Rod Photoreceptors Signal Fast Changes in Daylight Levels Using a Cx36-Independent Retinal Pathway in Mouse

Abstract

Temporal contrast detected by rod photoreceptors is channeled into multiple retinal rod pathways that ultimately connect to cone photoreceptor pathways via Cx36 gap junctions or via chemical synapses. However, we do not yet understand how the different rod pathways contribute to the perception of temporal contrast (changes in luminance with time) at mesopic light levels, where both rods and cones actively respond to light. Here, we use a forced-choice, operant behavior assay to investigate rod-driven, temporal contrast sensitivity (TCS) in mice of either sex. Transgenic mice with desensitized cones (GNAT2 ^cpfl3 line) were used to identify rod contributions to TCS in mesopic lights. We found that at low mesopic lights (400 photons/s/μm² at the retina), control and GNAT2 ^cpfl3 mice had similar TCS. Surprisingly, at upper mesopic lights (8000 photons/s/μm²), GNAT2 ^cpfl3 mice exhibited a relative reduction in TCS to low (<12 Hz) while maintaining normal TCS to high (12-36 Hz) temporal frequencies. The rod-driven responses to high temporal frequencies developed gradually over time (>30 min). Furthermore, the TCS of GNAT2 ^cpfl3 and GNAT2 ^cpfl3 ::Cx36^-/- mice matched closely, indicating that transmission of high-frequency signals (1) does not require the rod-cone Cx36 gap junctions as has been proposed in the past; and (2) a Cx36-independent rod pathway(s) (e.g., direct rod to OFF cone bipolar cell synapses and/or glycinergic synapses from AII amacrine cells to OFF ganglion cells) is sufficient for fast, mesopic rod-driven vision. These findings extend our understanding of the link between visual circuits and perception in mouse.SIGNIFICANCE STATEMENT The contributions of specific retinal pathways to visual perception are not well understood. We found that the temporal processing properties of rod-driven vision in mice change significantly with light level. In dim lights, rods relay relatively slow temporal variations. However, in daylight conditions, rod pathways exhibit high sensitivity to fast but not to slow temporal variations, whereas cone-driven responses supplement the loss in rod-driven sensitivity to slow temporal variations. Our findings highlight the dynamic interplay of rod- and cone-driven vision as light levels rise from night to daytime levels. Furthermore, the fast, rod-driven signals do not require the rod-to-cone Cx36 gap junctions as proposed in the past, but rather, can be relayed by alternative Cx36-independent rod pathways.

Keywords: gap junctions; mesopic vision; mouse vision; operant behavior; rod pathways; temporal contrast sensitivity.

Figure 1.

Rod circuit schematics of control and transgenic mice. A–E, Circuit diagrams depicting WT (A) and targets of disruption in transgenic mice (B–E). B, G2 mice (cpfl3 and KO) have disrupted cone circuits leaving only functional rod circuits. C, G2::Cx36 mice have both disrupted cone circuits and disrupted Cx36-dependent rod pathways (secondary pathway and ON branch of the primary pathway). D, G1 mice have disrupted rod circuits leaving only functional cone circuits. E, G1::G2 mice have disrupted rod and cone circuits. R, rod; C cone; black resistors, Cx36 gap junctions; RB, rod bipolar; AII, amacrine AII; ON CB, ON cone bipolar; OFF CB OFF cone bipolar; GC ganglion cell; green, primary path circuit; blue, secondary path and cone path circuit; pink, tertiary path circuit.

Figure 2.

