Figure 7.—

Disruption of GABAergic signaling or synaptic transmission in MB reduces the bang sensitivity of kcc^DHS1 flies. (A) Expression of the UAS-kcc⁺ transgene in GABAergic neurons reduces their bang sensitivity. Expression of the wild-type kcc⁺ gene in the neurons targeted by either of two GAL4 drivers (Rdl, those expressing the Rdl GABA_A receptor and Gad1, those producing GABA) produced marked reductions in bang sensitivity. Expression of kcc⁺ in neurons targeted by the Cha GAL4 driver (neurons producing acetylcholine) resulted in more modest, albeit statistically significant, reductions in bang sensitivity. In these experiments, virgin females carrying the kcc^DHS1 mutation as well as a particular GAL4 driver (i.e., D670, D590, or D589) were crossed to D634 males carrying the kcc^DHS1 mutation and a UAS-kcc⁺ transgene over a TM6B balancer chromosome. The percentage bang sensitivity of the kcc^DHS1 + Driver-GAL4 + UAS-kcc⁺ test progeny is given relative to that of their kcc^DHS1 + Driver-GAL4 control siblings. n > 80; (**) P < 0.001, (*) P < 0.05. (B) Reducing the dosage of the Rdl GABA_A receptor by mutation or RNAi reduces the bang sensitivity of kcc^DHS1 flies. kcc^DHS1 flies heterozygous for either of two Rdl mutations (Rdl¹ or Rdl^MD-RR) displayed almost one-third the bang sensitivity (39% or 35% relative BS, respectively) of their control siblings. Mushroom body expression of an RNAi construct that reduces the level of Rdl produces an even more marked reduction in the level of bang sensitivity of kcc^DHS1 mutant flies (25% relative bang sensitivity). In the first set of experiments, D506 virgin females homozygous for kcc^DHS1 were crossed to males carrying kcc^DHS1 and the corresponding Rdl mutation over a TM6B balancer. The percentage bang sensitivity of the kcc^DHS1; Rdl/+ test progeny is presented relative to that of the corresponding kcc^DHS1 control siblings. In the last experiment, D689 virgin females carrying kcc^DHS1 and the MB GAL4 driver c772 were crossed to D691 males carrying kcc^DHS1 as well as an Rdl RNAi construct over a TM6B balancer. The percentage bang sensitivity of the kcc^DHS1 + c772 + Rdl-RNAi test progeny is presented relative to that of the corresponding kcc^DHS1 + c772 control siblings. n > 220; (**) P < 0.001. (C) Reducing the level of the Ncc69 Na⁺–K⁺–Cl⁻ cotransporter specifically in MB reduces bang sensitivity. kcc^DHS1 flies carrying either of two lesions in Ncc69 [Ncc69^PL00618 or Df(3L)eyg^C1] displayed a modest reduction in bang sensitivity compared to their control siblings (lightly shaded bars; 65–66% relative bang sensitivity). By contrast, MB expression of an Ncc69 RNAi construct produces a marked reduction in the level of bang sensitivity of kcc^DHS1 mutant flies (dark-shaded bars; 31% relative bang sensitivity). In the first set of experiments, D506 virgin females homozygous for kcc^DHS1 were crossed to males carrying kcc^DHS1 and an Ncc69 mutation over a TM6B balancer. The percentage bang sensitivity of the kcc^DHS1; Ncc69/+ test progeny is presented relative to that of the corresponding kcc^DHS1 control siblings (n > 75). In the last experiment, D661 virgin females carrying kcc^DHS1 and the MB GAL4 driver c772 was crossed to D690 males carrying kcc^DHS1 as well as an RNAi construct for Ncc69 over a TM6B balance. The percentage bang sensitivity of the kcc^DHS1 + c772 + Ncc69-RNAi test progeny is presented relative to that of the corresponding kcc^DHS1 + c772 control siblings. n > 360; (**) P < 0.001. (D) Blocking synaptic transmission in MB ameliorates the bang sensitivity of kcc^DHS1 flies. Tetanus toxin (TNT) blocks synaptic transmission by cleaving synoptobrevin, which is necessary for synaptic vesicle fusion (Martin et al. 2002). Flies that express tetanus toxin specifically in their mushroom bodies show a significant reduction in bang sensitivity (35% of control levels, dark-shaded bar); no such reduction was observed when synaptic transmission was instead blocked in the antennal neurons that project to the MB (97% of control, lightly shaded bar). In the first experiment, D661 flies carrying kcc^DHS1 and the MB driver MB247 were crossed to either D700 males carrying kcc^DHS1 and a UAS-TNT transgene or D506 males carrying only kcc^DHS1; percentage bang sensitivity of kcc^DHS1 + MB247 + UAS-TNT progeny from the first cross is presented relative to that of kcc^DHS1 + MB247 progeny from the second cross. In the second experiment, D695 flies carrying kcc^DHS1 and the GH146 GAL4 driver were crossed to either D700 or D506; percentage bang sensitivity of kcc^DHS1 + GH146 + UAS-TNT progeny from the first cross is presented relative to that of kcc^DHS1 + GH146 progeny from the second cross. n > 110; (**) P < 0.001.