Supplemental Information. Sleep Drive Is Encoded by Neural. Plastic Changes in a Dedicated Circuit. Sha Liu, Qili Liu, Masashi Tabuchi, and Mark N.

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1 Cell, Volume 165 Supplemental Information Sleep Drive Is Encoded by Neural Plastic Changes in a Dedicated Circuit Sha Liu, Qili Liu, Masashi Tabuchi, and Mark N. Wu

2 Supplemental Experimental Procedures Fly Strains Flies were maintained on standard food containing molasses, cornmeal, and yeast at room temperature. UAS-IVS-Syn21-GFP-p10, UAS-myrGCaMP5, CaLexA (LexAop-CD2::GFP; UASmLexA-VP16-NFAT, LexAop-CD8::GFP-2A-CD8::GFP), LexAop2-TNT::HA, UAS-IP3R-RNAi1 UAS-IP3R-RNAi2, UAS-EGFP-L10a, UAS-dsNR1 and UAS-dsNR2 flies, were obtained from Drs. Gerald Rubin, Troy Littleton, Jing Wang, Chi-hon Lee, Jean-Rene Martin, Rob Jackson, and Chia-Lin Wu. UAS-CD4-tdTomato (#35837), UAS-TNT (#28838), STaR (UAS-FLP, brp(frt.stop)v5-2a-lexa-vp16, #55751), UAS-GCaMP6s (#42746), and the Rubin Janelia Farm Gal4 lines were obtained from the Bloomington Drosophila Stock Center. The R58H05- DBD and R30G03-AD transgenic lines were generated using standard techniques (Rainbow Transgenics), after subcloning their respective enhancer regions into pbpzpgal4dbduw or pbpp65adzpuw. Female flies were used for all experiments. Additional Sleep Methods For UAS-dTrpA1 experiments, flies were raised at 22 C and 1 day of baseline sleep was recorded at 22 C. Flies were then shifted to 29 C for 12 hrs during the night (ZT12-ZT24), in order to activate the neurons. Arousal threshold was measured at the indicated time by providing stimuli of different intensity and duration using mechanical stimulation. For mechanical stimulation, a vortexer mounting plate and multi-tube vortexer (Trikinetics) were used to apply 0.1g-, 0.5g- or 1.2g-force for 1 sec to the flies. Flies that were inactive for 5 min before a stimulus and exhibited beam crossings within 3 min after the mechanical stimulus were identified as aroused. Sleep propensity was measured by examining the % flies falling asleep within 5 minutes after being aroused, and also assessing sleep latency after flies were aroused. Sleep latency after arousal was defined as the time from arousal to the first sleep bout. Data from flies that were aroused by moderate stimuli (0.5g mechanical stimulation) were analyzed.

3 Immunostaining Immunostaining of whole-mount brains was performed as previously described (Liu et al., 2014). Brains were fixed in 4% PFA for ~40 mins. After washing in PBST (PBS+0.3% Triton X- 100), brain samples were incubated with rabbit anti-gfp (Invitrogen) at 1:1000, mouse anti-gfp (Invitrogen) at 1:500, mouse anti-v5 (Invitrogen) at 1:400, mouse nc82 (Development Studies Hybridoma Bank) at 1:50, rabbit anti-dnr2 (Wu et al., 2007) at 1:1000 at 4 C for 72 hours. The following secondary antibodies were used: Alexa 488 anti-rabbit (Invitrogen, 1:1000), Alexa 488 anti-mouse (Invitrogen, 1:500), Alexa 568 anti-rabbit (Invitrogen, 1:1000) and Alexa 568 antimouse (Invitrogen, 1:500). Brain samples were incubated with secondary antibodies overnight at 4 C. Images were obtained on a Zeiss LSM700 confocal microscopy with 1 µm thick sections under 25x or 63x magnification. Nomenclature The sleep-drive neurons identified in this study, which innervate the most anterior ring of the EB, have recently been referred to as R2 (Lin et al., 2013) and EB A (Wolff et al., 2015) ring neurons. It should be noted that other ring neurons have also previously been named R2 neurons, such as the neurons in the C42 driver (Renn et al., 1999) or the EB1 driver (Young and Armstrong, 2010), which are distinct from the ring neurons in this study. Driver Screen 505 Rubin/Janelia Farm Gal4 drivers were manually selected and obtained from the Bloomington Drosophila Stock Center. The criteria for selection were a sparse expression pattern in the brain, and weak or absent expression in the thoracic ganglia. These drivers were crossed to UAS-dTrpA1 flies. At least 8 female flies for each Rubin-Gal4>UAS-dTrpA1 genotype were assessed in the primary screen. To calculate Sleep during neural activation, the sleep amount for each Gal4 driver during dtrpa1 activation at 29 C was measured (ZT12-ZT24) and subtracted from the averaged sleep amount during the same period from all drivers (sleep mean ), and then divided by sleep mean. For Sleep after neural activation, the difference between daytime sleep amount (ZT0-12) after dtrpa1 activation and daytime sleep amount (ZT0-12) of the previous day before dtrpa1 activation (sleep difference ) was calculated. To calculate Sleep after neural activation, sleep difference for each driver was subtracted from the

