Please cite this article in press as: Sadaf et al., Neural Control of Wing Coordination in Flies, Current Biology (2015), http://dx.doi.org/ 10.1016/j.cub.2014.10.069 Current Biology 25, 1–7, January 5, 2015 ª2015 Elsevier Ltd All rights reserved
http://dx.doi.org/10.1016/j.cub.2014.10.069
Report Neural Control of Wing Coordination in Flies Sufia Sadaf,1 O. Venkateswara Reddy,1,2 Sanjay P. Sane,1 and Gaiti Hasan1,* 1National Centre for Biological Sciences, TIFR, Bellary Road, Bangalore 560065, India 2Manipal University, Manipal, Karnataka 576104, India
Summary At the onset of each flight bout in flies, neural circuits in the CNS must rapidly integrate multimodal sensory stimuli and synchronously engage hinges of the left and right wings for coordinated wing movements. Whereas anatomical and physiological investigations of flight have been conducted on larger flies [1], molecular genetic studies in Drosophila have helped identify neurons that mediate various levels of flight control [2–7]. However, neurons that might mediate bilateral coordination of wing movements to precisely synchronize left and right wing engagement at flight onset and maintain their movement in perfect coordination at rapid frequencies during flight maneuvers remain largely unexplored. Wing coordination could be directly modulated via bilateral sensory inputs to motoneurons of steering muscles [6, 8] and/or through central interneurons. Using a Ca2+-activity-based GFP reporter, we identified three flight-activated central dopaminergic interneurons in the ventral ganglion, which connect to and activate motoneurons that innervate a pair of direct-steering flight muscles. The activation of these newly identified dopaminergic interneurons is context specific. Whereas bilateral wing engagement for flight requires these neurons, they do not control unilateral wing extension during courtship. Thus, independent central circuits function in the context of different natural behaviors to control the motor circuit for Drosophila wing movement. Results Genetic and pharmacological perturbations have implicated monoamines, including serotonin, octopamine, and dopamine in the neuromodulation of insect flight [9–14]. Hence, we hypothesized that monoaminergic interneurons modulate the activity of wing-coordinating direct flight motor neurons. To test this hypothesis, we first identified dopaminergic neurons that are activated during flight bouts stimulated by an air puff and investigated the effect of altered neural activity in these interneurons on wing coordination during flight. Initially, dopaminergic neurons were targeted with a Drosophila strain (THGAL4) in which the yeast transcription factor GAL4 is expressed under control of the tyrosine hydroxylase gene (TH) promoter [15]. In the adult Drosophila nervous system, TH expresses specifically in dopaminergic interneurons [15] of the brain and ventral ganglion [16]. To identify putative dopamine neurons of the flight circuit, we used the recently developed calcium-dependent nuclear import of LexA (CaLexA) system [17], which consists of a genetically encoded GFP reporter whose expression is limited to neurons exhibiting sustained
*Correspondence: [email protected]
Ca2+ activity in response to a stimulus. THGAL4 flies were mated with flies containing the CaLexA transgenic constructs (UAS-mLexA-VP16-NFAT, LexAop-CD2-GFP; LexAop-CD8GFP-2A-GFP; subsequently referred to as CaLexA). A single gentle air puff was delivered to tethered flies with CaLexA expression in dopaminergic neurons (TH>CaLexA), and their flight bouts, typically ranging from 30 to 300 s, were observed. Flight evoked a response in just three dopaminergic neurons of the ventral ganglion in 10/10 flies, as evident by GFP expression 8 hr after a flight bout (Figures 1A and 1B). The three GFP-expressing neurons were located in the mesothoracic segment (T2). Of these, one neuron was unpaired (ventral unpaired medial [VUM]) whereas two (a, a0 ; our nomenclature) were paired. GFP expression in dopaminergic neurons of the brain was absent at 8 hr (Figure 1A) and visible faintly in some neurites at 24 hr (data not shown). Control flies of the experimental genotype were tethered but were not stimulated for flight with an air puff (Figure S1A available online). Synaptic Activity in Three Dopaminergic Neurons Is Required for Wing Coordination To test requirement of the three dopaminergic neurons for flight, we identified GAL4 strains with restricted expression in TH neurons. Based on our recent work [18], we identified FMRFGAL447715, with exclusive expression in the a-a0 pair of mesothoracic dopaminergic neurons (Figures 1C and S1B; 0 henceforth referred to as FMRFaa ) and the recently published 0 TH-C GAL4 strain [19], which drives expression in a THGAL4 neuronal subset including the T2 VUM and b-b0 pair (Figures 1D and S1C). Flight was significantly compromised either by expression of a temperature-sensitive dynamin mutant transgene (UASShibirets or UASShits), which prevents recycling of synaptic vesicles at 29 C [20], or by expression of an inwardrectifying potassium channel (Kir2.1) [21] in combination with a GAL80ts transgene [22], in either the a-a0 neurons or in THC0 -expressing cells (Figures S1D and S1E). For these experiments, flies were grown at 18 C and shifted to 29 C as 2-day-old adults, and the flight ability of 5- or 6-day-old flies was tested by the cylinder drop assay [12, 23]. Flight deficits were confirmed by electrophysiological recording of flightinduced action potentials from the dorsal longitudinal indirect flight muscles (DLMs). The DLMs receive neuronal inputs 0 at a frequency of w10 Hz from motoneurons [24]. In FMRFaa >Shits flies, with reduced synaptic vesicle recycling in the a-a0 neurons, we observed electrophysiological responses ranging from rhythmic activity for less than 10 s up to 30 s (Fig0 ure S1E). Electrophysiological recordings from FMRFaa >Kir2.1 flies, where both electrical and chemical transmission from a-a0 neurons should be compromised, yielded half the flies with normal trains of DLM spike activity, but the other half generated no spikes in response to the air puff (Fig0 ure S1E). The difference in flight ability of FMRFaa >Kir2.1 aa0 ts and FMRF >Shi adults is likely due to reduced efficacy of the Shits transgene at 25 C, the temperature at which we carried out the recordings. Because Drosophila interneurons sometimes express dual neurotransmitters, the role of dopamine as a neuromodulator for flight in TH-expressing neu0 rons was confirmed by knockdown of TH in FMRFaa and TH marked neurons by RNAi (dsDTH; Figures S1D and S1E). TH
Please cite this article in press as: Sadaf et al., Neural Control of Wing Coordination in Flies, Current Biology (2015), http://dx.doi.org/ 10.1016/j.cub.2014.10.069 Current Biology Vol 25 No 1 2
Figure 1. Three Dopaminergic Neurons in the Ventral Ganglion Coordinate Bilateral Wing Engagement for Air Puff Elicited Flight (A) Immunostaining with anti-GFP on the complete CNS of a representative animal expressing the CaLexA transgenes in all dopaminergic neurons. CaLexAdriven GFP expression was observed in the mesothoracic segment (T2) 8 hr post-tethered flight. Ten independent animals were tested in this assay. (B) Magnified images of the T2 segment with three air-puff-activated dopaminergic neurons that are both CaLexA (anti-GFP) positive and TH positive (antiTH). VUM, ventral unpaired medial. 0 (C) Expression of the FMRFaa strain is exclusively in the paired a-a0 dopaminergic neurons of the mesothorax. (legend continued on next page)
