and Postsynaptic Plasticity in the Drosophila Brain

Cell Reports Supplemental Information

Optical Dissection of Experience-Dependent Pre- and Postsynaptic Plasticity in the Drosophila Brain Ulrike Pech, Natalia H. Revelo, Katharina J. Seitz, Silvio O. Rizzoli, and André Fiala

Supplemental Figure 1: Expression of pre- and postsynaptic reporter proteins does neither alter neuronal morphology nor endo/exocytosis rates. Related to Figure 1 and 2.

(A) Expression of syp-GCaMP in boutons of motor neurons innervating muscles of the larval body wall in comparison with anti-syt immunoreactivity. (B) Expression of syp-pHTomato in boutons of motor neurons in comparison with anti-syt immunoreactivity. (C) Expression of homer-GCaMP in boutons of motor neurons and the larval ventral nerve cord in comparison with anti-syt immunoreactivity. (D) Schematic illustration of the preparation. Indicated in red is the site of analysis at muscles 6/7 in segment A3 (left). Anti-DLG immunoreactivity and anti-syt immunoreactivity are shown for w1118 animals and larvae expressing syp-GCaMP or syp-pHTomato in motor neurons using D42-Gal4, or homer-GCaMP postsynaptically in muscles using 24B-Gal4, respectively. The morphology of synaptic boutons is not different among the four genotypes. Scale bars: 5 µm in A-C, 50 µm in image of the brain in C, 20 µm in D. (E) Quantification of bouton numbers (top panel) and bouton diameters (bottom panel) between the four genotypes for type Ib and type Is boutons. Bars represent mean and SEM. No significant difference between the four genotypes is observed (p > 0.05, one-way-ANOVA; bouton numbers: n = 2 animals for w1118, n = 4 for syp-GCaMP, n = 5 for syp-pHTomato and homer-GCaMP; bouton size: n = 23 – 73 for Ib boutons, n = 21 – 78 for Is boutons). (F) Quantification of FM dye basal fluorescence in boutons of larval motor neurons following high potassium stimulation. Fluorescence values of animals that express cytosolic GCaMP, syp-GCaMP, syp-pHTomato or homer-GCaMP in all cells using tubulin-Gal4 are normalized to the mean fluorescence of w1118/tubulin-Gal4 animals for each dye used. Larvae expressing cytosolic GCaMP constitute the control for targeted GCaMP versions. n ≥ 22 boutons from at least three different animals each. Box plots indicate medians, means, interquartile ranges and Min/Max range. * - p < 0.05, n.s. – p > 0.05 with one-way-ANOVA and post-hoc Bonferroni pairwise comparisons. (G) Quantification of FM dye destaining in boutons of larval motor neurons during repetitive stimulation. Indicated is the loss of

fluorescence in comparison to basal fluorescence over time. Data are mean and SEM, n ≥ 22 boutons from at least three different animals. (p > 0.05, one-way-ANOVA for each time point)

Supplemental Figure 2: Expression of pre- and postsynaptic reporter proteins does not cause behavioral impairment. Related to Figure 1 and 2. (A) Larval morphology and ubiquitous expression of syp-GCaMP, syp-pHTomato or homerGCaMP using tubulin-Gal4. (B) Larvae expressing syp-GCaMP, syp-pHTomato or homerGCaMP using tubulin-Gal4 are indistinguishable from genetic control larvae in an olfactory preference assay. n.s. – p > 0.05, one-way-ANOVA with post-hoc Bonferroni pairwise comparisons (n = 8). (C) Brain and fly morphology and ubiquitous expression pattern of sypGCaMP, syp-pHTomato or homer-GCaMP using tubulin-Gal4. (D) Flies expressing sypGCaMP, syp-pHTomato or homer-GCaMP using tubulin-Gal4 are indistinguishable from genetic control flies in a negative geotaxis assay. Box plots indicate medians, means, interquartile ranges and 90% / 10% range n.s. – p > 0.05 with one-way-ANOVA and post-hoc Bonferroni pairwise comparisons (n = 8-9). Scale bar: 100 µm.

