Open Channel Block by Ca2+ Underlies the Voltage Dependence of Drosophila TRPL Channel

The light-activated channels of Drosophila photoreceptors transient receptor potential (TRP) and TRP-like (TRPL) show voltage-dependent conductance during illumination. Recent studies implied that mammalian members of the TRP family, which belong to the TRPV and TRPM subfamilies, are intrinsically voltage-gated channels. However, it is unclear whether the Drosophila TRPs, which belong to the TRPC subfamily, share the same voltage-dependent gating mechanism. Exploring the voltage dependence of Drosophila TRPL expressed in S2 cells, we found that the voltage dependence of this channel is not an intrinsic property since it became linear upon removal of divalent cations. We further found that Ca2+ blocked TRPL in a voltage-dependent manner by an open channel block mechanism, which determines the frequency of channel openings and constitutes the sole parameter that underlies its voltage dependence. Whole cell recordings from a Drosophila mutant expressing only TRPL indicated that Ca2+ block also accounts for the voltage dependence of the native TRPL channels. The open channel block by Ca2+ that we characterized is a useful mechanism to improve the signal to noise ratio of the response to intense light when virtually all the large conductance TRPL channels are blocked and only the low conductance TRP channels with lower Ca2+ affinity are active

Glial ER and GAP junction mediated Ca 2+ waves are crucial to maintain normal brain excitability

Astrocytes play key roles in regulating multiple aspects of neuronal function from invertebrates to humans and display Ca2+ fluctuations that are heterogeneously distributed throughout different cellular microdomains. Changes in Ca2+ dynamics represent a key mechanism for how astrocytes modulate neuronal activity. An unresolved issue is the origin and contribution of specific glial Ca2+ signaling components at distinct astrocytic domains to neuronal physiology and brain function. The Drosophila model system offers a simple nervous system that is highly amenable to cell-specific genetic manipulations to characterize the role of glial Ca2+ signaling. Here we identify a role for ER store-operated Ca2+ entry (SOCE) pathway in perineurial glia (PG), a glial population that contributes to the Drosophila blood-brain barrier. We show that PG cells display diverse Ca2+ activity that varies based on their locale within the brain. Ca2+ signaling in PG cells does not require extracellular Ca2+ and is blocked by inhibition of SOCE, Ryanodine receptors, or gap junctions. Disruption of these components triggers stimuli-induced seizure-like episodes. These findings indicate that Ca2+ release from internal stores and its propagation between neighboring glial cells via gap junctions are essential for maintaining normal nervous system function

<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

<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

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>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 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