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immunostaining of peptidergic and octopaminergic GAL4-expressing strains indicates that the VUM neuron is uniquely dopaminergic whereas the a-a0 pair is also peptidergic (data not shown). These data demonstrate a requirement for synaptic function in the TH-aa0 neurons and implicate the identified TH-VUM neuron in air-puff-induced flight. To determine how the three identified TH-positive interneurons might affect flight circuit function, two high-speed cameras (4,000 frames per second) were used to film the wing movement of flies with reduced synaptic function in either 0 the a-a0 neurons (FMRFaa >Shits adults) or in the T2-VUM and other neurons marked by TH-C0 (TH-C0 >Shits adults). Calibrated images from each camera were then used to reconstruct the individual wing strokes in three dimensions. Initially, we isolated nonfliers and fliers from controls and GAL4>Shits adults by the cylinder drop assay [23]. In both fliers and the few nonfliers obtained from control genotypes, bilateral wings were coordinated to millisecond precision during initiation and cessation of tethered flight, as well as during continuous flapping (Figure 1E; Movie S1). Thus, control flies that failed the cylinder drop test were apparently capable of flapping their wings normally in the tethered flight assay. Wing amplitude was low (40 degree) during the first two or three cycles of flight onset and during the last two or three cycles before flight cessation in all control flies (Figure 1E). At flight cessation, both wings were held in an elevated position for w30 s before they folded completely (Figure 1E). In contrast, nonfliers with reduced neurotransmitter release in the a-a0 neurons 0 (FMRFaa >Shits adults) had wing coordination defects for 2–4 wing beat cycles during flight initiation, cessation, or both (Figures 1F and 1I; Movie S2). We quantified the error in wing coordination by counting the number of uncoordinated wing beat cycles between the left and right wings in all flies tested 0 for a particular genotype (Figure 1I). Several FMRFaa >Shits nonflier adults (13/15) were unable to coordinate their wings at cessation of short flight durations. The delay in relative movement of the left or right wing was random and not restricted to the same wing (Figure S1F). We observed similar deficits in wing kinematics in flies with reduced neurotransmitter release in TH-C0 neurons (Figure 1G), in all TH neurons in adults (TH>Shits; Figure S1G), and upon TH knockdown in 0 adults in either the a-a0 neurons (FMRFaa >dsDTH; Figures 1H and 1I) or in all TH neurons (TH>dsDTH; Figure S1G). In fliers selected from Shits control and experimental adults, normal wing coordination was observed in all (10/10) adults tested (Figures 1I and S1G). Notably, wing coordination defects were observed specifically in flies with reduced neurotransmitter release from dopaminergic neurons. Flies with reduced neurotransmitter release from serotonergic neurons exhibit flight deficits [12], but we have not observed a lag between the two wings at initiation and cessation (data not shown).
Dopaminergic Neurons of the Mesothorax Synapse with the b1Mn, a Steering Muscle Motoneuron The consistent lack of wing coordination observed suggested that the identified TH-expressing neurons could modulate activity of direct flight muscle motoneurons. To identify the synaptic partners of TH VUM and a-a0 , we obtained two glutamatergic neuron GAL4 strains [25] with expression in either multiple motoneurons (9281GAL4) [25] or a single pair of peripherally located motoneurons with contralateral projections in T2 (VGlut9281(2)GAL4; Figures 2A, 2B, and S2A). The single pair of neurons appeared morphologically similar to the b1 direct flight muscle motoneurons (b1Mns) whose activity controls steering maneuvers during flight in Diptera [26]. Contralateral projections with a posterior branch are a unique morphological feature of the b1Mns [27]. To confirm the marked neurons as b1Mns, the b1 muscle was back-filled with Texas Red, a fluorescent dye [28], in flies expressing a membrane-bound GFP (mGFP) in VGlut9281(2)GAL4-marked neurons. Colocalization of the Texas Red and mGFP signals were observed in the b1 muscle (Figure S2C) and in the b1Mn (Figure 2C), confirming that the single pair of marked GFP cells were indeed the b1 motoneurons in the VGlut9281(2)GAL4 strain. We observed flight deficits in the cylinder drop assay (Figure S2D) and variable responses from DLM recordings (Figure S2E) when activity was reduced in 9281(2) GAL4-marked glutamatergic neurons of adult flies by expression of mutant dynamin (Shits) or by expression of the Kir2.1 transgene. Presence of both fliers and nonfliers among flies expressing Kir2.1 in either dopaminergic or VGlut9281(2)GAL4marked neurons, which includes the b1Mns, is very likely due to differential GAL4 expression in individual animals. Because the VGlut9281(2)GAL4 strain also expressed in a few central brain neurons (Figure S2A), a tshGAL80 transgene that limits expression of Kir2.1 to the brain was included in the 9281(2)>Kir2.1, GAL80ts strain (Figure S2B). Flies with Kir2.1 expression in the tshGAL80 strain exhibited normal cylinder drop flight (Figure S2D), indicating that reduced flight in 9281(2)>Kir2.1 adults was due to Kir2.1 expression in the b1Mns located in the ventral ganglion. We selected flies from among the 9281(2)>Shits animals identified as flightless in the cylinder drop assay [12] and quantified their wing kinematics during tethered flight. Significant errors were observed in wing coordination at both the onset and cessation of flight (Figures 2D–2F and S2F; Movie S3). Because reducing synaptic activity in both dopaminergic and b1Mns caused similar errors in wing coordination, we investigated anatomical connectivity between the newly identified dopaminergic neurons and b1Mns by immunohistochemistry and confocal imaging. TH immunoreactivity in a-a0 neurons was always reduced as compared to the VUM neuron and the TH b-b0 pair (Figures 3A and S3A). Posterior projections from the TH a-a0 pair loop upward and meet a centrally located dendritic projection from the VUM neuron (Figures 3A and 3B). Dendrites were marked by a dendritic marker,
(D) Expression of the TH-C0 >mGFP strain with anti-GFP and anti-TH immunostaining in the mesothorax is restricted to the paired b-b0 neurons and the VUM neuron. (E) Snapshots from a calibrated high-speed video recording of flight initiation and cessation in a normal fly. Lower panel shows a representative trace of the calculated stroke amplitudes for both wings. 0 (F) Reduced synaptic vesicle recycling through expression of a dominant-negative dynamin mutant in the a-a0 neurons (FMRFaa > Shits) leads to errors in 0 wing coordination at flight initiation and cessation (arrows). A representative trace of stroke amplitudes of the left and right wing in a FMRFaa >Shits fly is shown in the lower panel. (G) A representative trace of stroke amplitudes of the left and right wing in a TH-C0 >Shits fly. 0 (H) A representative trace of stroke amplitudes of the left and right wing in a FMRFaa >dsdTH fly, with reduced TH expression in a-a0 neurons. (I) Quantification of relative lag in left-right wing strokes during flight initiation and cessation from 15 flies of the indicated genotypes selected as fliers and nonfliers from the cylinder drop test (open circles represent individual data points; *p < 0.05; one-way ANOVA as compared with appropriate controls in Figure S1). See also Figure S1 and Movies S1 and S2.
