Optogenetic Control of Neuronal Network Activity on MEA
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1
Forschungszentrum Jülich GmbH, ICS/PGI-8 Bioelectronics, Germany
Motivation
Neurons can precisely encode information from external stimulation. Lasting changes in this coding can be considered memory. Memory and learning are procedures of information processing represented by changes of network firing patterns. Dissociated neuronal networks cultured on MEAs allowed us to investigate the firing patterns of networks over different time scales [1, 2]. Experiments performed on neurons expressing the ChR2 variant and grown on MEAs provide a highly reliable system for monitoring spontaneous or light correlated spiking and bursting both at the cellular and network level [3, 4]. Here, we present preliminary tests of manipulating network firing patterns by optical stimulation of neurons genetically modified with ChR2opt. Single cell stimulation was shown to evoke local network activity. Random stimulation sequences were able to induce persistent short-term (<10 min) and long-term (>1 day) changes in network dynamics.
Material and Methods
Dissociated E18 rat neurons were grown on MEAs as cortical-striatal co-cultures. These custom MEAs have 60- platinum electrodes with openings of 12 to 64 µm in a SU8 passivation (QWANE Biosciences, Lausanne, Switzerland). At DIV8-10, neurons were transduced with AAV6 psc-hsyn-ChR2opt-mKate (Lei Jin et al., unpublished data, packaged by Vector Biolabs, Philadelphia, PA, USA). To manipulate network activity, ChR2opt-expressing neurons were illuminated with a 473 nm laser (spot diameter 25 µm, 407 or 1630 mW/mm2, Rapp OptoElectronic) in 50 ms pulses at DIV18-23. The two stimulation protocols were: 1. single cell stimulation (407 mW/mm2): 50 ms light, 250 ms dark for 10 pulses (x3 runs, separated by 2-4 s); 2. random stimulation sequence (1630 mW/mm2): sequential illumination of 8 neurons at 4 Hz with 50 ms light and 200 ms break (x10 runs). Extracellular signals were recorded with an in-house developed 64 channel amplifier system “BioMAS” (analog bandwidth of 1 Hz - 3 kHz, AC coupled, total gain 1000x). MEA recordings were performed before, during, 7-10 min after, and 1 day after laser stimulation. We examined the spontaneous firing patterns vs. light altered activity patterns. As controls, non-transduced and transduced cultures without laser stimulation were assessed. Off-line analysis included filtering, threshold detection of spikes and bursts according to published methods [1, 2].
Results and Discussion
Single cell manipulation induced local network activation with robust laser responses (Fig.1A). Because of the risk of cell ablation and damage to the local network, repetition of this protocol was limited. No long-term changes in network dynamics were observed.
Random stimulation sequence was able to evoke changes that persisted throughout the network (Fig. 1B-F). Alterations showed different durations of persistence. Short-term (<10 min, Fig. 1E) and long-term (>1 day, Fig. 1F) changes in spiking and bursting dynamics were observed after laser manipulation. Silencing of some channels in the long-term observation (Fig. 1F) suggests possible neuronal excitotoxicity due to high-frequency stimulation and therefore could be critical neurodegenerative complications in optogenetic applications. No significant changes of firing pattern were detected in either control groups.
Conclusion
Neurons encode external stimuli as inputs into their firing patterns. Here, we present a strategy combining optogenetic stimulation and MEA recording for examining the spontaneous firing patterns vs. light altered network activity. This model allows the study of stimuli induced memory and learning process in neuronal cultures. The observed short-term (<10 min) and long-term (>1 day) changes in network dynamics suggested that learning and long-term memory were inducible and controllable using optogenetic tools.
References
[1] M. Chiappalone, M. Bove, A. Vato, M. Tedesco, S. Martinoia: Dissociated cortical networks show spontaneously correlated activity patterns during in vitro development. Brain Research 2006, 1093 41-53.
[2] D. A. Wagenaar, J. Pine and S. M. Potter: An extremely rich repertoire of bursting patterns during the development of cortical cultures. BMC Neuronscience 2006, 7:11.
[3] M. R. Dranias, H. Ju, E. Rajaram, and A. M. J. VanDongen: Short-Term Memory in Networks of Dissociated Cortical Neurons. The Journal of Neuroscience, January 30, 2013, 33 (5):1940-1953.
[4] G. Nagel: Channelrhodopsin-2, a directly light-gated cation-selective membrane channel. PNAS 2003, 100(24), 13940-5.
Figure Legend
Figure 1: Light-evoked changes in network firing patterns. (A) Single cell stimulation induced postsynaptic signals. Only local activity was observed. (B)- (F) One representative random stimulation experiment. (B) Fluorescence image of ChR2opt-mKate transduced network on MEA. Stimulation positions are shown in red (P: pulse 50 ms, D: dark 200 ms). Scale bar: 100 µm. (C) Raster plot of spontaneous activity before laser stimulation. (D) Activity during random stimulation, stimulation pulses shown in Ch 1. (E, F) Raster plots of firing patterns 7-10 min and 1 day after stimulation, respectively.
Acknowledgements
We thank B. Breuer and R. Fricke for support in cell culture, N. Wolters and D. Lomparski for electronics, ICS 4 - fz juelich for support and discussions.
This project was supported by Helmholtz project oriented funding BioSoft.
Keywords:
network dynamics,
MEA,
channelrhodopsin,
Optical stimulation,
Network activities
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:
Li
W,
Chen
L,
Schnitker
J,
Brings
F,
Maybeck
V and
Offenhäusser
A
(2016). Optogenetic Control of Neuronal Network Activity on MEA.
Front. Neurosci.
Conference Abstract:
MEA Meeting 2016 |
10th International Meeting on Substrate-Integrated Electrode Arrays.
doi: 10.3389/conf.fnins.2016.93.00051
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Received:
22 Jun 2016;
Published Online:
24 Jun 2016.
*
Correspondence:
Dr. Vanessa Maybeck, Forschungszentrum Jülich GmbH, ICS/PGI-8 Bioelectronics, Jülich, Germany, v.maybeck@fz-juelich.de