Supplemental Figure 3: Functionality of the targeted sensors. Related to Figures 3 and 4. (A) Wide field in vivo fluorescence and KCl-induced fluorescence change in the calyx of a fly that expresses either cytosolic GCaMP (cyt GCaMP, top, black line) or syp-GCaMP (bottom, red line) in olfactory projection neurons (OPNs). The maximal fluorescence change and the ratio between KCl-induced fluorescence change and standard deviation of the basal fluorescence (signal/noise) are compared (n = 5 each). (B) Wide field in vivo fluorescence and KCl-induced fluorescence change in the antennal lobe of a fly that expresses either cyt GCaMP (top, black line) or homer-GCaMP (bottom, red line) in OPNs. The maximal fluorescence change and the signal noise ratio are compared (n = 5 each).

(C) Wide field in vivo fluorescence and KCl-induced fluorescence change in the calyx of a fly that expresses either synapto-pHluorin (top, black line) or syp-pHTomato (bottom, red line) in OPNs. The maximal fluorescence change and the signal noise ratio are compared (n = 5 each). The evaluated region is indicated by the orange dotted circle. Traces indicate values for one exemplary fly each. Bars represent mean and SEM. Scale bars: 20 µm; LH lateral horn, m – medial, p – posterior; n.s.- p > 0.05, ** - p < 0.01, two-sample t-test.

Supplemental Figure 4: Two-photon imaging of odor-induced synaptic signaling. Related to Figures 3 and 4. (A-E) Odor-induced fluorescence changes of syp-GCaMP in presynaptic boutons of olfactory projection neurons in the calyx. (A) Fluorescence changes in different boutons in response to mineral oil (oil), methyl cyclohexanol (MCH) and 3-Octanol (3Oct). Scale bar: 20 µm. Three bouton regions are indicated in different colors, the respective dynamics of fluorescence change (mean and SEM of three stimulations) is shown in (B). (C) Temporal dynamics of fluorescence changes in 20 individual boutons (indicated by numbers). Boutons are numerically ordered in the false-color coded heatmap. Each row represents one bouton, each column a 250 ms time frame. Pixel values represent means of three stimulations. Arrowheads demarcate the three boutons shown in (B). (D) Comparative kinetics and amplitudes of MCH-induced fluorescence changes in a MCH-responding bouton in four different animals (mean of three stimulations each). (E) Ca2+ dynamics within a single bouton, indicated by the orange box. Scale bar: 2 µm. (F-J) Odor-induced fluorescence changes of syp-pHTomato in presynaptic boutons in the calyx. (F) Fluorescence changes in different boutons of a representative animal in response to oil, MCH and 3Oct. Scale bar: 20 µm. One bouton region is indicated by an orange circle, the respective dynamics of fluorescence changes (three repetitive stimulations and mean) are shown in (G). (H) Dynamics of fluorescence changes in 20 individual boutons. Each row in the heatmap represents one bouton, each column a 500 ms time frame. Pixel values represent means of three repetitions. Arrowheads demarcate three boutons discussed in the text. (I) Kinetics and amplitudes in a MCH-responding bouton in four different animals (mean of three stimulations each). (J) MCH-induced fluorescence changes of syp-pHTomato within the single bouton indicated by the orange box. Scale bar: 2 µm.

(K-N) Odor-induced fluorescence changes of homer-GCaMP in glomeruli of the antennal lobe. (K) Fluorescence changes in glomeruli in response to oil, MCH and 3Oct. Scale bar: 20 µm. Three glomerular regions are indicated in different colors, the respective time trace of fluorescence changes (mean and SEM of three stimulations) are shown in (L). (M) Dynamics of fluorescence changes in 10 identified glomeruli. In the heatmap, each row represents one glomerulus, each column one 250 ms time frame. Pixel values represent means of three stimulations. (N) Kinetics of MCH-induced fluorescence changes in glomerulus DL4 in four different animals. Values indicate means of three odor stimulations in each fly. Odor stimulation is indicated as grey bars. p – posterior, l – lateral.

Supplemental Figure 5: Syp-pHTomato reports synaptic vesicle release. Related to Figure 4. (A) Setup to control the temperature of the fly's brain during optical imaging. The fly is headfixed inside a chamber; odors are delivered to the antenna through a constant air flow. The brain is directly exposed to a constant flow of Ringer's solution which is heated or cooled using computer-controlled Peltier elements.

