Histochemical differences between motoneurons supplying multiply and singly innervated extraocular muscle fibers

The extraocular muscle fibers of vertebrates can be classified into two categories: singly innervated fibers (SIFs) and multiply innervated fibers (MIFs). In monkeys, the motoneurons of SIFs lie within the oculomotor, trochlear, and abducens nucleus, whereas the motoneurons of MIFs appear in separate subgroups in the periphery of the classical nuclei borders. In the present study, we investigated the histochemical properties of SIF and MIF motoneurons by using combined tract-tracing and immunofluorescence techniques. 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. Alternatively, the motoneurons were identified by choline acetyltransferase immunostaining. These techniques were combined with the detection of histochemical markers for perineuronal nets, nonphosphorylated neurofilaments, parvalbumin, or cytochrome oxidase. The experiments revealed that the MIF motoneurons in the periphery of the motonuclei do not contain nonphosphorylated neurofilaments or parvalbumin and lack perineuronal nets. In contrast, SIF motoneurons express all markers at high intensity. Cytochrome oxidase immunostaining was found in both motoneuron populations. An additional population of motoneurons with "MIF properties" was identified within the boundaries of the abducens nucleus, which could represent the motoneurons innervating MIFs in the orbital layer of lateral rectus muscle. Our data provide evidence that SIF and MIF motoneurons, which can be correlated with twitch motoneurons and presumed non-twitch motoneurons, differ in their histochemical properties. The absence of perineuronal nets, nonphosphorylated neurofilaments, and parvalbumin may help to identify the homologous MIF motoneurons in other species, including humans.