A STIM dependent dopamine-insulin axis maintains the larval drive to feed and grow in Drosophila

Appropriate nutritional intake is essential for organismal survival. In holometabolous insects such as Drosophila melanogaster, the quality and quantity of food ingested as larvae determines adult size and fecundity. Here we have identified a subset of dopaminergic neurons (THD’) that maintain the larval motivation to feed. Dopamine release from these neurons requires the ER Ca2+ sensor STIM. Larvae with loss of STIM stop feeding, whereas expression of STIM in THD’ neurons rescues feeding, growth and viability of STIM null mutants. Moreover STIM is essential for maintaining excitability and release of dopamine from THD’ neurons. Optogenetic stimulation of THD’ neurons identified connectivity to neuropeptidergic cells, including median neuro secretory cells that secrete insulin-like peptides. Loss of STIM in THD’ cells alters the developmental profile of specific insulin-like peptides including ilp3. Loss of ilp3 partially rescues STIM null mutants and inappropriate expression of ilp3 in larvae affects development and growth. In summary we have identified a novel STIM-dependent dopamine-ILP circuit that regulates developmental changes in larval feeding behaviour. Author summary The ability to feed appropriately when hungry is an essential feature for organismal survival and is under complex neuronal control. An array of neurotransmitters and neuropeptides integrate external and internal signalling cues to initiate, maintain and terminate feeding. In adult vertebrates and invertebrates dopamine serves as a reward cue for motor actions, including feeding. Larvae of holometabolous insects, including Drosophila melanogaster, feed and grow constantly followed by gradual cessation of feeding, once sufficient growth is achieved for transition to the next stages of development. Here we identified a subset of larval dopaminergic neurons in Drosophila melanogaster, activity in which maintains continuous feeding in larvae. By analysis of a null mutant we show that these neurons require the Stromal Interaction Molecule (STIM) an ER Ca2+ sensor, to maintain excitability. In turn they modulate activity of certain neuropeptidergic cells. Among these are the median neurosecretory cells (MNSc) that synthesize and secrete insulin-like peptides including ilp3. The identified dopaminergic neurons dysregulate the normal pattern of larval ilp3 expression leading to premature cessation of feeding and growth. Overall, our study identified a simple dopamine-insulin feeding circuit whose manipulation could be useful for model organism studies related to feeding disorders, obesity and diabetes.

116 changed significantly in 86h aged STIM KO larvae (Fig 1F and 1G). To identify the cause

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Among other factors, the exit of quiescence and maintenance of neuroblast 125 proliferation at the early second instar stage depends on nutritional intake [29,30]. The slow 126 growth and delayed exit from quiescence suggested that STIM KO larvae may lack adequate 127 nutritional inputs. As a first step staged larvae were placed on yeast, mixed with a blue dye 128 and tested for ingestion of food. Even as early as 40-44h AEL there was a significant reduction 129 of food intake in STIM KO larvae (Fig 1H, quantification in Fig 1I). By 80-84h AEL two classes 130 of STIM KO larvae were evident. One with reduced food intake and others with no food intake 131 (Fig 1H and S1D Fig). The proportion of STIM KO larvae with no food intake reached ~70% 132 by 82-86h AEL (Fig 1J). Based on these results it appears that STIM KO [33]. Rescue of STIM KO larvae from 2 nd to 3 rd instar 145 5 (~90%) was evident upon over-expression of STIM + in THD' marked neurons (Fig 2A and (Fig 2D). Expression of STIM + in the VG localised THD' neurons (THD'GAL4, 157 THGAL80) reduced the rescue of STIM KO larvae significantly (S2A Fig and Fig 2E) and was 158 absent in adults (Fig 2F). Thus, the rescue of viability in STIM KO animals derives to a significant 159 extent from brain-specific THD' dopaminergic neurons.    control (58-62h AEL) and STIM KO (70-74h AEL) larvae. We chose these time points because 6 at 72h STIM KO larvae appear healthy and developmentally similar to control larvae at 60h (Fig   183   1D). THD' cells responded with similar changes in GCaMP intensity, in control and STIM KO 184 larvae at these time points (Fig 3A-3C). However, the ability to evoke and maintain cytosolic 185 Ca 2+ transients upon KCl depolarisation was lost in THD' neurons of STIM KO larvae at 76-80h 186 AEL (Fig 3A-3C). Overexpression of STIM + in THD' cells of STIM KO larvae rescued the KCl 187 evoked Ca 2+ response in larvae as late as 80-84h AEL (Fig 3A-3C).

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STIM requirement for maintaining excitability of THD' neurons was tested further by     Neuronal excitability is required for neurotransmitter release at presynaptic terminals.

