Parameters of optical stimulation and the strength of depolarization in ChR2-transfected neurons

Składowska A1., Bekisz M1 , Majkowska M1., Łempicka M.1,2, Kublik. E1.
1) Department of Neurophysiology, Nencki Institute of Experimental Biology, Warsaw, Poland
2) Faculty of Physics, Warsaw University of Technology, Warsaw, Poland

With the use of optogenetics, neurons genetically modified to produce membrane channel sensitive to defined wavelength of light, can be activated with high temporal precision and unmatched cellular specificity. In this report we summarize the results of experiments testing the dependence of neural membrane depolarization on the different parameters of light stimulation. Modification of neurons in prelimbic cortex and/or central nucleus of amygdala of rats was achieved by an injection of AAV-hSyn-ChR2-EYFP viral vector introducing channelrhodopsin, a light sensitive sodium channel that opens in response to blue light (473 nm wavelengths) and causes excitatory response of modified neurons.
In vitro patch-clamp recording allowed for direct measurement of membrane depolarization in response to light emitted from LED source generating about 50 mW of light power (adequate to 0.26 lm) when supplied with a maximum current of 1 A. In consecutive steps of experiment we were modifying 1) light power; 2) duration of light impulse; 3) frequency of light impulses.
1) Cells were stimulated with a train of 2 ms light impulses delivered at 20 Hz with a driving current amplitude varying from 0.1 A to 1A . Observed relation between light power and resulting membrane depolarization can be approximated by a logarithmic function: 2.2 ln(x) + 13) (at -50 mV resting potential) and 8 ln(x) + 28 at membrane potential kept at -60 mV. The dependency of the latency of first action potential on the light intensity was found to be approximated by a power function: 1.2 x-1.3.
2) Cells were stimulated with a train of light impulses of varying duration (range 2 ms - 20 ms) delivered at 20 Hz, a current set at 1000 mA and resting membrane potential kept at 50 mV. We have found that the relation between light impulse duration and resulting membrane depolarization can be approximated by a logarithmic function 3 ln(x) + 4).
3) Cells were stimulated with a train of light impulses driven by 2 ms square pulses with a current set at 1000 mA and a frequency varying in a range between 20 Hz and 200 Hz. Membrane resting potential was kept at -50 mV. Relation between light impulse duration and resulting membrane depolarization can be approximated by a logarithmic function: 2.7 ln (x)+3).

[Supported by Polish National Science Centre grant 2013/08/W/NZ4/00691]

Author: Aleksandra Składowska
Conference: Title