Transmitter inputs to different motoneuron subgroups in the oculomotor and trochlear nucleus in monkey

Abstract

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.

Keywords: C-group; GABA; Glycine; extraocular muscles; vGlut.

Figure 1

Coronal section through the oculomotor nucleus (nIII) of a monkey, who had received an injection with wheat germ agglutinin-horseradish peroxidase (WGA-HRP) into the medial rectus (MR) muscle of the right eye. Retrogradely labeled neurons are found at three locations: ventral in the A-group, dorsolateral in the B-group and in the periphery dorsomedial to nIII in the C-group.Within the C-group motoneurons of multiply-innervated muscle fibers (MIF) in the MR are located in the medial part. Scale bar = 500 μm.

Figure 2

Overview of coronal sections through the oculomotor (nIII) and trochlear nucleus (nIV) in monkey to demonstrate the immunostaining for gamma-aminobutyric acid (GABA). A dense labeling of GABA-positive terminals is found in nIV. (A) All subgroups in nIII express a similar strong GABA-immunoreactivity, except the MR A and B-group, where a weaker labeling is observed (B–D). Note numerous GABAergic fibers are present in the medial longitional fascicle (MLF) next to the middle part of nIII containing motoneurons of vertically pulling eye muscles (B, upper inset), whereas much fewer GABA-positive fibers are found within the MLF portion adjacent to the MR A-group (B, lower inset). Detailed views of motoneuronal groups for vertical (E,F) and horizontal eye movements (G) reveal that most GABA immunoreactive profiles represent traversing fibers and cut axons (arrows) and only weakly stained puncta may form terminals (arrow heads) around motoneuronal somata (E–G, asterisks). Aq, aqueduct; CCN, central caudal nucleus, NIII, oculomotor nerve; INC, interstitial nucleus of Cajal; IO, inferior oblique muscle; RN, red nucleus; SR, superior rectus muscle; MIF, multiply innervated muscle fibers. Scale bar = 500 μm in (D) (applies to A–D); 30 μm in inset of (B); scale bar = 30 μm in (G) (applies to E–G).

Figure 3

Detailed view of coronal paraffin sections of the oculomotor (nIII) and trochlear nucleus (nIV) in the midbrain stained for glutamate decarboxylase (GAD) in black and choline acetyltransferase (ChAT) in brown (A–F). Numerous GAD-positive puncta outline most of the motoneuronal somata (asterisks) of the superior oblique (SO) (A) the levator palpebrae muscle (LP) (B) and the subgroup containing superior rectus (SR) and IO muscles. (D) In the MR subgroups fewer GAD-positive puncta are attached to the somata (C, asterisk), but are found in the neuropile contacting cut dendrites. A considerable number of GAD-positive puncta is found around MIF motoneurons in the C-group and S-group (E,F, asterisks indicate motoneurons; arrows). Detailed views of confocal images in (G,H) show tracer labeled MR MIF motoneurons (green) in the C-group that are in close association with GAD-positive (red) puncta suggestive for direct synaptic inputs (arrowheads). MIF, multiply innervated muscle fibers. Scale bar = 25 μm in (F) (applies to A–F); Scale bar = 25 μm in (G); Scale bar = 25 μm in (H).

Figure 4

Coronal sections of the oculomotor (nIII) and trochlear nucleus (nIV) of monkey stained for GABA-A receptor (GABA-A) in black and choline acetyltransferase (ChAT) in brown. The overview in (A,B) shows a strong GABA-A immunoreactivity associated with the motoneurons of the levator palpebrae muscle (LP) in the CCN, with those of the superior oblique in nIV (A) and SR and IO motoneurons. (B) The detailed views demonstrate the strong GABA-A expression (arrows) around motoneurons (asterisks) of the LP and SR/IO (C,F) and MIF-subgroups C and S (D,E) compared to MR SIF motoneurons of the A-group (G) GABA, gamma-aminobutyric acid; MIF, multiply innervated muscle fibers; SIF, singly innervated muscle fibers. Scale bar = 400 μm in (B) (applies to A,B); 30 μm in (G) (applies to C–G).

Figure 5

Histogram demonstrating the quantitative analysis of glutamate decarboxylase (GAD) input to motoneurons of oculomotor (nIII) and trochlear nucleus (nIV). The mean terminal density of stained puncta and the standard error of the mean were calculated for all motoneural subgroups. Note a similar strong input to almost all motoneuronal subgroups in nIII and nIV. Only the motoneurons of MR (A- and B-group) receive a weaker supply by GAD-positive terminals. The table shows the results of the ANOVA and following Bonferroni ś Multiple Comparison Test.

