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. Author manuscript; available in PMC: 2022 Dec 27.
Published in final edited form as: Mol Cell Neurosci. 2022 Apr 27;120:103730. doi: 10.1016/j.mcn.2022.103730

Skeletal muscle sympathetic denervation disrupts the neuromuscular junction postterminal organization: A single-cell quantitative approach

Zhong-Min Wang a,1, María Laura Messi a,1, Anna Carolina Zaia Rodrigues a,b,1, Osvaldo Delbono a,b,c,*,1
PMCID: PMC9793435  NIHMSID: NIHMS1859373  PMID: 35489637

Abstract

The sympathetic nervous system (SNS) regulates skeletal muscle motor innervation and stabilizes the NMJ in health, disease and aging. Previous studies using both chemical (6-hydroxydopamine, 6-OHDA) and microsurgically-induced sympathetic denervation examined the NMJ organization and transmission in the mouse; however, a detailed quantification of the postterminal on larger hindlimb muscles involved in gait mechanics and posture is lacking. The purpose of this study was to determine whether targets of the sympathetic neuron (SN) exhibiting different intrinsic composition such as the fast-twitch extensor digitorum longus (EDL) and the slow-twitch soleus muscles differ in their response to SN deprivation, and to develop a strategy to accurately quantify the impact of sympathectomy on the NMJ postterminal including those fibers located deeper in the muscle. This approach included muscle fixed ex vivo or through transcardial perfusion in mice treated with 6-OHDA or control ascorbic acid. We measured NMJ postterminal mean terminal total area, number of postterminal fragments, mean fragment area, and mean distance between fragments in free-floating alpha-bungarotoxin-stained in 1038 isolated muscle fibers. We found that muscle fiber sympathetic innervation plays a crucial role in the structural organization of the motorneuron-myofiber synapse postterminal and its deprivation leads to AChR cluster dispersion or shrinking as described in various neuromuscular diseases and aging.

Keywords: Skeletal muscle, Sympathetic neuron, Neuromuscular junction postterminal

1. Introduction

The stability of the neuromuscular junction (NMJ) across the entire animal lifespan is crucial to maintaining mammalian myofiber motor innervation, but increasing evidence confirms diminished neural influence on skeletal muscle at older ages (Delbono, 2003; Gonzalez-Freire et al., 2014; Larsson et al., 2019). Thus, targeting the mechanisms that drive neuromuscular junction (NMJ) instability and muscle motor denervation, will lead to more effective interventions to prevent or reverse clinical conditions characterized by loss in skeletal muscle mass such as aging sarcopenia, the age-related decline in skeletal muscle mass and force (Delbono, 2003; Delbono et al., 2021).

Predominant denervation of type-2 fibers, accompanied by myofiber atrophy and a motor unit remodeling (Delbono, 2003; Larsson and Ansved, 1995; Lexell, 1995, 1997) favors reinnervation of slow fibers by axonal sprouting (Frey et al., 2000; Kadhiresan et al., 1996; Larsson, 1995; Lexell, 1995). We reported a higher percentage of partially and completely denervated fibers in aging mice than previously thought and defined the extent of skeletal muscle denervation in older adults (Messi et al., 2016; Wang et al., 2005). Although we demonstrated the plasticity of muscle innervation in older adults practicing a resistance training regimen, the benefits were partial, variable, and difficult to sustain for most (Messi et al., 2016). Until we can target the mechanisms of NMJ instability and muscle denervation, we cannot develop interventions to prevent or reverse sarcopenia.

A role of the SNS in stabilizing the NMJ has been recently reported in animal models in which muscle fiber sympathetic innervation was experimentally induced (Khan et al., 2016; Rodrigues et al., 2018). Recently, we demonstrated that expression of the heart and neural crest derivative 2, a critical transcription factor for post-mitotic maintenance of SNs, declines over time, but inducing its expression in old mice pre-serves (a) the number and size of SNs; (b) muscle sympathetic innervation; (c) muscle weight and force and whole-body strength; (d) myofiber size; (e) NMJ transmission and nerve-evoked muscle force; and (f) motor innervation. Because the SNS controls a set of genes to reduce inflammation and to promote transcription factor activity, cell signaling, and synapse in the skeletal muscle, we examined and concluded that preserving sympathetic innervation would prevent or ameliorate sarcopenia in old and geriatric mice (Rodrigues et al., 2021; Rodrigues et al., 2020). These results indicate that skeletal muscle innervation is crucial to maintain the nerve-muscle connectivity and functional transmission unscathed.