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Figure 2. Reduced Synaptic Function in b1 Motoneurons Leads to Errors in Wing Coordination during Flight (A) mGFP expression driven by a glutamatergic neuron subdomain GAL4 (vGlut9281(2)>mGFP). A single pair of motoneurons (b1 soma) is seen in the T2 segment with contralateral projections and a posterior branch. b1Mn, b1 motor neuron; CP, contralateral projection; PB, posterior branch; SS, soma stalk. (B) A 3D reconstruction of the image in (A) obtained using the AMIRA software. (C) Backfill of the b1 direct flight muscle with Texas Red labels the motoneuron marked by VGlut9281(2)>GFP (arrow). Colocalization of the Texas Red and GFP is visible in the right T2 hemisegment of T2 (merge panel; n = 13). (D) Snapshots of high-speed video recordings with error in wing coordination in VGlut9281(2)>Shits adults. (E) The left and right wings are uncoordinated during flight initiation and cessation in a VGlut9281(2)>Shits fly. (F) Quantification of relative lag in left-right wing strokes during flight initiation and cessation of the indicated genotypes selected as either nonfliers or fliers from the cylinder drop test (*p < 0.05, as compared with appropriate controls in Figure S2E; one-way ANOVA; n R 5). See also Figure S2 and Movie S3.
DenMark (Figure 3B) [29]. Bilateral axonal projections (BP) from the dopaminergic VUM neuron extended toward the peripherally located b1Mns (Figures 3B and 3C, arrowheads). A 3D reconstruction of lateral axonal projections from the TH VUM indicates their close proximity to the b1Mn soma (Figure 3C). Contralateral projections of the b1Mn are positioned more dorsal to the TH-VUM neuron and its axonal projections located in the central T2 neuropil. These data suggest that the b1Mns receive inputs from the central aa0 and VUM circuit. Functional connectivity between aa0 -VUM and b1Mns was tested next. Initially, TrpA1 [30] was expressed in all dopaminergic neurons (THLexA>LexAopTrpA1), and a Ca2+ activity reporter GCaMP6m [31] was expressed in the b1Mns (VGlutGAL49281(2)>UASGCaMP6m). From these flies, ventral ganglia were dissected and placed in a thermally controlled imaging chamber. TrpA1 activation of dopaminergic neurons was achieved by raising the temperature from 23 C to 29 C, which elicited a robust Ca2+ response from the b1Mns (Figure 3D),
confirming that TH-expressing neurons in the ventral ganglia are functionally connected to the b1Mns. Next, we expressed 0 TrpA1 in the a-a0 neurons (FMRFaa GAL4>UASTrpA1) and GCaMP6m in glutamatergic neurons including the b1Mns (VGlutLexA54886>LexAopGCaMP6m). Expression of VGlutLexA54886 in the b1Mns was confirmed by observing the characteristic laterally located b1Mn soma with contralateral projections across the T2 segment (Figure S3B). Moreover, adults with reduced synaptic function in glutamatergic neurons marked by VGlutLexA54886 (VGlutLexA54886>LexAopShits) exhibit wing coordination defects (Figures S3C and S3D). Importantly, TrpA1 activation of the a-a0 neuron pair elicited GCaMP responses in the b1Mns (Figure 3E) and its extended neurites (Movie S4). This activation presumably occurs through synaptic connections between the bilateral axonal projections of the TH-VUM and dendritic neurites of the b1Mn. When GCaMP was expressed in multiple motor neurons, with an alternate glutamatergic subset GAL4 driver
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Figure 3. The a-a0 Neuron Pair Connects with and Activates the b1 Motoneurons (A) The mesothorax (T2) of a TH>mGFP fly exhibits five TH-positive neurons; the unpaired medial VUM, a and a0 , which stain faintly with anti-TH; b and b0 , which stain very faintly with anti-GFP. A 3D reconstruction of the GFP image of a-a0 and VUM is shown on the extreme right. (B) Dendritic and axonal projections and soma of the five dopaminergic neurons marked with anti-TH (green) for axonal projections (arrowheads) and antiDsRed (red) for dendrites (arrows), encoded by UASDenMark [29], to visualize dendrites. A 3D reconstruction of a-a0 and VUM from the merge panel is shown on the extreme right. BP, bilateral axonal projections of the VUM neuron. (C) Dopaminergic projections (anti-TH, red, arrow) and varicosities (anti-TH, red, arrowheads) lead to the b1Mn (VGlut9281(2)>mGFP, green) in the T2 segment. Insets show magnified images of the b1Mn region. Bottom panel shows a 3D reconstruction of the merged image. (D) Thermal activation of a Ca2+ channel, TrpA1, in dopaminergic neurons of the ventral ganglia leads to increased fluorescence of a genetically encoded Ca2+ reporter, GCaMP, in the b1Mns (TH-LexA>LexAop-TrpA1; VGlut9281(2)GAL4>UASGCaMP6m). Thoracic ganglia (sans brain) were dissected and suspended in PBS. Basal GCaMP fluorescence was collected at room temperature (w23 C; T = 0 s). TrpA1 was activated by raising the temperature to 29 C and GCaMP imaging performed at intervals of 2.375 s. GCaMP fluorescence was quantified in b1Mns of the indicated genotypes (n = 10). Snapshots of THLexA>TrpA1; VGlut9281(2)>GCaMP6m at the indicated time points exhibit elevation of the GCaMP signal in the b1Mn. A quantification of changes in GCaMP fluorescence in the indicated genotypes is shown in the trace on the right. Trace for VGlut9281(2)GAL4>UASGCaMP6m serves as a control. 0 (E) Snapshots of a mesothoracic hemisegment in which thermal activation of the TrpA1 channel (at T = 0 s) in the a-a0 neurons (FMRFaa GAL4>UASTrpA1) drives a GCaMP response in the b1Mns (VGlut54886LexA>LexAop-GCaMP6m). Quantification of changes in GCaMP fluorescence in the indicated genotypes (n = 6) is shown in the graph on the right. See also Figure S3 and Movie S4. Error bars denote SEM.
