. Incubation and maintenance

1. Leave the brain slices slightly submerged in the incubation chamber at 35 °C for 30 min. Then, remove the incubation chamber from the water bath and allow it to return to room temperature (20 - 25 °C). Wait 1 h for the brain slices to recover before performing electrophysiological recordings.
   Note: Ensure there are no air bubbles that collect underneath the brain slices in the incubation chamber. Air bubbles are air interfaces that cause tissue damage. The mouse brain slices can be maintained for 6 - 8 h in a well-carbogenated incubation chamber with ACSF.

6. Electrophysiological recordings of the superficial cortical layer

Note: Ictal events are observed in the extracellular local field potential (LFP) recordings from brain slices. The LFP of the brain slice can be observed and recorded with either an interface type chamber or a submerged multi-electrode array (MEA) system.

1. Use a wide-bore transfer pipette or a detailing brush to move a brain slice onto slightly larger pre-cut lens paper that is held in place using a dental tweezer. Transfer the lens paper (that the brain slice is resting upon) to the recording chamber and secure it in position with a harp screen.
2. Run warm (35 °C), carbogenated ACSF perfusate through the recording chamber over the brain slice at a rate of 3 mL/min (~1 drip/s). Use a digital thermometer to ensure the recording chamber is 33 - 36°C.
3. Pull glass electrodes with an impedance of 1 - 3 M‎Ω from borosilicate glass tubing (with an outer diameter of 1.5 mm) using a puller. Backfill the glass electrodes with ACSF (~10 µL) using a Hamilton syringe. Immediately discard the electrode if the tip is damaged.
   Note: Bleach the silver wire (5 min) and only allow a minimal portion (i.e., the tip) of the silver wire to be submerged in the ACSF back-filled glass electrodes to minimize noise and drift during the recordings. Remove any excess ACSF from the glass electrode with a Hamilton syringe if necessary.
4. Use a 20X stereo microscope to accurately guide the recording glass electrode into the superficial cortical layer (2/3) using manual manipulators. Record/view the electrical activity of the brain slice on a computer with standard software.
   Note: Viable (high-quality) brain slices will exhibit a robust evoked potential in response to the applied electrical stimuli (100 µs, 30 - 300 μA) or light pulses (30 ms, 10 mW/mm²) for optogenetic tissue.

7. Induction of seizure-like activities

1. Perfuse ACSF containing 100 µM 4-aminopyrimidine (4-AP) over the brain slice. Dissolve 80 mg of 4-AP in 8.5 mL of water to make a stock solution of 100 mM 4-AP. Add 100 µL of 100 mM 4-AP stock solution to 100 mL of ACSF to achieve an ACSF perfusate with 100 µM 4-AP.
   Note: Alternatively, use Zero-Mg²⁺ ACSF (Table 3), a modified ACSF solution containing no added Mg²⁺, to perfuse the brain slice. The average time for ictal events to appear is 15 min; however, it may take up to 40 min for some brain slices.

8. On-demand seizure generation: an optogenetic strategy for optogenetic mice

1. Apply a brief (30 ms) pulse of blue (470 nm) light (with a minimum 1 mW/mm² output intensity) to initiate an ictal event. Use a manual manipulator to position a 1,000 µm core diameter optical fiber (0.39 NA) directly above the recording region.
   Note: Set the rate of the photo-stimulation to match the desired rate of the ictal event occurrence (i.e., 1 pulse every 50 s). However, the rate cannot be overly exaggerated from the intrinsic rate at which ictal events occur.

Table 1: Recipe for dissection solution. These are instructions to make 1 L or 2 L volumes. MW = the molecular weight of the solute.

#	Reagent	Conc. [mM]	MW (g/mol)	1L (g)	2L (g)
1	Sucrose	248	342.3	84.89	169.78
2	Sodium Bicarbonate (NaHCO2)	26	84.01	2.18	4.37
3	Dextrose (D-glucose)	10	180.16	1.8	3.6
4	Potassium Chloride (KCl)	2	74.55	0.15	0.3
5	Magnesium Sulfate (MgSO4·7H2O)	3	246.47	0.74	1.48
6	Sodium phosphate monobasic monohydrate (H2NaPO4·H2O)	1.25	137.99	0.17	0.34
7	Calcium Chloride (CaCl2·2H2O)	1	147.01	0.15	0.29

Table 2: Recipe for rodent artificial cerebral spinal fluid (ACSF). These are instructions to make 2 L or 4 L volumes. MW = the molecular weight of the solute.

