Octopamine integrates the status of internal energy supply into the formation of

The brain regulates food intake in response to internal energy demands and the


Introduction 34
The internal energy status of an animal needs to be adjusted to the energy expenditure 35 of the organism and the availability of external food to ensure survival. Increased 36 storage of energy correlates with increased food intake in the past. Dysregulation of 37 food intake might result in diseases such as obesity and diabetes. Sucrose is a 38 carbohydrate enriched in Western diets, and the breakdown product of sucrose -39 glucose -can be stored in the organism as glycogen mainly in the liver and muscles. 40 Increased glycogen levels are a hallmark of glycogen storage diseases that are 41 accompanied by defects in the liver, muscles and brain. (Ellingwood and Cheng, 2018) 42 Similar to vertebrates, the fruit fly Drosophila melanogaster uses glucose as a primary 43 energy source and stores glycogen mainly in the muscles-the major site of energy 44 expenditure-and the fat body-the equivalent of the vertebrate liver. (Galikova and 45 Klepsatel, 2023; Wigglesworth, 1949) As in vertebrates, glycogen levels are also found 46 in the brain. (Yamada et al., 2018) 47 The peptide hormone insulin regulates glycose homeostasis at the cellular 48 level. (Saltiel and Kahn, 2001) Insulin and its Drosophila melanogaster counterpart, 49 insulin-like peptides, perform their functions through well-conserved signal transduction 50 cascades. (Chatterjee and Perrimon, 2021;Inoue et al., 2018) In vertebrates, in addition 51 to its function in fat tissue and muscles, the insulin receptor is broadly expressed in the 52 brain and regulates neuronal plasticity. (Nakai et al., 2022) For example, in rats, reduced 53 insulin receptor function in the hypothalamus results in loss of long-term potentiation 54 and impaired spatial memory. (Grillo et al., 2015) In Drosophila melanogaster, the insulin 55 receptor substrate Chico is required for the development of mushroom bodies, a brain 56 flies. In contrast to 16 h starved controls, 16 h starved Tβh nM18 mutants developed long-149 term memory that was sensitive to cold shock directly after training and cold shock 150 insensitive shortly before the test. Longer periods of starvation resulted in Tβh nM18 151 mutants in anesthesia-resistant memory. Thus, with increasing length of starvation, 152 memory becomes more stable. Depending on the duration of starvation, animals first 153 form STM memory, then cold-shock-sensitive LTM and later ARM. The Tβh nM18 mutants 154 that were starved for 16 h formed similar memories to the 40 h starved control flies. 155 156

Octopamine is a negative regulator of memory 157
Starvation induces the formation of protein synthesis-dependent long-term 158 memory. (Krashes and Waddell, 2008) Thus, it is likely that the emerging memory 6 h 159 after training in Tβh nM18 mutants is long-term memory. To address this, we abolished a 160 mechanism specifically required for long-term memory in Tβh nM18 mutants and analyzed 161 whether this interferes with emerging memory (Figure 2). The R15A04-Gal4 driver 162 targets dopaminergic neurons of the PAM cluster specifically required for appetitive 163 long-term memory but not short-term memory. (Yamagata et al., 2015) Blocking the 164 function of these dopaminergic neurons directly after training using a temperature-165 sensitive shibire transgene (UAS-shi ts ) and a 30 min long heat pulse of 31°C resulted in 166 loss of memory in Tβh nM18 mutants (Figure 2A). This is consistent with the fact that 167 emerging memory is long-term memory. Since the Tβh nM18 mutants lack the 168 neurotransmitter octopamine, we next investigated whether octopamine normally 169 suppresses LTM. To address this, we first blocked the function of octopamine receptors 170 in controls directly after the training by feeding the octopamine antagonist epinastin for 1 171 h and analyzed memory 5 h later ( Figure 2B). If the function of the octopamine receptor 172 is required for long-term memory, longer lasting memory should also appear in control 173 flies that are starved for 16 h prior to the training, which indeed was the case. 174 Consistent with the idea that octopamine is a negative regulator of long-term memory, 175 feeding octopamine to Tβh nM18 mutants directly after training blocked long-term memory 176 ( Figure 2B). To analyze whether octopamine is also able to block STM, we fed 177 octopamine prior to training to control flies ( Figure 2C). A short pulse of octopamine 178 before the training inhibits the STM. Thus, octopamine is a negative regulator of 179 appetitive dopaminergic neuron-dependent long-term memory and can block STM. 180 181

