Synapse-glia interactions at the mammalian neuromuscular junction

Abstract

Perisynaptic Schwann cells (PSCs) play critical roles in regulating and stabilizing nerve terminals at the mammalian neuromuscular junction (NMJ). However, although these functions are likely regulated by the synaptic properties, the interactions of PSCs with the synaptic elements are not known. Therefore, our goal was to study the interactions between mammalian PSCs in situ and the presynaptic terminals using changes in intracellular Ca(2+) as an indicator of cell activity. Motor nerve stimulation induced an increase in intracellular Ca(2+) in PSCs, and this increase was greatly reduced when transmitter release was blocked. Furthermore, local application of acetylcholine induced Ca(2+) responses that were blocked by the muscarinic antagonist atropine and mimicked by the muscarinic agonist muscarine. The nicotinic antagonist alpha-bungarotoxin had no effect on Ca(2+) responses induced by acetylcholine. Local application of the cotransmitter ATP induced Ca(2+) responses that were unaffected by the P2 antagonist suramin, whereas local application of adenosine induced Ca(2+) responses that were greatly reduced by the A1 receptor antagonist 8-cyclopentyl-1,3-dimethylxanthine (CPT). However, the presence of the A1 antagonist in the perfusate did not block responses induced by ATP. Ca(2+) responses evoked by stimulation of the motor nerve were reduced in the presence of CPT, whereas atropine almost completely abolished them. Ca(2+) responses were further reduced when both antagonists were present simultaneously. Hence, PSCs at the mammalian NMJ respond to the release of neurotransmitter induced by stimulation of the motor nerve through the activation of muscarinic and adenosine A1 receptors.

Fig. 1.

Identification of PSCs at the mammalian NMJ.A, False color confocal image of a mammalian NMJ loaded with the fluorescent Ca²⁺ indicator fluo-3 AM. The range of false colors reflects the level of Ca²⁺level, where blue indicates a low level of Ca²⁺, and green–red indicates a high level. Two PSCs (arrows) at rest before local application of muscarine. B, The same PSCs as inA, after application of muscarine (20 μm). Note the rise in fluorescence induced by muscarine. C, Labeling of PSCs of the same NMJ with anti-S100, a calcium binding protein used as a general marker for Schwann cells. Note that the two cells that responded to muscarine were also labeled by the S100 antibody. Scale bar, 20 μm.

Fig. 2.

Nerve-evoked Ca²⁺ responses in PSCs. Ca²⁺ responses obtained using the fluorescent Ca²⁺ indicator fluo-3 AM and monitored using a Bio-Rad 600 confocal microscope. Blue indicates low level of calcium, and red indicates a high level.A, Changes in fluorescence (%ΔF/F) before, during (bar), and after motor nerve stimulation (50 Hz, 30 sec). Note that relative increase in Ca²⁺fluorescence to a second train of stimuli applied 20 min later (break in the x-axis) was much reduced. B, Ca²⁺ response in the same PSC as inA, before (1), during (2), and after (3–5) transmitter release induced by repetitive stimulation of the motor nerve (50 Hz, 30 sec). The corresponding time at which each image was taken is illustrated on the graph in A with the corresponding number of the figure above. C, Changes in fluorescence (%ΔF/F) before, during (bar), and after motor nerve stimulation (50 Hz, 30 sec) in the presence of ω-conotoxin MVIIC (1 μm). Note that the increase in the intensity of the fluorescence was strongly reduced when transmitter release was blocked by ω-conotoxin MVIIC, a P/Q-type Ca²⁺ channel blocker. Different preparation than A and B.D, Mean ± SEM of amplitude of Ca²⁺ responses in PSCs elicited by nerve stimulation in the absence and in the presence of ω-conotoxin MVIIC (p = 0.001; Student's pairedt test). Scale bar: B, 10 μm.

Fig. 3.

Ca²⁺ responses of PSCs to local application of ACh. A, Changes in fluorescence over time caused by the local application of ACh (20 μm) for 200 msec in normal physiological solution. Application of ACh caused a Ca²⁺ response in all PSCs studied. A second application 20 min later (break in the x-axis) induced smaller changes in fluorescence. B, Changes in fluorescence caused by the local application of ACh (20 μm, 200 msec) in the presence (solid line) and in the absence (dashed line) of external Ca²⁺ for the same PSCs. Ca²⁺ was replaced by 5 mm Mg²⁺. Note that ACh application induced larger changes in fluorescence in the absence of external Ca²⁺. C, Mean ± SEM of relative changes in Ca²⁺ responses induced by a first and a second application of ACh (20 μm) and for application of ACh in absence of external Ca²⁺.

Fig. 4.

