Combined Use of Optogenetics and Genetically Encoded Fluorescence Imaging in Primary Cortical Cultures on Micro-electrode Arrays
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1
University Medical Center Mainz, Institute of Physiology, Germany
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2
University Medical Center Mainz, Institute of Physiology, Germany
Motivation
Combining extracellular electrical recordings with optogenetics and genetically encoded fluorescence microscopy enables
correlation of electrical activity patterns with cell- and network physiology and morphology. Here, we describe MEA-based
recordings of electrical activity from developing primary cortical cultures, transduced with channelrhodopsin2-H134R
(ChR2-H134R) for optogenetic stimulation, and the cell nucleus marker, histone cluster 1, H2bb, fused to mCherry (H2B-mCherry)
for chronic visualization of living neurons. Co-transduction of ChR2-H134R and H2B-mCherry served a dual purpose. It allowed
us to determine the earliest time point at which the expression of ChR2-H134R reached functional levels, and it enabled us
to evaluate the effectiveness of light stimulation, at an early stage in development.
Material and Methods
Dissociated cortical neurons from newborn C57BL/6 mice were cultured with a high cell density on 120-channel micro electrode
arrays (Multichannel Systems), as described previously. At days in vitro 2 (DIV2) the neurons were transduced with ChR2-H134R
and H2B-mCherry, using recombinant adeno-associated virus vector chimeric 1/2 (rAAV1/2) for gene delivery. Light activation of
ChR2-H134R results in larger photocurrents compared to the classical channelrhodopsin, which would, as we postulated, make it
more effective in young, not yet fully differentiated neurons. H2B-mCherry is a protein from the histone family tagged with
fluorescent mCherry, chronically visualizing the chromatin in the cell nucleus, and thus enabling us to quantify the number
of living neurons and to determine cell faith of individual neurons over time. Recordings were performed on DIV8 with a sampling
rate of 50 kHz and high-pass filtered at 200 Hz on a MEA2100 System (Multichannel Systems). A custom made LED circuit driving a
blue high power LED (minimum 460 nm λ) was used to activate neurons (Chr2-H134R peak activation 470 nm λ). Recorded multi-unit
activity was sorted and single unit activity was analyzed for different parameters in a custom Matlab routine (e.g. number of
active neurons and mean firing frequency). Bright field and fluorescence imaging was performed on an upright epifluorescence
microscope (Olympus BX61WI), coupled with a digital camera (Hamamatsu C10600 Orca-R2) and a xenon arc lamp (Olympus MT20) with
a fluorescent filter cube turret. Images were processed using custom-designed ImageJ macro’s.
Results
Already 6 days after transduction of cortical cultures with rAAV 1/2 constructs, fluorescence imaging revealed expression of
fluorescent reporter genes (EYFP for Chr2-H134R and nuclear mCherry for H2B-mCherry) (Fig.1). Although identification of
individual neurons, solely based on EYFP fluorescence in the membrane of the soma, axon and dendrites, proved to be difficult
(Fig.1.A), we were able to deduce from the high expression levels of H2B-mCherry (Fig. 1.B and C) that also the expression
levels of ChR2-H134R were at a functional level at DIV8, before the expression of EYFP reached its functional level. This
corresponded with our findings that neurons could be activated at DIV 8. A short episode of repetitive LED-illumination at
DIV8 of the whole recording area of the MEA at a frequency of 1 Hz, did significantly increase the mean firing frequency,
compared to control conditions (unpaired t test, p-value 0.0026 (p < 0.05), N = 6, N = 7 resp.) (Fig. 2.A, B and 3.A).
Light stimulation did not increase the number of active neurons, indicating that the increase in firing frequency resulted
from activation of already spontaneously active neurons (unpaired t test, p-value 0.2229, N = 6, N = 7 resp.) (Fig. 3.B).
Thus, while the expression of optogenetic elements, as assessed by imaging of the EYFP-reporter gene seemed still comparably
low in the first week after transduction, expression levels of the light-activated ion channel was already sufficient to drive
functional activation of cortical neurons. Chronic visualization of living neurons with H2B-mCherry further allowed us to quantify
cell death/cell survival. Neurons that expressed mCherry at DIV8, but lost this signal at DIV10, were considered to have died.
