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. 2022 Dec 9:16:1033738.
doi: 10.3389/fncel.2022.1033738. eCollection 2022.

High spatial resolution artificial vision inferred from the spiking output of retinal ganglion cells stimulated by optogenetic and electrical means

Andreea Elena Cojocaru  1 , Andrea Corna  1 , Miriam Reh  2 , Günther Zeck  1
Affiliations

High spatial resolution artificial vision inferred from the spiking output of retinal ganglion cells stimulated by optogenetic and electrical means

Andreea Elena Cojocaru et al. Front Cell Neurosci. .

Abstract

With vision impairment affecting millions of people world-wide, various strategies aiming at vision restoration are being undertaken. Thanks to decades of extensive research, electrical stimulation approaches to vision restoration began to undergo clinical trials. Quite recently, another technique employing optogenetic therapy emerged as a possible alternative. Both artificial vision restoration strategies reported poor spatial resolution so far. In this article, we compared the spatial resolution inferred ex vivo under ideal conditions using a computational model analysis of the retinal ganglion cell (RGC) spiking activity. The RGC spiking was stimulated in epiretinal configuration by either optogenetic or electrical means. RGCs activity was recorded from the ex vivo retina of transgenic late-stage photoreceptor-degenerated mice (rd10) using a high-density Complementary Metal Oxide Semiconductor (CMOS) based microelectrode array. The majority of retinal samples were stimulated by both, optogenetic and electrical stimuli using a spatial grating stimulus. A population-level analysis of the spiking activity of identified RGCs was performed and the spatial resolution achieved through electrical and optogenetic photo-stimulation was inferred using a support vector machine classifier. The best f1 score of the classifier for the electrical stimulation in epiretinal configuration was 86% for 32 micron wide gratings and increased to 100% for 128 microns. For optogenetically activated cells, we obtained high f1 scores of 82% for 10 microns grid width for a photo-stimulation frequency of 2.5 Hz and 73% for a photo-stimulation frequency of 10 Hz. A subsequent analysis, considering only the RGCs modulated in both electrical and optogenetic stimulation protocols revealed no significant difference in the prediction accuracy between the two stimulation modalities. The results presented here indicate that a high spatial resolution can be achieved for electrical or optogenetic artificial stimulation using the activated retinal ganglion cell output.