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Figure 4. Dopaminergic Neurons of the Mesothorax Coordinate Bilateral Wing Movement for Flight, but Not Unilateral Wing Extension during Courtship (A) Male courtship progresses normally in nonfliers with reduced neural excitability in the a-a0 0 neurons (FMRFaa >Kir2.1). Snapshots of a courting male during a single wing extension cycle (top panels). In contrast, males with reduced activity in the b1Mns (VGlut9281(2)>Kir2.1) exhibit aberrant wing extension patterns including extension of either left, right, or both wings. Snapshots show wing extension pattern at the indicated time intervals in seconds. (B) Wing extension events that occurred during 1 min calculated from 20 courting males of the indicated genotypes. BW, simultaneous extension of both wings; LW, extension of the left wing; RW, extension of the right wing; UW, unilateral wing extension of either left or right wing. (C) Quantification of number of wing vibrations per wing extension bout in the indicated genotypes (n = 4 animals; vibrations in three wing extension bouts were counted manually after visualizing the vibration bouts at 2,500 fps; mean 6 SEM; *p < 0.05, one-way ANOVA as compared with appropriate controls). (D) Quantification of latency to copulate, measured as the time taken for males to copulate after being introduced in the courtship chamber (n = 20; mean 6 SEM). (E) A schematic of the three dopaminergic neurons (DA) activated after tethered flight, and their proposed connectivity with the glutamatergic b1Mns. This connectivity is likely required for coordination of the clutch in right and left wing hinges. b1MN, first basalar direct flight muscle motor neuron; Glut, glutamate.
(9281GAL4; Figure S3E) [25], Ca2+ elevation after TrpA1 activation in dopaminergic neurons was observed in just the b1Mn (9281GAL4>GCaMP; Figure S3F). With this, we concluded that the b1Mns receive dopaminergic inputs from the aa0 VUM circuit. The Dopaminergic VUM and a-a0 Pair Is Required for Bilateral Wing Coordination, but Not Unilateral Wing Movement To test if the dopaminergic-b1Mn connection described here coordinates wing movements in behavioral contexts other than flight, we measured wing movements during courtship behavior [32]. Although flight-related coordinated wing movements were affected in males with Kir2.1 expression in the a-a0 0 neurons (FMRFaa >Kir2.1; Figure 1), the number of unilateral wing extensions during courtship, wing vibrations in a single wing extension bout (required for species-specific courtship songs), and finally the latency to copulate appeared normal (Figures 4A–4D) as compared with controls (Figures S4A–
S4D). The pattern of high-frequency wing vibrations after unilateral wing extension was visualized by highspeed videography (2,500 frames per second [fps]) of extended wings. Courtship behavior was however severely impaired by Kir2.1 expression in the b1Mns of adult males. Both unilateral extension of left and right wing in the same animal and bilateral wing extensions were observed in VGlut9281(2)>Kir2.1 males (Figures 4A and 4B). Wing vibrations of extended wings were reduced in VGlut9281(2)>Kir2.1 animals (Figure 4C) as compared with controls (Figure S4C), and successful mating was not observed in this genotype (Figure 4D). Discussion In agreement with previous observations in other Diptera and Drosophila, the b1Mns are an integral component of both the flight motor circuit [26] and unilateral wing extension during courtship [32, 33]. Our study establishes the presence of independent circuits for modulating b1Mn function for unilateral versus bilateral wing flapping. In the context of flight, where bilateral wing coordination is critical, recent studies have demonstrated mechanical linkages connecting the two wings of a dipteran fly to perfectly synchronize their motion (T. Deora, A.K. Singh, and S.P.S., unpublished data). Although these linkages mediate bilateral wing coordination during flight, it
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remains an open question how the wing hinges at the base of each wing are actively engaged (or disengaged) during the onset (or offset) of flight and remain engaged through flight. We propose a model in which mechanosensory inputs such as a gentle air puff activate paired dopaminergic neurons (a and a0 ) which convey this stimulus to the central dopaminergic VUM neuron. The VUM integrates the bilaterally received sensory information and helps coordinate firing of the paired and contralaterally connected b1Mns such that they remain in phase with each other and with the wing beats. This allows simultaneous engagement of the left and right wing hinges by the b1 direct flight muscle for initiation and their disengagement for flight cessation (Figure 4E). However, because unilateral wing extension and vibration during courtship requires coordination of other sensory and motor functions, modulation of b1Mn activity in this context likely occurs through a parallel neural circuit. Supplemental Information Supplemental Information includes Supplemental Experimental Procedures, four figures, and four movies and can be found with this article online at http://dx.doi.org/10.1016/j.cub.2014.10.069. Author Contributions S.S. and G.H. designed the experiments. S.S. performed experiments and analyzed the data. O.V.R. generated the VGlut9281(2)GAL4 strain. S.P.S. supervised high-speed tethered flight and courtship videography and analysis. S.S., S.P.S., and G.H. wrote the manuscript. Acknowledgments S.S. was supported by a research fellowship from the CSIR (http://rdpp.csir. res.in/csir_acsir/Home.aspx). The project was supported by grants from the DST (http://www.dst.gov.in/) and NCBS, TIFR (http://www.ncbs.res.in) to G.H. and by the Ramanujan Fellowship, DST, and a grant from the Air Force Office of Scientific Research (AFOSR) to S.P.S. The NCBS Central ImageFlow Facility helped with confocal imaging. Received: April 4, 2014 Revised: October 13, 2014 Accepted: October 29, 2014 Published: December 11, 2014 References 1. Borst, A., and Haag, J. (2002). Neural networks in the cockpit of the fly. J. Comp. Physiol. A Neuroethol. Sens. Neural Behav. Physiol. 188, 419–437. 2. Gorczyca, M., and Hall, J.C. (1984). Identification of a cholinergic synapse in the giant fiber pathway of Drosophila using conditional mutations of acetylcholine synthesis. J. Neurogenet. 1, 289–313. 3. Elkins, T., and Ganetzky, B. (1988). The roles of potassium currents in Drosophila flight muscles. J. Neurosci. 8, 428–434. 4. Engel, J.E., and Wu, C.F. (1992). Interactions of membrane excitability mutations affecting potassium and sodium currents in the flight and giant fiber escape systems of Drosophila. J. Comp. Physiol. A Neuroethol. Sens. Neural Behav. Physiol. 171, 93–104. 5. Oh, C.E., McMahon, R., Benzer, S., and Tanouye, M.A. (1994). bendless, a Drosophila gene affecting neuronal connectivity, encodes a ubiquitinconjugating enzyme homolog. J. Neurosci. 14, 3166–3179. 6. Fayyazuddin, A., and Dickinson, M.H. (1996). Haltere afferents provide direct, electrotonic input to a steering motor neuron in the blowfly, Calliphora. J. Neurosci. 16, 5225–5232. 7. Fayyazuddin, A., Zaheer, M.A., Hiesinger, P.R., and Bellen, H.J. (2006). The nicotinic acetylcholine receptor Dalpha7 is required for an escape behavior in Drosophila. PLoS Biol. 4, e63. 8. Fayyazuddin, A., and Dickinson, M.H. (1999). Convergent mechanosensory input structures the firing phase of a steering motor neuron in the blowfly, Calliphora. J. Neurophysiol. 82, 1916–1926.
