FMRFa receptor stimulated Ca2+ signals alter the activity of flight modulating central dopaminergic neurons in Drosophila melanogaster

Neuropeptide signaling influences animal behavior by modulating neuronal activity and thus altering circuit dynamics. Insect flight is a key innate behavior that very likely requires robust neuromodulation. Cellular and molecular components that help modulate flight behavior are therefore of interest and require investigation. In a genetic RNAi screen for G-protein coupled receptors that regulate flight bout durations, we earlier identified several receptors, including the receptor for the neuropeptide FMRFa (FMRFaR). To further investigate modulation of insect flight by FMRFa we generated CRISPR-Cas9 mutants in the gene encoding the Drosophila FMRFaR. The mutants exhibit significant flight deficits with a focus in dopaminergic cells. Expression of a receptor specific RNAi in adult central dopaminergic neurons resulted in progressive loss of sustained flight. Further, genetic and cellular assays demonstrated that FMRFaR stimulates intracellular calcium signaling through the IP3R and helps maintain neuronal excitability in a subset of dopaminergic neurons for positive modulation of flight bout durations.


Author summary
Neuropeptides play an important role in modulating neuronal properties such as excitability and synaptic strength and thereby influence innate behavioral outputs. In flying insects, neuromodulation of flight has been primarily attributed to monoamines. In this study, we have used the genetically amenable fruit fly, Drosophila melanogaster to identify a neuropeptide receptor that is required in adults to modulate flight behavior. We show from both knockdown and knockout studies that the neuropeptide receptor, FMRFaR, present on a few central dopaminergic neurons, modulates the duration of flight bouts. Overexpression of putative downstream molecules, the IP 3 R, an intracellular Ca 2+ -release channel, and CaMKII, a protein kinase, significantly rescue the flight deficits induced by knockdown of the FMRFaR. Our data support the idea that FMRFaR and CaMKII help maintain optimal membrane excitability of adult dopaminergic neurons required to sustain longer durations of flight bouts. We speculate that the ability to maintain longer flight Introduction Neuromodulation of animal behavior by neuropeptides is ubiquitous among vertebrates and invertebrates [1,2]. Unlike fast acting neurotransmitters, neuropeptides and their receptors influence neuronal activity and circuit dynamics by modulating presynaptic neurotransmitter release. The mechanisms for doing this include changes in ion channel and transporter function as well as regulation of gene expression [1,2]. The neural action of neuropeptides can either be local or at long distances by release into circulation and can influence intrinsic behaviors such as feeding, mating, sleep and aggression [3,4]. An important and critical behavior in flying insects is flight. Altered flight behavior, in particular the inability to maintain long durations of flight bouts, can impinge on the fly's ability to optimally search and reach food sources as well as sites suitable for egg laying. Neuromodulation of insect flight has thus far been attributed primarily to biogenic amines [5][6][7].
A role for neuropeptide-based modulation of flight behavior has remained largely unexplored. In invertebrates, neuropeptides activate G-protein coupled receptors (GPCRs) [8] followed by generation of soluble second messengers such as cAMP [9][10][11][12] or inositol 1,4,5-trisphosphate (IP 3 ) [13][14][15]. IP 3 binds to and activates the endoplasmic reticulum (ER) localized Ca 2+ channel, the IP 3 receptor (IP 3 R), resulting in release of calcium from ER-stores [16]. Invertebrate neuropeptide receptors that stimulate IP 3 -mediated Ca 2+ release include the Pigment Dispersing Factor Receptor (PdfR) [14], FMRFaR [13][14][15] amongst others [8,17]. We identified the PdfR and FMRFaR in a genetic screen for neuronal GPCRs that regulate flight in Drosophila through IP 3mediated Ca 2+ release [14]. Further investigation of PdfR demonstrated a role for this receptor in both the developing and adult flight circuit, though the identity of Pdf responsive neurons that affect flight remained ambiguous [14]. FMRFaR has been described earlier in the context of an escape response to intense light in Drosophila larvae [13] and the larval to pupal transition under nutrient-limiting conditions [15]. Adult behaviors implicating the FMRFaR include startle-induced locomotor activity [18] and adaptive sleep following heat stress [19].
In the context of flight behavior, a neuronal requirement for IP 3 -mediated Ca 2+ release was initially described in flight deficient IP 3 R mutants [20]. Subsequent cellular and molecular studies identified a role for IP 3 -mediated Ca 2+ release in dopaminergic neurons during pupal stages, when the flight circuit matures [7]. Interestingly though, flight deficits were also observed upon temporal knockdown of the FMRFaR exclusively in mature neurons [14]. Thus far, flight deficits arising from adult specific reduction of IP 3 /Ca 2+ signaling have remained unexplored. Here we have investigated if FMRFaR mediated Ca 2+ signaling is required for Drosophila flight by generating new CRISPR-Cas9 mediated mutants for the FMRFaR. Flight deficits arising from specific knockdown in adult dopaminergic neurons suggests a role for FMRFaR in modulating adult flight and implicate the IP 3 R and possibly CaMKII as downstream signaling components. Genetic and cellular assays indicate that the FMRFaR on adult dopaminergic neurons helps maintain optimal membrane excitability which could potentially be required for synaptic release of dopamine.

