Reduced odor responses from antennal neurons of G<SUB>q</SUB>&#945;, phospholipase C&#946;, and rdgA mutants in Drosophila support a role for a phospholipid intermediate in insect olfactory transduction

Mechanisms by which G-protein-coupled odorant receptors transduce information in insects still need elucidation. We show that mutations in the Drosophila gene for Gq&#945; (dgq) significantly reduce both the amplitude of the field potentials recorded from the whole antenna in responses to odorants as well as the frequency of evoked responses of individual sensory neurons. This requirement for Gq&#945; is for adult function and not during antennal development. Conversely, brief expression of a dominant-active form of Gq&#945; in adults leads to enhanced odor responses. To understand signaling downstream of Gq&#945; in olfactory sensory neurons, genetic interactions of dgq were tested with mutants in genes known to affect phospholipid signaling. dgq mutant phenotypes were further enhanced by mutants in a PLC&#946; (phospholipase C&#946;) gene, plc21C. Interestingly although, the olfactory phenotype of mutant alleles of diacylglycerol kinase (rdgA) was rescued by dgq mutant alleles. Our results suggest that Gq&#945;-mediated olfactory transduction in Drosophila requires a phospholipid second messenger the levels of which are regulated by a cycle of phosphatidylinositol 1,4-bisphosphate breakdown and regeneration

Mutants in Drosophila TRPC channels reduce olfactory sensitivity to carbon dioxide.

BACKGROUND: Members of the canonical Transient Receptor Potential (TRPC) class of cationic channels function downstream of Gαq and PLCβ in Drosophila photoreceptors for transducing visual stimuli. Gαq has recently been implicated in olfactory sensing of carbon dioxide (CO(2)) and other odorants. Here we investigated the role of PLCβ and TRPC channels for sensing CO(2) in Drosophila. METHODOLOGY/PRINCIPAL FINDINGS: Through behavioral assays it was demonstrated that Drosophila mutants for plc21c, trp and trpl have a reduced sensitivity for CO(2). Immuno-histochemical staining for TRP, TRPL and TRPγ indicates that all three channels are expressed in Drosophila antennae including the sensory neurons that express CO(2) receptors. Electrophysiological recordings obtained from the antennae of protein null alleles of TRP (trp(343)) and TRPL (trpl(302)), showed that the sensory response to multiple concentrations of CO(2) was reduced. However, trpl(302); trp(343) double mutants still have a residual response to CO(2). Down-regulation of TRPC channels specifically in CO(2) sensing olfactory neurons reduced the response to CO(2) and this reduction was obtained even upon down-regulation of the TRPCs in adult olfactory sensory neurons. Thus the reduced response to CO(2) obtained from the antennae of TRPC RNAi strains is not due to a developmental defect. CONCLUSION: These observations show that reduction in TRPC channel function significantly reduces the sensitivity of the olfactory response to CO(2) concentrations of 5% or less in adult Drosophila. It is possible that the CO(2) receptors Gr63a and Gr21a activate the TRPC channels through Gαq and PLC21C

Stimulation of angiogenesis using single-pulse low-pressure shock wave treatment

Endothelial cells respond to mechanical stimuli such as stretch. This property can be exploited with caution to induce angiogenesis which will have immense potential to treat pathological conditions associated with insufficient angiogenesis. The primary aim of this study is to test if low-pressure shock waves can be used to induce angiogenesis. Using a simple diaphragm-based shock tube, we demonstrate that a single pulse of low pressure (0.4bar) shock wave is enough to induce proliferation in bovine aortic endothelial cells and human pulmonary microvascular endothelial cells. We show that this is associated with enhanced Ca++ influx and phosphorylation of phosphatidylinositol-3-kinase (PI3K) which is normally observed when endothelial cells are exposed to stretch. We also demonstrate the pro-angiogenic effect of shock waves of single pulse (per dose) using murine back punch wound model. Shock wave treated mice showed enhanced wound-induced angiogenesis as reflected by increased vascular area and vessel length. They also showed accelerated wound closure compared to control mice. Overall, our study shows that just a single pulse/shot (per dose) of shock waves can be used to induce angiogenesis. Importantly, we demonstrate this effect using a pulse of low-pressure shock waves (0.4bar, in vitro and 0.15bar, in vivo).Key messagesLow-pressure single-pulse shock waves can induce endothelial cell migration and proliferation.This effect is endothelial cell specific.These shock waves enhance wound-induced angiogenesis in vivo.These shock waves can also accelerate wound healing in vivo

Electrophysiological recordings from the antennae of <i>plc21C</i> mutants.