A detailed quantification of the impact of skeletal muscle deprivation of sympathetic innervation on the NMJ postterminal is lacking, probably due to technical limitations, including interrupted continuity of a number of NMJs due to muscle longitudinal or transversal sections, and poor staining of a significant fraction of NMJs due to incomplete penetration of labeled alpha-bungarotoxin into core muscle layers. Quantifying the morphological disruption of the NMJ induced by depletion of muscle sympathetic innervation is clinically relevant since its substitution with sympathomimetic agents improves NMJ transmission and kinetics (Bukharaeva et al., 2021; Petrov et al., 2022; Rodrigues et al., 2019) and increases the amplitude of the muscle compound action potential in the mouse (Khan et al., 2016; Padilla et al., 2021). Whether this is achieved by rearranging the NMJ postterminal organization is unknown. Therefore, studies intended to intervene in the NMJ structure demand a precise quantification of its components.

To quantify the impact of skeletal muscle sympathetic denervation on the organization of the AChR clusters in the NMJ postterminal, we developed an approach consisting of staining a large number of individual muscle fibers from the EDL and soleus muscles. These muscles participate in distinct functions, hindlimb spatial displacement and posture, respectively. This study shows that muscle sympathetic denervation differentially disrupts the NMJ postterminal organization in both muscles by applying a quantitative single-cell approach. We also conclude that this research strategy can be extended to any muscle to investigate the influence of physiological or pathological conditions affecting the NMJ postterminal cytoarchitecture.

2. Material and methods

2.1. Animals and ethics statement

Male and female C57BL/6 mice (3–4 months) were obtained from the National Institute on Aging (NIA)/Charles River colony and housed in the pathogen-free Animal Research Program of the Wake Forest School of Medicine (WFSM). Mice were maintained at 21 °C and a 12:12-hour dark/light cycle. All mice were fed chow ad libitum and had continuous access to drinking water. All experimental procedures were conducted in compliance with National Institutes of Health laboratory animal care guidelines. We made every effort to minimize their suffering. The WFSM Institutional Animal Care and Use Committee approved protocol A18–204 for this study.

2.2. Chemical sympathectomy

For chemical sympathectomy, 6-hydroxydopamine (6-OHDA, Sigma-Aldrich, St. Louis, MO, USA) was diluted in 0.3% ascorbic acid (AA) in sterile oxygen-free water. Since IV administration for more than a week resulted in deterioration of overall health status, 6(OH)DA (100 mg/kg) or vehicle (ascorbic acid) alone were intramuscularly injected into the hindlimb of mice every other day for 1 week. Oxygen-free water was obtained by gassing water with N2 for 20 min (Finch et al., 1973). All solutions were prepared fresh before injection and protected from light. Mice received 2 injections, one into proximal (quadriceps or hamstring), the other into distal (tibialis anterior or gastrocnemius) muscles in both hindlimbs. The muscles under study, the EDL and soleus, were not injected to avoid mechanical injury. We previously verified that the same chemical sympathectomy protocol used here induces sharp, extensive decline in tyrosine hydroxylase immunoreactivity and significant changes in muscle transcripts (Rodrigues et al., 2018).

2.3. Single muscle fiber procurement

Mice were euthanized and their EDL and soleus muscles were rapidly and carefully dissected, fixed ex vivo in 2% paraformaldehyde (PFA) in PBS for 20–30 min, and then transferred to PBS. EDL and soleus single muscle bundles and fibers were manually dissected and processed for NMJ postterminal staining as described below.

Since it has been postulated that optimal NMJ structural preservation can be achieved by whole animal perfusion (Tse et al., 2014), we applied a second approach consisting of mouse transcardial perfusion using a 2% PFA in phosphate buffer (8% PFA in double distilled water stock further diluted in 0.2 M sodium phosphate, pH 7.4). EDL and soleus muscles were dissected and pinned in a dish coated with Sylgard 184 silicone (Dow Corning, Midland, MI). Under sterescopic visualization, we carefully pulled single muscle fibers away from the muscle bundle in phosphate-buffer solution (PBS) and transferred them to a 48-well dish for NMJ postterminal staining. This technique was successful in the EDL, but only partially in the soleus muscle because we were able to pull single fibers with a considerable membrane margin free of AChR clusters in a small number of fibers. Therefore, data for single soleus muscle fibers are reported only for those undergoing an ex vivo fixative procedure.

