Acetylcholine negatively regulates development of the neuromuscular junction through distinct cellular mechanisms

Abstract

Emerging evidence suggests that the neurotransmitter acetylcholine (ACh) negatively regulates the development of the neuromuscular junction, but it is not clear if ACh exerts its effects exclusively through muscle ACh receptors (AChRs). Here, we used genetic methods to remove AChRs selectively from muscle. Similar to the effects of blocking ACh biosynthesis, eliminating postsynaptic AChRs increased motor axon branching and expanded innervation territory, suggesting that ACh negatively regulates synaptic growth through postsynaptic AChRs. However, in contrast to the effects of blocking ACh biosynthesis, eliminating postsynaptic AChRs in agrin-deficient mice failed to restore deficits in pre- and postsynaptic differentiation, suggesting that ACh negatively regulates synaptic differentiation through nonpostsynaptic receptors. Consistent with this idea, the ACh agonist carbachol inhibited presynaptic specialization of motorneurons in vitro. Together, these data suggest that ACh negatively regulates axon growth and presynaptic specialization at the neuromuscular junction through distinct cellular mechanisms.

Fig. 1.

Postsynaptic transmission deficiency, muscle hyperinnervation, and increased motor neuron number in AChRα1 mutant mice. (A) Spontaneous miniature endplate potentials (MEPPs) were observed in control (^+/+) diaphragm but not in AChRα1 mutant (AChRα1^−/−) diaphragm. One MEPP is expanded below. (B) Nerve-evoked endplate potentials (EPPs) were observed in control but not in mutant muscle. (C–F) E17.5 whole-mount diaphragm muscles from controls and AChRα1 mutants were immunostained with antineurofilament (anti-NF) antibodies (green). Both (C and D) low- and (E and F) high-power magnifications showed that the phrenic nerve is highly branched in mutant muscle (D and F). (Scale bars: 200 μm for C and D; 100 μm for E and F.) (G) A similar increase in the number of lumbar motor neurons was observed in ChAT (ChAT^−/−) and AChRα1 mutants compared with controls. **, P < 0.01.

Fig. 2.

Presynaptic nerve terminals differentiate in AChRα1 mutants. Control (^+/+; A, C, and E) and AChRα1 mutant (AChRα1^−/−; B, D, and F) E17.5 whole-mount diaphragm muscles were immunostained with antisynapto-physin antibodies (A, B, and green in E and F) and costained with Texas Red-conjugated α-bungarotoxin (C, D, and red in E and F). Synaptophysin immunoreactivity is accumulated at the nerve terminals (arrows in A) and colocalized with receptor clusters in control diaphragms. Mutant diaphragms lack receptor clusters but maintain synaptophysin accumulations at the nerve terminals. (Scale bar: 50 μm.)

Fig. 3.

Absence of presynaptic differentiation in AChRα1/agrin double mutants. (A) Diaphragm muscles from control, agrin-deficient (AGD), AChRα1, and AChRα1/AGD mutants were immunostained with antibodies against synaptophysin. Presynaptic specialization is not observed in either AGD single or AChRα1/AGD double mutants. (Scale bar: 100 μm.) (B) Absence of AChE clusters in AGD and AChRα1/AGD mutants. (Scale bar: 200 μm.) (C) Summary analysis of accumulation of synaptic vesicles in control, AGD, AChRα1^−/−, AChRα1^−/−, AGD, and ChAT^−/−, AGD mutants.

Fig. 4.

Induced FGF mRNA expression by agrin in C2C12 myotubes and decreased FGF mRNA expression in agrin mutant muscle. (A) RT-PCR showed that FGF7 and FGF9 mRNAs are expressed in E18.5 diaphragm muscles. The results illustrate that the primer sets used for the RT-PCR experiments are specific because no signal is detected when reverse transcriptase (RT) is omitted (No RT) in the reaction. For the simplicity of presentation, we cropped the original scan to show only FGF7 and FGF9 expression in control samples. (B) Real-time quantitative RT-PCR showed that agrin induces expression of FGF7 and FGF9 mRNA in C2C12 myotube cultures. Expression level of control without agrin treatment is set as 100%. The results were expressed as percentage of control (n = 4). *, P < 0.05; **, P < 0.01. (C) RNA was isolated from E18.5 controls or agrin mutant muscles for real-time quantitative RT-PCR analysis. Expression level of control embryos is set as 100%. The results were expressed as percentage of control embryos. The results showed reduced levels of FGF7 and FGF9 mRNA in muscle from agrin mutants, relative to controls (n = 4). *, P < 0.05; **, P < 0.01.

Fig. 5.

Dispersion of FGF-induced aggre-gates of synaptic vesicles by the ACh agonist carbachol in ES-derived motor neurons. (A) Compared with control cultures of ES cell-derived motor neurons (Control), treatment with the ACh agonist CCh did not induce aggregation of synaptic vesicles in motor neurons (CCh). ES-derived motor neurons treated with FGF9 exhibited synaptic vesicle-rich varicosities (FGF9; arrows). CCh destabilized FGF-induced aggregation of synaptic vesicles (FGF9/CCh). (B) Quantitative analysis of the effect of CCh in the maintenance of synaptic varicosity (n = 4). **, P < 0.01.