<i>Obp69a</i> transcription is dimorphically regulated in response to male scents, and exposure to the male pheromone cVA.

<p>Total RNA extracted from heads of male and female flies exposed to male scents (<b>A,B</b>) or to cVA (<b>C,D</b>) for three days was analyzed for <i>Obp69a</i> mRNA levels by RT-qPCR. Statistical significance was determined by one-way ANOVA with Tukey post-hoc analysis. Error bars signify SEM. ***<i>P</i><0.001, (A) F(2, 6) = 28.5, (B) F(2, 6) = 44.1, (C) F(2, 6) = 24.3, (D) F(2, 6) = 28.3. n = 3 independent experiments of 15–20 fly heads/sample.</p

Obp69a links prior social interaction to modulation of social responsivity.

<p><b>A</b>. Obp69a was knocked-down by RNAi in male flies and number of lunges was scored. Statistical significance was determined by one-way ANOVA with Tukey post-hoc analysis, F(2, 69) = 25.4 ***<i>P</i><0.001, n = 24. <b>B, C</b>. Obp69a was over-expressed in male flies and the number of lunges was scored (<b>B</b>) or the time until the first lunge (latency) was measured. Statistical significance was determined by one-way ANOVA with Tukey post-hoc analysis. (B) F(2, 63) = 8.5, (C) F(2, 62) = 6.2 **<i>P</i><0.01, ***<i>P</i><0.001, n = 24 <b>D.</b> Female flies were exposed to male scents prior to the courtship assay, and the time to copulation with WT virgin male flies was measured. Statistical significance was determined by Students t-test. **P<0.01, n = 17. <b>E.</b> RNAi to Obp69a was expressed in females that were previously exposed to male scents, and the time to copulation with WT virgin males was measured. Statistical significance was determined by one-way ANOVA with Tukey post-hoc analysis, F(4, 42) = 3.55, *<i>P</i><0.05, n = 18. <b>F.</b> Obp69a-GFP was expressed in female flies, which were exposed to 1μg cVA prior to mating with WT virgin males. The time to copulation was measured. Statistical significance was determined by one-way ANOVA with Tukey post-hoc analysis, F(2, 44) = 4.9, **P<0.01, n = 34, 42 and 31 for <i>Obp69aMi-GAL4</i>, <i>UAS-Obp69a-GFP</i> and <i>Obp69aMi</i>-<i>GAL4</i>; <i>UAS-Obp69a-GFP</i> respectively.</p

<i>Odorant binding protein 69a</i> connects social interaction to modulation of social responsiveness in <i>Drosophila</i>

<div><p>Living in a social environment requires the ability to respond to specific social stimuli and to incorporate information obtained from prior interactions into future ones. One of the mechanisms that facilitates social interaction is pheromone-based communication. In <i>Drosophila melanogaster</i>, the male-specific pheromone cis-vaccenyl acetate (cVA) elicits different responses in male and female flies, and functions to modulate behavior in a context and experience-dependent manner. Although it is the most studied pheromone in flies, the mechanisms that determine the complexity of the response, its intensity and final output with respect to social context, sex and prior interaction, are still not well understood. Here we explored the functional link between social interaction and pheromone-based communication and discovered an odorant binding protein that links social interaction to sex specific changes in cVA related responses. <i>Odorant binding protein 69a</i> (<i>Obp69a</i>) is expressed in auxiliary cells and secreted into the olfactory sensilla. Its expression is inversely regulated in male and female flies by social interactions: cVA exposure reduces its levels in male flies and increases its levels in female flies. Increasing or decreasing Obp69a levels by genetic means establishes a functional link between Obp69a levels and the extent of male aggression and female receptivity. We show that activation of cVA-sensing neurons is sufficeint to regulate Obp69a levels in the absence of cVA, and requires active neurotransmission between the sensory neuron to the second order olfactory neuron. The cross-talk between sensory neurons and non-neuronal auxiliary cells at the olfactory sensilla, represents an additional component in the machinery that promotes behavioral plasticity to the same sensory stimuli in male and female flies.</p></div

<i>Obp69a</i> transcriptional regulation requires active neurotransmission of the information from the sensory neuron to the second order olfactory neuron.

