Temporal calcium profiling of specific circadian neurons in freely moving flies

Abstract

There are no general methods for reliably assessing the firing properties or even calcium profiles of specific neurons in freely moving flies. To this end, we adapted a GFP-based calcium reporter to luciferase that was expressed in small subsets of circadian neurons. This Tric-LUC reporter allowed a direct comparison of luciferase activity with locomotor activity, which was assayed in the same flies with video recording. The LUC profile from activity-promoting E cells paralleled evening locomotor activity, and the LUC profile from sleep-promoting glutamatergic DN1s (gDN1s) paralleled daytime sleep. Similar profiles were generated by novel reporters recently identified based on transcription factor activation. As E cell and gDN1 activity is necessary and sufficient for normal evening locomotor activity and daytime sleep profiles, respectively, we suggest that their luciferase profiles reflect their neuronal calcium and in some cases firing profiles in wake-behaving flies.

Keywords: Drosophila; calcium; circadian; optogenetics; sleep.

Fig. 1.

Identifying sleep-promoting and activity-promoting circadian neurons. (A) Schemes of Flybox, which has red and green LEDs to provide optogenetic activation and inhibition, and a camera that can record behavior of flies in a 96-well plate in real time. (Right) Raw and processed images recorded from Flybox. (B) Optogenetic activation of gDN1s promotes persistent sleep. (Left) Expression pattern of the split GAL4 line that labels gDN1s (Spl-gDN1) in one brain hemisphere. (Right) Sleep and activity profile of Spl-gDN1 > CsCrimson flies under optogenetic activation. Error bars represent SEM. n = 24 for each group. (C) Optogenetic activation of E cells results in persistent activity. (Left) Expression pattern of split GAL4 line that labels E cells (Spl-E) in one brain hemisphere. (Right) Sleep and activity pattern of Spl-E cell > CsCrimson flies under optogenetic activation. Error bars represent SEM. n = 23 for each group.

Fig. S1.

Anatomic characterization of Spl-E cell and Spl-gDN1 expression. (A) Representative GFP (green), PDF (red), and PER (magenta) costaining in a brain of Spl-gDN1 > UAS-CsCrimson fly. (B) Representative GFP (green), PDF (red), and PER (magenta) costaining in the brain of an Spl-E cell > UAS-CsCrimson fly. (C) GFP (green) and PDF (red) costaining of Spl-gDN1 > UAS-CsCrimson (Upper) and Spl-E cell > UAS-CsCrimson (Lower) brains. Spl-gDN1 GAL4 labels three to four DN1s in each hemisphere, and Spl-E cell GAL4 labels three LNds as well as the fifth s-LNv in each hemisphere.

Fig. S2.

Effects of optogenetic activation and inhibition on activity and sleep profiles. (A) Locomotor activity profile of Spl-E cells > UAS-CsCrimson (Left, red) and Spl-gDN1 > UAS-CsCrimson (Middle, green) on the baseline day (Upper) and LED day (Lower). (Right) Quantification of 24-h sleep level on the baseline and LED days. The black boxes and white boxes indicate dark and light periods, respectively. The pink boxes denote the red light (637-nm) stimulation window. Genotypes of each group are labeled above the panel. Error bars correspond to SEM. n = 24 for each group. (B) Percentage of daytime sleep change during the LED day compared with the baseline day of E cell and gDN1-inhibited flies. Genotypes of each group are listed below the panel. n = 16 for Spl-gDN1 > UAS-GtACR1 and Spl-E cell > UAS-GtACR1 groups; n = 12 for three control groups. **P < 0.001, one-way ANOVA. Error bars represent SEM.

Fig. 2.

Effect of silencing E cells and gDN1s on sleep profiles. (A) Temperature-induced expression of Kir2.1 in different circadian neurons affects sleep and locomotor activity at different circadian times. (Upper) Sleep profiles of Spl-E cell > Tub-GAL80^ts+UAS- Kir2.1 (green), Tub-GAL80^ts/+; UAS- Kir2.1/+ (blue), and Spl-E cell/+ (orange) at 29 °C. (Lower) Sleep profiles of Spl-DN1 > Tub-GAL80^ts+UAS- Kir2.1 (green), Tub-GAL80^ts/+; UAS- Kir2.1/+ (blue), and Spl-DN1/+ (orange) at 29 °C. (Right) Quantification of relative sleep and activity change during the time window. The dashed box indicates the time window (ZT2-9 for Upper; ZT9-12 for Lower) within which the relative change of sleep and locomotor activity was calculated. n = 16 for each group. **P < 0.001, two-tailed Student’s t test. Error bars represent SEM. (B) Optogenetic inhibition of DN1s by expression of GtACR1 (driven by Clk4.1M-GAL4) reduces daytime sleep (Left), and optogenetic inhibition of E cells (driven by DvPDF-GAL4;PDF-GAL80) increases sleep during the evening (Right). The green box indicates the time window when the 530-nm green LEDs were turned on. n = 16 for each group. Error bars represent SEM. The quantification of experimental and control groups is shown in Fig. S2B. (C) Sleep change profiles of gDN1-inhibited flies and E cell-inhibited flies during LD. (Left) Profile calculated from the data in A. (Right) Profile calculated from the data in B. Red and brown arrows point to the putative peaks of DN1 and E cell neuronal activity in LD. The genotypes used to calculate the profiles are labeled below the panels.

Fig. 3.

