Table 1.
List of the antibodies used in this study.
Figure 1.
Distribution of six core mammalian circadian clock proteins in the mouse retina.
Typical examples of vertical sections of C57Bl/6 mouse retinas collected in the middle of the day during a LD cycle (ZT02-06) are illustrated for CLOCK (A), BMAL1 (B), NPAS2 (C), PER1 (D), PER2 (E), and CRY2 (F). Expression of each clock protein is clearly prominent in the inner nuclear layer (INL) and most cells in the ganglion cell layer (GCL). However, expression is also detected in few cells in the outer nuclear layer (ONL) (vertical arrows). These cells were later identified as cones. OPL: outer plexiform layer; IPL: inner plexiform layer. Optical sections 3×0.4 µm. Bar is 10 µm.
Table 2.
Proportion of cells among the retinal nuclear layers that express the core circadian clock components.
Figure 2.
Expression of the six core mammalian circadian clock proteins CLOCK, BMAL1, NPAS2, PER1, PER2 and CRY2 in mouse retina during LD and DD cycles.
Typical examples of vertical sections of C57Bl/6 mouse retinas collected in the middle of the day (ZT06; A–F) or subjective day (CT06; A”–F”) or middle of the night (ZT18; A’–F’) or subjective night (CT18; A”’–F”’) are illustrated for CLOCK (A–A”’), BMAL1 (B–B”’), NPAS2 (C–C”’), PER1 (D–D”’), PER2 (E–E”’), and CRY2 (F–F”’). Optical sections 3×0.4 µm. Bar is 10 µm. Temporal expression profiles of CLOCK, BMAL1, NPAS2, PER1, PER2, and CRY2 in mouse retinal sections under LD and DD conditions (A””–F””). Mouse retinas were collected every 4 h during a LD cycle or a DD cycle, and clock protein expression was quantified by measuring the mean fluorescence per section within a squared area whose upper and lower boundaries were the outer and inner limiting membranes, respectively (see Materials and Methods for details). COSINOR regression was performed only when variations in clock protein expression with time of day were detected (one-way ANOVA; P<0.05). Note that only PER1 expression is significantly rhythmic under either LD or DD conditions (D””). Each data point represents the mean fluorescence/section +/− SEM of 5/6 animals (5 pictures from 3–4 sections/animal). ONL: outer nuclear layer; OPL: outer plexiform layer; INL: inner nuclear layer; IPL: inner plexiform layer; GCL: ganglion cell layer.
Figure 3.
Mammalian core circadian clock protein expression in cones, horizontal cells and rod bipolar cells of the mouse retina.
Typical examples of vertical sections of mouse retinas collected between ZT02 and ZT06 and double labeled for one of the following clock proteins: CLOCK (A–C), BMAL1 (A’–C’), NPAS2 (A”–C”), PER1 (A’”–C’”), PER2 (A””–C””), and CRY2 (A’””–C’””) and one of the following protein markers: cARR (cones; A), CALD28 (horizontal cells; B), and PKCα (rod bipolar cells; C). (see Table 1 for details about the antibodies). Note that in the outer nuclear layer (ONL) clock protein expression was evident in cones but was weak or absent in rods. Some double-labeled retinal neurons are shown (arrows). OPL: outer plexiform layer; INL: inner nuclear layer; IPL: inner plexiform layer. Optical sections 3×0.4 µm. Bar is 10 µm.
Table 3.
Proportion of cells among identified retinal neurons that express the core circadian clock components at ZT02/06.
Figure 4.
Mammalian core circadian clock protein expression in bipolar, amacrine and ganglion cells of the mouse retina.
Typical examples of vertical sections of mouse retinas collected between ZT02 and ZT06 and double labeled for one of the following clock proteins: CLOCK (A–C), BMAL1 (A’–C’), NPAS2 (A”–C”), PER1 (A’”–C’”), PER2 (A””–C””), and CRY2 (A’””–C’””) and one of the following protein markers: Chx10 (bipolar cells; A), Pax6 (most amacrine cells and ganglion cells; B), and TH (dopaminergic amacrine cells; C) (see Table 1 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.
Figure 5.
Mammalian core circadian clock protein expression in amacrine and ganglion cells of the mouse retina.
Typical examples of vertical sections of mouse retinas collected between ZT02 and ZT06 and double labeled for one of the following clock proteins: CLOCK (A–C), BMAL1 (A’–C’), NPAS2 (A”–C”), PER1 (A’”–C’”), PER2 (A””–C””), and CRY2 (A’””–C’””) and one of the following protein markers: ChAT (starburst amacrine cells; A), Brn3b (most ganglion cells; B), and eGFP (ipRGCs; C) (see Table 1 for details about the antibodies). The concurrent expression of CLOCK, BMAL1, NPAS2, PER1, PER2, and CRY2 was found in all identified neurons. Note also that all the clock proteins are expressed in the ON starburst amacrine cells whose cell body is located in the ganglion cell layer (GCL), indicating that both GCs and displaced amacrine cells in the GCL express the core components of the mammalian clock. Clock protein expression in ipRGCs was confirmed with the AB-N38 antibody (data not illustrated), although this antibody only labeled M1 and M2 ipRGC subtypes. Some double-labeled retinal neurons are shown (arrows). Abbreviations and bar as in Fig. 3.
Figure 6.
Core circadian clock protein expression in wild-type, coneless and rodeless retinas.
Typical examples of vertical sections of wild-type C57Bl/6J (A–A’””), coneless (B–B’””), and rodeless (C–C’””) retinas immunolabeled for each of the following core clock proteins: CLOCK (A–C), BMAL1 (A’–C’), NPAS2 (A”–C”), PER1 (A’”–C’”), PER2 (A””–C””), and CRY2 (A’””–C’””). Retinal tissue was collected around ZT09. For a given clock protein antibody, confocal settings were adjusted on the brightest picture and the 2 other sections were taken at the same settings. Note that clock protein expression in the outer nuclear layer (ONL) is detected in a few cells in the wild-type retina (vertical arrows) and in most cells in the rodless retina (oblique arrows), but is very weak in the coneless retina. OPL: outer plexiform layer; INL: inner nuclear layer; IPL: inner plexiform layer; GCL: ganglion cell layer. Optical sections 3×0.4 µm. Bar is 10 µm.
Table 4.
COSINOR analysis of core circadian clock component expression in cones and dopaminergic amacrine cells.
Figure 7.
Circadian clock core component expression in mouse rod and cone photoreceptors and dopaminergic amacrine cells under LD and DD conditions.
Typical examples of clock protein immunostaining in cones (A–A””’) and dopaminergic cells (D–D’””) obtained from retinas collected in the middle of the day (ZT06) or subjective day (CT06) or middle of the night (ZT18) or subjective night (CT18) are illustrated for CLOCK (A,D), BMAL1 (A’,D’), NPAS2 (A”,D”), PER1 (A’”,D’”), PER2 (A””,D””), and CRY2 (A’””,D’””). COSINOR regression analysis (cosine curves) was performed only for clock protein levels displaying significant temporal variation (as determined by one-way ANOVA; P<0.05). The results from the COSINOR analysis are shown in Table 4. Note that the expression of all six clock proteins is rhythmic under LD and DD conditions in cones (B–B’””) but arrhythmic under the same conditions in dopaminergic amacrine cells, except for CRY which is rhythmic under both LD and DD conditions (E–E’””). Clock protein expression in rods remained low at any time point under both LD and DD conditions (C–C’””). Each data point represents the mean fluorescence/identified neuron +/− SEM of 5/6 animals (5 pictures from 3–4 sections/animal).