Age associated arrhythmicity is dependent on ena function.

(A) Rescue of ENA function on the PDF circuit restores behavioral rhythmicity in aged flies. Recombinant pdf-gal4, ena^rev carrying one copy of UAS-ena were indistinguishable from control UAS-ena. Representative double plotted actograms for aged (24–28d) flies are shown. Overexpression of ENA in young flies do not affect locomotor activity rhythms (data not shown). (B) The percentage of rhythmicity for aged flies for each strain is shown. pdf-gal4, ena^rev/++ is significantly different from both UAS-ena/+ (control) and pdf-gal4, ena^rev/UAS-ena (*** p<0.001). (C) Representative actograms of young (3d) and aged (21d) flies carrying one or two copies of ena^rev, along with the transheterozygotes ena^rev/ena^GC5. Both homozygous ena^rev and ena^rev/ena^GC5 exhibit a decline on rhythm strength. (D) Summary of behavioral data for flies of the indicated genotypes. Control ena^rev/+ flies stay rhythmic throughout lifespan. Aged ena^rev is significantly different from its younger counterpart (* p<0.05). Both aged ena^rev and ena^rev/ena^GC5 are different from old ena^rev/+ (* p<0.05). Experiments summarized in B and D were repeated at least 3 times. (E) Whole mount brain immunofluorescense of 10 day-old adult y w flies were stained with a specific antibody against ENA. A general staining of neuropils in the adult brain was observed. Single confocal planes (2 µm thick) are shown at two depths (8 and 22 µm) to highlight different brain areas. Some of the neuropils labeled with ENA are the outer (o me) and inner medulla (i me), lobula (lo) and lobula plate (lo p) within the optic lobe, the protocerebral bridge (pr br) in the central complex as well as other regions in the protocerebrum such as the lateral horn (l ho). Other structures, as the protolateral deutocerebrum (p l deu), superior lateral protocerebrum (s l pr), the peduncles of the mushroom body (pe), pars intercerebralis (pars in), suboesophageal ganglion (su oes g) and oesophagus (oes) are also shown in the figure. (F) ena levels are reduced in ena^rev mutants compared to the control y w. Images were taken with the same confocal settings for direct comparison; projections of 2.3 µm depth are shown. ENA immunohistochemistry was repeated at least three times. (G) RT-PCR analysis was performed in adult ena^rev, ena^rev/+ and control total RNA. The ratio between ena and actin expression levels for each genotype is shown. Quantification of RNA levels showed significant changes in ena^rev homozygous (* p<0.05) whereas a minor (non significant) decrease was seen in ena^rev/+ heterozygous when compared to the y w control line. The experiment was repeated three times employing independent RNA preparations.