An investigation of the sensory and motor innervation of extraocular muscles in monkey and rat with combined tract-tracing and immunofluorescence methods: evidence for a dual motor innervation as common concept in mammals

The oculomotor system is one of the best studied motor systems. Afferents from a variety of premotor areas converge on the motoneurons in the three oculomotor nuclei to produce the different types of eye movements. All oculomotor motoneurons participate in all types of eye movements, and it was generally accepted, that these motoneurons form a relative homogenous group which provides the final common pathway for extraocular muscle (EOM)-motor innervation. The EOM in mammals, the effector organs of the oculomotor system, are fundamentally different from skeletal muscle. They have two functionally different layers, global and orbital layer, and are composed of two major muscle fibre classes, singly-innervated (SIF) and multiply innervated fibres (MIF). Previous studies in monkey revealed that SIF and MIF motoneurons are anatomically separated and have different premotor inputs, which support the idea of a dual motor innervation of EOM rather than a final common pathway from motoneuron to EOM. Up to date, neither motoneuron type has been further characterized nor has any study proven their presence in other species to support the hypothesis of the dual motor innervation as a common concept in mammals. The functional implication of this system remains speculative, though a role of MIFs together with their motoneurons in a sensory feedback system controlling the EOMs is quite possible and heavily debated. However, the lack of a common proprioceptor in eye muscles does not support this theory. In monkeys SIF and MIF motoneurons of extraocular muscles were identified by tracer injections into the belly or the distal myotendinous junction of the medial or lateral rectus muscle and further characterized by combined tracer detection and immunohistochemical methods. The experiments revealed that the MIF motoneurons in the periphery of the motor nuclei lack non-phosphorylated neurofilaments, parvalbumin and perineuronal nets, whereas SIF motoneurons intensively express all three markers. In addition to the histochemical differences, the MIF motoneurons are on average significantly smaller in size than the SIF motoneurons. Analogous to the study in monkey, the SIF and MIF motoneurons of the medial and lateral rectus muscle of rats were identified with tracer injections and further characterized by immunolabelling. For the first time it was shown that both motoneurons types are present in rat as well. The MIF motoneurons lie mainly separated from the SIF motoneurons, and are different in size and histochemical properties. As in monkey, the smaller MIF motoneurons lack non-phosphorylated neurofilaments and perineuronal nets, both of which are definite markers for the larger SIF motoneurons. A possible proprioceptive control of eye movements requires the presence of proprioceptive structures. The palisade endings represent the best candidate for an EOM-proprioceptor. They were analysed using antibody stains against the synaptosomal associated protein of 25kDA, SNAP-25. With this robust method palisade ending-like structures were identified for the first time in the extraocular muscles of the rat. Furthermore the rat palisade endings show characteristics of sensory structures thereby supporting their role in proprioception. In conclusion, the EOM of both monkey and rat are innervated by two sets of motoneurons which differ in localization, morphology and molecular components. These findings further support the presence of a dual motor control of EOM that may apply widely to mammals, since it was verified in monkey and rat. Palisade endings are a ubiquitous feature of mammal EOM and most likely provide sensory information used for the proprioceptive control of eye movements.