Neuronal and Glial Clocks Underlying Structural Remodeling of Pacemaker Neurons in Drosophila

Abstract

A number of years ago we reported that ventral Lateral Neurons (LNvs), which are essential in the control of rest-activity cycles in Drosophila, undergo circadian remodeling of their axonal projections. This structural plasticity gives rise to changes in the degree of connectivity, which could provide a means of transmitting time of day information. Thus far, work from different laboratories has shown that circadian remodeling of adult projections relies on activity-dependent and -independent mechanisms. In terms of clock- dependent mechanisms, several neuronal types undergoing circadian remodeling hinted to a differential effect of clock genes; while per mutants exhibited poorly developed axonal terminals giving rise to low complexity arbors, tim mutants displayed a characteristic hyper branching phenotype, suggesting these genes could be playing additional roles to those ascribed to core clock function. To shed light onto this possibility we altered clock gene levels through RNAi- mediated downregulation and expression of a dominant negative form exclusively in the adult LNvs. These experiments confirmed that the LNv clock is necessary to drive the remodeling process. We next explored the contribution of glia to the structural plasticity of the small LNvs through acute disruption of their internal clock. Interestingly, impaired glial clocks also abolished circadian structural remodeling, without affecting other clock-controlled outputs. Taken together our data shows that both neuronal and glial clocks are recruited to define the architecture of the LNv projections along the day, thus enabling a precise reconfiguration of the circadian network.

Keywords: LNvs; cell autonomous clocks; circadian remodeling; structural plasticity.

Figure 1

Impaired LNv clocks result in disrupted locomotor activity patterns. (A) Representative double-plotted actograms and their respective periodograms of the different genotypes: UAS-dcr2, UAS-per^RNAiI (#40878), UAS-tim^RNAiI (#29583), and UAS-cyc^DN under the control of pdf-Gal4. Locomotor activity of individual flies was recorded for 4 days under 12:12 LD cycles and then transferred to DD (gray area) for 9 additional days. In the actograms, white bars represent day, black bars represent night. For every fly actogram, periodograms of the free-running rhythm in DD are shown (bottom). Rhythmic behavior of a typical male control is shown (orange). per^RNAi (in blue), tim^RNAi (in pink), and cyc^DN (in green) expressing animals are shown. All genetic manipulations gave rise to a largely arrhythmic locomotor behavior. (B) Percentage of rhythmicity. Data represents at least three independent experiments; over 44 flies were analyzed. (C) Validation of the effectiveness of the RNAis employed. Both UAS-per^RNAi and UAS-tim^RNAi were expressed under tim-Gal4. Levels are normalized to rpl49. Both tim and per mRNA levels are reduced compared to their respective control. Student's t-test showed a significant difference in levels of expression. Triple asterisks (^***) indicate significant differences with p < 0.001. Three independent experiments were performed.

Figure 2

Different clock components trigger dampened molecular oscillations. For each gene, the ratio describing higher/lower mRNA levels is plotted (that is, CT14/CT2 for per, tim and cyc; and CT2/CT14 for clk). Levels are normalized to the reference gene rpl49. Statistical analysis was performed comparing individual transcript levels (indicated by a dashed line). The genotypes analyzed are as follows: control (orange), per^RNAiI (blue), tim^RNAiI (pink), and cyc^DN (green), under tim-Gal4. Different letters indicate statistically significant differences with a p < 0.05 (One-way ANOVA with a Tukey post-hoc test). Three independent experiments were performed.

Figure 3

Circadian structural plasticity is differentially altered by clock genes. (A) Schematic diagram illustrating the standard protocol. “Veh” and “RU” stand for “vehicle” and “RU486” containing fly food. (B) Representative confocal images of GFP immunoreactivity at the dorsal protocerebrum at the early subjective day (CT2, gray bar) and early subjective night (CT14, black bar) during the 3rd day of constant darkness (DD3). (C) Quantitation of total axonal crosses. Control flies display circadian structural remodeling of axonal terminals while animals with a deregulated clock show no differences across the day. Data represents the average of 3 experiments; a minimum of 27 brains were analyzed per CT/genotype. Different letters indicate statistically different treatments with a p < 0.05 (Two-way ANOVA with a Tukey post-hoc test, n = 8–10, N = 3). Controls in vehicle are not different from controls in RU containing food (Depetris-Chauvin et al., 2011). (D) Quantitation of PDF immunoreactivity at the dorsal protocerebrum at CT2 and CT14 on DD3. For a more direct comparison, PDF levels are shown as the ratio between CT2 and CT14. Control flies (orange), exhibit circadian oscillation of PDF levels, while different clock deregulation genotypes were significantly different from the control. Different letters indicate statistical differences with a p < 0.05 (Kruskal–Wallis One-way ANOVA, followed by Conover Post-hoc test, n = 8–10, N = 3).

Figure 4

Clocks in glia are required for circadian remodeling of neuronal terminals. (A) Schematic diagram illustrating the standard protocol; the restrictive condition is highlighted in light-blue (23°C), and the permissive condition is shown in orange (30°C). (B) Representative confocal images of dsRed immunoreactivity at the dorsal protocerebrum of flies containing UAS-cyc^DN under repo-Gal4;tub-Gal80^TS; pdf RED enables visualization of the axonal terminals. Brains were dissected at the early subjective day (CT2, gray bar) and early subjective night (CT14, black bar) during the 2nd day of constant darkness (DD2), which corresponds to the 3rd day of permissive condition (30°C). Control flies (always maintained at 23°C) are indicated in light-blue. (C) Quantitation of total axonal crosses of repo-Gal4;tub-Gal80^TS > cyc^DN. Control flies (kept at 23°C) display circadian structural remodeling of axonal terminals while animals induced at 30°C show no differences along the day. Data represents the average of 3 experiments; a minimum of 27 brains were analyzed per CT/genotype. Different letters indicate statistical differences with a p < 0.05 (Two-way ANOVA with a Tukey post-hoc test, n = 8–10, N = 3). (D) Quantitation of PDF immunoreactivity at the dorsal protocerebrum from brains dissected at CT2 and CT14 on DD3. Control flies (23°C), exhibit circadian oscillation of PDF levels; those expressing cyc^DN at 30°C were not significantly different from controls. Same letters indicate no statistically different conditions (p > 0.05) (Kruskal–Wallis One-way ANOVA, followed by a Conover post-hoc test, n = 8–10, N = 2). Data represents the average of 2 experiments; a minimum of 16 brains were analyzed per CT/genotype.