FMRFaR is required in dopaminergic neurons for maintenance of flight
The neuropeptide receptor, FMRFaR, was identified amongst other G-protein coupled receptors (GPCRS) as a positive regulator of Drosophila flight in a pan-neuronal screen, where genetic data supported IP 3 R mediated Ca 2+ signaling and store-operated calcium entry (SOCE) as the down-stream effectors of receptor activation [14]. We confirmed these initial observations by RNAi mediated knockdown of the FMRFaR with another pan-neuronal GAL4 (nSybGAL4) in the central nervous system (CNS), followed by measurement of flight bout durations in tethered flies in response to a gentle air-puff. Unlike previous measurements where flight times were recorded for a maximum of 30 seconds [14], in the current study we monitored flight for 15 minutes (900 seconds), which allows for resolution of longer flight bout durations. Upon FMRFaR knockdown in nSyb positive neurons, significantly shorter flight bouts were observed as compared to controls (FMRFaR RNAi /+, nSyb/+; Fig 1A). The efficacy of FMRFaR knockdown by the RNAi strain was confirmed in the adult CNS by pan-neuronal expression of the RNAi with nSybGAL4. A significant reduction in FMRFaR mRNA levels was observed (Fig 1B).
Similar to the pan-neuronal knockdown of FMRFaR, flight deficits were also observed in flies with FMRFaR RNAi targeted to a large number of dopamine-synthesizing cells driven by THGAL4 (Fig 1A). Because non-neuronal expression of THGAL4 has been documented [21], we tested GAL4s with restricted expression in neurons [22]. For this, we used the THD and THCGAL4 driver lines that mark non-overlapping subsets of central dopaminergic neurons [22]. Knockdown of FMRFaR using either the THD1GAL4 or the THD'GAL4 resulted in flight deficits that were equivalent to the phenotype observed with THGAL4-driven knockdown ( Fig  1A and 1C, S1A Fig; TH>FMRFaR RNAi vs. THD1>FMRFaR RNAi or TH>FMRFaR RNAi vs THD'>FMRFaR RNAi are not significantly different from each other; p>0.05; Mann-Whitney U-test). However, the THDGAL4 strains are also known to express in some non-dopaminergic neurons [22]. To test the role, if any, of FMRFaR function in non-dopaminergic neurons marked by THDGAL4s, expression of the FMRFaR RNAi was blocked specifically in TH-expressing neurons with the THGAL80 transgene [23]. THGAL80 was made with the same promoter region of the TH gene as that employed for THGAL4 and has been shown to effectively suppress THGAL4 driven GFP expression [23]. In presence of the THGAL80 transgene, THD1 and THD' driven GFP expression was selectively blocked in dopaminergic neurons, as identified by the presence of anti-TH immunostaining and the absence of GFP in a majority of neurons (S1B Fig). However, a few GFP positive and TH negative neurons remain in both THD1 and THD'GAL4 strains even in the presence of THGAL80 (S1B Fig; white arrows). Presumably, the shorter genomic regions used for making THD1 and THD' lend themselves to the influence of neighbouring genomic regions resulting in some ectopic expression in non-TH neurons. Importantly, the flight deficits observed in THD>FMRFaR RNAi flies were reversed upon introduction of THGAL80 confirming that such flight deficits arose exclusively from dopaminergic neurons marked by these GAL4s (Fig 1A; blue bar, S1A Fig; blue bar). On the other hand, flies with knockdown of the FMRFaR using the THC1 or THC'GAL4s did not result in significant flight deficits, indicating that dopaminergic neurons unique to THD1 or THD'GAL4 require FMRFaR mediated signaling to regulate flight bout durations (S1A Fig). THDGAL4 strains express in central dopaminergic neurons comprising the Protocerebral Posterior Lateral 1 (PPL1) and Protocerebral Posterior Medial 3 (PPM3) clusters and these two neuronal clusters are not marked by the THC'GAL4 [7,22]. Furthermore, the reduced flight deficit observed with FMRFaR knockdown in dopaminergic neurons was not a consequence of overall reduction in motor activity because THD1>FMRFaR RNAi flies showed normal locomotor activity as compared to their genetic controls (S1C Fig). Having identified the dopaminergic subsets where FMRFaR is required for flight, subsequent experiments were performed with THD1GAL4 as the dopaminergic expression driver.
RNAi knockdown of FMRFaR with nSybGAL4 reduced FMRFaR levels by about 30% ( Fig  1B). To further refine our understanding of the FMRFaR's role in Drosophila flight, a complete knockout of the FMRFaR (ΔFMRFaR) was generated by the CRISPR-Cas9 method [24]. The strategy employed, removed nearly 1.5 kb of the 1.6 kb coding region of FMRFaR (Fig 1D). Primers spanning the FMRFaR locus (5'F+3'R) amplified only about~400bp genomic fragment from FMRFaR homozygous and heterozygous knockouts, thereby confirming FMRFaR gene knockout (Fig 1D). Other primer pair combinations (5'F+5'R and 3'F+3'R) helped us distinguish between the homozygotes and heterozygotes. Homozygous knockouts of the FMRFaR showed near complete loss of FMRFaR mRNA as well as reduced flight bout durations as compared to the heterozygotes (Fig 1E and 1F).
Next, we tested dopaminergic neuron specific knockout of FMRFaR by expression of UAS-cas9 in the genetic background of flies with ubiquitous expression of guide RNAs that target the gene for FMRFaR (THD1>FMRFaR dual ;cas9; see Materials and methods). Flies with knockout of the FMRFaR in THD1 cells flew for significantly shorter durations as compared to the appropriate genetic controls (FMRFaR dual /+;cas/+- Fig 1F; 4 th bar in black and THD1/+- Fig 1A; last bar in black; p<0.01; Mann-Whitney U-test) (Fig 1F). To account for non-specific effects of cas9 expression, we tested flies expressing only the cas9 transgene in THD1 neurons (THD1>cas9). The flight deficit observed with FMRFaR knockout in THD1 cells was significantly different from THD1>cas9 flies (Fig 1F; THD1>cas9

