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Optogenetic methods

Various microorganisms have developed light sensitive protein channels and pumps in order to respond to their environment. Among these channelrhodopsins are Channelrhodopsin 2, from C. reinhardtii, and GtACR1 from G. theta. ChR2 opens in response to blue light and conducts cations.
We are using a mutated version of channelrhodopsin (ChR2opt) to stimulate cardiomyocyte-like cells and neurons with light. Alternatively, GtACR1 opens upon exposure to green light and conducts Cl- allowing us to suppress cell signals.



Photo-responsive ion channels can be selectively expressed in cortical neurons using recombinant adeno-associated virus particles to deliver genetic material. Cell activity can then be measured using our chip based technology for days without harming the cell while the activity of the network is manipulated with light. We've further shown the advantages in terms of spatial resolution of optogenetic stimulation over extracellular electrical stimulation. Though the size of the area stimulated relative to the size of the stimulation source in both optical and electrical stimulation, technical factors allow much smaller areas of light to be applied and still have sufficient charge injection to trigger action potentials. This light based stimulation compliments the high temporal accuracy of extracellular recordings, making optogenetics and on-chip recordings complimentary techniques.

We are using our ability to pattern cell growth via microcontact printing to investigate the strength and dynamics of light based stimulation dependent on cell morphology and neurite branching. The use of patterned networks further can be used to define connectivity, suggesting the combined use of optogenetics and cell patterning to investigate defined biological computations. This combined system has already been used to validate biochemically identified axon guidance with directional signal propagation in patterned networks.


Optogenetics_3Laser-gated neurophysiology/2 color setup



Diverse genetically engineered ‘optogenetic tools’ are used to activate or inactivate neurons by light stimulation of encoded proteins. Our two laser-illuminating system allows precise targeting of cultured neurons in both temporal and spatial scales.

To investigate information processing in neural circuits, which are represented by changes of network firing patterns, we presented preliminary tests of manipulating network dynamics by optical stimulation of neurons genetically modified with ChR2opt.

A. Two Laser-illuminating System
B. Fluorescence image of ChR2opt-mKate transduced neuronal network on MEA.

Stimulation positions are shown in red (473 nm, P: pulse 50 ms, D: dark 200 ms, spot diameter: 25 µm). Scale bar: 100 µm.


Optogenetics_4

Additional Information

Contact:

Dr. Vanessa Maybeck

Tel.:  +49-2461-61-3285
e-mail: v.maybeck@fz-juelich.de

More Information

Publications:

An evaluation of extracellular MEA versus optogenetic stimulation of cortical neurons
Maybeck et al., Biomed. Phys. Eng. Express, 2016, 2, 055017.


High-efficiency transduction and specific expression of ChR2opt for optogenetic manipulation of primary cortical neurons mediated by recombinant adeno-associated viruses
Jin et al., J. Biotechnol., 2016, 233, 171–180.

 

Optogenetic Control of Neuronal Network Activity on MEA
Li et al., Front. Neurosci. Conference Abstract: MEA Meeting 2016 | 10th International Meeting on Substrate-Integrated Electrode Arrays.


Recombinant Adeno-associated virus (rAAV)-mediated transduction and optogenetic manipulation of cortical neurons in vitro
Lange et al., Proc. SPIE 8928, Optical Techniques in Neurosurgery, Neurophotonics, and Optogenetics, 89282S, vol. 8928, p. 89282S.


Light induced stimulation and delay of cardiac activity
Hofmann et al., Lab Chip, 2010, 10, 2588–96.


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