Distinct muscarinic acetylcholine receptor subtypes contribute to stability and growth, but not compensatory plasticity, of neuromuscular synapses

Abstract

Muscarinic acetylcholine receptors (mAChRs) modulate synaptic function, but whether they influence synaptic structure remains unknown. At neuromuscular junctions (NMJs), mAChRs have been implicated in compensatory sprouting of axon terminals in paralyzed or denervated muscles. Here we used pharmacological and genetic inhibition and localization studies of mAChR subtypes at mouse NMJs to demonstrate their roles in synaptic stability and growth but not in compensatory sprouting. M(2) mAChRs were present solely in motor neurons, whereas M(1), M(3), and M(5) mAChRs were associated with Schwann cells and/or muscle fibers. Blockade of all five mAChR subtypes with atropine evoked pronounced effects, including terminal sprouting, terminal withdrawal, and muscle fiber atrophy. In contrast, methoctramine, an M(2/4)-preferring antagonist, induced terminal sprouting and terminal withdrawal, but no muscle fiber atrophy. Consistent with this observation, M(2)(-/-) but no other mAChR mutant mice exhibited spontaneous sprouting accompanied by extensive loss of parental terminal arbors. Terminal sprouting, however, seemed not to be the causative defect because partial loss of terminal branches was common even in the M(2)(-/-) NMJs without sprouting. Moreover, compensatory sprouting after paralysis or partial denervation was normal in mice deficient in M(2) or other mAChR subtypes. We also found that many NMJs of M(5)(-/-) mice were exceptionally small and reduced in proportion to the size of parental muscle fibers. These findings show that axon terminals are unstable without M(2) and that muscle fiber growth is defective without M(5). Subtype-specific muscarinic signaling provides a novel means for coordinating activity-dependent development and maintenance of the tripartite synapse.

Figure 1.

Atropine causes diverse and severe effects on NMJ organization. Confocal images of LAL muscles treated twice daily for 7 d with saline or atropine. Preterminal and terminal axons were immunolabeled for neurofilaments (NF) and a synaptic vesicle protein, SV2 (green), nAChRs were labeled for α-bungarotoxin (red), and Schwann cells were labeled for S100 (blue). A, Low-magnification images of saline- or atropine-treated LAL muscles. In saline-treated muscles, axon terminals (green) precisely overlap brightly labeled nAChRs (red), making NMJs appear yellow. In atropine-treated muscles, axon terminals sprout (arrows) or retract (arrowheads), and postsynaptic nAChRs are faintly labeled at some NMJs. B, High-magnification images of NMJs showing diverse patterns of synaptic disorganization evoked by atropine. Saline-treated NMJs (e.g., junction 1) show precise alignment of axon terminals (green), tSCs (blue), and postsynaptic clusters of nAChRs (red). In atropine-treated muscles, at some NMJs (e.g., junction 2), nerve terminals extended sprouts (white arrows) that elongated along activated tSC processes (blue arrows), whereas nAChRs are unaffected. Another set of NMJs (e.g., junction 3) demonstrated selective, profound loss of postsynaptic nAChRs. Other NMJs (e.g., junction 4) displayed selective, complete loss of axon terminals and abnormally quiescent tSCs that formed no processes even in the absence of axon terminals. C–F, Quantification of diverse effects evoked by atropine at various doses. C, The percentage of NMJs exhibiting terminal sprouts. D, The percentage of NMJs exhibiting uniform, profound loss of postsynaptic nAChRs. E, The percentage of NMJs exhibiting selective loss of terminal arbors. F, The average diameter of muscle fibers. Atropine-treated muscle fibers are substantially atrophied. *p < 0.01. Scale bars: A, 30 μm; B, 10 μm.

Figure 2.