Starvation influences sucrose consumption preference 182
Starvation reduces internal energy storage. The reduction might result in reevaluation of 183 external food cues and increased food consumption to restore the energy supply. To 184 investigate whether starvation depletes glycogen storage, we measured glycogen levels 185 in whole animals ( Figure 3A). In controls and Tβh nM18 mutants, starvation reduces 186 glycogen levels; however, non-starved Tβh nM18 mutant males started out with 187 significantly higher glycogen levels. After 40 h of starvation, the glycogen levels of 188 Tβh nM18 mutants were still higher than those of controls that were starved for the same 189 amount of time. The reevaluation of external food cues might be reflected in the choice 190 of food. Adult Tβh nM18 mutants have a reduced sucrose intake. (Li et al., 2016;Scheiner 191 et al., 2014) To investigate whether starvation influences the reallocation of an external 192 food cue, we starved flies and determined the preference to consume sucrose to 193 protein-enriched food using the capillary feeder assay (CAFE (Ja et al., 2007)). After 194 starvation, control flies and Tβh nM18 mutants chose between 5% sucrose and 5% yeast 195 ( Figure 3B). Prolonged starvation resulted in a decreased sucrose preference in 196 controls and Tβh nM18 mutants, but Tβh nM18 mutants started with a lower sucrose 197 preference. Only after 40 h of starvation did Tβh nM18 mutants show a similar preference 198 to control sucrose consumption. To further investigate whether Tβh nM18 mutants have 199 defects in the regulation of internal sucrose homeostasis, we deprived male flies of 200 sucrose-or protein-enriched food by letting them feed on 5% sucrose, 5% yeast or 201 standard food for 3 days and analyzed their food preference after deprivation (Figure 202 Tβh nM18 mutant showed a significantly reduced preference for sucrose, and sucrose-207 deprived mutants had sucrose preferences similar to those of controls. In mated 208 females, no differences in food preference between controls and the Tβh nM18 mutant 209 were observed. In summary, the reduced preference to consume sucrose correlated in 210 Tβh nM18 with increased glycogen levels. In addition, Tβh nM18 mutants can sense the 211 reduction in specific internal energy supplies and change their food preferences 212 accordingly. 213 214

Internal glycogen storage influences sucrose-related memories 215
Since memory performance increases upon reduction of the internal energy supplies 216 due to starvation, we wanted to investigate whether the internal energy supply 217 influences memory performance. In Drosophila, glycogen is mainly found in the fat 218 bodies -the major energy storage organ -and the muscles, a major site of energy 219 expenditure. (Wigglesworth, 1949  Increasing glycogen levels in the muscles did not change short-term memory in 231 16 h starved flies, but the reduction in glycogen significantly improved memory strength 232 ( Figure 4B). Increasing or decreasing glycogen levels in the fat bodies had no effect on 233 memory performance ( Figure 4C). When the glycogen levels were significantly 234 increased in the muscles and fat bodies, flies showed a reduced memory to odorants 235 paired with sucrose. An increase in memory performance was observed when glycogen 236 levels were significantly reduced in both tissues ( Figure 4D The memory was still increased ( Figure S3). Reciprocally, the reduction of GlyS HMS01279 -242 RNAi using the mb247-Gal4 driver targeting the mushroom bodies (Zars et al., 2000) did 243 not change short-term memory ( Figure S3). Thus, low levels of glycogen in the muscles 244 upon starvation positively influence appetitive short-term memory, while high levels of 245 glycogen in the muscles and fat body reduce short-term memory. 246 247