Figure 6

Overviews of coronal sections through the monkey trochlear (nIV) and oculomotor nucleus (nIII) immunostained for glycine transporter 2 (GlyT2). A dense glycinergic input is found to levator palpebrae motoneurons (LP) in the CCN (A,D) and to (A) and (B) groups of the MR muscle (B,C,F) compared to a few GlyT2-positive fibers and puncta (arrow) scattered within nIV (A,E) and the superior rectus/inferior oblique (SR/IO) motoneuronal group (B,C,G). (H) Shows a detailed view of the C-group (left side) with a MIF MR motoneuron (asterisk) covered with numerous GlyT2-positive puncta (arrows) next to a MIF inferior rectus (IR) motoneuron (asterisk) associated with only few GlyT2-positive puncta (arrows). Similarily, neurons in the S-group (asterisks) are associated with GlyT2-positive puncta and fibers (I, arrow). Aq, aquaeduct; IR, inferior rectus; SO, superior oblique. Scale bar = 500 μm in (C) (applies to A–C); Scale bar = 30 μm in (I) (applies to D–I).

Figure 7

Overviews and detailed views of immunoperoxidase labeling for glycine receptor 1 (GlyR1) in coronal sections of the oculomotor nucleus (nIII) in monkey. The strongest GlyR1 expression is present in the CCN as puncta profiles (A,B, stars and arrows). Unlike the motoneurons of the superior rectus and inferior oblique subgroup (SR/IO; F, asterisk) the MR SIF motoneurons (E, asterisks) show few GlyR1-positive puncta (E, arrows). The MIF motoneurons in the C-group (C, lines; D, asterisk) are associated with numerous GlyR1-positive puncta (D, arrows). MIF, multiply innervated muscle fibers; SIF, singly innervated muscle fibers. Scale bar = 200 μm in (A) (applies to A,C); Scale bar = 30 μm in (F) (applies to B,D–F).

Figure 8

Histogram demonstrating the quantitative analysis of the glycine transporter 2 (GlyT2) input to motoneurons in the trochlear (nIV), oculomotor (nIII) and CCN. The glycinergic input to all motoneural groups was quantified by counting immunoreactive puncta along the measured length of the contour of a motoneuron. The mean terminal density of inputs and the standard error of the mean were calculated for all motoneural subgroups. The strongest supply by GlyT2-positive puncta is seen to levator palpebrae motoneurons (LP), and fewer puncta are found around MR motoneurons of the A-, B- and C-group. The density of GlyT2-positive puncta around motoneurons for horizontal eye movements (MR) was significant stronger compared to those for vertical eye movements. The added table shows the results of the ANOVA and following Bonferroni ś Multiple Comparison Test.

Figure 9

Overviews of coronal paraffin sections through the monkey trochlear (nIV) and oculomotor nucleus (nIII) immunostained for the simultaneous detection of vesicular glutamate transporter1 (vGlut1) in black and choline acetyltransferase (ChAT) in brown (A–C). Note that SIF motoneuron subgroups in nIV (A,D) and nIII (B,C,E) are devoid of vGlut1-positive neuronal structures. Detailed views confirm the complete lack of vGlut1-positive puncta in nIV (D) and nIII with the B-group as examples (E). Note that numerous vGlut1-positive puncta are attached only to MIF motoneurons in the C-group—and there confined to the MR motoneurons (F, arrows), but not present at IR motoneurons (F, asterisk). MIF motoneurons in the S-group are associated with few vGlut1-positive puncta (G, arrows). EWpg; preganglionic Edinger-Westphal nucleus; MIF, multiply innervated muscle fibers; SIF, singly innervated muscle fibers. Scale bar = 500 μm in (C) (applies to A–C), Scale bar = 30 μm in (G) (applies to D–G).

Figure 10

Coronal section of the oculomotor nucleus (nIII) in monkey showing the expression of vesicular glutamate transporter 2 (vGlut2). (A) No differences were noted in the vGlut2 puncta labeling in different motoneuronal subgroups. The detailed views in (B,C) demonstrate the similar dense input of vGlut2-positive puncta (black) around cholinergic motoneurons (brown, asterisks) in the C-group and around motoneurons of the vertically pulling superior rectus/ inferior oblique (SR/IO) muscles (B,C, arrows). EWpg, preganglionic Edinger-Westphal nucleus. Scale bar = 250 μm in (A, 30 μm in (C) (applies to B,C).