This article details the methodology for isolating the medial thalamus-anterior cingulate cortex (MT-ACC) pathway from mouse brain tissue. The process involves careful dissection and slicing of brain tissue to maintain viability for subsequent studies.
Begin with a severed mouse head. Remove the skin to expose the skull, and open the skull to access the brain.
Remove the olfactory bulbs and sever any nerve connections to extract the brain into ice-cold oxygenated artificial cerebrospinal fluid, or aCSF.
Take the brain, and make two vertical cuts on each half to expose the internal anatomy.
Make two angled cuts to isolate the region containing the medial thalamus-anterior cingulate cortex or MT-ACC pathway. The MT, a part of the thalamus, acts as a relay station via neuronal projections extending to the ACC in the frontal cortex.
Fix the brain on an angled plate using an adhesive, make a cut to unfold the brain and expose the pathway, and then attach it to a vibratome specimen stage.
Place the stage in the vibratome, add a buffer, and generate slices containing the MT-ACC pathway.
Keep the brain slices in a continuous flow of oxygenated aCSF to maintain their viability.
Expose the skull of the animal, and trim off the remaining muscle. Next, peel away the dorsal surface of the skull using rongeurs and trim away the sides. Subsequently, using a spatula, cut the olfactory bulbs and nerve connections along the ventral surface of the brain before removing it.
Quickly transfer the brain to a beaker filled with ice-cold oxygenated ACSF. To prepare slices containing the MT-ACC pathway, make a sagittal cut 2 millimeters lateral to the midline in each hemisphere to display the subcortical anatomy.
Then, make an angled crosscut parallel to the visible fiber tract in the striatum. Make the second angled cross-cut from the connection between the cerebellum and visual cortex, to the midpoint between the anterior commissure and the optic tract that are ventral and parallel to the thalamus cingulate pathway. After that, attach the brain block to an angular plate with a cyanoacrylate adhesive.
Make a cut just above the turning point of the pathway. After that, unfold the plate, flatten it, and glue it onto the chamber stage of a vibratome. Subsequently, section the brain slices at 500 micrometers thickness and keep them in ice-cold oxygenated ACSF. Transfer a slice to the recording chamber at 32 degrees Celsius with continuous perfusion of oxygenated ACSF for one hour.
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