Korean J Physiol Pharmacol.  2023 Nov;27(6):541-553. 10.4196/kjpp.2023.27.6.541.

Multiple consecutive-biphasic pulse stimulation improves spatially localized firing of retinal ganglion cells in the degenerate retina

• Ahn J
• Yoo Y
• Goo YS

• KMID: 2547320
• DOI: http://doi.org/10.4196/kjpp.2023.27.6.541

Abstract

Retinal prostheses have shown some clinical success in restoring vision in patients with retinitis pigmentosa. However, the post-implantation visual acuity does not exceed that of legal blindness. The reason for the poor visual acuity might be that (1) degenerate retinal ganglion cells (RGCs) are less responsive to electrical stimulation than normal RGCs, and (2) electrically-evoked RGC spikes show a more widespread not focal response. The single-biphasic pulse electrical stimulation, commonly used in artificial vision, has limitations in addressing these issues. In this study, we propose the benefit of multiple consecutive-biphasic pulse stimulation. We used C57BL/6J mice and C3H/HeJ (rd1) mice for the normal retina and retinal degeneration model. An 8 × 8 multi-electrode array was used to record electrically-evoked RGC spikes. We compared RGC responses when increasing the amplitude of a single biphasic pulse versus increasing the number of consecutive biphasic pulses at the same stimulus charge. Increasing the amplitude of a single biphasic pulse induced more RGC spike firing while the spatial resolution of RGC populations decreased. For multiple consecutive-biphasic pulse stimulation, RGC firing increased as the number of pulses increased, and the spatial resolution of RGC populations was well preserved even up to 5 pulses. Multiple consecutive-biphasic pulse stimulation using two or three pulses in degenerate retinas induced as much RGC spike firing as in normal retinas. These findings suggest that the newly proposed multiple consecutive-biphasic pulse stimulation can improve the visual acuity in prosthesis-implanted patients.

Keyword

Electrically-evoked spikes; Multiple consecutive-biphasic pulse; stimulation; Retinal degeneration; Retinal ganglion cell; Retinal prosthesis

Figure

• 

  Fig. 1 Stimulus protocols used in the experiment. (A) Single-pulse stimulation. Pulse amplitudes range from 10 to 50 μA with pulse duration fixed at 250 μs are applied once per second. (B) Burst stimulation. The number of pulses per burst ranges from 1 to 5, with pulse duration fixed at 250 μs. (C) Burst stimulation. 1–20 pulses per burst. Pulse amplitude: 30 μA. Pulse duration: 100 μs. (D) Burst stimulation. 1–3 pulses per burst. Pulse amplitudes: 30 or 50 μA. Pulse duration: 100 μs.

• 

  Fig. 2 Comparison of electrically-evoked RGC spikes between single-pulse and burst stimulation in the WT retina. (A) Left inset: MEA positions of the stimulation (channel 26, marked with a red dot) and recording (channel 36, marked with a yellow dot) electrodes spaced 200 μm apart. Left panel: Typical responses of the WT RGC to single-pulse stimulation are shown as raster plots. Electrically-evoked RGC spikes are highlighted. The time window for the raster plots is set to ± 0.1 sec, with 0 sec representing the stimulus onset (red dashed line). For each raster plot (10 trials), the pulse amplitude increases from top to bottom (10–50 μA). The pulse duration is fixed at 250 μs/phase. Middle panel: Typical responses of the WT RGC to burst stimulation are shown as raster plots. For each raster plot, the pulse number increases from top to bottom (#1–#5). Right panel: RGC response curves to single-pulse and burst stimulation in WT RGCs located 200 μm away from the stimulation electrode. Error bars represent the standard error of the mean (SEM). Statistical differences in response curves are indicated by asterisks (*p < 0.05). (B) Left and Middle panels: same representative raster plots of WT RGCs located 600 μm away from the stimulation electrode. Right panel: RGC response curves to single-pulse and burst stimulation in WT RGCs located 600 μm away from the stimulation electrode. Statistical differences in the graphs are indicated by asterisks (*p < 0.05, ***p < 0.001). (C) Changes in normalized responses with the distance between stimulation and recording electrodes. Left panel: single-pulse stimulation. The inset (spatial MEA color map) shows the normalized RGC responses in one representative retinal patch upon electrical stimulation with a pulse amplitude of 50 μA. The red dot indicates the location of the stimulation electrode. Right panel: burst stimulation. The inset (spatial MEA color map) shows the normalized RGC responses in one representative retinal patch upon electrical stimulation with five pulses per burst. RGC, retinal ganglion cell; WT, wild-type; MEA, multi-electrode array.

