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. 2020 Apr 15;123(5):1864–1869. doi: 10.1152/jn.00095.2020

Impaired neuromuscular transmission of the tibialis anterior in a rodent model of hypertonia

Matthew J Fogarty 1,4, Gary C Sieck 1,2,✉, Joline E Brandenburg 2,3
PMCID: PMC7444917  PMID: 32292122

Abstract

Early-onset hypertonia is characteristic of developmental neuromotor disorders, including cerebral palsy (CP). The spa transgenic mouse displays early-onset spasticity, abnormal gait, and motor impairments that are remarkably similar to symptoms of human CP. Previously, we showed that spa mice have fewer motor neurons innervating the tibialis anterior (TA). An expanded innervation ratio may result in increased susceptibility to neuromuscular transmission failure (NMTF). We assessed NMTF in an ex vivo TA muscle nerve preparation from spa and wild-type (WT) mice by comparing forces elicited by nerve versus muscle stimulation. TA muscle innervation ratio was assessed by counting the number of muscle fibers and dividing by the number of TA motor neurons. Muscle fiber cross-sectional areas were also assessed in the TA muscle. We observed that NMTF was immediately present in spa mice, increased with repetitive stimulation, and associated with increased innervation ratio. These changes were concomitant with reduced TA muscle fiber cross-sectional area in spa mice compared with WT. Early-onset hypertonia is associated with increased innervation ratio and impaired neuromuscular transmission. These disturbances may exacerbate the underlying gait abnormalities present in individuals with hypertonia.

NEW & NOTEWORTHY Nerve-muscle interaction is poorly understood in the context of early-onset spasticity and hypertonia. In an animal model of early-onset spasticity, spa mice, we found a marked impairment of tibialis anterior neuromuscular transmission. This impairment is associated with an increased innervation ratio (mean number of muscle fibers innervated by a single motor neuron). These disturbances may underlie weakness and gait disturbances observed in individual with developmental hypertonia and spasticity.

Keywords: innervation ratio, muscle fibers, neuromotor, spastic cerebral palsy

INTRODUCTION

Early-onset hypertonia is characteristic of a variety of developmental neuromotor disorders, including hereditary spastic paraplegia, leukoencephalopathy, and, notably, cerebral palsy (CP), the most common motor disability of childhood (Brandenburg et al. 2019; Huntsman et al. 2015). Symptoms and signs of CP include spasticity, neuromotor impairments, and hyper-reflexia (Brandenburg et al. 2019; Rosenbaum et al. 2007). These symptoms unequivocally involve dysfunction of the spinal cord and motor unit, as spasticity is related to disinhibition of the motor neuron (MN) (Brandenburg et al. 2019; Katz and Rymer 1989).

The spa transgenic mouse displays a phenotype remarkably similar to symptoms of human CP, namely early-onset motor impairment, spasticity, abnormal gait, myoclonic jerks, and exaggerated startle response (Becker et al. 1986; Heller and Hallett 1982; Simon 1997), due to reduced glycinergic neurotransmission on MNs (Graham et al. 2006; Heller and Hallett 1982; Tadros et al. 2014). Previously, we found that spa mice have fewer MNs innervating the tibialis anterior (TA) muscle, which disproportionally affects larger TA MNs, resulting in a more homogeneous MN pool with more uniform and smaller somal surface areas (Brandenburg et al. 2018). Together, the smaller MN surface area and removal of glycinergic-mediated inhibition in spa mice is likely to increase MN excitability and thereby contribute to spasticity.

It is currently unknown whether the reduction in the number of TA motor neurons results in expanded motor unit size (innervation ratio; i.e., the number of muscle fibers innervated by each MN) or affects neuromuscular transmission in spa mice. We hypothesize that an expanded innervation ratio in spa mice increases susceptibility to neuromuscular transmission failure (NMTF). Motor neuron loss is consistent with increased branch point failure, where NMTF occurs at axonal branch bifurcations that are required to innervate the conserved number of muscle fibers (Sieck and Prakash 1995). These changes will be assessed in relation to the cross-sectional areas of TA muscle fibers in spa and wild-type mice.

METHODS

Ethical approval and animals.

