Nov 3, 2016 - tests were performed using Prism (GraphPad Software Inc.), and the figures were arranged in Illustrator (Adobe Systems Inc.). relative mRNA.
Supporting Information Buhl et al. 10.1073/pnas.1606547113 SI Materials and Methods Animals. Flies were raised with a 12-h:12-h LD cycle on standard Drosophila medium (0.7% agar, 1.0% soy flour, 8.0% polenta/ maize, 1.8% yeast, 8.0% malt extract, 4.0% molasses, 0.8% propionic acid, 2.3% nipagen) at 25 °C and were collected ∼3–5 d post eclosion. The following flies used in this study were described previously or obtained from the Bloomington, Vienna, or NIG fly stock center: gl60j (15), norpAP41 (37), HdcJK910 (38), and cry02 (56). For wild-type control recordings either Pdf-RFP (8) or Pdf-gal4 (57) flies crossed to uas-mCD8-RFP (BL27392) or uas-mCherry (BL52268) flies were used. For the behavioral controls wildtype (y w) and tim-gal4:16 (58) flies were used. Experimental genotypes including uas-qsm#5 (qsmOX) (16), uas-qsmRNAi (VDRC15394) and uas-qsmRNAi(2) (qsm RNAi ) (16), uasShawWT12B (ShawOX) (21), uas-ShawRNAi (ShawRNAi) (21), and uas-CG31547RNAi (NKCCRNAi, NIG2509R-2) were crossed to tim-gal4:16 for behavioral tests and to either Pdf-gal4; uasmCD8-RFP or Pdf-gal4; uas-mCherry flies for electrophysiological recordings. qsm105, a gal4 line inserted in the qsm first intron that reduces qsm expression (16), was used to test for behavioral interactions of qsm with Shaw and NKCC. For respective tests of physiological interaction, Pdf-RFP; tim-gal4:16 uas-qsmRNAi(2) flies were crossed to either uas-mCD8-GFP (BL30001) or uasShawWT12B and uas-CG31547RNAi flies. Note that in both sets of electrophysiology experiments (i.e., those involving Pdf-gal4 and tim-gal4), the ratio of gal4 to uas constructs was always 1:2. To test for Cry-dependent effects, Pdf-RFP was crossed into the cry02 background (56). The light sensitivity of wild-type flies is influenced by the naturally occurring s-tim/ls-tim polymorphism (35, 36). ls-tim flies are less light sensitive than s-tim flies, and in conjunction with jetlag mutants ls-tim elicits LL rhythmicity (35, 36). Therefore all fly stocks used in this study were genotyped for timeless and jetlag polymorphisms as described (35, 36) and, if necessary, were crossed into the ls-tim background to allow comparison between genotypes (all stocks were jetlag+). To reveal the effect of the s/ls-tim polymorphism, selected genotypes were also analyzed in an s-tim background (Table S1). Generation of Transgenic Flies. A flag-HA–tagged uas construct of CG31547-PB (UFO03679) was obtained from the Drosophila Genetics Resource Center (59) (www.fruitfly.org/EST/proteomics. shtml). uas-CG31547-flgHA was integrated into the ZH-attP-86Fb landing site using the ΦC31 integrase system to generate NKCCOX (52). The eye-expressed 3xP3-RFP cassette present in the landing site was eliminated by Cre-mediated excision as described (52). RNA Isolation and RT-qPCR. To test the efficiency of NKCCRNAi, 5- to
10-d-old flies were frozen at ZT 2, and 20 heads of each genotype were collected over dry ice. The total RNA was extracted using an RNeasy kit (QIAGEN) according to the manufacturer’s instructions and was finally eluted in RNase-free water and stored at −80 °C. cDNA synthesis was performed with the Reverse Transcription Reagents Kit (Applied Biosystems) in 20-μL reactions using 1 μg of total RNA. To verify the level of NKCC mRNA expression, dilutions of cDNA were used for PCR with rp49 primers, followed by DNA electrophoresis on 2% agarose gels to visualize the PCR products. Taqman probes for NKCC (catalog no. 4351372; Thermo Fisher) were applied to determine the amount of mRNA. Real-time assays were performed using an ABI GeneAMP PCR System 9700 using the standard program, and threshold cycle (CT) values were applied to determine the amount of RNA in each genotype. The relative concentrations were Buhl et al. www.pnas.org/cgi/content/short/1606547113
calculated using the comparative CT method, and RPL32 was used as control. Behavior. Analysis of the locomotor activity of 4- to 5-d-old male flies was performed using the Drosophila Activity Monitor System (DAM2; Trikinetics Inc.) with individual flies in recording tubes containing food (2% agar, 4% sucrose). The DAM monitors and an environmental monitor (DEnM; Trikinetics Inc.) were located inside a light- and temperature-controlled incubator (Percival Scientific Inc.) in which the fly’s activity was monitored for 4 d in a 12-h:12-h LD cycle followed by 7 d under LL (10 μW/cm2) at 25 °C. Plotting of behavioral activity, period calculations, and the determination of rhythmic statistics were performed using either a signal-processing tool-box (53) implemented in Matlab (MathWorks) or the ImageJ (https://imagej.nih.gov/ij/) plug-in ActogramJ (54). LL rhythmicity was determined on the basis of a rhythmic statistics value >1.5 and was classified according to the period length of individual flies as being either bimodal and circadian (∼12 h or ∼24 h, respectively) or ultradian (
tim
N
KC
CR
tim
N
Ai
>
>
+
+
0
Fig. S1. RT-qPCR verification of NKCCRNAi knockdown (related to Figs. 1–5). qPCR analysis of relative mRNA levels in whole heads showing the efficiency of RNAi-mediated knockdown of NKCC (tim > NKCCRNAi) compared with controls (either gal4 or uas alone). Bars represent means; whiskers represent minimum and maximum.
