High-Frequency Neuronal Bursting is Essential for Circadian and Sleep Behaviors in Drosophila

I_h is important for high-frequency bursting of LNvs. A, Representative traces of whole-cell patch clamp recordings of lLNvs of control (pdf-RFP, top) and I_h homozygote mutant genotypes (I_h^f03355; pdf-RFP, bottom). B, Representative trace of a recording of a sLNv control (pdf-RFP) in whole-cell patch clamp configuration. C, Representative traces of cell-attached recordings of sLNvs of control (pdf-RFP, top) and I_h homozygote mutant genotypes (I_h^f03355; pdf-RFP, bottom). D, Box plot showing the median and interquartile range of the bursting frequency quantification of lLNvs and sLNvs of control (pdf-RFP) and I_h homozygote mutant genotypes (I_h^f03355; pdf-RFP). All quantifications were done at exactly 23 min postdissection. Different letters indicate significant differences (p < 0.05) after a one-way ANOVA with Tukey's test for means comparisons. n: lLNvs_CONTROL = 14, lLNvs_Ihf03355 = 12, sLNvs_CONTROL = 10, sLNvs_Ihf03355 = 7.

Mutation of I_h does not significantly affect other electrophysiological parameters of LNvs. A, No statistical significant differences were found in action potential firing rate of lLNvs when comparing control (pdf-RFP) and I_h homozygote mutant genotypes (I_h^f03355; pdf-RFP). B, No statistical significant differences were found in membrane potential (measured as the trough between bursts) of lLNvs when comparing control (pdf-RFP) and I_h homozygote mutant genotypes (I_h^f03355; pdf-RFP). C, No statistical significant differences were found in action potential firing rate of sLNvs when comparing control (pdf-RFP) and I_h homozygote mutant genotypes (I_h^f03355; pdf-RFP). Membrane potential was not quantified in sLNvs as recordings were made in cell-attached configuration, and it is not possible to measure this parameter under this configuration. All quantifications were done at exactly 23 min postdissection. In all cases, p > 0.05 after Student's t test. n: lLNvs_CONTROL = 7, lLNvs_Ihf03355 = 8, sLNvs_CONTROL = 7, sLNvs_Ihf03355 = 6.

sLNvs bursting depends on synaptic inputs. As has been demonstrated before for lLNvs (Muraro and Ceriani, 2015), we show here that sLNvs bursting frequency also decays as a function of the time ex vivo. A, The number of bursts in the initial minute of recording of nine individual control (pdf-RFP) sLNvs recorded at different times postdissection is shown. For the late points the preparation was left in the chamber on purpose before establishing the recording. B, Shows the bursting frequency of five individual control (pdf-RFP) sLNvs where the recordings were long enough to appreciate the decay in this parameter as a function of time postdissection not only as a population as in A, but as individual cells. C, Shows 30-s windows of cell-attached recording of a representative sLNv (sLNv3 in B) at different times postdissection. From top to bottom, the 30 s starting at 25, 35, 45, and 55 min postdissection are shown. At the beginning of the recording, all action potentials are organized in bursts. As time passes, action potentials become less organized in bursts, going through a phase of bursting-tonic firing and becoming purely tonic toward the end. This figure shows that the fact that in A, B the neurons have a tendency toward the cero bursting frequency does not mean that the neurons are not firing, but that they are doing so in a tonic mode. D, Box plot showing the median and interquartile range of the bursting frequency quantification of lLNvs and sLNvs of control (pdf-RFP) and I_h homozygote mutant genotypes (I_h^f03355; pdf-RFP), these quantifications were done at exactly 33 min postdissection. Different letters indicate significant differences (p < 0.05) after a one-way ANOVA with Tukey's test for means comparisons. n: lLNvs_CONTROL = 7, lLNvs_Ihf03355 = 8, sLNvs_CONTROL = 8, sLNvs_Ihf03355 = 5.

I_h downregulation affects PDF levels and structural plasticity. A, Confocal images of representative sLNvs dorsal projections of individual flies of control (pdfGS/+) and I_h downregulation (pdfGS>I_h^RNAi) at day (top) and night (bottom) showing their PDF content. Flies were kept in LD 12:12 at 25°C for 7 d in food containing RU486. Brains were dissected at ZT02 and ZT14 and standard anti-PDF immunofluorescence detection was performed. The bar indicates 10 µm. B, PDF quantitation of the sLNvs dorsal projections for the four conditions mentioned before. Circles represent day time, squares, night time; empty symbols are the control genotype (pdfGS/+) and filled symbols, the experimental one (pdfGS>I_h^RNAi). Different letters indicate significant differences, analysis included a two-way ANOVA (genotype and time of day; F_(3,150) = 18.58 p < 0.0001 with Tukey's post hoc test, α = 0.05), n = 35–43 per group. C, Confocal images of sLNvs projections illustrating their complexity at ZT02 and ZT14 for both in the control and I_h downregulated genotypes. Procedure as in A but with immunofluorescence against GFP. The bar indicates 10 µm. D, Complexity quantitation was asses by Sholl analysis (ImageJ) corroborated by visual inspection of each picture. Symbols as in B, analysis included a two-way ANOVA (F_(3,123) = 4.24 p < 0.01 with Tukey's post hoc test, α = 0.05). In B and D the mean ± SEM are shown. Different letters indicate significant differences. n = 34–38 per group.

