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DE GRUYTER Current Directions in Biomedical Engineering 2022;8(3): 33-36 Andrea Corna*, Timo Lausen, Roland Thewes, and Günther Zeck Electrical imaging of axonal stimulation in the retina https://doi.org/10.1515/cdbme-2022-2009 the signal to noise ratio are required. This is especially true for tissue or possibly in vivo application, where an optimal inter- Abstract: Stimulation of axons or its avoidance plays a cen- face is challenging to achieve. The most common approach to tral role for neuroprosthetics and neural-interfaces research. electrically image signal propagation relies on spike triggered One peculiar example constitutes retinal implants. Retinal im- average (STA) algorithms. STA consists in aligning multiple plants aim to artificially activate retinal ganglion cells (RGCs) snippets of extracellular recording on the somatic spike. Aver- via electrical stimulation. Such stimulation, however, often aging of snippets reduces the noise and visualizes the axonal generates undesired stimulation of RGC axon bundles, which AP. This is a very effective technique as shown previously also leads to distorted visual percepts. In order to establish stim- by us . The main limitation of the technique is the neces- ulation strategies avoiding axonal stimulation it is necessary sary detection of somatic spikes and consequent spike sorting to image the evoked activity in single axons. In this work we to perform the STA. More recently imaging of axons has been electrically imaged axonal stimulation in ex vivo mouse retina demonstrated with stimulus triggered average (StiTA) in cell using a high-density CMOS-based microelectrode array. We culture with pulsatile stimulation . In this work we utilized demonstrate signal propagation tracking via stimulus triggered a HD CMOS based MEA comprising 4225 recording elec- average during high frequency (100 Hz) sinusoidal electrical trodes and 1024 stimulation electrodes  to investigate ax- stimulation. onal propagation during electrical stimulation of the ex vivo Keywords: electrical stimulation, electrical imaging, retinal mouse retina. We first localized high adhesion areas of the implant, microelectrode array, MEA. retina sample on the sensor array analyzing the standard de- viation (SD) of the recorded extracellular voltage. As a proof- of-concept we electrically imaged axonal activation and signal 1 Introduction propagation during sinusoidal stimulation at 100 Hz. Axons are considered stable neuronal transmission cables, which convey information from the site of initiation to synap- tically connected cells. In neuroprosthetic applications axon 2 Methods stimulation is of general interest, predominantly in peripheral nerve interfaces. For retinal prostheses, which aim to replace 2.1 Retina preparation and extracellular lost photoreceptor input[1, 2], axonal stimulation causes a dis- recording using CMOS-based MEAs torted visual percept in blind patients and therefore needs to be avoided . Stimulation at one spatial location may acti- Experiments were performed using ex vivo retinae from vate axons of passage, which originate from remote locations. either adult wild-type (C57BL6/J) or from photoreceptor- The development of stimulus protocols avoiding axonal stim- degenerated mice (rd10). The experimental procedures for ulation requires techniques allowing imaging of the stimulated preparation of the ex vivo retina were reported and approved by action potentials (AP). High-density CMOS-based microelec- the Center for Biomedical Research, Medical University Vi- trode arrays have been used to image spontaneously occurring enna, Austria. Retina preparation and recording were done us- axonal signals in the retina  and axonal and potentially den- ing carbogenated (95% O2, 5% CO2) Ames’ medium (Ames dritic signals in elaborate neurites of dissociated neurons . A 1420, Sigma Aldrich + NaHCO ). The preparation was per- In general extracellular axonal signals have a much lower am- formed at room temperature while for the recording the tem- plitude compared to the somatic one, and strategies to enhance perature was adjusted to 32-35°C . The retina preparation was previously described here . In detail: a portion of the retina *Corresponding author: Andrea Corna, TU Wien, Institute of is separated from the enucleated eye using a scalpel, detached Biomedical Electronics, Gusshausstraße 27-29, Vienna, Austria, from the pigment epithelium and the vitreous is removed; Fi- e-mail: firstname.lastname@example.org nally the sample is placed on the MEA sensor with the RGC Timo Lausen, Roland Thewes, TU Berlin, Faculty of EECS, layer directly facing the electrodes. Retina samples were dark Chair of Sensor and Actuator Systems, Berlin, Germany. adapted for 30-45 min before recording. Recordings involv- Günther Zeck, TU Wien, Institute of Biomedical Electronics, Gusshausstraße 27-29, Vienna, Austria. Acesso aberto. © 2022 O Autor(s), publicado por De Gruyter. Este trabalho é licenciado sob a atribuição creative commons 4.0 Internacional Licença. 33 A.Corna et al., Electrical imaging of axonal stimulation in the retina ing electrical stimulation were performed in dark, while light stimuli were presented during the spontaneous activity record- ing (Figure C,D). A CMOS-MEA comprising 4225 record- ing electrodes was used for the recording (CMOS-MEA 5000, Multi Channel Systems MCS GmbH), with an electrode pitch of 16 or 36 µm (65 × 65 pixels and a total area: 1 or 5.3 mm ) and 1024 stimulation electrodes. The chip surface was cleaned with detergent (Tickopur R60, 5%, Stamm/Berlin, 80 °C), rinsed with bi-distilled water and coated with ≈ 100 𝜇l (1 mg/ ml) poly-L-lysine (P1399, MW 150–300 kDa, Sigma, Germany) before the recording. Recordings were performed at 20 kHz using CMOS-MEA Control software (Multi Channel Systems MCS GmbH). 