Muscarinic control of cytoskeleton in perisynaptic glia

Abstract

Similar to astrocytes at CNS synapses, perisynaptic Schwann cells (PSCs) surround nerve terminals at the neuromuscular junction (NMJ). These special teloglial cells are sensitive to neurotransmitters and upregulate glial fibrillary acidic protein (GFAP) when deprived of synaptic activity. We found that activation of muscarinic acetylcholine receptors (mAChRs) at PSCs, but not purinergic (ATP and adenosine) or peptidergic [substance P (SP) and calcitonin gene-related peptide (CGRP)] receptors, prevented this upregulation. When applied onto single PSCs, muscarine evoked Ca2+ responses that fatigued but prevented upregulation of this glial cytoskeletal protein. Application of ATP onto single PSCs evoked Ca2+ signals that showed little fatigue, and GFAP upregulation occurred. Thus, Ca2+ signals alone cannot prevent GFAP upregulation in the PSCs. After blockade of cholinergic receptors by gallamine, neuronal activity was not effective in maintaining low GFAP levels in the perisynaptic glia. Last, immunohistochemistry disclosed mAChRs on PSCs and nearby fibroblasts. Thus, acetylcholine secreted by the nerve terminal acts on the PSCs via mAChRs to regulate GFAP. Cytoskeletal changes may influence perisynaptic glial functions, including growth, remodeling, and modulation of the synapse.

Fig. 1.

Muscarine prevents GFAP upregulation. Shown are simultaneously acquired confocal images from muscles excised and incubated for 6 hr in FRS, then double-labeled using PNA-TRITC (top panels) and anti-GFAP, revealed with goat anti-mouse FITC (bottom panels). Scale bars, 20 μm.A, Two sets of images from separate muscles, each showing three PSCs outlined by PNA (arrows), that express GFAP. The fields also contain GFAP-labeled perisynaptic fibroblasts (asterisks) that have processes contacting the synapse. B, The contralateral muscles toA were incubated in 20 μm muscarine. Note that in the top panel, the two PSCs outlined by PNA (arrows) had no corresponding expression of GFAP. At the synapses shown in the bottom panels, one of two PSCs in the field has upregulated GFAP along most of its entire length (arrowheads). In contrast, the other PSC has little GFAP, mostly in the form of periodic dots, and does not meet the criterion of GFAP positive.

Fig. 2.

ATP fails to prevent GFAP upregulation. Shown are simultaneously acquired confocal images from muscle treated for 6 hr in 50 μm ATP and subsequently labeled using PNA-TRITC (left; appears in red) and anti-GFAP revealed with goat anti-mouse FITC (right; shown ingreen). Note that several PSCs outlined by PNA have expressed GFAP. Scale bar, 20 μm.

Fig. 3.

Ca²⁺ responses evoked by ATP differ from those evoked by muscarine. Averaged peak Ca²⁺ responses evoked by consecutive local application of either ATP or muscarine (see inset). Results are pooled from experiments summarized in Table 2 (also see Figs. 4, 5). Note the attenuation of Ca²⁺ responses that occurred with repetitive application of muscarine. In contrast, ATP, which was applied twice as frequently (15 vs 30 min intervals), evoked Ca²⁺ responses that were relatively resistant to fatigue. Note that the first application of transmitter occurred between 1.5 and 2 hr after muscle excision (see Materials and Methods), and this corresponds to time = 0 min in the plot.

Fig. 6.

Expression of mAChRs at PSCs and fibroblasts. Shown are four pairs of simultaneously acquired confocal images from neuromuscular junctions identified using PNA-TRITC (top panels) and labeled for mAChRs using clone M35 (pan-mAChR) antibodies revealed with FITC-goat anti-mouse IgM (bottom panels). Scale bars, 20 μm. A, Images revealing synapses from two separate normal (quickly excised and fixed) preparations. PSCs were immunoreactive as judged by staining at the level of PSC cell bodies (arrows); fluorescence elsewhere within the PNA outline corresponds to PSC and possibly also nerve terminal staining. Fibroblast-like cells also had mAChR staining.B, Images from two separate muscle preparations previously denervated (2 weeks). PSCs and fibroblasts continued to express mAChRs.

Fig. 7.

Muscarinic receptor antibody induces Ca²⁺ transients in PSCs. Results from an experiment on a PSC from a denervated muscle preparation (A) and plots of normalized Ca²⁺ fluorescence in response to successive (5 min apart) microelectrode application of various agents (B–G). A, Confocal image of a PSC loaded with fluo-3 AM showing fluorescence attributable to resting levels of Ca²⁺ and fluorescence after application of M35 antibody. Scale bar, 20 μm. B, Application of ascites fluid containing nonspecific IgM did not evoke a Ca²⁺ response. C–E, Successive additions of M35 pan-muscarinic antibody (200 nm) induced a Ca²⁺ signal that became attenuated in amplitude.F, Subsequent addition of muscarine (40 nm) failed to evoke a Ca²⁺ response. G, The PSC was still responsive to ATP (50 μm).