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Published in final edited form as: Curr Biol. 2015 Apr 23;25(10):1270–1281. doi: 10.1016/j.cub.2015.03.027

(A) No improvement in STM is observed in 3–5 day old Cs, w¹¹¹⁸ , Ore-R, ry⁵⁰⁶ or Berlin flies maintained on 0.1mg/mL of THIP compared to vehicle-fed controls. A 5 (Genotype) x 2 (Veh, THIP) ANOVA failed to identify any main effects nor a Genotype X Drug interaction, F_[4,69]=1.4,p=0.22; (n=8/group); nonsignificant (n.s.) modified Bonferroni test. (B,C) rut²⁰⁸⁰ and rut¹ mutants exhibit deficits in STM (veh) which are reversed following 48 h of sleep induced by THIP (0.1T); mutants maintained on THIP but sleep deprived are learning impaired (0.1T^SD) (n=>8/group). One way ANOVA for rut²⁰⁸⁰ F_[2,21] = 4.09; p=0.03 and for rut¹ F_[2,21] = 5.35; p=0.01;*P<0.05, modified Bonferroni test. For comparison, the symbol indicates wild-type performance. (D) Individual rut²⁰⁸⁰ maintained on vehicle reliably choose the lighted vial on two trials spaced two days apart (V1 and V2). (E) Individual rut²⁰⁸⁰ flies showed performance decrements while on vehicle (V1) and these decrements were reversed following 2 days of THIP-induced sleep (0.1T2). (F) Mean performance scores ± SEM for rut²⁰⁸⁰ maintained on vehicle (V1, V2) or switched from vehicle (V1) to THIP for 2-days (T2); paired t-test, *p<0.05. (G,H). Neither RU nor THIP influence STM in DaGsw/+ or rut^RNAi/+ parental controls; main effect for RU (F_[1,28] = 0.21; p=0.64, and F_[1,28] = 0.16; p=0.69, respectively), and THIP(F_[1,28] = 0.21; p=0.64, F_[1,28] = 0.16; p=0.69, respectively). (I) RU disrupts STM in DaGsw/+>UAS-rut^RNAi/+ flies; main effect for RU (F_[1,28] = 11.06; p=0.002). THIP restores STM to RU fed DaGsw/+>UAS-rut^RNAi/+ flies (RU^01T); main effect for THIP (F_[1,28] = 6.6; p=0.02); n=8 flies/group, *P<0.05, modified Bonferroni test. (J) STM impairments are reversed in rut²⁰⁸⁰ mutants after 24 h, but not 12 h, of THIP-induced sleep, One way ANOVA F_[3,29] = 3.0; P=0.04; n>=8 flies/group, *P<0.05, modified Bonferroni test. (K)rut²⁰⁸⁰ mutants continue to exhibit STM for 48 h after being removed from THIP, One way ANOVA F_[3,33] = 8.4; P=0.0002; n>=8 flies/group, *P<0.05, modified Bonferroni test. (L)rut²⁰⁸⁰;104y–GAL4/+>UAS-NaChBac/+ and rut²⁰⁸⁰;;C5-GAL4/+>UAS-NaChBac/+ lines display normal STM; in contrast, performance is impaired in all parental controls, One way ANOVA F_[4,33] = 7.01; p=3.380^E-004, *P<0.05, n=8 flies/group, modified Bonferroni test. (M)rut²⁰⁸⁰;104yGAL4/+>UAS-TrpA1/+ flies display normal STM following sleep induction for 24 h at 31°C compared to siblings maintained at 25°C; STM remains impaired in parental controls at 25°C and 31°C. A 3(genotype) X 2 (temperature) ANOVA revealed a significant genotype X temperature interaction F_[2,42] = 16.4; p= 5.39^E-06, *P<0.05, n=8 flies/group, modified Bonferroni test. (N) w (isoCJ1) background controls exhibit similar daytime sleep at both 20°C and 30°C; p>0.05, ttest, n=16 flies/condition. w (isoCJ1) flies display similar performance scores in the APS at 20°C and after being maintained at 30°C for 2 days ; p>0.05, ttest, n=8 flies/condition. (O) dFabp/+ flies sleep more at 30°C than at 20° consistent with previous reports; *p<.05, ttest, n=15–16 flies/condition. Increasing sleep by placing dFabp/+ flies at 30°C for two days does not improve STM; p>0.05, ttest, n=8–10 flies/condition. (P) Placing rut²⁰⁸⁰;;dFabp/+ at 30°C increases sleep compared to siblings maintained at 20°C, *p<0.05, ttest, n=15–16 flies/condition. At 20 °C, rut²⁰⁸⁰;;dFabp /+ exhibit STM impairments which are reversed when sleep is increased by placing flies at 30°C; the improvements in STM are not observed in the absence of sleep (30°C SD). One way ANOVA for condition :F_[2,25] = 3.4; p=0.05, *p<0.05 modified Bonferroni test, 8–10 flies/condition. (Q) Flies were maintained on vehicle or THIP for 2 days. THIP-fed flies removed from THIP and placed onto normal food at 10am sleep less than vehicle-fed controls (n=16). (R) Schematic of the protocol used for courtship conditioning. (S) No change in the Performance Index (PI) is observed in vehicle-fed rut²⁰⁸⁰mutants following training; in contrast increasing sleep with 0.1T results in LTM; Krustal-Wallis p=0.007. n=16–20 flies/group. Error bars, s.e.m.;*P<0.05.

