Drosophila ebony activity is required in glia for the circadian regulation of locomotor activity

Abstract

Previous studies suggest that glia may be required for normal circadian behavior, but glial factors required for rhythmicity have not been identified in any system. We show here that a circadian rhythm in Drosophila Ebony (N-beta-alanyl-biogenic amine synthetase) abundance can be visualized in adult glia and that glial expression of Ebony rescues the altered circadian behavior of ebony mutants. We demonstrate that molecular oscillator function and clock neuron output are normal in ebony mutants, verifying a role for Ebony downstream of the clock. Surprisingly, the ebony oscillation persists in flies lacking PDF neuropeptide, indicating it is regulated by an autonomous glial oscillator or another neuronal factor. The proximity of Ebony-containing glia to aminergic neurons and genetic interaction results suggest a function in dopaminergic signaling. We thus suggest a model for ebony function wherein Ebony glia participate in the clock control of dopaminergic function and the orchestration of circadian activity rhythms.

Figure 1. Ebony gene expression is under clock control

(A) Ebony mRNA cycles in abundance in a light:dark (LD) cycle and in constant darkness (DD). Total RNA samples were prepared from heads of control yw and yw;tim⁰¹ flies collected every 4 hours in LD and DD. Real-time quantitative RT-PCR was performed on RNA samples using primers specific for ebony (see Methods). Relative abundance indicates the amount of ebony transcript normalized to rp49 mRNA abundance which is known to remain constant throughout the cycle. Ebony RNA cycles in yw but not in tim⁰¹(A) flies. (B) Ebony protein abundance changes in a diurnal manner in wild-type (yw) brains. Hand-dissected brains were collected every 4 hours in an LD cycle. Protein blots were immunostained with rabbit anti-MAPK antibodies. Each gel lane contained approximately 5μg protein. The graph shows the ratio of Ebony to MAPK protein as a function of time of day. The horizontal bars in panels A and B indicate the light:dark schedules.

Figure 2. Ebony protein is localized exclusively to glia of the larval and adult brains

(A-F) Ebony can be detected with a cytoplasmic distribution in cells of the wild-type (Canton-S) adult brain (A), the adult ventral nervous system (B), and the larval brain and ventral nervous systems (C), but is not specifically detected in the e¹ mutant (E, F; note the lack of cell body staining in the mutant). Panel D shows co-localization of Ebony and Repo proteins in glial cells of the optic lobes. Repo can be detected in every Ebony-containing glial cell. Staining with the neuronal marker ELAV did not detect any Ebony-positive cells in brain tissues (data not shown).

Figure 3. The Ebony protein oscillation can be detected by immunostaining in wild-type brains but is damped or eliminated in the tim⁰¹ mutant

(A) Ebony immunofluorescence at two times of the cycle in wild-type brains. Brains were dissected at ZT2 and ZT21 from flies maintained in LD or at CT3.5 and CT21 in DD conditions. Ebony fluorescence was observed to be more intense during the day (or subjective day) than at night (Arrows indicate sites of prominent rhythmicity) (B) Ebony fluorescence intensity is weaker in tim⁰¹ than in the wild type during the day, and rhythmicity is damped in the mutant. (C) Average normalized fluorescence (pixel) intensity for different brain regions (optic lobes and protocerebrum) in wild type and tim⁰¹. In all cases, Ebony fluorescence was normalized to Repo antibody staining intensity. * p<0.01, ** p<0.05 for differences in fluorescence intensity at two times of day in wild type (two-tailed student’s t-test). Differences were not significant for tim⁰¹.

Figure 4. Expression of ebony+ in glia is sufficient to rescue the rhythm phenotype of the e¹ mutant. Overexpression of ebony+ in a wild-type background has no obvious effects

(A) Actograms for representative control flies, e¹ mutants, rescued mutants, and individuals overexpressing Ebony in an e+ background. Data are from the DD portions of the records starting with the first day after lights-off. The horizontal bars beneath records indicate subjective day and night. The overexpression of Ebony in an e¹ mutant background, using a glial cell driver (repo-Gal4), rescued circadian behavior. Overexpression of Ebony in a wild-type background had no effect on rhythmicity. All flies were in a w¹¹¹⁸ genetic background. Repo > ebony+ = w¹¹¹⁸; repo-Gal4, e¹/UAS-ebony+, e¹. (B) Body color phenotypes of various genotypes. i) w¹¹¹⁸. ii) w¹¹¹⁸; e¹/e¹. iii) w¹¹¹⁸; actin-Gal4/+; UAS-ebony+, e¹/e¹. iv) w¹¹¹⁸; repo-Gal4, e¹/UAS-ebony+, e¹.