Subtype-specific inhibition of mAChRs elicits subsets of atropine-evoked effects. Confocal views of representative synapses in the LAL muscles treated twice daily for 7 d with methoctramine or 4-DAMP. A, Methoctramine, an M_2/4 receptor-preferring antagonist, induces terminal sprouting, tSC activation, and partial terminal loss. Note terminal sprouts (arrowheads; green) associated with leading tSC processes (arrowheads; blue). At some NMJs with sprouting (bottom), axon terminals are partially retracted as evidenced by clusters of nAChRs (arrow; red) unoccupied by terminal branches (arrow; green). B, 4-DAMP, which blocks the odd-numbered mAChRs and M₄ receptors with high affinity, induces dramatic dying back of nerve terminal accompanied by abnormally quiescent tSCs. Retracting axons are present at the vacant endplates (arrowheads; green) or along the preterminal Schwann cells (arrows; green). Note that tSCs are abnormally quiescent as evidenced by bright S100 labeling without process extension (blue arrows). Postsynaptically, muscle fibers are normal and there is no loss of nAChRs or fiber atrophy. Schwann cells are presented in black and white (single images) as well as in blue (merged images) to illustrate better fine processes formed by tSCs, a hallmark of activated tSCs. Scale bar, 10 μm. NF, Neurofilaments.

Figure 3.

Spontaneous sprouting and terminal loss at M₂^−/− NMJs. A, Low-magnification confocal views of M₂^+/+ and M₂^−/− NMJs. Terminal arbors of many M₂^−/− NMJs (arrows; green) do not have the compact oval shape of wild-type NMJs but display unusually long and dispersed branches in contact with fragmented patches of nAChRs. B, High-magnification view of M₂^−/− NMJs. Terminal sprouts form varicosities directly apposed to islands of nAChR patches (arrowheads), whereas parental terminal branches that covered original synaptic contacts are retracted, leaving abandoned, faintly labeled branches of parental clusters of nAChRs (e.g., area marked by an asterisk). Some M₂^−/− NMJs with no sprouting exhibit vacant branches of nAChRs and tSCs that are unoccupied by terminal branches (arrows in bottom), indicating that terminal loss precedes terminal sprouting in M₂^−/− muscles. Scale bars: A, 30 μm; B, 10 μm. NF, Neurofilaments.

Figure 4.

Unlike paralysis-induced sprouting, sprouting in M₂^−/− NMJs is accompanied by parental terminal loss. Confocal views of a representative junction from an M₂^−/− LAL muscle (top) and a junction from an LAL muscle paralyzed for 7 d by botulinum toxin. Original synaptic contacts are identified by the presence of preterminal axons (arrowheads), terminal sprouts (arrows), and compact oval-shaped, postsynaptic clusters of nAChRs (asterisks in nAChR panels). Note that M₂^−/− NMJs display a cluster of nAChRs completely unoccupied by axons at original synaptic contacts (asterisks, top). In contrast, nerve terminals in paralyzed muscles sprout but maintained parental terminal branches (asterisks, bottom). Scale bars, 10 μm.

Figure 5.

A subset of M₂^−/− NMJs shows markedly abnormal synaptic function. A, Mean endplate current amplitude for LAL muscles from seven M₂^−/− and four M₂^+/+ mice. Approximately 17 muscle fibers were recorded in each mouse. In two of the M₂^−/− mice, endplate current amplitude was reduced by one-third relative to M₂^+/+ mice. B, The time constant of endplate current decay was greatly prolonged in the two affected M₂^−/− mice. C, Representative EPCs and MEPCs for a wild-type endplate and an endplate from an affected M₂^−/− mouse. EPCs and MEPCs from unaffected M₂^−/− NMJs were indistinguishable from M₂^+/+ NMJs.

Figure 6.