(B) In vivo fluorescence in the antennal lobe in a fly that expresses both homer-GCaMP (green) and syp-pHTomato (magenta) in olfactory projection neurons. Indicated in black is glomerulus DL5, which shows fluorescence changes upon stimulation with banana odor, as shown in false colors for homer-GCaMP. (C) Simultaneously monitored, banana-induced fluorescence changes of homer-GCaMP and syp-pHTomato, measured at different temperatures in DL5. Temporal dynamics of fluorescence change are shown for five individuals in grey. The mean is shown as colored line. The left three traces indicate values of flies that do not express shibirets, the three traces on the right indicate values of flies that express shibirets. (D) Mean fluorescence changes and SEM during the three frames directly following odor onset, measured with homer-GCaMP (left) and syp-pHTomato (right) at restrictive and permissive temperatures. Scale bar: 20 µm; l – lateral, p – posterior; n.s.- p > 0.05, *** - p < 0.001, two-sample t-test.

Supplemental Figure 6: Comparison of cytosolic and postsynaptically targeted GCaMP following apple exposure. Related to Figure 7. (A) Odor-evoked cytosolic GCaMP fluorescence changes in DC1 for MCH, in DM3 for 3Oct, and in DL5 for apple and banana (top row) and the respective maximal fluorescence changes during odor stimulation (bottom row). n = 5. (B) Odor-evoked homer-GCaMP fluorescence changes in DC1 for MCH, in DM3 for 3Oct, and in DL5 for apple and banana (top row) and the respective maximal fluorescence changes during odor stimulation (bottom row). n = 5. Traces show mean values and SEM; box plots indicate medians, interquartile ranges and 10% / 90% range. n.s.- p > 0.05, ** - p < 0.01, *** - p < 0.001, two-sample t-test.

Supplemental Experimental Procedures

Generation of DNA constructs and transgenic flies The DNA of synaptophysin-GCaMP3 (Li et al., 2011) was obtained from S. Voglmaier. Synaptophysin-GCaMP3 was subcloned into the pUAST vector (Brand and Perrimon, 1993), using NotI and XbaI restriction enzyme sites. The DNA of pRham-pHTomato (Li and Tsien, 2012) was obtained from Y. Li. The DNA of pcDNA3synaptophysin-4xpHluorin (addgene #37005, Zhu et al., 2009) was obtained from S. Heinemann. Synaptophysin_4xpHluorin was amplified using linker PCR and subcloned into the pUAST vector using the NotI restriction enzyme site. Subsequently, the 4xpHluorin sequence was replaced by the pHTomato DNA sequence using the SpeI and AgeI restriction enzyme sites. To amplify the cDNA of dHomer total RNA was extracted from w1118 flies and transcribed into cDNA using the RNeasy kit (Qiagen, Duesseldorf, Germany) and the iScript cDNA Synthesis Kit (Bio-Rad, Hercules, CA).

The

cDNA

of

dHomer

ATGGGCGAACAACCGATTTTCA-3’

was

obtained

and

by

PCR

using

the

primers

5’-

5’-TCAAGTGCACTTGGCAATTTGTATAA-3’.

Restriction enzyme sites were added and the stop codon replaced by subsequent linker PCR. The DNA of GCaMP3 (plus AAAT-linker sequence) was obtained by linker PCR from genomic DNA of UAS:GCaMP3 flies (Tian et al., 2009). Both DNA fragments were inserted into the pUAST vector using the EcoRI/BglII and BglII/XbaI restriction enzyme sites. All constructs were sequenced and germline transformation was carried out by the BestGene Inc. (CA). For each construct several different fly strains with random insertion sites were obtained, out of which one strain was chosen for further experiments based on expression levels and health of the animals.

Fly strains UAS:GCaMP3 (Tian et al., 2009) and UAS:synapto-pHluorin (Ng et al., 2002) were used as reference. To express the constructs in olfactory projection neurons GH146-Gal4 (Stocker et al., 1997) was used. To express the constructs in motor neurons D42-Gal4 (Yeh et al., 1995) was used. To express the constructs in muscle cells 24B-Gal4 (Brand and Perrimon, 1993) was used. To express the constructs ubiquitously in all cells tub-Gal4 (Lee and Luo, 1999) was used. To disrupt synaptic transmission, 20xUAS:shibirets was used (Pfeiffer et al., 2012). All flies were reared on standard corn meal medium at 25° C (unless noted otherwise), 60% humidity and a 12/12 h light/dark cycle.

