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Lienbacher, Karoline (2012): The source of innervation and function of palisade endings in the extraocular muscles of Macaca mulatta. Dissertation, LMU München: Fakultät für Biologie



Vertebrate extraocular muscles show a highly complex anatomy, which differs in many respects from skeletal muscles. Furthermore, there is a considerable variation among different species with regard to the presence of proprioceptive organs. Whereas muscle spindles and Golgi tendon organs are well developed in sheep and pig, neither are found in cat, and only poorly developed muscle spindles are present in human and monkey. In all vertebrates studied so far cuffs of nerve terminals around multiply innervated muscle fibers of the global layer, termed palisade endings (PE) are present at the myotendinous junction. Palisade endings (PE) are specialized nerve endings unique to extraocular muscles. There is still an ongoing debate on PE function. A proprioceptive function is supported by their ultrastructural morphology, and the location of the majority of their terminals within the muscle tendon. A motor function of PEs is suggested by the expression of different cholinergic markers, and the binding of α-bungarotoxin to at least a small proportion of PE nerve endings. So far the location of the somata giving rise to the PEs is unknown. After eye muscle injections with different tracers, and the investigation of retrogradely labeled cells after three days of survival, there are two possible options for the location of the cell bodies of PEs: The trigeminal ganglion (TG), where almost all of the sensory afferents of eye muscles come from, or the motor neurons of the abducens (nVI), trochlear (nIV) and oculomotor nucleus (nIII) in the brainstem, where the motor neurons of the extraocular muscles are situated. In the first part of this thesis the histochemical and morphometric properties of TG cells projecting to the extraocular muscles were studied and related to different nerve fibers and terminals in these muscles, to obtain more information about their function. Retrogradely labeled TG neurons and eye muscle terminals were processed for the presence of substance P (SP), nitric oxide synthase (NOS), calretinin (CR) and cholinacetyltransferase (ChAT). Injections of the tracer were placed into the medial rectus (MR) or the lateral recuts muscle (LR), either in the belly or in the distal, myotendinous part of the muscle. PEs were only labeled by CR or ChAT and none of the other markers that were used. Furthermore, CR positive retrogradely labeled tracer cells in the TG were rarely found (under 1 % of all tracer cells). The results indicate that the possible function of TG afferents could be mainly vasodilatation or nociception whereas the probability of proprioception via PEs is very low. These results point to the fact, that the source of the PE cell body is probably localized in the brainstem. In a second attempt to solve this problem, rhesus monkey received tract-tracer injections (WGA or CT) into the oculomotor nucleus, which contains the motor neurons of the medial, inferior and superior recti and inferior oblique muscles, as well as the trochlear nucleus, which contains the motor neurons of the superior oblique. All extraocular muscles were processed for the combined immunocytochemical detection of the tracer and non-phosphorylated neurofilament for the visualization of the complete muscle innervation. In all muscles (except lateral rectus) numerous anterogradely tracer labeled PEs were found, as well as tracer-filled tendon organs. In addition the en plaque and en grappe motor endings, were also strongly tracer positive. Double immunolabeling revealed that all types of nerve endings including tendon organs were anterogradely labeled, except the thin tyrosine hydroxylase positive autonomic nerve fibers of the sympathetic system. No anterograde labeling was found within the trigeminal ganglia. These results suggest that the somata of palisade endings are located within the brainstem, in or around the oculomotor nuclei, and confirm several previous studies. In how far the multiple nerve endings of non-twitch muscle fibers and the PEs form an anatomical entity with one parent soma in the periphery of the oculomotor nuclei was studied by morphological and histochemical analysis of the peripheral neurons around the motor nuclei. This data revealed two populations: one group of cholinergic multipolar neurons represent the motor neurons supplying the multiple motor innervation, one group of round calretinin positive cholinergic neurons giving rise to PEs. If the palisade endings do have a sensory function, then their cell body location amongst the non-twitch motor neurons would be an ideal location to control the tension in the non-twitch extraocular muscle fibers, and the data brought together in this thesis points strongly in this direction. To fully establish this hypothesis more physiological experimental data is required.