Internal glycogen levels reduce sucrose-related memories in Tβh mutants 248
The elevated glycogen levels in Tβh nM18 mutants might be responsible for the reduced 249 STM. To determine whether the reduction in glycogen levels in the muscles or fat 250 bodies restores STM, we expressed GlyS HMS01279 -RNAi under the control of the mef2-251 Gal4 or FB-Gal4 driver in Tβh nM18 mutants and analyzed STM ( Figure 5A). Neither the 252 reduction in the muscles nor the reduction in fat bodies of Tβh nM18 mutants improved 253 STM. Only when glycogen was reduced in both tissues did the Tβh nM18 mutants show 254 improved STM compared to controls. Thus, Tβh nM18 mutant flies can form appetitive 255 STM similar to controls when energy storage is sufficiently reduced. Next, we analyzed 256 whether male Tβh nM18 mutants can form STM to other nutrients than carbohydrates by 257 using a protein-enriched diet in the form of 5% yeast as a positive reinforcer ( Figure  258 5B). To evaluate whether there is a difference in the evaluation of protein as a food 259 source between male flies and Tβh nM18 mutants, we determined yeast intake in non-260 starved and starved flies ( Figure S4). Non-starved Tβh nM18 mutant males have a 261 significantly higher protein intake than controls. However, after 16 h of starvation, the 262 level of protein intake was comparable to that of the controls. Using 5% yeast as a food 263 reward, male Tβh nM18 mutants showed comparable levels of STM to controls ( candidate that links the internal energy level to reinforcing neurons. First, we analyzed 283 whether the insulin receptor is expressed in octopaminergic reward neurons in the brain 284 ( Figure 6). To detect the expression of an activated insulin receptor, we used an insulin 285 antibody that recognizes the phosphorylated form of the insulin receptor (InR). This 286 region is highly conserved between humans and flies. First, we tested whether the 287 antibody indeed recognizes the activated insulin receptor. Therefore, we overexpressed 288 the activated insulin receptor using the UAS-InR.A1325D transgene under the control of 289 the dTdc2-Gal4 driver ( Figure S5). The InR.A1325D protein variant mimics the human 290 V938D protein variant that is constitutively active, (Longo et al., 1992)   To investigate whether the improved STM also affects the emerging LTM of the 303 Tβh nM18 mutants, we performed cold-shock experiments in Tβh nM18 mutants in which 304 InR DN was expressed in octopaminergic reward neurons ( Figure 6D). The 3 h memory 305 in controls is cold-shock sensitive, but the memory in Tβh nM18 mutants is cold-shock 306 insensitive, supporting the idea that the mutants formed anesthesia-resistant memory. 307 Given that octopamine is a negative regulator of memory and that it is still missing in 308 Tβh nM18 mutants with blocked insulin signaling on octopaminergic reward neurons, it is 309 not surprising that ARM is still observed in the mutants. Thus, two functionally distinct 310 memory traces were formed after training, an insulin receptor-sensitive appetitive short-311 term memory and a long-term memory that can be blocked by octopamine. 312 313