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We hypothesized that dopamine release from THD' neurons might be affected in STIM KO 209 larvae. To test this idea, we identified the pre-synaptic (green) and post-synaptic (red) terminal 210 regions of THD' neurons by marking them with SyteGFP and Denmark respectively (Fig 4A)

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[38]. Analysis of pre-synaptic regions (SyteGFP expression) identified three distinct areas in 212 the CNS. One at the centre of the CNS (Fig 4A; asterisk), the second as a branched form 213 (Fig 4A; arrowhead) and the third one in the brain-gut interaction area as punctae (Fig 4A; 214 hash).

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Next, dopamine release was measured in the most prominent presynaptic area of though with altered dynamics from control animals (Fig 4B and 4C). We chose to measure 221 dopamine release in 76-80h STIM KO larvae because THD' neurons in their brains no longer 222 responded to KCl evoked depolarization (Fig 3B) even though the larvae appear normal (Fig   223   1D). Dopamine release was stimulated by Carbachol (CCh), an agonist for the muscarinic 224 acetylcholine receptor (mAChR), that links to Ca 2+ release from ER-stores through the ER-

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During normal larval development ilp3 transcript levels are low in actively feeding larvae (L2 295 and L3, 12h) and are gradually upregulated in later stages of L3 when larvae stop feeding ( Fig   296   6C; DGET [49] ). Based on the up-regulation of ilp3 in STIM KO larvae (Fig 6A and 6B), we 297 hypothesised that knock down of ilp3 in MNSc might rescue STIM KO larvae. Indeed, partial 298 rescue of larval lethality in 2 nd instar larvae followed by their transition to 3 rd instar larvae 299 (5+0.5) was observed (Fig 6D and F). A few of the rescued 3 rd instar larvae pupariated (Fig.   300 6F, L3a type larvae) and some even eclosed as adults (2. 3+0.3; S6A and S6B Fig). The 301 partial rescue observed may be due to lower expression of ilp2 and ilp5 in STIM KO animals 302 because both ilps are growth signals in 2 nd and 3 rd instar larvae (Fig 6A and 6B). A small 303 proportion of STIM KO larvae rescued by ilp3 knockdown appear significantly larger than control 304 larvae (Fig 6F and 6G), suggesting excess feeding in the absence of ilp3. This idea is further 305 supported by over-expression of ilp3 in MNSc [50] where we observed smaller sized larvae 306 (Fig 6H and 6L), suggesting reduced feeding due to untimely over-expression of ilp3, and a 307 delay in larval moults from 2 nd to 3 rd instar stage (Fig 6J). Conversely, knock-down of ilp3 308 resulted in larger sized pupae (Fig 6K and 6L), suggesting prolonged feeding in 3 rd instar 309 larvae, accompanied by an ~24h delay in pupariation (Fig 6M). Taken together, these data including the MNSc (Fig 5D) to regulate appropriate changes in larval feeding behaviour 324 through development. channel NaChBac (Fig 3D-3G) and restoration of dopamine release upon rescue by STIM + 336 ( Fig. 4B and 4C) supports the idea that STIM-dependant SOCE maybe required for 337 appropriate function and/or expression of ion channels and synaptic components in THD' 338 neurons. Changes in ER-Ca 2+ (Fig 4D and 5E) suggest that STIM is also required to maintain 339 neuronal Ca 2+ homeostasis.  Interestingly, dopamine is also required for reward-based feeding, initiation, and

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Of specific interest is the untimely upregulation of ilp3 transcripts in STIM KO larvae.

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Rescue of lethality in STIM KO larvae either by bringing back activity to THD' neurons or by 385 reducing ilp3 levels suggests an interdependence of Dopamine-Insulin signaling that is likely 386 conserved across organisms [60-65]. Our data suggest that ilp3 expression is suppressed 387 during the feeding stages of larvae (Fig 6H-6M), and once enough nutrition accumulates 388 expression of ilp3 is up-regulated, concurrent with a reduction in carbachol-induced Ca 2+ 389 signals in THD' neurons, possibly followed by release of ilp3. The idea of ilp3 as a metabolic 390 signal that is required to end persistent feeding prior to pupariation is supported by the 391 observation that knock-down of ilp3 in the MNSc leads to larger pupae in wild type animals 392 and larger larvae in STIM KO (Fig 6F and G) . To our knowledge this is the first report of ilp3 393 as a larval satiety signal. Expression of other neuropeptides did not show significant changes 394 in STIM KO larval brains (S1

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Optical density (OD) at 625nm as a measure of ingested blue dye was measured from 30µl of 453 the homogenate. Due to variation in larval sizes between control and experimental samples,

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the OD was normalized to whole larval protein concentration (µg/µl). OD was obtained using 455 the SkanIt Software 6.

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Confocal images were acquired by using FV3000 LSM and the Fluoview imaging software.

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Changes in ER-Ca 2+ were measured using an ER-GCaMP-210 strain(42). The brain 510 sample was prepared as above. Images were acquired as a time series on an XY plane at an