Immunohistochemistry Brains of 3 – 6 days-old female flies were removed from the head capsule or larval filets of third instar wandering larvae were prepared as described (Jan and Jan, 1976) under Ringer’s solution (Estes et al., 1996) and fixed in 4% paraformaldehyde dissolved in phosphatebuffered saline (PBS), pH7.4, for 2 h at 4° C. The samples were washed three times in PBS containing 0.6% Triton X-100 and subsequently incubated in block solution containing 1% bovine serum albumine (BSA) and 5% normal goat serum for 2 h at 4° C. The samples were then incubated with the primary antibody diluted in block solution at 4° C overnight. The following antibodies were used: rabbit anti-GFP (Invitrogen, Carlsbad, CA; dilution, 1:1000), rat anti-RFP (Chromotek, Munich; dilution 1:200), mouse anti-syt (3H2 2D7, DSHB, Iowa; dilution 1:50), mouse anti-DLG (4F3, DSHB, Iowa; 1:200) and mouse anti-nc82 (gift from E.Buchner, Würzburg; 1:50). After the samples had been washed at least three times for 20 minutes each with PBS containing 0.6% Triton and 1% BSA (PAT), they were incubated in secondary antibodies diluted in PAT at 4° C overnight.

The following secondary antibodies were used: Anti-mouse Alexa633 (Invitrogen; A21050), anti-rabbit Alexa488 (Invitrogen; A11034) and anti-rat Cy3 (Invitrogen; A10522), all diluted 1:250. Anti-mouse atto647N (Sigma, 50185) diluted 1:500 and anti-rabbit chromeo494 (Active Motif, 15042) diluted 1:100. Samples were washed subsequently in PBS containing 0.6% TritonX-100, later in PBS, and mounted in Vectashield (Vector, Burlingame, CA; H1000) for confocal imaging or processed further for STED imaging.

Confocal imaging and data processing Confocal images were obtained using a Leica TC SP2 confocal microscope equipped with a Leica Apochromat x20 water-immersion objective (NA=0.7) for whole brain scans, and a x63 oil-immersion objective (NA=1.4) for scans of individual neuropils or of larval motor neuron boutons. All preparations were scanned in 1 µm z-steps. Images were processed using Fiji (Schindelin et al., 2012). For display, either single optical slices or maximal intensity projections of stacks through the entire brain or neuromuscular junction were used, contrast and brightness adjusted, and a median filter with pixel range of 1 applied. For quantification of the staining intensity, an unprocessed maximal projection of ~20 µm was used that covered either calycal boutons and adjacent axonal tracts or antennal lobe glomeruli and adjacent axonal tracts. Pixel intensity was measured within a 1x1 µm region in either a bouton or glomerulus and in the respective adjacent axonal tract.

STED imaging and data processing For STED microscopy (Hell and Wichmann, 1994), immunohistochemichally stained brains were embedded in the polymer resin melamine (TCI Europe), which was prepared as described in Revelo et al., (2014).

Brains were placed on an 18mm glass coverslip, a bottomless BEEM capsule (Beem Inc., West Chester, PA, USA) was placed around it, and the freshly prepared melamine was poured inside the capsule until covering the brain completely. Subsequent sample treatment was as described in Revelo et al., (2014). Briefly, the samples were kept overnight at room temperature and then heated to 40° C for 24 h. Subsequently, the BEEM capsule was filled to the top with Epon resin (Epo Fix kit, 40200029, Struers, Ballerup, Denkmark) and incubated at 60° C for 48 h. Melamine blocks were trimmed and were again incubated at 60° C for 48 h. Finally, melamine blocks were cut into 50 to 60 nm sections with an ultramicrotome (Leica EM UC6, Leica Microsystems GmbH). Two-color STED microscopy was performed using a Leica TCS SP5 STED microscope (Leica Microsystems GmbH) equipped with a 100x oil immersion objective (NA=1.4). Chromeo494 and Atto647N were excited with pulsed diode lasers (PDL 800-D, PicoQuant, Berlin, Germany) at 531 nm and 640 nm respectively. The STED beam was generated by a Ti:Sapphire laser (MaiTai, Spectra-Physics, Darmstadt, Germany) emitting at 750 nm. Acquired images were deconvolved using Huygens Essential 4.4, from Scientific Volume Imaging (Hilversum, The Netherlands), using the in-built deconvolution functions of the software, adjusted to the imaging parameters of the STED microscope described above.

