Inducing sleep by remote control facilitates memory consolidation in Drosophila

Abstract

Sleep is believed to play an important role in memory consolidation. We induced sleep on demand by expressing the temperature-gated nonspecific cation channel Transient receptor potential cation channel (UAS-TrpA1) in neurons, including those with projections to the dorsal fan-shaped body (FB). When the temperature was raised to 31°C, flies entered a quiescent state that meets the criteria for identifying sleep. When sleep was induced for 4 hours after a massed-training protocol for courtship conditioning that is not capable of inducing long-term memory (LTM) by itself, flies develop an LTM. Activating the dorsal FB in the absence of sleep did not result in the formation of LTM after massed training.

Fig. 1

Chronic sleep induction. (A and B) Expressing UAS-NaChBac using C5-GAL4 or 104y-GAL4 increased sleep. Data presented as sleep in min/hour (n = 12 to 16 per group). Error bars indicate xxxxx; ZT, zeitgeber time. (C) C5/+>UAS-CD8::GFP and 104y/+>UAS-CD8::GFP expression. (D) Total sleep was increased in C5/+>UAS-NaChBac/+ and 104y/+>UAS-NaChBac/+ flies; *P < 0.05, modified Bonferroni test. (E and F) Neuronal activation with C5 and 104y increased daytime and nighttime sleep bout duration. (G) Intensity of waking activity was increased in C5/+>UAS-NaChBac/+ and 104y/+>UAS-NaChBac/+ flies. (H) A light pulse during the night (gray) awakened all genotypes compared with undisturbed siblings. χ², n = 23 to 29 each group, *P < 0.05; Mann-Whitney U test comparing each group to untreated siblings. (I) All genotypes exhibited a sleep rebound after 12 hours of sleep deprivation. (J and K) Expressing UAS-NaChBac using NP6510-GAL4 or C232-GAL4 did not change sleep. (L) NP6510/+>UAS-CD8::GFP/+ labeled the ventral FB, whereas C232/+>UAS-CD8::GFP/+ was expressed in the EB.

Fig. 2

Inducing sleep on demand. (A) Capsaicin-fed 104y/+>UAS-TrpVr1/+ flies increased sleep. (B to D) Transferring C5/+>UAS-TrpA1/+ and 104y/+>UAS-TrpA1/+ flies to 31°C for 6 hours increased sleep. *P < 0.05 modified Bonferroni test. (E) Mean sleep bout duration was increased in C5/+>UAS-TrpA1/+ and 104y/+>UAS-TrpA1/+ flies at 31°C. (F) Counts per waking minute increased at 31°C in C5/+>UAS-TrpA1/+ and 104y/+>UAS-TrpA1/+ flies. (G) Awakenings increased with perturbation number. SNAP, sxxxx nxxxx axxxxx pxxxxx. (H) 104y/+>UAS-TrpA1/+ flies fed caffeine (2.5 mg/ml or 7.5 mg/ml) 1 hour before 31°C exposure display weakened sleep induction.

Fig. 3

Inducing sleep reverses deficits in LTM after social enrichment. (A) Quantification of dlg-positive terminals expressed as percent of isolated siblings (n = 13 to 16 brains per group). (B) Images of dlgGFP- positive terminals from LN_Vs in isolated flies and enriched siblings (~95 flies). (C) Schematic for evaluating LTM after social enrichment. (D) Isolated (ISO) flies reduced courtship 48 hours after training (Student’s t test P = 0.008, n = 40 to 42). Enriched siblings lacked LTM (ENR+0 days, Student’s t test P = 0.77, n = 28 or 29). Enriched flies that were isolated for 1 day before training showed no LTM (ENR+1 day, Student’s t test P = 0.61, n = 25 to 29). Enriched flies that were isolated for 3 days exhibited LTM (ENR+3 days, Student’s t test P = 0.029, n = 12 or 13). (E) Schematic for testing LTM recovery after sleep induction. (F) Isolated males displayed LTM. (G) Enriched males showed no LTM when housed at 25°C. (H) 104y/+UAS-TrpA1/+ flies that were enriched, housed at 31°C for 4 hours, then trained exhibited LTM; *P < 0.05 planned comparisons. Error bars indicate xxxx.

Fig. 4

Massed-training generates LTM when accompanied by sleep induction. (A) Schematic of experimental design. SD, sleep deprived. (B, D, and F) No LTM was detected in flies at 25°C. Planned comparisons between naïve and trained siblings found no significant differences. CI, confidence interval. (C) Activating the EB did not alter LTM. (E and G) Placing C5/+>UAS-TrpA1/+ and 104y/+>TrpA1/+ flies at 31°C for 4 hours after massed-training induced LTM. (H) Mechanical perturbation prevents sleep in 104y/+>UAS-TrpA1/+ flies at 31°C. (I) 104y/+>UAS-TrpA1/+ males sleep deprived at 31°C after massed training show no LTM (Student’s t test P = 0.54, n = 13 to 15).