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A method for single-neuron chronic recording from the retina in awake mice

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Science  29 Jun 2018:
Vol. 360, Issue 6396, pp. 1447-1451
DOI: 10.1126/science.aas9160
  • Fig. 1 Noncoaxial intravitreal injection and conformal coating of mesh electronics on the mouse retina.

    (A) I: Schematic showing the layout of mesh electronics comprising 16 recording electrodes (green dots indicated by a green arrow) and I/O pads (red dots indicated by a red arrow). II: Schematic showing noncoaxial intravitreal injection of mesh electronics onto the RGC layer. Multiplexed recording electrodes are shown as yellow dots. III: Schematic of noncoaxial injection that allows controlled positioning of mesh electronics on the concave retina surface (cyan arc). The blue and red dotted arrows indicate the motion of the needle and desired trajectory of the top end of the mesh, respectively (see fig. S1 for details) (22). (B) In vivo through-lens images of the same mouse eye fundus on days 0 and 14 after injection of mesh electronics, with electrode indexing in the day 14 image (22). (C) Ex vivo imaging of the interface between injected mesh electronics (red, mesh polymer elements) and the retina (green dots, RGCs) on days 0 (I) and 7 (II) after injection. The inset of II shows the region indicated by a yellow arrow where the high-resolution image was taken (22). (D) Comparison of pupillary reflex (n = 3), OKR (n = 5), and visual acuity (n = 3) between control and injected mouse eyes. Error bars denote SD; NS, not significant (P > 0.05) by one-way ANOVA test.

  • Fig. 2 Chronic 16-channel in vivo electrophysiology of single RGCs measured with mesh electronics.

    (A and B) Representative 16-channel recordings from the same mesh electronics delivered onto a mouse retina on day 3 (A) and day 14 (B) after injection. (C and D) Light modulation of two representative channels (Ch2 and Ch8) in red dashed boxes in (A) and (B) on day 3 (C) and day 14 (D) after injection. The red shaded and unshaded regions indicate the light ON and OFF phases, respectively. Representative sorted spikes assigned to different neurons on both days are shown in the rightmost column for each channel. Each distinct color in the sorted spikes represents a unique identified neuron. (E) Firing rates of all sorted neurons from Ch2 and Ch8 during light modulations on days 3 and 14 after injection (22). Error bars denote SEM. **P < 0.01, ***P < 0.001, ****P < 0.0001 (one-way ANOVA); NS, not significant (P > 0.05). Five mice were used for multiplexed recordings.

  • Fig. 3 Chronic in vivo recording and tracking of the same DSGCs.

    (A) Photograph showing a mouse immediately after mesh injection. The red and white arrows indicate part of mesh electronics outside of the eye and a head plate for head fixation, respectively. (B) Red-light photograph showing in vivo recording of DSGCs in response to moving grating stimulations (22). (C) Raster (left), polar plots (center), and overlaid spike waveforms (right) of single-unit firing events of three neurons (with corresponding colors) from Ch8 in response to moving grating stimulations on days 7 and 14 after injection. In the raster plots, the pink shaded regions correspond to times when gratings were displayed on the screen, with moving directions indicated by arrows on the bottom (22). Only the raster plots on day 7 are shown. In the polar plots, the DSi for each cell on different days is labeled with corresponding colors. (D) Bar chart summarizing numbers of identified DSGCs, OSGCs, and non-DSGCs on day 7 (red bars) and day 14 (green bars) after injection. (E) Bar chart with overlaid scatterplot of DSi or OSi of all RGCs on days 7 and 14, with thin lines of corresponding colors connecting the same neurons identified on both days. The bar height and the whisker indicate the mean and maximum of DSi and OSi values, respectively. Four mice were used for direction and orientation selectivity studies; data shown are from one representative mouse.

  • Fig. 4 Chronic circadian modulation of individual RGC activity.

    (A) Representative polar plots of a DSGC at different times in one complete circadian cycle on days 4 and 5 after injection. All graphs are plotted in same range of firing frequencies. (B) I: Firing rates of the same DSGC in (A) averaged over preferred directions in three complete circadian cycles on days 1 and 2, 4 and 5, and 6 and 7 after injection. II: Mean firing rate by taking the average over these three circadian cycles. III: This DSGC is identified as an ON-OFF transient type. (C) I: Firing rates of another DSGC averaged over preferred directions on three complete circadian cycles on days 1 and 2, 4 and 5, and 6 and 7 after injection. II: Mean firing rate by taking the average over these three circadian cycles. III: This DSGC is identified as an OFF transient type. In (B) and (C), I and II, yellow and gray shaded regions indicate diurnal and nocturnal circadian times, respectively; in (B) and (C), III, the red shaded and white regions indicate light ON and OFF phases, respectively. Red and blue shaded regions in (B) and (C), II, denote SEM. (D) Bar chart with overlaid scatterplot of the CMi of diurnal cells (red bars), nocturnal cells (blue bars), and circadian independent cells (green bars) (22). The bar height and the whisker indicate the mean and maximum of CMi values, respectively. (E) Plots showing the evolution of CMi values for four representative cells (three diurnal cells and one nocturnal cell) that were recorded for three complete circadian cycles. Red and blue dashed lines in (D) and (E) indicate the threshold for defining diurnal and nocturnal cells, respectively. Three mice were used for circadian modulation study of RGC activity.

Supplementary Materials

  • A method for single-neuron chronic recording from the retina in awake mice

    Guosong Hong, Tian-Ming Fu, Mu Qiao, Robert D. Viveros, Xiao Yang, Tao Zhou, Jung Min Lee, Hong-Gyu Park, Joshua R. Sanes, Charles M. Lieber

    Materials/Methods, Supplementary Text, Tables, Figures, and/or References

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    • Materials and Methods
    • Figs. S1 to S12
    • Tables S1 and S2
    • Captions for Movies S1 and S2
    • References

    Images, Video, and Other Media

    Movie S1
    NIR pupil imaging of mouse eye injected with mesh electronics. This video shows the video-rate NIR imaging and tracking of mouse pupil size changes in response to ambient light intensity modulation. The frame rate is 25 frames per second (fps) and the video is played at 1× real time. The mesh electronics was injected and fixed to the lateral canthus of the eye (right side of the eye), and the pupil boundary is shown as the red dashed circle in the video. This video was taken on Day 1 post-injection of mesh electronics.
    Movie S2
    Visual stimulation with moving light gratings to a head-fixed mouse. This video shows the setup of moving grating visual stimulation to a mouse restrained in a Tailveiner® restrainer with the head-plate fixed to an optical-table-mounted frame. The frame rate is 30 frames per second (fps) and the video is played at 1× real time. This video covers the duration of gratings moving in eight different directions in one complete stimulation trial. During visual stimulation, single-neuron firing activity of RGCs is recorded through the FFC cable to external recording instrumentation and the size and central location of the pupil are tracked with a NIR camera.

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