<p>(<b>A</b>) Male flies expressing <i>UAS-Cs-Chrimson</i> and <i>UAS-Shibire</i>^<i>ts</i> in Or65a neurons where subjected to three 15 minutes long optogenetic activations as a positive control, synaptic release blocking, or both. Obp69a relative expression was then measured using RT-qPCR. (<b>B</b>) Female flies expressing <i>UAS-Cs-Chrimson</i> and <i>UAS-Shibire</i>^<i>ts</i> in Or67d neurons where subjected to three 15 minutes long optogenetic activation as a positive control, synaptic release blocking, or both. Obp69a relative expression was then measured using RT-qPCR. Statistical significance was determined by One-way ANOVA with Tukey post-hoc analysis. Error bars signify SEM. (A) F(2,6) = 21.18, (B) F(2, 6) = 7.9, *<i>P</i><0.05, **<i>P</i><0.01 n = 3 independent experiments of 10–15 fly heads/sample. (<b>C</b>) Proposed model in which exposure to cVA regulates the production of Obp69a in accessory cells oppositely in male and female flies, via a mechanism that depends on relaying the information from the sensory neurons to the second order olfactory neurons in the brain, and eventually back to Obp69a producing cells.</p

Obp69a is expressed in cells within the third antennal segment and is exported to the lymph.

<p><b>A-C</b>. Relative Obp69a expression levels in male heads and bodies (<b>A</b>), and between heads without antennae or maxillary palps in males (<b>B</b>) and females (<b>C</b>). Statistical significance was determined using Student’s t-test (<b>A</b>) ***<i>P</i><0.001, or One-way ANOVA with Tukey post-hoc analysis, Error bars signify SEM. (<b>B</b>)F(3,8) = 120, (<b>C</b>)F(3,8) = 124 **P<0.01. n = 3 independent experiments of 10–15 fly heads/sample. <b>D-H.</b> Confocal images of a membrane bound GFP (mcd8-GFP) in Obp69a expressing cells (<i>Obp69a-GAL4</i>), marking the 3^rd antennal segment. Note the GFP expression in the eyes is a marker of the <i>minos</i> element. (D). Arrowheads mark individual cells in antenna. E, F. Confocal images of a membrane-bound GFP (mcd8-GFP) in Obp69a expressing cells (using <i>Obp69a-GAL4</i> and <i>Minos Obp69a-GAL4</i> accordingly), marking the 3^rd antennal segment and presumably auxiliary cells. <b>G, H.</b> Confocal images of a transgenic Obp69a fused to GFP (<i>UAS-Obp69a-GFP</i>) expressed in Obp69a-expressing cells (Obp69a-GAL4). Asterisks mark Obp69a-GFP expression within the cells, Arrowheads mark exported Obp69a-GFP in the lymph. <b>I.</b> Western blot analysis of antennae and heads of <i>Obp69a-GAL4/+; UAS-Obp69a-GFP</i> flies using anti-GFP antibodies. <b>J</b>. RNAi to Obp69a was expressed in Or67d neurons, Lush, Obp69a, and Obp28a and nompA cells. <i>Obp69a</i> mRNA levels assessed by RT-qPCR. Statistical significance between relative mRNA levels in control and KD in each cell type was determined by Student’s T-test with Bonferroni correction for multiple hypothesis testing, Error bars signify SEM. *<i>P</i><0.05, n.s., not-significant. n = 3 independent experiments of 10–15 fly heads/sample.</p

<i>Odorant binding protein 69a</i> (<i>Obp69a</i>) exhibits sexually dimorphic expression levels and is regulated inversely by social conditions in male and female flies.