<p>A) Representative traces of field recordings obtained from the basiconica rich region of the 3^rd antennal segment of 3 to 4 days old flies. Individual genotypes are indicated. Both the <i>plc21C</i> mutants show reduced electrophysiological responses to the three concentrations of CO₂ tested as compared to the wild type flies (n = 10, <i>p<</i>0.0001). <i>Gr63a</i> null mutants (<i>Gr63a−/−</i>) and an RNAi knockdown of Gαq in CO₂ sensitive neurons (<i>Gr21aGAL4>UASGαq^1F1</i>) were included as test controls. B) Quantification of the field recordings for the genotypes tested (n = 10; <i>p<</i>0.0001). Error bars indicate SEM.</p

Expression of TRPC proteins in CO₂ sensing neurons located in the third antennal segment of adult <i>Drosophila</i>.

<p>TRP, TRPL and TRPγ are expressed in CO₂ responsive neurons in the adult <i>Drosophila</i> antenna. A) Frozen antennal sections (14 µm thick) from <i>Gr21aGAL4/UASH2bRFP</i> animals stained with anti-TRP, anti-TRPL and anti-TRPγ antibodies showing expression of TRP, TRPL and TRPγ respectively along the membranes of the Gr21a receptor neurons, marked by anti- RFP staining in red. The first panel shows the localization of Gr21a neurons in the antenna after staining with anti- RFP. B) Frozen antennal sections (14 µm thick) from the null mutants of <i>trpl</i> and <i>trp</i> stained with anti-TRPL and anti-TRP antibodies respectively. No expression of TRPL and TRP proteins could be observed in the respective mutant strains. mAb22C10 (anti-futch, microtubule protein) staining in red served as a neuronal marker.</p

Null mutants of <i>trp</i> and <i>trpl</i> show reduced behavioral avoidance towards CO₂.

<p>A) The mean avoidance index towards 5% CO₂ in a Y-maze behavioral assay is shown for the indicated genotypes. The ability of <i>trpl</i> null homozygotes (<i>trpl³⁰²/trpl³⁰²</i>), to discriminate between 5% CO₂ and air is significantly reduced (<i>p<</i>0.0001) as compared to the heterozygous control. The phenotype of the null mutant is rescued by expressing a wild type <i>trpl</i> transgene in Gr21a receptor neurons (<i>trpl³⁰²/trpl³⁰²; Gr21a GAL4/UAS trpl⁺</i>) (<i>p<</i>0.0001). B) Null mutant of <i>trp</i> (<i>trp³⁴³/trp³⁴³</i>) has reduced avoidance to 5% CO₂ in the Y-maze assay. The avoidance response of the double null mutant (<i>trpl³⁰²/trpl³⁰²; trp³⁴³/trp³⁴³</i>) is also reduced but not significantly different from the single null homozygotes (<i>p></i>0.05). Error bars indicate SEM in A and B.</p

Reduced sensitivity to CO₂ is not a developmental defect.

<p>A) RNAi lines grown at the restrictive temperature of 18°C (active GAL80) show normal electrophysiological responses to CO_2, since the CO₂ receptor neuron specific GAL4 remains inactive (absence of RNAi expression; <i>p></i>0.05). RNAi lines grown at the permissive temperature of 29°C (inactive GAL80) show reduced electrophysiological responses to CO₂ due to active GAL4 and RNAi expression (n = 10; <i>p<</i>0.0001). The RNAi heterozygotes in the absence of <i>Gr63aGAL4</i> show normal responses to CO₂ at 29°C. Error bars indicate SEM. B) Whole antennal mounts showing CO₂ sensory neurons marked using <i>UAS RedStinger</i> driven by <i>Gr21aGAL4</i> in wild type, <i>plc21C^P319/plc21C^P319</i> and <i>trpl³⁰²/trpl³⁰²</i> mutant lines. C) Quantification of CO₂ sensory neurons in the adult antennae of the same lines (n = 14; <i>p</i> value not statistically significant).</p