Serotonergic neurons of the Drosophila air-puff-stimulated flight circuit

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1 Serotonergic neurons of the Drosophila air-puff-stimulated flight circuit SUFIA SADAF and GAITI HASAN* National Centre for Biological Sciences, Tata Institute of Fundamental Research, Bellary Road, Bangalore, India *Corresponding author ( , Monoaminergic modulation of insect flight is well documented. Recently, we demonstrated that synaptic activity is required in serotonergic neurons for Drosophila flight. This requirement is during early pupal development, when the flight circuit is formed, as well as in adults. Using a Ca 2+ -activity-based GFP reporter, here we show that serotonergic neurons in both prothoracic and mesothoracic segments are activated upon air-puff-stimulated flight. Moreover ectopic activation of the entire serotonergic system by TrpA1, a heat activated cation channel, induces flight, even in the absence of an air-puff stimulus. [Sadaf S and Hasan G 2014 Serotonergic neurons of the Drosophila air-puff-stimulated flight circuit. J. Biosci ] DOI /s Introduction This article is dedicated to the memory of Obaid Siddiqi primarily with a sense of loss but also a sense of continuity for carrying on with the tradition of neurogenetics. Obaid Siddiqi established the field of neurogenetics at TIFR, Mumbai, and subsequently at NCBS, TIFR, Bangalore, the Institute of which he was the founder director. His group began studying Drosophila to identify behavioural genes almost as soon as this idea arose from molecular genetics in the second half of the 20th century. Initial successes in this field were the ability to relate the loss of gene function through single gene mutants to an altered behavioural response. Obaid s group generated several such behavioural mutants in Drosophila which have been studied all over the world and which have influenced our knowledge of neural function and behavior (Hasan 2012). Meanwhile, neurogenetics as a scientific field has grown steadily, and an important conceptual advance has been the ability to relate the molecular and cellular function of a gene and its encoded protein on the development and activity of the cognate neural circuit and finally relate all of this to behaviour of the whole organism. Molecular genetic tools that aid in the study of behaviour by this paradigm have grown, and here we describe the use of a few of these recently developed genetic tools to understand Drosophila flight behaviour. Neural circuits for insect flight integrate sensory stimuli with motor activity to produce tightly regulated spatiotemporal responses. Connections for the adult Drosophila flight circuit form during early pupal development (Consoulas et al. 2005). Neurotransmitters such as octopamine, tyramine, dopamine and serotonin have been implicated in both development of the flight circuit and modulation of acute flight (Claassen and Kammer 1986; Stevenson and Meuser 1997; Banerjee et al. 2004; Brembs et al. 2007; Sadaf et al. 2012). Information from Drosophila and other insects suggests that mechanosensory neurons at the base of the antenna project to regions in the antennal mechanosensory and motor centre (AMMC) and the mesothoracic segments to coordinate air-puff-stimulated flight (Dickinson and Tu 1997; Saneet al. 2007; Buhl et al. 2008). However, specific identities of neurons in the flight circuit that respond during voluntary or air-puff-stimulated Keywords. Biogenic amine; leg movement; neurotransmitter; tethered flight , * Indian Academy of Sciences 575 Published online: 7 July 2014

2 576 Sufia Sadaf and Gaiti Hasan flight have not been shown. Recently, we established that synaptic activity in serotonergic neurons is required both during early pupal development and in adults, for sustained flight in Drosophila (Sadaf et al. 2012). In this study, using a Ca 2+ -activity-based GFP reporter, CaLexA (Masuyama et al. 2012), we have investigated the identity of serotonergic neurons that are activated during air-puff-stimulated tethered flight and further analysed the role of serotonergic neurons in Drosophila flight. 2. Material and methods 2.1 Fly strains Cell-specific expression strains: TRHGAL4 (Sitaraman et al. 2012), with regulatory region of the Tryptophan Hydroxylase gene present upstream of yeast GAL4; to mark serotonergic neurons (Serge Birman, ESPCI, Paris, France), DdcGAL4 (Li et al. 2000); with region of the Dopa decarboxylase gene, to mark dopaminergic and serotonergic neurons (Jay Hirsh, UVA, VA, USA). UAS effector genes: UASTrpA1 for heat activation of neurons by the dtrpa1 channel (Bloomington Stock Centre, Bloomington, IN, USA), (Hamada et al. 2008). For the CaLexA experiment we used a strain with the following transgenes: UASmLexA- VP16-NFAT (Masuyama et al. 2012), LexAop-CD8-GFP- 2A-GFP (Masuyama et al. 2012) andlexaop-cd2-gfp (Lai and Lee 2006) (Jing W. Wang, UCSD, CA, USA). 