Heterogeneity in form and function of the rat extensor digitorum longus motor unit

Abstract

The motor unit comprises a variable number of muscle fibres that connect through myelinated nerve fibres to a motoneuron (MN), the central drivers of activity. At the simplest level of organisation there exist phenotypically distinct MNs that activate corresponding muscle fibre types, but within an individual motor pool there typically exists a mixed population of fast and slow firing MNs, innervating groups of Type II and Type I fibres, respectively. Characterising the heterogeneity across multiple levels of motor unit organisation is critical to understanding changes that occur in response to physiological and pathological perturbations. Through a comprehensive assessment of muscle histology and ex vivo function, mathematical modelling and neuronal tracing, we demonstrate regional heterogeneities at the level of the MN, muscle fibre type composition and oxygen delivery kinetics of the rat extensor digitorum longus (EDL) muscle. Specifically, the EDL contains two phenotypically distinct regions: a relatively oxidative medial and a more glycolytic lateral compartment. Smaller muscle fibres in the medial compartment, in combination with a greater local capillary density, preserve tissue O₂ partial pressure (PO₂ ) during modelled activity. Conversely, capillary supply to the lateral compartment is calculated to be insufficient to defend active muscle PO₂ but is likely optimised to facilitate metabolite removal. Simulation of in vivo muscle length change and phasic activation suggest that both compartments are able to generate similar net power. However, retrograde tracing demonstrates (counter to previous observations) that a negative relationship between soma size and C-bouton density exists. Finally, we confirm a lack of specificity of SK3 expression to slow MNs. Together, these data provide a reference for heterogeneities across the rat EDL motor unit and re-emphasise the importance of sampling technique.

Keywords: capillary supply; motoneuron; oxygen modelling; skeletal muscle; work loop.

FIGURE 1

Phenotypical arrangement of the extensor digitorum longus (EDL) muscle. (a) Histological micrographs taken in the medial and lateral compartments showing Type I (red), Type IIa (green) and Type IIb/x (unstained) fibres; scale bar = 100 µm. (b) Probability density distribution of fibre cross‐sectional area across the EDL with Gaussian plots for the whole muscle (black), the medial fibres (blue) and the lateral fibres (red). (c) Numerical composition of fibres contained within the different portions of the EDL. Mean ± standard error of the mean (SEM), *p < 0.05

FIGURE 2

Heterogeneity in capillary supply. (a) Capillary domain areas (white) overlaid onto histological micrographs of the medial and lateral compartment. Using the publicly available oxygen transport modeller (Al‐Shammari et al., 2019) mathematical predictions of tissue PO₂ are calculated using individual capillary locations as a modelled point source of oxygen, with muscle fibre type‐specific oxygen demands. The figures are the resultant oxygen tension presented as heatmaps of PO₂ across the muscle fibres (white boundaries); scale bar = 100 µm. Frequency distribution of the capillary domain area across the whole extensor digitorum longus (EDL) (b) with Gaussian plots for the whole EDL (black), the medial fibres (blue) and the lateral fibres (red). Local indices of capillary supply are presented as the average capillary domain area (c), plus the relationship between fibre cross‐sectional area and local capillary to fibre ratio (LCFR) (d) and local capillary density (LCD) (e). Finally, predictions for mean fibre PO₂ across all three major fibre types within each compartment (f). Mean ± standard error of the mean (SEM), *p < 0.05

FIGURE 3

Mechanical properties of the extensor digitorum longus (EDL). (a) The compartmental arrangement of the EDL and sites of retrograde tracer injection. (b) The force–length relationship for the medial (blue) and lateral (red) compartment, with both active (solid lines) and passive (dashed lines) force profiles. (c) Peak length for (d) maximum isometric tetanic stress. (e) An example work loop for the medial (blue) and lateral (red) compartment muscle preparations and (f) the net power generated. Mean ± standard error of the mean (SEM), *p < 0.05. L ₀, optimum muscle length to generate force; P/P ₀, force relative to the peak force during the work loop cycle

FIGURE 4

Diversity in morphometrics of a single motor pool. (a) Example low magnification micrograph of spinal cord white matter highlighting fast blue labelled (blue) medial cells and cholera toxin subunit b (CTB) labelled (red) lateral motoneurons. Higher magnification of individual cells co‐stained with vesicular acetylcholine transporter (VAChT, green). (b) Probability density of motoneuron cross‐sectional area (CSA) for the whole extensor digitorum longus (EDL) with Gaussian plots for the whole EDL (black), medial cells (blue) and the lateral cells (red) (b), with (c) the average motoneuron cross‐sectional area. (d) Frequency distribution of VAChT density for the whole EDL with Gaussian plots for the whole EDL (black), medial cells (blue) and the lateral cells (red), with (e) the average density plotted. (f) The significant negative relationship between VAChT and motoneuron surface area across EDL labelled cells. (g) The percentage of positive/negative labelled motoneurons with SK3 in the medial and lateral compartments. (h) Probability density for SK3 density across the entire EDL with Gaussian plots for the whole EDL (black), medial cells (blue) and the lateral cells (red). Finally (i), the lack of any significant relationship between SK3 density and cell surface area. Scale bar = 150 µm. Mean ± standard error of the mean (SEM)

FIGURE 5

Overview of the extensor digitorum longus (EDL) motor unit heterogeneities. Labelled motoneurons across the EDL motor pool highlight the negative relationship between soma area and VAChT Density and SK3 Density. These motoneurons innervate phenotypically distinct portions of the EDL muscle that is synchronously recruited to provide power for movement. At the level of individual fibres and capillaries heterogeneities in oxygen supply and demand are evident, which when scaled up combine to meet the demands of the muscle, principally maintenance of oxidative phosphorylation in the medial and removal of lactate in the lateral compartment