Wide field imaging and data processing For wide-field imaging, flies were heterozygous for both GH146-Gal4 and the respective sensor construct. Female transgenic flies (5 days-old) were briefly immobilized on ice, placed in a custom-built fly holder and fixed using adhesive tape. A window was cut into the tape with a fine razorblade (0.1 mm), the head capsule underneath that window was opened and fat and tracheae were carefully removed with forceps.

The head opening was covered with Ringer's solution (5 mM HEPES, pH 7.4, 130 mM NaCl, 5 mM KCl, 2 mM CaCl2, 2 mM MgCl2, 36 mM sucrose). Imaging was performed using an fluorescence microscope (Zeiss Axioscope 2 FS) equipped with a xenon lamp (Lambda DG4, Sutter Instruments), a 14 bit CCD camera (Coolsnap HQ, Photometrics), a 20x/NA=1 water-immersion objective and filter sets for GFP and RFP. Image acquisition was controlled using the software Metafluor (Visitron Systems, Puchheim). Images were acquired at a frame rate of 5 Hz, green sensors were excited for 100 ms at 488 nm, pHTomato was excited for 100 ms at 560 nm. The focus was adjusted to capture all neuropils that are targeted by GH146-Gal4 at once (i.e. that all, calyx, lateral horn and antennal lobes were visible). After recording of some initial frames, 50 µl 1 M KCl were injected into the Ringer's solution covering the brain (final concentration at the brain ~0.05 M). Image processing and analysis was performed with Fiji software. To correct for potential small shifts in x-y direction, recorded images were aligned using a TurboReg (Thévenaz et al., 1998) based customwritten plugin. Circular regions of interest (ROIs, diameter 20 µm) were placed in the center of the calyx, the lateral horn and antennal lobe. Background fluorescence outside the neuropils was subtracted from each ROI, "F0" was determined by the average pixel intensity of five frames just before stimulus onset, ΔF is the difference between the fluorescence in each frame and F0, and resulting values were normalized to a common scale by dividing F 0. Since synapto-pHluorin and pHTomato showed pronounced stimulus-induced bleaching, individual traces were bleach-corrected by subtracting the best least square fit (y=ax+bx²+c) of ten frames before stimulus onset and all remaining frames 125 frames after stimulus onset.

Two-photon imaging and data processing For two-photon microscopy (Denk et al. 1990), flies were heterozygous for both GH146-Gal4 and the respective sensor construct, except for flies that carried UAS-syp-pHTomato: two copies (i.e., flies homozygous for UAS-syp-pHTomato), were used for two-photon imaging to achieve sufficient emission at the conditions used. Female transgenic flies (3–6 days old) were prepared as described above. To stabilize the brain and to reduce movements, the head capsule was filled with 1.5% low melting agarose solution (Sigma-Aldrich) diluted in Ringer’s solution and the head opening was embedded in Ringer's solution. Although prepared flies reliably showed leg/proboscis movements and odor-responses for up to 12 h, imaging sessions were limited to 2 h to ensure comparable conditions. Imaging was performed using an LSM 7 MP two-photon microscope (Zeiss) equipped with a mode-locked Ti-sapphire Chameleon Vision II laser (Coherent, CA) and with a Plan-Apochromat 20x water-immersion objective (NA=1, Zeiss). All constructs were excited at 950 nm, and a dichroic mirror was combined with a 500-550 nm and a 575-610 nm bandpass filter to record green fluorescence constructs or pHTomato, respectively. Attached to the microscope was a custom-built device to supply odorous air with a constant flow rate of 1 ml/s directly to the fly’s antennae as described previously (Riemensperger et al., 2005). Throughout the study, MCH was diluted 1:750, 3Oct 1:500 in mineral oil. Onset and duration of the odor stimulus were controlled using a custom-written LABVIEW program (National instruments, TX). Each odor presentation was separated by a 20 s break, and each odor was presented three times to the animal for each optical plane measured. Unless noted otherwise, calyces were recorded at a resolution of 0.2 µm/pixel and 5 Hz, antennal lobes were recorded at 0.3 µm/pixel and 4 Hz. Image processing and analysis was performed using Fiji software.

