Blocking skeletal muscle DHPRs/Ryr1 prevents neuromuscular synapse loss in mutant mice deficient in type III Neuregulin 1 (CRD-Nrg1)

Schwann cells are integral components of vertebrate neuromuscular synapses; in their absence, pre-synaptic nerve terminals withdraw from post-synaptic muscles, leading to muscle denervation and synapse loss at the developing neuromuscular junction (NMJ). Here, we report a rescue of muscle denervation and neuromuscular synapses loss in type III Neuregulin 1 mutant mice (CRD-Nrg1−/−), which lack Schwann cells. We found that muscle denervation and neuromuscular synapse loss were prevented in CRD-Nrg1−/−mice when presynaptic activity was blocked by ablating a specific gene, such as Snap25 (synaptosomal-associated 25 kDa protein) or Chat (choline acetyltransferase). Further, these effects were mediated by a pathway that requires postsynaptic acetylcholine receptors (AChRs), because ablating Chrna1 (acetylcholine receptor α1 subunit), which encodes muscle-specific AChRs in CRD-Nrg1−/−mice also rescued muscle denervation. Moreover, genetically ablating muscle dihydropyridine receptor (DHPR) β1 subunit (Cacnb1) or ryanodine receptor 1 (Ryr1) also rescued muscle denervation and neuromuscular synapse loss in CRD-Nrg1−/−mice. Thus, these genetic manipulations follow a pathway–from presynaptic to postsynaptic, and, ultimately to muscle activity mediated by DHPRs and Ryr1. Importantly, electrophysiological analyses reveal robust synaptic activity in the rescued, Schwann-cell deficient NMJs in CRD-Nrg1−/−Cacnb1−/−or CRD-Nrg1−/−Ryr1−/−mutant mice. Thus, a blockade of synaptic activity, although sufficient, is not necessary to preserve NMJs that lack Schwann cells. Instead, a blockade of muscle activity mediated by DHRPs and Ryr1 is both necessary and sufficient for preserving NMJs that lack Schwann cells. These findings suggest that muscle activity mediated by DHPRs/Ryr1 may destabilize developing NMJs and that Schwann cells play crucial roles in counteracting such a destabilizing activity to preserve neuromuscular synapses during development.

Emerging evidence suggests that NRG1/erbB expression is correlated with the state of skeletal muscle innervation/denervation [36]. Deficiencies in NRG1/erbB signaling-as shown in CRD-Nrg1 −/− , erbB2 −/− and erbB3 −/− mutant mice-lead to a loss of Schwann cells and, consequently, a retraction of motor nerve terminals from diaphragm muscle, resulting in muscle denervation and neuromuscular synapse loss [37][38][39][40][41][42][43]. These defects are likely due to the loss of Schwann cells, rather than a loss of NRG1/erbB signaling from motor neurons to muscles, since deleting erbBs specifically in muscles does not affect NMJ formation and function [44]. How an absence of Schwann cells may cause muscle denervation and neuromuscular synapse loss, however, remains unclear.
Synaptic activity plays crucial roles in sculpting neural circuits [45,46]. Terminal Schwann cells are known to play important roles in regulating synaptic activity at the NMJ [5,[47][48][49], suggesting possible relationships among Schwann cells, activity and synapse formation. In this study, we ask the question if an absence of specific activity at pre-synaptic nerve terminals, post-synaptic acetylcholine receptors (AChRs), or muscle fibers, may affect NMJ formation in the absence of Schwann cells. To address this question, we blocked pre-synaptic activity in CRD-Nrg1 −/− mice, which lack Schwann cells, by ablating specific genes known to be required for transmitter release from the nerve terminal. Surprisingly, blocking neurotransmitter release results in a rescue of muscle denervation and prevents the neuromuscular synapse loss that normally occurred in CRD-Nrg1 −/− mice. We further show that these effects were mediated by postsynaptic acetylcholine receptors (AChRs), because genetic elimination of muscle-specific AChRs in CRD-Nrg1 −/− mice also rescued muscle denervation. And finally, we show that these effects were mediated through muscle activity because genetically ablating either dihydropyridine receptors (DHPRs) or ryanodine receptor 1 (Ryr1) also rescues muscle denervation and neuromuscular synapse loss in CRD-Nrg1 −/− mice. Together, these results demonstrate that bipartite NMJs lacking Schwann cells can be established if muscle activity is blocked, suggesting that muscle activity mediated by DHPRs/Ryr1 plays a key role in preserving Schwann cell-deficient NMJs.