4 mean sleep difference for all drivers (sleep meandifference ), and then divided by sleep meandifference. Thus, Sleep after neural activation considers both the sleep from all drivers and withingenotype changes, while Sleep before neural activation only considers sleep from all drivers. Scatterplot of Sleep during neural activation vs Sleep after neural activation of all 505 drivers was plotted, and a linear regression analysis was performed by a least squares approach using Prism 5 (Graphpad). To identify drivers that induced sleep after dtrpa1 activation without sleep loss during activation, we selected those drivers with the largest residual values (2.5%) that also exhibited a Sleep during neural activation value 0 (the 8 magenta dots in Figure 1A). Brain registration and averaging High-resolution images of the 8 drivers denoted by magenta in Figure 1A were downloaded from Fly Light ( (Jenett et al., 2012). Using IGSRegistrationTools (Jefferis et al., 2007), a standard reference brain was generated by averaging 17 co-registered nc82 stained brains (randomly selected, and not among in the 505 drivers) from the Fly Light image database. Images of the 8 drivers were registered using IGSRegistrationTools to this standard reference brain using the nc82 channel. Anti-GFP signal from the 8 registered brains were averaged using a MATLAB-based program to generate a heat map. Video measurement of behavior R69F08-Gal4>UAS-dTrpA1 were loaded into DAM (Trikinetics) locomotor tubes. After 12 hours dtrpa1 activation at 29 C from ZT12-ZT24, 2 hr video recordings were made from ZT0 to ZT2 using a Logitech Pro 9000 web camera. The time individual animals spent moving and grooming were manually scored. Feeding behavior was scored when flies were immobile on the food. Functional connectivity measurements R69F08-Gal4>UAS-P2X2; R72G06-LexA>LexAop-GCaMP6s and control flies (UAS-P2X2; R72G06-LexA>LexAop-GCaMP6s) were used to perform functional connectivity experiments as previously described (Yao et al., 2012). In brief, fly brains were dissected in the Adulthemolymph like saline (AHLS) containing 2mM Ca 2+, 108mM NaCl, 5mM KCl, 8.2mM MgCl 2, 2mM CaCl 2, 4mM NaHCO 3, 1mM NaH 2 PO 4, 5mM HEPES, 10mM Sucrose, 5mM Trehalose,

