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

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

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