Age-matched wild-type (WT) and homozygous B6.Cg-Glrbspa/J-knockout (spa) mice (male and female, aged 9 to 12 mo) were used in this study. All mice were genotyped by PCR analysis of DNA isolated from tail snips at weaning (Brandenburg et al. 2018; Graham et al. 2006). All procedures were approved by the Institutional Animal Care and Use Committee at the Mayo Clinic (protocol no. A23215). All animals were heavily anesthetized with intraperitoneal injection of ketamine (100 mg/kg) and xylazine (10 mg/kg) before the experiments.

Ex vivo nerve-TA muscle preparation.

The distal tendon of the TA muscle was sectioned and tied off with 6.0 silk. All other muscles of the lower hindlimb were sectioned distally. The sciatic nerve was identified and sectioned as far proximally as possible. The hindlimb was then sectioned about one-third of the distance proximal to the knee, with care taken not to disturb the reflected sciatic nerve.

Assessment of NMTF in the TA muscle.

Following dissection, the preparation was transferred to a bath containing Reese-Simpson buffer (pH 7.4) bubbled with carbogen gas (95% O2 and 5% CO2) and maintained at 26°C. The bony insertion of the TA was fixed with pins, the distal end was attached with suture to a force transducer (6350; Cambridge Technology), and optimal muscle length for force generation was determined. We then assessed NMTF as previously described in detail (Fogarty et al. 2019; Fournier et al. 1991; Johnson and Sieck 1993; Kuei et al. 1990; Rizzuto et al. 2017; Sieck and Prakash 1995; Sieck et al. 2012) by comparing submaximal tetanic forces evoked by repetitive (40 Hz) nerve versus forces evoked every 15 s by superimposed direct muscle stimulation. The sciatic nerve was repetitively (at 40 Hz) stimulated (701C; Aurora Scientific) via a suction electrode using supramaximal (125% of maximal force response) 0.05-ms duration, 50-μA current pulses delivered in 330-ms duration trains repeated every second for 60 s. Every 15 s, the muscle was supramaximally stimulated using 0.5-ms duration, 50-mA current pulses delivered in a 330-ms duration train via platinum plate electrodes placed on either side of the TA. All force measurements were digitized (400-Hz sampling rate) and recorded using LabChart software (ADInstuments, Dunedin, New Zealand). Following these experiments the TA muscle was dissected, dried, and weighed. The extent of NMTF was calculated using the following equation: NMTF = [MF/MFinit – NF/NFinit]/MF/MFinit × 100), where MF = muscle force, MFinit = initial muscle force, NF = nerve force, and NFinit = initial nerve force in a manner identical to past studies (Fogarty et al. 2019; Fournier et al. 1991; Johnson and Sieck 1993; Kuei et al. 1990; Sieck and Prakash 1995; Sieck et al. 2012).

With NMTF, muscle fibers are not activated by nerve stimulation and thus spared from muscle-derived contributions to fatigue (Rowley et al. 2005, 2007). In these ex vivo studies, NMTF reflects differences between forces evoked by nerve compared with muscle stimulation (i.e., %NMTF is the percentage of force loss that is related to neuromuscular transmission, not DIAm fatigue), with increased NMTF indicating increased force loss due to perturbed axonal or synaptic deficits.

Estimating innervation ratio and fiber cross-sectional areas of TA muscle.

TA muscle was rapidly frozen in melting isopentane at optimal length. Cross-sections were cut at 10 μm for histological staining with hematoxylin and eosin in a manner previously described (Greising et al. 2013). Bright-field mosaic images (Olympus IX71; Olympus America, Melville, NY) using a ×20 objective were used to quantify the total number of TA muscle fibers. A mean count from two serial sections from a point half-way along the belly of the TA muscle was used for the estimation of total TA muscle fibers. The innervation ratio was calculated by dividing the number of TA muscle fibers by the mean number of TA MNs previously established for each genotype (Brandenburg et al. 2018). Briefly, these MN numbers were obtained using retrograde rhodamine labeling of the entire TA MN pool with via a nerve dip, allowing 72 h to successfully transport to the spinal cord (Mantilla et al. 2009; Novikova et al. 1997; Richmond et al. 1994). This method offers unambiguous identification of every TA MN (Novikova et al. 1997; Richmond et al. 1994), bypassing the neuromuscular junction to avoid confounding due to nerve/muscle abnormalities (Brandenburg et al. 2019, 2020; Fogarty et al. 2018). Mosaic images of TA muscle were also used to quantify the size distributions of muscle fiber cross-sections.

