Sleep restores behavioral plasticity to Drosophila mutants

Abstract

Given the role that sleep plays in modulating plasticity, we hypothesized that increasing sleep would restore memory to canonical memory mutants without specifically rescuing the causal molecular lesion. Sleep was increased using three independent strategies: activating the dorsal fan-shaped body, increasing the expression of Fatty acid binding protein (dFabp), or by administering the GABA-A agonist 4,5,6,7-tetrahydroisoxazolo-[5,4-c]pyridine-3-ol (THIP). Short-term memory (STM) or long-term memory (LTM) was evaluated in rutabaga (rut) and dunce (dnc) mutants using aversive phototaxic suppression and courtship conditioning. Each of the three independent strategies increased sleep and restored memory to rut and dnc mutants. Importantly, inducing sleep also reverses memory defects in a Drosophila model of Alzheimer's disease. Together, these data demonstrate that sleep plays a more fundamental role in modulating behavioral plasticity than previously appreciated and suggest that increasing sleep may benefit patients with certain neurological disorders.

Figure 1. THIP induces sleep in Drosophila

(A) Cs females were maintained on vehicle (veh), 10% ethanol (ETOH), 40 µM of the GABA-B agonist SKF97541, 20µM of the vesicular monoamine transporter inhibitor reserpine (res), or 0.1mg/mL of the GABA-A agonist THIP (0.1T) for 48 h. Compared to vehicle-fed controls, Cs flies maintained on ETOH, SKF97541, res and THIP showed significant increases in Daytime quiescence ANOVA F_[3,99] = 12.9; p= 3.35^E-7; the data are presented as difference from vehicle fed controls (ΔDaytime Sleep). *p<0.05 modified Bonferroni test, n=14–30 flies/group. (B) Short-term memory was significantly impaired in SKF97541, reserpine and ETOH fed Cs flies but was unchanged in flies fed THIP, ANOVA F_[3,25] = 27.6; p= 4.21^E-8; *p<0.05 modified Bonferroni test, n=5–9 flies/genotype. (C) THIP increases quiescence (min/h) in a dose-dependent manner in Cs flies. Data are presented as sleep in minutes/hour. Repeated measures ANOVAs reveals a significant Dose (4) X Hour (24) interaction (Cs: F_(69,1265)=5.15, p= 9.99^E-16 n=23–30/group). (D) Relative transcript levels of Amylase, Homer, Synaptotagmin (syt), bruchpilot (brp), Syntaxin18 (syx18), Metchnikowin (Mtk), Attacin-B (AttB), Drosocin (Dro), Immune induced molecule 23 (IM23), and Drosomycin (Drs) are upregulated following 12 h of sleep deprivation and reduced following 48 h of THIP (0.1T) feeding. (E) DISCS-LARGE (DLG) levels are significantly increased following 12 h of sleep deprivation (left) but reduced by 48 h of THIP treatment as revealed by Western blots (right) (n=3, 6 brains /group). (F) Representative traces of local field potentials from individual vehicle-fed (Left) and THIP-fed (right) flies during waking and quiescence. (G) Representative power spectra during waking and sleep from the flies presented in 1F: vehicle-fed (left) and THIP-fed fly (right). (H) Schematic of the training protocol. (I) Cs flies maintained on vehicle (veh) post-training do not have an LTM (black bars) while flies whose sleep was increased with THIP for 4 h immediately following training resulted in an LTM, (white bars); Krustal-Wallis, p= 0.008, n=16–20 flies/group, Performance Index (PI). (J) No memory is detected when Cs flies are fed either veh (black bars) or SKF97541 (white bars) following training; n=17–20 flies/group. Error bars, s.e.m.;*P<0.05.

Figure 2. Inducing sleep in rutabaga mutants restores short term memory and long term memory

(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.

Figure 3. Inducing sleep in dunce mutants restores short term memory and long term memory

(A) dnc¹ mutants exhibit deficits in STM (veh) which are reversed following 48 h of THIP-induced sleep (0.1T); mutants maintained on THIP but sleep deprived are learning impaired (0.1T^SD) (n=>8/group). One-way ANOVA F_[2,21] = 9.5; p=0.001; *P<0.05, modified Bonferroni test. (B) Individual dnc¹ flies maintained on vehicle exhibit disrupted STM when tested on two trials spaced two days apart (V1 and V2). (C) Individual vehicle-fed dnc¹ flies showed impaired STM which is reversed following 2 days of THIP-induced sleep (0.1T2). (D) Mean performance scores ± SEM for dnc¹ maintained on vehicle (V1, V2) or switched from vehicle (V1) to THIP for 2-days (T2); paired t-test, *p<0.05. (E) RU-fed DaGsw/+>UAS-dnc^RNAi/+ flies display impaired STM that is reversed by 48 h of THIP-induced sleep (RU^0.1T); vehicle-fed flies on and off THIP (veh^0.1T, veh) display normal STM; A 2(Vehicle, RU) x 2 (Vehicle, THIP) ANOVA yields a significant interaction F_[1,30] = 10.13; p=0.003; n=8 flies/group, *P<0.05, modified Bonferroni test. (F)dnc¹;104y–GAL4/+>UAS-NaChBac/+ and dnc¹;;C5-GAL4/+>UAS-NaChBac/+ lines display normal STM; in contrast, performance is impaired in all parental controls, One way ANOVA F_[4,35] = 8.75; p= 5.26^E-05, *P<0.05, n=8 flies/group modified Bonferroni test. (G) dnc¹;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, main effect for Genotype F_[2,45] = 6.2; p= 0.004, n=8 flies/group *P<0.05, modified Bonferroni test. (H) dnc¹;;dFabp /+ sleep more at 30°C than their siblings maintained at 20°C, *p<0.05, ttest, n=15–16 flies/ condition. (I) When dnc¹;;dFabp /+ flies are maintained at 20°C, they display impairments in STM; these impairments are reversed when sleep is increased for 2 days by placing the flies at 30°C. Importantly no improvements in STM are observed in the absence of sleep. A oneway ANOVA yielded a significant effect for condition F_[2,30] = 7.5; p=0.002 modified Bonferroni Test ,n=8–12 flies /condition.(J) STM impairments are reversed in dnc¹ mutants after 12 h of THIP-induced sleep, One-way ANOVA F_[3,30] = 5.99; P=0.002; n>=8 flies/group, *P<0.05, n=8 flies/group modified Bonferroni test. (K) dnc¹ mutants continue to exhibit STM for 24 h after being removed from THIP, One-way ANOVA F_[3,30] = 5.06; P=0.003; n>=8 flies/group, *P<0.05, modified Bonferroni test. (L) Schematic of the protocol used for courtship conditioning. (M) No change in the Performance Index (PI) is observed in vehicle-fed dnc¹ mutants following training; in contrast increasing sleep with 0.1T results in LTM; Krustal-Wallis p=0.026, n=16–20 flies/group. Error bars, s.e.m.;*P<0.05.

