Identification of a Circadian Clock-Controlled Neural Pathway in the Rabbit Retina

Background: Although the circadian clock in the mammalian retina regulates many physiological processes in the retina, it is not known whether and how the clock controls the neuronal pathways involved in visual processing. Methodology/Principal Findings: By recording the light responses of rabbit axonless (A-type) horizontal cells under darkadapted conditions in both the day and night, we found that rod input to these cells was substantially increased at night under control conditions and following selective blockade of dopamine D2, but not D1, receptors during the day, so that the horizontal cells responded to very dim light at night but not in the day. Using neurobiotin tracer labeling, we also found that the extent of tracer coupling between rabbit rods and cones was more extensive during the night, compared to the day, and more extensive in the day following D 2 receptor blockade. Because A-type horizontal cells make synaptic contact exclusively with cones, these observations indicate that the circadian clock in the mammalian retina substantially increases rod input to A-type horizontal cells at night by enhancing rod-cone coupling. Moreover, the clock-induced increase in D2 receptor activation during the day decreases rod-cone coupling so that rod input to A-type horizontal cells is minimal. Conclusions/Significance: Considered together, these results identify the rod-cone gap junction as a key site in mammals through which the retinal clock, using dopamine activation of D2 receptors, controls signal flow in the day and night fro

Circadian clock regulation of cone to horizontal cell synaptic transfer in the goldfish retina.

Although it is well established that the vertebrate retina contains endogenous circadian clocks that regulate retinal physiology and function during day and night, the processes that the clocks affect and the means by which the clocks control these processes remain unresolved. We previously demonstrated that a circadian clock in the goldfish retina regulates rod-cone electrical coupling so that coupling is weak during the day and robust at night. The increase in rod-cone coupling at night introduces rod signals into cones so that the light responses of both cones and cone horizontal cells, which are post-synaptic to cones, become dominated by rod input. By comparing the light responses of cones, cone horizontal cells and rod horizontal cells, which are post-synaptic to rods, under dark-adapted conditions during day and night, we determined whether the daily changes in the strength of rod-cone coupling could account entirely for rhythmic changes in the light response properties of cones and cone horizontal cells. We report that although some aspects of the day/night changes in cone and cone horizontal cell light responses, such as response threshold and spectral tuning, are consistent with modulation of rod-cone coupling, other properties cannot be solely explained by this phenomenon. Specifically, we found that at night compared to the day the time course of spectrally-isolated cone photoresponses was slower, cone-to-cone horizontal cell synaptic transfer was highly non-linear and of lower gain, and the delay in cone-to-cone horizontal cell synaptic transmission was longer. However, under bright light-adapted conditions in both day and night, cone-to-cone horizontal cell synaptic transfer was linear and of high gain, and no additional delay was observed at the cone-to-cone horizontal cell synapse. These findings suggest that in addition to controlling rod-cone coupling, retinal clocks shape the light responses of cone horizontal cells by modulating cone-to-cone horizontal cell synaptic transmission

Tracer coupling between rabbit rod and cone photoreceptor cells varies with the time of day and D₂ receptor activity.

<p><i>A-E</i>, Typical examples of photoreceptor cell tracer coupling obtained under dark-adapted conditions during the day (<i>A</i>), night (<i>B</i>), and day in the presence of spiperone (10 µM) (<i>C</i>), night in the presence of quinpirole (1 µM) (<i>D</i>), and day in the presence of SCH23390 (10 µM) (<i>E</i>). Shown are confocal images of whole-mount rabbit retinas taken parallel to the retinal surface at the level of the photoreceptor inner segments near the cut (<i>Ai-Ei</i>) and detailed perpendicular views at higher magnification of the 3D reconstruction of the labeled photoreceptor cells (<i>Aii-Eii</i>). The micrographs in <i>Aii-Eii</i> show labeled photoreceptor cells in images that range along the horizontal axis from the cuts (leftmost edge of the micrographs) to 50 µm from the cuts (rightmost edge). In addition, at the bottom of the micrographs cone pedicles are visible in <i>Aii</i>, <i>Dii</i> and <i>Eii</i>, and horizontal cells/bipolar cells are indicated (asterisks) in <i>Bii</i> and <i>Cii</i> proximal to the photoreceptors. Large vertical arrows indicate the location of the cuts in <i>Ai-Ei</i>. Some cones (small arrows) and rods (arrowheads) are indicated in <i>Aii-Eii</i>. Rod cell bodies are located in the innermost half of the outer nuclear layer, whereas cone cell bodies are typically located in the outermost half of the outer nuclear layer <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0011020#pone.0011020-Nikonov1" target="_blank">[49]</a>. Scale bar = 50 µm (<i>Ai-Ei</i>); 20 µm (<i>Aii-Eii</i>). <i>F</i>, Averaged normalized fluorescence in the photoreceptor cell layer as a function of the distance from the cut under dark-adapted conditions during the day (open circles; <i>n</i> = 6), night (filled circles; <i>n</i> = 4), and during the day in the presence of spiperone (open diamonds; <i>n</i> = 4) or SCH23390 (open squares; <i>n</i> = 2), and night in the presence of quinpirole (filled diamonds; <i>n</i> = 4). Curves generated from the non-linear analysis of the data during the day (grey curve) and night (black curve) are also shown. Data points represent averaged data from <i>n</i> experiments (1 retina/condition/experiment) ± SEM.</p

Tracer coupling between rabbit A-type horizontal cells does not vary with the time of day.

