TITLE: Mouse Brain Tissue Collection and Analysis 
AUTHORS: Robert Edwards, Shweta Jain

[DESCRIPTION]
This protocol describes the dissection and collection of coronal sections of the striatum and midbrain from a mouse brain. The tissue can be used in a number of applications and here we describe two: the measurement catecholamine levels using high-performance liquid chromatography (aided by a neurochemistry core) and the distribution of the vesicular monoamine transporter (VMAT2) using a radioligand binding assay.

[STEPS]
SECTION: Tissue dissection
2. Euthanize mice by inhalation of CO2
SECTION: Tissue dissection
3. Decapitate mouse and remove brain
SECTION: Tissue dissection
4. Place brain into a rodent brain matrix (RBM-2000C, Protech International Inc.)
SECTION: Tissue dissection
5. By inserting razor blades into the slots of the brain matrix, collect two 1 mm coronal sections containing the striatum

 

SECTION: Tissue dissection
6. Remove the cortical tissue and cut out striatum, collecting tissue in cold solution (HBSS or SHT depending on application)
SECTION: Tissue dissection
7. From the same brain, dissect the midbrain using the brain matrix
Transfer this region onto parafilm. Remove top 70% of the dorsal side and collect the ventral side (containing the VTA and substantia nigra) into cold solution (HBSS or SHT depending on application). Do not separate hemispheres; there will be one sample per mouse.
SECTION: Measurement of catecholamine levels by HPLC
8. Transfer tissue to Eppendorf tubes and flash freeze in liquid nitrogen. Immediately transfer to -80 °C .

SECTION: Measurement of catecholamine levels by HPLC
9. Tissue is shipped on dry ice to the Vanderbilt Neurochemistry Core (https://lab.vanderbilt.edu/vbi-core-labs/neurochemistry-core/), where tissue catecholamine levels is measured by HPLC with coupled electrochemical detection.
SECTION: Tissue dissection
1. Prepare solutions
SECTION: Tissue dissection
1.1. For measurement of brain catecholamine by HPLC, use cold Hank's Balanced Salt Solution (HBSS) containing 10 mM HEPES and 20 mM glucose
SECTION: Tissue dissection
1.2. For [3H]-Dihydrotetrabenazine binding, dissection into cold SHT buffer (320 mM sucrose, 10 mM HEPES/Tris, pH 7.4) with 0.4 mM EDTA and Complete Protease Inhibitor Cocktail (Roche)
SECTION: [3H]-Dihydrotetrabenazine binding
10. Disrupt midbrain and striatal tissue with 12 strokes of a Dounce homogenizer at 500 RPM in cold SHT buffer
SECTION: [3H]-Dihydrotetrabenazine binding
11. Sonicate tissue for 30 seconds
SECTION: [3H]-Dihydrotetrabenazine binding
12. Sediment the debris in a centrifuge at 2000 g for 2 minutes
SECTION: [3H]-Dihydrotetrabenazine binding
13. Collect supernatant
SECTION: [3H]-Dihydrotetrabenazine binding
14. Measure protein content with bicinchoninic (BCA) assay 
SECTION: [3H]-Dihydrotetrabenazine binding
15. Dilute 50 µg protein into SHT buffer and add 10 nM (+)-a-dihydrotetrabenazine [9-O-methyl-3H] (ARC; 80 Ci/mmol); incubate at 30 °C for 30 minutes
Binding should be performed in triplicate for each sample; measure non-specific binding by adding 10 µM non-radioactive benazine in the assay


SECTION: [3H]-Dihydrotetrabenazine binding
16. Stop reaction by filtration through a Supor 200 0.2 µm filter (PALL); wash 3 times in ice-cold SHT buffer with 20 mM tetrabenezine (Fluka)
SECTION: [3H]-Dihydrotetrabenazine binding
17. Measure radioligand signal and normalize specific binding to the amount of membrane protein added to the reaction