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. 2018 Jul 1;19(7):1935.
doi: 10.3390/ijms19071935.

Postnatal Development and Distribution of Sympathetic Innervation in Mouse Skeletal Muscle

Tatjana Straka  1   2   3 , Veronica Vita  4 , Kaltrina Prokshi  5   6 , Sarah Janice Hörner  7   8 , Muzamil Majid Khan  9   10   11 , Marco Pirazzini  12 , Marion Patrick Ivey Williams  13 , Mathias Hafner  14 , Tania Zaglia  15   16   17 , Rüdiger Rudolf  18   19   20
Affiliations

Postnatal Development and Distribution of Sympathetic Innervation in Mouse Skeletal Muscle

Tatjana Straka et al. Int J Mol Sci. .

Abstract

Vertebrate neuromuscular junctions (NMJs) have been conceived as tripartite synapses composed of motor neuron, Schwann cell, and muscle fiber. Recent work has shown the presence of sympathetic neurons in the immediate vicinity of NMJs and experimental and clinical findings suggest that this plays an eminent role in adult NMJ biology. The present study examined the postnatal development and distribution of sympathetic innervation in different muscles using immunofluorescence, confocal microscopy, and Western blot. This demonstrates the proximity of sympathetic neurons in diaphragm, extensor digitorum longus, tibialis anterior, soleus, and levator auris longus muscles. In extensor digitorum longus muscle, sympathetic innervation of NMJs was quantified from perinatal to adult stage and found to increase up to two months of age. In diaphragm muscle, an extensive network of sympathetic neurons was prominent along the characteristic central synapse band. In summary, these data demonstrate that an elaborate sympathetic innervation is present in several mouse skeletal muscles and that this is often next to NMJs. Although the presence of sympathetic neurons at the perisynaptic region of NMJs increased during postnatal development, many synapses were already close to sympathetic neurons at birth. Potential implications of these findings for treatment of neuromuscular diseases are discussed.

Keywords: endplate; neuromuscular junction; neuropeptide Y; sympathetic neuron; tyrosine hydroxylase.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Sympathetic innervation is richly developed in mouse hindleg muscle. Tibialis anterior muscles of adult wildtype mice were cryosectioned longitudinally, then stained with antibodies against TH (A–C, green) and, in addition, against α-actinin (B,C, red). Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI; B,C, blue). Pictures show single confocal sections, (C) depicts the detail in the boxed region of (B). Arrowheads and arrows in (A) indicate alignment of sympathetic neurons with longitudinally and vertically cut muscle fibers, respectively. Scalebars show 500 µm, 50 µm, and 5 µm in (A–C), respectively.
Figure 2
Figure 2
Sympathetic innervation approaches NMJs in various skeletal muscles. Diaphragm muscle was from P30, tibialis anterior, soleus, and levator auris longus muscles were from P90 wildtype mice. Except for diaphragm muscle, which was prepared as a whole mount, fiber bundles were teased from these muscles as indicated and stained for NMJs (BGT, red in overlays) and sympathetic neurons (TH, green in overlays). Images show maximum z-projections of confocal image stacks. Scalebars, 20 µm.
Figure 3
Figure 3
Sympathetic innervation of NMJs increases during postnatal development in mouse hindlimb muscle. Mouse extensor digitorum longus muscles from animals aged zero days (P0) to six months (adult) were sampled, fixed, transversally sectioned, and stained for NMJs (BGT, red in merges) and various marker proteins (green in merges). Therefore, antibodies against TH (A), NPY (B), β2-adrenergic receptor (C, β2AR), and vesicular acetylcholine transporter (D, VAChT) were employed. Labeled sections were imaged by confocal microscopy and colocalization between BGT-signals and immunolabelling was determined. Fluorescence micrographs show single optical sections of representative tissue slices from P0, P21, and adult. Scalebars, 10 µm. Bar graphs show quantitative analysis of immunopositive NMJs as percent of all analyzed NMJs (mean ± SEM; n = 3). In total, more than 8400 NMJs were analyzed. * p < 0.05, ** p < 0.01.
Figure 3
Figure 3
Sympathetic innervation of NMJs increases during postnatal development in mouse hindlimb muscle. Mouse extensor digitorum longus muscles from animals aged zero days (P0) to six months (adult) were sampled, fixed, transversally sectioned, and stained for NMJs (BGT, red in merges) and various marker proteins (green in merges). Therefore, antibodies against TH (A), NPY (B), β2-adrenergic receptor (C, β2AR), and vesicular acetylcholine transporter (D, VAChT) were employed. Labeled sections were imaged by confocal microscopy and colocalization between BGT-signals and immunolabelling was determined. Fluorescence micrographs show single optical sections of representative tissue slices from P0, P21, and adult. Scalebars, 10 µm. Bar graphs show quantitative analysis of immunopositive NMJs as percent of all analyzed NMJs (mean ± SEM; n = 3). In total, more than 8400 NMJs were analyzed. * p < 0.05, ** p < 0.01.
Figure 4
Figure 4
Total amounts of TH and β2AR in hindleg muscles do not vary significantly during postnatal development. Mouse tibialis anterior muscles from animals aged zero days (P0) to six months (adult) were sampled, lysed, and subjected to SDS-PAGE followed by Western blot analysis. In each lane, the same amount of protein was loaded. Western blot used antibodies against TH (A) or β2AR (B). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was employed as a loading control. Bar graphs depict TH or β2AR band intensities relative to their corresponding GAPDH band (mean ± SEM; n = 4 mice for each time point).
Figure 5
Figure 5
Sympathetic innervation of mouse diaphragm muscle gains complexity during postnatal development. Mouse diaphragm muscles from P0 (A) and P30 (B) mice were fixed, stained with BGT against NMJs (red) and antibodies against TH (green). Then, muscles were imaged by high-resolution tile scanning confocal microscopy with a lateral resolution of 758 nm and a vertical step size ranging between 1 and 1.2 µm (P0) and 7 µm (P30). Image acquisition was followed by deconvolution. Large pictures in (A,B) depict maximum z-projections of hemidiaphragms. Insets 1, 2, 3 show single optical sections (A) or enhanced details (B) from correspondingly numbered boxed regions in the overview images. Arrowheads in (B) indicate a zone, where a main branch of the phrenic artery is lined by TH label.
Figure 6
Figure 6
Quantitative analysis confirms interaction of NMJs with sympathetic ramifications in postnatal diaphragm muscles. Mouse diaphragm muscles from P0 mice were fixed, stained with BGT against NMJs (red signals in A–C) and antibodies (green signals in A–C) against VAChT (A) or neurofilament L (B). In (C), primary antibody was omitted. Then, muscles were imaged by high-resolution tile scanning confocal microscopy with a lateral resolution of 758 nm and a vertical step size ranging between 1 and 1.2 µm. Image acquisition was followed by deconvolution. Large pictures in (A–C) depict maximum z-projections of hemidiaphragms. Insets 1–3 show single optical sections from the correspondingly numbered boxed regions in the overview images. (D) shows a distribution plot indicating the distance of individual NMJs to the nearest corresponding immunofluorescence signal. Image data for P0—TH were as shown in Figure 5A. In total, 5986 NMJs were analyzed.
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