2.2 Temperature shift experiments Flies were allowed to grow at 18 C throughout development and adult animals were maintained at this temperature for three days prior to the experiment. For activation of the TrpA1 cation channel in neurons, the ambient temperature was raised to 29 C for the duration of the experiment. Single flight assays were performed with tethered flies maintained at 20 C and later shifted to 29 C. Videos at 29 C were captured at 25 frames per second through a Nikon camera (Nikon Coolpix P6000, Nikon Inc., NY, USA). 2.3 Climbing assay Climbing assay was performed on 3-day-old flies in batches of 10. Flies were put in a measuring cylinder of 2 cm diameter and left to acclimatize for 1 min. The measuring cylinder was banged thrice in rapid successions. The animals that were able to cross a marked point of 8 cm within 12 s were scored as climbers. The data was plotted as percentage of mean climbers in Origin 7.5 (MicroCal, Origin Lab, Northampton, MA, USA). 2.4 Quantification of single animal flight The flight times of both male and female flies were similar in all genotypes. Animals were anesthetized on ice and tethered (Sadaf et al. 2012). The ambient temperature during tethering was 20 C. Flies were allowed to hold small pieces of paper to avoid spontaneous flight. Tethered animals were shifted to the experimental room that was maintained at 29 C. Flight duration was monitored in response to temperature elevation. For each genotype studied, flight durations were recorded for minimum of 30 flies and quantified as described in the section on statistical tests. Leg movements were counted for 5 animals and plotted as the mean and S.E.M. in Origin 7.5 (MicroCal). 2.5 CaLexA imaging Animals of the genotype UAS-mLexA-VP16-NFAT, LexAop- CD2-GFP; TRHGAL4/LexAop-CD8-GFP-2A-GFP were raised at 18 C. Pupae were collected in batches of three to four and each batch was placed in a single vial. The vials were covered with aluminum foil to avoid light-based activity and maintained at 18 C. Post eclosion, males and females were separated and put in fresh vials, where a cotton plug was placed close to the surface of the food to restrict mobility and the vials were covered with aluminum foil. Animals were aged for 3 days at 25 C and anaesthetized on ice for 20 min prior to tethering. Tethering was done as described above. Post-tethering, the flies were given bits of paper to hold with their legs so as to prevent spontaneous flight. A mouth blown air-puff stimulus was delivered to the animals and they were allowed to fly till cessation of voluntary flight was observed. Animals were de-tethered using acetone, stored in separate vials in similar conditions as described above, at 25 C. Dissections were carried out either 8 h or 24 h post-tethered flight. No-stimulus controls were treated in an identical manner, except that they were not given an airpuff stimulus while holding onto the bits of paper and hence did not fly while tethered. 2.6 Immunohistochemistry on adult brains Immunohistochemistry was performed on Drosophila adult brains, expressing the CaLexA transgenes with TRHGAL4, after fixing the dissected tissue in 4% paraformaldehyde. The following primary antibodies were used: mouse monoclonal anti-5ht (1:50; #MS1431S, NeoMarkers, Fremont, CA), rabbit anti-gfp antibody (1:10,000; #A6455, Molecular Probes, Eugene, OR, USA). Fluorescent secondary antibodies (Molecular Probes) were used at a dilution of 1:400 as follows: anti-rabbit Alexa Fluor 488 (#A11008) and anti-mouse Alexa Fluor 568 ((#A11004). Confocal analysis

3 5-HT neurons activated by flight 577 was performed on an Olympus Confocal FV1000 microscope (Olympus Corporation, Tokyo, Japan) using either a 20X objective with a numerical aperture (NA) of 0.9. Confocal data were acquired as image stacks of separate channels and combined and visualized as three-dimensional projections using the FV10-ASW 1.3 viewer (Olympus Corporation). 2.7 Statistical tests The mean flight duration and S.E.M values for TrpA1 induced tethered flight were computed using Origin 7.5 software (MicroCal) on raw data obtained from 30 animals. Statistically significant differences were calculated using one-way ANOVA tests between the genotype of interest and the corresponding genetic control as mentioned in individual figure legends. All graphs were plotted using either the Origin 7.5 software (MicroCal). 