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12 13
13 0
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0 time (h)
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0
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0 time (h)
6
Fig. S2. LL behavior of flies carrying the s-tim allele (related to Fig.1 and Table S1). Double-plotted actograms of exemplary individual flies of the indicated genotypes recorded for the first 4 d in LD (gray, lights off; white, lights on) followed by 9 d of constant dim light.
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A
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C
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60 s Fig. S3. Whole-cell recording preparation and response of l-LNvs to acute light (related to Figs. 2, 4, and 5 and Table S2). (A) Whole-brain preparation showing the Pdf-RFP–labeled l-LNvs for recording (the band in the middle is a nylon thread holding the brain in the recording chamber). (B) Detail of the l-LNvs with the recording electrode (viewed from below). (C) In wild-type control flies green light (555 nm) had no effect on wild-type l-LNvs either in the daytime or at nighttime. (D) cry02 l-LNvs did not respond to blue light (470 nm) with a change of firing either in the daytime or at nighttime. (E–H) l-LNvs with altered Shaw or NKCC levels (Pdf-gal4) showed no differential response in recordings taken during the daytime (Left) or at nighttime (Right). Note that the scale of the graphs in G differs from the scale used in the graphs in the other panels. In each panel the traces show an example of a current-clamp recording of an l-LNv for 1 min before, 30 s during, and 2.5 min after exposure to light (indicated by the green or blue bar). The graphs show a quantification of the light response from multiple recordings. N.B.: In the genotypes not responding to acute light (qsmOX, ShawOX, NKCCRNAi), 18 neurons did not spike or spiked only very occasionally; the neurons that did spike regularly did not change their firing rate. However, some neurons (n = 8) showed a startle response with briefly increased or decreased spiking (e.g., left panels in F and Fig. 4C). The examples shown are from occasionally spiking neurons. In the graphs the solid line shows the mean, and the gray shading shows the SD. The number of recorded neurons of each genotype is indicated.
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Table S1. Circadian behavior of flies in LL Genotype
tim allele
% rhythmic
Period, h (SD)
Rhythmic statistics
n
tim tim tim tim tim tim tim tim
> > > > > > > >
+ (wild-type) qsmOX qsmRNAi qsmRNAi(2) ShawOX ShawRNAi NKCCOX NKCCRNAi
ls ls ls ls ls ls ls ls
4.5 75.0 89.7 98.1 72.7 91.3 83.0 73.9
— 13.4 (1.8) 26.6 (1.6) 27.6 (2.0) 4.2 (0.6) 12.5 (0.8) 28.9 (0.8) 13.3 (1.1)
— 2.2 (0.7) 2.4 (0.6) 3.3 (1.0) * 2.0 (0.7) 2.4 (1.0) 2.3 (0.8)
44 32 29 104 22 23 53 23
tim tim tim tim tim
> > > > >
qsmOX qsmRNAi qsmRNAi(2) ShawRNAi NKCCOX
s s s s s
34.0 31.4 40.0 58.3 26.6
30.3 30.2 24.6 29.8 34.1
2.1 2.5 2.3 2.5 2.4
s ls s s/ls
96.6 0 0 16.6
ls ls ls ls ls
36.8 2.2 54.2 81.3 2.2
cry02 gl60j norpAP41 HdcJK910 qsm105 qsm105 qsm105 qsm105 qsm105
> > > > >
+ ShawOX ShawRNAi NKCCOX NKCCRNAi
(5.6) (5.4) (2.5) (5.4) (3.9)
(0.4) (1.2) (0.6) (1.2) (1.2)
50 35 20 24 30
24.3 (1.3) – – 25.6 (4.3)
3.1 (1.0) – – 2.3 (0.8)
29 22 32 36
27.4 (1.9) – 26.6 (2.9) 29.1 (1.2) –
2.4 (0.8) – 2.3 (0.7) 3.3 (0.8) –
19 45 24 16 45
This table is related to Fig. 1 and Fig. S2. For each genotype, the percentage of the overall rhythmic flies, the mean (SD) of the prevalent rhythmic category, the rhythmic statistics, and the number of flies, n, are given. *Rhythmic statistics values could not be calculated for periods of this length using the signal-processing tool-box in MatLab. Instead, rhythmicity for each fly was deemed significant based on periodogram analysis using Actogram J with a P value cut-off set to 0.001. Each period was subsequently verified by manual inspection of individual actograms.