2.4. NMJ postterminal staining

Myofibers from both mouse groups, cardiac and non-cardiac perfused, were permeabilized in 1% Triton X-100 (Sigma, St. Louis, MO) for 30 min and stained with 1:250 in PBS tetramethylrhodamine 554-α-bungarotoxin-conjugate (T1175, Invitrogen, ThermoFisher Scientific, Waltham, MA) in a dark, humid chamber at room temperature for 1 h in free-floating cells. Fibers were washout 3 times in PBS, and mounted on a glass slide using S3023 Dako fluorescence mounting medium (Agilent Technologies, Santa Clara, CA).

2.5. Muscle fiber imaging and NMJ postterminal analysis

Stained fibers were visualized with a fluorescence microscope (Olympus IX81, Tokyo, Japan) and images digitized using an Orca R2 Hamamatsu camera (Hamamatsu, Japan). The camera driver and image acquisition were controlled with a MetaMorph Imaging System software (Olympus). Images were acquired at high resolution, 0.52 μm/pixel in the x and y axes. We used MetaMorph and the NIH Image-J/Fiji software for image quantification.

Specific measures included: (a) NMJ postterminal mean terminal total area, which was calculated as the background subtracted area surrounding all AChR clusters; (b) the number of postterminal fragments, as the number of AChR clusters equal or larger than 7μm2; (c) mean fragment area (μm2), as the sum of pixels outlining a single AChR cluster converted to square microns; and (d) mean distance between fragments (μm) as the shortest linear distance measured between the most proximal borders of a dominant and a surrounding AChR cluster. The larger, more central, and better defined structure, was defined as the dominant AChR cluster. Image brightness and contrast were enhanced to improve the signal-to-noise ratio. The image threshold used to detect AChR fluorescence was established for each batch of fibers subjected to the same staining procedure.

2.6. EDL and soleus muscles preparation for confocal microscopy

EDL and soleus muscles were dissected and freed from surrounding tissues, the tendons pinned at the bottom of a plastic histology mold, the muscle cryopreserved in 5% sucrose overnight, followed by 20% sucrose overnight, then washed in PBS and covered with optimal cutting temperature compound (O.C.T.), and frozen at −80 °C until used. In preliminary experiments, we exposed muscles sections to a buffer containing the calcium chelator 3% ethylenediaminetetraacetic acid (EDTA) in PBS to prevent contraction; however, they did not show significant differences in the muscle and NMJ organization compared to those in control conditions devoid of EDTA. Muscles were longitudinally sectioned (25 μm) with a Leica CM3050S cryostat, mounted on glass slides, fixed in 2% PFA for 30 min at room temperature, washed in PBST, then in PBS, incubated in 1:250 in PBS tetramethylrhodamine 554-α-bungarotoxin-conjugate for 3 h, rinsed in PBS, and mounted using Dako mounting medium. The preparation was imaged using an Olympus FV1200 spectral laser scanning confocal microscope, an UPLSAPO20X NA: 0.75 objective, and Olympus MetaMorph software, at 2 μs/pixel, and 0.31 μm/pixel.

2.7. Statistical analysis

All experimental recordings and analyses were conducted blind to muscle, fixative procedure, and treatment groups. No statistical methods were used to predetermine sample sizes; however, our sample sizes are similar to those reported in recently published studies.(Rodrigues et al., 2018; Rodrigues et al., 2020) Sigma Plot version 12.5 (Systat Software, Inc., San Jose, CA) and Microsoft Excel software were used for statistical analyses. All data were expressed as mean ± S.E.M. Analysis of variance (ANOVA) followed by Bonferroni’s post-hoc analysis were used to compare three or more mouse groups. All muscle fibers from a mouse were averaged and the mouse was considered the unit for statistical comparison. Since data from male and female did not show significant differences, their values were pooled based on the treatment (6-OHDA or AA), fixative procedure (ex vivo or cardiac perfusion), and muscle type (EDL or soleus). We measured the strength and direction of linear relationships between pairs of continuous variables by the Bivariate Pearson correlation method. Values are expressed as the mean ± S.E.M. A p-value < 0.05 was considered significant.