No overt degeneration or remodeling in G2 mouse retinas. A, Similar thickness in the ONL and OPL of 6 month WT and G2 retinas. Top, Representative vertical sections labeled with DAPI. Optic nerve head labeled ON. Thickness measurements were performed in two distinct regions: (1) 500 and (2) 800 μm from optic nerve (white boxes). Bottom, ONL and OPL thickness is not significantly different (N.S.) at 500 μ or 800 μm (two-way RM ANOVA; see Results). n = 4 mice, 3–4 sections/mouse. Scale bar, 100 μm. B–D, Confocal images of immunolabeled retinal sections show normal retinal structures in 6 month WT (B–D), G2 (B′–D′) and G2::Cx36 retinas (B″–D″). B–B″, Calbindin (green) labels horizontal cells (arrow) and some types of amacrine cells (arrowhead). C–C″, Immunolabeling of rod bipolar cells with antibody against PKCα (green), ribbon synapses in photoreceptor terminals with antibody against CtBP2 (red) and cone terminals labeled with PNA (blue). Arrows point to representative extended rod bipolar cell dendrites rarely observed in both WT and G2 retinas. Insets show detailed synaptic structure of boxed regions. D–D″, Immunolabeling of Cx36 gap junctions with antibody against Connexin 36 (Cx36, red) and cone terminals with antibody against cone arrestin (CAR, cyan). Scale bars, 20 μm. E, Amplitudes of dark-adapted flash ERG b-waves as a function of rod photoisomerizations elicited by brief flashes for WT (black circles; n = 10), G2 (red triangles; n = 10), and G2::Cx36 mice (purple diamonds; n = 10) at 3 months of age. G2 and G2::Cx36 mice exhibit normal rod-driven flash ERG responses and reduced cone-driven flash ERG responses. Statistical analysis: two-way RM ANOVA; see text for details. Error bars: SEM is indicated but generally smaller than the size of the symbols. F, b-wave amplitudes of WT, G2, and G2::Cx36 mice measured at 3 and 6 months of age. Minor changes in amplitude suggest no significant degeneration or remodeling during the time course of experiments. Statistical analysis: two-way RM ANOVA, *p < 0.05; see text for details.

Figure 3.

Desensitized cone responses of G2 mice do not mask rod responses at the mesopic light levels used in this study. A, Optomotor contrast sensitivity of WT (black circles; n = 4) and G2 mice (red triangles; n = 6) plotted as a function of rod photoisomerizations. Grating parameters: f_t = 3 Hz, fs = 0.128 cyc/deg. B, Optomotor contrast sensitivity of WT (black bars; n = 5), G2 (red bars; n = 5) and G1::G2 mice (blue bars; n = 6) measured at backgrounds levels of 1500 R*/rod/s and 50,000 R*/rod/s. Grating parameters: f_t = 1.5 Hz, fs = 0.128 cyc/deg. C, Amplitudes of dark-adapted flash ERG b-waves as a function of R*/rod for WT (black circles; n = 5), G2 (red triangles; n = 6), G1 (green inverted triangles; n = 5), and G1::G2 mice (blue diamonds; n = 6). D, Magnitudes of the response at the fundamental frequency in the flicker ERG response of WT (black circles; n = 3) and G2 mice (red triangles; n = 3) in response to a 16 Hz sinusoidal stimulus, 100% in contrast and indicated mean retinal irradiance. Region between dashed lines represents the intensity range over which subsequent operant behavior experiments were performed. All mice were 2–4 months old.

Figure 4.

Rods are sufficient to mediate TCS to high but not low temporal frequencies in bright mesopic light levels. A–D, TCSFs of WT (black circles) and G2 mice (red triangles) measured at mean retinal irradiance values of: (A) 400 ph/s/μm², WT (n = 4), and G2 mice (n = 3–6); (B) 8000 ph/s/μm², WT (n = 4–9), and G2 mice (n = 4–8); (C) 20,000 ph/s/μm², WT (n = 4), and G2 mice (n = 4–5); and (D) 35,000 ph/s/μm², WT (n = 3–5), and G2 mice (n = 3). Mice were 3–6 months old. Closed symbols represent mean ± SEM, whereas the smaller, lighter symbols are responses of individual mice (WT, gray; G2, light red). Statistical analysis: two-way ANOVA (see text for details), frequency × genotype interactions: *p < 0.05 and +p < 0.01, all other interactions p > 0.05. E, F, TCSFs over increasing background intensities for (E) WT and (F) G2 mice. Retinal irradiance values of WT and G2 mice were estimated using the pupil area values of freely behaving mice as described in Materials and Methods.

