Acute control of the sleep switch in Drosophila reveals a role for gap junctions in regulating behavioral responsiveness
- The University of Queensland, Australia
- King's College London, United Kingdom
- University of Cambridge, United Kingdom
- Washington University School of Medicine, United States
Figures
Figure 1 with 1 supplement
The sleep switch modulates behavioral responsiveness.
(A) Flies in glass tubes were filmed from above and DART was used to track activity and to test behavioral responsiveness using a mechanical vibration. (B) Sleep duration was measured using 5 min …
Figure 1—figure supplement 1
Correlation between responsiveness and sleep duration following activation of the dFB in various Gal4 drivers during the day (yellow) and night (grey).
(A) Left, correlation between the peak response speed (mm/s) and sleep duration (min/hr) for R23E10-Gal4/+control flies (n = 49) during the day (yellow R=- 0.082, p=0.57) and night (gray R = −0.371, …
Figure 2 with 3 supplements
Behavioral effects of acutely activating R23E10 neurons.
(A–D) Effects on sleep and responsiveness following the activation of R23E10 neurons (blue, UAS-CsChrimson/+;R23E10-Gal4/+ with ATR, n = 102) compared to those in control flies (black, no ATR …
Figure 2—figure supplement 1
Measuring sleep intensity.
(A) Schematic showing fly activity prior to and in response to a mechanical stimulus (orange dashed line). Flies can be inactive (grey shading) or active (green shading) prior to stimulus delivery. …
Figure 2—figure supplement 2
Acute effects in awake flies.
(A) Mean speed of all flies for the minute prior to stimulation during acute CsChrimson activation. n.s. represents not significantly different in a t-test. (B) Average stimulus response for …
Figure 2—figure supplement 3
1 Hz optogenetic activation of the dFB.
(A)-(D) Effects on sleep and responsiveness following 1 Hz activation of R23E10 neurons (blue, UAS-CsChrimson/+;R23E10-Gal4/+ with ATR, n = 115) compared to control flies (black, no ATR feeding, n = …
Figure 3 with 2 supplements
Electrophysiological effects of acutely activating R23E10 neurons.
(A) Setup for recording in vivo adult Drosophila electrophysiology with whole-cell patch clamp (orange). (B) dFB neuron schematic showing whole-cell recordings targeted to R23E10-Gal4 cell bodies. …
Figure 3—figure supplement 1
1Hz stimulation.
(A) Example traces (left) of a CsChrimson-expressing spiking cell (top), a CsChrimson-expressing non-spiking cell (middle), and a non-CsChrimson-expressing cell (bottom) when exposed to 1 Hz light …
Figure 3—figure supplement 2
Whole-cell patch physiology in wildtype and INX6 knockdown flies.
Top panels show combined traces (yellow lines indicate mean values) for all 120 pulses for all CsChrimson-expressing R23E10 neurons that showed the presence of evoked spikes (red arrows) in response …
Figure 4 with 2 supplements
INX6 dFB localization.
(A) INX6 antibody staining (red) in wildtype Canton-S flies at 20x (top) and 60x (middle). White arrow indicates the location of the dFB and white dashes outline it. Staining using the secondary …
Figure 4—figure supplement 1
Acute synaptic manipulations.
(A–B) Mean day sleep duration (min/hr), mean peak responsiveness (mm/s) and sleep intensity (% responding) at 23C and 31C for flies expressing UAS-Syntaxin3-69 (green, n = 71) or UAS-ShibireTS …
Figure 4—figure supplement 2
INX6 in dFB neurons.
(A) INX6 antibody labeling shows reactivity in the dFB (outlined) and in PI cells (arrow). (B) R23E10-Gal4 cell bodies (green) showed no overlap with INX6 staining. Scale bar: 25 μm. (C) Images …
Figure 5 with 2 supplements
INX6 effects on behavior.
(A) Mean sleep duration (min/hr) and (B) mean peak responsiveness (mm/s) and sleep intensity (% responding) for R23E10-Gal4/+;UAS-INX6-RNAi/+ (red, n = 85) compared to R23E10-Gal4/+ (gray, n = 83) …
Figure 5—figure supplement 1
INX6 RNAi effectiveness.
(A) qRT-PCR shows relative amount of inx6 mRNA normalized to the housekeeping gene Actin in pan-neuronal knockdown using elav-Gal4. **p<0.01, t-test. (B) Average intensity ratio (± SEM) in the dFB …
Figure 5—figure supplement 2
Behavioral effects of acute downregulation of INX6 in adult flies.
