Mouse Brain Tissue Collection and Analysis
Robert Edwards, Shweta Jain
Abstract
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
Tissue dissection
Prepare solutions
For measurement of brain catecholamine by HPLC, use cold Hank's Balanced Salt Solution (HBSS) containing 10 mM HEPES and 20 mM glucose
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)
Euthanize mice by inhalation of CO2
Decapitate mouse and remove brain
Place brain into a rodent brain matrix (RBM-2000C, Protech International Inc.)
By inserting razor blades into the slots of the brain matrix, collect two 1 mm coronal sections containing the striatum
Remove the cortical tissue and cut out striatum, collecting tissue in cold solution (HBSS or SHT depending on application)
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.
Measurement of catecholamine levels by HPLC
Transfer tissue to Eppendorf tubes and flash freeze in liquid nitrogen. Immediately transfer to -80°C .
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.
[3H]-Dihydrotetrabenazine binding
Disrupt midbrain and striatal tissue with 12 strokes of a Dounce homogenizer at 500 RPM in cold SHT buffer
Sonicate tissue for 30 seconds
Sediment the debris in a centrifuge at 2000 g for 2 minutes
Collect supernatant
Measure protein content with bicinchoninic (BCA) assay
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
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)
Measure radioligand signal and normalize specific binding to the amount of membrane protein added to the reaction
