All studies were performed in accordance with the NIH Guide for the Care and Use of Laboratory Animals and were approved by our Institutional Animal Care and Use Committee. Six young male C57BL/6J mice were obtained from Jackson Laboratories at 8 weeks of age. Six old (20 months) male C57BL/6 mice were obtained from the National Institute of Aging rodent facility. Animals were housed in our vivarium for 1 month in microisolator cages (Lab Products Inc., Seaford, DE). Mice experienced a 12:12 lighting cycle (lights on 0600 CST) and were provided with chow (Envigo Teklad #7012) and water ad libitum.
Mice were euthanized with acute CO2 asphyxiation, followed by rapid decapitation. Brain was isolated from the skull and placed into ice-cold PBS (Hyclone SH30256.01). Under a dissecting microscope, cerebellum was separated from brain and flash-frozen in isopentane for 5-10 s. Frozen cerebellums were stored at -80°C in canonical tubes until shipping, at which time the tissues/tubes were packed in dry ice and shipped overnight to Brains Online for further analysis.
Mouse cerebella were homogenized using a Misonix Sonicator 3000 ultrasonic cell disruptor (Qsonica, LLC, Newton, USA). Per mg cerebellum 4 μL of 0.5 M perchloric acid was added as tissue destruction matrix. After homogenization, the samples were spun down and supernatant was processed further for the quantification of glutamate, GABA, glycine, glutamine, tryptophan, dopamine, norepinephrine, serotonin, alanine, asparagine, and threonine.
Supernatant was further processed and 20 μL of the processed sample was mixed with 4 μL stable labeled internal standards mix of the analytes of interest. The mixture was derivatized with the proprietary SymDAQ reagent (BrainLink, Groningen, NL) in the autosampler part of an integrated LC system (prominence series; Shimadzu, Japan) by addition of 30 μL SymDAQ reagent solution to the sample vial. After the reaction, 45 μL of the mixture was injected onto the HPLC-MS system. Chromatographic separation was performed on a reverse-phase column (100×3.0 mm, 2.5 μm particle size; Synergi MAX-RP, Phenomenex, Torrance, USA) at 30°C. Sample analytes were separated using a 100 to 0% gradient of mobile phase A (ultrapure water/acetonitrile (98/2), 0.1% formic acid) to mobile phase B (ultrapure water/acetonitrile (30/70), 0.1% formic acid) at a flow rate of 300 μl/min. A post-column make-up flow of 150 μL/min consisting of 90% acetonitrile and 10% water, was added to the flow of the HPLC, prior to entering the MS for analyte detection. MS analysis was performed using a system consisting of an API 4000 triple quadropole detector and a Turbo V Ion Spray interface (AB Sciex, Redwood City, USA). The acquisitions were performed in positive ionization mode, with ionization spray voltage set at 3 kV and a probe temperature of 200°C. The instrument was operated in multiple reaction monitoring mode. The collision gas (nitrogen) pressure was held at 2 psig. Data was calibrated and quantified using the Analyst™ version 1.4.2 data system (AB Sciex, Redwood City, USA).
Neurotransmitter/amino acid concentrations were compared over the two cohorts using one-way analysis of variance, with Bonferroni corrections applied to adjust the critical p value for eight simultaneous comparisons (we did not include alanine or threonine in our calculations since these amino acids are not neurotransmitters or neurotransmitter precursors in the cerebellum). Statistics were calculated using MATLAB R (anova1; Mathworks, Natick, MA).