(A) Mean sleep duration (min/hr), (B) peak responsiveness (mm/s) (C) and sleep intensity (% responding) for tubpGAL80TS/+;R23E10-GAL4/UAS-INX6-RNAi (red, n = 34) compared to negative tubpGAL80TS/+;UA…
Figure 6 with 1 supplement
Electrophysiological effects of INX6 knockdown in activated R23E10 neurons.
(A) In vivo adult Drosophila electrophysiology recording setup with a whole-cell patch clamp (orange) targeting R23E10 cell bodies and a local field potential (blue) electrode targeting the dFB. (B) …
Figure 6—figure supplement 1
Combined traces (yellow lines indicate mean values) for all 120 pulses for each of the recorded CsChrimson-expressing R23E10 neurons with INX6 knockdown.
All neurons responded to 5 ms light stimulus with burst firing in the absence of stereotypical subsequent firing in the 1,000 ms that follows. See also Figure 3—figure supplement 2.
Figure 7
Behavioral effects of INX6 knockdown in activated R23E10 neurons.
(A–D) Three different behavioral conditions comparing flies with R23E10 neurons that can be activated (UAS-CsChrimson/+; R23E10-Gal4/+with ATR, blue, n = 50) with flies where R23E10 neurons cannot …
Author response image 1
Acute inhibition of the dFB.
(A) Example average activity trace (speed ± SEM) of ATR-fed R23E10/GtACR-3M flies responding to stimuli 15 minutes apart (gray dashed lines); 5min GtACR activation (green shading), with 1min prior …
Videos
Video 1
3D reconstruction of dye coupling experiment.
https://doi.org/10.7554/eLife.37105.014Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Genetic reagent (D. melanogaster) | R23E10-Gal4 | Bloomington | RRID:BDSC_49032 | |
| Genetic reagent (D. melanogaster) | C5-Gal4 | doi: 10.1002/ ssscne.22284 | Paul Shaw Lab | |
| Genetic reagent (D. melanogaster) | 104y-Gal4 | Paul Shaw Lab | ||
| Genetic reagent (D. melanogaster) | UAS-CsChrimson | doi: 10.1038/ nmeth.2836 | provided by Vivek Jarayaman Lab | |
| Genetic reagent (D. melanogaster) | UAS-2eGFP | Bloomington | RRID:BDSC_32186 | |
| Genetic reagent (D. melanogaster) | UAS-NaChBac | Bloomington | RRID:BDSC_9469 | |
| Genetic reagent (D. melanogaster) | UAS-syntaxin3-69 | Fly Base | FBal0092503 | |
| Genetic reagent (D. melanogaster) | UAS-shibireTS | Paul Shaw Lab | Gene ID: 45928 | |
| Genetic reagent (D. melanogaster) | tubpGAL80ts | Bloomington | RRID:BDSC_7108 | |
| Genetic reagent (D. melanogaster) | UAS-INX6 RNAi | VDRC | v8638 | Provided by the Chia-Lin Wu Lab |
| Antibody | Rabbit anti -INX6 | Provided by the Chia-Lin Wu Lab | 1:1,000 | |
| Antibody | Goat anti-mouse AlexaFluor488 | Invitrogen | Catalog # A-10680 | 1:200 |
| Antibody | Goat anti-rabbit AlexaFluor568 | Invitrogen | Catalog # A-11011 | 1:200 |
| Antibody | Goat anti-rabbit AlexaFluor647 | Invitrogen | Catalog # A-21244 | 1:200 |
| Antibody | Mouse anti-NC82 | DSHB | AB_2314866 | 1:10 |
| Antibody | Goat anti-rabbit AlexaFluor488 | Invitrogen | Catalog # A-11008 | 1:200 |
| Chemical compound, drug | Neurobiotin | Vector Labs | Cat. No: SP-1120 | |
| Software, algorithm | DART | bfklab | http://www.bfklab.com/ | |
| Software, algorithm | MATLAB code | This paper | 142faca | https://github.com/melvynyap/gap-junction-sleep-control (copy archived at https://github.com/elifesciences-publications/gap-junction-sleep-control) |
| Chemical compound, drug | All-trans retinal | SIGMA-Aldrich | SID 24899355 | |
| Chemical compound, drug | Vectashield | Vector Labs | Cat. No: H-1000 | |
| Chemical compound, drug | Streptavidin | Invitrogen | Catalog number: S32357 | 1:200 |
Additional files
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Transparent reporting form
- https://doi.org/10.7554/eLife.37105.021
