Novel integrated microdialysis–amperometric system for in vitro detection of dopamine secreted from PC12 cells: Design, construction, and validation

Abstract

a b s t r a c tA 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.Ó A wide variety of techniques have been used for studying in vitro DA secretion/release, and most of them use PC12 cells because they are capable of synthesizing, secreting, and metabolizing DA. The PC12 cell line, obtained from rat pheochromocytoma of the adrenal medulla, is also used as a tool to understand the biochemical mechanisms underlying the physiology and degeneration of central dopamine neurons [12][13][14]. In previous studies, we successfully used PC12 cells to investigate the mechanism of nitric oxide (NO) donor-induced DA secretion [7] and DA oxidative homeostasis in vitro [6,8]. In such studies, DA was quantified in microdialysates by high-performance liquid chromatography with electrochemical detection (HPLC-EC). The aim of the current research was to combine a modified device for in vitro microdialysis with a novel, dual channel amperometric system for the online detection of DA secreted from PC12 cells. The new electronics de-0003-2697/$ -see front matter Ó

LRRK2 Affects Vesicle Trafficking, Neurotransmitter Extracellular Level and Membrane Receptor Localization

“…The capillary tube for microdialysis of PC12 cell lines is an adaptation of an in vitro device described previously [28], [29]. The microdialysis probe was constructed using two sections of plastic-coated silica tubing (150 µm o.d., 75 µm i.d., Scientific Glass Engineering, Milton Keynes, UK), each placed in the centre of a semipermeable polyacrylonitrile dialysis fiber (AN-69, Hospal Industrie, Meyzieu, France).…”

“…Cell viability was assessed before the start and at the end of each experiment by trypan blue exclusion. The viability rate was given as the difference between final and initial percentage of non-viable cells [29], [30].…”

Real-Time Monitoring of Brain Tissue Oxygen Using a Miniaturized Biotelemetric Device Implanted in Freely Moving Rats

“…Prior to the start of any experimental procedure, the health of the animals was assessed according to published guidelines. 15 16 Approximately 1 mm of the wire was exposed and inserted into a silica capillary tube (10 mm in length; i.d. ) 180 µm, Polymicro Technologies, Phoenix, AZ) partly filled with graphiteloaded (55% w/w) epoxy resin (Araldite-M, Sigma-Aldrich, Milan, Italy).…”

“…180 µm, Polymicro Technologies, Phoenix, AZ) partly filled with graphiteloaded (55% w/w) epoxy resin (Araldite-M, Sigma-Aldrich, Milan, Italy). A preliminary 180 µm diameter carbon-composite disk electrode (area, 2.5 × 10 -4 cm 2 ) was fabricated by mixing 850 mg of graphite with 500 mg of Araldite-M and 200 mg of harderer 16 and filling the silica capillary tubing with the mixture. The silver wire guaranteed a good electrical contact.…”

“…No significant interference signals were observed on exposing the sensors to other electroactive molecules (ascorbic acid (AA); uric acid (UA); dopamine (DA); 3,4-dihydroxy-phenylacetic acid (DOPAC); homovanillic acid (HVA)) present in the striatal extracellular fluid, even at pharmacologically relevant concentrations (0.5 mM for AA and up to 5 µM for the other neurochemicals). 16 Sensor selection was performed connecting microelectrodes to a three-electrode system (BAS CV37) and exposing them to variable O 2 concentrations (0%-100%) by means of the apparatus illustrated in Figure 2; only sensors having a O 2 sensitivity higher than 200 pA µM -1 were selected ( Figure 3A). A more accurate calibration was performed at low oxygen concentrations ( Figure 3B) connecting the microsensor to the biotelemetric device (two-electrode system) and adding known volumes of a standard O 2 solution (100%) to nitrogen-saturated PBS.…”