FMRFaR is present on dopaminergic neurons and is functionally active
Owing to the lack of either an antibody for FMRFaR or GAL4 strains that reliably mark FMRFaR expressing neurons, we chose to employ molecular and functional assays to confirm the presence of FMRFaR on dopaminergic neurons. Towards this end, we sorted adult brain dopaminergic neurons marked by THGAL4 driven eGFP, using fluorescence-activated cell sorting (FACS) and measured transcript levels of a few selected genes. Two markers of dopaminergic neurons, ple (encoding the enzyme Tyrosine Hydroxylase or TH) and dDAT (encoding a dopamine transporter) were highly expressed in GFP +ve cells, confirming homogeneity of the sorted population (Fig 2A). FMRFaR expression, as measured by qPCR, was significantly enriched in TH expressing neurons (GFP +ve), whereas expression of calcium signaling molecules like dSTIM and dOrai was similar in dopaminergic and non-dopaminergic neurons (Fig 2A).
The presence of active FMRFaR on central dopaminergic neurons was tested next. THD1 neurons with expression of a genetically encoded calcium sensor, GCaMP6m [25] were tested for receptor activation in adult brain explants and calcium responses were specifically monitored from the PPL1 and PPM3 clusters, previously implicated in the regulation of flight bout durations ( Fig 1A, S1A Fig) [7]. Stimulation with one of the most abundantly expressed neuropeptide ligands of FMRFaR, DPKQDFMRFa (henceforth referred to as FMRFa) [26,27], resulted in a slow calcium rise (Fig 2B, 2C, 2E and 2F). FMRFa peptide stimulated GCaMP6m response was significantly attenuated upon knockdown of the FMRFaR using either the RNAi or FMRFaR dual ;cas9 (Fig 2B, 2C, 2E and 2F). Peptide stimulation of THD1 cells in the presence of a sodium channel inhibitor, 2 μM Tetrodotoxin (TTX) resulted in calcium responses that were comparable to that obtained in the absence of TTX (Fig 2D, 2E and 2F). This suggested that the rise in FMRFa-stimulated Ca 2+ is due to direct activation of the FMRFaR and not a consequence of synaptic inputs from other neurons to the THD1 neurons. Moreover, FMRFa stimulated Ca 2+ signals attenuated significantly upon knockdown of itpr in THD1 marked dopaminergic neurons (Fig 2D, 2E and 2F). Taken together, these data suggest the presence of FMRFaR on central brain dopaminergic neurons and that receptor activation likely leads to Ca 2+ release from the IP 3 R.

FMRFaR is required in adult dopaminergic neurons for sustained flight
Pan-neuronal knockdown of the FMRFaR in an earlier study suggested a requirement of the receptor both during pupal development and in adult Drosophila neurons [14]. To investigate the developmental stage(s) when FMRFaR signaling is required in dopaminergic neurons for maintaining flight, the TARGET (Temporal And Regional Gene Expression Targeting) system [28] was used for stage-specific FMRFaR knockdown. This system employs a temperature sensitive GAL80 element (TubGAL80 ts ) which represses GAL4 expression at 18˚C. At 29˚C the GAL80 ts is inactivated, thus allowing GAL4 driven expression of the UAS transgene. Flight durations of flies with FMRFaR knockdown in dopaminergic neurons throughout development (29˚C) were reduced to less than 300 seconds as compared to genetic controls that flew for a median time of about 600 seconds ( Fig 3A, S2A Fig). Flies with knockdown of the FMRFaR in larval stages displayed normal flight bouts (29˚C Larval), whereas a modest reduction in flight bout durations was observed with pupal knockdown (29˚C Pupal) ( Fig 3A). Interestingly, knockdown of the FMRFaR in adult stages affected flight duration maximally, with exacerbation of the flight deficit over time, such that flies with knockdown for eight days flew for less than 150 seconds (29˚C Adults) ( Fig 3A). In contrast, the control genotypes maintained at 29˚C for the same adult period could fly for significantly longer and showed a median flight time of over 600 seconds (S2A Fig). Next, we tested the ability of IP 3 R overexpression to compensate for loss of FMRFaR in adult dopaminergic neurons. Indeed, flight deficits observed with FMRFaR knockdown in adults could be rescued to a significant extent by simultaneous overexpression of an itpr + transgene (Fig 3A; blue bar; p<0.01; Mann-Whitney U-test), but not another UAS transgene, GCaMP6m (Fig 3A; orange bar; p>0.05; Mann-Whitney U-test). Rescue of FMRFaR knockdown by overexpression of itpr + supports earlier findings that IP 3 R-mediated Ca 2+ release functions downstream of FMRFaR [13] and is consistent with observations in Fig 2D, 2E and 2F. Temporal and cell specific knockout of the FMRFaR with FMRFaR dual was attempted, but was not pursued because control flies with expression of cas9 in adult THD1;TARGET cells exhibit flight deficits when maintained at 29˚C for 8 days (S2A Fig, last lane in pink). Consistent adult specific phenotypes were observed with the THD1GAL4 strain which has been used for subsequent experiments.
The cellular basis for flight deficits arising from loss of FMRFaR in THD1 marked neurons was investigated next. Expression of endogenous TH and membrane-bound GFP driven by THD1GAL4 was visualized in the PPL1 and PPM3 clusters of adult Drosophila brains at 8 days after knockdown of FMRFaR. Both TH and GFP immunoreactivity appeared as in a control genotype, and there was no apparent loss of dopaminergic neurons or their neurite projections ( Fig 3B, 3C and 3D, S2B Fig). Taken together these data suggest that the FMRFaR is required in adult THD1 neurons for sustaining flight bout durations. The increasing disability to maintain flight bouts for longer durations upon manipulation of FMRFaR levels in adult THD1 neurons could likely be a consequence of increased RNAi expression over time.