#	Reagent	Conc. [mM]	MW (g/mol)	2L (g)	4L (g)
1	Sodium Chloride (NaCl)	123	58.4	14.37	28.73
2	Sodium Bicarbonate (NaHCO2)	26	84.01	4.37	8.74
3	Dextrose (D-glucose)	10	180.16	3.6	7.21
4	Potassium Chloride (KCl)	4	74.55	0.6	1.19
5	Magnesium Sulfate (MgSO4·H2O)	1.3	246.47	0.64	1.28
6	Sodium phosphate monobasic monohydrate (HNaPO4·H2O)	1.2	137.99	0.33	0.66
7	Calcium Chloride (CaCl2·2H2O)	1.5	147.01	0.44	0.88

Table 3: Recipe for Zero-Mg²⁺ rodent artificial cerebral spinal fluid (Zero-Mg²⁺ ACSF). These are instructions to make 2 L or 4 L volumes. MW = the molecular weight of the solute.

#	Reagent	Conc. [mM]	MW (g/mol)	2L (g)	4L (g)
1	Sodium Chloride (NaCl)	123	58.4	14.37	28.73
2	Sodium Bicarbonate (NaHCO2)	26	84.01	4.37	8.74
3	Dextrose (D-glucose)	10	180.16	3.6	7.21
4	Potassium Chloride (KCl)	4	74.55	0.6	1.19
5	Magnesium Sulfate (MgSO4·H2O)	Nominally Free	246.47	0	0
6	Sodium phosphate monobasic monohydrate (HNaPO4·H2O)	1.2	137.99	0.33	0.66
7	Calcium Chloride (CaCl2·2H2O)	1.5	147.01	0.29	0.59

Materiales

Name	Company	Catalog Number	Comments
Sodium pentobarbital	N/A	N/A	Purchased through the Toronto Western Hospital's Suppliers
1 mm syringe	N/A	N/A	Purchased through UT Med Store
25G 5/8" sterile needle	N/A	N/A	Purchased through UT Med Store
Single edge razor blade (2x)	N/A	N/A	Purchased through UT Med Store
Instant adhesive glue	N/A	N/A	Purchased through UT Med Store
Lens paper	N/A	N/A	Purchased through UT Med Store
Glass petri dish (2x)	N/A	N/A	Purchased through UT Med Store
Splinter forceps (2x)	N/A	N/A	Purchased through UT Med Store
PVC handle micro spatula	N/A	N/A	Purchased through UT Med Store
Micro spoon with flat end	N/A	N/A	Purchased through UT Med Store
Detailing brush 5/0	N/A	N/A	Purcahsed from a boutique art store
Wide bore transfer pipette	N/A	N/A	Purchased through UT Med Store
Dental Tweezer	N/A	N/A	Purchased through UT Med Store
Thermometer (digital)	N/A	N/A	Purchased on Amazon.ca
Check carbogen tank (95%O2/5%CO2)	N/A	N/A	Purchased through the Toronto Western Hospital's Suppliers
Vibratome	Leica	N/A	Purchased through the Toronto Western Hospital's Suppliers
brain slice incubation chamber (a.k.a. brain slice keeper)	Scientific Systems Design Inc	N/A
Sodium Chloride (NaCl)	N/A	N/A	Purchased through UT Med Store
Sodium Bicarbonate	N/A	N/A	Purchased through UT Med Store
Dextrose	N/A	N/A	Purchased through UT Med Store
Potassium Chloride (KCl)	N/A	N/A	Purchased through UT Med Store
Magnesium Sulfate (MgSO4.H2O)	N/A	N/A	Purchased through UT Med Store
Sodium phosphate monobasic monohydrate (HNaPO4·H2O)	N/A	N/A	Purchased through UT Med Store
Calcium Chloride (CaCl2·2H2O)	N/A	N/A	Purchased through UT Med Store
Sucrose	N/A	N/A	Purchased through UT Med Store