3. Results

As an initial approach to investigate the impact of chemical sympathectomy on the NMJ postterminal organization, we used longitudinally sliced EDL and soleus muscles, stained with alpha-bungarotoxin and imaged with a confocal microscope (Material and methods). Fig. 1 shows muscle images from the surface (a, d) and two deeper layers in the soleus and EDL muscles, b–e (12.5 μm) and c–f (25 μm), respectively. While superficial NMJ postterminals could be clearly delineated, two deeper layers showed fewer and less defined NMJ postterminals despite the focus depth of the confocal microscope. This was confirmed in three 3 EDL and 3 soleus muscles from three healthy, young (3–4 months) mice, which showed a mean reduction of 70% in the number of NMJ postterminals at 25 μm from the surface.

Fig. 1.

Fig. 1.

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Soleus and EDL muscle NMJ postterminals examined by confocal microscopy. Superficial NMJ postterminals are clearly outlined images in the soleus and EDL muscles longitudinal sections, while two deeper layers shows fewer and less well-defined NMJ postterminals. Three EDL and soleus muscles from 3 mice were examined. Images are 1024 × 1024 pixels or 317 × 317 μm in the x-y axes. The optical slice thickness was 1.26 μm.

To improve our ability to analyze the influence of sympathectomy on NMJ postterminal organization in a larger number of fibers, single free-floating muscle fibers were stained with rhodamine-conjugated alpha-bungarotoxin and imaged using epifluorescence microscopy. We also used two procedures to determine whether the technical approach employed to procure single muscle fibers, ex vivo muscle fixation and whole body mouse cardiac perfusion, has any impact on the NMJ postterminal organization and/or quantitative assessment.

Single myofiber images showed markedly differences in the NMJ postterminal organization between the EDL and soleus muscles. Fig. 2a shows a single AChR cluster in an enhanced background contrast image of a soleus fiber from a mouse treated with ascorbic acid (AA, control). Fig. 2b and c shows cropped images of a and a 6-OHDA-treated fiber, respectively. It appears that sympathectomy shrinks the NMJ postterminal area with no obvious effects on the number of AChR clusters. Although some soleus fibers displayed more than one AChR cluster (below), 6-OHDA-treatment decreased rather than fragment their area.

Fig. 2.

Fig. 2.

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Skeletal muscle sympathectomy modifies the NMJ postterminal organization.

a. Enhanced background image of a soleus muscle myofiber from an ascorbic acid-treated mouse. The fiber was stained with rhodamine-conjugated alpha-bungarotoxin to visualize the NMJ postterminal and the myofiber outline. b. Background corrected and cropped image in panel a. Postterminal in a soleus myofiber from a 6-OHDA-treated mouse (c). EDL muscle fibers from ascorbic acid (d) and 6-OHDA (e)-treated mice, respectively were ex-vivo fixed. EDL muscle fibers from ascorbic acid (f) and 6-OHDA (g)-treated mice, respectively, were fixed by cardiac perfusion. h. Enhanced background of the image in “e” to visualize the NMJ postterminal fragmentation and fragments distance. Calibration bar in a, g, and h = 30 μm.

In contrast, EDL fibers from control- (Fig. 2d) and 6-OHDA (e)-treated mice showed stark differences in the NMJ postterminal architecture, consisting of fragmentation and distancing of the AChR clusters, in sympathectomized fibers. To determine whether EDL postterminal desintegration reflects a real phenomenon in the muscle in vivo and was not induced by ex vivo fixation, we applied a second fixative procedure consisting of mouse transcardial perfusion. Fig. 2f shows that control EDL fibers treated with ascorbic acid display a morphology similar to ex-vivo fixed EDL fibers (d), but sympathectomized fibers (g) confirmed the finding on 6-OHDA-treated and ex-vivo fixed fibers (e), which indicates that sympathectomy induces disarray of AChR clusters after completing mouse 6-OHDA-treatment in a technique-independent fashion. A larger area of the fiber in (h) shows that cropped images used for the analysis (e) do not leave fragments of the NMJ postterminal undetected.