Nerve terminal shifting and fragmentation in M_2/4^−/− dKO NMJs. Confocal views of LAL muscles of 3-week-old (A, B) and 3-month-old (C, D) M_2/4^−/− dKO mice. Muscles were labeled as described in Figure 1. A, Portions of endplates are commonly unoccupied by terminal arbors (e.g., NMJs marked by arrows, asterisk area in A). B, Some terminal arbors appear to have shifted, leaving previous endplates completely abandoned (e.g., junctions 1 and 2 in B). The synaptic morphology of abandoned endplates closely resembles that of shifted terminal arbors. C, An example of 3-month-old M_2/4^−/− NMJs exhibiting extensively fragmented clusters of postsynaptic nAChRs. D, Relative distribution of NMJs displaying particular numbers of nAChR patches in M_2/4^+/+ and M_2/4^−/− dKO mice. Scale bars: A, B, 20 μm; C, 10 μm.

Figure 7.

Parallel reduction of muscle fiber and synaptic size in M₅^−/− muscles. LAL muscles of adult M₅^+/+ and M₅^−/− mice were labeled as described in Figure 1. A, Low-magnification confocal views show numerous unusually small NMJs (arrows) in M₅^−/− muscles. Inset denotes an exceptionally small M₅^−/− NMJ comprising a terminal bouton and a tiny patch of nAChRs (arrows in inset). B, At some small M₅^−/− NMJs, postsynaptic clusters of nAChRs are fragmented and only faintly labeled (arrowheads), indicating postsynaptic disassembly. C, Comparative analysis of synaptic and muscle fiber size in M₅^+/+ and M₅^−/− muscles. Many muscle fibers and NMJs in M₅^−/− muscles, except slow-twitch soleus muscles, are substantially smaller than those of M₅^+/+ muscles. In addition, synaptic size is highly correlated with the size of parental muscle fibers, indicating that muscle fiber growth is the primary limitation in M₅^−/− muscles. Scale bars: A, 100 μm; B, 20 μm. STM, Sternomastoid muscle; SOL, soleus muscle; NF, neurofilaments.

Figure 8.

Paralysis initiates normal sprouting in mAChR KO mice. LAL muscles of wild-type (+/+), M_1/3^−/−, M_2/4^−/−, and M₅^−/− mice were paralyzed for 7 d and labeled as described in Figure 1. A, Representative junctions of +/+, M_2/4^−/−, and M₅^−/− NMJs exhibiting sprouting. Paralysis induced normal sprouting in M_1/3^−/− (data not shown) and M_2/4^−/− NMJs. In M₅^−/− NMJs, terminal sprouts form numerous secondary and tertiary branches, elongating far more extensively than those observed at +/+ or M_1/3^−/− or M_2/4^−/− NMJs. B–E, Quantitative and comparative analysis of the sprouting in terms of the percentage of the junctions displaying sprouts (B), the number of primary branches at NMJs with sprouts (C), relative distribution of NMJs with different lengths of sprouts (D), and the average length of sprouts (E). Terminal sprouts become exceptionally lengthy in M₅^−/− NMJs attributable to elongation of secondary and tertiary branches. The initiation of paralysis-induced sprouting, however, was normal in M_1/3^−/−, M_2/4^−/−, and M₅^−/− NMJs. n = 3 or more LAL muscles, >100 NMJs per muscle were analyzed. **p < 0.0001. Scale bar, 30 μm.

Figure 9.

Localization of mAChR subtypes at NMJs. A, Although an anti-M₂ antibody labels axon terminals, tSCs, and postsynaptic gutters in wild-type NMJs, nerve terminal-associated labeling (arrow, inset) disappears in M₂^−/− NMJs, whereas labeling associated with tSC cell bodies and postsynaptic gutters remains. Thus, M₂ mAChRs appear to be expressed selectively in motor neurons because only this binding is specific. B, Laser-assisted microdissection of spinal motor neurons (MN) and NMJs. C, RT-PCR analyses show that motor neurons express only M₂ mAChRs, whereas Schwann cells and/or muscle fibers at NMJs are likely to express multiple odd-numbered receptors (M₁, M₃, M₅). Scale bars: A, 10 μm; B, 50 μm. IR, Immunoreactivity; NF, neurofilaments.