5 ph 7.5. Then, brains were transferred to a custom-made chamber with AHLS. Brains were allowed to settle in the AHLS for ~5 mins for optimum baseline fluorescence stabilization. Perfusion flow was established over the brain with a gravity-fed perfusion system (Warner instrument VC-6). Brains were imaged by a Zeiss AX10 fluorescence microscope under 40x objective lens. Regions of interest (ROIs) were selected over the cell body region of ExFl2 neurons. GCaMP signals were recorded at 2 Hz for 5 min. 5 mm ATP was delivered during the second minute to the brains by switching perfusion flow from AHLS to AHLS with 5mM ATP. The average intensity of the ROIs from the first 30 sec of the recording were used as the F 0. ΔF/F 0 of each frame were calculated by (F n -F 0 )/F 0, where F n is the raw intensity of ROIs of each frame. Electrophysiological recordings Brains were removed and dissected in a Drosophila physiological saline solution (101 mm NaCl, 3 mm KCl, 1 mm CaCl 2, 4 mm MgCl 2, 1.25mM NaH 2 PO 4, 20.7 mm NaHCO 3, and 5 mm glucose; ph 7.2), which was pre-bubbled with 95% O 2 and 5% CO 2. To better visualize the recording site, the glial sheath surrounding the brain was focally and carefully removed after treating with an enzymatic cocktail, collagenase (0.5 mg/ml) and dispase (1.5 mg/ml), at 22 C for 3-4 min and cleaning with a small stream of saline pressure-ejected from a large diameter pipette using a 1 ml syringe. In addition, prior to recording, cell surfaces were cleaned with saline pressure-ejected from a small diameter pipette, using a 1 ml syringe connected to the pipette holder. The preparation was immobilized on the bottom of a recording chamber using a custom-made platinum anchor. The recording chamber was placed on an X-Y stage platform (PP ; Scientifica, UK), and the cell bodies of R2 neurons were visualized with GFP fluorescence on a fixed-stage upright microscope (BX51WI; Olympus, Japan) and viewed with a 40 water immersion objective lens (LUMPlanFl, NA: 0.8, Olympus). As the recording electrode approached the R2 neurons, visualization was achieved by IR-DIC optics. Whole-cell recordings using the perforated patch configuration of the patch-clamp technique were performed at ZT0-ZT2 (+/- 12 hr sleep deprivation) or ZT13-ZT15. Patchpipettes (8-10 MΩ) for perforated patch clamp were fashioned from borosilicate glass capillary (without filament) by using a Flaming-Brown puller (P-1000; Sutter Instrument), and further polished with a MF200 microforge (WPI) prior to filling internal pipette solution (102 mm

6 potassium gluconate, mm CaCl 2, 0.94 mm EGTA, 8.5 mm HEPES, 4 mm Mg-ATP, 0.5mM Na-GTP, 17 mm NaCl; ph7.2). To prepare for the perforated patch recording procedure, β-escin was prepared as a 50- mm stock solution in water (stored up to 2 weeks at 20 C) and was added fresh into the internal pipette solution to a final concentration of 50 μm. As β-escin is light-sensitive, filling syringes were covered with aluminum foil. During the filling process, pipette tips were dipped briefly for 1 sec or less into a small container with β-escin-free internal pipette solution, and then were back-filled with the β-escin-containing solution from the filling syringe. Air bubbles were removed by gentle tapping. β-escin pipette solutions remained stable for several hrs after mixing in the filling syringe, with no evidence of precipitate formation. Recordings were acquired with an Axopatch 200B amplifier (Molecular Devices) or a Model 2400 amplifier with 100 MΩ headstage (A-M systems), and sampled with Digidata 1440A interface (Molecular Devices). These devices were controlled on a computer using pclamp 10 software (Molecular Devices). The signals were sampled at 20 khz and low-pass filtered at 2 khz. Junction potentials were nullified prior to high-resistance (GΩ) seal formation. After establishing a GΩ seal, perforated patches were allowed to develop spontaneously over time (usually ~1 8 min) without any suction pulse applied in the pipette. After breakthrough became evident, as determined by the gradual development of a large capacitance transient in the seal test window of pclamp 10 software, access resistance monitoring was initiated employing the membrane test function. After that point, access resistance was monitored continuously during the final completion of perforation process, until it reached a minimal steady state (access resistance stably < 40 MΩ). Cells showing evidence of mechanical breakthrough, as assessed by the abrupt generation of a large capacitance transient (versus the more progressive, gradual one generated by chemical perforation), were excluded. In addition, cells were also excluded if inflowing cytosolic GFP florescence into the pipette was visually detected during or after recording. One neuron per brain was recorded. During the recording, the bath solution was continuously perfused with saline by means of a gravity-driven system. For identification of bursts in a spike strain, the following criteria were used: burst onset, ISI < 80 ms; burst offset, ISI > 160 ms (Grace and Bunney, 1984). Electrophysiological analysis was performed in Igor software (WaveMetrics), MATLAB (MathWorks), and Clampfit (Molecular devices).

