Defocused Images Change Multineuronal Firing Patterns in the Mouse Retina

Abstract

Myopia is a major public health problem, affecting one third of the population over 12 years old in the United States and more than 80% of people in Hong Kong. Myopia is attributable to elongation of the eyeball in response to defocused images that alter eye growth and refraction. It is known that the retina can sense the focus of an image, but the effects of defocused images on signaling of population of retinal ganglion cells (RGCs) that account either for emmetropization or refractive errors has still to be elucidated. Thorough knowledge of the underlying mechanisms could provide insight to understanding myopia. In this study, we found that focused and defocused images can change both excitatory and inhibitory conductance of ON alpha, OFF alpha and ON-OFF retinal ganglion cells in the mouse retina. The firing patterns of population of RGCs vary under the different powers of defocused images and can be affected by dopamine receptor agonists/antagonists' application. OFF-delayed RGCs or displaced amacrine cells (dACs) with time latency of more than 0.3 s had synchrony firing with other RGCs and/or dACs. These spatial synchrony firing patterns between OFF-delayed cell and other RGCs/dACs were significantly changed by defocused image, which may relate to edge detection. The results suggested that defocused images induced changes in the multineuronal firing patterns and whole cell conductance in the mouse retina. The multineuronal firing patterns can be affected by dopamine receptors' agonists and antagonists. Synchronous firing of OFF-delayed cells is possibly related to edge detection, and understanding of this process may reveal a potential therapeutic target for myopia patients.

Keywords: amacrine cell; ganglion cell; gap junction; myopia; retina.

Figure 1

Major types of cells’ responses were identified based on their response profiles to light in the mouse retina. Raster plots and peristimulus time histograms (PSTHs) of cells’ responses were recorded with a multi-electrode array (MEA): (A) ON-transient retinal ganglion cell (RGC) to a 1-s full-field light stimulus with its Raster plots (upper part) and PSTH (lower part), (B) ON-sustained RGC, (C) ON-delayed RGC, (D) ON–OFF RGC, (E,F) OFF-transient RGC and OFF-sustained RGC and (G) OFF-delayed RGC or displaced amacrine cell (AC). A 525-nm full-field (light intensity 1311 Rh*/rod/sec) 1-s light stimulus was applied. (H) Summary of numbers inside histograms represent recorded cells.

Figure 2

RGC firing responses to different defocused images on MEA recording: Firing pattern recorded from MEA can reflect the image projected on the mouse retina. First, 5 × 5 images (diameter 0.6 mm (height) × 0.646 mm (width), 0.5 cycle/degree (light intensity 7.4 × 10⁴ Rh*/rod/sec); stimuli time 1 s in 6-s circle for 10 min; there were more stripes compared with 0.2 C/D) were projected (A). Different colors labeled the ON, OFF, ON–OFF and ON/OFF-delayed cell responses at the position of MEA arrays and were mapped (B). The map of the firing pattern changed after image stimulation switch to 0.2 C/D (light intensity 9.1 × 10⁴ Rh*/rod/sec) (C). Merging these two maps showed the co-localization of cells’ responses (E). Only 30% of cells had responses at the same position. Then, the same 0.5 C/D image stimulation was projected again onto the retina (D). For the same position of the first 0.5 C/D projection, 50% of cells had responses. The firing pattern was similar to the former 0.5 C/D image projection (F). From Figure 2G–K, the images were programmed with diameter of 1.804 mm; 0.2 C/D, square-wave grating; light intensities varied from 1.5 × 10⁵ Rh*/rod/sec to 1.1 × 10⁵ Rh*/rod/sec under defocus. Firing patterns of RGCs or dACs in the mouse retina changed among the focused images (G) and different dioptric powers of optical defocus (+10D/+20D/−10D/−20D) (H–K). (L) Summary of numbers representing the recording from four different types of cell responses (ON, OFF, ON–OFF and ON/OFF-delayed cells). The bar graph shows the difference in the number of these four types of cell responses under focus and optical defocus (+10D/+20D/−10D/−20D). Different color asterisks represent the statistical significance (p < 0.01).

