For all comparisons, age df = 1, error df = 10, total df = 11. Respective F and p values are as follows: Glu F1,10 = 1.13, p = 0.3131; GABA F1,10 = 1.64, p = 0.229; Gln F1,10 = 0.38, p = 0.5495; Gly F1,10 = 6.85, p = 0.0257; Ala F1,10 = 0.44, p = 0.52; Thr F1,10 = 3.8, p = 0.0799; Trp F1,10 = 3.13, p = 0.1073; Asn F1,10 = 2.9. p = 0.1192; DA F1,10 = 0.08, p = 0.7863; NE F1,10 = 0.03, p = 0.8665; 5-HT (*) F1,10 = 13.53, p = 0.0043. Critical p = 0.0063 to achieve α = 0.05 for Bonferroni correction overcomparisons.
The predicted outcome of increasing cerebellar serotonergic activity may thus be to increase granule cell input sensitivity to mossy fiber input, decrease Purkinje cell sensitivity to parallel fiber input, and increase inhibition of Purkinje cell firing. These effects would combine to expand mossy fiber input participating in center-surround inhibition. In other words, increased serotonergic activity may allow a larger set of afferent sensory inputs concurrent access to the Purkinje cell network while preserving overall Purkinje cell firing properties. Of note, in normal human volunteers, increased extracellular serotonin (through a single dose of a selective serotonin uptake inhibitor) evoked a significant increase in resting-state fMRI centrality measures across the cerebellum, consistent with the hypothesis that increased serotonergic activity enhance cerebellar functional connectivity.It is thus interesting to note in this context that inhibition of serotonergic tone (by treatment with the partial 5 HT1AR agonist buspirone) improved cerebellar tremor. Further, prominent cerebellar ataxia has been observed in aged (21-24 mo old) compared to young (2-3 mo old) C57BL/6 mice . Locomotor ataxia thus accompanies the increased cerebellar serotonin content observed in C57BL/6 mice, but not in BALB mice with no age-related changes in cerebellar serotonin content. Gait ataxia may potentially be a behavior evoked by serotonergic increases in cerebellar functional connectivity. Further studies to determine if age-associated increases in cerebellar serotonin content are reflected in extracellular serotonin concentrations, and to determine the specific serotonin receptor subtypes associated with Luga o cell activation, will better define the mechanisms through which serotonergic activity may influence cerebellar tremor, as well as identify potential therapeutic targets.
All studies were performed in accordance with the NIH Guide for the Care and Use of Laboratory Animals and were approved byInstitutional Animal Care and Use Committee. Six young male C57BL/6J mice were obtained from Jackson Laboratories at 8 weeks of age. Six old (20 mo ) male C57BL/6 mice were obtained from the National Institute of Aging rodent facility. Animals were housed in vivarium for 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.
Mouse cerebella were homogenized using a Misonix Sonicator 3000 ultrasonicell disruptor (Qsonica, LLC Newton, USA). Per mg cerebellum 4 μ 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 μ100×3.0 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 μ/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 w calibrated and quantified using the Analyst™ version 1.4.2 data system (AB Sciex, Redwood City, USA).of the processed sample was mixed with 4 μ 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 μ SymDAQ reagent solution to the sample vial. After the reaction, 45 μ of the mixture was injected onto the HPLC-MS system. Chromatographic separation was performed on a reverse phase column (
Neurotransmitter/amino acid concentrations were compared over the two cohorts using, with Bonferroni corrections applied to adjust the critical p value for 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 ( Mathworks, Natick MA).
No fraudulence is committed in performing these experiments or during processing of the data. We understand that in the case of fraudulence, the study can be retracted by Matters.
Create a Matters account to leave a comment.