Research ArticleCELLULAR NEUROSCIENCE

Synaptic silencing of fast muscle is compensated by rewired innervation of slow muscle

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Science Advances  08 Apr 2020:
Vol. 6, no. 15, eaax8382
DOI: 10.1126/sciadv.aax8382
  • Fig. 1 Generation of γ/ε DKO zebrafish.

    (A) Schematic diagram of targeted genes. Arrowheads indicate targeted regions of genome editing. Each box and line indicates an exon and an intron, respectively. Alignment of genomic DNA sequences of WT and KO lines showed a 7–base pair (bp) insertion in the AChR γ subunit gene chrng and a 1-bp insertion in the AChR ε subunit gene chrne. (B) Photograph showing WT and γ/ε DKO larva at 6 dpf. Notice the lack of swim bladder (arrowheads) in DKO. Scale bar, 1 mm. (C) Trunk regions of a WT larva (6 dpf) and a DKO larva (6 dpf) were stained with α-BTX conjugated with Alexa Fluor 488 (green). In WT, AChRs were distributed in myoseptal regions (arrows) and in punctae in middle regions (arrowhead). DKO had α-BTX signals only in myoseptal regions. Scale bars, 100 μm.

  • Fig. 2 Synaptic functions of the DKO line.

    (A) mEPC traces from fast or slow muscles of WT and DKO larvae (6 dpf) by whole-cell patch-clamp recordings. Fast muscle cells in DKO failed to exhibit mEPCs. (B and C) Frequencies (B) and amplitudes (C) of mEPCs were plotted for each muscle (n = 8 cells). (D) Representative traces of voltage-clamped slow and fast muscles in DKO larvae in response to the application of 30 μM ACh. Calibration: 1 s, 500 pA. Amplitudes of ACh-induced currents in slow (n = 7 cells) and fast muscles (n = 7 cells) are shown. Each dot represents a muscle cell. (E) Construct used for Ca2+ imaging. Top: The GCaMP7a coding sequence was fused to the promoter region of the α-actin promoter pαact. Bottom: Schematic illustration showing the experimental procedure. The gene construct was injected into eggs of DKO at the one cell stage. Ca2+ response was analyzed at 6 dpf. Representative traces showing the increase of ΔF/F in a fast muscle (black line) and a slow muscle (red line) during spontaneous contractions. (F) Overexpression of the ε subunit fused with an EGFP (ε-EGFP) in WT (3 dpf). Top panels: ε-EGFPs were expressed under the control of a slow muscle–specific promoter, psmyhc. EGFP signals (green), expressed in the superficial slow muscles, filled the cytoplasm and did not colocalize with α-BTX (magenta) signals. Bottom panels: ε-EGFPs were expressed under the regulation of pαact. In deeper layer fast muscles, the clusters of EGFP and α-BTX colocalized (arrowheads). Scale bars, 50 μm.

  • Fig. 3 Locomotion of WT and KO lines.

    (A) Escape behaviors in WT and DKO lines at 6 dpf in response to tactile stimuli. Images of representative larva on the left show superimposed frames of the complete escape response (the duration of movement is indicated in the top right corner). Scale bars, 2 mm. Kinematics for representative traces of 10 larvae are shown for the initial 50 ms of the response. Middle panels represent averaged traces. In the right panels, each trace represents a different larva. Body angles are shown in degrees, with 0 indicating a straight body, and positive and negative values indicating body bends in opposite directions. Scale bars, 10 ms. (B to D) Maximum turn angles, time to reach the maximum angle, and post-startle swimming speed were calculated for each group of fish (6 dpf). In DKO, the turn angle and the swimming speed were notably reduced, and it took longer to reach maximum angles (n = 10 fish). (E and F) Analyses of spontaneous locomotion. Images of representative larva (left) for WT or DKO showed superimposed frames of spontaneous swim bouts (the duration of movement indicated in the bottom right corner). Swimming speed was calculated for WT (n = 5 fish) and DKO (n = 5 fish), which showed no significant difference. Scale bars, 2 mm.

  • Fig. 4 AChR expression restricted to slow muscles of 3- to 5-month-old adult εKO zebrafish.

