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Determining Serotonin and Dopamine Uptake Rates in Synaptosomes Using High-Speed Chronoamperometry.

Editors

In: Michael AC, Borland LM, editors.

Source

Electrochemical Methods for Neuroscience. Boca Raton (FL): CRC Press; 2007. Chapter 7.

Excerpt

The methodology for using chronoamperometry to measure transporter-mediated uptake into synaptosomes is described here. Alterations in uptake in brain tissue from mice lacking one or both (SERT−/−) intact copies of the SERT gene are detailed and compared with previous studies using either radiometric methods in synaptosomes [38] or chronoamperometry in vivo [61]. The factors underlying the discrepancies between the results of radiochemical and chronoamperometric serotonin uptake experiments are also explored. We show that oxygen saturation of the assay buffer greatly influences clearance rates and that a clear incremental relationship exists between serotonin transporter gene expression and serotonin uptake rates [65,66]. We have discovered that mechanical disruption of synaptosomes by stirring or filtration leads to significant loss of transported serotonin resulting in misleadingly low maximal uptake rates (Vmax) and transporter substrate affinities (Km), as well as a loss of sensitivity for determining biologically important alterations in uptake rates [66]. Finally, we show how chronoamperometry has been used to determine decreases in dopamine uptake in striatal synaptosomes prepared from a transgenic mouse model expressing a mutant form of human α-synuclein, which is associated with a hyperactive phenotype [16].

Copyright © 2007, Taylor & Francis Group, LLC

PMID:
21204396
[PubMed]
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