Figure 5.

Rod-driven TCS to high temporal frequencies develops gradually over time. A, B, Values of d′m measured during corrective trials plotted as a function of time after the start of the trial. WT (A) and G2 (B) mice were dark-adapted for 1 h before the trial. Measurements were performed at indicated mean retinal irradiance levels. Flicker contrast was 70% in all cases except for the tests at 20,000 ph/s/μm², which were performed with 90% contrast flicker. Continuous lines represent fits with rising exponential functions (all curves in A; and response at 10 and 400 ph/s/μm² in B) and s-shaped hyperbolic functions (B; 8000 and 20,000 ph/s/μm²). Following the initial 60 min of each trial during which the corrective protocol (measured d′m) was run, we switched to the normal protocol (without corrective trials) and determined the average d′ value over the subsequent 90 min. The corresponding value of d′ is indicated at time t = 150 min. Symbols in these plots and in C, E, and F represent mean ± SEM; n = 4–6 runs in each condition. C, The time course of d′m plotted as a function of time for a G2 mouse tested at 8000 ph/s/μm² and flicker contrasts 80 and 70%. Flicker frequency was 18 Hz. D, Experimental design applied to studies described in E and F. In Condition 1, mice are tested immediately after a 60 min dark-adaptation period (E, F, closed symbols); in Condition 2, mice undergo a 60 min light adaptation period before testing (E, F, open symbols). Retinal irradiance during the light adaptation period was 20,000 ph/s/μm². Flicker frequency was 21 Hz, and the contrast was adjusted individually to elicit a d′ value ranging from 2 to 2.5. E, F, Values of d′m plotted as a function of time measured with Condition I (closed symbols) and Condition 2 (open symbols). E, d′m values for two representative WT mice (WT M1, WT M2) do not vary with experimental condition; (F) d′m values for G2 mice (G2 M1, G2 M2) rise later in Condition 1 compared with Condition 2. TTR was determined at the intersection of the fitting functions with the line representing d′m = 1 (see G). Fitting functions were rising exponential for all data of (E) and data determined under condition 2 in (F). Hyperbolic functions fit the responses to condition 1 in (F). G, TTR for four WT and four G2 mice measured under Conditions 1 and 2. Analysis: two-way RM ANOVA, Holm–Sidak method; corresponding p values for pairwise comparisons are indicated on graph. See text for details.

Figure 6.

Cx36-independent rod pathways are sufficient to relay rod-driven TCS to high temporal frequencies. A, B, TCSFs of G2 (red triangles) and G2::Cx36 mice (purple diamonds) measured at mean retinal irradiance values of (A) 400 ph/s/μm², G2 (n = 3–6) and G2::Cx36 mice (n = 3); (B) 8000 ph/s/μm², G2 (n = 4–9) and G2::Cx36 mice (n = 4). Mice were 3–6 months old. Closed symbols represent mean ± SEM, whereas the smaller, lighter purple symbols are responses of individual G2::Cx36 mice. Data for individual G2 mice are shown in Figure 4. Statistical analysis: two-way or three-way ANOVA; see text for details. Retinal irradiance values of G2 and G2::Cx36 mice were estimated using the pupil area values of freely behaving mice as described in Materials and Methods.

Figure 7.