Increased starvation results in overconsumption in Tβh nM18 mutants 314
To analyze whether the increased glycogen levels and the reduced sucrose reward 315 correlate with food consumption, the energy demands in flies under different starvation 316 conditions were analyzed by measuring food intake (Figure 7). When control flies were 317 starved for 16 h or 40 h, they consumed similar amounts of 5% sucrose. In contrast, 16 318 h-starved Tβh nM18 mutants consumed significantly less sucrose, but after 40 h of 319 starvation, they consumed approximately 34% more sucrose ( Figure 7B). After 40 h of 320 starvation, the glycogen levels in Tβh nM18 mutants were still higher than those in controls 321 ( Figure 3A). Thus, they overconsumed sucrose. The overconsumption was independent 322 of the diet, as they showed similar overconsumption when fed with a solution containing 323 5% yeast and 5% sucrose ( Figure 7B). To investigate whether the integration of the 324 internal energy status is also integrated into feed behavior by octopaminergic reward 325 neurons, we blocked insulin signaling in reward neurons in 16 h-starved Tβh nM18 cells 326 and analyzed sucrose consumption ( Figure 7C). The reduced sucrose consumption of 327 Tβh nM18 cells was significantly improved to control levels when insulin signaling was 328 blocked in reward neurons. Thus, the regulation of food consumption also requires the 329 integration of internal energy levels into the reward system via octopaminergic neurons. with CO 2 anesthesia and were kept for 2 days at 25°C to recover from CO 2 sedation. 469 Briefly, three-to five-day-old male flies were starved for 16 h or 40 h in vials with water-470 soaked filter paper at the bottom. Flies were transferred to training tubes and exposed 471 to the first odorant for 2 min, either 3-octanol (3-OCT diluted 1:80 in paraffin oil) or 4-472 methylcyclohexanol (MCH diluted 1:100 in paraffin oil). After that, they were transferred 473 to a second tube and exposed to the second odorant in the presence of filter paper 474 Food intake was displayed as the mean ± s.e.m. For learning and memory experiments, 535 the data were displayed as boxplot ± minimum (Q1-1.5*IQR) and maximum (Q1 + 536 1.5*IQR). 537 The nonparametric one-sample sign test and the parametric one-sample t test were 538 used to analyze whether behavior was based on random choice. Differences between 539 two groups were determined with Student's t test, and more groups were compared with 540 one-way ANOVA with Tukey's post hoc HSD test. Statistical analysis was performed 541 with Statistica 9.1 (StatSoft, Tulsa, OK, USA). Boxplots were generated with Microsoft 542 Excel 2016 and GIMP 2.10.12. 543 544

Data availability 545
All data related to figures are included in the supplement Table S2. Controls were water-fed. One-way ANOVA with Tukey's HSD post hoc test was used to 585 determine differences between three groups, and Student's t tests were used for two 586 groups. The letter "a" marks a significant difference from random choice as determined 587 by a one-sample sign test (P* < 0.05; P**< 0.01). muscles have no effect on STM, whereas reduced muscle glycogen increases 609 appetitive STM. C, Increased or decreased glycogen levels in the fat bodies did not 610 interfere with STM. D, A combined increase in glycogen in muscles and fat bodies 611 reduced STM, and a decrease in glycogen increased STM. Student's t tests were used 612 to determine differences between two groups, and one-way ANOVA with post hoc 613 Tukey's HSD was used to determine differences between three or more groups. The 614 letter "a" marks a significant difference from random choice as determined by a one-615 sample sign test (P* < 0.05; P** < 0.01). Numbers below box blots indicate one pair of 616 reciprocally trained independent fly groups. and Tβh nm18 displayed STM, whereas mated females of both genotypes did not. 624 Differences between two groups were determined using Student's t tests, and 625 differences among more than two groups were determined with one-way ANOVA with 626 Tukey's HSD post hoc test. Differences from random choice were determined using a 627 one-sample sign test and marked with the letter "a". P* < 0.05; P** < 0.01. Numbers 628 below box blots indicate one pair of reciprocally trained independent fly groups. shock did not disrupt emerging memory in Tβh nM18 mutants with blocked InR under the 638 control of the Tdc2-Gal4 driver. Student's t test was used to determine differences 639 between two groups, and one-way ANOVA with Tukey's post hoc HSD test was used to 640 determine differences between three or more groups. The letter "a" marks a significant 641 difference from random choice as determined by a one-sample sign test (P < 0.05). n.s. 642 is not significant; P* < 0.05; P** < 0.01.