Abstract
Motor neurons are the final common pathway1 through which the brain controls movement of the body, forming the basic elements from which all movement is composed. Yet how a single motor neuron contributes to control during natural movement remains unclear. Here we anatomically and functionally characterize the individual roles of the motor neurons that control head movement in the fly, Drosophila melanogaster. Counterintuitively, we find that activity in a single motor neuron rotates the head in different directions, depending on the starting posture of the head, such that the head converges towards a pose determined by the identity of the stimulated motor neuron. A feedback model predicts that this convergent behaviour results from motor neuron drive interacting with proprioceptive feedback. We identify and genetically2 suppress a single class of proprioceptive neuron3 that changes the motor neuron-induced convergence as predicted by the feedback model. These data suggest a framework for how the brain controls movements: instead of directly generating movement in a given direction by activating a fixed set of motor neurons, the brain controls movements by adding bias to a continuing proprioceptive–motor loop.
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Data availability
Data to reproduce the figures in this paper are available at bitbucket.org/stephenhuston/code_data_gorko_et_al. The expression patterns and flies for split-GAL4 lines generated in this study are available online at splitgal4.janelia.org (release: ‘Gorko et al 2024’), except for JR153 and JR161 whose expression patterns can be downloaded at bitbucket.org/stephenhuston/code_data_gorko_et_al; flies available upon request. Electron microscopy data are available at fafb.catmaid.virtualflybrain.org (SKIDs: 1167858 (CvN6) and 1337777 (CvN7)).
Code availability
Code to reproduce the figures in this paper is available at bitbucket.org/stephenhuston/code_data_gorko_et_al.
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Acknowledgements
The Janelia Project Technical Resources team (including K.C., C. Christoforou, G.I., C. Managan and Y. He) performed central nervous system dissections, histological preparations and confocal imaging. Members of the Janelia Fly Facility (including T. Laverty, A. Cavallaro, G. Zheng, J. Kao, S. Pitts, M. Mercer, J. McMahon, B. Sharp and G. Gonzalez) constructed the stocks used. The Janelia Media Facility (B. Rebhorn, R. Simmons and H. Naz) provided fly food. S. Imtiaz contributed to the electron microscopy tracing. H. Dionne provided the PJFRC315-10XUAS-IVS-tdtomato::Kir2.1 construct. The LED arena display and cameras used were a gift from the Reiser Laboratory and W. Korff, respectively. We thank S. Laughlin, L. Abbott and V. Jayaraman for feedback on the manuscript.
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B.G., S.J.H., S.Y.L. and A.B.C. carried out behavioural experiments. I.S., K.C., C. Christoforou and G.I. carried out all anatomical imaging. J.P. and S.J.H. performed electrophysiology. S.N., C. Chen, K.L.H., J.C.T. and S.J.H. generated and characterized the fly lines. D.D.B. generated the electron microscopy dataset. M.K., A.L. and K.B. wrote the behavioural tracking software. H.R. and S.J.H. modelled the data. B.G. and S.J.H. analysed the data and wrote the paper.
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Extended data figures and tables
Extended Data Fig. 1 Comparison of light and electron microscopy images of two neck motor neurons.
(a-b) Comparison of light microscopy data (a) and electron microscopy (b) data for the CvN6 neck motor neuron. (a) Confocal image of CvN6. Grey = JRC2018 male standard brain template which the confocal image of the BRP neuropil stain was aligned to47,48. Black = CsChrimson-mVenus expression in CvN6. Scale bar = 50 µm. (b) Skeleton of the manually traced and proofread same CvN6 neuron in the FAFB whole-brain Electron Microscopy volume16 (catmaid-fafb.virtualflybrain.org, SKID: 1167858). (c) Confocal image of CvN7, colours as for (a). (d) Skeleton of the manually traced and proofread same CvN7 neuron in the FAFB whole-brain Electron Microscopy volume (SKID: 1337777). Posterior and lateral views are shown for each dataset.
Extended Data Fig. 2 Individual trial data used to generate CvN7 action fields.
