Expression of Channelrhodopsin-2 Using in Suspension Electroporation for Studying the Monosynaptic Transmission in Neuronal Culture
- 81 Downloads
Understanding the mechanism of synaptic transmission and its plasticity is one of the central goals of modern neuroscience. Neuronal culture is a very convenient model for studying mechanisms of synaptic transmission. However, investigation of two or more synaptically connected neurons in culture by conventional methods is a difficult task. In this study, we describe new protocol for studying the synaptic transmission between cultured neurons using in suspension electroporation for the expression of channelrhodopsin-2 (ChR2) which was applied to only certain subpopulation of cultured cells, leaving the majority of neurons completely untreated. We show that this technique allows reliable long-lasting (hours) recording of monosynaptic excitatory postsynaptic potentials (EPSPs) in cultured hippocampal neurons using a repeated light stimulation of neighboring ChR2-expressing neurons.
KeywordsElectroporation Neuronal culture Channelrhodopsin Optogenetics Synaptic transmission
This research was funded by the Russian Science Foundation grant 14-25-00072 (neuronal cell culture and development of the electroporation protocol) and by RFBR grant no. 15-04-06286 (whole cell recording with optical stimulation).
Compliance with Ethical Standards
All experimental procedures were conducted in accordance with the European Communities Council Directive of 24 November 1986 (86/609/ EEC) on the protection of animals used for scientific purposes. The study protocol was approved by the Ethics Committee of the Institute of Higher Nervous Activity and Neurophysiology of RAS.
- 2.Sombati, S., & Delorenzo, R. J. (1995). Recurrent spontaneous seizure activity in hippocampal neuronal networks in culture. Journal of Neurophysiology, 73, 1706–1711.Google Scholar
- 3.Bi, G. Q., & Poo, M. M. (1998). Synaptic modifications in cultured hippocampal neurons: dependence on spike timing, synaptic strength, and postsynaptic cell type. Journal of Neuroscience, 18, 10464–10472.Google Scholar
- 5.Hayakawa, K., & Yasuhiko, H. (2013). Foreign gene transfer method by electroporation technique. Patent US 2013/0122592 A1.Google Scholar