CaMKII in dopaminergic neurons is required for flight
Downstream signaling mechanisms that require FMRFaR activation in THD1 dopaminergic neurons were investigated next. There is evidence demonstrating that FMRFa evoked modulation of excitatory junction potentials (EJPs) at Drosophila neuro-muscular junctions are Individual data points are shown as open diamonds. Numbers below indicate total number of cells imaged. Each bar is compared to the Control peptide stimulated condition shown in black ( ÃÃ p<0.01; ns-not significant; Mann-Whitney Utest).
https://doi.org/10.1371/journal.pgen.1007459.g002 dependent on the protein kinase, CaMKII [13,29]. Hence, we tested whether overexpression of wild-type CaMKII (WT-CaMKII) in dopaminergic cells rescued the deficit in flight bout durations observed upon reduced FMRFaR signaling. Overexpression of WT-CaMKII during pupal stages was insufficient to rescue flight deficits in adults ( Fig 4A). However overexpression of WT-CaMKII in adults modestly rescued FMRFaR RNAi induced flight deficits on day 4, 6 and 8 ( in THD1 neurons using the TARGET system (THD1;TubGAL80 ts >FMRFaR RNAi ), which allows effective expression of transgene at 29˚C, but not at 18˚C. Animals maintained at 18˚C or 29˚C throughout development and as adults are indicated as 18˚C and 29˚C, respectively. Knockdown of FMRFaR only during the larval, pupal or adult stages are shown as 29˚C Larval, 29˚C Pupal or 29˚C Adult, respectively (red). Knockdown of FMRFaR in adult stages (29˚C Adult) reduced flight bout durations significantly. The phenotype observed upon FMRFaR knockdown in adults for 8 days could be rescued by overexpression of the itpr + transgene, but not with another UAS transgene, GCaMP6m (THD1;TubGAL80 ts >FMRFaR RNAi ;itpr + shown in blue and THD1;TubGAL80 ts >FMRFaR RNAi ;GCaMP6m shown in orange; n!30, ÃÃ p<0.01, ns-not significant; Mann-Whitney U-test). All comparisons are to the control condition, THD1;TARGET>FMRFaR RNAi at 18˚C (grey bar; THD1; TubGAL80 ts >FMRFaR RNAi ; 18˚C) or as indicated by horizontal lines above the graph (n!30, ÃÃ p<0.01, Mann-Whitney U-test). (B) Immunohistochemistry of representative adult brain samples corresponding to 8 day adult controls (above; THD1; TubGAL80 ts >mCD8GFP Control) and FMRFaR knockdown (below; THD1;TubGAL80 ts >mCD8GFP;FMRFaR RNAi ). Anti-TH antibody was used to mark the various dopaminergic clusters, amongst which PPL1 and PPM3 clusters are indicated. Scale bars represent 100 μm.  presumably via modulation of potassium currents [30] (also see Discussion). Overall the partial rescue of flight deficits in the FMRFaR knockdown animals by expression of a WT-CaMKII transgene indicates a genetic interaction between them.
To test directly if CaMKII is required in dopaminergic neurons to modulate flight bout durations, we expressed a synthetic peptide inhibitor of CaMKII (Ala) [31] in dopaminergic cells of interest. Ala is a peptide analog of the CaMKII autoinhibitory domain that can bind to the catalytic domain of CaMKII, thereby functioning as an exogenous inhibitor. Inhibition of CaMKII in pan-dopaminergic (TH) or the subset dopaminergic neurons (THD1) resulted in flies with significantly reduced durations of flight bouts (Fig 4B). Knockdown of CaMKII with a CaMKII RNAi also elicited a significant reduction of flight bout durations (S3B Fig). Inhibition of CaMKII activity by the Ala peptide in THD1 neurons did not affect their general locomotor ability, indicating that CaMKII function in THD1 neurons is likely to be flight specific (S3C Fig). Additionally, TARGET experiments demonstrated that THD1-driven expression of Ala specifically in either pupal or adult stages significantly compromised the duration of flight bouts, as compared to genetic controls (Fig 4C, S2A Fig-THD1;TARGET controls, S3D Fig-Ala/+ controls). Given that CaMKII is involved in several developmental processes, for example, axon terminal growth [32], a pupal requirement for CaMKII is not surprising. Maximal reduction in flight bout duration was observed in flies wherein CaMKII was inhibited by Ala peptide expression in THD1 cells for 8 days as adults ( Fig 4C).
These data support a role for CaMKII activity in the THD1 subset of dopaminergic neurons in the specific context of maintaining flight bout durations. Similar to FMRFaR knockdown, CaMKII inhibition with Ala in adult neurons did not affect levels of TH mRNA (S3E Fig). Thus, unlike previous findings where reduced Ca 2+ signaling in pupal dopaminergic neurons resulted in reduced expression of the gene encoding TH (ple or TH) [6,7], FMRFaR and CaM-KII in adult dopaminergic neurons appear to modulate flight by an alternate cellular mechanism.
To test if CaMKII activity affects FMRFaR induced Ca 2+ release, THD1 neurons expressing Ala peptide were stimulated with the FMRFa peptide. FMRFa-stimulated cellular calcium responses were not affected by Ala expression in THD1 neurons (Fig 4D, 4E and 4F). These data suggest that the function of CaMKII is either downstream or parallel to FMRFaR induced Ca 2+ release.
The ability of FMRFa to activate CaMKII in Drosophila central brain neurons was tested next by immunostaining for pCaMKII, a CaMKII modification that occurs upon prolonged calcium elevation in the cell [30,33]. For technical reasons, this immunostaining was performed on primary neuronal cultures from larvae, wherein we overexpressed the FMRFaR + in all nSybGAL4 positive neurons [34]. In addition, the same neurons were marked with mCD8GFP to normalize the pCaMKII signal and to account for variability in strength of expression of the overexpressed FMRFaR + . A significant increase in pCaMKII/GFP staining was observed upon stimulation of larval nSyb>FMRFaR + ;mCD8GFP positive neurons with FMRFa as compared to cells with solvent addition (S3F- S3H Fig). These data support the idea that FMRFa stimulated Ca 2+ release activates CaMKII in central brain neurons. Even though a direct test for FMRFa-stimulated CaMKII activation in THD1 neurons has not been possible so far due to technical reasons, the ability of WT-CaMKII to partially rescue the flight deficit in FMRFaR knockdown animals (Fig 4A), taken together with these cellular data suggest that CaMKII may function downstream of FMRFaR signaling. Nevertheless, an FMRFaR-independent and parallel role for CaMKII in THD1 neurons remains possible. The underlying cellular basis for the flight deficits observed upon FMRFaR knockdown and Ala expression in dopaminergic neurons was investigated next.

FMRFaR and CaMKII modulate calcium entry and membrane potential in central dopaminergic neurons
To measure neuronal activity of THD1 neurons, we tested their response to a depolarizing stimulus. Ex vivo brain preparations from THD1 marked dopaminergic neurons were stimulated with 70 mM KCl, a condition known to raise the resting membrane potential and activate voltage-gated calcium channels on the plasma membrane [35]. The stimulation was followed by optical measurements of calcium dynamics. Firstly, using the TARGET system, we expressed GCaMP6m in THD1 marked cells, specifically in adults. Animals reared at 29˚C for 8 days (also the time corresponding to maximal flight loss upon FMRFaR knockdown or Ala expression; Figs 3A and 4C), were used in these experiments. Robust Ca 2+ elevations were observed in THD1 marked cells from control animals upon addition of KCl (Fig 5A-5D; black trace). Ca 2+ responses from neurons with either FMRFaR knockdown or Ala expression, were however significantly reduced as compared to controls (Fig 5A-5D; red and blue traces). Similar experiments on ex vivo brains obtained after FMRFaR knockdown in adults for 2 days, exhibit a minimal decrease in Ca 2+ responses upon depolarization with KCl, in agreement with their weak flight deficits (S4A- S4C Fig, Fig 3A). These data suggest that FMRFaR and CaMKII help maintain normal cellular responses to changes in membrane excitability in THD1 neurons.  Interestingly, in the FMRFaR knockdown condition, most cells exhibit a dampened Ca 2+ response (Fig 5D, 2 nd row-pink arrow), but a few cells responded normally (Fig 5D, 2 nd rowyellow arrow). Their normal response may be due to insufficient receptor knockdown. Alternately all THD1 marked cells might not express the FMRFaR (see Discussion).

