Neuromuscular NMDA Receptors Modulate Developmental Synapse Elimination

Abstract

At birth, each mammalian skeletal muscle fiber is innervated by multiple motor neurons, but in a few weeks, all but one of those axons retracts (Redfern, 1970) and differential activity between inputs controls this phenomenon (Personius and Balice-Gordon, 2001; Sanes and Lichtman, 2001; Personius et al., 2007; Favero et al., 2012). Acetylcholine, the primary neuromuscular transmitter, has long been presumed to mediate this activity-dependent process (O'Brien et al., 1978), but glutamatergic transmission also occurs at the neuromuscular junction (Berger et al., 1995; Grozdanovic and Gossrau, 1998; Mays et al., 2009). To test the role of neuromuscular NMDA receptors, we assessed their contribution to muscle calcium fluxes in mice and tested whether they influence removal of excess innervation at the end plate. Developmental synapse pruning was slowed by reduction of NMDA receptor activation or expression and by reduction of glutamate production. Conversely, pruning is accelerated by application of exogenous NMDA. We also found that NMDA induced increased muscle calcium only during the first 2 postnatal weeks. Therefore, neuromuscular NMDA receptors play previously unsuspected roles in neuromuscular activity and synaptic pruning during development.

Significance statement: In normal adult muscle, each muscle fiber is innervated by a single axon, but at birth, fibers are multiply innervated. Elimination of excess connections requires neural activity; because the neuromuscular junction (NMJ) is a cholinergic synapse, acetylcholine has been assumed to be the critical mediator of activity. However, glutamate receptors are also expressed at the NMJ. We found that axon removal in mice is slowed by pharmacological and molecular manipulations that decrease signaling through neuromuscular NMDA receptors, whereas application of exogenous NMDA at the NMJ accelerates synapse elimination and increases muscle calcium levels during the first 2 postnatal weeks. Therefore, neuromuscular NMDA receptors play previously unsuspected roles in neuromuscular activity and elimination of excess synaptic input during development.

Keywords: competition; glutamate; neuromuscular junction; polyneuronal; synapse elimination.

Figure 1.

Reduction of NMDA receptor activity slows synapse elimination. A, Pharmacological reduction of NMDA and AMPA receptor activity with AP5 and CNQX between P4 and P11. Left, Whole-mount NMJ immunolabeling in anterior tibialis (AT) muscle of P11 pups. Asterisks show polyinnervated end plates. Red, Rhodamine-α-bungarotoxin labels ACh receptors. Green, Antibodies SV2 and 2H3 label synaptic vesicles and neurofilaments. Right, Means and distributions of percentage of multiply innervated muscles. Open symbols indicate control; filled symbols, treated; horizontal lines, means. The treated muscles contained significantly more multiply innervated fibers than the contralateral internal control muscles (EDL: 22 ± 3% control limb vs 52 ± 4% treated limb, p = 0.00006, n = 12, 12; soleus: 35 ± 3% control limb vs 53 ± 2% treated limb, p = 0.00001, n = 11, 13). B, Vivo-morpholino downregulation of GluN1 expression between P4 and P11. A vivo-morpholino was pressure injected into the AT muscle end plate band in P4 pups. Conventions are as in A. The AT muscle demonstrated significant retention (9 ± 5% internal control limb vs 24 ± 8% treated limb, p = 0.001, n = 8, 8). Scale bars, 50 μm.

Figure 2.

GCPII antagonist slows synapse elimination. A, Model of glutamate production and induction of calcium level influx at the NMJ. The motor neuron terminal (gray) releases ACh and NAAG. GCPII on the Schwann cell (cyan) cleaves NAAG into NAA and glutamate. Glutamate then activates AMPA and NMDA receptors on the muscle (orange), leading to further calcium influx. B, Presynaptic expression of the enzyme GCPII during the period of synapse elimination. Whole mount of a P11 EDL muscle immunolabeled for GCPII, AChR, and terminal Schwann cells using the antibody GCP-04 against GCPII (green), rhodamine-α-bungarotoxin (red), and S100 against Schwann cells (blue). Scale bar, 25 μm. C, GCPII antagonist slows synapse elimination by reducing glutamate at the NMJ. Muscles treated with 2-PMPA-infused Elvax contained higher proportions of multiply innervated fibers than internal control muscles (EDL: 4 ± 1% control limb vs 15 ± 2% treated limb, p = 0.0004, n = 9, 10; soleus: 9 ± 2% control limb vs 26 ± 3% treated limb, p = 0.004, n = 11, 11). Scale bar, 50 μm.

Figure 3.

Exogenous NMDA accelerates removal of excess innervation. Left, Examples of multiply innervated (asterisks) and singly innervated end plates in control and NMDA-treated muscles at P8. Scale bar, 50 μm. Right, NMDA-treated muscles at P8 had significantly fewer multiply innervated fibers than control. Open symbols indicate control; filled symbols, treated (EDL: 48 ± 2%, control limb vs 38 ± 4% vs NMDA-treated limb, p = 0.05, n = 8, 9; soleus: 63 ± 2%, control limb vs 49 ± 4%, NMDA-treated limb, p = 0.014, n = 7, 8).

Figure 4.

NMDA raises muscle calcium levels in early postnatal muscle. A, Ca²⁺ response to bath-applied NMDA (200 μm) in an EDL of a P11 CD-1 pup. AP5 (200 μm) blocked most of the response to NMDA. Scale bar, 50 μm. B, NMDA significantly evoked Ca²⁺ responses in P4–P14 pups compared with control. AP5 blocked the NMDA Ca²⁺ response (relative brightness (ΔF/F); P4–P7 NMDA, 78 ± 30, AP5, 43 ± 24, control, 24 ± 19, p < 0.001-; P11–P14 NMDA, 95 ± 24, AP5, 43 ± 10, control, 40 ± 8, p < 0.001; P28 NMDA, 50 ± 20, AP5, 35 ± 17, control, 29 ± 10, p = 0.104; adult NMDA, 56 ± 29, AP5, 47 ± 2, control, 49 ± 7, p = 0.789, ANOVA; n = 7, 6, 6, 4).