3. Results 3.1 Serotonergic neurons of the thoracic ganglion form part of the air-puff-stimulated flight circuit Synaptic activity is required in serotonergic neurons both during early pupal development and in adults (Sadaf et al. 2012). To identify serotonergic neurons of the air-puff stimulated flight circuit in adults, we expressed a Ca 2+ activity reporter, CaLexA in serotonergic neurons (TRHGAL4/ UAS-mLexA-VP16-NFAT, LexAop-CD2-GFP; LexAop- CD8-GFP-2A-GFP; subsequently referred to as TRH>CaLexA) (Masuyamaet al. 2012). Animals were raised in the dark with restricted mobility. Tethered flight was stimulated in 3-day-old flies by a gentle air-puff stimulus (Sadaf et al. 2012). In this paradigm, animals could show sustained flight for durations lasting from 30 s up to 5 min. Brains and thoracic ganglia were dissected from these animals 8 h post air-puff stimulated flight and immunostained for GFP (to detect CaLexA based activity) and serotonin (to identify serotonergic neurons). We observed GFP and serotonin positive neurons in the prothoracic (T1) and mesothoracic segments (T2) of ventral ganglia (figure 1A B; table 1). The prothoracic segment is thought to control coordination of leg movements for walking, whereas flightcoordinating interneurons are housed in the mesothoracic segment. In T1, four serotonergic neurons, a, b, c and d expressed GFP (figure 1C) (nomenclature in accordance to our previous study (Sadaf et al. 2012)). Additionally, a nonserotonergic neuron was GFP positive in T1 (arrow head, figure 1B). Presumably, this neuron is part of the flight circuit, and is one amongst the few non-serotonergic neurons that express TRHGAL4 (Sitaraman et al. 2012). In T2, the a, b, c and d serotonergic neurons (Sadaf et al. 2012) all exhibit CaLexa based GFP expression indicating that they are activated during tethered flight (table 1; three neurons seen in figure 1B). Control animals, which did not receive an air-puff stimulus but were treated in an identical manner, show normal serotonergic neurons in the T1 and T2 segment but without any GFP expression. A minimum basal level of GFP was observed in the brain and in some neurons of the abdominal segments of both control (figure 1C) and air-puffstimulated animals (arrows, figure 1A and C). These abdominal neurons may be activated in response to egg laying in controls and air-puff-stimulated animals both of which were females. In Drosophila and other insects, serotonin modulates feeding, reproduction and locomotion (Kamyshev et al. 1983; Barreteau et al. 1991; NovakandRowley1994; Neckameyer et al. 2007). 3.2 Serotonergic neurons control concerted leg-movements during tethered flight Because serotonergic neurons of the T1 segment were also activated in response to air-puff-stimulated flight (figure 1A, B), we hypothesized that leg movements at the onset of airpuff-stimulated tethered flight may be modulated by the T1 serotonergic neurons. To test this hypothesis, we re-visited tethered flight videos of TRH>Kir2.1 (with hyperpolarized serotonergic neurons and thus reduced synaptic activity) and TRH>TNT (reduces synaptic vesicle recycling in serotonergic neurons), published previously (Sadaf et al. 2012). In control animals it was observed that 3s prior to the air-puff stimulus, the prothoracic legs moved 5 times on an average, whereas the meso- and metathoracic legs moved 8-9 times in the same time interval (figure 2A, B). Interestingly, in animals with reduced neural activity in serotonergic neurons, leg movements prior to air-puff delivery were either absent (TRH>Kir2.1) or occasional (TRH>TNT) (figure 2A, B). Next we analysed leg movements during tethered flight. In control animals the number of leg movements persisted to the same extent, both before and during flight (figure 2C, D). However, during tethered flight the leg movements were primarily restricted to small joint movements of the legs. This was in contrast to the full leg extensions from body plane that were observed prior to the airpuff. In animals where synaptic activity was reduced in serotonergic neurons (TRH>Kir2.1), and where flight activity lasted for 2 s after an air-puff stimulus (Sadaf et al. 2012), movement of the prothoracic and mesothoracic legs was significantly reduced (0 3 times) as was movement of the metathoracic legs (3 5 times; figure 2C, D). Although, the TRH>TNT animals exhibit no flight, occasional metathoracic leg movement was observed (figure 2C, D). The most consistent observation was the inability of TRH>Kir2.1 and TRH>TNT animals to extend all the three pair of legs in response to an air-puff, as compared with the control, where the animal thrust out all three pairs of legs (figure 2E).

