Cholecystokinin receptor type A are involved in the circadian rhythm of the mouse retina

Abstract

The retina is the only organ projecting external light to the suprachiasmatic nucleus. Cholecystokinin receptor type A (Cckar/Cckar) is one of the essential factors for light reception in retinal cells. As there was a lack of literature on the matter, we aimed to elucidate the cause of the time-dependent phase change in clock gene expression. We found that Cckar mRNA expression in retinal cells exhibited diurnal variations. The rhythm of expression of the clock gene Per1/Per2 in retinal cells was altered in Cckar ^-/- mice. The light sensitivity of retinal cells was evaluated in wild-type mice, which showed c-Fos was activated in the ganglion cell layer more than in the inner granular layer. This increase in the number of c-Fos-positive cells was suppressed by lorglumide, a Cckar antagonist. Treatment of rat retina primary cells with lorglumide suppressed Per2 transcription, which was altered in a time-dependent manner relative to the Per2 expression. Light irradiation studies in Cckar ^-/- mice did not exhibit an increase in Period expression in the suprachiasmatic nucleus. These results indicate that Cckar is among the factors that regulate the cycle of clock genes on the retina. Cckar knockout attenuates the light responsiveness of suprachiasmatic nucleus and reduces the expression amplitude of Period genes in the retina. Thus, Cckar may contribute to entrainment of the light environment and maintenance of the expression cycle of Period gene, which is one of the core clock genes.

Graphical abstract

Fig. 1

Influence of Cckar^−/−mice on the amplitude of clock gene expression in the retina. (A) Temporal-expression profiles of Cckar mRNA in the retina of wild-type mice. Each value represents the mean with S.E.M. of results for six mice. There were significant time-dependent variations in Cckar mRNA levels in wild-type mice (P < 0.01; cosinor method). (B, C) Temporal-expression profiles of Per1 mRNA in the retina of wild-type (B) and Cckar^−/− mice (C). (D, E) Temporal-expression profiles of Per2 mRNA in the retina of wild-type (D) and Cckar^−/− mice (E). Each value represents the mean with S.E.M. of results for six mice. The probe list used for quantification is described in Table 1. P-values in the graph were the result of the cosinor method.

Fig. 2

Light-induced alterations in clock gene expression in the SCN of wild-type and Cckar^−/−mice. (A) Influence of light intensity on the number of c-fos positive cells in the suprachiasmatic nucleus (SCN) of wild-type and Cckar^−/− mice. The number of c-fos positive cells did not significantly change with light intensity. Each value represents the mean with S.E.M. of results for six to eight mice (wild-type: six, Cckar^−/−: eight). (B, C) Light-induced changes in Per1 mRNA (B) and Per2 mRNA (C) expression in the SCN of wild-type and Cckar^−/− mice. Each value represents the mean with S.E.M. of results for eight mice. The probe list used for quantification is described in Table 1. *P < 0.05, ***P < 0.001; significant difference between the two groups (two-way ANOVA with Tukey's post-hoc test).

Fig. 3

Light-induced c-fos positive cell expression in the retina of mice with lorglumide. (A) Microscopic images of c-fos-positive cells in the mouse retina; arrowheads in the GCL indicate c-fos-positive cells. A 200 μm wide region was randomly selected, and the number of cFos-positive cells expressed in GCL and INL of that region was counted. (B, C) Effect of lorglumide on light-induced c-fos-positive cells in GCL (B) and INL (C). Each value represents the mean with S.E.M. of results for six mice. **P < 0.01, ***P < 0.001; significant difference between the two groups (two-way ANOVA with Tukey's post-hoc test). GCL: ganglion cell layer, INL: inner granular layer.

Fig. 4

Influence of CCK receptor ligands on Per2 expression activation. (A) Effects of CCK-8s and lorglumide on Per2 mRNA expression. Each value represents the mean with SEM of results for four samples. **P < 0.01; significant difference between the two groups; #P < 0.05; significant difference from 0 h at corresponding groups (two-way ANOVA with Tukey's post-hoc test). (B) Temporal expression profiles of the Per2 mRNA in rat retinal cells after DEX treatment with DMSO or CCK-8s. Each value represents the mean with S.E.M. of results for three samples. The mean of 20 h Per2 expression levels in DMSO group were set to 1. (C) Temporal expression profiles of the Per2 mRNA in rat retinal cells after DEX treatment with DMSO or lorglumide. Each value represents the mean with S.E.M. of results for three samples. The mean of 20 h Per2 expression levels in DMSO group were set to 1. The results for the DMSO-treated groups in Fig. 4B and C are represented identically. The primer list used for quantification is described in Table 2. **P < 0.01, ***P < 0.001; significant difference between the two groups at the corresponding time (two-way ANOVA with Tukey's post-hoc test). The results of the analysis by the Cosinor method are listed in Table 3. DMSO: Dimethyl sulfoxide, CCK-8s: Cholecystokinin Octapeptide, DEX: Dexamethasone.