Palisade endings in extraocular eye muscles revealed by SNAP-25 immunoreactivity

Abstract

Palisade endings form a cuff of nerve terminals around the tip of muscle fibres. They are found only in extraocular muscles, but no definite evidence for their role in eye movements has been established. Palisade endings have been reported in all species so far investigated except the rat. In this study we demonstrate that antibodies against SNAP-25, the synaptosomal associated protein of 25 kDa, reliably visualize the complete motor, sensory and autonomic innervation of the extraocular muscles in human, monkey and rat. The SNAP-25 antibody can be combined with other immunofluorescence procedures, and is used here to study properties of palisade endings. With SNAP-25 immunolabelling putative palisade endings are identified in the rat for the first time. They are not well branched, but fulfil several criteria of palisade endings, being associated with non-twitch fibres as shown by double labelling with 'myosin heavy chain slow-twitch' antibodies. The putative palisade endings of the rat lack alpha-bungarotoxin binding, which implies that these synapses are sensory. If palisade endings are sensory then they could function as an eye muscle proprioceptor. They seem to be a general feature of all vertebrate eye muscles, unlike the other two extraocular proprioceptors, muscle spindles and Golgi tendon organs, the presence of which varies widely between species.

Fig. 1

Laser scanning photomicrographs of longitudinal sections of extraocular eye muscles. (A–C) Double immunofluorescence labelling of a rat inferior rectus muscle with antibodies for SNAP-25 (red) and NF-M (green). A motor nerve is entering the eye muscle and branches into several ‘en plaque’ endings. SNAP-25 antibodies visualize both the nerve fibres and the ‘en plaque’ endings (A) whereas NF-M immunoreactivity is present only in the majority of the nerve fibres (B). This is clearly shown in the overlay of both markers in C. (D–I) Double immunofluorescence labelling of a monkey inferior rectus muscle with antibodies for SNAP-25 (red) and synaptophysin (green). Detailed view of an ‘en plaque’ ending on a twitch muscle fibre (D–F) and ‘en grappe’ endings (arrows) on a multiply innervated muscle fibre (G–I). In both types of endings, SNAP-25 and synaptophysin immunoreactivity clearly outline the morphology of these endings and are co-localized in all terminals, as indicated in the overlays (F,I). Note that the motor nerve giving rise to the ‘en grappe’ endings lacks synaptophysin and is only visualized with SNAP-25 antibodies (H,I). Scale bar, 50 μ.

Fig. 2

Photomicrographs of neural structures in longitudinal sections of sheep and human extraocular eye muscles labelled with SNAP-25 antibodies and counterstained with hemalaun. (A) Identified muscle spindle in a sheep inferior rectus muscle. With SNAP-25 immunolabelling, a nerve fibre which enters the collagenous capsule is visualized. On the intrafusal muscle fibres an annulospiral ending which winds around the fibres is clearly SNAP-25 positive (arrow). (B) Blood vessels in the tendon of a human extraocular muscle. Using SNAP-25 antibodies, a fine network of varicose nerve fibres surrounding the lumen (L) of the vessels could be identified. (C) A palisade ending at the myotendinous junction of a human extraocular eye muscle. SNAP-25 antibodies completely label the palisade ending (PE), the axon which enters from the tendon (td) as well as the bunch of branches and small terminals which terminate on a muscle fibre. Scale bar, 50 μm.

Fig. 3

Laser scanning photomicrographs of longitudinal sections of extraocular eye muscles. (A–C) Double immunofluorescence labelling of a palisade ending at the myotendinous junction of a monkey inferior rectus muscle with antibodies for SNAP-25 (red) and NF-M (green). The afferent axon and the bunch of axonal branches are both visible in the SNAP-25 (A) stain and the NF-M (B) stain. In contrast, the cuff of terminals lacks NF-M immunoreactivity and can only be identified with SNAP-25 antibodies (B), which is clearly demonstrated in the overlay of both markers in C. (D–F) Double (D) or triple (E,F) immunofluorescence labelling of a rat inferior rectus muscle with antibodies for SNAP-25 (green), myosin heavy chain slow-twitch (MHCslow-twitch; blue) and additional applied α-bungarotoxin (red). Panels D–F show overlays of the applied markers. (D) At the myotendinous junction (tendon left, muscle right), a putative palisade ending is identified with SNAP-25 antibodies. A nerve fibre enters from the distal tendon (arrow) and forms a clump of terminals on a muscle fibre. At higher magnification, a similar clump of terminals of the putative palisade ending in a different section can be clearly identified as several individual terminals, which form neuromuscular contacts on an MHC slow-twitch positive multiply innervated muscle fibre (E). In the same section, two α-bungarotoxin and SNAP-25 positive ‘en grappe’ endings (arrows) on an MHC slow-twitch-positive multiply innervated muscle fibre are clearly visible (F). Note that in contrast to these ‘en grappe’ endings, the terminals of the putative palisade endings in D and E do not show red any α-bungarotoxin binding. Scale bars: 50 µm (A–C), 200 µm (D), 20 m (E,F).