4 578 Sufia Sadaf and Gaiti Hasan Interestingly, the climbing activity of TRH>Kir2.1 and TRH>TNT animals remained normal (figure 2F), suggesting that the motor output of the walking circuit is not affected. These results support the idea that serotonergic neurons in the T1 and T2 segment coordinate leg movements with wing movement after air-puff stimulation for tethered flight.

5 5-HT neurons activated by flight 579 Figure 1. CaLexA-based activation of serotonergic neurons in during tethered flight in response to an air-puff stimulus. (A) Representative image of a brain and thoracic ganglion (TG) from a TRH>CaLexA dissectedfly8haftertetheredflight(n=5).anti-gfp immunostaining was done to mark GFP expression in response to Ca 2+ activation; anti-5-ht staining was carried out to confirm the serotonergic identity of GFP-positive neurons. Arrow marks the GFP positive abdominal segment neurons present in both controls (see below) and air-puff-stimulated animals. (B) Magnified image of the T1 and T2 segment from (A). An additional GFP positive and non-5- HT neuron is seen in the T1 segment (arrow head). (C) Representative image of the brain and thoracic ganglion of TRH>CaLexA control animals, which were not presented with an air-puff (n=5). See also table Activation of aminergic neurons induces flight, even without an air-puff To test if activation of the aminergic system in adult flies is sufficient to induce flight, we expressed the heat-activated cation channel, TrpA1 (Hamada et al. 2008), in aminergic neurons, using DdcGAL4 (Li et al. 2000), which expresses in both serotonergic and dopaminergic neurons. Activation of TrpA1 by shifting Ddc>TrpA1 tethered flies to 29 C induced flight activity in all animals with flight durations that ranged from 20 s to 6 min (Ddc>TrpA1, figure 3A). Because we have recently demonstrated a role for dopaminergic neurons in flight (Sadaf et al., under review) we asked if the DdcGAL4 driven neuronal activation of flight observed occurs through serotonergic neurons. For this purpose we took tethered flies of the genotype TRH>TrpA1 and tested flight durations in these animals after shifting to 29 C. Out of 30 animals tested, 13 animals showed flight which ranged from 20 s to 6 min, 3 animals showed flight for 3 4 s and the remaining 14 TRH>TrpA1 animals did not initiate flight (figure 3A, B). Thus, the effect of TrpA1 activation in serotonergic neurons was less as compared to activation of all aminergic neurons, supporting an additional role for dopaminergic neurons in flight. In control animals with either the GAL4 driver or the TrpA1 transgene, no flight was observed upon transfer to 29 C (figure 3A, B). Experimental animals when tested at 20 C did not exhibit any flight (data not shown). The effect of TrpA1 activation Table 1. Cell numbers in T1 and T2 in TRH>CaLexA during airpuff flight Sample number Number of 5-HT cells in T1 Number of 5-HT cells in T2 1. (shown in figure 1A, B) 4 (a, b, c, d) 3 (a, b, c ) 2. 4 (a, b, c, d) 4 (a, b, c, d ) 3. 3 (a, b, c) 4 (a, b, c, d ) 4. 4 (a, b, c, d) 3 (a, b, c ) 5. 4 (a, b, c, d) 4 (a, b, c, d ) Number of serotonergic neurons observed in T1 and T2 segments across five samples in TRH>CaLexA animals 8h after air-puff stimulated tethered flight (see also figure 1). in serotonergic neurons upon leg movement could not be tested because controls and experimental animals at 20 C and 29 C showed the same extent of leg movements (data not shown). We therefore conclude that activation of serotonergic neurons in the entire CNS can be sufficient to induce flight and that serotonergic neurons are either a part of the circuit or modulate the circuit for concerted leg-movement during active flight. 4. Discussion Previous studies have shown that insect flight can be modulated by serotonergic neurons (Claassen and Kammer 1986; Parker 1995; Sadaf et al. 2012). However, the identity of these neurons was not known. The CaLexA -based experiments have now helped identify 3 4 serotonergic neurons, referred to as a, b, c and d in the T2 segment, that are activated upon tethered flight. The connectivity of these serotonergic interneurons to flight motor neurons will help establish their precise role in insect flight. Activation of either the entire aminergic system (Ddc>TrpA1) or the serotonergic system (TRH>TrpA1) led to flight in the absence of an air-puff stimulus. Taken together these data suggest that activation of multiple serotonergic neurons, including the ones in the T2 segment, can stimulate flight. An air-puff is one sensory