(a) Video frames of nine flies receiving a CvN7 stimulus when their head is initially pitched upward. The grayscale video frame is taken at the end of the 300 millisecond stimulus period, while the underlaid green image is from one frame before the stimulus started. The difference between the two frames illustrates the pitch downward head movement elicited by the CvN7 stimulus when the head starts in an upward posture. (b) Similar to panel a, this panel displays video frames of nine flies of the same genotype, but with the stimulus occurring when their heads were initially pitched downward. The difference between the overlaid grey (end of stimulus period) and green (before stimulus) frames illustrates the small pitch upward movement of the head elicited by the same CvN7 stimulus as in panel a. Symbols in the lower left hand corners of a & b correspond to the same symbols in panel e and indicate which data bins the videos frames were drawn from. See Supplementary Videos 1-2 for the source videos. (c) Coordinate system used in this paper. All head movement data (except Extended Data Fig. 4e,f) in this paper are presented in the global, lab-fixed frame coordinate system convention shown here. The X, Y and Z axis show the axes roll, pitch and yaw occur about respectively according to the right-hand rule. The origin is in the fly’s neck, located at the computationally estimated pivot point of the head. The X axis is defined as tilted downward 40° from the tethered fly’s long body axis, reflecting the resting head posture of a typical fly during flight. For plots of the visual responses of motor neurons we define positive elevation as moving upward from the X to the Z axis and positive azimuth as moving from the fly’s front to its left, i.e. from the X axis to the Y axis. This azimuth/elevation convention is chosen to be consistent with our right-hand coordinate system for describing rotations of the head, but it does not match that previously used in the literature for describing visual receptive fields15. (d) The rotational trajectories of the head for all 2442 trials (32 flies) that were used to generate the CvN7 action field shown in Fig. 1j. Each line represents the trajectory of rotational postures occurring during one stimulation trial, with black dots indicating the head’s starting posture. The colour of the lines corresponds to the starting posture and the axes are as in Fig. 1j. (e) The same data as in panel d, but with the trajectories grouped by the starting posture bin they were assigned to when generating the average trajectories plotted in Fig. 1j. The locations of the starting posture bins have been expanded by a factor of x3.5, moving them apart to prevent the data groups from obscuring each other. Black arrows indicate the quaternion averages of each bin. (f) The same plot as panel e but with the individual trajectories colour-coded by time since the stimulus was turned on. (g) depicts the relationship between the initial head posture and the CvN7 stimulus-induced change in head posture, using the individual trials underlying the means shown in Fig. 2a&c. The first column represents data from 3912 individual trials, illustrating the association between the head posture recorded just before stimulus onset and the subsequent change in head posture between the first and last frame of the stimulus. In the second column, we employ the additional processing step depicted in Fig. 2c. This involves calculating the ‘rotational difference’ between the no-stimulus and stimulus conditions. Specifically, we plot the rotations required to align each frame’s head posture with the corresponding frame of the mean no-stimulus trajectory from the closest bin (depicted in Fig. 2b). In agreement with the mean data displayed in Fig. 2a–c, individual trials exhibit a correlation between initial head posture and stimulus-induced movement (first column, R2 = 0.45). However, when plotting the ‘rotational difference’ between stimulus and no-stimulus conditions (second column), this correlation is reduced (R2 = 0.22).
Extended Data Fig. 3 Calibration of optogenetic stimulus strength.
(a) Example trace of the membrane potential (vertical scale bar = 10 mV) of the CvN7 motor neuron in response to a 300 ms flash of 625 nm, 0.04 mW/mm2 light. Underneath the voltage trace is a raster indicating the timing of action potentials from multiple trials. (b) An action field plotting the head movements resulting from stimulating CvN7 with the same 0.04 mW/mm2 light pulse. (c) A plot of the ‘rotational difference’ between the action field in b and the equivalent data that occurs during the no-stimulus control condition – see Fig. 2a–c for more detail. (d) The neural response of CvN7 to a 0.09 mW/mm2 stimulus. (e) The CvN7 action field resulting from a 0.09 mW/mm2 stimulus. (f) A plot of the ‘rotational difference’ between the action field in e and the equivalent data that occurs during the no-stimulus control condition. (g) The neural response of CvN7 to a 0.37 mW/mm2 stimulus. (h) The CvN7 action field resulting from a 0.37 mW/mm2 stimulus. (i) A plot of the ‘rotational difference’ between the action field in h and the equivalent data that occurs during the no-stimulus control condition. (j) The neural response of CvN7 to a 1.43 mW/mm2 stimulus. (k) The CvN7 action field resulting from a 1.43 mW/mm2 stimulus. (l) A plot of the ‘rotational difference’ between the action field in k and the equivalent data that occurs during the no-stimulus control condition. Genotype for all panels: SS00754 x w +, norpA, 20XUAS-CsChrimson-mVenus (attP18).
