Delineation of motoneuron subgroups supplying individual eye muscles in the human oculomotor nucleus

Abstract

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.

Keywords: GABA; calretinin; central caudal nucleus; extraocular muscles; eye movements; glycine; motoneurons; nucleus of Perlia.

FIGURE 1

Transverse sections of the human trochlear nucleus (nIV) demonstrating the cytoarchitecture in cresyl violet (A), Gallyas fiber staining (B), and immunostaining for non-phosphorylated neurofilaments (NP-NF) (C). The nIV is devoid of calretinin (CR) expressing neurons and fiber profiles (D,G),and it does not express immunoreactivity for the glycine receptor (GlyR) (E,H). The nIV shows a modest supply by GABAergic punctate profiles revealed with antibodies against glutamate decarboxylase (GAD) (F). Panels (A–C, F) show neighboring 40 μm frozen sections of one case, panels (D,E) show neighboring 10 μm paraffin sections of another case. Panels (G–I) are detailed views from (D–F). A line drawing the midbrain section at this level is given at the bottom. DR, dorsal raphe nucleus; IC, inferior colliculus; ML, medial lemniscus; MLF, medial longitudinal fascicle; PAG, periaqueductal gray; PN, pontine nuclei; SCP, superior cerebellar peduncle. Scale bar: (A–F) 500 μm; (G–I,C) inset 30 μm.

FIGURE 2

Transverse sections through the caudal plane of the oculomotor nucleus (nIII). Cresyl violet (A), Gallyas fiber staining (B), and immunostaining for non-phosphorylated neurofilaments (NP-NF) (C) reveal several subnuclei of the oculomotor nucleus complex that exhibit different staining patterns for calretinin (CR) (D), glycine receptor (GlyR) (E), and glutamate decarboxylase (GAD) (F). The central caudal nucleus (CCN) appears as a separate nucleus embedded in the medially descending fibers (A–C). The CCN is high-lighted by its GlyR expression (E) and shows a moderate supply by CR- and GAD-positive profiles (D,F). A dorsolateral group (DL) of nIII is separated by encircling fibers (A–C). DL is devoid of CR-positive profiles (D), but rich in GlyR- and GAD-positive profiles (E,F). A similar pattern is seen for the ventral group (VEN) and lateral group (LAT), which forms an island of cells within the medial longitudinal fascicle (MLF) (A–F). A central group (CEN) is high-lighted by its strong expression of CR (D), but shows less staining for GAD (F) and almost none for GlyR (E). Panels (A–C,F) show neighboring 40 μm frozen sections of one case, (D,E) neighboring 10 μm paraffin sections of another case. Panels (G–I) are detailed views from (D–F). Asterisks label corresponding blood vessels in neighboring frozen sections of one case (A–C,F), stars label those in neighboring paraffin sections from a different case (D,E). Detailed views of levator palpebrae (LP) motoneurons in CCN are shown for CR (G), GlyR (H), and GAD-immunoreactivity (I). A line drawing at the bottom shows the midbrain section at this level. IPN, interpeduncular nucleus; ML, medial lemniscus; PAG, periaqueductal gray; SC, superior colliculus; SCP, superior cerebellar peduncle; scale bars: (A–F) 500 μm; (G–I) 30 μm.

FIGURE 3

Transverse sections at the level of the mid oculomotor nucleus (line drawing at the bottom). Cresyl violet (A), fiber staining (B), and immunostaining for non-phosphorylated neurofilaments (NP-NF) (C) reveal six subnuclei of the nIII complex at this level that exhibit different staining patterns for calretinin (CR) (D), glycine receptor (GlyR) (E), and glutamate decarboxylase (GAD) (F). The nucleus of Perlia (NP) forms an elongated midline cell group separated from the main nucleus by dorsoventrally traversing fibers (A,B), some expressing CR-immunoreactivity (D, arrows, inset). At the dorsomedial border of nIII, a compact cell group forms the centrally projecting non-preganglionic part of the Edinger–Westphal nucleus (EWcp), which contain urocortin (UCN)-positive and some scattered CR-positive neurons (D,G). The EWcp does not contain NP-NF (C), is devoid of GlyR (E), but receives a strong GAD input (F). High power magnification of two adjacent 5 μm paraffin sections immunostained for CR and UCN reveal only few UCN-positive neurons expressing CR (H,I, arrows). Corresponding blood vessels are indicated by asterisks. PAG, periaqueductal gray; RN, red nucleus; SC, superior colliculus; SN, substantia nigra (reticulata and compacta). Scale bar: (A–F) 500 μm; (G) 100 μm; (H,I) 50 μm.

