Large field-of-view HD-MEA setup for ex vivo mouse retina electrophysiological recordings
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1
ETH Zürich, Department of Biosystems Science and Engineering, Switzerland
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2
Friedrich Miescher Institute for Biomedical Research, Switzerland
Motivation: Ex vivo retina has been a common model for studies of neural networks since the 1960s. However, for a long time, electrophysiological studies had experimental access to only single or, at most, a few neurons at a time, which severely limited our ability to assess dynamic architectures of the neural networks or neuronal-population coding. With the latest generation of complementary metal oxide semiconductor high-density microelectrode array (CMOS HD-MEA) chips, which comprise thousands of densely packed electrodes, the number of simultaneously observable neurons could be increased by a few orders of magnitude. Yet, to realize the full potential of this tool, two technical challenges have to be overcome: (i) precise visual stimulation and (ii) good tissue-to-chip adhesion over a large area. Here, we present a large-field-of-view CMOS HD-MEA setup for ex vivo recording of retinal activity. We briefly introduce the setup components and address their contribution to the overall recording quality.
Biological background: The retina is the light-sensitive tissue of the eye, which converts the photon energy into electrical signals that are ultimately transmitted to the brain. The structure of the retina consists of three distinct neuronal layers, namely light-detecting photoreceptor cells, intermediate bipolar cells, and a heterogeneous population of around 30 types of retinal ganglion cells (RGCs). Each RGC type extracts specific information, such as contrast, the direction of motion, or the angle of a contrast edge, from the visual scene and transmits it to the brain. The spiking activity of the RGCs defines the entire information that is sent, as a train of action potentials, via the optic nerve to the higher visual areas in the brain. Which RGC type transmits which type of information and how this information depends on retinal location is, by and large, an open question.
The setup: To record the spiking activity of a large population of RGCs with an HD-MEA, the retina is first dissected from the eye cup of a mouse and cut so that it can be flattened on the chip, where it is positioned with the RGC layer facing down towards the recording array. In our work, we used a CMOS HD-MEA chip developed in our group [1], which has been previously successfully applied in diverse in vitro neurophysiological experiments [2], [3], as well as for ex vivo retinal recordings on a smaller scale [4], [5]. The overall electrode area measures 3.85 x 2.10 mm or 8.09 mm2, which corresponds to roughly 50% of the entire mouse retinal surface [6]. To control the retinal input, the chip was mounted on an upright microscope (Olympus BX51WI) equipped with a beam-splitter, through which light stimuli from a projector (Acer K10) were projected onto the retina. The projector beam was focused with a standard photography lens (AF-S Micro NIKKOR 60mm f/2.8 ED), and the light intensity was regulated with a set of neutral density (ND) filters. The sample was visualized with a mounted microscope camera (Leica DFC280), which facilitated the alignment of a light stimulus with the area to be recorded. In order to make the stimulation area as large as possible, the light was projected through a 2.5x single-lens objective from Thorlabs (AC254-075-A-ML Achromatic Doublet), resulting in a stimulus covering a rectangular or a square area of up to 5.09 mm2 (63% of the chip) or 3.84 mm2 (48% of the chip). Thus, with our setup, we can optically stimulate the photoreceptor layer and at the same time record the spiking activity of the RGCs from up to a third of the entire mouse retina. The size of the stimulated area obtained from a single projected pixel was 10.63 µm2.
Challenges: The reliability of the electrophysiological recordings of the retina strongly depended on the quality of tissue preparation, the experimental conditions (stable temperature, continuous perfusion) and on the tight contact between the retina and the recording array. The latter, especially in the large field-of-view setups, presented a major challenge, since pressing of the retina against the MEA with too low force will result in a poor signal-to-noise ratio, if in any signal will be obtained at all. In turn, a too large pressing force will damage the tissue and induce aberrant neuronal activity. Furthermore, the means to apply the optimal pressure should not interfere with tissue perfusion or the optical path required for light stimulation. This challenge was addressed by using a transparent dialysis membrane, attached to an in-house designed 3D-printed holder, which provided a fine control over the applied pressing force. In addition, quality control of our recordings was ensured through monitoring the fitness of the retina across the entire chip area, specifically through measurements of the frequency and amplitude of the spiking events and through monitoring of tissue adhesion.
Acknowledgements
This work was founded by National Science Foundation Ambizione Grant PZ00P3_167989
References
[1] U. Frey et al., “Switch-matrix-based high-density microelectrode array in CMOS technology,” IEEE J. Solid-State Circuits, vol. 45, no. 2, pp. 467–482, 2010.
[2] M. Radivojevic, F. Franke, M. Altermatt, J. Müller, A. Hierlemann, and D. J. Bakkum, “Tracking individual action potentials throughout mammalian axonal arbors,” Elife, vol. 6, pp. 1–23, 2017.
[3] D. J. Bakkum et al., “Tracking axonal action potential propagation on a high-density microelectrode array across hundreds of sites,” Nat. Commun., vol. 4, 2013.
[4] K. Yonehara et al., “Congenital Nystagmus Gene FRMD7 Is Necessary for Establishing a Neuronal Circuit Asymmetry for Direction Selectivity,” Neuron, vol. 89, no. 1, pp. 177–193, 2016.
[5] M. Fiscella et al., “Visual coding with a population of direction-selective neurons.,” J. Neurophysiol., vol. 114, no. 4, pp. 2485–99, 2015.
[6] S. Remtulla and P. E. Hallett, “A schematic eye for the mouse, and comparisons with the rat,” Vision Res., vol. 25, no. 1, pp. 21–31, 1985.
Keywords:
ex vivo retina,
Electrophysiology,
Retinal Ganglion Cells,
large-field-of-view,
CMOS HD-MEA
Conference:
MEA Meeting 2018 | 11th International Meeting on Substrate Integrated Microelectrode Arrays, Reutlingen, Germany, 4 Jul - 6 Jul, 2018.
Presentation Type:
Poster Presentation
Topic:
Neural Networks
Citation:
Žnidarič
M,
Bucci
A,
Diggelmann
R,
Hierlemann
A and
Franke
F
(2019). Large field-of-view HD-MEA setup for ex vivo mouse retina electrophysiological recordings.
Conference Abstract:
MEA Meeting 2018 | 11th International Meeting on Substrate Integrated Microelectrode Arrays.
doi: 10.3389/conf.fncel.2018.38.00100
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Received:
18 Mar 2018;
Published Online:
17 Jan 2019.
*
Correspondence:
Mr. Matej Žnidarič, ETH Zürich, Department of Biosystems Science and Engineering, Zurich, Switzerland, matej.znidaric@bsse.ethz.ch
Ms. Annalisa Bucci, ETH Zürich, Department of Biosystems Science and Engineering, Zurich, Switzerland, annalisa.bucci@bsse.ethz.ch