Planar multipolar cells in the cochlear nucleus project to medial olivocochlear neurons in mouse

Abstract

Medial olivocochlear (MOC) neurons originate in the superior olivary complex and project to the cochlea, where they act to reduce the effects of noise masking and protect the cochlea from damage. MOC neurons respond to sound via a reflex pathway; however, in this pathway the cochlear nucleus cell type that provides input to MOC neurons is not known. We investigated whether multipolar cells of the ventral cochlear nucleus have projections to MOC neurons by labeling them with injections into the dorsal cochlear nucleus. The projections of one type of labeled multipolar cell, planar neurons, were traced into the ventral nucleus of the trapezoid body, where they were observed terminating on MOC neurons (labeled in some cases by a second cochlear injection of FluoroGold). These terminations formed what appear to be excitatory synapses, i.e., containing small, round vesicles and prominent postsynaptic densities. These data suggest that cochlear nucleus planar multipolar neurons drive the MOC neuron's response to sound.

Figure 1

Neural pathway of the medial olivocochlear (MOC) neurons projecting to the left cochlea (Ipsilateral side). Also shown are the stages of the reflex pathway leading to the MOC neurons: the auditory nerve fibers that project to the ventral cochlear nucleus (VCN), and the VCN multipolar cells that presumably project to MOC neurons. To test the hypothesis that multipolar cells project to MOC neurons, we labeled them via their collaterals using biotinylated dextran amine (BDA) injected into the dorsal cochlear nucleus (DCN). Multipolar cell axons were followed into the superior olivary complex (SOC), where many form branches to the lateral superior olive (LSO) on the ipsilateral side as reported previously (Doucet and Ryugo, 2003). Results of the present study show that they also form branches to the ventral nucleus of the trapezoid body (VNTB) on the contralateral side, the location of most MOC neurons in the mouse. In some experiments, a second injection of Fluorogold (FG) was made into the cochlea to retrogradely label MOC neurons.

Figure 2

Light micrograph of a transverse section containing the DCN injection site. More ventral is the band of labeling in the VCN (arrow) that contains labeled multipolar cells, their dendrites, and auditory nerve fibers. Scale bar = 250 μm.

Figure 3

A. Light micrograph showing the pattern of multipolar-cell labeling in a band (arrow) in the PVCN. Labeled processes are mainly branches of auditory nerve fibers. Smaller cells and granule cells are labeled at the lateral margin (left side of micrograph) and in the lamina dividing the PVCN from the DCN. Scale bar = 100 μm. B. Atlas drawings showing positions of multipolar cell (red circles) and small and granule cells (black dots). Labeled multipolar cell axons (red lines) project out of the nucleus in a ventromedial direction. Each panel of the atlas shows the labeled neurons in single 80-μm sections with half (every other one) of the sections omitted for clarity. Scale bar = 500 μm.

Figure 4

Camera lucida drawing of presumed multipolar cell axons projecting from the PVCN through the SOC. The three axons illustrate branching to: 1) ipsilateral LSO (purple and red fibers), and 2) contralateral VNTB, (red and orange fibers). The branches to the LSO could not be completely reconstructed because of intermingling with other labeled branches that are not illustrated, but the branches to the VNTB were spatially separated and could be fully reconstructed. All axons continue on in the lateral lemniscus towards the inferior colliculus (not drawn). MNTB: medial nucleus of the trapezoid body. Scale bar = 500 μm.

Figure 5

A: Electron micrograph of a labeled axon from presumed multipolar cell axon in the trapezoid body near the contralateral VNTB, showing the axon’s myelination and reaction product in the axoplasm. Scale bar = 0.5 μm. Inset: myelin lamellae at higher magnification. B: Micrograph of a labeled branch to contralateral VNTB showing reaction product in axoplasm but lack of myelination. The branch forms a swelling (right) but analysis of serial sections through this swelling failed to find a synapse. Scale bar = 1 μm.

Figure 6

Light and electron micrographs of a labeled branch in the contralateral VNTB. A: Light micrograph and reconstruction of a labeled branch (red). The branch has a large swelling that contacts (arrow) a neuron (N). Scale bar = 20 μm. B: Electron micrograph of the same neuron (N) in a section containing the nucleus (Nuc). The neuron is contacted (arrow) by the labeled swelling (note black reaction product), which forms a synapse (see C). Surrounding this neuron are numerous axons and small amounts of neuropil. Scale bar = 5 μm. C, D: High-magnification micrographs of different sections of the synapse (denoted by arrowheads) from the swelling. The labeled swelling contains synaptic vesicles and this one has a dense core vesicle (DCV, panel D). In the upper portion of C, two unlabeled terminals, denoted by open circles, are seen to form synapses onto the neuron (N). Scale bars = 1 μm for C, 0.2 μm for D. E: Mean vesicle circularity versus mean vesicle area (see Methods) from unlabeled and labeled synaptic terminals contacting this neuron. Terminals used for measurements were from the labeled branch (closed circles) or from unlabeled terminals (open circles) onto neuron N.

Figure 7

Light micrograph of the contralateral VNTB showing boutons of presumed multipolar cells (red) that contact MOC neurons (blue) on their somata (arrowheads) and dendrites (arrows). In this double-injected case, fluorescently tagged DA (red) was injected into the DCN and Fluorogold (blue) was injected in the cochlea. Scale bar = 15 μm.

Figure 8

Electron microscopy of a labeled branch from a different case than Figure 6. A: Low-magnification electron micrograph shows a labeled branch winding through the contralateral VNTB neuropil adjacent to a neuron (N). The neuron was not a postsynaptic target in our material, but nearby postsynaptic targets included two dendrites (Dend, one of them indicated in two segments in the center and confirmed to be contiguous in serial sections) and a spine (sp). In the neuropil, an axon of unknown origin forms a large ending as it loses its myelin (myel). Scale bar = 5 μm. B–D: Higher-magnifications of the synapses on dendrites (Dend) and a dendritic spine (sp). Panel B reveals a dendrite intervening between the labeled branch and the neuron. Scale bars = 1 μm.