Generation of Medial Thalamus-Anterior Cingulate Cortex Slices from Mouse Brains

Protocolo

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. Brain Slice Preparation

1. Use male C57BL/6J mice, 4-8 weeks old. House the animals in an air-conditioned room (21-23 °C; 50% humidity; 12 hr/12 hr light /dark cycle, lights on at 8:00 AM) with free access to food and water.
2. Take a 250 ml aliquot of the artificial cerebrospinal fluid (aCSF) and place it in a beaker that contains ice. At the same time, supply continuous gas that is composed of 95% O₂ and 5% CO₂.
3. Surgery
   1. Anesthetize the animal with 4% isoflurane in a glass box for approximately 3 min. Once the animal reaches a surgical depth of anesthesia (indicated by the lack of a response to toe pinch), place it on a shallow tray that is filled with crushed ice, and remove the head using scissors.
   2. Expose the skull, and trim off the remaining muscle. Next, using rongeurs, peel away the dorsal surface of the skull from the brain. Trim away the sides of the skull using rongeurs. Sterilize all of the surgical instruments with a 75% ethanol solution.
   3. Using a spatula, cut the olfactory bulbs and nerve connections along the ventral surface of the brain, and remove the brain. After decapitation, quickly transfer the brain to a beaker filled with ice-cold oxygenated aCSF.
4. Preparation of Medial Thalamus (MT)-Anterior Cingulate Cortex (ACC) Brain Slice
   Note: Prepare slices that contain the pathway from the MT to ACC.
   1. Hand-cut the brain block with two sagittal cuts 2.0 mm lateral to the midline in each hemisphere to display the subcortical anatomy. Then make two angled cuts. Make the first cross-cut parallel to the visible fiber tract in the striatum.
   2. Make the second cross-cut from the connection between the cerebellum and visual cortex to the midpoint between the anterior commissure and optic tract that are ventral and parallel to the thalamocingulate pathway.
   3. Attach the brain block to an angular plate (~120°) with cyanoacrylate adhesive and make a cut just above the turning point of the pathway. Unfold the plate, flatten it, and glue it onto the chamber stage of a vibratome.
   4. Make medial thalamus-ACC brain slices (500 µm thick) and then immerse them in ice-cold oxygenated aCSF. Transfer slices to the recording chamber, and keep at 32 °C under continuous perfusion (12 ml/min) with oxygenated aCSF for 1 hr.

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Materiales

Name	Company	Catalog Number	Comments
Anesthetic:
Isoflurane	Halocarbon Products Corporation	NDC 12164-002-25	4%
aCSF (total:1L):
D(+)-Glucose	MERCK	1.08337.1000	10 mM
Sodium hydrogen carbonate	MERCK	1.06329.0500	25 mM
Sodium chloride	MERCK	1.06404.1000	124 mM
(+)-Sodium L-ascorbate, >=98%	SIGMA	A4034-100G	0.15 g / 2 c.c
Magnesium sulfate, anhydrous, ReagentPlus	SIGMA	M7506-500G	2 mM
Calcium chloride dihydrate	MERCK	1.02382.1000	2 mM
Sodium dihydrogen phosphate monohydrate	MERCK	1.06346.1000	1 mM
Potassium chloride	May & Baker LTD Dagenham England	MS 7616	4.4 mM