Figure 1.
Generation of the DmIh null mutant.
(A) Diagram of the DmIh gene showing the position of the two piggyBac transposon insertion sites. The dotted line at the bottom scheme corresponds to the deleted region upon recombination. Black arrows indicate the position of the primers used to amplify the newly formed piggyBac element (amplicon size 7.5 Kb). Gray arrows indicate the position of the primers inside the deleted region (amplicon size 242 bp). (B) PCR results show lack of amplification of the presumptive deleted region in the Ih− line, further confirmed with the positive amplification of the newly formed piggyBac element.
Figure 2.
Circadian levels of dopamine in control flies.
Dopamine and activity was measured in 6 to 8 days old flies at day 3 or 4 in each condition. Error bars indicate SEM. (A) In LD conditions, control flies show daily cycling of dopamine amount with two peaks at daytime and reduced levels at nighttime. Bars at the top indicate significant difference between two points (*p<0.05, **p<0.01, post hoc Bonferroni test). (B) In this condition the total daily activity plot (n = 32) reveals the typical bimodal pattern of activity with two maximums at the moment of lights on (ZT0) and of lights off (ZT12). (C) When transferred to constant darkness, the cycling of dopamine in control flies is attenuated. (D) Total daily activity plot (n = 47) in DD conditions shows the characteristic sustained activity plateau during the subjective day (CT0-CT12).
Figure 3.
Ih gene is expressed in dopaminergic neurons.
(A) Confocal projection of adult brain showing dopaminergic neurons labeled with GFP using the Tyrosine Hydroxilase Gal4 driver (THG4;UAS:GFP). D, dorsal, L, lateral, OE, oesophagus, OL, optic lobe. (B) RT-PCR amplification of Ih (112 bp) and ple (133 bp) RNAs from 200 isolated dopaminergic and non-dopaminergic neurons sorted by Fluorescence Activated Cell Sorting (FACS). Image shows amplification of Ih RNA in dopaminergic, ple-expressing neurons. Failure to PCR-amplify the neuronal ple RNA isoform in non-dopaminergic neurons (GFP negative cells) confirms that cells have been correctly sorted. Asterisks in lanes 1, 2, and 5 point to nonspecific bands, which were obtained in the negative controls (DmIh mutant brain-RNA extract for Ih –lane 1- and GFP-negative cell-RNA extract for ple –lane 5-).
Figure 4.
Lack of Ih current alters dopamine levels.
Dopamine and activity was measured in 6 to8 days old flies at day 3 or 4 in each condition. Error bars indicate SEM. (A) In LD conditions, DmIh mutant flies (black dotted line) lose the characteristic cyclic pattern of dopamine at daytime, and show a notable increase in dopamine levels at night time. To facilitate comparison, dopamine in control flies is displayed with a gray dotted line. (B) Correspondingly, total activity plot of mutant flies (n = 31; bottom black actogram) reveals subtle changes in the circadian locomotor activity when compared with control flies (top gray actogram): activity peak at lights on looks wider and blunted, while peak at lights off ends abruptly. In addition, mutant flies show increased activity during the night compared to control flies. (C) In DD conditions, the level of dopamine in DmIh mutant flies (black dotted line) increases drastically throughout the 24 hour period when compared to control flies (gray dotted line). (D) Total daily activity plot of mutant flies (n = 53; bottom black actogram) shows that activity during the second half of the subjective day is reduced compared to control flies (top gray actogram).
Table 1.
Rhythmicity parameters.
Figure 5.
DmIh mutant flies lose the characteristic bimodal activity pattern in LD.
Average actograms show the typical bimodal pattern of activity in LD in most of the control flies (98.4%, n = 53; top) and in 58.9% of the DmIh mutant flies (average of 33 flies; bottom “Ih− Average with a normal pattern”). However, the rest of the DmIh mutant flies (41.1%, n = 23) display an altered pattern characterized by a plateau of sustained activity during the light period (middle “Ih− Average with a plateau pattern”). Mutant flies also show a (plateau of?) sustained activity during the night.
Figure 6.
Loss of Ih current affects sleep consolidation.
(A) Daily time course (30 min interval) of the amount of sleep in 6 to 8 day-old males of control (n = 64) and DmIh mutant (n = 64) genotypes in LD conditions. White and gray areas indicate light and dark periods, respectively. Data points represent mean ± SEM. (B) Rest∶activity parameters of both genotypes in the light period. Mutant flies are hypoactive as infer from the decreased beam crossing counts per active minute. These flies have the same amount of total sleep during the day, but with significantly more sleep bouts of shorter duration. (C) At nighttime total activity of mutant flies is not different from controls, but beam crossing counts per active minute are significantly higher in mutant flies. Total sleep is the same for both genotypes, but, as in the light phase, DmIh mutants have more sleep bouts of a shorter duration. Error bars represent two SEMs. Asterisks denote significant differences based on the proper test performed by FlySiesta software (*p<0.05; **p<0.01; ***p<0.001; n.s., not significant).
Figure 7.
Decreasing dopamine levels by 3IY in DmIh null mutant rescues sleep phenotype.
Sleep parameters of control and mutants flies under control conditions (flies fed with gelatin solution) or drug treatment (flies fed with 10 mg/ml 3YI in gelatin solution). Drug treatment considerably increases sleep consolidation in control flies (gray vs white bars) both at day and night. In DmIh mutant flies, drug treatment rescues the sleep fragmentation phenotype, especially at night time (black vs dark gray bars), when the number of sleep periods and their duration show values similar to non-treated control flies (see text for details). Error bars represent two SEMs. Asterisks denote significant differences based on the proper test performed by FlySiesta software (*p<0.05; **p<0.01; n.s., not significant).
Figure 8.
The absence of Ih current shortens lifespan but does not affect resistance to oxidative stress.
(A) Lifespan determination of DmIh mutant males (black line, n = 234) compared to controls (gray line, n = 145). Survival curves of the two genotypes are significantly different (Mantel-Cox Statistic = 32.281, df = 1, p<0.0001). (B) Resistance to oxidative stress of DmIh mutant males (black line, n = 511) is not different from the controls (gray line, n = 384).
Figure 9.
In constant dark, DmIh mutant flies shorten the circadian period.
Average (top) and single fly representative (middle and bottom) double-plotted actograms of control and DmIh mutant flies in DD conditions. Flies were entrained for three days in LD conditions prior to being released to DD. The first day of each actogram corresponds to the last day in LD. Mutant flies included rhythmic flies (Ih-R), weakly rhythmic flies (Ih-WR), and arrhythmic flies (Ih-A) (see Table 1 for circadian parameters). Rhythmic mutant flies display a shorter period and a failure to maintain the activity plateau during the subjective day.
Figure 10.
Lack of Ih current does not disrupt PDF clock output.
Ih null mutants show normal circadian release of pigment dispersing factor (PDF) in DD conditions. (A) Anti-PDF staining of brains showing the LNv cells and their dorsal projections. Dotted line shows the area selected for analyses. (B) PDF immunostaining in the dorsal projections shows normal oscillation of this factor in wild type (wt) and Ih null mutants (Ih−), with high levels in the early day (CT2) and low levels in the early night (CT14). (C) Quantification of PDF immunoflorescence (Integrated Density) in wt and Ih− at CT2 and CT14 (Error bars indicate standard deviation) There are no differences between genotypes at any time point (t13 = −1.121, p = 0.283 for CT2; t11 = −1.794, p = 0.100 for CT14).