Starvation influences the strength of memory

A, Appetitive olfactory learning and memory paradigm. B, Appetitive 2 min STM, 6 h and 24 h memory of w1118 or TβhnM18 that were starved either for 16 h (white bars) or 40 h (dark gray bars) before the training. 2 M sucrose was used as reward. C, 0.15 M sucrose was used as reinforcer. Prolonged starvation increases memory performance. Prolonged starvation from 16 h to 40 h leads to the formation of STM in TβhnM18. Independent of the duration of starvation, 6 h after training, memory appeared in the mutants. C, TβhnM18 mutants starved for 16 h developed an aversive STM memory to 0.15 M sucrose. After 40 h of starvation, the mutants developed an appetitive STM. Numbers below box plots indicate pairs of reciprocally trained independent groups of male flies. The letter “a” marks a significant difference from random choice as determined by a one-sample sign test (P < 0.05). The Student’s t tests were used to determine differences between two groups. For differences between more than two groups one-way ANOVA with Tukey’s post hoc HSD test was used. (P* < 0.05; P** < 0.01; P*** < 0.001).

Starvation influences the type of memory

A, Appetitive STM training with 2 M sucrose and cold shock directly or 2 h after training. Mildly starved control flies exhibit an appetitive memory sensitive to cold shock 3 h after training. Severely starved control flies develop a memory that is initially sensitive to cold shock, but becomes insensitive after 2 h. This phenotype is shared with mildly starved TβhnM18 mutants. Prolonged starvation in the mutants shifts memory to cold shock insensitive memory. B, To block neuronal activity and the formation of LTM, a 30-min heat shift was applied immediately after training to flies expressing a temperature-sensitive shibire transgene under the control of the R1504-Gal4 driver. The block results in TβhnM18 mutants losing LTM. Numbers below box plots indicate pairs of reciprocally trained independent groups of male flies. The letter “a” marks a significant difference from random choice as determined by a one-sample sign test (P < 0.05). One-way ANOVA with Tukey’s post hoc HSD test or for data in A, and Kruskal-Wallis followed by post-hoc Dunn’s test and Bonferroni correction was used to determine differences in B. (P* < 0.05; P** < 0.01). C, Model summarizing memory performance of control and mutant flies, that were either starved for 16 h (blue line) or 40 h (orange line). The dots present the average of the data presented in Figure 1 and Figure 2. Anesthesia sensitive memory (ASM), anesthesia resistant memory (ARM), intermediate memory (ITM) and long-term memory (LTM).

Octopamine suppresses memory

A, Feeding 3 mM of the octopamine receptor antagonist epinastine for 1 h after training resulted in memory 6 h later in w1118 flies. Feeding 3 mM octopamine for 6 h after training suppresses LTM in TβhnM18. B, A 3 mM octopamine feeding pulse 30 min before training inhibits STM in TβhnM18 mutants. Controls were water-fed. A, B, Flies were starved for 16 h and 2 M sucrose was used as reinforcer. The letter “a” marks a significant difference from random choice as determined by a one-sample sign test (P < 0.05). Student’s t tests were used to determine differences between two groups. (P* < 0.05). Numbers below box plots indicate pairs of reciprocally trained independent groups of male flies. C, Model for memory suppression.

Elevated glycogen levels correlate with reduced sucrose preference

A, Analysis of whole-body glycogen levels in w1118 and Tβhnm18 flies. In TβhnM18 flies, glycogen content is significantly higher than 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. Tβhnm18 showed a significantly reduced preference for sucrose in comparison to control flies, but not after 40 h starvation. N = 20 - 26 groups of eight flies. C, Feeding male flies for 3 days on standard fly food (food), 5% sucrose or 5% yeast resulted in control flies preferring to consume sucrose. Male Tβhnm18 mutants fed normal food, sucrose and yeast showed a significant reduction in sucrose preference. Female flies of controls and mutants did not differ in their preferences. N = 14 - 28 groups of eight flies. To determine differences between two groups, the Mann-Whitney U test was used. P* < 0.05; P** < 0.01, P*** < 0.001.

Carbohydrate storage influences appetitive STM

A, Schemata 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 (Figure S3). 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. Flies were starved for 16 h and 2 M sucrose was used as reinforcer. Numbers below box plots indicate one pair of reciprocally trained independent fly groups. The letter “a” marks a significant difference from random choice as determined by a one-sample sign test (P < 0.05). The one-way ANOVA with post hoc Tukey’s HSD was used to determine differences between three groups (P* < 0.05; P** < 0.01).

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. Flies were starved for 16 h and 2 M sucrose was used as reinforcer. B, 16 h starved w1118 and Tβhnm18 flies formed similar levels of appetitive STM when 5% yeast was used as a reinforcer. C, 16 h starved virgin females of w1118 and Tβhnm18 displayed STM, whereas mated females of both genotypes did not. Differences from random choice were determined using a one-sample sign test and marked with the letter “a” (P < 0.05). Differences between two groups were determined using Student’s t tests, and differences among four groups were determined with one-way ANOVA with Tukey’s HSD post hoc test. P* < 0.05; P** < 0.01. Numbers below box plots indicate one pair of reciprocally trained independent fly groups.

Insulin signaling in reward neurons regulates STM

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 using 2 M as reinforcer. 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 TβhnM18 mutants 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 to determine differences between three or more groups. n.s. is not significant; P* < 0.05; P** < 0.01, P*** < 0.01. The letter “a” marks a significant difference from random choice as determined by a one-sample sign test (P < 0.05). E, Model of memory regulation in TβhnM18 mutants. Long-term memory (LTM), intermediate-term memory (ITM).

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. After 40 h of starvation, TβhnM18 mutants significantly consumed more sucrose and sucrose with yeast. 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. n.s. = non-significant; P* < 0.05; P** < 0.01, P*** < 0.001.