Keywords: CMOS-based microelectrode array; channelrhodopsin-2; epiretinal electrical stimulation; optogenetics; retinal ganglion cell.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
Overview of the experimental protocol used to infer the spatial resolution of the stimulus based on the RGC spiking. (A) Photograph of ex vivo rd10 retina interfaced to a CMOS-MEA. The central square (1 × 1 mm2) comprises 1,024 capacitive stimulation electrodes and 4,225 recording sites. (B) Schematic of the stimulation array with 1,024 stimulation electrodes. The recording sites are in the same area, with each stimulation electrode being enclosed by four recording sites (not shown here). Cell positions (orange circles) and inferred axon pathways are shown. In the background (blue and gray) the two phases of the reversed grating stimulus are shown. (C) Microscopic image of the retina on the CMOS MEA with the optogenetic photo-stimulation pattern superimposed. Scale bar: 100 μm. (D) The electrical stimulation protocol exemplified using the raw voltage of one recording sensor (recording site a). Upper panel: Four sinusoidal stimulation waveforms were applied to “blue” stimulation electrodes (panel B) between 0 and 100 ms, followed by four stimulation waveforms applied to the “gray” electrodes (panel B) between 350 and 450 ms. The selected recording sensor records a higher extracellular voltage artifact for stimulation with gray stimulation electrode as compared to stimulation with blue electrodes. Note that the same stimulus amplitude was applied to “blue” and “gray” electrodes. Scale bar: 10 mV. Lower panel: High-pass filtered extracellular voltage of the signal shown in the upper panel reveals spiking in each cycle of the four sinusoidal waveforms for stimulation with the gray electrodes only. Scale bar: 1 mV. (E) Detection of selective electrical activation revealed by a second sensor (“recording site b”). Description of the traces as in (D). (F) The optogenetic stimulation protocol exemplified using the raw voltage of one recording sensor (recording site c). Upper panel: Spatially patterned photo-stimuli with fine gratings were projected onto selected regions of the CMOS sensor array (see panel C) between 0 and 200 ms, followed by a reverse grating between 200 and 400 ms. Dashed line in the raw extracellular voltage trace marks a sensor reset. Scale bar: 10 mV. Lower panel: High-pass filtered extracellular voltage of the signal shown in the upper panel reveals spiking during one of the stimulation phases (marked with “blue”). (G) Detection of selective optogenetic activation revealed by another sensor (“recording site d”). Description of the traces as in (F). RGC, retinal ganglion cell; CMOS MEA, Complementary Metal-Oxide-Semiconductor microelectrode array.
Figure 2
Figure 2
Optogenetic stimulation with fine gratings evokes spatially restricted RGC spiking. All results presented here originate from one retina. (A) Raster plots of two exemplary RGCs stimulated for 24 s with an alternating stimulus at a spatial frequency of 50 μm. Every 200 ms, the spatial pattern was switched. A preferential activation is detected for both cells. (B) Raster plots of the very same RGCs, for a stimulus presented at 10 Hz temporal switching and a 50 μm grating width. Both RGCs cells show a preferential response to one of the two stimulation phases. (C,D) Spatial mapping of identified RGCs for the 2.5 Hz pattern switching with a 50 μm (C) and 500 μm (D) grating width. Color coded is the relative change in firing rate between the two stimulation phases. Arrows indicate the two selected cells’ positions depicted in panel (A). RGC axons are inferred from the spike-triggered averaging; only a few of them are shown here for visualization purposes. (E,F) Spatial mapping of identified RGCs for the 10 Hz pattern reversal with a spatial frequency of 50 μm (E) and 500 μm (F). Color coded is the relative change in firing rate between the two stimulation phases. Arrows indicate the two selected cells’ positions depicted in panel (B).
Figure 3
Figure 3
Electrical stimulation with fine gratings evokes spatially restricted RGC spiking. All results presented here originate from one retina. (A) Rasterplot of two selected RGCs upon stimulation with fine grating stimuli (bar width: 32 μm). Cell 3 is stimulated only in phase 1 (0–100 ms) while cell 4 is stimulated in phase 2 (100–200 ms). The cell positions are marked with arrows in panel (C). (B) Rasterplot of two selected RGCs upon stimulation with grating stimuli (bar width: 128 μm). The spiking pattern for each cell is confined to one phase. The cell positions are marked with arrows in panel (D). (C) Positions of the RGC on the stimulation array. Color-coded is the relative change in firing rate. The background colors of the grating mark the electrodes used in phase 1 and phase 2. The results presented here were obtained after stimulation with 32 μm narrow gratings (panel A). For visualization purposes, only a few axons are shown. (D) Positions of the RGC on the stimulation array, with the corresponding color coding of the relative change in firing rate. Note the dominant red colors on top of the gray stimulation electrodes indicating selective activation by this stimulus.
Figure 4
Figure 4
Inferring the spatial resolution of optogenetically and electrically stimulated RGCs from a support vector machines classifier. (A) Classification results are shown as f1 score values for data obtained from optogenetic experiments. The different colors denote different experimental days. On the upper side of the plot are data from the 2.5 Hz switching frequency and on the lower side are data from the 10 Hz switching frequency. A classifier’s performance is considered to be significant when the f1 score is above the “chance level” which, in this case, we considered to be at 0.5. With the exception of one recording day, the metrics show a notably high value for all of the spatial frequencies considered, reaching saturation at around 30 μm for 2.5 Hz and 50 μm for 10 Hz. (C) Classification results for data obtained from electrical stimulation experiments. The different colors correspond to different experimental days. The saturation is reached at 64 μm for all datasets considered. However, for some experimental days, we note particularly high values for the 32 μm grating width as well. (B,D) Feature importance plot showing the most significant 20 features (vectors containing firing rate values for individual stimulus repetitions) for the classifier. In panel (B) there is an exemplary plot for good classification results, for the case of the 50 μm grating width, with a 10 Hz pattern reversal from an optogenetic stimulation dataset, while in panel (D) the plot shows an example of the feature importance for the 32 μm grating width, in the case of an electrical stimulation dataset.
Figure 5
Figure 5
Comparing the spatial resolution of electrically or optogenetically stimulated RGCs in the same retinal sample. (A) Classification results of the grating pattern reversal stimuli inferred from a subset of RGCs responding to both stimulation types, electrical (yellow) and optogenetic (blue). In the upper part of the graph, the yellow color-coded bars show the results obtained from four different electrically stimulated retinae. The blue scheme shows the results for the same responsive RGCs in the same four retinae upon optogenetic stimulation. Each bar for the electrical stimulation corresponds to a bar on the optogenetic stimulation at the same x-position on the graph, indicating that the same experimental day and the same sample was used in the analysis. We note that for gratings wider 32 μm not all protocols were applied or activated sufficient RGCs. (B) Classification results for the full datasets in the four retinae stimulated by electrical and optogenetic means, i.e., with RGCs responding to either electrical or optogenetic stimulation. In the upper part of the graph, the yellow color-coded bars show the results from the RGC datasets upon electrical stimulation, while in the lower part the prediction results inferred from the optogenetic stimulation are shown. Each bar for the electrical stimulation corresponds to a bar on the optogenetic stimulation at the same x-position on the graph, indicating that the same sample was considered in both cases.
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