Circadian regulation of the Na+/K+-ATPase alpha subunit in the visual system is mediated by the pacemaker and by retina photoreceptors in Drosophila melanogaster

Abstract

We investigated the diurnal oscillation in abundance of the catalytic α subunit of the sodium/potassium pump (ATPα) in the brain of Drosophila melanogaster. This rhythm is bimodal and is particularly robust in the glia cells of the lamina, the first optic neuropil. We observed loss of ATPα cycling in lamina glia in behaviourally arrhythmic per(01) and tim(01) mutants and in flies overexpressing the pro-apoptotic gene hid in the PDF-positive clock neurons. Moreover, the rhythm of ATPα abundance was altered in cry(01) and Pdf(0) mutants, in flies with a weakened clock mechanism in retina photoreceptor cells and in those subject to downregulation of the neuropeptide ITP by RNAi. This complex, rhythmic regulation of the α subunit suggests that the sodium/potassium pump may be a key target of the circadian pacemaker to impose daily control on brain activities, such as rhythmic changes in neuronal plasticity, which are best observed in the visual system.

Figure 1. α5 immunolabeling of Na⁺/K⁺-ATPase α-subunit (ATPα) in the optic lobe of CantonS flies at specific time points under LD 12:12.

The intensity of immunofluorescence in the lamina differs at different time points: A – ZT1, B – ZT4, C – ZT13, D – ZT16. The fluorescence signal is maximal in the lamina and in the medulla neuropils.

Figure 2. Rhythmic immunoreactivity of ATPα in wild-type CantonS flies under LD 12:12 (A) and DD (B).

The fluorescence index ± SE is shown as a function of time. Under LD 12∶12 statistically significant differences were detected between ZT1 and ZT4, ZT1 and ZT16, ZT13 and ZT4, ZT13 and ZT16. The fluorescence index was highest at ZT13 and then lowered by 10.4% at ZT1, 63.7% at ZT4 and 82% at ZT16. In DD the fluorescence index was significantly higher during the subjective night (CT16 and CT13), than the subjective day (48.4% reduction at CT1 and 59.8% reduction at CT4). Parametric ANOVA Tukey's test; p<0.05.

Figure 3. Arrhythmic ATPα immunoreactivity in mutants that affect the clock (A) or the viability of clock cells (B).

The fluorescence index ± SE is shown as a function of time (A) tim⁰¹, (B) Pdf-GAL4> UAS-hid flies. There are not statistically significant differences between time points. Parametric ANOVA Tukey's test; p<0.05.

Figure 4. Pattern of ATPα immunoreactivity in cry⁰¹ mutants under LD (A) and DD (B) and in rescue flies under LD (C).

The fluorescence index ± SE is shown as a function of time. (A) cry⁰¹ mutants under LD. ZT1 and ZT4 are significantly different from the other time points. The immunoreactivity of the α subunit was highest at ZT16 and decreased at other time points (ZT13 by 16%, ZT1 by 50.08%, and ZT4 by 56.4%). (B) cry⁰¹ mutants under DD. The pattern of immunoreactivity was similar to LD conditions. The highest level of immunoreactivity was reached at CT16 and decreased by 15.3%, 55.8% and 63.7% at CT13, CT1 and CT4, respectively. (C) cry rescue under LD. Peak immunofluorescence was at ZT1 and then decreased by 70%, 20% and 82% at ZT4, ZT13 and ZT16, respectively. There are statistically significant differences between ZT1 and ZT4, ZT1 and ZT16, ZT13 and ZT4, ZT13 and ZT16. Parametric ANOVA Tukey's test; p<0.05. The two stars symbols indicate statistically significant differences between the experimental strains and CantonS controls at different time points.

Figure 5. Pattern of ATPα immunoreactivity in flies with reduced CRY expression in photoreceptors (gmr-GAL4>UAS-cry-RNAi) (A) and glia (repo-GAL4>UAS-cry-RNAi) (B) under LD.

The fluorescence index ± SE is shown as a function of time. (A) In gmr-GAL4>UAS-cry-RNAi flies the higher levels of immunoreactivity were observed at ZT1 and ZT13. The immunosignal decreased by 66.3% at ZT4 and by 58.3% at ZT16. Statistically significant differences were observed between ZT1 and ZT4, ZT1 and ZT16, ZT13 and ZT4, ZT13 and ZT16. (B) In repo-GAL4>UAS-cry-RNAi flies higher immunosignal was observed at ZT1 and ZT13 and then decreased by about 30% in ZT4 and ZT16. There were statistically significant differences between ZT1 and ZT4, ZT1 and ZT16, ZT13 and ZT4, ZT13 and ZT16. Parametric ANOVA Tukey's test; p<0.05. The two stars symbols indicate statistically significant differences between the experimental strains and CantonS controls at different time points.

Figure 6. Pattern of ATPα immunoreactivity in flies with reduced clock activity in photoreceptors ( gmr-GAL4>UAS-Δcyc) (A) and glia (repo-GAL4>UAS-Δcyc) (B).

The fluorescence index ± SE is shown as a function of time. (A) In gmr-GAL4>UAS-Δcyc flies immunofluorescence levels were high at ZT1, ZT13 and ZT16 and lowered by about 50% at ZT4. There were statistically significant differences between ZT4 and the other time points. (B) In repo-GAL4>UAS-Δcyc flies the immunofluorescence index was higher at ZT1 and ZT13 and lowered by 45.1% at ZT4 and by 56.4% at ZT16. There were statistically significant differences between ZT1 and ZT4, ZT1 and ZT16, ZT13 and ZT4, ZT13 and ZT16. Parametric ANOVA Tukey's test; p<0.05. The two stars symbols indicate statistically significant differences between the experimental strains and CantonS controls at different time points.

Figure 7. Pattern of ATPα immunoreactivity in Pdf⁰ mutants under LD (A) and DD (B) and in rescue flies under LD (C).

The fluorescence index ± SE is shown as a function of time. (A, B) In Pdf⁰ mutants under both LD and DD conditions, the highest levels of immunofluorescence were observed at ZT13. At other time points the levels lowered by about 30% in LD and by about 50% in DD. There were statistically significant differences between ZT13 and the other time points under both LD and DD conditions. (C) In Pdf rescue flies the highest immunofluorescence level was at ZT13 that lowered by 15.6% at ZT1, by 57.6% at ZT4 and by 66.1% at ZT16. Statistically significant differences were seen between ZT1 and ZT4, ZT1 and ZT16, ZT13 and ZT4, ZT13 and ZT16. Parametric ANOVA Tukey's test; p<0.05. The two stars symbols indicate statistically significant differences between the experimental strains and CantonS controls at different time points.

Figure 8. Pattern of ATPα immunoreactivity in flies with reduced ITP in CRY expressing cells under LD (A) and DD (B).

The fluorescence index ± SE is shown as a function of time. (A) Under LD conditions the immunofluorescence index was high at ZT1, ZT4 and ZT13. It was lowered by 47.7% at ZT16. There were statistically significant differences between ZT16 and the other time points. (B) Under DD high immunosignal levels were observed at CT4 and CT13. There was a reduction of about 60% at the other time points There were statistically significant differences between CT1 and CT4, CT1 and CT13, CT16 and CT4, CT16 and CT13. Parametric ANOVA Tukey's test; p<0.05. The two stars symbols indicate statistically significant differences between the experimental strains and CantonS controls at different time points.