Figure 4. THIP requires the Fan Shaped body to increase sleep

(A) Expressing UAS-Kir2.1 in 104y–GAL4 expressing cells disrupts sleep in a rut²⁰⁸⁰ mutant background. Both rut²⁰⁸⁰;104y/+ and rut²⁰⁸⁰; UAS-Kir2.1/+ parental controls sleep normally. A 3(genotype) X 24 (Time) ANOVA revealed a significant Genotype X Time interaction F_[46,966] = 6.68; p=9.99^E-016 consistent with previous reports (n=14–16 flies/group). (B) THIP does not result in an increase in Daytime sleep in rut²⁰⁸⁰;104y/+> UAS-Kir2.1/+ flies; while both rut²⁰⁸⁰;104y/+ and rut²⁰⁸⁰; UAS-Kir2.1/+ parental controls increase sleep as expected. ΔSleep is calculated by subtracting sleep in THIP fed flies from vehicle-fed siblings. A One way ANOVA for Genotype: F_[2,43] = 76.2; p=7.24^E-15, *p<0.05 modified Bonferroni test, n=15–16 flies /group. (C) THIP does not restore STM to rut²⁰⁸⁰;104y/+> UAS-Kir2.1/+ flies but returns STM to normal in parental controls that increase their sleep. A 3(Genotype) X 2 (Drug) ANOVA revealed differential responses to THIP: F_[1,49] = 15.98; p=2.14^E-004, *p<0.05 modified Bonferroni test, n=8–12 flies /group. (D) THIP (0.1mg/ml) treated Cs, rut²⁰⁸⁰ and dnc¹ flies and their vehicle-fed siblings were collected for Western blot analysis (n=4 brains/condition). Experiments were run in triplicate, a representative blot is shown. The graphs are the quantification (mean ± SEM) expressed as % change relative to vehicle (t-test *, p < 0.05). (E) Compared to rut²⁰⁸⁰, both aru^8.128/+ and rut²⁰⁸⁰; aru^8.128 /+ mutants exhibit a significant increase in DLG protein, ttest *p<.05. (F) Single mutants for either rut²⁰⁸⁰or aru^8.128/+ display impairments in STM (black and white bars, respectively); however, rut²⁰⁸⁰;aru^8.128/+ flies (gray bar) have normal STM. *p<0.05 ttest, n=8–9 flies/genotype. (G)DaGsw/+>UAS-aru^RNAi/+ flies fed RU486 (RU)display significant memory impairments compared to vehicle fed controls (Veh); *p<0.05, ttest. (H) Knocking down aru using DaGsw does not restore STM in a dnc¹ mutant background p>0.05, ttest n=8 flies/group. (I) Vehicle–fed rut²⁰⁸⁰;DaGsw/+>UAS-aru^RNAi/+ flies display STM impairments while RU-fed siblings exhibit STM; *p<0.05, ttest, n=8 flies/group. Error bars, s.e.m.;*P<0.05.

Figure 5. Sleep Induction fully restores LTM to Presenilin mutants

(A,E) Young 7-d old Psn^B3/+, Psn^C4/+ and Cs flies, show similar sleep profiles. (B,F) Young Psn^B3/+ (n=16/naïve and n=14/trained) and Psn^C4/+ (n=10/naïve and n=11/trained) flies display normal LTM as assessed using courtship conditioning; Krustal-Wallis p=0.007 Performance Index (PI). (C,G) 30 day old Psn^B3/+ and Psn^C4/+ flies increase sleep in response to 0.1T. (D,H) No LTM is observed in vehicle-fed 30-d old Psn^B3/+ (n=16 for both groups) and Psn^C4/+ (n=22/naïve and n=27/trained) flies after spaced training (black bars). Increasing sleep with 0.1T results in LTM in 30-d old Psn^B3/+ (n=16 for both groups) and Psn^C4/+ flies (n=15/naïve and n=21/trained); white bars. Error bars, s.e.m.;*P<0.05.