Effects Of Ethanol On Light-Activated Current Flow In Hek293 Cells Expressing Channelrhodopsin-2

Julia Sequerth

School Name

Governor's School for Science and Math

Grade Level

12th Grade

Presentation Topic

Cell and Molecular Biology

Presentation Type

Mentored

Mentor

Mentor: Dr. Woodward; Department of Psychiatry, Medical University of South Carolina

Abstract

Optogenetics is a novel technique used in research to understand the function of the brain. It can be theorized to aid in mapping neural circuitry and understanding human brain disorders such as addiction. ChR2 is a light-activated, nonspecific cation channel from the algae Chlamydomonas reinhardtii. When neurons expressing ChR2 are subjected to blue light stimulus, the neurons are activated. Currently optogenetics uses Channelrhodopsin-2 (ChR2) DNA transfection in neurons of mice. Past research has demonstrated that neuron channels, such as the GABA and NMDA receptors, exhibit channel inhibition under high concentrations of alcohol. The aim of this research is to test if ChR2 channels are affected in the presence of alcohol so they may be cleared for future use in alcohol addicts by optogenetics. The hypothesis is that ChR2 channels are not be affected by blue light in the presence of alcohol. This will be achieved by transfecting HEK293 cells with ChR2-GFP plasmid DNA, treating the cells with 100mMol and 300mMol ethanol (EtOH), followed by patch clamping in two blue light stimulant protocols, each for a duration of ten seconds. Results by Evos microscopy indicated that the transfected HEK293 cells had higher than 60% expression of ChR2 channels. Patch clamping results of the HEK293 cells showed inhibited negative current in the presence of EtOH under light-activation. However, the results of inhibition in 300mMol EtOH might be due to damage to HEK293 cells. ChR2 channel is not affected in the presence of alcohol so they can be used to treat alcohol addiction.

Location

Owens 202

Start Date

4-16-2016 8:30 AM

COinS
 
Apr 16th, 8:30 AM

Effects Of Ethanol On Light-Activated Current Flow In Hek293 Cells Expressing Channelrhodopsin-2

Owens 202

Optogenetics is a novel technique used in research to understand the function of the brain. It can be theorized to aid in mapping neural circuitry and understanding human brain disorders such as addiction. ChR2 is a light-activated, nonspecific cation channel from the algae Chlamydomonas reinhardtii. When neurons expressing ChR2 are subjected to blue light stimulus, the neurons are activated. Currently optogenetics uses Channelrhodopsin-2 (ChR2) DNA transfection in neurons of mice. Past research has demonstrated that neuron channels, such as the GABA and NMDA receptors, exhibit channel inhibition under high concentrations of alcohol. The aim of this research is to test if ChR2 channels are affected in the presence of alcohol so they may be cleared for future use in alcohol addicts by optogenetics. The hypothesis is that ChR2 channels are not be affected by blue light in the presence of alcohol. This will be achieved by transfecting HEK293 cells with ChR2-GFP plasmid DNA, treating the cells with 100mMol and 300mMol ethanol (EtOH), followed by patch clamping in two blue light stimulant protocols, each for a duration of ten seconds. Results by Evos microscopy indicated that the transfected HEK293 cells had higher than 60% expression of ChR2 channels. Patch clamping results of the HEK293 cells showed inhibited negative current in the presence of EtOH under light-activation. However, the results of inhibition in 300mMol EtOH might be due to damage to HEK293 cells. ChR2 channel is not affected in the presence of alcohol so they can be used to treat alcohol addiction.