Increased membrane excitability can partially compensate for loss of FMRFaR and CaMKII function
Based on the reduced Ca 2+ elevation upon depolarization with KCl, observed in THD1 marked dopaminergic neurons of flies with either FMRFaR knockdown or CaMKII inhibition, we hypothesized that such neurons might have reduced membrane excitability. To ascertain if reduced FMRFaR signaling indeed alters the ability of THD1 neurons to undergo membrane depolarization, a genetically encoded fluorescent voltage indicator, Arclight [36], was expressed in dopaminergic neurons of THD1GAL4. Stimulation of 8 day old adult brains with KCl showed a robust change in Arclight fluorescence corresponding to neuron depolarization ( Fig  6A-6D; black trace). As proof of concept, expression of Kir2.1 in THD1 cells for 2 days nearly abolished KCl induced membrane depolarization (S5A- S5C Fig; blue trace). Dopaminergic neurons with FMRFaR knockdown for 8 days displayed a significantly reduced ability to depolarize after KCl addition (Fig 6A-6D; red trace). As was observed previously (Fig 5D), membrane depolarization induced changes in Arclight fluorescence were attenuated in most THD1 neurons (Fig 6D, 2  Because knockdown of FMRFaR reduced KCl induced Ca 2+ entry in dopaminergic neurons (Fig 5A-5D), attenuated membrane depolarization (Fig 6A-6D) and correlated with shortened flight bout durations (Fig 3A), we hypothesized that FMRFaR function is likely required to maintain optimal excitability in these neurons. Consequently, increasing neuronal excitability might compensate for reduced FMRFaR function and thereby rescue the flight deficits observed in FMRFaR knockdown flies. To test this idea, we introduced a temperature sensitive cation channel, dTrpA1 [37] in flies with adult specific FMRFaR knockdown. Activation of the dTrpA1 calcium channel can compensate in part for reduced Ca 2+ entry through voltage-gated channels [15,38]. Indeed, expression of dTrpA1 significantly improved the maintenance of flight bouts in 6 and 8 day old FMRFaR knockdown adults (Fig 6E, S2A Fig-THD1 (S5D and S5F Fig). Taken together, these data demonstrate that FMRFaR helps maintain optimal excitability of flight bout extending central dopaminergic neurons.

Synaptic vesicle recycling in dopaminergic neurons is essential for sustained flight
We hypothesized that changes in membrane excitability are likely to affect the release of dopamine containing synaptic vesicles from THD1 neurons. A temperature-sensitive mutant transgene for dynamin (UAS-Shibire ts or Shi ts ) [40] was used to block synaptic vesicle recycling by shifting flies transiently to the non-permissive temperature of 30˚C. Indeed, when synaptic vesicle recycling was inhibited in THD1 neurons of adult flies, reduced flight bout durations were observed as compared with controls ( Fig 7A). Flies of the same genotype (THD1>Shi ts ) maintained at 22˚C throughout showed normal flight bout durations (Fig 7A). Thus acute manipulation of synaptic function of adult THD1 neurons affects the maintenance of flight bout duration in the tethered flight paradigm. Finally, we tested requirement for the neurotransmitter, dopamine in THD1 neurons, for modulating the duration of adult flight bouts. Adult-specific expression of an RNAi transgene for the dopamine-synthesizing enzyme Tyrosine Hydroxylase (TH RNAi ; [41]) in THD1 neurons significantly impaired the maintenance of longer flight bouts from as early as 2 days of TH RNAi expression (Fig 7B), suggesting that dopamine release from synapses of THD1 neurons is required for maintaining longer flight bouts.

