Sviluppo di un nuovo sistema voltammetrico microdialitico per lo studio delle variazioni neurochimiche in coltura

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 medium perfusion (PBS) was carried out using a dual channel miniaturized peristaltic pump. One capillary was perfused with normal PBS, whereas extracellular Ca2+ 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 Ca2+-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 optimized for dual sensor CPA applications. The microdialysis system was tested and validated in vitro, and DA secretion was confirmed by 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.