stimulus that activates serotonin-modulated flight, but other stimuli such as vision and olfaction may also function in this context. Further, identification of sensory neurons and other aminergic interneurons, which ultimately communicate with the flight motor neurons for airpuff-stimulated flight, will be of interest. Leg movements also accompany flight. In Drosophila, leg extension is seen prior to flight during the giant-fibremediated escape response (Hammond and O'Shea 2007). Because animals with reduced synaptic activity did not extend the three pairs of legs with a thrust in response to an airpuff as compared with controls, the jump response of TRH>Kir2.1 animals will be investigated further. It is likely that in freely behaving animals, synaptic activity in serotonergic neurons modulates the jump response prior to flight. In locusts, serotonin has been shown to modulate fast extensor and tibiae motor neurons (Parker 1995). In moths, locusts and cockroaches, octopamine and tyramine have been

6 580 Sufia Sadaf and Gaiti Hasan Figure 2. Reduced synaptic activity in serotonergic neurons abolishes concerted leg movements during active flight. (A) Snapshot of control (Kir2.1/+), TRH>Kir2.1 and TRH>TNT animals 3 s before air-puff stimulus. (B) Quantification of pro, meso and metathoracic leg movements in animals of the indicated genotypes (n=5) obtained by manually counting the movements for 3 s before air-puff stimulus (movies were recorded at 25 frames per s). (C) Snapshot of the same animals as in (A), 2 s after air-puff-induced flight. (D) Quantification of leg movements for the three pairs of legs for 2 s after air-puff-induced flight. (E) Snapshot of the same animals as in (A) and (B), upon air-puff stimulus delivery (to observe leg extension). (F) Quantification of percentage of climbers of indicated genotypes. Results were obtained from 10 batches of 10 flies each and expressed as mean ±S.E.M. implicated in leg movements (Sombati and Hoyle 1984; Strauss 2002; Rillich et al. 2013; Ritzmann and Zill 2013). Exclusive walking pattern generators have been shown in insects, including Drosophila, where the protocerebral bridge in the central complex is important for proper forward walking (Strauss 2002) and descending neurons in the subesophageal ganglion for backward walking (Bidaye et al. 2014). Injection of serotonin in 3-day old adult Drosophila

7 5-HT neurons activated by flight 581 Figure 3. TrpA1-based activation of serotonergic neurons induces flight in tethered animals. (A) Quantification of total flight duration in tethered animals (n=30) by TrpA1 activation of either aminergic neurons (Ddc>TrpA1) or serotonergic neurons (TRH>TrpA1). TrpA1 activation was achieved by transfer to 29 o. GAL4 and the TrpA1 transgenic controls did not show flight. Results are shown as mean ±S.E.M. with individual data points for the 30 individuals tested. (B) Representative snapshots of a TRH>TrpA1 animal and a control (TrpA1/+) at 20 (left) and 8 s after shifting to 29 (right). Temperature elevation from 20 C to 29 C could induce flight in TRH>TrpA1 animals, without an air-puff. induces increased locomotor activity (Kamyshev et al. 1983). These studies, however, did not analyze if concerted leg-movement occur during flight. In locusts, different circuits have been implicated in walking and flight (Rillich et al. 2013). Although tyramine treatment can readily induce walking behaviour, octopamine induces fictive flight and additionally, couples leg movements with flight in tethered animals (Rillich et al. 2013). Leg movements in freely behaving insects occur during walking (Ritzmann and Zill 2013) or during grooming behaviour (Vandervorst and Ghysen 1980; Berkowitz and Laurent 1996). During free flight, leg movements have not been reported to accompany wing movements. It is possible that leg movements are seen only during tethered flight (Lorez 1995; Rillich et al. 2013). Nonetheless, the a, b, c and d 5-HT neurons in T1 segment appear to modulate concerted leg movements during airpuff-stimulated flight in tethered animals. The role of serotonergic interneurons in specific walking behaviours,

