Starvation influences the strength and stability of memory

A, Appetitive olfactory learning and memory paradigm. B, Appetitive 2 min STM and 6 h memory in w1118 and TβhnM18 after 16 h (white bars) or 40 h starvation (dark gray bars) before the training using 2 M sucrose as reinforcer. Prolonged starvation increases memory performance. Prolonged starvation of TβhnM18 for 40 h resulted in STM. Independent of the duration of starvation, 6 h after training, memory appeared in the mutants. E, Appetitive STM training with 2 M sucrose and cold shock directly or 2 h after training. Prolonged starvation shifts in control STM to LTM and in TβhnM18 LTM to ARM. Numbers below box blots indicate pairs of reciprocally trained independent groups of male flies. Student’s t tests were used to determine differences between two groups, and one-way ANOVA with Tukey’s post hoc HSD test was used to determine differences between three groups. The letter “a” marks a significant difference from random choice as determined by a one-sample sign test (P* < 0.05; P**< 0.01).

Octopamine acts upstream of dopamine and negatively regulates memory

A, A 30 min block of dopaminergic PAM neurons required for LTM after training using a temperature-sensitive shibire transgene under the control of the R1504-Gal4 driver results in TβhnM18 mutants losing LTM. B, Feeding 3 mM of the octopamine receptor antagonist Epinastine for 1 h directly after training resulted in w1118 flies in memory 6 h later. Feeding 3 mM octopamine for 6 h after training suppresses LTM in TβhnM18. C, A 3 mM octopamine feeding pulse 30 min before training inhibits STM in TβhnM18 mutants. Controls were water-fed. One-way ANOVA with Tukey’s HSD post hoc test was used to determine differences between three groups, and Student’s t tests were used for two groups. The letter “a” marks a significant difference from random choice as determined by a one-sample sign test (P* < 0.05; P**< 0.01).

Elevated glycogen levels correlate with reduced sucrose preference

A, Analysis of whole-body glycogen levels in w1118 and TβhnM18 flies. Glycogen levels in TβhnM18 flies are significantly higher than those in w1118 flies under similar starvation conditions. N = 3 groups of 5 male flies. B, Flies were starved 18 h or 40 h before food intake was measured for 24 h. Flies chose between 5% sucrose and 5% yeast. The preference was determined. All flies showed a significant preference for sucrose consumption. Starvation reduced the preference. TβhnM1818 showed a significantly reduced preference for sucrose after 18 h. N = 20-26 groups of eight flies. C, Feeding flies for 3 days on standard, 5% sucrose or 5% yeast resulted in control flies preferring to consume sucrose. TβhnM1818 mutants fed normal food and sucrose showed a significant reduction in sucrose preference but not when fed 5% yeast for three days. N = 20-28 groups of eight flies. To determine differences between two groups, Student’s t test was used. P* < 0.05; P**< 0.01, P***< 0.001.

Carbohydrate storage influences appetitive STM

A, Schema of glycogen synthesis. The expression of GlyP-RNAi reduced glycogen phosphorylase and increased glycogen levels, whereas GlyS-RNAi reduced glycogen synthase and decreased glycogen levels in target tissues. B-D, PAS was used to visualize glycogen levels in larval muscle or fat bodies. B, Increases in glycogen in the muscles have no effect on STM, whereas reduced muscle glycogen increases appetitive STM. C, Increased or decreased glycogen levels in the fat bodies did not interfere with STM. D, A combined increase in glycogen in muscles and fat bodies reduced STM, and a decrease in glycogen increased STM. Student’s t tests were used to determine differences between two groups, and one-way ANOVA with post hoc Tukey’s HSD was used to determine differences between three or more groups. The letter “a” marks a significant difference from random choice as determined by a one-sample sign test (P* < 0.05; P** < 0.01). Numbers below box blots indicate one pair of reciprocally trained independent fly groups.

Reducing glycogen in Tβhnm18 improves appetitive STM

A, Decreasing glycogen concentration using UAS-GlySRNAi in the muscles or fat bodies in TβhnM18 mutants did not improve STM, but decreasing glycogen in both tissues improved STM to control levels. B, w1118 and TβhnM18 flies formed similar levels of appetitive STM when 5% yeast was used as a reinforcer. C, Virgin females of w1118 and TβhnM18 displayed STM, whereas mated females of both genotypes did not. Differences between two groups were determined using Student’s t tests, and differences among more than two groups were determined with one-way ANOVA with Tukey’s HSD post hoc test. Differences from random choice were determined using a one-sample sign test and marked with the letter “a”. P* < 0.05; P** < 0.01. Numbers below box blots indicate one pair of reciprocally trained independent fly groups.

Insulin signaling in reward neurons regulates STM performance

A, The activated form of the InR is expressed in punctuate manner throughout the brain (in magenta) and is also detected in octopaminergic reward neurons visualized by using the UAS-mcd8::GFP transgene under the control of the Tdc2-Gal4 driver (in green). B, Blocking InR signaling in Tdc2-Gal4-targeted octopaminergic neurons does not change appetitive STM in 16 h starved flies. C, Blocking InR signaling in Tdc2-Gal4-targeted octopaminergic neurons in TβhnM18 mutants restored STM to control levels. D, A cold shock did not disrupt emerging memory in TβhnM18mutants with blocked InR under the control of the Tdc2-Gal4 driver. Student’s t test was used to determine differences between two groups, and one-way ANOVA with Tukey’s post hoc HSD test was used to determine differences between three or more groups. The letter “a” marks a significant difference from random choice as determined by a one-sample sign test (P < 0.05). n.s. is not significant; P* < 0.05; P** < 0.01.

Prolonged starvation results in rebound sucrose intake in hyperglycemic TβhnM18 mutants

A, Capillary feeder assay used to determine food intake. B, Flies were starved 16 h or 40 h before 24 h food intake was measured. After 16 h of starvation, TβhnM18 mutants significantly consumed less 5% sucrose or 5% sucrose with 5% yeast, and after 40 h of starvation, TβhnM18 mutants significantly consumed more sucrose. N = 20-26 groups of eight flies. C, Blocking InR signaling in Tdc2-Gal4-targeted octopaminergic neurons in TβhnM18 mutants significantly increased 5% sucrose consumption. N = 20-28 groups of eight flies. To determine differences between two groups, Student’s t test was used, and to determine differences between three or more groups, one-way ANOVA with post hoc Tukey’s HSD was used. P* < 0.05; P**< 0.01, P***< 0.001.