    (A) Quantification of γ or ε subunit mRNA in adult muscles. γ Subunit was not detected in WT. γ or ε subunit mRNA was not detected in εKO (n = 6 fish in WT, n = 5 fish in εKO). Sample numbers are shown in parentheses. (B) mRNA expression of γ subunit in 1-dpf larvae. γ Subunit was highly up-regulated in the εKO (n = 5 fish) compared to WT (n = 5 fish). Sample numbers are shown in parentheses. (C) Schematic illustration of a transverse section of the trunk region. The area shown in micropictograms is indicated with a box. The distribution of AChRs in adults, WT or εKO, was visualized by α-BTX conjugated with Alexa Fluor 488 (green). Broken lines indicate the boundary of fast muscle area (arrowheads). Fast muscles in the εKO fish lack α-BTX signals. (D) Sections of adult fast muscles of WT and εKO, stained with the fast muscle–specific F310 antibody. Fast muscles in εKO fish did not display atrophy. In the right panel, diameters of fast muscles in WT and εKO were calculated (87 fibers, n = 3 fish). There was no significant difference. Scale bars, 100 μm.

  • Fig. 5 Locomotion of the εKO fish in the adult stage.

    (A) Escape behaviors in WT and εKO adults (3 to 4 months old). The startle response was induced by dropping objects on water. Images of representative fish to the left show superimposed frames of the complete escape response (the duration of movement is indicated in the bottom right corner). Kinematics for representative traces from 10 or 9 fish are shown for the initial 50 ms of response. Middle panels represent averaged traces. In right panels, each trace represents a different fish. Body angles are shown in degrees, with 0 indicating a straight body. Positive and negative values indicate body bends in opposite directions. (B) First turn angles were calculated for each group of fish (n = 10 fish in WT, n = 9 fish in εKO). Turn angles were reduced in the εKO fish. Sample numbers are shown in parentheses. (C) Post-startle swimming speed and total distance traveled were calculated for the first 120 ms. There was no significant difference between WT (n = 10 fish) and εKO (n = 9 fish) adults.

  • Fig. 6 Retrograde labeling of motor neurons show changed innervation.

    (A) Schematic illustration of a transverse section of the trunk region showing the sites of dye injections. Right panels showing cell bodies of labeled motor neurons (arrowheads) in spinal cords. Broken lines indicating outlines of spinal cords. Scale bars, 50 μm. (B) A graph showing the distance from the center of the spinal cord to cell bodies of motor neurons. In WT, motor neurons located close to the center innervate fast muscles, and ventrolateral motor neurons innervate slow muscles. In εKO, slow muscles were innervated by motor neurons located close to the center. Numbers of labeled cells are shown in parentheses. (C) Graph showing the size of cell somas of motor neurons. In WT, large motor neurons innervate fast muscles, and smaller neurons innervate slow muscles. In εKO, slow muscles were innervated by large motor neurons. (D) Schematic illustration of a transverse section of the trunk region showing the locations of the DiI crystal insertion. The right panel displays cell body of labeled pMN (arrowhead) in the spinal cord. The broken line indicates the outline of the spinal cord. Scale bar, 50 μm. (E) Presynaptic structures were visualized by SV2A antibody. Broken lines indicate the boundary of slow muscle area (left side). Note the reduced signal in the fast muscles of the εKO fish. Scale bars, 100 μm. (F and G) Fast muscle–specific myosins labeled by F310 antibody in WT (F) and εKO (G). In (G), the boxed area is enlarged in the right panel. Broken lines indicate the boundary of slow muscle area (left side). Arrowheads indicate muscle cells with F310 signals in the slow muscle region. While a small number of slow muscle cells in WT sometimes showed immunoreactivity, the cell number was markedly increased in εKO. Scale bars, 100 μm. (H and I) Glycolytic muscle fibers were visualized by αGPD staining in WT (H) and εKO (I). Black broken lines indicate the boundary between slow and intermediate muscles, and the red broken line indicates the boundary between intermediate and fast muscles. Fast, intermediate, and slow muscle areas are labeled with F, I, and S, respectively. Note that the intermediate muscle region in εKO is hard to distinguish from the fast muscle region, blurring the boundary (I). Arrowheads in the right panel indicate muscle cells with αGPD signals in the slow muscle region. Scale bars, 100 μm. (J) Schematic illustration showing the rerouted innervation of pMNs. In εKO adults, synaptic silencing of fast muscles led to the innervation of fast muscle–specific pMNs on slow muscle. This reinnervation caused conversion of slow to fast muscles. The projections of sMNs that innervate fast muscles may not change.

Supplementary Materials

  • Supplementary Materials

    Synaptic silencing of fast muscle is compensated by rewired innervation of slow muscle

    Buntaro Zempo, Yasuhiro Yamamoto, Tory Williams, Fumihito Ono

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