Extended Data Fig. 4 Action fields are robust to stimulation length, saccade removal and reference frame.
(a-b) To confirm that the motor neuron-elicited convergence of head pose we observed is not an artefact of our 300 millisecond stimulation times, we plot just the first 80 milliseconds of the head trajectory when the angular velocity of the head is still increasing (Fig. 1h–i). (a) shows the head movements resulting from the first 80 milliseconds of CvN7 stimulation. (b) Shows the first 80 milliseconds of DProN2 stimulation. While there is, by definition, less time for the head to converge, the early head movements are still highly posture dependent and moving towards a point of convergence. Compare the different movements in the green and magenta circled trajectories in b, which are dominated by roll and pitch respectively. (c-d) To confirm that the mean head trajectories plotted in our action fields are representative of the underlying data and not biased by outlier events such as fast head saccades, we removed trials containing head saccades from the data. (c) is the action field of the CvN7 neuron replotted from Fig. 1j. (d) shows the same data but excluding any trial containing head saccades: defined as any trial containing yaw movements faster than 200 °/second. (e) All rotations in this study were measured in the lab reference frame. To confirm that the choice of reference frame did not substantially alter the results22 we plot here the head rotations resulting from CvN7 stimulation measured in the reference frame of the head at the time of stimulus onset. By definition all head rotations start at the origin. (f) To allow comparison of the head reference frame data in e with that measured in the lab reference frame in c we moved the start points of all trajectories in e from the origin to their respective start locations in the lab frame.
Extended Data Fig. 5 Proprioceptive manipulations alter the action field of a motor neuron.
(a–d) central anatomy of the neck chordotonal organ afferents. (a) The central projection pattern of the Lateral Neck Chordotonal neurons (LNCs) within the ventral nerve cord. Purple: anti-BRP neuropil stain, green: anti-GFP. Genotype: JR153 x pJFRC2-10XUAS-IVS-mCD8::GFP (attP2). (b) Example of a single neuron from within the LNC population, genotype: JR153 x Multicolour flip out reporter (MCFO-160). (c) The central projection pattern of the Medial Neck Chordotonal neurons (MNCs) within the ventral nerve cord. Purple: anti-BRP neuropil stain, green: anti-GFP. Genotype: JR161 x pJFRC2-10XUAS-IVS-mCD8::GFP (attP2). (d) Example of a single neuron from within the MNC population, genotype: JR161 x Multicolour flip out reporter (MCFO-160). (f-k) Peripheral anatomy of the neck chordotonal afferents. (f) Peripheral expression of the same LNCs shown in (a-b). Large magenta structure: neck muscles, small magenta structure: actin rich scolopale cells of the chordotonal organ (F-actin binding Phalloidin), green: LNCs (anti-GFP). (g) Peripheral expression of the same MNCs as shown in c-d. Large magenta structure: neck muscles, small magenta structure: actin rich scolopale cells of the chordotonal organ (F-actin binding Phalloidin), green: MNCs (anti-GFP). (h) Magnification of the dendrites of the LNCs shown in f. LNC dendrites (green) can be seen to invade the magenta cylindrical scolopale cells at the lateral edge of the chordotonal organ. (i) Diagram of the chordotonal organ and the LNC neuron innervation. (j) Magnification of the dendrites of the MNCs shown in g. MNC dendrites (green) can be seen to invade the magenta cylindrical scolopale cells at the medial edge of the chordotonal organ. Scale bars in a-j are 50 µm. (k) Diagram of the chordotonal organ and the MNC neuron innervation. (l) Action field resulting from activation of the CvN7 motor neuron replotted from Fig. 3. Colour indicates the head’s angular velocity. (m) Action