Ultrastructural and molecular biologic comparison of classic proprioceptors and palisade endings in sheep extraocular muscles

Abstract

Purpose: To analyze and compare the structural and molecular features of classic proprioceptors like muscle spindles and Golgi tendon organs (GTOs) and putative proprioceptors (palisade endings) in sheep extraocular muscle (EOMs).

Methods: The EOMs of four sheep were analyzed. Frozen sections or wholemount preparations of the samples were immunohistochemically labeled and analyzed by confocal laser scanning microscopy. Triple labeling with different combinations of antibodies against neurofilament, synaptophysin, and choline acetyltransferase (ChAT), as well as alpha-bungarotoxin and phalloidin, was performed. Microscopic anatomy of the nerve end organs was analyzed by transmission electron microscopy.

Results: The microscopic anatomy demonstrated that muscle spindles and GTOs had a perineural capsule and palisade endings a connective tissue capsule. Sensory nerve terminals in muscle spindles and GTOs contained only a few vesicles, whereas palisade nerve terminals were full of clear vesicles. Likewise, motor terminals in the muscle spindles' polar regions were full of clear vesicles. Immunohistochemistry showed that sensory nerve fibers as well as their sensory nerve terminals in muscle spindles and GTOs were ChAT-negative. Palisade endings were supplied by ChAT-positive nerve fibers, and the palisade complexes including palisade nerve terminals were also ChAT-immunoreactive. Motor terminals in muscle spindles were ChAT and alpha-bungarotoxin positive.

Conclusions: The present study demonstrated in sheep EOMs that palisade endings are innervated by cholinergic axons exhibiting characteristics typical of motoneurons, whereas muscle spindles (except the polar regions) and GTOs are supplied by noncholinergic axons. These results raise the question of whether palisade endings are candidates for proprioceptors in EOMs.

Fig. 1

Light microscope image of a muscle spindle. The image shows a cross section through a muscle spindle in the equatorial region. The muscle spindle contains eight intrafusal muscle fibers (IF) which are separated from the capsule by a subcapsular space (asterisk). One thick and several thin nerve fibers (N) are inside the muscle spindle. Capsule (C). Scal bar, 100 μm.

Fig. 2

TEM micrographs of muscle spindles. (A) Cross section through the muscle spindle’s capsule (C). The capsule consists of perineural cells which are covered on both sides with a basal lamina (arrows). (B) Longitudinal section through two nuclear chain fibers (NC) being richly endowed with sensory nerve terminals (T). (C) Detail of a sensory nerve terminal (T). The nerve terminal contains mitochondria and the synaptic cleft (arrow) is free from a basal lamina. Muscle fiber (MF). (D) Longitudinal section through a motor terminal (MT). The nerve terminal contains mitochondria and aggregations of clear vesicles. A basal lamina fills the synaptic cleft (arrow). Inset showing a detail of a motor terminal. Muscle fiber (MF). Scale bars, 1 μm.

Fig. 3

CLSM images of muscle spindles showing the equatorial regions (A, C, E) and the polar regions (B, D, F). The equatorial regions are shown in longitudinal sections whereas the polar regions are shown in cross section (B, D) or oblique section (F) respectively. Triple fluorescent images were overlaid with a transmission light image. (A, B) Labeling with anti-synaptophysin (green), α-bungarotoxin (red) and phalloidin (blue). (A) Sensory nerve endings in the equatorial region are only positive for synaptophysin whereas a motor terminal (arrow) outside the spindle is synaptophysin/α-bungarotoxin-positive. (B) In the muscle spindle’s polar region, motor terminals exhibit synaptophysin/α-bungarotoxin reactivity. (C, D) Labeling with anti-ChAT (green), anti-synaptophysin (red), and phalloidin (blue). (C) Sensory anulospiral nerve endings in the equatorial regions are synaptophysin-positive but ChAT-negative. ChAT-positive nerve fibers are seen alongside the spindle. Two ChAT-positive axons (arrow) running towards the spindle’s pole are visible inside the spindle. (D) In the polar region ChAT-positive nerve fibers establish motor terminals on the intrafusal muscle fibers. Motor terminals co-localize ChAT and synaptophysin. (E, F) Labeling with anti-ChAT (green), anti-neurofilament (red), and phalloidin (blue). (E) In the muscle spindles’ equatorial region nerve fibers solely positive for neurofilament and nerve fibers positive for ChAT/neurofilament are visible. Only neurofilament-positive axons enwrap intrafusal muscle fibers and are thereby forming anulospiral endings in the equatorial region. Inset showing the mixed innervation of a muscle spindle in a cross section. (F) In the polar region axons stain double positive for ChAT and neurofilament. Scale bars, 100 μm.