Morphometric analysis of digitized fiber images was performed on 186 and 192 ex vivo fixed EDL fibers from 6 mice treated 6-OHDA (mean 31 fibers per mouse) and 6 mice treated with ascorbic acid alone (mean 32 fibers per mouse), respectively. We also examined 162 and 198 ex vivo fixed soleus fibers from 6 mice treated 6-OHDA (mean 27 fibers per mouse) and 6 mice treated with ascorbic acid alone (mean 33 fibers per mouse), respectively. For the cardiac perfused group, we analyzed 144 and 156 EDL fibers from 6 mice treated with 6-OHDA (mean 24 fibers per mouse) and 6 mice treated with ascorbic acid alone (mean 26 fibers per mouse), respectively.

Quantitative assessment showed that, compared to control mice, those treated with 6-OHDA showed a significant reduction in the soleus and EDL fiber NMJ postterminal mean terminal total area (Fig. 3A), while their fragmentation was obvious in the EDL but not in soleus fibers (B). To distinguish a AChR cluster from artifactual fluorescence spots, we used an arbitrary cut-off area value of 7μm2 based on our experience on muscle fluorescence images. This cut-off value eliminates a large percentage of artifacts without compromising analysis of the areas of interest (Bonilla et al., 2020) Consistently, cumulative percent of AChR fragments per junction show no obvious differences in soleus myofibers (C); however, an increased terminal fragmentation is apparent in sympathectomized compared to control ex-vivo fixed EDL fibers (D). Surprisingly, this difference is more noticeable in EDL fibers collected after cardiac perfusion. Posterminals exhibiting 2–3 fragments represent 50% of all analyzed fibers in EDL from mice treated with ascorbic acid, while that percent is reached with 5–6 fragments in 6-OHDA-treated mice (E).

Fig. 3.

Fig. 3.

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Analysis of muscle fiber NMJ postterminal mean terminal total area and number of fragments.

A. Mean postterminal total area measured in the soleus and EDL muscles from mice treated with ascorbic acid (AA, black columns) or 6-OHDA (red columns). Soleus were ex vivo (EV) fixed while EDL muscles were ex vivo (EV) fixed or dissected after cardiac perfusion (CP) and whole body fixation. B. Number of NMJ postterminal fragments in the same experimental groups. ANOVA shows statistically significant differences between the three experimental groups and both measures except for the number of postterminal fragments in soleus muscle fibers. C–E. Cumulative percent of AChR fragments per NMJ junction in the soleus (C), ex-vivo fixed EDL (D), and EDL fixed muscles from cardiac perfused mice (E).

Further analysis showed that the NMJ mean fragment area was smaller in myofibers from both EDL and soleus muscles, and the two treatments in EDL muscles (Fig. 4A), while the mean distance between NMJ fragments did not change significantly with sympathectomy in the soleus, but increased notably in the EDL muscle, fixed using the two fixative techniques from 6-OHDA-compared to ascorbic acid-treated mice (B).

Fig. 4.

Fig. 4.

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Analysis of muscle fiber NMJ postterminal mean fragment area and mean distance between fragments.

A. Mean NMJ postterminal fragment area in the soleus and EDL muscles from mice treated with ascorbic acid (AA, black columns) or 6-OHDA (red columns). Soleus were ex vivo (EV) fixed while EDL muscles were EV fixed or dissected after cardiac perfusion (CP) and whole body fixation. ANOVA shows significant difference between experimental groups. ANOVA shows statistically significant differences between control and sympathectomized groups. B. Mean distance in fibers from control and sympathectomized NMJ postterminal fragments. ANOVA shows statistically significant differences between both treatments in the EDL muscle, but not in the soleus muscle fibers.

Next, we examined whether sympathectomy-induced changes in NMJ mean terminal total area, number of postterminal fragments, or mean distance between fragments correlate with myofiber diameter in EDL and soleus muscle myofibers procured both ex-vivo and through cardiac perfusion. Our analysis showed no statistical significance for any of the variables except the number of postterminal fragments in ex-vivo fixed (Pearson coefficient, r = −0.58; p < 0.05) and cardiac-perfused (r = −0.62; p ≤ 0.01) EDL muscle from mice treated with 6-OHDA.