field of the same CvN7 motor neuron when the LNCs are hyperpolarized due to expression of Kir2.1, replotted from Fig. 3. (n) Action field of the same CvN7, with the MNCs hyperpolarized due to expression of Kir2.1 (43 flies, 5160 trials, JR161 x w+ norpA, 13XLexAop2>dsFRT>CsChrimson-mVenus (attP18), BPhsFlpPest-OPt (attP3); 81B12LexAp65 (VK00022), PJFRC315-10XUAS-IVS-tdtomato::Kir2.1 (su(Hw)attp5)). (o) Action field of the same CvN7 again, with the sensory neurons of the Prosternal Organ25 head proprioceptor hyperpolarized due to expression of Kir2.1 (12 flies, 1632 trials, SS47486 x w+ norpA, 13XLexAop2>dsFRT>CsChrimson-mVenus (attP18), BPhsFlpPest-OPt (attP3); 81B12LexAp65 (VK00022), PJFRC315-10XUAS-IVS-tdtomato::Kir2.1 (su(Hw)attp5)). (p-s) 2D plots of the same data plotted in 3D in Fig. 3g–m. (p) The CvN7 action field. Equivalent to Fig. 3g. (q) 2D plot of the results of using a feedback model to simulate the CvN7 action field. Equivalent to Fig. 3i. (r) 2D plot of the CvN7 action field when the LNC proprioceptive neurons are expressing the Kir2.1 potassium channel and thus hyperpolarized. Equivalent plot to Fig. 3k. (s) 2D plot of the CvN7 action field simulated by the same model shown in q but with the pitch velocity feedback parameter reduced to match the data in r. Equivalent plot to Fig. 3m. (t) Distributions of head rotations recorded when no stimulus was present for flies where the LNC neurons were either unperturbed (blue, empty split-Gal4 x w+ norpA, 13XLexAop2>dsFRT>CsChrimson-mVenus (attP18), BPhsFlpPest-OPt (attP3); 81B12LexAp65 (VK00022), PJFRC315-10XUAS-IVS-tdtomato::Kir2.1 (su(Hw)attp5)) or where the LNC neurons were hyperpolarized due to expression of Kir2.1 (red, JR153 x w+ norpA, 13XLexAop2>dsFRT>CsChrimson-mVenus (attP18), BPhsFlpPest-OPt (attP3); 81B12LexAp65 (VK00022), PJFRC315-10XUAS-IVS-tdtomato::Kir2.1 (su(Hw)attp5)). The mean roll, pitch and yaw head angles for control flies were 0, 8, −1° respectively with standard deviations of 8, 12, 4° (N = 36 flies). The equivalent mean values for the LNC-hyperpolarized flies were 1,5 and 0°, with standard deviations of 9,15 and 5 (N = 43 flies). A Two-sample Kolmogorov–Smirnov test could not reject the null hypothesis that the data from the two genotypes came from the same distribution (p-values 0.3, 0.9 and 0.2 for roll, pitch and yaw respectively, corresponding effect sizes: 0.12°, 0.09°, −0.09°). (u) Time series of the head pitch velocity during CvN7 stimulation for flies where LNC neurons were hyperpolarized due to expression of Kir2.1 (red, genotypes same as panel t) or unperturbed (blue). Shaded error bars are standard deviations, thick lines are means. Black bar indicates stimulus time course. (v) Pitch velocity distributions for LNC-hyperpolarized (red) and unperturbed (blue) flies (genotypes same as t-u). Only the velocities at the average time of peak velocity (135 ms) are plotted. (w) illustrates the velocity dependence of the model described in Fig. 2. The position dependence of the same model is shown in Fig. 2e. Arrows plot the acceleration vector that, in the absence of any stimulus, the feedback loop alone applies to the head at each velocity. (x) illustrates the same velocity dependence of the model but now for the model fit to the proprioceptor perturbed (JR153 Kir2.1) flies with reduced pitch velocity damping.
Extended Data Fig. 6 Expression patterns of split-Gal4 stocks generated as part of this study.
The full expression patterns of the split-Gal4 stocks generated and used in this study. Full stacks are available at splitgal4.janelia.org (stock names beginning “SS”) and bitbucket.org/stephenhuston/code_data_gorko_et_al (stock names beginning “JR”). A neuropil stain (anti-BRP nc82 stain) is shown in magenta. The expression of each stock when crossed to UAS- CsChrimson-mVenus (20XUAS-CsChrimson-mVenus(attP18)) is shown in green (anti-GFP stain). Scale bars = 50 µm.
