Systematic investigation of the response reliability of optogenetically enforced activity patterns in developing cortical networks in vitro as a prerequisite for studying the effects of modified electrical activity patterns on cell death in developing cortical networks
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1
Universitätsmedizin Mainz, Institute of Physiology, Germany
Developmental cell death is a determinant factor in the formation of mature neuronal networks. Synchronous network-wide neuronal activity, typical for developing neuronal networks, has been shown to be crucial to control levels of apoptosis during development. Neuron density, in turn, is known to be a major determinant in the development of structural and functional connectivity. We postulate that not only the mere levels of electrical activity but also specific activity patterns are essential in counterbalancing default pro-apoptotic signals during early development. Our aim is to modify activity patterns in cortical cultures by applying a wide variety of stimulation protocols ranging from non-physiological patterns such as white noise and continuous, tonic, low-frequency stimulation to patterns resembling physiological activity.
In order to investigate the time-related aspects of these activity patterns, tight temporal control is imperative. For this purpose we built custom-made LED circuits controlled by a programmable Arduino board (a microcontroller), driving a blue high power LED (peak wavelength 470 nm λ) to be used in a patch clamp setup and MEA setup, respectively. We transduced dissociated cortical cultures plated on coverslips and MEAs with rAAV-2 vectors (recombinant Adeno Associated Virus, serotype 2) carrying ChR2(H134R)-EYFP. In comparison to the classical ChR, the H134 mutation causes the channel to be slightly less prone to desensitization and slightly more sensitive to light, resulting in increased photocurrents. Expression of the ChR2(H134R)-EYFP is driven by the CamKII promoter, which restricts expression to excitatory glutamatergic neurons. Synaptic transmission was attenuated by glutamatergic and GABAergic receptor antagonists.
We assessed the efficacy of optogenetic stimulation on neuronal activity by evaluating the reliability of the response to a tailored test stimulation protocol through patch clamp and microelectrode array (MEA) recordings. Whole-cell patch clamp experiments revealed that ChR-positive cells were reliably depolarized by light stimulation, that a stimulus duration of 5 ms was sufficient to induce action potentials and that neurons could follow frequencies reliably up to 33 Hz with action potential generation.
We used this information to optimize the stimulus parameters in planar MEA. Repetitive light pulse stimulation with increasing pulse durations (1 up to 100 ms at 1 Hz) were applied to identify the optimal stimulus duration that would repeatedly result in a reliable response but would not evoke multiple action potentials per stimulus in a neuron. Subsequently, we tried to identify the maximum frequency up to which neurons could follow repeated optogenetic stimulation. Network-wide bursts in developing cortical cultures, at DIV14, consist on average of 13 spikes per burst. Therefore, repetitions of 13 pulses were applied at a fixed pulse width, with intra-burst frequencies between 1 and 50 Hz. The MEA-based experiments showed that light pulses of 10 and 20 ms result in accurate, single action potential responses. Shorter pulses resulted in failures and longer pulses in multiple action potentials. Repeated stimulation at 32 Hz with 10 ms light pulses, resulted in a response probability of 0.6. Higher response probabilities were achieved at lower frequencies. In order to prove that the derived optimal parameters were effective to mediate physiologically relevant activation patterns we finally stimulated neurons with repetitions of 13 pulses with a duration of 10 ms at a frequency of 34 Hz.
These experiments demonstrated that it is possible to optogenetically enforce physiologically relevant, as well as unphysiological activity patterns in dissociated cortical cultures.
Acknowledgements
Supported by DFG grant CRC 1080, project A01.
Keywords:
developmental cell death,
electrical network activity,
optogenetics,
stimulation protocols,
Response reliability
Conference:
MEA Meeting 2018 | 11th International Meeting on Substrate Integrated Microelectrode Arrays, Reutlingen, Germany, 4 Jul - 6 Jul, 2018.
Presentation Type:
Poster Presentation
Topic:
Stimulation strategies
Citation:
Wong Fong Sang
I,
Halbhuber
L,
Kilb
W,
Sinning
A and
Luhmann
HJ
(2019). Systematic investigation of the response reliability of optogenetically enforced activity patterns in developing cortical networks in vitro as a prerequisite for studying the effects of modified electrical activity patterns on cell death in developing cortical networks.
Conference Abstract:
MEA Meeting 2018 | 11th International Meeting on Substrate Integrated Microelectrode Arrays.
doi: 10.3389/conf.fncel.2018.38.00068
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Received:
16 Mar 2018;
Published Online:
17 Jan 2019.
*
Correspondence:
Prof. Heiko J Luhmann, Universitätsmedizin Mainz, Institute of Physiology, Mainz, Rheinland Pfalz, 55128, Germany, luhmann@uni-mainz.de