Discussion
In this study we demonstrate that the FMRFaR and CaMKII signaling in specific central dopaminergic neurons of the adult Drosophila brain helps maintain optimal membrane excitability (Figs 5A-5D and 6A-7D). Previous work by our group demonstrated that loss of IP 3 R mediated Ca 2+ signaling in central dopaminergic neurons during pupal stages similarly affects flight circuit function in adults [6,7]. In parallel, neuropeptidergic modulation of flight by the Pdf receptor was investigated, but the exact class of neurons that required this receptor or the downstream effectors remained elusive [14]. For the first time, we describe here a neuropeptidergic signaling mechanism in adult brain neurons that is required for sustained flight (S6A Fig). Our data support a model where FMRFa-modulated activity in central dopaminergic neurons belonging to the PPL1 and PPM3 clusters extends the duration of flight bouts, a behavior that is likely to have significant effects on optimal sourcing of nutrients, finding sites for egg laying and identifying mates in the wild.
The role of neuropeptide signaling in modulating animal behavior is well documented [2,4]. Indeed, rescue of flight deficits by overexpression of the cation channel dTrpA1 in dopaminergic neurons of FMRFaR knockdown flies directly addresses the importance of peptide driven neuronal excitability, for flight behavior (Fig 6E). Reduced cellular responses to a depolarizing stimulus observed upon FMRFaR knockdown in THD1 neurons may arise from functional modification, possibly by CaMKII, of plasma membrane resident ion channels, or their expression levels (Figs 5A-5D and 6A-6D). However, our data do not exclude other kinases or modifiers as functioning downstream of the FMRFaR. We predict that FMRFaR in dopaminergic neurons is a key neuromodulator of membrane excitability which stimulates release of dopamine containing synaptic vesicles. In fact, stimulation of synaptic transmission downstream of FMRFaR activation has been described previously in Drosophila larval neuromuscular junctions [13]. Though we have not tested synaptic vesicle release directly upon FMRFaR stimulation, our data support an essential role for synaptic transmission and dopamine synthesis in adult dopaminergic neurons for maintenance of flight bout durations (Fig 7A and 7B).
Identification of the precise ion channels that are affected by FMRFaR signaling, leading to changes in membrane excitability and very likely synaptic transmission in flight modulating dopaminergic neurons, needs further investigation.
Multiple FMRFa peptides exist in Drosophila [26,27]. Cells positive for FMRFa peptides have been characterized in the Drosophila central nervous system using antisera specific to some of the FMRFa peptides [42][43][44][45]. Immunostaining of adult brains, especially against the peptide, DPKQDFMRFa (also used in this study) have revealed extensive neuronal varicosities in the anterior and lateral regions of the protocerebrum [44,45]. It is hence conceivable that FMRFa released through these projections activates the FMRFaR on the anatomically proximal PPL1 and PPM3 clusters of dopaminergic neurons. The PPL1 neurons are known to innervate the superior protocerebrum, a region considered to function at the interface of olfactory input and motor output modules of the brain [46]. Thus activation of the FMRFaR on dopaminergic neurons might stimulate dopamine release, which could then reinforce a dopaminergic circuit, required for sustained flight. Furthermore, co-expression of the dopamine synthesizing enzyme Tyrosine Hydroxylase (TH) in a few neurons marked by an FMRFaGAL4 [47], suggests the existence of an autocrine signaling mechanism within the flight modulating dopaminergic neurons. This idea is supported by recent studies of single cell sequencing of Drosophila neurons where FMRFa transcripts were seen in a small number of central brain dopaminergic neurons [48,49]. Interestingly, a recent report from Manduca demonstrated that an FMRFa positive neuron lies at the center of a putative sensory-motor circuit for integration of olfactory stimuli with wing movements during flight [50]. The natural context in which FMRFa release is triggered for modulation of flight in Drosophila remains to be identified. Neuromodulatory signals from other receptors in central dopaminergic neurons is also a possibility and would widen the scope of sensory inputs received by these cells for integration with flight behavior.
FMRFaR activation by the peptide DPKQDFMRFa stimulates intracellular Ca 2+ release through the IP 3 R (Fig 2D-2F) [13] and enhances synaptic transmission at the larval neuromuscular junction and thereby modulates muscle contraction [13,29,45]. Previous work from our lab has demonstrated that in Drosophila neurons, subsequent to intracellular Ca 2+ release, cytosolic calcium is further elevated by store-operated Ca 2+ entry (SOCE) through dOrai [51]. Thus, we predict that the rise in Ca 2+ we see upon FMRFa stimulation of THD1 neurons could possibly be a combination of both IP 3 R mediated Ca 2+ release and SOCE. Rescue of flight deficits in the FMRFaR knockdown flies by an itpr + transgene ( Fig 3A) and loss of peptide induced Ca 2+ rise in an itpr knockdown background (Fig 2D-2F) support the idea that IP 3 R-mediated Ca 2+ release is likely downstream of FMRFaR activation. Activation of overexpressed IP 3 Rs in the FMRFaR knockdown condition might occur through alternate receptors. It is also possible that overexpression of the IP 3 R allows for more ER-Ca 2+ release from the residual FMRFaRs after RNAi knockdown. Thus, in the context of flight as well, FMRFaR signaling in dopaminergic neurons appears to function upstream of IP 3 R-mediated Ca 2+ release (Figs 2D-2F and  3A). The adult requirement of FMRFaR on dopaminergic neurons supports a primary role for this receptor in mature neurons for modulation of flight, but not as much for maturation of the flight circuit in pupal stages (Fig 3A). In contrast, the dFrizzled2 receptor, which also functions upstream of the IP 3 R in central dopaminergic neurons, was shown to be required exclusively during the pupal stages, albeit in a different dopaminergic cluster of the brain [6]. These observations support the existence of specific receptors that stimulate IP 3 -mediated Ca 2 + release and function either during flight circuit maturation in pupae or in a neuromodulatory role in adults, to influence flight circuit dynamics. A range of flight deficits in IP 3 R mutants with differing allelic strength support this idea [20].
As described previously by other groups in larval motor neurons and in larval neuromuscular junctions [13,29], our genetic and cellular data also suggests that CaMKII might be a downstream effector of FMRFaR signaling in neurons (Fig 4A, S3F-S3H Fig). Although we have shown increased pCaMKII immunostaining in larval CNS neurons upon FMRFa stimulation (S3F-S3H Fig), direct activation of either CaMKII or other Ca 2+ dependent kinases by FMRFa in adult THD1 neurons, remains to be tested rigorously. CaMKII, thus far has been known to contribute to synaptic plasticity in the context of learning and memory [52,53]. Our data, for the first time, demonstrate that CaMKII is required in dopaminergic neurons for maintenance of Drosophila flight over periods of several minutes, possibly by modulating neuronal firing during flight. Multiple mechanisms for CaMKII dependent modulation of membrane excitability have been described in Drosophila such as phosphorylation of the potassium channel, Eag, leading to an increase in the Eag current [54]. In another study, CaMKII dependent phosphorylation of the Ca 2+ activated potassium channel binding protein, Slob was shown to favor its binding to 14-3-3, that eventually altered the voltage sensitivity of slowpoke channels [55]. In both cases, CaMKII acted as a negative regulator of neuronal excitability, and support our data demonstrating flight deficits observed upon overexpression of WT-CaMKII and CaM-KII T287D (S3A Fig). However, the more interesting and compelling explanation for our data demonstrating reduced KCl-induced depolarization upon CaMKII inhibition by Ala comes from in vitro work that has described a role for CaMKII in decelerating the inactivation of voltage sensitive calcium channels, thereby enhancing transmitter release [56]. This was however shown to be independent of its kinase activity. More recently, there is evidence to suggest CaMKII-dependent activation of transcription factors in Drosophila neurons [57,58]. Hence, the possibility of transcriptional regulation of voltage-gated membrane channel genes by CaM-KII cannot be excluded and needs further investigation.

