We raised a novel antibody (code-numbered 2166) and used immunostaining to study a hitherto neglected cell type located at neuromuscular junctions.

Mice and rats were humanely killed by cervical dislocation and standard techniques were used for immunostaining and confocal microscopy of isolated whole-mount preparations of triangularis sterni and/or transverse sections of hind limb muscles.

The 2166-positive cells exhibit a distinct morphology, and screening against a panel of antibodies showed an immunohistochemical profile different from those of terminal Schwann cells (tSC) and muscle satellite cells. The identity of junctional 2166-positive cells as fibroblasts was confirmed by immunostaining for prolyl-4-hydroxylase. Although perijunctional fibroblasts have been described before (Gatchalian et al. 1989), immunohistochemistry using the 2166 antibody suggests that these junctional cells represent a distinct sub-population of fibroblast. Previous studies identified tSC as the ones at neuromuscular junctions expressing neuregulin GGFIIβ3 (Trinidad et al. 2000). However, immunostaining with HM-24 antibody shows that 2166-positve junctional fibroblasts express this neuregulin isoform and terminal Schwann cells do not. The junctional fibroblast population arises from a more diffuse, larger population during postnatal development. To test for a possible functional role, we performed partial denervation of adult mouse triangularis sterni muscles under ketamine-xylazine anaesthesia (86 and 13 mg kg^-1, respectively, I.P.). One day after denervation, junctional fibroblasts had proliferated and extended processes between the motor endplates of adjacent fibres, while terminal Schwann cells remained in an inactivated state on the denervated endplates. By 3 days post denervation, tSC became activated (identified by nestin protein expression; Kang et al. 2001) and extended processes which associated with the pre-formed junctional fibroblast sprouts.

These observations suggest that reinnervation following partial denervation may not be completely dependent on terminal Schwann cells (Son et al. 1996), and that junctional fibroblasts provide cellular bridges for denervation-induced terminal Schwann cell sprouting. We propose that junctional fibroblasts are a specific cellular component, with possible roles in development, maintenance and plasticity of neuromuscular synapses.

This work was supported by the Wellcome Trust. Dr J.B. Cohen kindly donated HM-24 antibody.