Using in vivo calcium reporters to monitor temporal patterns within circadian pacemakers. (A) Schemes of CaLexA-LUC and Tric-LUC calcium reporters. (B) Comparing CaLexA-LUC and Tric-LUC responses to CsChrimson activation. The fold change of luminescence was calculated as the ratio of the luminescence level after CsChrimson activation to the baseline luminescence level. The red shaded box indicates the 2 V (0.59∼0.63 mW/mm²), 10-min, 5-Hz, 627-nm light pulse. The genotypes of each line are shown below. n = 8 for R18H11-GAL4 > Tric-LUC+CsCrimson; n = 15 for R18H11-GAL4 > Tric-LUC; n = 16 for other groups. Shading represents SEM. (C) Comparing the Tric-LUC response between different circadian neurons. The maximum fold changes of luminescence after optogenetic stimulation are plotted. The blue bars indicate GAL4 > Tric-LUC+CsCrimson, and the orange bars indicate GAL4 > Tric-LUC only. The GAL4 lines used in each group and the voltages of LED stimulation (2 V: 0.59∼0.63 mW/mm²; 5 V: 0.9∼1 mW/mm²) are shown below each histogram. n = 16 for each group. **P < 0.001, two-tailed Student’s t test. Error bars represent SEM.

Fig. 4.

Exploring the Tric-LUC patterns under different conditions. (A) The Tric-LUC pattern in the two circadian neuron groups. The normalized LUC levels of Spl-gDN1 > Tric-LUC (red curve, Left) and Spl-E cell > Tric-LUC (blue curve, Middle) from a 2-d recording are plotted. The light and dark periods are indicated by the white and gray backgrounds. The red arrows indicate peaks of LUC activity, and the black arrows point to the troughs of LUC activity. n = 24 for each group. Shading corresponds to SEM. (Right) The ratio of daytime LUC activity divided by nighttime LUC activity. **P < 0.001, two-tailed Student’s t test. Error bars represent SEM. (B) Environment input changes the calcium pattern of the circadian neurons. LUC activity from DvPDF-GAL4 > Tric-LUC flies were recorded in either 12:12 LD cycles (Left) or 16:8 LD cycles (Right). The red arrows point to the evening peak of DvPDF-GAL4 > Tric-LUC flies. n = 16 for each group. Shading corresponds to SEM. (Right) Peak times of the M and E LUC profiles in 12:12 LD (Upper) and 16:8 LD (Lower). **P < 0.001, two-tailed Student’s t test. Error bars correspond to SEM. (C) Real-time unfiltered raw data recordings of Spl-E-cell > Tric-LUC (Upper) and per^S; Spl-E-cell > Tric-LUC flies (Lower). Mean LUC levels and locomotor activities from WT and per^S groups are plotted. Tric-LUC levels (blue) and locomotor activities (orange) were recorded at the same time. The plate was placed into the TopCount NXT recording chamber every 30 min. The period of these flies is shown below the panel. Black boxes and white boxes indicate dark and light periods in LD, respectively. Gray boxes and black boxes indicate subjective day and night in DD, respectively. Red arrows point to the major locomotor activity peaks in DD, and the blue arrows point to the evening activity peaks in LD.

Fig. S3.

Daytime activity/sleep patterns track calcium levels within the two different circadian neuron groups. The red curve represents the real-time Tric-LUC level monitored by the TopCount NXT plate reader at 30-min intervals, and the black and purple curves represent the activity and sleep levels, respectively, recorded simultaneously by a webcam in the 96-well plate format. (Upper) The GAL4 expression patterns used in the experiment. The white and gray boxes below the figures indicate dark and light periods, respectively. A full 12-h light period is shown, followed by the beginning of the dark period. n = 20 for the Spl-E cell > Tric-LUC group (Left); n = 16 for the Spl-gDN1 > Tric-LUC group (Right). Error bars represent SEM.

Fig. 5.

Comparing neuronal activity reporters and calcium reporters within the circadian neurons. (A) The neuronal activity reporters have a different pattern than Tric-LUC in the gDN1s. (Upper) Schematic of the neuronal activity reporters. (Lower) Comparison of the Tric-LUC reporter and the Relish-LUC as well as Lola-LUC reporters in gDN1s. Light and dark periods are indicated by white and gray backgrounds, respectively. n = 24 for the Spl-gDN1 > Tric-LUC group; n = 16 for the other groups. Shading correspond to SEM. (B) Real-time recording of gDN1 LUC and locomotor activity of individual flies. The comparison shows that DN1 LUC activity is negatively associated with fly daytime activity level. The locomotor activity (orange histogram) and the LUC activity (blue curve) of individual CLK4.1m(DN1s) > UAS-FLP,Lola > stop > LUC flies were recorded simultaneously for 4 d in LD. (Upper) Recordings from individual flies with relatively high locomotor activity levels. (Lower) Recordings from individual flies with relatively low locomotor activity levels. Black boxes and white boxes indicate dark and light periods, respectively. (C) The neuronal activity reporter patterns are indistinguishable from the Tric-LUC pattern in E cells. The panels show normalized LUC activity from Spl-E cell > UAS-Tric-LUC, Spl-E cell > UAS-FLP Lola > stop > LUC, and Spl-E cell > UAS-FLP Relish > stop > LUC groups. Light and dark periods are indicated by white and gray backgrounds, respectively. n = 24 for the Spl-E cell > Tric-LUC group; n = 16 for the other groups. Shading corresponds to SEM. (D) The neuronal activity reporter patterns are also indistinguishable from the Tric-LUC pattern in the PDF neurons. The panels show normalized LUC activity from PDF-GAL4 > UAS-Tric-LUC, PDF-GAL4 > UAS-FLP Lola > stop > LUC, and PDF-GAL4 > UAS-FLP Relish > stop > LUC groups. Light and dark periods are indicated by the white and gray backgrounds, respectively. n = 16 for each group. Shading corresponds to SEM.