2.2 Retina stimulation, data analysis and stimulus triggered average The signal was high-pass filtered at 1 kHz (butterworth 4th order). Adhesion was computed as the SD of the filtered elec- trode voltage and visualized as a heat-map. The HD MEA al- lows arbitrary selection of the stimulation pattern and wave- form. We tested different stimulation protocols to find the op- timal parameters. For the sample shown in Figure 1B we stim- ulated with a 100Hz sinus for 100 ms, and repeated the stimuli for 30 repetitions with a 500 ms pause between repetition. For the stimulation we used 4 adjacent stimulating electrodes for a total area of 0.02 mm . To compute the Stimulus triggered average we high-pass filtered the signal at 1 kHz. We then cut 10 ms snippets aligned with the start and the end of the sinus cycle. We average between the 300 snippets for each channel Fig. 1: A) Heat-map of the standard deviation (SD) of the extra- cellular voltage recorded by an array of 65x65 electrodes of the to obtain the final stimulus triggered average. Sorting and STA HD CMOS MEA, showing the adhesion of a retina sample during analysis was done with the CMOS-MEA Tool software (Multi spontaneous activity. Yellow-green color marks higher SD as com- Channel Systems MCS GmbH). pared to the surround, which indicates better tissue adhesion. B) Stimulus Triggered Average (StiTA) for a 100 Hz sinusoidal stimu- lation, for the sample shown in A. At the center of the heatmap the artifact generated by the stimulating electrodes is visible as well as 3 Results two branches of signal propagation in the axon. Yellow indicates positive extracellular voltage, blue negative. C) Sinusoidal Stimula- tion artifact in a recording electrode in proximity of the stimulation. This work demonstrates signal propagation imaging in the The insert shows the evoked activity (spikes) of a retinal ganglion retina upon sinusoidal high frequency stimulation. This project cell (RGC) in phase with the stimulus. D) Electrode voltage com- had two main goals: first, to image the axonal signals propaga- puted with StiTA for a single electrode located under the axon tion from the extracellular recording without additional knowl- showing the axonal extracellular action potential, with a tri-phasic edge of single cell location or activity; second, we wanted to waveform. The duration of one stimulation cycle (10 ms) is shown. investigate the characteristic, antidromic or orthodromic, of E) Single cells and axon computed via spike sorting and spike triggered average (STA) from the sample shown in A. Red dots the elicited signal. In order to track the axons and study sig- indicate the soma locations and black lines the axons. F) Picture nal propagation it is necessary to know the location and the of an exemplary retina sample placed in epiretinal configuration adhesion of the tissue on the MEA sensor array. To calculate over the 5.3 mm CMOS sensor array. the area of high adhesion of the retina sample, we computed the SD of the filtered signal of each electrode. The results are visualized as a heat-map in Figure 1A. Strong adhesion means 34 A.Corna et al., Electrical imaging of axonal stimulation in the retina high cleft resistance (the resistance of the electrolyte between ration of the recording sensor at a distance of 72 𝜇m from the electrode and the tissue), which translates in an increased the stimulation electrode (Figure 1B) for the 5.3 mm sen- SD of the recorded voltage signal [9, 10]. In Figure 1A the sor or no saturation for the 1 mm sensor (Figure 1C). An green-yellow areas indicate good sample adhesion. The valid- open question is if sinusoidal stimulation can, and in which ity of the results is confirmed when comparing the adhesion regimes, avoid axonal stimulation. While the axonal activa- map with the results of the spike sorter spontaneous activ- tion upon 100 - 80 Hz sinusoidal stimuli (1.3 nC, stimulation ity (Figure 1E). All the detected cells are located in the area electrode area: 0.02 mm ) could be imaged electrically, lower with computed high adhesion. Once the position of the tis- stimulation frequencies (i.e. 40 Hz) did not show axonal prop- sue was identified, we proceeded to deliver a stimulation in agation with this approach. A possibility is that phase-lock for a central area of the sample (Figure 1B). We applied a 100 40 Hz is not precise enough to allow StiTA, and the StiTA Hz sinusoidal stimulation as shown in the artifact generated cannot provide a definitive answer for low frequency stimu- in the recording electrode (Figure 1C). High frequency sinu- lation. In our previous studies, comprising thousands of cells, soidal stimulation causes the excitation of APs in RGC that are we did not detect axonal stimulation with 40 Hz stimulation in phase-lock with the stimulation waveform. This is shown in via somatic activation . In the result shown here the prop- the insert of Figure 1C, where it is possible to see a stimulated agation proceeds both in antidromic or orthodromic direction. spike in concomitance with the cathodic phase of the sinus. Therefore another possibility is that elicited axonal AP prop- The stimulus triggered average (StiTA) is computed averaging agates antidromically (towards the soma), but without activat- between the recording snippets of a stimulation cycle aligned ing the soma. Antidromic propagation, however, should lead to with the stimulation phase. The result is a mean response to the somatic activation, as previously reported by different groups stimulation characterized by a lower electrical noise. A single [7, 12, 13]. The underlying mechanism is currently investi- frame of the StiTA is shown in Figure 1B as a heat-map of the gated and should be clarified in future experiments. voltage. The image shows the artifact of the stimulation in the Author Statement center and the axonal signal propagating both antidromically Research funding: Funding was partially provided by the Eu- or orthodromically to the edges of the sample. The StiTA de- ropean Union’s Horizon 2020 research and innovation pro- tected axonal propagation follows the same path of the STA gramme under the Marie Skłodowska-Curie grant agreement calculated axons (Figure 1E). In figure 1D we show the StiTA No 861423.Conflict of interest: Authors state no conflict of in- computed signal of a single electrode located under the axon. terest The image shows the electrode voltage during the total dura- tion (10ms) of a 100Hz sinusoidal stimuli. 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Current Directions in Biomedical Engineering – de Gruyter
Published: Sep 1, 2022
Keywords: electrical stimulation; electrical imaging; retinal implant; microelectrode array; MEA
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