4. Discussion

This study quantifies the NMJ postterminal disarrangement evoked by muscle sympathetic denervation after mouse treatment with 6-OHDA for one week. Sympathetic denervation has been described in aging muscle, which is more subtle, but relentless, leading to distortion of both the NMJ pre- and postsynapse (Delbono et al., 2021; Rodrigues et al., 2021; Rodrigues et al., 2020). Since a previous work reported that the nerve terminal remodels neuromuscular synapses in mice following regeneration of the postsynaptic muscle fiber (Li and Thompson, 2011), we focused our research on the NMJ postterminal in the present study. Since the decline in nerve-muscle connectivity is multifactorial in aging organisms, we evoked sympathetic denervation in young, healthy C57BL6 mice, as a model in which the participation of previously discussed confounding factors in NMJ postterminal organization (Delbono et al., 2021) was omitted.

4.1. The EDL and soleus muscles differ in the pattern of their response to sympathectomy

We found that the EDL muscle shows an extensive NMJ postterminal breakdown evoked by sympathetic denervation, which is also obvious in the cumulative percentage analysis of the number of AChR fragments per NMJ. The mechanism leading to fast muscles response was examined in detail before. We concluded that the SNS regulates NMJ transmission and the stability of the AChR, by maintaining optimal β2-adrenergic receptor-associated Gαi2 expression, and preventing any increase in histone deacetylase-4 (Hdac4), myogenin, muscle RING-finger protein-1 (MuRF1), and miR-206, and that SNS ablation leads to upregulation of MuRF1, muscle atrophy, and downregulation of postsynaptic AChR (Rodrigues et al., 2018). Sympathectomy may also prevent SN trophic support of NMJs by downregulating tibialis anterior myofiber cAMP signaling (Khan et al., 2016), or increasing AChR turnover and upregulating endo/lysosomal AChR vesicles as well as autophagic marker proteins in the tibialis anterior muscle (Straka et al., 2021). While the histological analysis and mechanistic studies were performed in fast-twitch muscles, the mechanisms leading to soleus muscle resistance to NMJ postterminal fragmentation in response to sympathectomy is unknown. Whether these mechanisms regulates AChR stability more tightly in slow or fast fibers is unknown and can be tested in future experiments using rat soleus muscle, which, in contrast to that in mouse, consists almost exclusively of slow-twitch fibers. This approach will permit us to define gene expression in whole muscle and immunohis-tochemistry in single slow-twitch myofibers with a high degree of certainty.

A previous study from our laboratory using both chemical (6-OHDA) and microsurgical sympathetic denervation examined the NMJ organization in the lumbricalis muscle because this muscle was also used for electrophysiological NMJ transmission recordings (Rodrigues et al., 2018); however, a detailed quantification on larger hindlimb muscles involved in gait mechanics and mobility is missing. In this study, we selected two hindlimb muscles, the EDL, a muscle almost exclusively composed of type-II fibers, and the soleus, a muscle consisting of a mixture of type-I and type-II fibers (Bloemberg and Quadrilatero, 2012; Rodrigues et al., 2018). The first, largely involved in limbs phasic movements, while the second in animal limb posture.

Similarly to the lumbricalis muscle, both EDL and soleus muscles exhibit modifications in the NMJ postterminal arrangement. Although most NMJs are in immediate proximity to tyrosine hydroxylase-positive SNs in the EDL and soleus muscles (Straka et al., 2018), the EDL showed marked postterminal fragmentation while the soleus muscle exhibited only shrinking of the main AChR cluster. The magnitude and extension of NMJ postterminal fragmentation and simplification recorded in the lumbricalis muscle was more similar to the EDL than the soleus muscle, which can be related to their myosin heavy-chain composition. The lumbricalis muscle is a fast-twich muscle composed mainly of type-IIx fibers, which are more abundant in the EDL than soleus muscle. Also, type-IIb fibers are predominant in the EDL, but rarely seen in the soleus muscle (Bloemberg and Quadrilatero, 2012; Rodrigues et al., 2018).

Since MuRF1 is located near the postterminal synapse and plays a role in AChR turnover (Rudolf et al., 2013), we previously examined whether MuRF1KO precludes the effect of SNS ablation on membrane AChR; in a mouse model of constitutive MuRF1 gene ablation, KO prevented the decline in postterminal membrane AChR expression. We also concluded that membrane’s AChR downregulation occurs at the protein, not the transcript, level (Rodrigues et al., 2018).