Figure 5. Many Ebony-containing cells stain positive for PER or are close to PER-containing neurons

(A-C) Double labeling for Ebony and PER shows that Ebony glia are adjacent to the sLNv, LNd, DN1, and DN3 neurons. (D, E) In the optic medulla, Ebony protein is localized to PER-containing glia (Red = PER, Green = Ebony); these two panels show different regions within the optic medulla. TIM protein was also co-localized with Ebony in many optic lobe glial cells (data not shown). (F-I) Localization of Ebony relative to PDF. (F) pdf-Gal4-driven expression of UAS-mCD8GFP in the adult brain. Ebony antibody was used to visualize the localization of Ebony protein relative to PDF-expressing cells and their projections. (G-H) Ebony-containing cells are in close proximity to PDF cell projections in the dorsal brain (G, arrow shows one Ebony cell) or in the medulla (H). (I) a high magnification picture of the region in panel H indicated by the dotted rectangle (Red = Ebony, Green= PDF).

Figure 6. Ebony glia are located near aminergic neurons

Ebony-containing cells are in close proximity to TH (Tyrosine Hydroxylase)-positive (A) or serotonergic (B) neurons of the larval and adult brains. In panel A, green = Ebony, Red = TH; in panel B, green = Ebony, Red = serotonin). Panel A shows a whole mount of the entire larval brain. Panel B shows the larval ventral nervous system (VNS) minus the brain lobes. The proximity of Ebony glia and 5-HT neurons is more easily visualized in the VNS. Insets show high magnification views of Ebony glia in proximity to TH-or 5-HT-positive neuronal processes of the adult brain.

Figure 7. Per/tim RNA and protein cycling are normal in the e¹ mutant

(A-B) Quantitative RT-PCR shows that per and tim RNAs cycle in abundance in head tissues of wild-type (C-S) and e¹ mutant flies. Values for per or tim RNA abundance were normalized to rp49 values (see Methods). Each curve represents 5 independent measures of RNA abundance from two independent fly head preparations (two measures from one, three measures from the other). Error bars indicate standard error. (C-D) TIM protein oscillates in abundance in C-S and e¹ heads. TIM protein amounts are normalized to MAPK values for all timepoints. Each curve in panel D represents 3 independent measures of protein abundance from two independent fly head extracts. Error bars indicate standard error.

Figure 8. e¹ suppresses the hyperactivity of the DAT^fmn mutant

Mean daily activity for e¹, DAT ^fmn and the double mutant. Plots show population averages (w n=32, w; e¹ n=32, w; DAT^fmn n=63, w; DAT^fmn; e¹ n=70). Flies were entrained to LD 12:12 at 23°C for 5-6 days and then transferred to constant darkness (DD) at the same temperature for an additional 10-14 days. Average activity level per 30 minute bin was calculated for each genotype. Error bars indicate standard error.

Figure 9. Hypothetical model for the control of the Ebony molecular rhythm and the regulation of adult locomotor activity

The model postulates that ebony expression may be regulated by autonomous glial oscillators and by output from clock neurons. In turn, rhythms in Ebony activity are postulated to control the excitability of dopaminergic or other neurons that regulate locomotor activity, perhaps by rhythmic production of NBAD. Presumably, dopamine (DA) and NBAD are actively synthesized during the daytime, as a consequence of rhythmic TH and Ebony activities, both of which are high during the day. According to the model, DA is released from dopaminergic terminals and taken up by ebony-containing glial cells. Ebony (BAS) activity then conjugates dopamine to β-alanine to produce N-β-alanine-dopamine (NBAD) which may be released from glia to regulate neuronal excitability and the activation of circuits controlling locomotor activity.