PDF transport is affected on I_h manipulation. A, B, Confocal images of representative sLNv projections (A) and somas (B) of individual flies of pdfGS/+ (control), pdfGS>I_h^RNAi (Ih), pdfGS>UAS-pdf (pdfOX) and pdfGS>UAS-pdf, I_h^RNAi (pdfOX, Ih) at day (left) and night (right) showing their PDF content. Flies were kept in LD 12:12 at 25°C for 7 d in food containing RU486. Brains were dissected at ZT02 and ZT14 and standard anti-PDF immunofluorescence detection was performed. Bars indicate 10 µm. C, E, PDF quantitation of the sLNv dorsal projections (C) or somas (E) for the four genotypes mentioned before. Circles represent day time, squares, night time; each color is a different genotype. Asterisks represent significant statistical differences. For the projections, a non-parametric ANOVA Kruskal–Wallis test and Dunn's comparisons test showed differences among the two time points in control, pdfOX and pdfOX, Ih groups but not in Ih group (Kruskal–Wallis statistic_(8,196) = 71.95, p < 0.0001, n = 18–28). Immunoreactivity from somas was analyzed with one-way ANOVA and Sidak's multiple comparisons test and revealed differences between the two time points in every genotype except pdfOX, although in Ih and pdfOX, Ih showed differences in the anti-phase direction compared with the control, ANOVA F_(7,120) = 10.95, p < 0.0001, n = 10–22 (each point is the average of three to four cell somas for one hemi-brain of an individual fly). D, F, Morning to evening PDF level ratios for axonal projections (D) or somas (F). G, Locomotor behavior under constant darkness of the same genotypes as before. Experiments were performed as in Figure 1 and Table 3. The rhythmicity measured as power-significance was analyzed by Kruskal–Wallis test followed by Dunn's comparisons test and showed a significant reduction of power-significance in Ih and pdfOX, Ih compared with control and pdfOX as indicated by different letters (Kruskal–Wallis statistic_(4,31) = 31.40, p < 0.0001, n = 65–72). H, Free running period values were analyzed as well. The same type of analysis reveals a reduction of tau in pdfOX, Ih compared with all the other genotypes as indicated by a different letter (Kruskal–Wallis statistic_(4,31) = 38.28, p < 0.0001, n = 45–58). In C, G and H the median ± confidence intervals are shown. In E the mean ± Standard Deviation is shown. ns, not significant.

Genetic manipulations of I_h increase sleep. A, E, I, Sleep ethograms for the indicated genotypes, quantification of the relative amount of sleep every 30 min as a function of the time of the day (starting at ZT = 0, when lights are turned on) and its standard deviation (shadowed area). Black and white bars at the bottom represent daytime (white) and nighttime (black). B, F, J, Boxplots showing the total amount of sleep minutes for each genotype. C, G, K, Boxplots showing the average duration of sleep episodes for each genotype. D, H, L, Boxplots showing the total amount of sleep episodes for each genotype. For all the boxplots, different letters indicate significant differences (p < 0.05) after non parametric Kruskal–Wallis statistical analysis with multiple comparisons (p adjustment method = BH). Box represents the median and interquartile range of each parameter. For more information on sleep parameters see Table 5.

Relative contribution of specific clusters to sleep control. Boxplots show daytime and nighttime sleep duration on RNAi-mediated I_h downregulation using c929-Gal4 (top panel), R6-Gal4 (middle panel), and pdf-Gal4 (bottom panel), along with their genetic controls. Adult-specific manipulations performed using the TARGET system are shown in the left and correspond to the second day at the permissive temperature of 30°C. Constitutive genetic manipulations are shown on the right, all performed at the standard temperature of 25°C. Different letters indicate significant differences (p < 0.05) after non parametric Kruskal–Wallis statistical analysis with multiple comparisons (p adjustment method = BH). Box represents the median and interquartile range of each parameter. For more detailed information on sleep parameters see Table 5.