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Abstract
Dopaminergic PC12 cells can synthesize and release dopamine, providing a good cellular model for investigating dopamine regulation. Optogenetic stimulation of channelrhodopsin-2 provides high spatial and temporal precision for selective stimulation as a powerful neuromodulation tool for neuroscience studies. The aim of this study is to measure dopamine release from dopaminergic PC12 cells under optogenetic stimulation using electrochemical recording of self-assembled monolayers modified microelectrode with amperometric measurement in real time. The activation of PC12 cells under various optogenetic stimulation schemes are characterized by measuring single-cell Ca2+ imaging. After 10 seconds of optogenetic stimulation, the evoked intracellular Ca2+ level and dopamine current of channelrhodopsin-2-transfected PC12 cells were 1.6- and 3.5-fold higher than those of the control cells. The optogenetic stimulation effects on Ca2+ influx and dopamine release were 81% and 63% inhibition by using a Ca2+ channel antagonist Nifedipine. The results indicate that optogenetic stimulation can evoke voltage-gated Ca2+ channel-dependent dopamine exocytosis from PC12 cells in a cell specific, temporally precise and dose-dependent manner. This proposed dopamine recording system can be developed to be a good cell model for dopamine regulation and drug screening in vitro, or dopaminergic cell implantation therapy in vivo using optogenetic stimulation in a precise and convenient way.
Highlights
Dopamine is an important catecholamine neurotransmitter implicated in physiological functions
The Cyclic voltammetry (CV) scans were applied from 20.2 V to 0.7 V in phosphate-buffered saline (PBS) containing dopamine at a rate of 10 V s21
It is important to develop a delicate tool for effective control of dynamic process of dopamine, release or reuptake, from cells and real-time monitoring of extracellular dopamine levels for neuroscience investigation or clinical application
Summary
Dopamine is an important catecholamine neurotransmitter implicated in physiological functions. Conventional approaches for regulating dopamine release in PC12 cells are pharmaceutical or electrical stimulation (ES) techniques, which have critical limitation on controlling dopamine release. Pharmaceutical stimulation such as KCl solution has poor temporal resolution and untargetable regulation of dopamine release from PC12 cells [15,16]. Only some studies have demonstrated the inhibition effect of ES on dopamine release [17,18]. These limitations restrict the application of dopamine release from PC12 cells as a precise cellular model for dopamine regulation
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