Fig. 4

Light microscopic image (A) and TEM micrographs (B, C, D) of GTOs (A) Oblique section through a GTO stained with Azan. The GTO is enclosed by a capsule (C). Inside the capsule collagen bundles are visible which are separated from the capsule by a subcapsular space (asterisk). (B) Cross section through the GTO capsule (C) consisting of four perineural cell layers. Perineural cells are ensheathed by a basal lamia (arrow). (C) Cross section through a GTO. Among the collagen bundles (COL) numerous nerve terminals (T) are visible. Capsule (C). (D) High resolution micrograph of a nerve terminal (T) contacting the neighboring collagen fibrils (COL). The nerve terminal is partly covered by a Schwann cell (S) and at the point of contact only a basal lamina (arrow) is interposed between the axolemma and the collagen. Scale bars, (A) 100 μm, (B, C, D) 1 μm.

Fig. 5

CLSM images of GTOs in whole mount preparations (A, D) and longitudinal frozen sections (B, C). In frozen sections the triple fluorescent images were overlaid with a transmission light image. (A) Labeling with anti-neurofilament (red), anti-synaptophysin (green) and phalloidin (blue). A single neurofilament-positive nerve fiber enters a GTO containing a muscle fiber. Inside the GTO the axon divides into nerve branches which establish synaptophysin-positive contacts. (B) Labeling with anti-synaptophysin (green), α-bungarotoxin (red) and phalloidin (blue). Nerve terminals inside the GTO are synaptophysin-immunoreactive but α-bungarotoxin-negative. Motor endplates outside the GTOs stain synaptophysin/α-bungarotoxin-positive. (C) Labeling with anti-ChAT (green), anti-synaptophysin (red), and phalloidin (blue). GTO-nerve terminals are synaptophysin positive but ChAT-negative. Motor nerve terminals contacting muscle fibers outside the GTO co-localize ChAT/synaptophysin. (D) Labeling with anti-ChAT (green), anti-neurofilament (red), and phalloidin (blue). In this staining combination the nerve fiber innervating the GTO exhibits solely neurofilament reactivity. Motor nerve fibers establishing neuromuscular contacts exhibit ChAT/neurofilament immunoreactivity. Scale bars, 100 μm.

Fig. 6

TEM micrographs of palisade nerve endings (A) Cross section through the capsule of a palisade ending. The capsule (C) consists of fibroctyes which lack a basal lamina. Inside the capsule, a palisade nerve terminal (T) contacting the collagen fibrils is visible. (B) High resolution micrograph of a neurotendinous contact containing mitochondria and a dense aggregation of clear vesicles. The nerve terminal is partly covered with a Schwann cell (S) and at the collagen contact site only a basal lamina (arrow) covers the terminal. Inset: Detail of the contact. (C) High resolution image of a palisade nerve terminal (T) contacting the muscle fiber (MF) which is covered with a basal lamina (arrow). The neuromuscular contacts contain mitochondria and clear vesicles. In the synaptic cleft (arrowhead) a basal lamia is absent. Inset: Detail of the contact. Scale bars, 1μm.

Fig. 7

CLSM images of palisade endings in whole mount preparations. The unlabeled tendon continues the muscle fibers to the right. (A) Labeling with anti-neurofilament (red), anti-synaptophysin (green) and phalloidin (blue). A neurofilament-positive nerve fiber forms a palisade ending at a muscle fiber tip. Palisade nerve terminals are synaptophysin-immunoreactive. (B, C) Labeling with anti-ChAT (green), anti-synaptophysin (red), and phalloidin (blue). ChAT-positive nerve fibers supplying palisade endings at the muscle fiber tips. All palisade nerve terminals co-localize ChAT/synaptophysin. (D) Labeling with anti-ChAT (green), anti-neurofilament (red), and phalloidin (blue). The nerve fiber forming a palisade ending exhibits ChAT/neurofilament immunoreactivity. Scale bars, 100 μm.