Fly rearing and stocks
Drosophila strains used in this study were maintained on cornmeal media, supplemented with yeast. Flies were reared at 25˚C, unless otherwise mentioned. WT strain of Drosophila used was Canton-S. The fly lines nSybGAL4 (BL51635), UASFMRFaR RNAi (BL25858), UAScas9 UASWT-CaMKII (BL29662), UASCaMKII T287D (BL29664), UASTH RNAi (BL25796), UASDicer (BL24648) were obtained from Bloomington Drosophila Stock Centre (BDSC). The UASitpr R-NAi (1063-R2) strain was from National Institute of Genetics (NIG) and the UASCaMKII RNAi (v38930) was from Vienna Drosophila Resource Center (VDRC). A strain with two copies of TubGAL80 ts on the second chromosome was generated by Albert Chiang, NCBS, Bangalore, India, and has been used for all TARGET experiments. The dopaminergic GAL4 driver, THGAL4 [21] and the THGAL80 strain [23] were kindly provided by Serge Birman (CNRS, ESPCI Paris Tech, France). All the TH subset GAL4s (THD1, THD', THC1, THC') were a gift from Mark N Wu (Johns Hopkins University, Baltimore) [22]. The UASeGFP transgene was provided by Michael Rosbash (Brandeis University, Waltham, MA). The UASShi ts strain was obtained from Toshihiro Kitamoto (University of Iowa, Carver College of Medicine, Iowa City). The generation and use of UASitpr + transgene has been described earlier [15,59]. The UASFMRFaR + strain was generated by microinjection of the pUAST-FMRFaR full-length cDNA into fly embryos and generating stable UAS transgenic lines. Standard fly genetics were followed for making strains and recombinants.
Temperature shift experiments were performed as described below. Briefly, larvae, pupae or adults were maintained at 18˚C and transferred to 29˚C only at the stage when the UAStransgene needed to be expressed. Firstly, as a control, flies of the genotype THD1;TAR-GET>UAS-transgene were maintained throughout at 18˚C, a condition where the UAS-transgene expression is suppressed (labeled as '18˚C' in figures). To observe maximum effect, flies of the same genotype were grown at 29˚C throughout development and as adults (labeled '29˚C'). For larval specific knockdown, animals of the desired genotypes developed at 29˚C from embryos to the wandering larval stage, following which they were maintained at 18˚C till the time when they were tested for flight (labeled as '29˚C Larval'). Likewise, for pupal specific knockdown, wandering third instar larvae were shifted from 18˚C to 29˚C. Pupae developed at 29˚C and were transferred to 18˚C, soon after eclosion (labeled as '29˚C Pupal'). In case of adult specific knockdown, larval and pupal development continued at 18˚C. Following this, adult flies were maintained at 18˚C for two days post eclosion and then transferred to 29˚C and used for experiments at 1, 2, 4, 6 or 8 days after transfer (labeled as '29˚C Adult').

Generation of a knockout for FMRFaR using the CRISPR-Cas9 system
To generate a FMRFaR null allele, we used the CRISPR-Cas9 methodology [60][61][62][63][64]. Two guide RNAs (gRNAs) were designed, at the 5' and 3'ends of the gene at the following sequences: (sg1-GGGAGCCATGAGTGGTACAGCGG, sg4-GATCTCTGCATTTCGCGGGCGGG) so as to delete~1.5 kb from the coding region of the 1.6 kb gene. A gRNA dual transgenic fly (FMRFaR dual ) was made first [24]. FMRFaR dual transgenic males were mated with Act5c-Cas9 virgins [65]. From the F1 progeny obtained, 16 were screened by PCR for the deletion. Because all flies tested were positive for the deletion, 4 F1 flies were individually crossed to balancers and 30 F2 progeny were screened for the deletion. Progeny, that were positive for deletion and negative for presence of dual gRNA (total 6 F2s), were maintained as stable lines. The deletion was confirmed by sequencing of the DNA from the FMRFaR knockout flies. The following primers were used for confirmation of deletion-5'F: GACATAGTCATCAGGTGCTC, 5'R: TGCACCTCCGTGTGGTTAAG, 3'F: GAACAACGGCGATGGAACTC, 3'R: GGTGCTCTA AGTCAACCCCT. For tissue specific deletions of FMRFaR gene, a fly strain containing FMRFaR dual and UAScas9 was made and subsequently mated with GAL4 strains of interest.

Single flight assay
Single flight tests were performed on adult flies 3-5 days post eclosion, unless specified differently. Flies were anaesthetized on ice for 2-3 minutes and then tethered using nail polish applied to the end of a thin metal wire. The tether was glued onto the region between the neck and thorax of adult flies. A gentle mouth blown air-puff was delivered to test for flight response. Flight time in tethered condition was recorded for a maximum of 15 minutes. In three independent batches of 10, a minimum of 30 flies were tested for each genotype. All control genotypes tested were obtained by crossing the GAL4 or UAS strain of interest to the wildtype strain, Canton-S. Raw data was plotted as box and whisker plots using Origin (OriginLab, Northampton, MA) software. Each bar represents 25 th to 75 th percentile. The solid diamond and horizontal line within the bars indicate mean and median, respectively. Individual values are shown as open diamonds. Because the distribution of flight data was not normal, we have used the non-parametric Mann-Whitney U-test, which compares medians across the datasets.
However, the mean is also represented in all bar graphs and is especially useful for data when flight times are shorter and the distributions are normal.

Locomotor assay
Locomotor activity was measured by modifying a previously published protocol [66]. Briefly, locomotor activity of 4-6 day old singly housed virgin males was tested in a circular chamber of diameter 4 cm. The chamber was placed on a sheet containing a specific pattern. Single flies were aspirated into each chamber and allowed to acclimatize for 5 minutes. Each fly was monitored separately for the number of times it crossed each line in the pattern, over a period of 10 seconds. For every experiment, six single flies were tested sequentially for the span of 10 seconds. This was repeated 5 times, i.e. we measured locomotor activity of every single fly five times in blocks of 10 seconds, so as to randomize activity measurements during the experimental time. Total locomotor activity is represented as the sum total of the number of lines crossed by each fly over the experimental duration of 50 seconds (plotted as Locomotor Activity Units). A minimum of 25 flies were tested from each genotype.

