Brain Slice Preparation for electrophysiology recording

On the prior day, prepare the working aCSF and sucrose aCSF solution in a 1:10 dilution from stock solutions (1 part stock, 1 part bicarbonate, 8 part Milli-Q water).

On day of cutting, place sucrose aCSF solution in -20°C freezer for 30-40 minutes.

Remove sucrose aCSF and place in large Styrofoam container of ice to remain cold.

Place two 95mm diameter glass petri dishes on top of ice bucket and fill with cold sucrose aCSF. Bubble the sucrose aCSF in the bottle and the two petri dishes.

Turn on water bath and set to 35°C.

Fill a 250 mL beaker with 150mL of working aCSF.

Place a second container that has a sieve bottom into the beaker containing the working aCSF.

Place the beaker with the aCSF and second container in water bath and begin bubbling working aCSF.

Fill a 95 mm diameter glass petri dish with working aCSF and set aside.

On the cutting head of the vibratome, rotate blade holder 45°-90° using a size 3 Allen key.

Open the blade holder using a size 3 Allen key and insert razor blade into blade holder.

Clamp down the blade holder using the size 3 Allen key until hand-tight.

Set parameters (speed, amplitude, and section thickness) on the Vibratome control panel.

Turn on Julabo FL300 (recirculating chiller/cooler) and set to desired temperature to cool buffer tray.

Cut a small piece of agarose (approximately 2x2cm square) and super glue to specimen plate so that it is standing on its side.

Fill one 10 mL syringe with cold sucrose aCSF, connect with tubing, and place 27G needle at end of tubing.

Fill metal tray with ice.

Weight the mouse to the nearest 0.1 gram. Anesthetize with Avertin (dosage: 0.3mL/10g body weight, i.p.). Place the mouse back to the home cage.

Use toe pinch-response method to determine depth of anesthesia.

Place the animals On ice in metal tray lying on the back with face upward.

Make an incision through the abdominal skin.

Make two additional skin incisions from the xiphoid process along the base of the ventral ribcage laterally.

Gently reflect the two flaps of skin to expose thoracic field completely.

Grasp the cartilage of the xiphoid process with blunt forceps and raise it slightly to insert pointed scissors. Cut through the thoracic musculature and ribcage between the breastbone and medial rib insertion points and extend the incision rostrally to the level of the clavicles.

Separate the diaphragm from the chest wall on both sides with scissor cuts.

Clamp the reflected ribcage laterally with a hemostat to expose the heart.

Secure the beating heart with fingers or blunt forceps, and immediately insert a blunt 27G syringe needle.

Cut the right atrium with scissors, and at the first sign of blood flow, begin the infusion of 1x sucrose aCSF at 2-4mL/min.

Continue perfusion with sucrose aCSF until the 10 mL syringe is empty.

Decapitate the mouse with large surgical scissors.

Place the decapitated head in one of the petri dishes containing cold, bubbling sucrose aCSF.

Cut down the midline to expose the skull.

Make two lateral and one dorsal cut using sharp surgical scissors on the base of the skull.

Cut the olfactory bulbs/optic nerve at rostral end of skull.

Gently peel off the skull using blunt forceps.

Once brain is fully exposed, make an incision along the coronal plane of the cerebellum to create a flat surface.

Super glue the flat section onto the specimen plate so that the vibratome cuts along coronal plane.

Place specimen plate in buffer tray and fill buffer tray with sucrose aCSF.

Place O₂/CO₂ tubing into buffer tray to bubble sucrose aCSF.

If using manual mode :

Press the “AUTO/MAN” button so that the bottom LED light is on, indicating semiautomatic/manual mode. In semiautomatic mode, only continuous slicing (SINGLE) is possible.

Position razor blade (using the blade forward/backward and “UP” / ”DOWN” buttons on control panel) at desired location and perform first cut by pressing the “RUN/STOP” button.

Press “RUN/STOP” on the control panel after blade has successfully cut through the entire brain.

Return the blade to the initial position using the blade backwards button on control panel.

Turn Dial 2 (location next to “Section Thickness” button on control panel) to desired slice thickness.

Press “RUN/STOP” to perform second cut.

Press “RUN/STOP” again after blade has successfully cut through the entire brain.

Repeat steps 40.3-40.6 until desired about of tissue or brain region(s) is cut.

If using continuous mode :

Press the “AUTO/MAN” button so that the top LED light is on, indicating automatic mode.

Press the “SINGLE/CONT” button to toggle between single stroke and continuous stroke.

Position razor blade (using the blade forward/backward and “UP”/”DOWN” buttons on the control panel) above the brain at the initial window edge.

Press the first cutting window button to set the first window edge.

Move the blade forward to the second window edge and press the second window cutting edge button.

Steps about continuous slicing.

After each slice, remove brain slice from buffer tray using a glass wide bore transfer pipette and place into the second petri dish On ice containing bubbling sucrose aCSF.

Once all brain slices are collected, move brain slices from the petri dish containing sucrose aCSF to a Room temperature petri dish containing working aCSF.

Move brain slices from the petri dish containing working aCSF to beaker in the water bath with bubbling working aCSF.

After 30 minutes, turn off water bath and leave brain slices in beaker for 1h 0m 0s to recover.