Inline graphic — (A) No improvement in STM is observed in 3–5 day old Cs, w¹¹¹⁸ , Ore-R, ry⁵⁰⁶ or Berlin flies maintained on 0.1mg/mL of THIP compared to vehicle-fed controls. A 5 (Genotype) x 2 (Veh, THIP) ANOVA failed to identify any main effects nor a Genotype X Drug interaction, F_[4,69]=1.4,p=0.22; (n=8/group); nonsignificant (n.s.) modified Bonferroni test. (B,C) rut²⁰⁸⁰ and rut¹ mutants exhibit deficits in STM (veh) which are reversed following 48 h of sleep induced by THIP (0.1T); mutants maintained on THIP but sleep deprived are learning impaired (0.1T^SD) (n=>8/group). One way ANOVA for rut²⁰⁸⁰ F_[2,21] = 4.09; p=0.03 and for rut¹ F_[2,21] = 5.35; p=0.01;*P<0.05, modified Bonferroni test. For comparison, the symbol indicates wild-type performance. (D) Individual rut²⁰⁸⁰ maintained on vehicle reliably choose the lighted vial on two trials spaced two days apart (V1 and V2). (E) Individual rut²⁰⁸⁰ flies showed performance decrements while on vehicle (V1) and these decrements were reversed following 2 days of THIP-induced sleep (0.1T2). (F) Mean performance scores ± SEM for rut²⁰⁸⁰ maintained on vehicle (V1, V2) or switched from vehicle (V1) to THIP for 2-days (T2); paired t-test, *p<0.05. (G,H). Neither RU nor THIP influence STM in DaGsw/+ or rut^RNAi/+ parental controls; main effect for RU (F_[1,28] = 0.21; p=0.64, and F_[1,28] = 0.16; p=0.69, respectively), and THIP(F_[1,28] = 0.21; p=0.64, F_[1,28] = 0.16; p=0.69, respectively). (I) RU disrupts STM in DaGsw/+>UAS-rut^RNAi/+ flies; main effect for RU (F_[1,28] = 11.06; p=0.002). THIP restores STM to RU fed DaGsw/+>UAS-rut^RNAi/+ flies (RU^01T); main effect for THIP (F_[1,28] = 6.6; p=0.02); n=8 flies/group, *P<0.05, modified Bonferroni test. (J) STM impairments are reversed in rut²⁰⁸⁰ mutants after 24 h, but not 12 h, of THIP-induced sleep, One way ANOVA F_[3,29] = 3.0; P=0.04; n>=8 flies/group, *P<0.05, modified Bonferroni test. (K)rut²⁰⁸⁰ mutants continue to exhibit STM for 48 h after being removed from THIP, One way ANOVA F_[3,33] = 8.4; P=0.0002; n>=8 flies/group, *P<0.05, modified Bonferroni test. (L)rut²⁰⁸⁰;104y–GAL4/+>UAS-NaChBac/+ and rut²⁰⁸⁰;;C5-GAL4/+>UAS-NaChBac/+ lines display normal STM; in contrast, performance is impaired in all parental controls, One way ANOVA F_[4,33] = 7.01; p=3.380^E-004, *P<0.05, n=8 flies/group, modified Bonferroni test. (M)rut²⁰⁸⁰;104yGAL4/+>UAS-TrpA1/+ flies display normal STM following sleep induction for 24 h at 31°C compared to siblings maintained at 25°C; STM remains impaired in parental controls at 25°C and 31°C. A 3(genotype) X 2 (temperature) ANOVA revealed a significant genotype X temperature interaction F_[2,42] = 16.4; p= 5.39^E-06, *P<0.05, n=8 flies/group, modified Bonferroni test. (N) w (isoCJ1) background controls exhibit similar daytime sleep at both 20°C and 30°C; p>0.05, ttest, n=16 flies/condition. w (isoCJ1) flies display similar performance scores in the APS at 20°C and after being maintained at 30°C for 2 days ; p>0.05, ttest, n=8 flies/condition. (O) dFabp/+ flies sleep more at 30°C than at 20° consistent with previous reports; *p<.05, ttest, n=15–16 flies/condition. Increasing sleep by placing dFabp/+ flies at 30°C for two days does not improve STM; p>0.05, ttest, n=8–10 flies/condition. (P) Placing rut²⁰⁸⁰;;dFabp/+ at 30°C increases sleep compared to siblings maintained at 20°C, *p<0.05, ttest, n=15–16 flies/condition. At 20 °C, rut²⁰⁸⁰;;dFabp /+ exhibit STM impairments which are reversed when sleep is increased by placing flies at 30°C; the improvements in STM are not observed in the absence of sleep (30°C SD). One way ANOVA for condition :F_[2,25] = 3.4; p=0.05, *p<0.05 modified Bonferroni test, 8–10 flies/condition. (Q) Flies were maintained on vehicle or THIP for 2 days. THIP-fed flies removed from THIP and placed onto normal food at 10am sleep less than vehicle-fed controls (n=16). (R) Schematic of the protocol used for courtship conditioning. (S) No change in the Performance Index (PI) is observed in vehicle-fed rut²⁰⁸⁰mutants following training; in contrast increasing sleep with 0.1T results in LTM; Krustal-Wallis p=0.007. n=16–20 flies/group. Error bars, s.e.m.;*P<0.05.