Ca²⁺ responses of PSCs to local application of muscarine. A, Changes in fluorescence caused by the local application of acetylcholine (20 μm, 200 msec) in the presence of α-BuTx (20 μm). The increase in the intensity of fluorescence induced by the local application of ACh was unaffected by α-BuTx. B, Comparison of mean ± SEM of Ca²⁺ responses induced by ACh alone, in the presence of α-BuTx, and in the presence of atropine. C, Changes in fluorescence over time caused by the local application of muscarine (20 μm) for 200 msec in normal physiological solution. Application of muscarine caused a Ca²⁺ response in all PSCs studied. A second application 20 min later (break in the x-axis) induced smaller changes in fluorescence. D, Changes in fluorescence caused by the local application of muscarine (20 μm) in the presence (solid line) and in the absence (dashed line) of external Ca²⁺ for the same PSCs. Ca²⁺ was replaced by 5 mm Mg²⁺. Note that muscarine induced larger changes in fluorescence in the absence of external Ca²⁺. E, Comparison of mean ± SEM of Ca²⁺ responses induced by a first and a second application of muscarine (left) and application of muscarine in presence and absence of external Ca²⁺ (right).

Fig. 5.

Ca²⁺ responses of PSCs to local application of ATP and adenosine. A, Changes in fluorescence over time caused by the local application of ATP (20 μm) for 200 msec in normal physiological solution. Application of ATP caused a Ca²⁺ response in all PSCs studied. A second application 20 min later (break in thex-axis) induced smaller changes in fluorescence.B, Changes in fluorescence over time caused by the local application of adenosine (20 μm) for 200 msec in normal physiological solution. Application of adenosine caused a Ca²⁺ response in all PSCs studied. A second application 20 min later (break in the x-axis) induced smaller changes in fluorescence. C, Changes in fluorescence caused by the local application of ATP (20 μm, 200 msec) in the presence (solid line) and in the absence (dashed line) of external Ca²⁺ for the same PSCs. Note that ATP induced larger changes in fluorescence in the absence of external Ca²⁺. D, Changes in fluorescence caused by the local application of adenosine in the presence (solid line) and in the absence (dashed line) of external Ca²⁺ for the same PSCs. Note that adenosine induced larger changes in fluorescence in the absence of external Ca²⁺. E, Comparison of mean ± SEM of Ca²⁺ responses induced by a first and a second application of ATP (left) and the application of ATP in the presence and in the absence of external Ca²⁺ (right).F, Comparison of mean ± SEM of Ca²⁺ responses induced by a first and a second application of adenosine (left) and application of adenosine in the presence and in the absence of external Ca²⁺ (right).

Fig. 6.

Ca²⁺ responses of PSCs to local application of ATP and adenosine in presence of purinergic antagonists.A, Changes in fluorescence caused by the local application of ATP before and after (break in thex-axis) a 20 min perfusion with suramin (100 μm), a nonspecific P2 antagonist. Note that the increase in the intensity of fluorescence induced by the local application of ATP was unaffected by the presence of suramin. B, Changes in fluorescence caused by the local application of adenosine before and after a 20 min perfusion with CPT (10 μm).C, Changes in fluorescence in four PSCs of an NMJ caused by the local application of ATP in the presence of the A1 receptor antagonist CPT (20 μm). Inset, False color confocal images of the four PSCs at rest (Rest), at the peak of the Ca²⁺ response (Peak), and after recovery (Recov). Note that the increase in the intensity of fluorescence induced by the local application of ATP was unaffected by the presence of CPT. Scale bar, 10 μm.

Fig. 7.

Nerve-evoked Ca²⁺ responses in PSCs in presence of atropine and CPT. A, Changes in fluorescence of a PSC before, during (bar), and after repetitive motor nerve stimulation (50 Hz, 30 sec) after 20 min perfusion with atropine (20 μm), a muscarinic antagonist. The boxed gray zone illustrates the mean ± SEM of nerve-evoked Ca²⁺ responses obtained from the 10 PSCs in absence of any antagonists (Fig. 2A).B, Changes in fluorescence of a PSC before, during (bar), and after repetitive motor nerve stimulation (50 Hz, 30 sec) after 20 min perfusion with CPT (10 μm), an A1 antagonist. The gray zone illustrates the mean ± SEM of nerve-evoked Ca²⁺ responses obtained from the 10 cells in absence of any antagonists. C,Changes in fluorescence of a PSC before, during (bar), and after repetitive motor nerve stimulation (50 Hz, 30 sec) after 20 min perfusion with CPT (10 μm) and atropine (20 μm). Note that the repetitive stimulation of the motor nerve did not induce any Ca²⁺ response in the PSC. After a recovery period of 5 min, ATP (20 μm) was locally applied on the PSC, which elicited a Ca²⁺response.