Table S2. Circadian behavior of flies in DD Genotype
tim allele
% rhythmic
> > > > > > >
ls ls ls ls ls ls ls
93.8 100 100 13 97 85.7 81.3
tim tim tim tim tim tim tim
+ (wild-type) qsmOX qsmRNAi ShawOX* ShawRNAi* NKCCOX NKCCRNAi
Period, h (SD) 23.6 24.1 24.1 24.4 24.3 24.2 24.6
(0.4) (0.2) (0.2) (0.4) (0.1) (0.3) (0.5)
Rhythmic statistics 2.8 3.9 4.0 2.5 4.8 2.9 2.7
(0.8) (0.4) (0.4) (0.2) (0.3) (0.6) (0.7)
n 16 8 8 39 32 14 16
This table is related to Fig. 1. For each genotype, the percentage of the overall rhythmic flies, the mean (SD) of the prevalent rhythmic category, the rhythmic statistics, and the number of flies, n, are given. *Shaw data are taken from ref. 21.
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Table S3. Physiological parameters (RMP, SFR) and acute light response (Fon − Foff) in daytime and nighttime Day or night
RMP, mV (SD)
SFR, Hz (SD)
Fon − Foff, Hz (SD)
n
Pdf > + (wild-type)
Day Night
−51.2 (4.6) −57.7 (2.7)
2.27 (0.90) 0.60 (0.41)
−0.23 (0.84) 1.61 (1.17)
15 11
Pdf > qsmOX
Day Night
−57.6 (5.3) −56.0 (2.6)
0.54 (0.46) 0.74 (0.73)
0.17 (0.22) 0.06 (0.52)
10 9
Pdf > qsmRNAi
Day Night
−51.6 (4.5) −52.9 (2.5)
1.56 (0.67) 2.15 (0.56)
1.50 (1.31) 1.41 (0.98)
11 10
Pdf > ShawOX
Day Night
−61.1 (6.3) −58.3 (4.3)
0.34 (0.47) 0.35 (0.39)
−0.02 (0.17) −0.06 (0.14)
9 6
Pdf > ShawRNAi
Day Night
−51.0 (3.4) −51.7 (3.3)
1.93 (0.82) 1.76 (0.68)
1.56 (1.61) 1.21 (0.69)
10 6
Pdf > NKCCOX
Day Night
−51.7 (4.1) −50.7 (2.7)
2.94 (1.66) 5.30 (1.74)
1.52 (0.99) 1.20 (1.14)
7 6
Pdf > NKCCRNAi
Day Night
−61.7 (7.1) −61.3 (6.4)
0.56 (0.87) 0.51 (0.61)
0.11 (0.15) −0.02 (0.21)
10 7
cry02
Day Night
−53.3 (5.1) −56.5 (2.4)
2.47 (1.11) 0.63 (0.53)
−0.06 (0.38) 0.11 (0.19)
4 4
tim > qsmRNAi/GFP
Day Night
−54.9 (2.7) −55.8 (2.1)
5.78 (0.84) 5.70 (1.26)
– –
9 8
tim > qsmRNAi/ShawOX
Day Night
−61.2 (3.1) −59.0 (2.5)
0.47 (0.57) 0.90 (0.84)
– –
9 7
tim > qsmRNAi/NKCCRNAi
Day Night
−57.6 (1.8) −55.7 (3.5)
1.90 (0.72) 2.56 (0.56)
– –
7 7
Genotype
This table is related to Figs. 2, 3, and 5. For each parameter, data are shown as mean (SD). n, the number of measured neurons.
Table S4. Sustained current density at +100 mV Genotype Pdf Pdf Pdf Pdf Pdf Pdf
> > > > > >
+ (wild-type) day + (wild-type) night ShawOX ShawRNAi qsmOX qsmRNAi
Current, pA/pF (SD) 37.1 69.9 81.9 46.1 82.5 45.8
(12.0) (21.6) (11.7) (16.7) (19.2) (12.4)
n 6 8 6 6 5 5
This table is related to Fig. 2. Data are shown as mean (SD). n, the number of measured neurons.
Table S5. EGABA Genotype Pdf Pdf Pdf Pdf Pdf Pdf
> > > > > >
+ (wild-type) day + (wild-type) night NKCCOX NKCCRNAi qsmOX qsmRNAi
EGABA, mV (SD) −63.8 −74.6 −59.0 −74.8 −72.5 −55.6
(4.3) (4.2) (5.0) (5.3) (7.2) (4.9)
n 7 8 5 5 5 5
This table is related to Fig. 2. Data are shown as mean (SD), n, the number of measured neurons.
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