The mesopic range shifts with temporal frequency. A, B, Contrast sensitivity as a function of retinal irradiance for (A) G2 mice at 6 Hz (filled red triangles; n = 4–9) and 21 Hz (open red squares; n = 3–8); and (B) G1 mice at 6 Hz (filled green triangles; n = 4–5) and 21 Hz (open green squares; n = 3–4). C, D, Contrast sensitivity as a function of retinal irradiance for WT (black; n = 4–11), G2 (red; n = 3–9), and G1 (green; n = 4–5) mice at: (C) 6 Hz and (D) 21 Hz. Sensitivity values in A–D are plotted as a function of the WT retinal irradiance (for WT and G2 mice) or corrected for phenotypic differences in pupil areas (for G1 mice). See Table 1 for respective pupil area and retinal irradiance values. Note: for light conditions where pupil areas were not measured, G1 retinal irradiance values were determined by interpolation of data. Filled pink squares in (D) represent dark-adapted (DA) G2 data and open red squares represent light-adapted (LA) G2 data as described in Figure 5. Red bars represent the intensity range over which rods are active in G2 mice, green bars represent the intensity range over which cones are active in G2 mice, and blue bars represent the mesopic range over which both rods and cones are active. Dashed red, green, and blue lines in (D) represent extrapolated values. Statistics: two-way ANOVA, p values for pairwise comparisons are shown in the figure.

Figure 8.

Map of rod and cone contributions to TCS. Irradiance versus Temporal Frequency adaptation map showing the irradiance-frequency combinations where contrast thresholds are driven largely by rods (pink), cones (green) and rod + cones (blue shading), loosely defining the conditions for scotopic, photopic, and mesopic temporal contrast vision in mouse, respectively. The transition between the pink and blue regions was inferred from the cone threshold values of G1 mice at 6 Hz (green triangle) and 21 Hz (green square), whereas the transition between blue and green regions was defined as the upper irradiance levels eliciting a behavioral response in G2 mice to 6 Hz (red triangle) and 21 Hz (red square) flicker respectively (Fig. 7). The blue region with stripes represents extrapolated sensitivity at high temporal frequencies (Fig. 6). The high-frequency boundary of the map (black circles) is determined by the CFF values for WT mice measured in this study (Fig. 4) and previously by Umino et al. (2018) (see CFF at 10 ph/s/μm²).

Figure 9.

Activation of S-opsin-expressing cones does not contribute significantly to TCS when coactivated with M-opsin-expressing cones. A, Normalized sensitivity for rhodopsin (R-opsin), M-opsin, and S-opsin plotted as a function of stimulus wavelength. Fits to mouse pigments were computed according to Lucas et al. (2014) using the Govardovskii nomograms (Govardovskii et al., 2000). Sensitivity after accounting for media losses (Jacobs et al., 2004) is shown with dashed lines. B, Estimated photoisomerization rates in pure M-opsin (green edge symbols) or S-opsin (blue edge symbols) -expressing cones plotted as a function of rod photoisomerization rates for 405 nm (filled blue symbols) and 505 nm (filled green symbols) narrowband stimuli (for details of calculations, see Materials and Methods). Photoisomerization rates for cones coexpressing M- and S-opsin at a given wavelength fall within the range delimited by the pure cones (gray arrows; see text for details). C, D, TCS to 405 and 505 nm plotted as a function of initial (before bleach) rod photoisomerization rates for (C) 6 Hz and (D) 21 Hz. TCS to 505 nm is same as in Figure 7 after converting retinal irradiance to photoisomerizations/s in rods. Symbols represent mean ± SD, n = 4–11 mice for 505 nm and n = 4 mice for 405 nm.

Figure 10.

Similar TCS in G2 and G2 KO mice. A, B, TCS of G2 and G2 KO mice in response to 505 nm stimuli plotted as a function of initial (before bleach) rod photoisomerizations in response to (A) 6 Hz and (B) 21 Hz. TCS of G2 mice is same as in Figure 7 after converting retinal irradiance to photoisomerizations/s in rods. TCS of G2 and G2 KO mice matched closely, suggesting that any residual cone function in G2 mice does not alter their TCS. Symbols represent mean ± SD; n = 3–9 for G2 mice and n = 4 for G2KO mice. Note: data points for G2 KO mice were shifted slightly along the x-axis for clarity.