Statistics.

All statistical analyses were performed using Prism 8.0 software (GraphPad, La Jolla, CA). Differences between groups were examined using unpaired Student’s t-tests when data were determined to be normally distributed according to Shapiro-Wilk normality tests. A Mann-Whitney test was used when the data were not normally distributed or if there were differences in variances between groups. Two-way ANOVA was used when comparing two factors, with post hoc tests where appropriate. Kolmogorov-Smirnov tests were used to compare two distributions. Statistical significance was established at the P < 0.05 level. Note that our a priori exclusion criterion was any data point greater than two standard deviations outside the mean. All values are reported as the mean ± 95% confidence intervals (CI), unless otherwise stated. The NMTF experiments were not blinded since obvious morphological and behavioral differences between spa and wild-type mice precluded such blinding. However, the imaging analyses of innervation ratio and TA fiber cross-sectional areas were performed in a blinded fashion.

RESULTS

Extent of NMTF in TA muscle.

In the spa mice, the force evoked by nerve stimulation was markedly less than that evoked by direct muscle stimulation in the first stimulation train (Fig. 1, A and B). In spa mice, the extent of NMTF in the initial stimulus train (29.1 ± 15.0%; n = 7) was approximately fivefold greater compared with WT mice (5.7 ± 2.5%; n = 9, P = 0.0002, Mann-Whitney test; Fig. 1C).

Fig. 1.

Fig. 1.

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Representative muscle- and nerve-evoked tibialis anterior (TA) contractions in wild-type (WT; A) and spa (B) mice. There is an increase in neuromuscular transmission failure (NMTF) in spa mice compared with WT (C); n = 9 for WT, n = 7 for spa, P = 0.0002; Mann-Whitney test. *P < 0.05, with precise values in results. CI, confidence interval.

During 60 s of repeated nerve stimulation, the extent of NMTF increased in spa (n = 7) compared with WT (n = 9) mice (Fig. 2, A and B), which depended on genotype [F(1, 14) = 15.2, P = 0.002] and time point (i.e., 0, 15, 30, 45 or 60 s) of repeated stimulation [F(2, 28) = 86.7, P < 0.0001; Fig. 2C]. The extent of NMTF increased progressively with time in both spa and WT mice, but to a greater extent in the spa TA (Fig. 2C). Following 60 s of repeated stimulation, the final extent of NMTF was 82.7 ± 7.6% in the spa TA muscle compared with 65.4 ± 5.0% in WT (P = 0.0004, Student’s t-test; Fig. 2D).

Fig. 2.

Fig. 2.

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A and B: representative force traces showing 60 s of continued nerve-evoked tibialis anterior (TA) muscle stimulation superimposed with muscle stimulation every 15 s in wild-type (WT; A) and spa (B) mice. C: during 60-s stimulation protocols, increased TA neuromuscular transmission failure (NMTF) was evident in all mice, with greater NMTF evident in spa mice; n = 9 for WT, n = 7 for spa, P = 0.002, 2-way ANOVA with Bonferroni post hoc tests. D: following the 60-s bout of TA muscle contractions, NMTF is more apparent in spa mice than WT; n = 9 for WT, n = 7 for spa, P = 0.0004, Student’s t-test. *P < 0.05, with precise values in results. CI, confidence interval.

Innervation ratio TA muscle.

There was a marked ∼20% reduction in the mass of the TA muscle of spa mice (0.042 ± 0.06 g; n = 7) compared with WT (0.051 ± 0.02 g; n = 9, P = 0.009; Student’s t-test). Despite this difference in total TA mass, there was no difference in the number of TA muscle fibers in spa (1,934 ± 441; n = 4) compared with WT (2,099 ± 238; n = 4, P = 0.69, Mann-Whitney test; Fig. 3C).

Fig. 3.

Fig. 3.