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http://dx.doi.org/10.1016/j.cub.2014.10.069
Report Neural Control of Wing Coordination in Flies Sufia Sadaf,1 O. Venkateswara Reddy,1,2 Sanjay P. Sane,1 and Gaiti Hasan1,* 1National Centre for Biological Sciences, TIFR, Bellary Road, Bangalore 560065, India 2Manipal University, Manipal, Karnataka 576104, India
Summary At the onset of each flight bout in flies, neural circuits in the CNS must rapidly integrate multimodal sensory stimuli and synchronously engage hinges of the left and right wings for coordinated wing movements. Whereas anatomical and physiological investigations of flight have been conducted on larger flies [1], molecular genetic studies in Drosophila have helped identify neurons that mediate various levels of flight control [2–7]. However, neurons that might mediate bilateral coordination of wing movements to precisely synchronize left and right wing engagement at flight onset and maintain their movement in perfect coordination at rapid frequencies during flight maneuvers remain largely unexplored. Wing coordination could be directly modulated via bilateral sensory inputs to motoneurons of steering muscles [6, 8] and/or through central interneurons. Using a Ca2+-activity-based GFP reporter, we identified three flight-activated central dopaminergic interneurons in the ventral ganglion, which connect to and activate motoneurons that innervate a pair of direct-steering flight muscles. The activation of these newly identified dopaminergic interneurons is context specific. Whereas bilateral wing engagement for flight requires these neurons, they do not control unilateral wing extension during courtship. Thus, independent central circuits function in the context of different natural behaviors to control the motor circuit for Drosophila wing movement. Results Genetic and pharmacological perturbations have implicated monoamines, including serotonin, octopamine, and dopamine in the neuromodulation of insect flight [9–14]. Hence, we hypothesized that monoaminergic interneurons modulate the activity of wing-coordinating direct flight motor neurons. To test this hypothesis, we first identified dopaminergic neurons that are activated during flight bouts stimulated by an air puff and investigated the effect of altered neural activity in these interneurons on wing coordination during flight. Initially, dopaminergic neurons were targeted with a Drosophila strain (THGAL4) in which the yeast transcription factor GAL4 is expressed under control of the tyrosine hydroxylase gene (TH) promoter [15]. In the adult Drosophila nervous system, TH expresses specifically in dopaminergic interneurons [15] of the brain and ventral ganglion [16]. To identify putative dopamine neurons of the flight circuit, we used the recently developed calcium-dependent nuclear import of LexA (CaLexA) system [17], which consists of a genetically encoded GFP reporter whose expression is limited to neurons exhibiting sustained
*Correspondence: [email protected]
Ca2+ activity in response to a stimulus. THGAL4 flies were mated with flies containing the CaLexA transgenic constructs (UAS-mLexA-VP16-NFAT, LexAop-CD2-GFP; LexAop-CD8GFP-2A-GFP; subsequently referred to as CaLexA). A single gentle air puff was delivered to tethered flies with CaLexA expression in dopaminergic neurons (TH>CaLexA), and their flight bouts, typically ranging from 30 to 300 s, were observed. Flight evoked a response in just three dopaminergic neurons of the ventral ganglion in 10/10 flies, as evident by GFP expression 8 hr after a flight bout (Figures 1A and 1B). The three GFP-expressing neurons were located in the mesothoracic segment (T2). Of these, one neuron was unpaired (ventral unpaired medial [VUM]) whereas two (a, a0 ; our nomenclature) were paired. GFP expression in dopaminergic neurons of the brain was absent at 8 hr (Figure 1A) and visible faintly in some neurites at 24 hr (data not shown). Control flies of the experimental genotype were tethered but were not stimulated for flight with an air puff (Figure S1A available online). Synaptic Activity in Three Dopaminergic Neurons Is Required for Wing Coordination To test requirement of the three dopaminergic neurons for flight, we identified GAL4 strains with restricted expression in TH neurons. Based on our recent work [18], we identified FMRFGAL447715, with exclusive expression in the a-a0 pair of mesothoracic dopaminergic neurons (Figures 1C and S1B; 0 henceforth referred to as FMRFaa ) and the recently published 0 TH-C GAL4 strain [19], which drives expression in a THGAL4 neuronal subset including the T2 VUM and b-b0 pair (Figures 1D and S1C). Flight was significantly compromised either by expression of a temperature-sensitive dynamin mutant transgene (UASShibirets or UASShits), which prevents recycling of synaptic vesicles at 29 C [20], or by expression of an inwardrectifying potassium channel (Kir2.1) [21] in combination with a GAL80ts transgene [22], in either the a-a0 neurons or in THC0 -expressing cells (Figures S1D and S1E). For these experiments, flies were grown at 18 C and shifted to 29 C as 2-day-old adults, and the flight ability of 5- or 6-day-old flies was tested by the cylinder drop assay [12, 23]. Flight deficits were confirmed by electrophysiological recording of flightinduced action potentials from the dorsal longitudinal indirect flight muscles (DLMs). The DLMs receive neuronal inputs 0 at a frequency of w10 Hz from motoneurons [24]. In FMRFaa >Shits flies, with reduced synaptic vesicle recycling in the a-a0 neurons, we observed electrophysiological responses ranging from rhythmic activity for less than 10 s up to 30 s (Fig0 ure S1E). Electrophysiological recordings from FMRFaa >Kir2.1 flies, where both electrical and chemical transmission from a-a0 neurons should be compromised, yielded half the flies with normal trains of DLM spike activity, but the other half generated no spikes in response to the air puff (Fig0 ure S1E). The difference in flight ability of FMRFaa >Kir2.1 aa0 ts and FMRF >Shi adults is likely due to reduced efficacy of the Shits transgene at 25 C, the temperature at which we carried out the recordings. Because Drosophila interneurons sometimes express dual neurotransmitters, the role of dopamine as a neuromodulator for flight in TH-expressing neu0 rons was confirmed by knockdown of TH in FMRFaa and TH marked neurons by RNAi (dsDTH; Figures S1D and S1E). TH
Please cite this article in press as: Sadaf et al., Neural Control of Wing Coordination in Flies, Current Biology (2015), http://dx.doi.org/ 10.1016/j.cub.2014.10.069 Current Biology Vol 25 No 1 2
Figure 1. Three Dopaminergic Neurons in the Ventral Ganglion Coordinate Bilateral Wing Engagement for Air Puff Elicited Flight (A) Immunostaining with anti-GFP on the complete CNS of a representative animal expressing the CaLexA transgenes in all dopaminergic neurons. CaLexAdriven GFP expression was observed in the mesothoracic segment (T2) 8 hr post-tethered flight. Ten independent animals were tested in this assay. (B) Magnified images of the T2 segment with three air-puff-activated dopaminergic neurons that are both CaLexA (anti-GFP) positive and TH positive (antiTH). VUM, ventral unpaired medial. 0 (C) Expression of the FMRFaa strain is exclusively in the paired a-a0 dopaminergic neurons of the mesothorax. (legend continued on next page)
