Data_Sheet_1.docx

<p>Here we report on ultrastructural features of brain synapses in the fly Drosophila melanogaster and outline a perspective for the study of their functional significance. Images taken with the aid of focused ion beam-scanning electron microscopy (EM) at 20 nm intervals across olfactory glomerulus DA2 revealed that some synaptic boutons are penetrated by protrusions emanating from other neurons. Similar structures in the brain of mammals are known as synaptic spinules. A survey with transmission EM (TEM) disclosed that these structures are frequent throughout the antennal lobe. Detailed neuronal tracings revealed that spinules are formed by all three major types of neurons innervating glomerulus DA2 but the olfactory sensory neurons (OSNs) receive significantly more spinules than other olfactory neurons. Double-membrane vesicles (DMVs) that appear to represent material that has pinched-off from spinules are also most abundant in presynaptic boutons of OSNs. Inside the host neuron, a close association was observed between spinules, the endoplasmic reticulum (ER) and mitochondria. We propose that by releasing material into the host neuron, through a process triggered by synaptic activity and analogous to axonal pruning, synaptic spinules could function as a mechanism for synapse tagging, synaptic remodeling and neural plasticity. Future directions of experimental work to investigate this theory are proposed.</p

Additional file 5: Figure S2. of Olfactory coding from the periphery to higher brain centers in the Drosophila brain

Similar spatial odor representation patterns between PNs and OSNs in the AL. (a) Spatial response patterns for each odor are reconstructed on a template AL using odor response intensity of the 31 PN classes. (b) Spatial response patterns for each odor are reconstructed on a template AL using odor response intensity of the 29 OSN classes. In each map, the AL is viewed from anterior (top) and posterior (bottom). Each glomerulus name is indicated on the template AL (bottom right). Scale bars = 20 μm. (PDF 105472 kb

Additional file 10: Figure S7. of Olfactory coding from the periphery to higher brain centers in the Drosophila brain

Axonal projections of PNs in the LH and in the MB. (a) The position of glomeruli are mapped on a template AL. (b) Reconstructed axonal projections in the MB calyx and LH. The PNs are labeled with the same color scheme as in (a). (c) Individual traces of reconstructed axonal projections for the 28 PN classes after registration to the template MB calyx and LH. PNs and glomeruli are colored according to the clusters in Fig. 4c, except for VM2 (magenta), which is included in all the three clusters, and D, DM6 (cyan), which are included in the second and third clusters. Anterior view (top) and dorsal view (bottom). Scale bars = 50 μm. (PDF 43110 kb

Additional file 12: Figure S9. of Olfactory coding from the periphery to higher brain centers in the Drosophila brain

Temporal dynamics of odor representation. (a) Represented trajectories of PN ensemble activities for three odors (red: ethyl butyrate, blue: acetophenone, green: 1-octen-3-ol) visualized in the three-dimensional PC space. Color is matched with Fig. 4a. Each trajectory is reconstructed with 100-ms steps indicated with circles, and filled circles indicate 150, 450, and 950-ms time frames. (b) Two-dimensional view of the odor response trajectories for all odors. The odors within the same cluster (colored in red, blue, green, and gray) in Fig. 4a have similar trajectories compared to those between different clusters. (c) Inter-odor distances between pairs of odors measured by ensemble PN odor responses with Euclidean distances. (d) Distance matrices of odor representations by PNs at different time frames. Odors are ordered in the same order as in Fig. 4b, to facilitate comparison to the pattern reconstructed with the mean firing rate for 1-s odor stimuli. Clustering of odors remain largely distinct during 1-s odor stimulus but disperse at 2 s. (e), (f) Distance matrices of odor representations in the LH (e) and MB (f) at different time frames. Odors are ordered in the same order as in Fig. 6b (for LH) and Fig. 7b (for MB) to facilitate comparison to the pattern reconstructed with the mean firing rate for 1-s odor stimuli. (PDF 994 kb

Deficiency ED5438 uncovers the <i>FoxP³⁹⁵⁵</i> self-learning phenotype.

<p><b>a</b>, Genomic region of <i>dFoxP</i> gene. The deficiency deletes all exons of the <i>dFoxP</i> locus up until the 5-SZ-3955 insertion, which was used to generate the deficiency, as well as 52 upstream genes. ED5438 leaves the downstream gene <i>hyperplastic disks</i> (<i>hyd</i>) intact. <b>b</b>, Operant self-learning performance indices in a two-minute test with the heat permanently switched off immediately after eight minutes of training showed a significant impairment of <i>FoxP³⁹⁵⁵</i>/ED5438 flies compared to control animals in which either the deficiency or a Canton S chromosome was crossed over the 5-SZ-3955 insertion (Kruskal Wallis ANOVA, H(2, N = 52) = 10.13; p<0.007; two-sided, Bonferroni-corrected post-hoc p-values indicated in the graph).</p