Blocking pre-synaptic activity in CRD-Nrg1 -/mice prevents neuromuscular synapse loss
To investigate how synaptic activity might impact the development of neuromuscular synapses in CRD-Nrg1 -/mice, we took several genetic approaches. First, we took advantage of previously characterized mutant mice that lack pre-synaptic activity. For example, evoked synaptic transmission is completely blocked in mutant mice deficient in synaptosomal-associated 25 kDa protein (SNAP25) [50], a key protein component of Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) complexes required for regulated exocytosis in all chemical synapses [51][52][53][54][55]. We therefore bred CRD-Nrg1 mutant mice with Snap25 mutant mice, and generated double mutant mice deficient in both CRD-Nrg1 and Snap25 (CRD-Nrg1 -/-Snap25 -/-).
These results indicated that muscle denervation and NMJ loss were rescued in CRD-Nrg1 mutant mice that were also deficient in Snap25 (CRD-Nrg1 -/-Snap25 -/-). At the NMJ, motor nerve terminals are known to co-release both acetylcholine (ACh) and adenosine 5 0 -triphosphate (ATP), which has been shown to regulate the expression and stability of post-synaptic AChRs [56][57][58][59]. Ablating Snap25 blocks vesicular exocytosis and thus blocks the release of both ACh and ATP. This raises the question whether the effects seen in CRD-Nrg1 -/-Snap25 -/mice were due to a blockade of the release of ACh, ATP, or both.
In CRD-Nrg1 -/mice, motor neuron numbers were markedly reduced in the spinal cord [38]. To determine if motor neurons were also rescued in CRD-Nrg1 -/-Chat −/− mice, we examined cervical segments of spinal cords using anti-choline transporter (CHT) antibodies, which label motor neurons [62]. We found that the somata of motor neurons in the ventral horn of the spinal cord were labeled CHT-positive in both control (S2A Fig, left panels) and CRD-Nrg1 -/-Chat −/− mice (S2A Fig, right panels). Furthermore, we counted the numbers of motor axons from phrenic nerves (S2B Fig) and found that the average motor axon numbers per phrenic nerve were similar between control (248 ± 8, N = 3 mice) and CRD-Nrg1 -/-Chat −/− (244 ± 11, N = 3 mice) (S2C Fig). Together, these results demonstrated that the loss of phrenic nerve innervation and neuromuscular synapses in single mutant mice deficient in CRD-NRG1 (CRD-Nrg1 -/-) were rescued in double mutant mice deficient in both CRD-NRG1 and ChAT (CRD-Nrg1 -/-Chat -/-).

Absence of Schwann cells in CRD-Nrg1 -/-Chat −/− mice
Using anti-S100β antibodies, which recognize developing Schwann cells [39], we confirmed that Schwann cells were present in control mice but absent in CRD-Nrg1 -/-Chat −/− mice ( Fig  3E). The absence of Schwann cells was further demonstrated by electron microscopy (EM). In control and Chat single mutant mice (i.e., CRD-Nrg1 +/-Chat −/− mice; Fig 3I and 3J), individual axons were regularly interspersed, and each axon was wrapped by Schwann cell processes. In contrast, phrenic nerves were completely devoid of Schwann cells in CRD-Nrg1 -/-Chat −/− mice (Fig 3I and 3J, right panels), and the axons were tightly packed together with very little extracellular space in between.
Ultrastructural analyses of the NMJs also showed that terminal Schwann cells were absent at the NMJs in CRD-Nrg1 -/-Chat −/− mice. Instead, the flanks of presynaptic nerve terminals were directly exposed to the interstitial space in double mutant mice ( Fig 4B). No other cell type was found as a substitute for Schwann cells. Occasionally, thin processes of fibroblast-like cells were seen in the interstitial space around the NMJs, but this feature was readily observed in both control and CRD-Nrg1 -/-Chat −/double mutant mice. Although these processes could be seen near the nerve terminal membrane in a few instances, in no case did they wrap, cap or flank the nerve terminals. Consistent with our results in light microscopy ( Fig 2D and 2E), no NMJs were observed under EM in E18.5 diaphragm muscles in CRD-Nrg1 −/− mice. In contrast, n = 201, N = 3, P = 0.0039), or CRD-Nrg1 -/mice (80.5 ± 2.2 μm 2 , n = 287, N = 3, P = 0.0036), but not significantly different from those in Snap25 -/mice (133.6 ± 13.9 μm 2 , n = 198, N = 3, P = 0.7670). Scale bars: A: 400 μm; B: 20 μm. Strikingly, despite the absence of terminal Schwann cells, the ultrastructure of presynaptic nerve terminals in CRD-Nrg1 −/− Chat −/− mice appeared similar to that of control mice: multiple nerve terminals, each with abundant synaptic vesicles, mitochondria, glycogen granules, and other membranous structures, were observed. The postsynaptic compartment also displayed typical ultrastructural features: electro-dense postsynaptic membranes and abundant sub-synaptic organelles, such as mitochondria and ribosomes (compare Fig 4A to Fig 4B). Thus, bipartite neuromuscular synapses composed of only presynaptic nerve terminals and postsynaptic muscle membrane were established in the absence of Schwann cells in CRD-Nrg1 -/-Chat −/mice.