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immunostaining of peptidergic and octopaminergic GAL4-expressing strains indicates that the VUM neuron is uniquely dopaminergic whereas the a-a0 pair is also peptidergic (data not shown). These data demonstrate a requirement for synaptic function in the TH-aa0 neurons and implicate the identified TH-VUM neuron in air-puff-induced flight. To determine how the three identified TH-positive interneurons might affect flight circuit function, two high-speed cameras (4,000 frames per second) were used to film the wing movement of flies with reduced synaptic function in either 0 the a-a0 neurons (FMRFaa >Shits adults) or in the T2-VUM and other neurons marked by TH-C0 (TH-C0 >Shits adults). Calibrated images from each camera were then used to reconstruct the individual wing strokes in three dimensions. Initially, we isolated nonfliers and fliers from controls and GAL4>Shits adults by the cylinder drop assay [23]. In both fliers and the few nonfliers obtained from control genotypes, bilateral wings were coordinated to millisecond precision during initiation and cessation of tethered flight, as well as during continuous flapping (Figure 1E; Movie S1). Thus, control flies that failed the cylinder drop test were apparently capable of flapping their wings normally in the tethered flight assay. Wing amplitude was low (40 degree) during the first two or three cycles of flight onset and during the last two or three cycles before flight cessation in all control flies (Figure 1E). At flight cessation, both wings were held in an elevated position for w30 s before they folded completely (Figure 1E). In contrast, nonfliers with reduced neurotransmitter release in the a-a0 neurons 0 (FMRFaa >Shits adults) had wing coordination defects for 2–4 wing beat cycles during flight initiation, cessation, or both (Figures 1F and 1I; Movie S2). We quantified the error in wing coordination by counting the number of uncoordinated wing beat cycles between the left and right wings in all flies tested 0 for a particular genotype (Figure 1I). Several FMRFaa >Shits nonflier adults (13/15) were unable to coordinate their wings at cessation of short flight durations. The delay in relative movement of the left or right wing was random and not restricted to the same wing (Figure S1F). We observed similar deficits in wing kinematics in flies with reduced neurotransmitter release in TH-C0 neurons (Figure 1G), in all TH neurons in adults (TH>Shits; Figure S1G), and upon TH knockdown in 0 adults in either the a-a0 neurons (FMRFaa >dsDTH; Figures 1H and 1I) or in all TH neurons (TH>dsDTH; Figure S1G). In fliers selected from Shits control and experimental adults, normal wing coordination was observed in all (10/10) adults tested (Figures 1I and S1G). Notably, wing coordination defects were observed specifically in flies with reduced neurotransmitter release from dopaminergic neurons. Flies with reduced neurotransmitter release from serotonergic neurons exhibit flight deficits [12], but we have not observed a lag between the two wings at initiation and cessation (data not shown).
Dopaminergic Neurons of the Mesothorax Synapse with the b1Mn, a Steering Muscle Motoneuron The consistent lack of wing coordination observed suggested that the identified TH-expressing neurons could modulate activity of direct flight muscle motoneurons. To identify the synaptic partners of TH VUM and a-a0 , we obtained two glutamatergic neuron GAL4 strains [25] with expression in either multiple motoneurons (9281GAL4) [25] or a single pair of peripherally located motoneurons with contralateral projections in T2 (VGlut9281(2)GAL4; Figures 2A, 2B, and S2A). The single pair of neurons appeared morphologically similar to the b1 direct flight muscle motoneurons (b1Mns) whose activity controls steering maneuvers during flight in Diptera [26]. Contralateral projections with a posterior branch are a unique morphological feature of the b1Mns [27]. To confirm the marked neurons as b1Mns, the b1 muscle was back-filled with Texas Red, a fluorescent dye [28], in flies expressing a membrane-bound GFP (mGFP) in VGlut9281(2)GAL4-marked neurons. Colocalization of the Texas Red and mGFP signals were observed in the b1 muscle (Figure S2C) and in the b1Mn (Figure 2C), confirming that the single pair of marked GFP cells were indeed the b1 motoneurons in the VGlut9281(2)GAL4 strain. We observed flight deficits in the cylinder drop assay (Figure S2D) and variable responses from DLM recordings (Figure S2E) when activity was reduced in 9281(2) GAL4-marked glutamatergic neurons of adult flies by expression of mutant dynamin (Shits) or by expression of the Kir2.1 transgene. Presence of both fliers and nonfliers among flies expressing Kir2.1 in either dopaminergic or VGlut9281(2)GAL4marked neurons, which includes the b1Mns, is very likely due to differential GAL4 expression in individual animals. Because the VGlut9281(2)GAL4 strain also expressed in a few central brain neurons (Figure S2A), a tshGAL80 transgene that limits expression of Kir2.1 to the brain was included in the 9281(2)>Kir2.1, GAL80ts strain (Figure S2B). Flies with Kir2.1 expression in the tshGAL80 strain exhibited normal cylinder drop flight (Figure S2D), indicating that reduced flight in 9281(2)>Kir2.1 adults was due to Kir2.1 expression in the b1Mns located in the ventral ganglion. We selected flies from among the 9281(2)>Shits animals identified as flightless in the cylinder drop assay [12] and quantified their wing kinematics during tethered flight. Significant errors were observed in wing coordination at both the onset and cessation of flight (Figures 2D–2F and S2F; Movie S3). Because reducing synaptic activity in both dopaminergic and b1Mns caused similar errors in wing coordination, we investigated anatomical connectivity between the newly identified dopaminergic neurons and b1Mns by immunohistochemistry and confocal imaging. TH immunoreactivity in a-a0 neurons was always reduced as compared to the VUM neuron and the TH b-b0 pair (Figures 3A and S3A). Posterior projections from the TH a-a0 pair loop upward and meet a centrally located dendritic projection from the VUM neuron (Figures 3A and 3B). Dendrites were marked by a dendritic marker,
(D) Expression of the TH-C0 >mGFP strain with anti-GFP and anti-TH immunostaining in the mesothorax is restricted to the paired b-b0 neurons and the VUM neuron. (E) Snapshots from a calibrated high-speed video recording of flight initiation and cessation in a normal fly. Lower panel shows a representative trace of the calculated stroke amplitudes for both wings. 0 (F) Reduced synaptic vesicle recycling through expression of a dominant-negative dynamin mutant in the a-a0 neurons (FMRFaa > Shits) leads to errors in 0 wing coordination at flight initiation and cessation (arrows). A representative trace of stroke amplitudes of the left and right wing in a FMRFaa >Shits fly is shown in the lower panel. (G) A representative trace of stroke amplitudes of the left and right wing in a TH-C0 >Shits fly. 0 (H) A representative trace of stroke amplitudes of the left and right wing in a FMRFaa >dsdTH fly, with reduced TH expression in a-a0 neurons. (I) Quantification of relative lag in left-right wing strokes during flight initiation and cessation from 15 flies of the indicated genotypes selected as fliers and nonfliers from the cylinder drop test (open circles represent individual data points; *p < 0.05; one-way ANOVA as compared with appropriate controls in Figure S1). See also Figure S1 and Movies S1 and S2.