Ex vivo live imaging of adult brains
Adult brains were dissected in adult haemolymph-like solution (AHL) [67] and embedded iñ 10 μl of 1% low melt agarose (Invitrogen) with anterior side facing upwards. Brains were then bathed in 100 μl AHL until imaging. Images were obtained on an Olympus FV1000 inverted microscope (Olympus Corp., Japan) in time lapse mode using the 20X objective. Both GCaMP6m and Arclight signals were captured using the 488 nm excitation laser line. Fifty frames of basal activity were recorded, followed by stimulation with either 5 μM Peptide (DPKQDFMRFa, NeoBioLab, Massachusetts, USA) or 70 mM KCl. A minimum of 5 brain explants were imaged for every experimental condition.
Raw fluorescence data for regions of interest were extracted using the Time Series Analyser plugin in Image J 1.47v. Change in fluorescence, ΔF/F was calculated using the following formula for each time (t): ΔF/F = (F t -F 0 )/F 0 , where F 0 is the average basal fluorescence of the first 10 frames. Area under the curve and Peak ΔF/F was calculated using Microsoft Excel considering all frames post-stimulation. Values were plotted as box plots.

Sorting of adult neurons by FACS
Twenty adult CNS (THGAL4>eGFP) were dissected in cold Schneider's medium (#21720-024, Life Technologies) followed by~45 minutes incubation in an enzymatic solution containing 0.75 μg/μl of Collagenase (Sigma-Aldrich) and 0.40 μg/μl Dispase (Sigma-Aldrich). Brain lysates were then spun at 3000 rpm for 3 minutes. The supernatant was discarded and the pellet was resuspended in cold Schneider's media. Single cell suspensions were obtained by gentle tituration and then passed through a 40 μm mesh filter. Fluorescence-activated cell sorting (FACS) of samples was carried out as described earlier [7]. A minimum of 4 biological replicates were sorted and~10,000 GFP +ve and GFP -ve cells were collected separately in TRIzol Reagent (Life Technologies) for RNA isolation.

RNA isolation and qPCR
Total RNA was extracted from sorted cells or dissected adult CNS using TRIzol RNA extraction protocol (Ambion, ThermoFischer Scientific). In case of dissected CNS, a minimum of three biological replicates, each containing 5 brains, was used per genotype. Approximately 500 ng of RNA was then used for DNAse treatment and first strand cDNA synthesis. Quantitative real-time PCR was performed on ABI7500 fast machine (Applied Biosystems) using Kapa SYBR FAST qCR Master mix (KAPA Biosystems, Wilmington, MA). rp49 was used as an internal control to quantify relative levels of the target transcripts. qPCR values are represented as normalized fold change of mRNA transcripts of the indicated genes. In case of sorted cells, normalized mRNA levels of genes in GFP -ve cells have been plotted relative to the GFP +ve population. Every qPCR run was followed by a melt curve analysis to confirm primer specificity. The following formula was used to calculate fold change: Fold change = 2 -ΔΔCt , where, ΔΔCt = (Ct( target gene )-Ct( rp49 )) Experimental -(Ct( target gene )-Ct ( rp49 )) Control Sequences of primers used are as follows: rp49:

Immunohistochemistry
Immunohistochemistry was performed on dissected adult CNS as described earlier [7]. Briefly, brains were dissected in 1x PBS, followed by fixation in 4% paraformaldehyde and blocking in 0.2% phosphate buffer, 0.2% Triton-X 100 and 5% normal goat serum. Overnight incubation with primary antibodies was followed by 2 hours of incubation with corresponding fluorescent secondary antibodies. The primary antibodies used were: mouse anti TH (1:50, #22941, Immu-noStar) and rabbit anti GFP (1:10,000, A6455, Life Technologies). The following were the fluorescent secondary antibodies used at 1:400: anti-mouse Alexa Fluor 568 (#A11004, Life Technologies) and anti-rabbit Alexa Fluor 488 (#A11008, Life Technologies). Images were obtained as confocal stacks of 1 μm thickness using 20X objective on Olympus FV1000 Confocal microscope.

Primary neuronal culture and immunostaining
Primary neuronal cultures and immunostaining of neurons was carried out as described previously [34]. Briefly, third instar larval CNS were dissected in Schneider's medium containing 50 μg/ml Streptomycin (Invitrogen), 10 μg/ml Amphotericin B (Invitrogen) and 50U/ml Penicillin (Invitrogen). Following this, brains were subjected to 40 minutes of enzyme treatment in 0.75 μg/ml Collagenase and 0.40 μg/ml Dispase. Neurons were dissociated, spun down and plated in growth medium, DMEM/F12-1065 (Gibco) supplemented with the antibiotics mentioned previously and 20mM HEPES. All cell culture reagents were procured from Sigma-Aldrich, unless specified differently. Fourteen-sixteen hours old cultures were washed twice with hemolymph-like saline, HL 3 and incubated with either the FMRFa peptide or a solvent control for 20 minutes. Following this, cells were fixed in 3.5% paraformaldehyde for 20 minutes at room temperature and then washed in wash buffer (1/10 th dilution of blocking buffer). Cells were then permeabilized for a total of half an hour (10 minutes Ã 3 times), blocked in blocking buffer (5% BSA, 0.5% Triton X in PBS) for 1 hour, at room temperature and subsequently incubated overnight in the primary antibody diluted in wash buffer (mouse anti pCaMKII-22B1; 1:100; #sc-32289, Santa Cruz). The next day, cells were washed thrice in wash buffer (for 10 minutes each) and incubated with anti-mouse Alexa Fluor 568 (#A11004, Life Technologies) secondary antibody for half an hour at room temperature. Control dishes for the secondary antibody were treated as the experimental dishes and were incubated with the fluorescent secondary, but not the primary antibody. Following another set of three washes (15 minutes each) in wash buffer, cells were covered in 60% glycerol before imaging. An inverted Olympus FV3000 confocal microscope with a 60X oil objective (1.42 NA), pinhole size 150 μm was used to image the cells. GFP and pCaMKII signals were captured with 488 and 561 laser lines respectively. The same settings were used to capture images from all experimental conditions, on any particular day of imaging. The entire cell volumes of cells were captured as optical slices of 0.60 μm thickness. Images were analyzed in Image J as described above. After applying background subtraction, fluorescence values of pCaMKII were divided over GFP values to obtain the ratios shown in S3G Fig.

Data representation and analysis
Origin (OriginLab, Northampton, MA) software was used for plotting raw data and calculation of statistical significance. Raw data were tested for normality. A Student t-test or an ANOVA statistical test was performed on normally distributed data. Non-normal data were tested for statistical significance using the Mann-Whitney U-test.