The circadian clock in the retina controls rod-cone coupling

Abstract

Although rod and cone photoreceptor cells in the vertebrate retina are anatomically connected or coupled by gap junctions, a type of electrical synapse, rod-cone electrical coupling is thought to be weak. Using tracer labeling and electrical recording in the goldfish retina and tracer labeling in the mouse retina, we show that the retinal circadian clock, and not the retinal response to the visual environment, controls the extent and strength of rod-cone coupling by activating dopamine D(2)-like receptors in the day, so that rod-cone coupling is weak during the day but remarkably robust at night. The results demonstrate that circadian control of rod-cone electrical coupling serves as a synaptic switch that allows cones to receive very dim light signals from rods at night, but not in the day. The increase in the strength and extent of rod-cone coupling at night may facilitate the detection of large dim objects.

Figure 1. Rod-cone Tracer Coupling Varies with Time of Day

(A–H) Following iontophoresis of biocytin into individual cones, the tracer remained in a few cells (indicated by arrows in A1, D1, E1, G1, H1) near the injected cone during the subjective day (A), during the subjective night in the presence of the D₂-like receptor agonist quinpirole (1 μM, D), and following dim light adaptation for > 60 min in the day (E) and bright light adaptation for > 60 min in the day (G) and night (H), but diffused into many rods and cones during the subjective night (B), during the subjective day in the presence of the D₂-like receptor antagonist spiperone (10 μM, C), and following dim light adaptation for > 60 min in the night (F). In each of A–H, confocal images of a whole-mount retina at the level of the rod inner segments are shown on the left and perpendicular views of the 3-D reconstruction of the photoreceptor cells from the same retina are shown on the right. Some cones (arrows) and rods (arrowheads) are indicated. Scale bars (A–H): 50 μm.

Figure 2. The Circadian Clock in the Goldfish Retina Controls Rod-cone Coupling by Activating Dopamine D₂-like Receptors in the Day

(A and B) Average numbers of stained cones (open bars) and rods (filled bars) following biocytin injections into individual cones (1 cone injected/retina) under dark-adapted conditions (A) during the day (n = 11) and subjective day (n = 5), night (n = 4) and subjective night (n = 5), subjective day in the presence of spiperone (n = 6), and subjective night in the presence of quinpirole (n = 6), and under dim light-adapted conditions (B-left) during the day (n = 6) and night (n = 3) and bright light-adapted conditions (B-right) during the day (n = 2) and night (n = 3). Under dark-adapted conditions, the number of tracer coupled rods and cones was significantly greater during the night (p < 0.001) and during the day following spiperone treatment (p < 0.001) than during the day under control conditions. Under dim light-adapted conditions, the number of tracer coupled rods and cones was significantly greater during the night (p < 0.001) compared to the day (Tukey post hoc analysis). Under bright light-adapted conditions, biocytin was restricted to the injected cone; no other cells were labeled. Error bars represent s.e.m.

Figure 3. Dark-adapted Cones Receive Very Dim Light Signals from Rods at Night, but not in the Day

(A and B) Representative examples of cone responses to a series of full-field white light stimuli of increasing intensity recorded under dark-adapted conditions (A) during the subjective day, subjective night, in the presence of spiperone (10 μM) during the subjective day, and in the presence of quinpirole (1 μM) during the subjective night, and under dim and bright light-adapted conditions (B) during the day and night.

Figure 4. The Retinal Circadian Clock Regulates Cone Light Responses and Receptive Field Size by Activating D₂-like Receptors in the Day so that Rod Input is Dominant at Night, but not Present in the Day

(A and B) Average normalized intensity-response curves of cones (1 cone/retina) recorded under dark-adapted conditions (A) during the day (n = 7) and subjective day (n = 9) (open circles), night (n = 7) and subjective night (n = 3) (filled circles), in the subjective day in the presence of spiperone (open diamonds, n = 5), and in the subjective night in the presence of quinpirole (filled diamonds, n = 9), and under light-adapted conditions (B). Shown are values obtained under dim light-adapted conditions during the day (open squares, n = 6) and night (filled squares, n = 6), and under bright light-adapted conditions in the day (n = 9) and subjective day (n = 2) (open triangles) and night (n = 6) and subjective night (n = 4) (filled triangles). (C) Average day/night and circadian rhythms of the cone light response threshold (i.e. intensity required to elicit a 0.5 mV response) under dark-adapted conditions. The average cone light response threshold (log intensity) was significantly higher during the day (p < 0.001) and subjective day (p < 0.001) than during the night and subjective night (Tukey post hoc analysis). Data points represent averages of 4 to 15 measurements. Error bars indicate s.e.m. (D) Average normalized response amplitudes of dark-adapted cones plotted against stimulus radius for a stimulus of intensity -5 log Io. These data indicate that the receptive field size of cones is larger at night than in the day. Measurements were performed during the day (open circles, n = 6) and night (filled circles, n = 6).