7 TRAP purification of translating RNAs Approximately 250 R2-Split-Gal4, UAS-EGFP-L10a homozygous flies were mechanically sleep deprived for 24 hours. After sleep deprivation the heads were collected and stored at -80 C for TRAP purification. The TRAP experiments were essentially performed following a previously described protocol in Drosophila (Jackson et al., 2015). In our experiments, we used a highaffinity GFP-nanobody GFP-Trap (Allele Biotechnology) for immunoprecipitation of EGFP labeled ribosomes. RNA was extracted from the GFP-Trap beads using Trizol reagent (Invitrogen). qpcr cdna was synthesized using SuperScript III First-Strand Synthesis System (Invitrogen). qpcr was performed using SYBR PCR master mix (Applied Biosystems) as previously described (Liu et al., 2012). Statistical analysis Statistical analyses were performed with Prism 5 (GraphPad). For comparisons of two groups of normally distributed data, Student s t tests were performed. For multiple comparisons, one-way ANOVAs followed by Tukey s post hoc test were performed. For non-normally distributed data, Mann-Whitney test were performed. For multiple comparisons of non-normally distributed data, Kruskal-Wallis test followed by Dunn s post hoc test were performed. Supplemental References Grace, A.A. and Bunney, B.S. (1984). The control of firing pattern in nigral dopamine neurons: burst firing. J. Neurosci. 4, Jackson, F.R., Ng, F.S., Sengupta, S., You, S., and Huang, Y. (2015). Glial cell regulation of rhythmic behavior. Methods Enzymol. 552, Jefferis, G.S.X.E., Potter, C.J., Chan, A.I., Marin, E.C., Rohlfing, T., Maurer, C.R., and Luo, L.Q. (2007). Comprehensive maps of Drosophila higher offactory centers: Spatially segregated fruit and pheromone representation. Cell 128,

8 Lin, C.Y., Chuang, C.C., Hua, T.E., Chen, C.C., Dickson, B.J., Greenspan, R.J., and Chiang, A.S. (2013). A comprehensive wiring diagram of the protocerebral bridge for visual information processing in the Drosophila brain. Cell Rep. 3, Liu, S., Lamaze, A., Liu, Q., Tabuchi, M., Yang, Y., Fowler, M., Bharadwaj, R., Zhang, J., Bedont, J., Blackshaw, S., et al. (2014). WIDE AWAKE mediates the circadian timing of sleep onset. Neuron 82, Renn, S.C., Armstrong, J.D., Yang, M., Wang, Z., An, X., Kaiser, K., and Taghert, P.H. (1999). Genetic analysis of the Drosophila ellipsoid body neuropil: organization and development of the central complex. J. Neurobiol. 41, Wolff, T., Iyer, N.A., and Rubin, G.M. (2015). Neuroarchitecture and neuroanatomy of the Drosophila central complex: A GAL4-based dissection of protocerebral bridge neurons and circuits. J. Comp. Neurol. 523, Yao, Z., Macara, A.M., Lelito, K.R., Minosyan, T.Y., and Shafer, O.T. (2012). Analysis of functional neuronal connectivity in the Drosophila brain. J. Neurophysiol. 108, Young, J.M., and Armstrong, J.D. (2010). Structure of the adult central complex in Drosophila: organization of distinct neuronal subsets. J. Comp. Neurol. 518,