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Figure 2. Reduced Synaptic Function in b1 Motoneurons Leads to Errors in Wing Coordination during Flight (A) mGFP expression driven by a glutamatergic neuron subdomain GAL4 (vGlut9281(2)>mGFP). A single pair of motoneurons (b1 soma) is seen in the T2 segment with contralateral projections and a posterior branch. b1Mn, b1 motor neuron; CP, contralateral projection; PB, posterior branch; SS, soma stalk. (B) A 3D reconstruction of the image in (A) obtained using the AMIRA software. (C) Backfill of the b1 direct flight muscle with Texas Red labels the motoneuron marked by VGlut9281(2)>GFP (arrow). Colocalization of the Texas Red and GFP is visible in the right T2 hemisegment of T2 (merge panel; n = 13). (D) Snapshots of high-speed video recordings with error in wing coordination in VGlut9281(2)>Shits adults. (E) The left and right wings are uncoordinated during flight initiation and cessation in a VGlut9281(2)>Shits fly. (F) Quantification of relative lag in left-right wing strokes during flight initiation and cessation of the indicated genotypes selected as either nonfliers or fliers from the cylinder drop test (*p < 0.05, as compared with appropriate controls in Figure S2E; one-way ANOVA; n R 5). See also Figure S2 and Movie S3.
DenMark (Figure 3B) [29]. Bilateral axonal projections (BP) from the dopaminergic VUM neuron extended toward the peripherally located b1Mns (Figures 3B and 3C, arrowheads). A 3D reconstruction of lateral axonal projections from the TH VUM indicates their close proximity to the b1Mn soma (Figure 3C). Contralateral projections of the b1Mn are positioned more dorsal to the TH-VUM neuron and its axonal projections located in the central T2 neuropil. These data suggest that the b1Mns receive inputs from the central aa0 and VUM circuit. Functional connectivity between aa0 -VUM and b1Mns was tested next. Initially, TrpA1 [30] was expressed in all dopaminergic neurons (THLexA>LexAopTrpA1), and a Ca2+ activity reporter GCaMP6m [31] was expressed in the b1Mns (VGlutGAL49281(2)>UASGCaMP6m). From these flies, ventral ganglia were dissected and placed in a thermally controlled imaging chamber. TrpA1 activation of dopaminergic neurons was achieved by raising the temperature from 23 C to 29 C, which elicited a robust Ca2+ response from the b1Mns (Figure 3D),
confirming that TH-expressing neurons in the ventral ganglia are functionally connected to the b1Mns. Next, we expressed 0 TrpA1 in the a-a0 neurons (FMRFaa GAL4>UASTrpA1) and GCaMP6m in glutamatergic neurons including the b1Mns (VGlutLexA54886>LexAopGCaMP6m). Expression of VGlutLexA54886 in the b1Mns was confirmed by observing the characteristic laterally located b1Mn soma with contralateral projections across the T2 segment (Figure S3B). Moreover, adults with reduced synaptic function in glutamatergic neurons marked by VGlutLexA54886 (VGlutLexA54886>LexAopShits) exhibit wing coordination defects (Figures S3C and S3D). Importantly, TrpA1 activation of the a-a0 neuron pair elicited GCaMP responses in the b1Mns (Figure 3E) and its extended neurites (Movie S4). This activation presumably occurs through synaptic connections between the bilateral axonal projections of the TH-VUM and dendritic neurites of the b1Mn. When GCaMP was expressed in multiple motor neurons, with an alternate glutamatergic subset GAL4 driver
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Figure 3. The a-a0 Neuron Pair Connects with and Activates the b1 Motoneurons (A) The mesothorax (T2) of a TH>mGFP fly exhibits five TH-positive neurons; the unpaired medial VUM, a and a0 , which stain faintly with anti-TH; b and b0 , which stain very faintly with anti-GFP. A 3D reconstruction of the GFP image of a-a0 and VUM is shown on the extreme right. (B) Dendritic and axonal projections and soma of the five dopaminergic neurons marked with anti-TH (green) for axonal projections (arrowheads) and antiDsRed (red) for dendrites (arrows), encoded by UASDenMark [29], to visualize dendrites. A 3D reconstruction of a-a0 and VUM from the merge panel is shown on the extreme right. BP, bilateral axonal projections of the VUM neuron. (C) Dopaminergic projections (anti-TH, red, arrow) and varicosities (anti-TH, red, arrowheads) lead to the b1Mn (VGlut9281(2)>mGFP, green) in the T2 segment. Insets show magnified images of the b1Mn region. Bottom panel shows a 3D reconstruction of the merged image. (D) Thermal activation of a Ca2+ channel, TrpA1, in dopaminergic neurons of the ventral ganglia leads to increased fluorescence of a genetically encoded Ca2+ reporter, GCaMP, in the b1Mns (TH-LexA>LexAop-TrpA1; VGlut9281(2)GAL4>UASGCaMP6m). Thoracic ganglia (sans brain) were dissected and suspended in PBS. Basal GCaMP fluorescence was collected at room temperature (w23 C; T = 0 s). TrpA1 was activated by raising the temperature to 29 C and GCaMP imaging performed at intervals of 2.375 s. GCaMP fluorescence was quantified in b1Mns of the indicated genotypes (n = 10). Snapshots of THLexA>TrpA1; VGlut9281(2)>GCaMP6m at the indicated time points exhibit elevation of the GCaMP signal in the b1Mn. A quantification of changes in GCaMP fluorescence in the indicated genotypes is shown in the trace on the right. Trace for VGlut9281(2)GAL4>UASGCaMP6m serves as a control. 0 (E) Snapshots of a mesothoracic hemisegment in which thermal activation of the TrpA1 channel (at T = 0 s) in the a-a0 neurons (FMRFaa GAL4>UASTrpA1) drives a GCaMP response in the b1Mns (VGlut54886LexA>LexAop-GCaMP6m). Quantification of changes in GCaMP fluorescence in the indicated genotypes (n = 6) is shown in the graph on the right. See also Figure S3 and Movie S4. Error bars denote SEM.
