Regulation of forward and backward locomotion through intersegmental feedback circuits in Drosophila larvae

Abstract

Animal locomotion requires spatiotemporally coordinated contraction of muscles throughout the body. Here, we investigate how contractions of antagonistic groups of muscles are intersegmentally coordinated during bidirectional crawling of Drosophila larvae. We identify two pairs of higher-order premotor excitatory interneurons present in each abdominal neuromere that intersegmentally provide feedback to the adjacent neuromere during motor propagation. The two feedback neuron pairs are differentially active during either forward or backward locomotion but commonly target a group of premotor interneurons that together provide excitatory inputs to transverse muscles and inhibitory inputs to the antagonistic longitudinal muscles. Inhibition of either feedback neuron pair compromises contraction of transverse muscles in a direction-specific manner. Our results suggest that the intersegmental feedback neurons coordinate contraction of synergistic muscles by acting as delay circuits representing the phase lag between segments. The identified circuit architecture also shows how bidirectional motor networks could be economically embedded in the nervous system.

Fig. 1

Identification of 2nd order premotor interneurons. Circuit mapping of PMSIs, A02e and A02g and their presynaptic partners from EM. a (Left) Schematic of larval muscles and the nervous system. Crawling behavior is generated by the propagation of muscular contraction along the length of the body, which is operated by the propagation neural activity within the central nervous system (VNC: ventral nerve cord). (Right) The body wall muscles are classified as longitudinal and transverse muscles by their orientations. They are innervated by distinct motor neurons, Longitudinal motor neurons (MNs), and Transverse MNs. b An image of the entire CNS reconstructed by electron microscopy. The black box indicates the region shown in (c). c, d Dorsal (c) and posterior (d) views of A02e and A02g (blue) in segment A1. Gray shading represents the outline of the nervous system. e, f Dorsal (e) and posterior (f) views of A02e and A02g (blue) and their presynaptic interneurons, Ifb-Fwd (A01d3 in a lineage-based nomenclature, green), Ifb-Bwd (A27k in a lineage-based nomenclature, magenta), and others (gray). g Posterior views showing the connection between PMSIs (A02e and g) and the presynaptic partner Ifb-Fwd and Ifb-Bwd. (h) A connectivity diagram

Fig. 2

Intersegmental projection of Ifb-Fwd and Ifb-Bwd interneurons. a–f Analyses of Ifb-Fwd interneuron. a–c Neuron reconstruction from EM. a Dorsal view of the Ifb-Fwd neuron (green) and its target A02e (blue). b Location of the synaptic inputs (cyan) and outputs (red) in Ifb-Fwd. c A synaptic contact between Ifb-Fwd and A02e (arrow). d The morphology of Ifb-Fwd as revealed by clonal analysis in SS02065>MCFO (multi-color flip-out). A dorsal view. e SS02065-Gal4 specifically targets Ifb-Fwd. A dorsal view of an SS02065-Gal4 > mCD8::GFP CNS stained for GFP. f Ifb-Fwd is ChAT-positive. Dorsal view of an SS02065 > mCD8::GFP CNS (n = 5 larvae). g–l Analyses of Ifb-Bwd interneuron. g–i Neuron reconstruction from EM. g Dorsal view of the Ifb-Bwd neuron (magenta) and its target A02e (blue). h Location of synaptic inputs (cyan) and outputs (red) in Ifb-Bwd. i Synaptic contacts between Ifb-Bwd and A02e (arrow). j The morphology of Ifb-Bwd as revealed by clonal analysis in SS026694>MCFO. A dorsal view. k SS026694-Gal4 specifically targets Ifb-Bwd. A dorsal view of an SS026694>mCD8::GFP CNS stained for GFP. l Ifb-Bwd is ChAT-positive. Dorsal view of an SS026694>mCD8::GFP CNS (n = 5 larvae). Black horizontal lines in (a, b, g, h) and white horizontal lines in (d, j) indicate the midlines. Scale bar, 20 µm (d, e, j, k) and 5 µm (f, l)

Fig. 3

Ifb-Fwd and Ifb-Bwd mediate intersegmental feedback signaling. a Temporally stacked images of dual-color calcium imaging (pseudocolored such that Ifb-Fwd>GCaMP is green and A02e>RGECO is blue). b An example of calcium imaging of Ifb-Fwd (green) and A02e (blue). Ifb-Fwd is active during forward (f) but not backward (b) fictive locomotion (64 forward waves and 8 backward waves from 8 larvae). A02e activity was used to monitor wave propagation. c Correlation coefficients of calcium signals between Ifb-Fwd and A02e across trials in forward and backward waves. Each horizontal line corresponds to a single trial. d Summary of the activity and connectivity of Ifb-Fwd. e Temporally stacked images of dual-color calcium imaging (pseudocolored such that Ifb-Bwd>GCaMP is magenta and A02e>RGECO is blue). f An example of calcium imaging of Ifb-Bwd (magenta) and A02e (blue). Ifb-Bwd is active during backward (b) but not forward (f) fictive locomotion (10 forward waves and 19 backward waves from three larvae). g Correlation coefficients of calcium signals between Ifb-Bwd and A02e across trials in forward and backward waves. Each horizontal line corresponds to a single trial. h Summary of the activity and connectivity of Ifb-Bwd. Scale bar, 40 µm (a, e)