As has been shown, increase of electrical activity using pharmacological stimulants rescues neurons from cell death during early
development. Here we show that, as proof of concept, the increase of electrical activity due to light stimulation also promotes
neuronal survival. The mean firing frequency was significantly increased during light stimulation and at the same time the number
of cells that died in a period of two days after a short episode of light stimulation was significantly decreased (unpaired t
test, p-value 0.0262 (p < 0.05), N = 6, N = 7 resp.) (Fig. 3.C). The firing frequency appeared to be negatively correlated with
cell death (Spearman r – 0.7593, p-value 0.0026 (p < 0.05)) (Fig. 3.D).
Discussion
We have attempted to evaluate the effectiveness of light stimulation at an early stage of development. Our results show that
light stimulation acutely affects the mean firing frequency significantly already at DIV8 during stimulation. Light stimulation
did not increase the number of active neurons, indicating that the increase in firing frequency resulted from activation of
already spontaneously active neurons. Our results show further, that, as proof of concept, the increase of electrical activity
due to light stimulation promotes neuronal survival, as already has been shown for increase of activity using pharmacological
stimulants. The number of cells that died in a period of two days after a short episode of light stimulation was significantly
decreased. The firing frequency appeared to be negatively correlated with cell death.
Conclusion
Here we show that optogenetic tools can already be applied at early developmental stages in cortical cultures to modulate
neuronal activity through light stimulation in vitro. Co-transduction of ChR2-H134R and H2B-mCherry allowed us to determine the
earliest time point at which the expression of ChR2-H134R reached functional levels, and it enabled us to evaluate the
effectiveness of light stimulation, at an early stage in development. A short episode of continuous light stimulation at 1 Hz
significantly increased the mean firing frequency during stimulation at DIV 8. Light stimulation did not increase the number
of active neurons, indicating that the increase in firing frequency resulted from activation of already active neurons. Our
results show further, that, as proof of concept, the increase of electrical activity due to light stimulation promotes neuronal
survival.
References
Sun, J.-J., Kilb, W. and Luhmann, H. J. (2010): Self-organization of repetitive spike patterns in developing neuronal networks
in vitro. European Journal of Neuroscience, 32: 1289–1299.
Golbs A., Nimmervoll B., Sun J.-J., Save I.E., Luhmann H.J. Cerebral Cortex. 2011, 1192-1202.
Heck N., Golbs A., Riedemann T., Sun J.-J., Lessman V., Luhmann H.J. Cerebral Cortex. 2008, 1335-1349.
Acknowledgements
We would like to thank Simone Dahms-Praetorius and Beate Krumm for their excellent technical assistance. The work was supported
by funding from the Collaborative Research Centre programme of the Deutsche Forschungsgemeinschaft.
Keywords:
Electrophysiology,
optogenetics,
Micro-Electrode Arrays,
dissociated cortical cell culture,
genetically encoded fluorescence imaging
Conference:
MEA Meeting 2016 |
10th International Meeting on Substrate-Integrated Electrode Arrays, Reutlingen, Germany, 28 Jun - 1 Jul, 2016.
Presentation Type:
Poster Presentation
Topic:
MEA Meeting 2016
Citation:
Wong
E,
Luhmann
HJ and
Sinning
A
(2016). Combined Use of Optogenetics and Genetically Encoded Fluorescence Imaging in Primary Cortical Cultures on Micro-electrode Arrays.
Front. Neurosci.
Conference Abstract:
MEA Meeting 2016 |
10th International Meeting on Substrate-Integrated Electrode Arrays.
doi: 10.3389/conf.fnins.2016.93.00026
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Received:
22 Jun 2016;
Published Online:
24 Jun 2016.
*
Correspondence:
Dr. Emma Wong, University Medical Center Mainz, Institute of Physiology, Mainz, Germany, emmawong@uni-mainz.de