title: Novel integrated microdialysis-amperometric system for in vitro detection of dopamine secreted from PC12 cells: design, construction, and validation.
abstract: A novel dual channel in vitro apparatus, derived from a previously described design, has been coupled with dopamine (DA) microsensors for the flow-through detection of DA secreted from PC12 cells. The device, including two independent microdialysis capillaries, was loaded with a solution containing PC12 cells while a constant phosphate-buffered saline (PBS) medium perfusion was carried out using a dual channel miniaturized peristaltic pump. One capillary was perfused with normal PBS, whereas extracellular calcium was removed from extracellular fluid of the second capillary. After a first period of stabilization and DA baseline recording, KCl (75 mM) was added to the perfusion fluid of both capillaries. In this manner, a simultaneous "treatment-control" experimental design was performed to detect K+-evoked calcium-dependent DA secretion. For this purpose, self-referencing DA microsensors were developed, and procedures for making, testing, and calibrating them are described in detail. The electronic circuitry was derived from previously published schematics and optimized for dual sensor constant potential amperometry applications. The microdialysis system was tested and validated in vitro under different experimental conditions, and DA secretion was confirmed by high-performance liquid chromatography with electrochemical detection (HPLC-EC). PC12 cell viability was quantified before and after each experiment. The proposed apparatus serves as a reliable model for studying the effects of different drugs on DA secretion through the direct comparison of extracellular DA increase in treatment-control experiments performed on the same initial PC12 cell population.
authors.affiliation: Department of Neuroscience, Medical School, University of Sassari, 07100 Sassari, Italy.
authors.forename: Rossana
authors.initials: R
authors.lastname: Migheli
authors.forename: Giulia
authors.initials: G
authors.lastname: Puggioni
authors.forename: Sonia
authors.initials: S
authors.lastname: Dedola
authors.forename: Gaia
authors.initials: G
authors.lastname: Rocchitta
authors.forename: Giammario
authors.initials: G
authors.lastname: Calia
authors.forename: Gianfranco
authors.initials: G
authors.lastname: Bazzu
authors.forename: Giovanni
authors.initials: G
authors.lastname: Esposito
authors.forename: John P
authors.initials: JP
authors.lastname: Lowry
authors.forename: Robert D
authors.initials: RD
authors.lastname: O'Neill
authors.forename: M S
authors.initials: MS
authors.lastname: Desole
authors.forename: Egidio
authors.initials: E
authors.lastname: Miele
authors.forename: Pier A
authors.initials: PA
authors.lastname: Serra
mesh_terms: D000818:Animals; D002118:Calcium / Q000494:pharmacology; D002138:Calibration; D002452:Cell Count; D002470:Cell Survival; D004298:Dopamine / Q000032:analysis* / Q000378:metabolism; D004563:Electrochemistry / Q000295:instrumentation / Q000379:methods*; D017551:Microdialysis / Q000295:instrumentation / Q000379:methods*; D016716:PC12 Cells; D011189:Potassium Chloride / Q000494:pharmacology; D051381:Rats; D015203:Reproducibility of Results; D012636:Secretory Rate / Q000187:drug effects; D012984:Software
chemical_list: D011189:Potassium Chloride; D002118:Calcium; D004298:Dopamine