Figure 5. Kinetics of Cone Light Responses during the Day and Night under Different Lighting Conditions

(A) Representative examples of cone responses to a light stimulus flashed (500 ms) at intensity -5 log Io during the subjective day and subjective night (grey trace), and of a rod horizontal cell response to the same stimulus. The amplitude of each trace has been normalized relative to its peak for better comparison. (B) Representative example of the responses of an individual cone to a light stimulus flashed (500 ms) at intensity – 2 log Io during the day immediately following 60 min of dark adaptation (grey trace), and following subsequent bright light adaptation (– 2 log Io, 500 ms stimuli at 0.125 Hz) for 6 min (black trace). Note that the cone response exhibited a prolonged plateau potential under dark-adapted conditions, but not following 6 min of bright light adaptation. (C–E) Average latency (C), time-to-peak (D), and duration of the hyperpolarization (E) of cone light responses recorded under dark-adapted conditions during the night (filled circles; n = 10 to 19), day (open circles; n = 12 to 26), subjective day in the presence of spiperone (10 μM) (open diamonds; n = 5), and subjective night in the presence of quinpirole (1 μM) (filled diamonds, n = 9) or under bright light-adapted conditions during the day (open triangles; n = 7 to 11) and night (filled triangles; n = 7 to 10). Error bars represent s.e.m. (F) Relationship between membrane current and membrane potential of dark-adapted cones during the day (open circles, n = 30) and night (n = 27). The peak current was measured when cones were voltage-clamped at −35 mV and stepped (duration 200 ms every 400 ms) from -90 mV to +30 mV in 10 mV increments.

Figure 6. Circadian Variations in Cone Spectral Sensitivity

(A) Average spectral sensitivity of cones recorded under dark-adapted conditions during the day or subjective day fit one of three nomograms (thin dotted curves) corresponding to the three major known types of goldfish cone pigments: L, M, and S. Data points represent average sensitivity ± s.e.m. from recorded red cones (open squares; n = 9), green cones (open circles; n = 6) and blue cone (open triangle; n = 1). In contrast, the spectral sensitivity of all dark-adapted cones recorded at night peaked at ~ 535 nm (filled circles; n = 10). Although cone spectral sensitivity at night under dark-adapted conditions closely fits a rod nomogram (solid thick line) for 400 nm < λ < 600 nm, it does not fit the nomogram as well for λ > 600 nm. Rather, the data points closely fit a modified nomogram that combines goldfish rod and L-cone pigment nomograms (dotted thick curve; λ_max = 537 ± 3 (s.d.) nm; r² = 0.91). Following application of spiperone (10 μM) (open diamonds; n = 2), cone spectral sensitivity in the subjective day resembled that observed during the subjective night and data points fit well the modified nomogram (λ_max = 537 ± 3 nm; r² = 0.96). (B) Following bright light adaptation at night or during the subjective night 3 groups of cones with different spectral sensitivities were observed: red cones (filled squares; n = 4), green cones (filled circles; n = 5) and blue cone (filled triangles; n = 1), whereas bright light adaptation during the day or subjective day did not affect the relative spectral sensitivity of the recorded cones (red cones: open squares; n = 2; green cones: open circles; n = 6) but slightly decreased the absolute sensitivity. Nomograms as in (A).

Figure 7. Day-night Difference in Photoreceptor Tracer Coupling in the Mouse Retina

(A–F) Representative examples of photoreceptor tracer coupling measured by intracellular propagation of Neurobiotin tracer after cut loading in goldfish (A–C) and mouse (D–F) retinas under dark-adapted conditions during the day (A and D), night (B and E), and in the presence of spiperone (10 μM) during the day (C and F). Similar results have been observed in 3 independent experiments (2 retinas/experiment). Shown are confocal images of whole-mount retinas at the level of the rod inner segments (A–C, D1, E1 and F1) and detailed perpendicular views of the 3-D reconstruction of the mouse photoreceptor cells adjacent to the cut at higher magnification (D2, E2 and F2). Large arrows (A-C, and D1-F1) indicate location of cut. Some cones (small arrows) and rods (arrowheads) are shown in D2-F2. Scale bars: 200 μm (A–C); 50 μm (D1, E1, F1); 10 μm (D2, E2, F2). (G, H) Relative fluorescent intensity as a function of the distance from the cuts in goldfish (G) and mouse (H) retinas under the three experimental conditions tested in A–F. Averaged data from 4 experiments (1 retina/experiment) are shown.

Figure 8. The Retinal Circadian Clock Controls Rod-Cone Coupling

The circadian clock in the retina increases dopamine release from dopaminergic neurons during the subjective day, thereby activating the D₂-like receptors on rods and cones so that the conductance of rod-cone gap junctions and the rod signal to cones and cone horizontal cells are decreased in the subjective day, compared to the subjective night. The traces shown are schematic representations of cone (top) and cone horizontal cell (bottom) responses to a 500 ms-light stimulus flashed at intensity −5 log Io during the subjective day (left) and subjective night (right). The cone traces were generated from the averaged response latency, time-to-peak, and response duration data shown in Fig. 5 and the cone horizontal cell traces were generated from similar averaged response kinetic data from Ribelayga et al., 2002, , as well as from unpublished data. In each case, the three data points were connected by a computer-generated, smoothing curve. The amplitude of each trace was normalized relative to its peak for better comparison of response kinetics. See Discussion for details.