Motoneurons of extraocular muscles are controlled by different premotor pathways, whose selective damage may cause directionally selective eye movement disorders. The fact that clinical disorders can affect only one direction, e.g., isolated up-/downgaze palsy or up-/downbeat nystagmus, indicates that up- and downgaze pathways are organized separately. Recent work in monkey revealed that a subpopulation of premotor neurons of the vertical eye movement system contains the calcium-binding protein calretinin (CR). With combined tract-tracing and immunofluorescence, the motoneurons of vertically pulling eye muscles in monkey were investigated for the presence of CR-positive afferent terminals. In the oculomotor nucleus, CR was specifically found in punctate profiles contacting superior rectus and inferior oblique motoneurons, as well as levator palpebrae motoneurons, all of which participate in upward eye movements. Double-immunofluorescence labeling revealed that CR-positive terminals lacked the γ-aminobutyric acid (GABA)-synthesizing enzyme glutamate decarboxylase, which is present in inhibitory afferents to all motoneurons mediating vertical eye movements. Therefore, CR-containing afferents are considered to be excitatory. In conclusion, a strong CR input is confined to motoneurons mediating upgaze, which derive from premotor pathways mediating saccades and smooth pursuit, but not from secondary vestibulo-ocular neurons in the magnocellular part of the medial vestibular nucleus. The functional significance of CR in these connections is unclear, but it may serve as a useful marker to locate upgaze pathways in the human brain.

The oculomotor nucleus (nIII) contains the motoneurons of medial, inferior, and superior recti (MR, IR, and SR), inferior oblique (IO), and levator palpebrae (LP) muscles. The delineation of motoneuron subgroups for each muscle is well-known in monkey, but not in human. We studied the transmitter inputs to human nIII and the trochlear nucleus (nIV), which innervates the superior oblique muscle (SO), to outline individual motoneuron subgroups. Parallel series of sections from human brainstems were immunostained for different markers: choline acetyltransferase combined with glutamate decarboxylase (GAD), calretinin (CR) or glycine receptor. The cytoarchitecture was visualized with cresyl violet, Gallyas staining and expression of non-phosphorylated neurofilaments. Apart from nIV, seven subgroups were delineated in nIII: the central caudal nucleus (CCN), a dorsolateral (DL), dorsomedial (DM), central (CEN), and ventral (VEN) group, the nucleus of Perlia (NP) and the non-preganglionic centrally projecting Edinger–Westphal nucleus (EWcp). DL, VEN, NP, and EWcp were characterized by a strong supply of GAD-positive terminals, in contrast to DM, CEN, and nIV. CR-positive terminals and fibers were confined to CCN, CEN, and NP. Based on location and histochemistry of the motoneuron subgroups in monkey, CEN is considered as the SR and IO motoneurons, DL and VEN as the B- and A-group of MR motoneurons, respectively, and DM as IR motoneurons. A good correlation between monkey and man is seen for the CR input, which labels only motoneurons of eye muscles participating in upgaze (SR, IO, and LP). The CCN contained LP motoneurons, and nIV those of SO. This study provides a map of the individual subgroups of motoneurons in human nIII for the first time, and suggests that NP may contain upgaze motoneurons. Surprisingly, a strong GABAergic input to human MR motoneurons was discovered, which is not seen in monkey and may indicate a functional oculomotor specialization.

In all vertebrates the eyes are moved by six pairs of extraocular muscles enabling horizontal, vertical and rotatory movements. Recent work showed 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 mediating a fast muscle contraction; and (2) motoneurons of multiply-innervated muscle fibers (MIF) in the periphery of motonuclei mediating a tonic muscle contraction. Currently only limited data about the transmitter inputs to the SIF and MIF motoneurons are available. Here we performed a quantitative study on the transmitter inputs to SIF and MIF motoneurons of individual muscles in the oculomotor and trochlear nucleus in monkey. Pre-labeled motoneurons were immunostained for GABA, glutamate decarboxylase, GABA-A receptor, glycine transporter 2, glycine receptor 1, and vesicular glutamate transporters 1 and 2. The main findings were: (1) the inhibitory control of SIF motoneurons for horizontal and vertical eye movements differs. Unlike in previous primate studies a considerable GABAergic input was found to all SIF motoneuronal groups, whereas a glycinergic input was confined to motoneurons of the medial rectus (MR) muscle mediating horizontal eye movements and to those of the levator palpebrae (LP) muscle elevating the upper eyelid. Whereas SIF and MIF motoneurons of individual eye muscles do not differ numerically in their GABAergic, glycinergic and vGlut2 input, vGlut1 containing terminals densely covered the supraoculomotor area (SOA) targeting MR MIF motoneurons. It is reasonable to assume that the vGlut1 input affects the near response system in the SOA, which houses the preganglionic neurons mediating pupillary constriction and accommodation and the MR MIF motoneurones involved in vergence.

The Y-group plays an important role in the generation of upward smooth pursuit eye movements and contributes to the adaptive properties of the vertical vestibulo-ocular reflex. Malfunction of this circuitry may cause eye movement disorders, such as downbeat nystagmus. To characterize the neuron populations in the Y-group, we performed immunostainings for cellular proteins related to firing characteristics and transmitters (calretinin, GABA-related proteins and ion channels) in brainstem sections of macaque monkeys that had received tracer injections into the oculomotor nucleus. Two histochemically different populations of premotor neurons were identified: The calretinin-positive population represents the excitatory projection to contralateral upgaze motoneurons, whereas the GABAergic population represents the inhibitory projection to ipsilateral downgaze motoneurons. Both populations receive a strong supply by GABAergic nerve endings most likely originating from floccular Purkinje cells. All premotor neurons express nonphosphorylated neurofilaments and are ensheathed by strong perineuronal nets. In addition, they contain the voltage-gated potassium channels Kv1.1 and Kv3.1b which suggests biophysical similarities to high-activity premotor neurons of vestibular and oculomotor systems. The premotor neurons of Y-group form a homogenous population with histochemical characteristics compatible with fast-firing projection neurons that can also undergo plasticity and contribute to motor learning as found for the adaptation of the vestibulo-ocular reflex in response to visual-vestibular mismatch stimulation. The histochemical characterization of premotor neurons in the Y-group allows the identification of the homologue cell groups in human, including their transmitter inputs and will serve as basis for correlated anatomical-neuropathological studies of clinical cases with downbeat nystagmus.

Examinations of eye movements offer an easy clinical method for the diagnosis of disturbances in the pathways for the generation of eye movements including the extraocular and inner eye muscles. A prerequisite is a good knowledge of the anatomy of the pathways for the generation of eye movements. The oculomotor nucleus represents an important relay station, which contains not only the motoneurons of four extraocular muscles and the levator palpebrae muscle, but also the preganglionic neurons of the ciliary ganglion for the mediation of the pupillary and accommodation response. Recent work about the special anatomy of the extraocular muscles and histochemical findings about the neurons in the Edinger-Westphal nucleus (EW), which indicated that this nucleus does not contain the preganglionic neurons of the ciliary ganglion, led to a new, modified map of the oculomotor nucleus complex. The most serious alteration refers to the location of the preganglionic neurons, which form a group of scattered neurons outside of the EW and now are termed EWpg. In contrast, the traditional cytoarchitectonically defined EW in the human eye contains peptidergic neurons with a completely different function, e.g., stress related, and is therefore termed EWcp (centrally projecting). A knowledge about the exact locations of extraocular motoneurons and preganglionic neurons is essential for the correct interpretation of clinico-anatomic findings.