<p>Extent of A-type horizontal cell tracer coupling under dark-adapted conditions in the subjective day (<i>n</i> = 5; filled light grey circles) and day (<i>n</i> = 14; open circles) and in the subjective night (<i>n</i> = 3; filled dark grey circles) and night (<i>n</i> = 5; filled black circles). Data were pooled into 2 groups (day-dark-adapted and night-dark-adapted) and averaged (horizontal bars). No difference was found between the 2 groups (Student's t-test; <i>P</i> = 0.201). Data points represent averaged number of coupled cells from <i>n</i> cells (1 cell/retina) ± SEM. For these experiments, light stimuli were never brighter than −5 log <i>I</i>_o.</p

The retinal circadian clock uses dopamine and D₂ receptors to control the light responses of rabbit A-type horizontal cells.

<p><i>A-G</i>, Representative examples of A-type horizontal cell responses to a series of 500 ms full-field white light stimuli of increasing intensity recorded under dark-adapted conditions during the subjective day (<i>A</i>), the subjective night (<i>B</i>), the day (<i>C</i>), the night (<i>D</i>), the day in the presence of the D₂ dopamine receptor antagonist spiperone (10 µM) (<i>E</i>), the night in the presence of D₂ dopamine receptor agonist quinpirole (1 µM) (<i>F</i>), and the day in the presence of the D₁ dopamine receptor antagonist SCH23390 (10 µM) (<i>G</i>). The light responses of only 1 cell per retina to the full series of light intensities were recorded.</p

Light response amplitude and sensitivity of rabbit A-type horizontal cells vary with the time of day and D₂ receptor activity.

<p><i>A,</i> Average normalized intensity-response curves of A-type horizontal cells recorded under dark-adapted conditions during the day (<i>n</i> = 8) and subjective day (<i>n</i> = 6) (open circles, solid line), night (<i>n</i> = 4) and subjective night (<i>n</i> = 3) (filled circles, solid line), and in the day in the presence of spiperone (10 µM; open diamonds; <i>n</i> = 9) or SCH23390 (10 µM; open squares; <i>n</i> = 8), and the night in the presence of quinpirole (1 µM; filled diamonds; <i>n</i> = 8). Two-way ANOVA analysis revealed both intensity and condition effects for each response property measured. Data points represent averaged data from <i>n</i> cells (1 cell/retina) ± SEM. <i>B</i>, Average light response threshold (i.e. intensity required to elicit a 0.5 mV response) of A-type horizontal cells under the conditions described in (<i>A</i>). Data points represent averages of 5 to 25 measurements. ***, <i>P</i><0.001 compared to day (Tukey's post test).</p

The circadian clock uses dopamine D₂ receptors to regulate the spectral sensitivity of rabbit A-type horizontal cells.

<p><i>A</i>, Relative spectral sensitivity of A-type horizontal cells recorded under dark-adapted conditions during the day (open circles; <i>n</i> = 19), and the night (filled circles; <i>n</i> = 8). <i>B</i>, Absolute spectral sensitivity of A-type horizontal cells recorded under dark-adapted conditions during the night (filled circles; <i>n</i> = 8), day (open circles; <i>n</i> = 19), and day in the presence of spiperone (10 µM; open diamonds; <i>n</i> = 6) or SCH23390 (10 µM; open squares, <i>n</i> = 3), and night in the presence of quinpirole (1 µM; filled diamonds; <i>n</i> = 5). Data points represent average sensitivity from <i>n</i> cells (1 cell/retina) ± SEM.</p

COSINOR analysis of core circadian clock component expression in cones and dopaminergic amacrine cells.

<p>COSINOR regression analysis was performed on the data illustrated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050602#pone-0050602-g007" target="_blank">Figure 7</a> and only for clock protein levels displaying significant temporal variation over the course of a LD or DD cycle (as determined by one-way ANOVA; <i>P</i><0.05). The regression coefficients (a, b, and c) are given with their respective standard error estimates.</p>*<p>: <i>P</i><0.05 compared to respective LD value (Student <i>t</i>-test).</p

Mammalian core circadian clock protein expression in bipolar, amacrine and ganglion cells of the mouse retina.

<p>Typical examples of vertical sections of mouse retinas collected between ZT02 and ZT06 and double labeled for one of the following clock proteins: CLOCK (<b><i>A–C</i></b>), BMAL1 (<b><i>A’–C’</i></b>), NPAS2 (<b><i>A”–C”</i></b>), PER1 (<b><i>A’”–C’”</i></b>), PER2 (<b><i>A””–C””</i></b>), and CRY2 (<b><i>A’””–C’””</i></b>) and one of the following protein markers: Chx10 (bipolar cells; <b><i>A</i></b>), Pax6 (most amacrine cells and ganglion cells; <b><i>B</i></b>), and TH (dopaminergic amacrine cells; <b><i>C</i></b>) (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050602#pone-0050602-t001" target="_blank">Table 1</a> for details about the antibodies). The analysis was restricted to type-1 catecholamine amacrine cells that express high levels of TH. Some double-labeled retinal neurons are shown (arrows). Abbreviations and bar as in Fig. 3.</p