Ablating post-synaptic AChRs preserves NMJs in CRD-Nrg1 -/mice
AChRs are expressed at multiple sites within the NMJ, including presynaptic nerve terminals, terminal Schwann cells and postsynaptic muscles [10,63]. Although Schwann cells were absent in CRD-Nrg1 -/mice, it remained possible that a blockade of cholinergic transmission (as in CRD-Nrg1 -/-Chat −/mice) may affect synaptic transmission at either pre-synaptic or post-synaptic sites, or both. To determine whether the effects of blocking cholinergic synaptic transmission were mediated through postsynaptic AChRs, we examined the NMJs of mice selectively lacking postsynaptic AChRs-mice lacking the gene encoding the AChR α1 subunit (Chrna1 -/mutants), which is selectively expressed in muscle but not in motor neurons or Schwann cells [64].

Deleting muscle DHPRs preserves NMJs in the absence of Schwann cells
Muscle AChRs are nonselective cation channels, and their activation leads to an influx of cations, including Na + and Ca 2+ , and triggers muscle action potentials (muscle electrical activity), mediated by the voltage-gated sodium channels. Muscle action potentials depolarize the sarcolemma and activate voltage-sensitive dihydropyridine receptors (DHPRs), the L-type Ca 2+ channels localized to the T tubules, and ultimately activate ryanodine receptors (RyR) in the sarcoplasmic reticulum [65,66]. It has been previously shown that genetic deletion of Cacnb1, the gene encoding the β1 subunit of DHPRs, blocks DHPR function [67] without affecting muscle electrical activity [68].

Schwann cell-deficient neuromuscular synapses exhibited increased spontaneous synaptic activity
Presynaptic nerve terminals at the NMJ release neurotransmitters spontaneously, leading to miniature end plate potentials (mEPPs) in post-synaptic muscle fibers [81,82]. To determine the levels of spontaneous synaptic activity at the NMJs lacking Schwann cells, we carried out electrophysiological analyses in acutely isolated diaphragm muscle/phrenic nerve preparations. No spontaneous synaptic activity was detected in CRD-Nrg1 −/− muscles (N = 6 mice, n = 36 cells), since the diaphragm muscles in CRD-Nrg1 −/− muscles were completely denervated due to withdrawal of the nerve terminals. However, spontaneous muscle action potentials were readily observed in CRD-Nrg1 −/− muscles-similar to spontaneous muscle action potentials recorded in control mice-and muscle contraction was observed following spontaneous muscle action potentials (Fig 9F). Spontaneous muscle action potentials were also observed in CRD-Nrg1 −/− Cacnb1 −/− and CRD-Nrg1 −/− Ryr1 −/− muscles, but these mutant muscles failed to contract after firing action potentials (Fig 9F).