• 

  Fig. 3 Comparison of electrically-evoked RGC spikes between single-pulse and burst stimulation in the RD retina. (A) Left inset: MEA positions of the stimulation (channel 52, marked with a red dot) and recording (channel 53, marked with a yellow dot) electrodes spaced 200 μm apart. Left panel: Typical responses of the RD RGC to single-pulse stimulation are shown as raster plots. Electrically-evoked RGC spikes are highlighted. The time window for the raster plots is set to ± 0.1 sec, with 0 sec representing the stimulus onset (red dashed line). For each raster plot (10 trials), the pulse amplitude increases from top to bottom (10–50 μA). The pulse duration is fixed at 250 μs/phase. Middle panel: Typical responses of the RD RGC to burst stimulation are shown as raster plots. For each raster plot, the pulse number increases from top to bottom (#1–#5). Right panel: RGC response curves to single-pulse and burst stimulation in RD RGCs located 200 μm away from the stimulation electrode. Error bars represent the standard error of the mean (SEM). Statistical differences in response curves are indicated by asterisks (*p < 0.05, **p < 0.01). (B) Left and Middle panels: same representative raster plots of RD RGCs located 600 μm away from the stimulation electrode. Right panel: RGC response curves to single-pulse and burst stimulation in RD RGCs located 600 μm away from the stimulation electrode. Statistical differences in the graphs are indicated by asterisks (*p < 0.05, **p < 0.01). (C) Changes in normalized responses with the distance between stimulation and recording electrodes. Left panel: single-pulse stimulation. The inset (spatial MEA color map) shows the normalized RGC responses in one representative retinal patch upon electrical stimulation with a pulse amplitude of 50 μA. The red dot indicates the location of the stimulation electrode. Right panel: burst stimulation. The inset (spatial MEA color map) shows the normalized RGC responses in one representative retinal patch upon electrical stimulation with five pulses per burst. RGC, retinal ganglion cell; RD, retinal degenerate; MEA, multi-electrode array.

• 

  Fig. 4 Increasing the number of pulses per burst in the WT retina. (A) Typical responses of the WT RGC to burst stimulation are shown as raster plots. Electrically-evoked RGC spikes are highlighted. The time window of the raster plot is set to ± 0.1 sec, with 0 sec representing the stimulus onset (red dashed line). In each raster plot (10 trials), the pulse number increases from top to bottom (#1–#5, #9–#13, #16–#20). The pulse amplitude and pulse duration are fixed with 30 μA and 100 μs/phase. Only RGCs closest 200 μm to the stimulation electrode were analyzed. (B) RGC response curves as a function of pulse number. Error bars represent the standard error of the mean (SEM). Statistical differences in the graphs are indicated by asterisks (*p < 0.05). RGC, retinal ganglion cell; WT, wild-type.

• 

  Fig. 5 Increasing the number of pulses per burst in the RD retina. (A) Typical responses of the RD RGC to burst stimulation are shown as raster plots. Electrically-evoked RGC spikes are highlighted. The time window of the raster plot is set to ± 0.1 sec, with 0 sec representing the stimulus onset (red dashed line). In each raster plot (10 trials), the pulse number increases from top to bottom (#1–#5, #9–#13, #16–#20). The pulse amplitude and pulse duration are fixed with 30 μA and 100 μs/phase. Only RGCs closest 200 μm to the stimulation electrode were analyzed. (B) RGC response curves as a function of pulse number. Error bars represent the standard error of the mean (SEM). Statistical differences in the graphs are indicated by asterisks (*p < 0.05). RGC, retinal ganglion cell; RD, retinal degenerate.

• 

  Fig. 6 Comparison of the number of electrically-evoked spikes between WT and RD RGCs for single-pulse and doublet or triplet pulse stimulation. Statistical differences in the graphs are indicated by asterisks (*p < 0.05). RGC, retinal ganglion cell; WT, wild-type; RD, retinal degenerate.

• 

  Fig. 7 Calcium-mediated RGC responses to burst stimulation in the RD retina. (A) Representative 1-second raw traces of RGCs in the RD retina without and with thapsigargin (Th) or Hi-Di solution. (B) Spontaneous firing rates of RGCs for RD, RD + Th, and RD + Hi-Di. Statistical differences in the graphs are indicated by asterisks (***p < 0.001). (C) Typical responses of RD RGCs to burst stimulation are shown as raster plots. Electrically-evoked RGC spikes are highlighted. The time window of the raster plot is set to ± 0.1 sec, with 0 sec representing the stimulus onset (red dashed line). In each raster plot (10 trials), the pulse number increases from top to bottom (#1–#5). The pulse amplitude and pulse duration are fixed with 30 μA and 100 μs/phase. Only RGCs 200 μm closest to the stimulation electrode are analyzed. (D) Normalized RGC response curves to burst stimulation. Error bars represent the standard error of the mean (SEM). Statistical differences in the response curves are indicated by asterisks (*p < 0.05, **p < 0.01, ***p < 0.001). RGC, retinal ganglion cell; RD, retinal degenerate; Hi-Di, high-divalent.

• 

  Fig. 8 Schematic diagram of calcium-mediated RGC firing to the burst stimulation. The burst stimulation strategy depends on an increase of intracellular calcium concentration, regulated by voltage-gated calcium channels (VGCCs) for calcium influx and sarco/endoplasmic reticulum Ca2+–ATPase (SERCA) for calcium reuptake. Burst stimulation activates VGCCs at bipolar cell (BC) synapses, boosting calcium influx and then glutamate release from the BC, therefore increasing RGC firing. Thapsigargin (Th) inhibits SERCA, preventing calcium reuptake, raising intracellular calcium concentration ([Ca2+]i), and then releasing glutamate from the bipolar cell, thus enhancing RGC firing. RGC, retinal ganglion cell; IP3R/RyR, inositol–1,4,5–triphosphate receptors/ryanodine receptors; ER, endoplasmic reticulum.

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