4.2. Advantages and limitations of quantifying a large number of NMJ postterminals using the single-cell approach

Quantifying the NMJ organization is normally limited to the most superficial layers of the muscle, which biases the analysis. We have used the lumbricalis muscle whole-mount technique (Wang et al., 2020), while others used flat muscles like the diaphragm or sternomastoid (Buffelli et al., 2003) to circumvent this issue; however, use of thicker hindlimb mouse muscles is often needed due to their distinct functional role.

Muscle clearing helped us and others to detect terminals previously inaccessible to confocal microscopy (Khan et al., 2016; Rodrigues et al., 2018). Direct comparison between our work and that of Williams et al. or Tu et al. is also problematic due to differences in the techniques and muscles employed. The Williams group developed a hydrogel procedure to improve NMJ labeling and detection in adult EDL muscles (Williams et al., 2019), while Tu and coworkers enhanced NMJ visualization using a sandwich-like apparatus (Tu et al., 2021) best applied to flat muscles like the diaphragm. Bolatto and coworkers used a technique similar to the one reported here, but in a mixture of rat single fibers and fiber bundles (Bolatto et al., 2021).

The approach described in this study ensures a thorough morphometric analysis of the NMJ postterminal in any type of muscle regardless of its size, shape or location. Compared to the whole muscle in situ NMJ analysis, our quantitative approach allows for: (1) a detail analysis of almost all muscle fibers, including those located in deeper layers of the tissue; (2) a muscle clearing-free analysis of deeper tissue layers because all myofibers are individually stained using a free-floating approach, and (3) one-photon or multi-photon confocal microscope-free image analysis due to NMJ postterminal accessibility by epifluorescence microscopy.

This study has some limitations. First, we do not know whether the isolated soleus fibers are either type-I or type-II. Future studies should define the myosin heavy-chain subtype by SDS-PAGE gel in a fragment of the fiber which NMJ postterminal was analyzed (Choi et al., 2012; Wang et al., 2019). Second, we tested two approaches, ex-vivo fixation and cardiac perfusion fixation in the EDL, but the second procedure worked only partially in the soleus muscle (Material and methods). The reason for this was that dissecting soleus fibers was more challenging than those from the EDL muscle due to their brittleness and abundance of connective tissue. Third, the technique described here is time consuming and demands some manual skills to examine a majority of NMJ postterminals in a particular muscle, but the study goals can determine the breadth of the analysis.

Systemic injection of alpha-bungarotoxin has been proposed as a means of improving the efficiency of motor endplate labeling (Chen et al., 2016); however, this technique requires tissue clearing and dissociation due to the limited depth of focus of current microscopy approaches.

5. Conclusions

The purpose of this study was two-fold, to determine whether SN targets exhibiting different intrinsic composition such as the EDL and soleus muscles differ in their response to SN deprivation, and to develop a strategy to accurately test this question. We conclude that muscle fiber sympathetic innervation plays a crucial role in the structural organization of the motorneuron-myofiber synapse postterminal, and that its acute deprivation leads to fragmentation and dispersion of the AChR clusters in the EDL, but only shrinking in the soleus muscle. Some questions remain: (a) why the soleus muscle NMJ postterminal shrinks, but is not fragmented by sympathectomy as the EDL even when these muscles exhibit some MHC similarities? (b) is the density of SN terminal network a key factor in the response of the target tissue to sympathetic ablation? and (c) does shrinking of NMJ postterminals in postural muscles, such as the soleus, account for the frequent falls reported in older adults?

Acknowledgments

This research was supported by National Institutes of Health grants R01AG057013, R01AG057013-02S1 to Osvaldo Delbono and the Wake Forest Claude Pepper Older Americans Independence Center (P30-AG21332).

Footnotes

CRediT authorship contribution statement

OD conceived the project, designed the research, and wrote the paper; MLM and ZMW performed the experiments; ACZR analyzed the experiments; all authors provided manuscript input.

Declaration of competing interest

The authors declare that they have no conflicts of interest.

Data availability statement

The data that support the findings of this study are available from the corresponding author upon request.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon request.

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