To correct for potential small shifts in x-y direction, recorded images were aligned using a TurboReg (Thévenaz et al., 1998) based custom written plugin. The same transformation matrix was used for both wavelength channels in dual color recordings. Subsequently, regions of interest (ROIs) were manually defined: in the calyx, individual presynaptic boutons were clearly visible and distinguishable when using the presynaptic sensors, but not when using homer-GCaMP. Hence, individual boutons were chosen as ROIs as long as the fly expressed a presynaptic sensor. In the antennal lobes, individual glomeruli could easily anatomically be distinguished when using either syp-GCaMP or homer-GCaMP. Due to the less defined anatomical pattern when using cytosolic GCaMP and due to the low fluorescence-levels of syp-pHTomato in the antennal lobe, activity profiles aided glomerulus identification when using the latter two constructs on its own. Stacks of the entire antennal lobes were recorded prior to measurements and used to identify glomeruli according to their localization and spatial integration. Glomeruli were identified with reference to a 3D atlas of the fly antennal lobe (Laissue et al., 1999; Flybrain on-line: [http://www.flybrain.org] Accession Number: AB00203). "F0" was determined as the average pixel intensity of five frames just before stimulus onset, ΔF is the difference between fluorescence and F 0, and resulting values were normalized to a common scale by dividing F 0. To compensate for the high intrinsic noise when measuring synaptic transmission, the average of two frames was used when displaying syp-pHTomato traces in Figure S4. False color coded images were produced accordingly by subtracting the average of three frames before stimulus onset from the average of three frames after stimulus onset (or from single frames in Figure S4E and J) and dividing the resulting image by the average of the before stimulus. For display, false color coded histological images are median filtered with a pixel-range of one or two.

Temperature-controlled optical imaging The flies in the experiments involving shibirets were prepared in a custom built flow-through system to achieve precise temperature control at the fly's brain during imaging. Flies in the apple-exposure experiment were also imaged in this system to achieve comparable imaging conditions. Preparation of flies was performed as described above (section two-photonimaging), except that the flies were additionally fixed with dental glue, and no agarose was used. The brain was exposed to a closed-loop constant flow of Ringer's solution and the temperature of the Ringer's solution was modified using Peltier elements controlled by a Peltier temperature control system (PTC20, npi electronics, Tamm, Germany). The temperature was measured using an independent thermometer directly before and after each measurement between the fly's brain and the immersed objective. Unless noted otherwise, flies were imaged at 23° C.

Deprivation experiments and apple-exposure experiment To image the acute disruption of vesicle release using shibirets, siblings that carried either the balancer TM3 on the third chromosome or the UAS-shibirets transgene were collected at the day of eclosion and raised together in mixed populations of ~10 flies for 10 days. The response to the aroma of a 2g piece of banana was imaged three times at 23° C; subsequently the temperature at the fly's brain was set to 32° C for 20 min. In the first ten minutes at 32° C, the flies were exposed to 40 short pulses of banana aroma to deplete the vesicles. After another 10 min (to reduce potential short time habituation effects) the response to the banana aroma was imaged three times at 32° C. Afterwards the temperature at the fly's brain was set back to 23° C, and after a 10 min break the response to the banana aroma was again measured three times.

In the deprivation experiment using shibirets, siblings that carried either the balancer TM3 on the third chromosome or the UAS-shibirets transgene were collected at the day of eclosion and raised together in mixed populations of ~10 flies. Some of these populations were kept for five days at 32° C to achieve chronic disruption of vesicle release; the others remained at 25° C. Flies raised at 32° C were allowed to recover for 1h at 25° C before imaging. To compensate for the accelerated aging at 32° C, flies raised at 25° C were imaged after 9 days. Each day the same amount of flies with TM3 or shibire ts were imaged, respectively, in an alternating order. In the apple-exposure experiment, siblings were collected at the day of eclosion and kept in mixed populations of ~10 animals. For some of those populations a 5g piece of apple was put into the food vial for five days. An equal amount of siblings that were raised on standard food or on standard food plus apple were imaged at the same day, in an alternating order. For imaging only female flies were used. In the respective experiments, the shibirets transgene insertion was confirmed using PCR. All apple and banana samples used for rearing the animals and for odor delivery derived from the same fruits, respectively. One ripe banana and one apple were portioned in 2g and 5g pieces, respectively, and kept in -20° C until usage. The evaluation of the imaging data in those experiments, including the determination of the bouton regions in the calyx, was performed blind to the genotype and condition.