Extended Data Fig. 7 Neck musculature and nerves of Drosophila melanogaster.
(a-c) Diagrams of the fly’s neck muscles. Grey = cuticle, red = muscle, orange = tendon, green = neck chordotonal organ. (a) A ventral slice through the fly’s neck, horizontal plane viewed from above. (b) Dorsal slice through the fly’s neck. (c) View of the same muscles in a-b from inside the fly’s thorax, facing towards the neck. SC-CO DV - dorsoventral muscles linking the cervical sclerite and the condyle. VL – ventral longitudinal muscles, DE - depressor muscles, SC-RO – sclerite rotator muscle, SC-RE - sclerite retractor muscle, LEV - levator muscle, TH - transverse horizontal muscles, OH - oblique horizontal muscles, AD - adductor muscle, ABD - abductor muscle. (d-f) Diagrams of the major cuticular structures within the fly’s neck. Grey= cuticle, transparent red = muscles, transparent green = chordotonal organ. (d) Ventral slice through the fly’s neck, (e) Dorsal slice through the fly’s neck, (f) view from inside the fly’s prothorax, facing towards the neck. T - tentorium, CO - condyle, SC – cervical sclerite, PO – prosternal organ, SA – sternal apodeme, PNA – pronotal apodeme, PA – pleuron apodem. Muscle and cuticle structure are named in a way that attempts to match, where possible, the terminology of Strausfeld9. (g) Diagram of the four neck motor outputs of the fly central nervous system after which we have named the motor neurons. CNS outline adapted from11. VCvN motor neurons exit through the Ventral Cervical Nerve (green) that branches from the subesophageal zone where it meets the cervical connective (called the VCN in Strausfeld9). DProN motor neurons exit the ventral nerve cord through the Dorsal Prothoracic nerve (purple, called frontal nerve/FN in Strausfeld9). CvN motor neurons exit through Cervical Nerve (blue, called CN in Strausfeld9), which branches from the cervical connective half way down its length. The CvN A1 and CvN A2 motor neurons (red) have the letter A before the neuron number to indicate that they ascend from the ventral nerve cord to reach the Cervical nerve, in contrast to the majority of CvN neurons which descend from the subesophageal zone. CvN A1-2 are likely homologous to ADN1-2 in the blowfly9 which, in the blowfly, exit from their own thin nerve that originates in the ventral nerve cord. In Drosophila melanogaster this nerve is missing and appears to be fused with the cervical connective and the Cervical Nerve to allow CvN A1-2 to exit the central nervous system via the Cervical Nerve.
Extended Data Fig. 8 The tendons of yaw muscles are coupled.
(a) Confocal image of the dorsal neck musculature showing the OH muscles and the large, curved tendon of the TH muscles. Red = muscles (F-actin stained with Phalloidin), blue = tendons and cuticle (chitin binding Calcofluor White). Yellow arrows indicate points of coupling between the OH and TH tendons. The three small OH muscles originate from the midline and travel anterolaterally to where they connect to the large, curved TH tendon via small tendon tendrils. (b) shows a higher magnification of the coupling between the OH 3 muscle’s tendon and that of the TH muscle. (c) Diagram of the image in a. Muscles are shown in red, the TH muscle tendon is shown in orange and the OH muscle tendons are shown in blue. Such an arrangement is likely to allow the OH muscles to change the line of action of the TH muscle by altering its tendon’s geometry. Such inter-muscle interactions will not be captured by our linear model.
Supplementary information
Supplementary Information
Supplementary Tables 1 and 2, Figs. 1–17, Methods and references.
Supplementary Video 1
The pitch movements elicited by CvN7 stimulation are dependent on the initial head pitch posture.
Supplementary Video 2
The roll movements elicited by CvN7 stimulation are dependent on the initial head roll posture.
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Gorko, B., Siwanowicz, I., Close, K. et al. Motor neurons generate pose-targeted movements via proprioceptive sculpting. Nature 628, 596–603 (2024). https://doi.org/10.1038/s41586-024-07222-5
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DOI: https://doi.org/10.1038/s41586-024-07222-5
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