Discussion
Terminal Schwann cells are normally required for the assembly of the classic tripartite structure of the NMJs. Mutant mice deficient in type III Neuregulin 1, as well as mutant mice deficient in erbB2 or erbB3, lack Schwann cells, and consequently, lose the NMJs in the diaphragm muscle, after a transient nerve-muscle contact during development [37][38][39][40][41]. In this study, we found that NMJs can be established in CRD-Nrg1 −/− mice, in the absence of Schwann cells, if either synaptic or muscle activity is blocked. These findings are surprising because it shows that bipartite NMJs can be established in vivo, in the absence of Schwann cells. Specifically, neuromuscular synapses are established in the absence of Schwann cells in CRD-Nrg1 −/− mice if one of the following genes is also ablated: Snap25 (neurotransmitter release), Chat (cholinergic transmission), Chrna1 (postsynaptic AChR), Cacnb1 (skeletal muscle DHPRs, the voltage sensor and L-type Ca 2+ channel on the muscle membrane) or Ryr1 (skeletal muscle ryanodine receptors). These genetic manipulations follow a pathway that ultimately leads to muscle activity mediated by (DHPR/RyR) (Fig 10). Importantly, electrophysiological analyses revealed robust synaptic activity in the rescued, Schwann-cell deficient NMJs in CRD-Nrg1 −/− Cacnb1 −/− or CRD-Nrg1 −/− Ryr1 −/− mutant mice. Thus, a blockade of synaptic activity, although sufficient, is not necessary to preserve NMJs that lack Schwann cells. Instead, a blockade of muscle activity mediated by DHRPs and Ryr1 is both necessary and sufficient for preserving NMJs that lack Schwann cells.
How might muscle inactivity contribute to the formation of neuromuscular synapses in the absence of Schwann cells? We can envision several distinct mechanisms. The first possibility is that muscle inactivity (i.e., a blockade of muscle activity) leads to increases in nerve branching, as shown previously [60,61,64,[83][84][85], which might accordingly increase the access of Schwann cell-deficient synapses to trophic factors that may preserve synapses [see review in These genetic rescues reveal a common pathway (B) that ultimately leads to muscle activity mediated by DHPR/Ryr1. Therefore, a blockade of muscle activity is the key to rescuing muscle denervation/synapse loss in the absence of Schwann cells. Together, these genetic manipulations indicate that the blockade of muscle activity prevents muscle denervation and neuromuscular synapse loss caused by CRD-NRG1 deficiencies in mice. https://doi.org/10.1371/journal.pgen.1007857.g010 Genetic rescue of neuromuscular synapse loss ref. [86]]. If this mechanism were to operative, one might expect to observe an increase in nerve branching and hence trophic access in mutants lacking both activity and Schwann cells. However, our data contradict this scenario because, despite marked increases in nerve defasciculation, no increases in nerve branching were detected in Schwann cell-deficient mutants, compared with mutant mice that retain Schwann cells (i.e., compare CRD- ; CRD-Nrg1 -/-Cacnb1 -/versus Cacnb1 -/-; and CRD-Nrg1 -/-Ryr1 -/versus Ryr1 -/mice). These data further indicate that the well-documented increases in nerve branching induced by inactivity [60,61,64,[83][84][85] require the presence of Schwann cells.
A second possibility is that, muscle fibers normally destabilize presynaptic nerve terminals during neuromuscular synaptogenesis. And, this negative and dynamic destabilizing factor(s) requires active nerve and, ultimately, muscle activity. Terminal Schwann cells play crucial roles to antagonize the destabilizing activity and thus to stabilize the NMJ. In the absence of Schwann cells, the destabilizing activity of such factors on presynaptic terminals is unopposed, which leads to synapse loss at the NMJ. In this scenario, blocking either nerve or muscle activity (as in CRD-Nrg1 -/-Snap25 -/-, CRD-Nrg1 -/-Chat -/-, CRD-Nrg1 -/-Chrna1 −/− , CRD-Nrg1 -/-Cacnb1 -/-, or CRD-Nrg1 -/-Ryr1 -/mice), would lead to a blockade of the muscle-derived destabilizing factor(s), which would allow Schwann cell deficient NMJs to form. Identification of the muscle-derived, muscle activity dependent factor(s) that regulate NMJ formation will provide important new insights into the mechanisms underlying neuromuscular synapse formation, maintenance and elimination.
A third, non-mutually exclusive possibility from the second possibility described above, is that Schwann cells may regulate synaptic activity itself. In this instance, the absence of Schwann cells would be predicted to lead to dysregulated synaptic activity. We have previously shown that spontaneous neuromuscular synaptic activity is markedly increased in Cacnb1 −/− and Ryr1 −/− mice, compared with control, likely due to precocious maturation of the NMJs in Cacnb1 −/− and Ryr1 −/− mice [68]. Remarkably, spontaneous synaptic activity is significantly further increased in Schwann cell deficient NMJs (i.e., in CRD-Nrg1 -/-Cacnb1 -/and CRD-Nrg1 -/-Ryr1 -/mice), compared with NMJs in Cacnb1 −/− or Ryr1 −/− mice, respectively. These further increases in spontaneous synaptic activity are likely due to the lack of Schwann cells at the NMJ in CRD-Nrg1 -/-Cacnb1 -/and CRD-Nrg1 -/-Ryr1 -/mice, suggesting that Schwann cells may regulate spontaneous synaptic activity during NMJ synaptogenesis. We do not know how evoked synaptic transmission is affected at the NMJs in CRD-Nrg1 -/-Cacnb1 -/and CRD-Nrg1 -/-Ryr1 -/mice since we were unable to obtain recordings of evoked end-plate potentials from Schwann cell deficient mutant mice due to technical difficulties. During our electrophysiological recordings, we have made numerous attempts to apply suction electrodes to the phrenic nerves in order to deliver electrical stimulation to the nerves in CRD-Nrg1 -/-Cacnb1 -/or CRD-Nrg1 -/-Ryr1 -/mice. However, we were unable to visualize live, unstained phrenic nerves under the microscope in double mutant mice, due to the fact that the phrenic nerves appeared transparent in the absence of Schwann cells. Nevertheless, the marked increases in spontaneous synaptic activity in NMJs lacking Schwann cells (i.e., CRD-Nrg1 -/-Cacnb1 -/versus Cacnb1 -/-; and CRD-Nrg1 -/-Ryr1 -/versus Ryr1 -/-) support the possibility, namely that Schwann cells may protect developing neuromuscular synapses by negatively regulating synaptic activity. Thus, the regulation of synaptic activity by Schwann cells appears to be critical for NMJ formation during development. Consistent with this idea, a recent Schwann cell ablation study demonstrates that these cells continue to regulate synaptic activity postnatally (Barik et al., 2016). These studies of synapses lacking Schwann cells give added support to the studies of intact synapses that demonstrate that the activation of Schwann cells by neural activity regulates presynaptic as well as postsynaptic function [48]. Previous studies have shown that Schwann cells are required for the maintenance of presynaptic structure in developing NMJs [11,14] and play important roles in synapse elimination [15,87]. It is possible that Schwann cells may preserve the NMJs, at least in part, by regulating the levels of synaptic activity (and therefore regulate muscle activity).
The results of the current study show that the level of muscle activity is a key regulator of neuromuscular synapse formation-NMJs can be established even in the absence of Schwann cells if muscle activity mediated by DHPR/Ryr1 is blocked. Our data are consistent with in vitro studies by O'Brien et al., [88,89], which show that excessive activity, either by the topical application of ACh and high [Ca 2+ ] to immature neuromuscular synapses, or by continuous stimulation of the nerve or the muscle, leads to muscle denervation. Importantly, our findings demonstrate that a blockade of muscle activity, instead of neuronal activity, is the key to preserving the developing neuromuscular synapses. We show that neuromuscular synapses are established in the absence of Schwann cells when muscle activity is eliminated. These findings further suggest that skeletal muscle activity might destabilize developing presynaptic nerves and that Schwann cells play crucial roles in counteracting such a destabilizing activity to preserve neuromuscular synapses during development.