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A and B: representative wild-type (WT; A) and spa (B) tibialis anterior (TA) muscle hematoxylin and eosin histology, with insets showing higher-magnification examples of fibers. C: there was no difference in the total TA muscle fiber number between WT and spa mice. P = 0.69, Mann-Whitney test. D: the TA innervation ratio [muscle fibers per motor neuron (MN)] was increased in spa mice compared with WT. P = 0.03, Mann-Whitney test. E: there was a marked reduction in the mean cross-sectional area of TA muscle fibers in spa compared with WT mice. P = 0.001, Student’s t-test. F: frequency distributions show that there is a substantially increased percentage of smaller TA muscle fibers in spa mice compared with WT. P < 0.0001, Kolmogorov-Smirnov test; n = 4 for both genotypes in all assessments. Note that our a priori exclusion criterion was any data point greater than 2 standard deviations outside the mean. Scale bar, 500 µm. *P < 0.05, with precise values in results. CI, confidence interval.

Using the mean number of TA MNs as previously determined for adult spa (73) and WT (183) mice (Brandenburg et al. 2018), we calculated the mean innervation ratio of TA motor units. The TA innervation ratio in spa mice (25.8 ± 5.0, n = 4) was 2.5-fold greater than that of WT (11.5 ± 1.2; n = 4, P = 0.03, Mann-Whitney test; Fig. 3D).

Cross-sectional areas of TA muscle fibers.

There was a marked ∼38% reduction in the mean cross-sectional areas of TA muscle fibers of spa mice (843 ± 198 μm2; n = 4) compared with WT (1,353 ± 157 μm2; n = 4, P = 0.001; Student’s t-test; Fig. 3E). There was a difference in the cumulative frequency distribution of TA muscle fibers between WT and spa mice, with spa mice showing a shift toward increased frequencies of smaller TA fiber sizes (P < 0.0001, Kolmogorov-Smirnov test; Fig. 3F).

DISCUSSION

The results of the present study demonstrate four main findings. 1) Initial neuromuscular transmission is impaired in the TA muscle of spa mice; 2) during 60 s of repeated stimulation, the extent of NMTF in the TA muscle increases in both spa and WT mice, but to a greater extent in spa mice; 3) the innervation ratio of spa mice is greater than that of WT; and 4) the mean TA muscle fiber cross-sectional area is reduced in spa compared with WT mice. Taken together, these results support the hypothesis that reduced MN number is associated with expanded motor unit innervation ratio and impaired neuromuscular transmission in spa mice.

Past studies assessing the initial train of neuromuscular transmission in mice and rats have shown negligible NMTF (Fogarty et al. 2019; Fournier et al. 1991; Johnson and Sieck 1993; Rizzuto et al. 2017). In spa mice, the initial extent of NMTF was substantial (∼30% failure; ∼5 times more than WT controls). In other cases where motor unit expansion is present due to MN loss [e.g., amyotrophic lateral sclerosis (Fogarty 2018b; Hegedus et al. 2008) and aging (Fogarty et al. 2018; Hepple 2018)], failure of neuromuscular transmission is also observed during the initial train (Fogarty et al. 2019; Rizzuto et al. 2017). This impairment may have two causes (Sieck and Prakash 1995): axonal propagation disturbances, such as increased branch point failures, or frank denervation and/or degeneration of neuromuscular junctions, which would depend on when motor neuron loss occurs (Fogarty et al. 2019; Smith 1979; Smith and Weiler 1987; Smith et al. 1990). Although we did not confirm diminished compound muscle action potentials in nerve-evoked contraction, we believe the former is more plausible. In our experiments, the latter is unlikely to occur at the younger age of animals assessed in the present study. Previous electron microscopy and fluorescent observations in limb muscles of mice with developmental reductions in MN number, including glycine-associated mutations, reported no degeneration at neuromuscular junctions (Banks et al. 2001, 2003, 2005; Fogarty et al. 2013, 2015).