Figure 3

Maps of firing patterns after clear and blurred images were projected on the mouse retina: 10 × 10 image arrays with 270 µm × 310 µm spaftial frequency 0.2 cycle/degree (C/D) (A,C) clear image, square-wave grating and 0.2 C/D (B,C) blurred image, Gaussian blur were programmed to project on the mouse retina. (D) Cells had maximal responses to 0.2 cycle/degree, square-wave grating with MEA recording. (E) Firing patterns of different RGCs/ACs can reflect the image projected for clear images (focused). (F) Projection of blurred images could not reflect in the mouse retina. Light intensity: 8.6 × 10⁴ Rh*/rod/sec in 0.2 C/D, square-wave grating to 6.1 × 10⁴ Rh*/rod/sec in 0.2 C/D, Gaussian blur. Light stimulus time was 1 s, at 5 s interval and recorded for 60 min.

Figure 4

Map of firing pattern changes under dopamine receptors 1 and 2 agonist and antagonist applications in the mouse retina: D1R antagonism SCH23390 5 μM (A,B) increased firing cell numbers. The same occurred for the D2 receptor blocker eticlopride (25 μM) (E,F). In the opposite, D1R agonist SKF38393 (10 μM) (C,D) and D2 receptor agonist Quinripole (100 μM) (G,H) decreased the firing cells number. Figure 4I summarizes the normalized firing cells number after different agonists and antagonists of dopamine receptor 1 and 2 application. (The dash line is 1 in normalized cells number.) For 5 × 5 image arrays, each image was programmed with diameter 0.6 mm (height) × 0.646 mm (width), 0.2 cycle/degree (square-wave grating); light intensity 9.1 × 10⁴ Rh*/rod/sec; and stimuli time 1 s in 6-s cycles.

Figure 5

ON alpha, OFF alpha and ON–OFF RGCs had varied cell responses to focused and defocused images (F). ON alpha, OFF alpha and ON–OFF RGCs were identified based on their response profiles to lights ON and OFF. Inhibitory (red) and excitatory (blue) currents measured in voltage-clamped ON alpha GC, OFF alpha GC and ON–OFF GC (A–C) holding potential −68mV and 0 mV in response to focused and equal to different dioptric powers of optical defocus ± 20D as indicated. Light stimuli of 1 s, 0.002 cycles/degree light stimuli (light intensity = 5.09 × 10⁴ Rh*/rod/sec) was projected on the outer segment of the photoreceptor layer. Defocused images had significantly different effects on EPSCs and IPSCs responses in these cells (D,E). ∗∗ p < 0.01; ∗ 0.01 < p < 0.05; n.s. p > 0.05.

Figure 6

OFF-delayed RGC light response and morphology. (A–B) The single cell recording of OFF-delayed RGC (525-nm full-field, light intensity 1311 Rh*/rod/sec, light stimulation time 1 s, at 5 s interval) and Peristimulus time histogram (PSTH) of cell response. (C) The cell was visualized by Neurobiotin injection (red) and double labelled with anti-ChAT antibody (blue). Scale bar 20 µm.

Figure 7

OFF-delayed RGCs/ displaced amacrine cell (dAC) synchronized firing may contribute to image edge detection. Two synchronized firing patterns (A,B) and the mapping of spatial firing pattern of focused image (C) and defocused image equivalent to −20D (D) and +5D (E) dioptric power were shown. The green colored box represents the reference cell, whereas the red colored box represents the synchronized cells. The highlighted blue part is the representation of edge of the image. In this experiment, only OFF-delayed RGCs/dACs were mapped. The gray part in Figure 7A,B showed shift predictor cross-correlogram profiles computed from the pairs of OFF-delayed RGCs/dACs that had no coherent firing. The image was projected on the mouse retina with diameter 1.804 mm; spatial frequency 0 cycle/degree; light intensity 1.6 × 10⁵ Rh*/rod/sec. MEA array size is 100/30 µm.