FM dye experiments All experiments were carried out at room temperature (22° C). Third instar larvae were dissected according to Jan and Jan, 1976, in standard Drosophila medium containing 130 mM NaCl, 26 mM sucrose, 5 mM KCl, 2 mM CaCl2, 2 mM MgCl2, 5 mM Hepes, pH 7.4 (Jan and Jan, 1976; Kuromi and Kidokoro, 1999).

The preparations were loaded with 10 µM FM 1-43 or 50 µM FM 4-64 (both Biotrend, Germany) in high potassium medium (90 mM KCl and 45 mM NaCl in standard medium) for 30 seconds and subsequently washed for 10 minutes in standard medium. Preparations were placed in standard medium inside a custom made platinum plate stimulation chamber (8 mm distance between electrodes). The 20 Hz stimulation trains of 100 mA (10 seconds each) were generated using an A310 AccupulserTM triggered by an A385 Stimulus Isolator (both World Precision Instruments, Germany). Images were acquired using an Axio Examiner.Z1 fluorescence microscope (Carl Zeiss AG, Germany) equipped with a 20x/NA=1 water immersion objective (Zeiss), a 100 W mercury lamp (Zeiss) and a EMCCD camera (QuantEM:512SC, Photometrics, USA). FM 1-43 fluorescence was detected using a 470/40 BP excitation filter, a 495 beamsplitter and a 525/50 BP emission filter. FM 4-64 fluorescence was detected using a 560/40 BP excitation filter, a 585 beamsplitter and a 630/75 BP emission filter (all from AHF, Germany). Images were analyzed using custom written MATLAB routines (The Mathworks, Inc., USA).

Larval chemotaxis assay Two caps (diameter 6 mm) were filled with 25 µl of either mineral oil or pentyl acetate (Sigma) diluted 1:50 in mineral oil and placed at the opposite sites of a Petri dish (diameter 92 mm) half-filled with 1% agarose. About 20 third instar wandering larvae were rinsed in tap water and placed in the center of the Petri dish. Larvae were allowed to crawl for 2 min in darkness and subsequently the number of larvae in each half of the Petri dish was scored. Performance index = (# of larvae at the side of the odor - # of larvae at the opposite side) / total # of larvae.

Geotaxis assay Geotaxis was quantified as described by Benzer, (1967) with modifications described by Inagaki et al., (2010). Briefly, a mixed population of 20 4-6 days-old flies was transferred in the countercurrent apparatus (equipped with a light source placed above) and tapped five times on the bench to induce locomotion, followed by a 30 s period in which flies could move into the upper tube. This procedure was repeated five times and a geotaxis index was calculated as described by Inagaki et al., (2010). Ambient conditions of 24° C and 60% humidity as well as Zeitgebertime were kept constant.

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Supplemental Movie 1: Odor-evoked presynaptic Ca2+ dynamics in the calyx visualized using syp-GCaMP. Related to Figure 3. The movie shows a raw image sequence of syp-GCaMP fluorescence in boutons of olfactory projection neurons in the calyx of the right brain hemisphere (dorsal view). Images were recorded using two-photon microscopy at 4 Hz through a window in the fly's head capsule. Stimulation with mineral oil (oil) and the odorants methyl cyclohexanol (MCH) and 3–octanol (3OCT) are indicated. Differential ensembles of boutons show odor-evoked increases in presynaptic Ca2+.

Supplemental Movie 2: Spontaneous and odor-evoked postsynaptic Ca2+ dynamics in the antennal lobe visualized using homer-GCaMP. Related to Figure 4. The movie shows a raw image sequence of homer-GCaMP fluorescence in olfactory projection neurons in one central focal plane of the antennal lobe of the left brain hemisphere (dorsal view). Images were recorded using two-photon microscopy at 4 Hz through a window in the fly's head capsule. Stimulation with mineral oil (oil) and the odorants methyl cyclohexanol (MCH) and 3–octanol (3OCT) are indicated. One glomerulus within the focal plane is responsive to 3OCT. Pronounced spontaneous Ca 2+ fluctuations that do not correlate with odor stimulation are visible.