This article details a method for isolating oxytocin receptor-rich nuclei from neonatal rat brains. The process involves precise anatomical identification and careful handling of brain slices to preserve neuronal viability.
The brain's oxytocin receptor-rich nuclei are clusters of neurons that express oxytocin receptors, which interact with oxytocin, a neuropeptide.
To isolate oxytocin receptor-rich nuclei, begin with a neonatal rat brain.
Place the brain in a mold with a slicing guide. Slice the entire brain to expose different regions with nuclei.
Transfer these slices to a dish and immobilize them.
Add artificial cerebrospinal fluid, or aCSF, to maintain neuronal viability and incubate under gentle rotation.
Using a published brain atlas as a reference, identify anatomical landmarks to locate oxytocin-receptor-rich nuclei within the slices.
Transfer an identified brain slice of interest to a new plate.
Under a microscope, locate the nuclei within the slice and punch it.
Repeat the process for other identified sections to isolate the oxytocin-rich nuclei.
Transfer the isolated nuclei to a tube containing ice-cold protein extraction buffer supplemented with protease and phosphatase inhibitors to preserve the oxytocin receptors.
After harvesting the brain, rapidly place the whole organ in a polymethylmethacrylate brain mold at room temperature, and immediately use a new razor blade to make 500-micrometer-thick slices of the tissue. Laying the slices rostral to caudal in a Petri dish as they are obtained, to maintain the correct orientation of the sections. When the entire brain has been sectioned, quickly add artificial cerebrospinal fluid without glucose to the dish and incubate the slices for 60 minutes at 28 to 30 degrees Celsius, with constant stirring on an orbital shaker.
Use a brain atlas and anatomic landmarks on each tissue to identify the brain nuclei to be punched, and place the slice with the nuclei of interest in a new Petri dish under a dissecting microscope. Once visualized, use a coring tool to quickly punch out four to six different nuclei, and rapidly immerse each punched nucleus in 0.06 milliliters of ice-cold protein extraction buffer in the appropriately labeled microcentrifuge tube, containing protease inhibitors and phosphatase inhibitors for 60 minutes.
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