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Figure 4. Dopaminergic Neurons of the Mesothorax Coordinate Bilateral Wing Movement for Flight, but Not Unilateral Wing Extension during Courtship (A) Male courtship progresses normally in nonfliers with reduced neural excitability in the a-a0 0 neurons (FMRFaa >Kir2.1). Snapshots of a courting male during a single wing extension cycle (top panels). In contrast, males with reduced activity in the b1Mns (VGlut9281(2)>Kir2.1) exhibit aberrant wing extension patterns including extension of either left, right, or both wings. Snapshots show wing extension pattern at the indicated time intervals in seconds. (B) Wing extension events that occurred during 1 min calculated from 20 courting males of the indicated genotypes. BW, simultaneous extension of both wings; LW, extension of the left wing; RW, extension of the right wing; UW, unilateral wing extension of either left or right wing. (C) Quantification of number of wing vibrations per wing extension bout in the indicated genotypes (n = 4 animals; vibrations in three wing extension bouts were counted manually after visualizing the vibration bouts at 2,500 fps; mean 6 SEM; *p < 0.05, one-way ANOVA as compared with appropriate controls). (D) Quantification of latency to copulate, measured as the time taken for males to copulate after being introduced in the courtship chamber (n = 20; mean 6 SEM). (E) A schematic of the three dopaminergic neurons (DA) activated after tethered flight, and their proposed connectivity with the glutamatergic b1Mns. This connectivity is likely required for coordination of the clutch in right and left wing hinges. b1MN, first basalar direct flight muscle motor neuron; Glut, glutamate.
(9281GAL4; Figure S3E) [25], Ca2+ elevation after TrpA1 activation in dopaminergic neurons was observed in just the b1Mn (9281GAL4>GCaMP; Figure S3F). With this, we concluded that the b1Mns receive dopaminergic inputs from the aa0 VUM circuit. The Dopaminergic VUM and a-a0 Pair Is Required for Bilateral Wing Coordination, but Not Unilateral Wing Movement To test if the dopaminergic-b1Mn connection described here coordinates wing movements in behavioral contexts other than flight, we measured wing movements during courtship behavior [32]. Although flight-related coordinated wing movements were affected in males with Kir2.1 expression in the a-a0 0 neurons (FMRFaa >Kir2.1; Figure 1), the number of unilateral wing extensions during courtship, wing vibrations in a single wing extension bout (required for species-specific courtship songs), and finally the latency to copulate appeared normal (Figures 4A–4D) as compared with controls (Figures S4A–
S4D). The pattern of high-frequency wing vibrations after unilateral wing extension was visualized by highspeed videography (2,500 frames per second [fps]) of extended wings. Courtship behavior was however severely impaired by Kir2.1 expression in the b1Mns of adult males. Both unilateral extension of left and right wing in the same animal and bilateral wing extensions were observed in VGlut9281(2)>Kir2.1 males (Figures 4A and 4B). Wing vibrations of extended wings were reduced in VGlut9281(2)>Kir2.1 animals (Figure 4C) as compared with controls (Figure S4C), and successful mating was not observed in this genotype (Figure 4D). Discussion In agreement with previous observations in other Diptera and Drosophila, the b1Mns are an integral component of both the flight motor circuit [26] and unilateral wing extension during courtship [32, 33]. Our study establishes the presence of independent circuits for modulating b1Mn function for unilateral versus bilateral wing flapping. In the context of flight, where bilateral wing coordination is critical, recent studies have demonstrated mechanical linkages connecting the two wings of a dipteran fly to perfectly synchronize their motion (T. Deora, A.K. Singh, and S.P.S., unpublished data). Although these linkages mediate bilateral wing coordination during flight, it
Please cite this article in press as: Sadaf et al., Neural Control of Wing Coordination in Flies, Current Biology (2015), http://dx.doi.org/ 10.1016/j.cub.2014.10.069 Wing-Coordinating Central Neurons in Drosophila 7
remains an open question how the wing hinges at the base of each wing are actively engaged (or disengaged) during the onset (or offset) of flight and remain engaged through flight. We propose a model in which mechanosensory inputs such as a gentle air puff activate paired dopaminergic neurons (a and a0 ) which convey this stimulus to the central dopaminergic VUM neuron. The VUM integrates the bilaterally received sensory information and helps coordinate firing of the paired and contralaterally connected b1Mns such that they remain in phase with each other and with the wing beats. This allows simultaneous engagement of the left and right wing hinges by the b1 direct flight muscle for initiation and their disengagement for flight cessation (Figure 4E). However, because unilateral wing extension and vibration during courtship requires coordination of other sensory and motor functions, modulation of b1Mn activity in this context likely occurs through a parallel neural circuit. Supplemental Information Supplemental Information includes Supplemental Experimental Procedures, four figures, and four movies and can be found with this article online at http://dx.doi.org/10.1016/j.cub.2014.10.069. Author Contributions S.S. and G.H. designed the experiments. S.S. performed experiments and analyzed the data. O.V.R. generated the VGlut9281(2)GAL4 strain. S.P.S. supervised high-speed tethered flight and courtship videography and analysis. S.S., S.P.S., and G.H. wrote the manuscript. Acknowledgments S.S. was supported by a research fellowship from the CSIR (http://rdpp.csir. res.in/csir_acsir/Home.aspx). The project was supported by grants from the DST (http://www.dst.gov.in/) and NCBS, TIFR (http://www.ncbs.res.in) to G.H. and by the Ramanujan Fellowship, DST, and a grant from the Air Force Office of Scientific Research (AFOSR) to S.P.S. The NCBS Central ImageFlow Facility helped with confocal imaging. Received: April 4, 2014 Revised: October 13, 2014 Accepted: October 29, 2014 Published: December 11, 2014 References 1. Borst, A., and Haag, J. (2002). Neural networks in the cockpit of the fly. J. Comp. Physiol. A Neuroethol. Sens. Neural Behav. Physiol. 188, 419–437. 2. Gorczyca, M., and Hall, J.C. (1984). Identification of a cholinergic synapse in the giant fiber pathway of Drosophila using conditional mutations of acetylcholine synthesis. J. Neurogenet. 1, 289–313. 3. Elkins, T., and Ganetzky, B. (1988). The roles of potassium currents in Drosophila flight muscles. J. Neurosci. 8, 428–434. 4. Engel, J.E., and Wu, C.F. (1992). Interactions of membrane excitability mutations affecting potassium and sodium currents in the flight and giant fiber escape systems of Drosophila. J. Comp. Physiol. A Neuroethol. Sens. Neural Behav. Physiol. 171, 93–104. 5. Oh, C.E., McMahon, R., Benzer, S., and Tanouye, M.A. (1994). bendless, a Drosophila gene affecting neuronal connectivity, encodes a ubiquitinconjugating enzyme homolog. J. Neurosci. 14, 3166–3179. 6. Fayyazuddin, A., and Dickinson, M.H. (1996). Haltere afferents provide direct, electrotonic input to a steering motor neuron in the blowfly, Calliphora. J. Neurosci. 16, 5225–5232. 7. Fayyazuddin, A., Zaheer, M.A., Hiesinger, P.R., and Bellen, H.J. (2006). The nicotinic acetylcholine receptor Dalpha7 is required for an escape behavior in Drosophila. PLoS Biol. 4, e63. 8. Fayyazuddin, A., and Dickinson, M.H. (1999). Convergent mechanosensory input structures the firing phase of a steering motor neuron in the blowfly, Calliphora. J. Neurophysiol. 82, 1916–1926.
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