8 582 Sufia Sadaf and Gaiti Hasan such as coordinated leg movements, forward walking, backward walking and correct turns during walking need further study. 5. Conclusion Our results show that the serotonergic neurons in the first and second thoracic segment are activated during air-puff-stimulated tethered flight (figure 1). Concerted leg-movements during active flight require synaptic activity in serotonergic neurons (figure 2), and activation of the aminergic neurons and specifically serotonergic neurons can induce flight in tethered animals (figure 3). Thus neural activity in serotonergic neurons is both necessary and sufficient for air-puff-stimulated tethered flight, which is a laboratory paradigm for voluntary flight. Acknowledgements This work was supported by a research fellowship from the Council of Scientific and Industrial research ( rdpp.csir.res.in/csir_acsir/home.aspx) to SS. The project was supported financially by core funding from the National Centre for Biological Sciences, Tata Institute of Fundamental Research ( to GH. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. We thank Dr Serge Birman (ESPCI, France) for providing reagents and Dr H Krishnamurthy and the NCBS Central Image-Flow Facility for help with confocal imaging. References Banerjee S, Lee J, Venkatesh K, Wu CF and Hasan G 2004 Loss of flight and associated neuronal rhythmicity in inositol 1,4,5-trisphosphate receptor mutants of Drosophila. J. Neurosci Barreteau H, Perriere C, Brousse-Gaury P, Gayral P, Jacquot C and Goudey-Perriere F 1991 Indolamines in the cockroach Blaberus craniifer Burm. nervous system I. Fed and crowded young females. Comp. Biochem. Physiol. C Berkowitz A and Laurent G 1996 Local control of leg movements and motor patterns during grooming in locusts. J. Neurosci Bidaye SS, Machacek C, Wu Y and Dickson BJ 2014 Neuronal control of Drosophila walking direction. Sci Brembs B, Christiansen F, Pfluger HJ and Duch C 2007 Flight initiation and maintenance deficits in flies with genetically altered biogenic amine levels. J. Neurosci Buhl E, Schildberger K and Stevenson PA 2008 A muscarinic cholinergic mechanism underlies activation of the central pattern generator for locust flight. J. Exp. Biol Claassen DE and Kammer AE 1986 Effects of octopamine, dopamine, and serotonin on production of flight motor output by thoracic ganglia of Manduca sexta. J. Neurobiol Consoulas C, Levine RB and Restifo LL 2005 The steroid hormone-regulated gene Broad Complex is required for dendritic growth of motoneurons during metamorphosis of Drosophila. J. Comp. Neurol Dickinson MH and Tu MS 1997 The function of dipteran flight muscle. Comp. Biochem. Physiol. Part A: Physiol Hamada FN, Rosenzweig M, Kang K, Pulver SR, Ghezzi A, Jegla TJ and Garrity PA 2008 An internal thermal sensor controlling temperature preference in Drosophila. Nature Hammond S and O'Shea M 2007 Escape flight initiation in the fly. J. Comp. Physiol. A Neuroethol. Sens. Neural. Behav. Physiol Hasan G 2012 The early years of Drosophila chemosensory genetics in Mumbai's Tata Institute of Fundamental Research. J. Neurogenet Kamyshev NG, Smirnova GP, Savvateeva EV, Medvedeva AV and Ponomarenko VV 1983 The influence of serotonin and p- chlorophenylalanine on locomotor activity of Drosophila melanogaster. Pharmacol. Biochem. Behav Lai SL and Lee T 2006 Genetic mosaic with dual binary transcriptional systems in Drosophila. Nat. Neurosci Li H, Chaney S, Roberts IJ, Forte M and Hirsh J 2000 Ectopic G- protein expression in dopamine and serotonin neurons blocks cocaine sensitization in Drosophila melanogaster. Curr. Biol Lorez M 1995 Neural control of hindleg steering in flight in the locust. J. Exp. Biol Masuyama K, Zhang Y, Rao Y and Wang JW 2012 Mapping neural circuits with activity-dependent nuclear import of a transcription factor. J. Neurogenet Neckameyer WS, Coleman CM, Eadie S and Goodwin SF 2007 Compartmentalization of neuronal and peripheral serotonin synthesis in Drosophila melanogaster. Genes Brain Behav Novak MG and Rowley WA 1994 Serotonin depletion affects blood-feeding but not host-seeking ability in Aedes triseriatus (Diptera: Culicidae). J. Med. Entomol Parker D 1995 Serotonergic modulation of locust motor neurons. J. Neurophysiol Rillich J, Stevenson PA and Pflueger HJ 2013 Flight and walking in locusts-cholinergic co-activation, temporal coupling and its modulation by biogenic amines. PLoS One 8 e62899 Ritzmann R and Zill SN 2013 Neuroethology of insect walking. Scholarpedia Sadaf S, Birman S and Hasan G 2012 Synaptic Activity in serotonergic neurons is required for air-puff stimulated flight in Drosophila melanogaster. PLoS One 7 e46405 Sadaf S, Reddy OV, Sane SP and Hasan G Context-dependent neural control of wing coordination in flies. Under Review Sane SP, Dieudonne A, Willis MA and Daniel TL 2007 Antennal mechanosensors mediate flight control in moths. Science

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