Ethics statement
All experimental protocols followed National Institutes of Health Guidelines and were approved by the University of Texas Southwestern Institutional Animal Care and Use Committee. The APN approval number is 2015-101081.

Electron microscopy
Diaphragm muscles with phrenic nerves attached were dissected in Ringer's solution from mouse embryos at E18.5. Samples were then fixed with 1% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4) and kept in the same fixative overnight at 4˚C. After a rinse in 0.1 M phosphate buffer, diaphragm muscles were trimmed into small pieces. Tissues were then post-fixed with 1% osmium tetroxide for 3 hr. on ice, dehydrated through a graded series of ethanol, infiltrated, and polymerized in Epon 812 (Polysciences, Warrington, PA, USA). Prior to embedding in Epon, the phrenic nerve trunks were cut half-way from the muscles, and the nerve trunks were embedded separately. These phrenic nerves were later cross sectioned for EM. Ultrathin sections (70 nm) were prepared and mounted on Formvar-coated grids, then stained with uranyl acetate and lead citrate. Electron micrographs were acquired using a Tecnai electron microscope (Netherlands) operated at 120 kV.

Statistical analysis
End-plate sizes were measured using ImageJ (NIH), with the measurements made blind with respect to the genotypes (at least 3 mice were analyzed in each group). Raw data were pooled and calculated as the mean ± standard error of the mean (SEM). Sigma Plot (version 11.0) was used to analyze statistics. Statistical differences among multiple groups were determined using one-way analysis of variance (ANOVA), followed by a Tukey post hoc test for pairwise multiple comparison between groups.