FIGURE 4

High-power photographs of the staining pattern with different markers in the oculomotor nucleus complex. Numerous calretinin (CR)-positive profiles are found in association with cell bodies only in CEN and NP (G,J), but not in DL, DM (A,D). CR-staining of somata is found in EWcp (M; see also Figure 3). Strong labeling for glycine receptors (GlyR) is found only in the dorsolateral group (DL) (B), and with some traversing fibers in the dorsomedial group (DM) (E), but none in CEN, NP and EWcp (H,K,N). Different density of glutamate decarboxylase (GAD)-positive puncta is seen around choline acetyltransferase (ChAT)-immunoreactive motoneurons in the dorsolateral (DL) (C), dorsomedial (DM) (F), central groups (CEN) (I), and the nucleus of Perlia (NP) (L). The strongest supply by GAD-positive punctate profiles is present around ChAT-negative non-preganglionic centrally projecting neurons in the Edinger–Westphal nucleus (EWcp) (O). Scale bar (A–O) 30 μm.

FIGURE 5

Transverse sections at the level of the rostral oculomotor nucleus (line drawing at the bottom). Cresyl violet (A), Gallyas-fiber staining (B) with detail (C), immunostaining for glutamate decarboxylase (GAD) (D), and non-phosphorylated neurofilaments (NP-NF) (E) with detail (F). Note, unlike the preganglionic neurons of the Edinger–Westphal nucleus (EWpg), the centrally projecting non-preganglionic neurons of EWcp do not express non-phosphorylated neurofilaments (NP-NF) (C). The strong labeling of GAD-positive puncta in the most rostral group in the nIII speaks for a continuation of the dorsolateral group (DL), which most likely corresponds to the medial rectus B-group in monkey (D). Note the presence of numerous crossing fibers between both nIII at this level (B,C,E,F, arrows). INC, interstitial nucleus of Cajal; MGB, medial geniculate body; PAG, periaqueductal gray; RN, red nucleus; SN, substantia nigra (reticulata and compacta). Scale bar: (A,B,D,E) 500 μm; (C,F) 100 μm.

FIGURE 6

Two parallel series of drawings of transversal sections through the trochlear (nIV) and oculomotor nucleus (nIII) complex arranged from caudal to rostral. Panels (A,B) correspond to the level of Figure 1, (C,D) to the level of Figure 2, (G,H) to the level of Figure 3, and (K,L) to the level of Figure 5. Panels (E,F and I,J) correspond to additional intermediate levels. On the left, the density of glutamate decarboxylase (GAD) positive puncta and the intensity of immunostaining for the glycine receptor (GlyR) in the different subgroups is indicated by blue hatching on the left and green hatching on the right, respectively. The right column shows the presence of neurons expressing immunoreactivity for urocortin (UCN, black), non-phosphorylated neurofilaments (NP-NF; gray) and calretinin (CR; red filled circles). The density of CR-positive profiles is indicated by different grades of red hatching.

FIGURE 7

Histogram of the quantitative analysis of GABAergic and calretinin (CR) input to motoneurons in the oculomotor and trochlear nucleus. The values are given in Table 3. (A) The GABA-input was quantified by counting glutamate decarboxylase-positive (GAD) puncta along the measured length of the contour of a given neuron. The mean terminal density of input and the standard error of the mean values were calculated for each subgroup. The one-way analysis of variance revealed a significant difference (p < 0.001). The strongest input is seen to non-preganglionic centrally projecting neurons in the Edinger–Westphal nucleus (EWcp; compare to Figure 4O), whereas the neurons of all other subgroups did not show major differences. (B) The strongest CR input is seen to neurons of the nucleus of Perlia (NP), the central group in oculomotor nucleus (CEN), and the central caudal nucleus (CCN). The number of counted CR-positive puncta associated with putative upgaze motoneurons is significantly higher compared to those around horizontal – and downgaze motoneurons (***p < 0.001).

FIGURE 8

Proposed map of the motoneurons for individual extraocular muscles in human shown at four representative planes from caudal to rostral. The right half shows corresponding sections in Nissl staining to demonstrate the cytoarchitecture. The central caudal nucleus at most caudal planes contains the motoneurons of the levator palpebrae muscle (LP) (A). The medial rectus muscles (MR) is represented in two groups, the dorsolateral B- and the ventral A-group (A,B). The central group represents the motoneurons of the inferior oblique (IO) and superior rectus (SR) muscle (A,B). The nucleus of Perlia (NP) is separated from the main nucleus, but may contain SR motoneurons as well (B,C). The dorsomedial group corresponds to the inferior rectus motoneurons (IR) (A–C). The centrally projecting neurons of the Edinger–Westphal nucleus (EWcp) appear as a single lateral group on caudal levels dorsal to nIII (A), adjoined by a medial group further rostrally (B), which both merge to a single dorsal group (C). Another ventral extension of the EWcp appears on rostral levels (D). Note that the preganglionic neurons in the EWpg do not form a compact nucleus (B). Scale bar: (A–D) 500 μm.