During repeated nerve stimulations, NMTF increased in TA of both spa and WT mice, but to a greater extent in spa mice. Substantial NMTF (∼50%) was observed in the TA of WT mice after 45 s, which lies intermediate to that reported for the extensor digitorum longus and diaphragm muscle, with 50% failure after 30 and 90 s, respectively (Fogarty et al. 2019; Pagala et al. 1984; Sieck et al. 2012). These differences are likely due to the different distributions of motor unit types within these muscles (Fogarty et al. 2019; Johnson and Sieck 1993; Pagala et al. 1984; Sieck et al. 2012). For repeated stimulation, desensitization of postsynaptic acetylcholine receptors and declines in the safety factor contribute to NMTF (Fogarty et al. 2019; Johnson and Sieck 1993; Pagala et al. 1984; Sieck and Prakash 1995; Smith and Weiler 1987; Smith et al. 1990).

Both pre- and postsynaptic mechanisms contribute to NMTF. Along with neuromuscular junction and acetylcholine receptor-associated deficits, each axon branch point is a locus for action potential propagation failure (Sieck and Prakash 1995). The spa mouse, noted for its early-onset spasticity and hypertonia, exhibits considerable TA MN loss. In other developmental models where survival of MNs is altered, neuromuscular junctions remain normal (Banks et al. 2005; Fogarty et al. 2013, 2015). Thus deficits may primarily be due to excessive presynaptic branch points. In agreement, we observed unchanged numbers of TA muscle fibers and an increased innervation ratio in spa mice compared with WT controls. The number of TA muscle fibers was consistent with reports in past studies of mouse TA (Hegedus et al. 2008) and with unchanged muscle fiber density in other models of altered MN development (Banks et al. 2001, 2003, 2005). Our results suggest that the greater initial NMTF in spa mice is directly associated with an increased innervation ratio.

The reduced cross-sectional areas of TA muscle fibers in spa mice are consistent with disproportionate reduction in the number of larger TA MNs in spa mice compared with WT. This MN loss and altered distribution of somal surface areas are not exclusive to the TA motor pool in similarly aged spa mice but are also observed in phrenic MNs (Brandenburg et al. 2020). In other conditions where larger MNs are selectively afflicted (such as amyotrophic lateral sclerosis and aging), loss of larger MNs (Dukkipati et al. 2018; Fogarty 2018a; Fogarty et al. 2018) is observed contemporaneously with reduced cross-sectional areas of muscle fibers (Atkin et al. 2005; Elliott et al. 2016; Hegedus et al. 2008; Khurram et al. 2018). However, it is currently unknown whether morphological abnormalities of the larger MNs and TA muscle fibers in spa mice are associated with MN death and denervation in adulthood or due to failures in early postnatal development, a period of rapid muscle fiber and motor neuron growth and muscle cross-sectional area increases in a variety of motor unit pools (Fogarty and Sieck 2019; Kanjhan et al. 2016; Prakash et al. 2000).

In conclusion, we show that early-onset hypertonia is strongly associated with impaired neuromuscular transmission. The deficits in motor control can be explained in part by an increase in the innervation ratio. Our results are of critical clinical importance, as motor units and neuromotor control have been overlooked in the etiology of developmental motor disorders, including CP and other hypertonic conditions (Brandenburg et al. 2019). Future investigations should take into consideration neuromuscular transmission deficits in the context muscle weakness and discoordination of gait in developmental motor disorders.

GRANTS

This work was supported by National Institutes of Health Grants R01-AG-044615 (G.C.S.) and R01-HL-96750 (G.C.S), a Mayo Clinic Children’s Research Center Pediatric Team Science Award (J.E.B.), a Mayo Clinic Office of Research Diversity and Inclusion Career Support and Advancement Award (J.E.B.), Mayo Clinic CTSA UL1 TR000135 (J.E.B.), and the National Health and Medical Research Council Early Career Fellowship (C.J. Martin) Scheme (M.J.F.).

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

M.J.F., G.C.S., and J.E.B. conceived and designed research; M.J.F. and J.E.B. performed experiments; M.J.F. and J.E.B. analyzed data; M.J.F., G.C.S., and J.E.B. interpreted results of experiments; M.J.F. and J.E.B. prepared figures; M.J.F., G.C.S., and J.E.B. drafted manuscript; M.J.F., G.C.S., and J.E.B. edited and revised manuscript; M.J.F., G.C.S., and J.E.B. approved final version of manuscript.

ACKNOWLEDGMENTS

We thank Becky Macken, Jeff Bailey, and Yun-Hua Fang for technical assistance in the completion of this work.

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