Histochemical characterization of inputs to motoneurons of extraocular muscles subserving different functions

Eye movements are important to aid vision, and they serve two main functions: to stabilize a moving visual target on the retina and to stabilize gaze during own body movements. Six types of eye movements have been evolved fulfilling this function: saccades, smooth pursuit, vestibulo-ocular reflex, optokinetic response, convergence and gaze holding. In all vertebrates the eyes are moved by six pairs of extraocular muscles that enable horizontal, vertical and rotatory eye movements. The motoneurons of these muscles are located in the oculomotor (nIII), trochlear (nIV) and abducens (nVI) nucleus in the brainstem. Motoneurons of the lateral rectus muscle (LR) in nVI and of the medial rectus muscle (MR) in nIII provide horizontal eye movements, those of inferior oblique (IO) and superior rectus muscle (SR) in nIII upward eye movements. Motoneurons of the superior oblique (SO) and the inferior rectus muscle (IR) in nIII convey downward eye movements. Recently, it was shown that each extraocular muscle is controlled by two motoneuronal groups: 1. Motoneurons of singly innervated muscle fibers (SIF) that lie within the boundaries of motonuclei providing a fast muscle contraction (twitch) and 2. motoneurons of multiply innervated muscle fibers (MIF) in the periphery of motonuclei providing a tonic muscle contraction (non-twitch). Tract-tracing studies indicate that both motoneuronal groups receive premotor inputs from different brainstem areas. A current hypothesis suggests that pathways controlling twitch motoneurons serve to generate eye movements, whereas the non-twitch system is involved in gaze holding. Lesions of inputs to the twitch motoneuron system may lead to supranuclear gaze palsies, whereas impairment of the non-twitch motoneuron system may result in gaze holding deficits, like nystagmus, or strabismus. Up to date only limited data are available about the histochemical characteristics including transmitters to the SIF- (twitch) and MIF (non-twitch) motoneurons. The present study was undertaken to investigate the histochemical profile of inputs to motoneuronal groups of individual eye muscles mediating horizontal and vertical eye movements including the inputs to MIF- and SIF motoneurons. The MIF motoneurons of the IR and MR are located in the periphery dorsolateral to nIII, close to the Edinger-Westphal nucleus (EW), which is known to contain preganglionic cholinergic neurons. Other scientists have found that the EW is composed of urocortin-positive neurons involved in food intake or stress. In order to delineate these different cell populations within the supraoculomotor area dorsal to nIII, a comparative study in different mammals was conducted to locate the cholinergic preganglionic neurons and urocortin-positive neurons. Only then, it became obvious that the cytoarchitecturally defined EW labels different cell populations in different species. In rat, ferret and human the cytoarchitecturally defined EW is composed of urocortin-positive neurons. Only in monkey the EW contains cholinergic preganglionic neurons, which lie close to the MIF-motoneurons of MR and IR in the C-group. In monkey, I performed a systematic study on the histochemical profile and transmitter inputs to the different motoneuron subgroups, including MIF- and SIF motoneurons. Brainstem sections containing prelabelled motoneurons were immunostained for the calcium-binding protein calretinin (CR), gamma-aminobutyric acid (GABA) or glutamate decarboxylase (GAD), glycine transporter 2, glycine receptor 1, and the vesicular glutamate transporters (vGlut) 1 and 2. The study on the histochemical profile of the motoneuron inputs revealed three main results: 1.The inhibitory control of SIF motoneurons for horizontal and vertical eye movements differs. Unlike previous studies in the primate a considerable GABAergic input was found to all SIF motoneuronal groups, but a glycinergic input was confined to motoneurons of the MR mediating horizontal eye movements. 2. The excitatory inputs to motoneurons for upgaze and downgaze differ in their histochemistry. A striking finding was that CR-positive nerve endings were confined to the motoneurons of muscles involved in upgaze, e.g. SR, IO and the levator palpebrae, which elevates the upper eyelid and acts in synchrony with the SR. Since double-immunoflourescence labelling with anti-GAD did not reveal any colocalization of GAD and CR, the CR-input to upgaze motoneurons is considered as excitatory. 3. The histochemistry of MIF- and SIF motoneurons differs only for vGlut1. Whereas SIF- and MIF motoneurons of individual eye muscles do not differ in their GABAergic, glycinergic and vGlut2 input, vGlut1 containing terminals were covering the supraoculomotor area and targeting only MR MIF motoneurons. It is reasonable to assume that the vGlut1 input affects the near response system in the supraoculomotor area, which houses the preganglionic neurons in the EW mediating pupillary constriction and accommodation and the MR MIF motoneurones involved in vergence. The histochemical data in monkey enabled the localization of the corresponding motoneuronal subgroups of individual eye muscles in human with the development of an updated nIII map. Taken together the present work provides new data on the histochemical properties of premotor inputs to motoneuronal groups of the twitch- and non-twitch eye muscle systems in primates. Especially the selective association of CR in premotor upgaze pathways may open the possibility for a targeted research of this system in human post-mortem studies of clinical cases with impairment of upward eye movements, such as progressive supranuclear palsy (PSP) or Niemann-Pick disease (NPC)