Fig. 4

Ifb-Fwd and Ifb-Bwd share postsynaptic target neurons. a–d Dorsal views of the reconstructed Ifb-Fwd and Ifb-Bwd and their postsynaptic targets A01c and A03g. Black horizontal lines indicate the midlines. e Downstream neurons shared by Ifb-Fwd and Ifb-Bwd (excitatory neurons, orange; inhibitory neurons, blue; neurotransmitter unknown, gray). We assigned their neurotransmitter phenotype as follows: A01c was reported to be cholinergic. A14b is putatively GABAergic since it belongs to a lineage of GABAergic neurons. A03g is shown to be cholinergic in (f). The neurotransmitter phenotype of the other two neurons (A07c4 and A07f2) could not be tested due to lack of specific Gal4 lines. The widths of arrows indicate synapse numbers. f Dorsal view of A03g (R36G02>mCD8::GFP) counterstained for ChAT (n = 5 larvae). Scale bar, 5 µm

Fig. 5

The shared module neurons innervate MNs in a synergistic manner. a–c Posterior views of the connectivity between the shared module neurons and the target MNs. a Excitatory neurons in the module (dark orange) and the target transverse MNs (light orange). b Inhibitory neurons in the module (dark blue) and the target longitudinal MNs (light blue). c Merged image of (a) and (b). Arrows indicate the presynaptic sites of excitatory interneurons (dark orange), inhibitory interneurons (dark blue), and postsynaptic sites of the interneurons receiving inputs from Ifb neurons (green and magenta). SN: segmental nerve; ISN: intersegmental nerve. d Connectome of the shared module. Excitatory neurons in the module innervate MNs targeting transverse muscles. Meanwhile, inhibitory neurons in the module innervate MNs targeting longitudinal muscles

Fig. 6

Temporal profiles of the activity of the shared module neurons. a–c Dual-color calcium imaging of A01ci and A02e. a Temporally stacked images of R75H04>GCaMP (expressed in A01ci; top) and R70C01>RGECO1 (expressed in A02e; bottom). b Plot of the calcium signal in the presynaptic sites of A01ci (green) and A02e (magenta) in the same neuromere during a forward (left) and backward (right) wave (44 forward waves and 22 backward waves from six larvae). c Histogram of the time delay in peak activity. A01ci activity compared with A02e during forward (left) and backward (right) waves. d–f Dual-color calcium imaging of A23a and A02e. d Temporally stacked images of SS04495>GCaMP (expressed in A23a; top) and R70C01>RGECO1 (expressed in A02e; bottom). e Plot of the calcium signal from presynaptic sites of A23a (green) and A02e (magenta) (71 forward waves and 23 backward waves from five larvae). f Histogram of the time delay as in (c). A23a compared with A02e. A23a follows A02e during forward waves, whereas A23a precedes A02e during backward waves. g Scatter plot of activity peaks of the shared module interneurons during forward and backward waves. The timing of the activity peaks in different neurons measured by GCaMP were plotted using the A02e activity, which was monitored by RGECO1, as a reference at each motor wave. Note that the spread of the A02e points is not always at zero phase, despite it being the reference, because peaks of GCaMP and RGECO1 signal in the same A02e do not perfectly coincide. Each horizontal line indicates a single trial. Sample size: 3–8 larvae for each neuron. h A wiring diagram of the shared module. A part of Fig. 5d is shown. Scale bar, 20 µm (a, d)

Fig. 7

Ifb-Fwd and Ifb-Bwd are required for the contraction of transverse muscles. a–d Muscle imaging using mhc-GFP during forward (a, b) and backward propagation (c, d). a, c The lengths of transverse muscles M23 are plotted. b, d Images of body wall muscles during wave propagations. White arrows indicate the contraction of the transverse muscle. Note the reduction in the contraction of transverse muscles during forward waves when the activity of Ifb-Fwd is blocked (a, b) and the reduction in the muscle contraction during backward waves when the activity of Ifb-Bwd is blocked (c, d). e Quantification of the contraction of transverse muscles. Data size: 10–18 waves from 4 to 6 larvae for each group. ***p < 0.005 (Ifb-Fwd > Kir: p = 5.6 × 10⁻⁹; Ifb-Fwd > Kir: p = 0.0031 by the two-sided Student’s t-test with the Bonferroni correction). See Methods section for statistical analysis. Center line, median; box limits, upper and lower quartiles; whiskers, 1.5× interquartile range; points, outliers. Scale bar, 200 µm (b, d)

Fig. 8

Summary of intersegmental feedback network in motor circuits. a Model of the roles of intersegmental feedback neurons Ifb-Fwd (left) and Ifb-Bwd (right) in transforming the progression of a wave front to the intersegmental delay in forward (left) and backward (right) propagations, respectively. b Schematic of connectivity from intersegmental feedback neurons (Ifb-Fwd and Ifb-Bwd) through the shared module (A02e and A03g, as examples) to MN synergy (longitudinal MNs and transverse MNs) in the nerve cord. Ifb neurons and the shared neurons form synaptic contacts at a centrolateral region of the neuropil (arrowhead). c Schematic of functional domains within the neuropil. Intersegmental feedback signals are conveyed at the centrolateral regions (arrowheads)