Quantifying Neurotransmitters in Mouse Brain

As previously described (Moon et al. 2014 (link)) mouse brain regions were solubilized using a Sonics & Materials, Inc ultrasonic tissue disruptor (Newtown, CT) in 0.1 N HClO₄ and 25 μM ascorbate. Solubilized brains were centrifuged at 12,000 × g for 10 min at 4 °C. Supernatants were collected and diluted in 0.02 N HClO₄ for analysis using an ESA Coulochem III detector with a 5041 Enhanced Analytical cell containing a +320 mV Thermo Scientific glassy carbon electrode (Sunnyvale, CA). Norepinephrine (NE), normetanephrine (NME), dopamine (DA), 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), 5-hydroxytrypatime (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) were measured using a pH 3.0 mobile phase (75 mM NaH₂PO₄, 25 μM EDTA, 0.45 mM octanesulfonic acid and triethylamine in acetonitrile:water (5:95, v:v)) pumped at 0.2 μL/min through a 2 mm × 150 mm Thermo Scientific ODS Hypersil column. Tissue pellets were homogenized in RIPA buffer (150 mM NaCl, 5 mM EDTA, 0.1% SDS, 1% Triton and 10 mM Tris, pH 7.4) and protein concentrations determined using the BioRad DC Protein Assay (Hercules, CA). Results were quantified as ng analyte/mg brain region protein. Values were expressed as raw values and as ratios.