Drosophila FoxP molecular, anatomical and behavioral raw data

<p>The anatomical and behavioral data providing evidence for the necessity of Drosophila FoxP for operant self-learning</p

<i>Drosophila</i> Avoids Parasitoids by Sensing Their Semiochemicals via a Dedicated Olfactory Circuit

<div><p>Detecting danger is one of the foremost tasks for a neural system. Larval parasitoids constitute clear danger to <i>Drosophila</i>, as up to 80% of fly larvae become parasitized in nature. We show that <i>Drosophila melanogaster</i> larvae and adults avoid sites smelling of the main parasitoid enemies, <i>Leptopilina</i> wasps. This avoidance is mediated via a highly specific olfactory sensory neuron (OSN) type. While the larval OSN expresses the olfactory receptor Or49a and is tuned to the <i>Leptopilina</i> odor iridomyrmecin, the adult expresses both Or49a and Or85f and in addition detects the wasp odors actinidine and nepetalactol. The information is transferred via projection neurons to a specific part of the lateral horn known to be involved in mediating avoidance. <i>Drosophila</i> has thus developed a dedicated circuit to detect a life-threatening enemy based on the smell of its semiochemicals. Such an enemy-detecting olfactory circuit has earlier only been characterized in mice and nematodes.</p></div

Innervation patterns of DL4 and DA2 PNs in MB and LH.

<p>(<b>A</b>) Reconstruction of two DA2 PNs. (<b>B</b>) Reconstruction of two DL4 PNs. (<b>C</b>) comparison of DA2 and DL4 domains after registration of datasets into a common reference space. DA2 and DL4 PNs overlap in the base of the MB and ventroposterior LH. a: anterior, d: dorsal, l: lateral, p: posterior v: ventral.</p

The ab10B neuron detects parasitoid odors.

<p>(<b>A</b>) Example spike traces of GC-coupled SSR with all <i>D</i>. <i>melanogaster</i> OSN types and the headspace of <i>L</i>. <i>boulardi</i> (note that the amount of odors within headspace is too low to be detected and analyzed by GC, but is still detected by ab10B). FID, flame ionization detector. (<b>B</b>) GC-coupled SSR with the ab10B neuron and the wash of <i>L</i>. <i>boulardi</i> (1st panel), as well as the identified active compounds (2nd–4th panel). (<b>C</b>) SSR dose-response curves of the ab10B neuron tested with active compounds. (<b>D</b>) GC-coupled SSR with mutant ab3A neuron ectopically expressing either Or49a or Or85f. Blue, green, and red lines indicate active compounds. (<b>E</b>) Tuning breadths of Or49a and Or85f. 232 odorants are displayed along the <i>x</i>-axis according to strengths of responses they elicit from each receptor. Odorants eliciting strongest responses are placed near the center of distribution. Negative values indicate inhibitory responses. For a list of compounds, see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002318#pbio.1002318.s005" target="_blank">S4 Fig</a>; for raw data see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002318#pbio.1002318.s001" target="_blank">S1 Data</a>. (<b>F</b>) Identification of glomeruli activated by parasitoid odors (-)-iridomyrmecin, (<i>R</i>)-actinidine, and nepetalactol (a mixture of 1S4aR7R7aS, 1R4aS7S7aS-nepetalactol and their enantiomers). 1st to 3rd columns, false color-coded images showing odorant-induced calcium-dependent fluorescence changes in OSNs expressing Or49a or PNs labeled by GH-146-Gal4 at the antennal lobe (AL) level. Flies express UAS-GCaMP3.0 under control of either Or49a-Gal4, or the GH146-Gal4 driver line. (<b>G</b>) GC-coupled extracellular recordings from larval dorsal organ and wash of <i>L</i>. <i>boulardi</i>. (for more GC-SSR traces of wildtype ab10B neurons and mutant ab3A neurons expressing Or49a or Or85f see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002318#pbio.1002318.s004" target="_blank">S3 Fig</a>)</p

Larvae and ovipositing flies are repelled by parasitoid odor.

<p>(<b>A</b>) Larval choice assay and preference indices when larvae were exposed to the wash of <i>L</i>. <i>boulardi</i>. (<b>B</b>) Different choice assays (T-maze, Trap assay, Oviposition assay) for adult flies and resulting preference indices when exposed to the wash of <i>L</i>. <i>boulardi</i>. PI = (number of larvae, flies, or eggs in odor side − number in control side) / total number. Bar plots indicate minimum and maximum values (whiskers), the upper and lower quartiles (boxes) and the median values (bold black line). Deviation of the indices against zero was tested with Wilcoxon rank sum test.</p