<p><b>A</b>. Male and female flies were housed separately in groups of 5 flies/vial for 3 days. Total RNA extracted from heads of grouped male and female flies was analyzed for mRNA levels of <i>lush</i>, <i>obp69a</i>, <i>cyp6a20</i>, <i>est-6 and obp28a</i> by RT-qPCR. Statistical significance was determined by Student’s T-test with Bonferroni correction for multiple hypothesis testing. Error bars signify SEM *<i>P</i><0.001, n.s., not significant, n = 6 independent experiments with 10–15 fly heads/sample. <b>B.</b> Schematic illustration of social conditions set-up. WT males (upper panel) or females (lower panel) were housed individually, in groups of five same sex flies/vial or in groups of five male and five female flies for 3 days. <b>C-L.</b> Total RNA extracted from heads of male and female flies under single housing, same sex group and mixed sex group was analyzed for mRNA levels of <i>lush</i>, <i>obp69a</i>, <i>cyp6a20</i>, <i>est-6</i> and <i>obp28a</i> by RT-qPCR. Statistical significance was determined by one-way ANOVA with Tukey post-hoc analysis and Bonferroni correction for multiple hypothesis testing. (D) F(2, 6) = 9.4 **<i>P</i><0.01. (E) F(2, 6) = 11.03 **<i>P</i><0.01. (I) F(2,6) = 12.2 **P<0.01. n = 6 independent experiments of 15–20 fly heads/sample.</p

The activity of cVA sensing neurons is necessary and sufficient for regulating <i>Obp69a</i> transcription.

<p><b>A, B.</b> Activity of Or65a and Or67d expressing neurons are necessary for Obp69a transcription changes in male and female flies. <b>A.</b> Relative <i>Obp69a</i> levels in single and Grouped housed male flies expressing <i>UAS-Kir2</i>.<i>1</i> in Or67d or Or65a neurons was analyzed using RT-qPCR. Log ratio represents the fold change in Obp69a relative levels of group divided by single housed. <b>B.</b> Relative <i>Obp69a</i> levels in female flies expressing <i>UAS-Kir2</i>.<i>1</i> in Or67d or Or65a neurons and subjected to group hosing or group housing with male flies was analyzed using RT-qPCR. Log ratio represents the fold change in Obp69a relative levels of grouped divided by grouped with males. Statistical significance was determined by One-way ANOVA with Tukey post-hoc analysis. Error bars signify SEM. (<b>A</b>) F(4,10) = 17.26, (<b>B</b>) F(4,10) = 15.5 **<i>P</i><0.01, n = 3 independent experiments of 10–15 fly heads/sample. <b>C-F</b>, activation of cVA sensory neurons is sufficient in eliciting Obp69a transcriptional changes. Male (<b>C,D</b>) and female (<b>E,F</b>) flies expressing <i>CsChrimson</i> in Or67d (<b>C,E</b>) or Or65a (<b>D,F</b>) neurons (<i>Or67d-GAL4</i>, <i>UAS-Cs-Chrimson</i> and <i>Or65a-GAL4</i>, <i>UAS-Cs-Chrimson</i> respectively), and genetic controls were either subjected to spaced three 15-min long optogenetic activation sessions or were kept in the dark, after which <i>Obp69a</i> transcript levels were analyzed using RT-qPCR. Statistical significance was determined by Student’s T-test with Bonferroni correction for multiple hypothesis testing. Error bars signify SEM. *P<0.05, **P<0.01 (C,E,F) n = 4, (D) n = 5. n represents the number of independent experiment of 10–15 fly heads/sample.</p

Additional file 6 of BBB opening by low pulsed electric fields, depicted by delayed-contrast MRI, enables efficient delivery of therapeutic doxorubicin doses into mice brains

Supplementary Material 6: Video 2b. Growth of U87 cells treated with 200nM Doxo

Additional file 2 of BBB opening by low pulsed electric fields, depicted by delayed-contrast MRI, enables efficient delivery of therapeutic doxorubicin doses into mice brains

Supplementary Material 2: Video 1a. Growth of GL261 cells without treatment

S7 Fig -

A. Behavioral signatures of male-male social interaction within the FlyBowl system during the optogenetic activation of Dh44 neurons. Data is represented as normalized Z scores of 60 behavioral parameters. Dh44 G4/+;UAS-csChrimson/+ males (green, n = 8) and their genetic controls Dh44 G4/+, UAS-csChrimson/+ (black, n = 7 and grey, n = 8, respectively). **pB. Starvation resistance assayed on Dh44 G4/NPFR RNAi (green, n = 70) flies and their genetic controls Dh44 G4/+ (blue, n = 66) and NPFR RNAi/+ (black, n = 64). No significant difference was observed among experimental flies and the controls, p>0.05. Pairwise log-rank test with FDR correction for multiple comparisons was performed. (EPS)</p