Dopaminergic innervation of the mouse inner ear: evidence for a separate cytochemical group of cochlear efferent fibers

Abstract

Immunostaining mouse cochleas for tyrosine hydroxylase (TH) and dopamine beta-hydroxylase suggests that there is a rich adrenergic innervation throughout the auditory nerve trunk and a small dopaminergic innervation of the sensory cell areas. Surgical cuts in the brainstem confirm these dopaminergic fibers as part of the olivocochlear efferent bundle. Within the sensory epithelium, TH-positive terminals are seen only in the inner hair cell area, where they intermingle with other olivocochlear terminals expressing cholinergic markers (vesicular acetylcholine transporter; VAT). Double immunostaining suggests little colocalization of TH and VAT; quantification of terminal volumes suggests that TH-positive fibers constitute only 10-20% of the efferent innervation of the inner hair cell area. Immunostaining of mouse brainstem revealed a small population of TH-positive cells in and around the lateral superior olive. Consistent with cochlear projections, double staining for the cholinergic marker acetylcholinesterase suggested that TH-positive somata are not cholinergic and vice versa. All observations are consistent with the view that a small dopaminergic subgroup of lateral olivocochlear neurons 1) projects to the inner hair cell area, 2) is distinct from the larger cholinergic group projecting there, and 3) may correspond to lateral olivocochlear "shell" neurons described by others (Warr et al. [1997] Hear. Res 108:89-111).

Fig. 1

TH immunostaining of cochlear sections (A) and whole mounts (B) reveals a TH-positive innervation of the modiolus, osseous spiral lamina, and organ of Corti. A: Immunostained cochlear section from the upper basal turn shows TH-positive beaded fibers in the osseous spiral lamina (OSL) and modiolus (e.g., solid arrow). B shows a higher magnification view of the immunostained puncta in the inner spiral bundle (ISB). The position of the inner hair cell nucleus is indicated by the arrowhead. This image is from the upper basal turn. C: Immunostained cochlear whole mount shows strings of TH-positive en passant swellings (three are indicated by open arrows) in the ISB beneath the inner hair cells. Nuclei of three adjacent inner hair cells are indicated by the arrowheads. Scale bars = 50 μminA;5 μminC.

Fig. 2

Double immunostaining for TH (green) and VAT (red) in a confocal z-series shows lack of colocalization of these two transmitter markers. A–C show xy, xz, and yz projections, respectively. All projections suggest that there is minimal colocalization of TH and VAT in efferent terminals. None shows evidence for spatial segregation of TH- and VAT-positive terminals. D–F show isosurfaces viewed in the xy plane as generated by Amira 3-D visualization software: isosur-faces generated in 3-D enclose all voxels with signal intensity >90 in eight-bit images. Red surfaces were generated from the VAT signal, green from the TH signal. See text for further details. All images are from the same confocal z-stack (114 slices at 0.25 μm per slice) from the lower second turn. Scale bar = 20 μm.

Fig. 3

Comparison of xy projection (A) and a single slice (B) from a double-immunostained confocal z-series shows that apparent colocalization in the projection view (yellow arrows in A) often arises from superposition of signal from different focal levels and thus different terminals: the source of the green channel (TH) signal in A is shown by the arrows in B; at that focal level, there is no red (VAT signal). All images are from the same confocal z-stack (114 slices at 0.25 μm per slice) from the middle of the second turn. Scale bar = 20 μm.

Fig. 4

Semiquantitative analysis of the density of VAT-positive (A) and TH-positive (B) terminals in the inner hair cell area shows that TH immunoreactivity in control ears is uniformly distributed along the cochlear spiral and that, after LOC lesion, both types of immunoreactivity decline to a similar degree. This analysis included 36 cochleas: the observer (blind to case history) separately rated the density of VAT-positive or TH-positive puncta on a four-point scale (see Materials and Methods) at 10 positions along the cochlear spiral in each case. These cochleas were from 17 animals with unilateral brainstem lesion; based on analysis of brainstem sections, the LOC cells were at least partially destroyed in 10 of 17 cases (“LOC lesion”). Control data are from the 17 opposite ears and two ears from animals without any brainstem surgery. Mean data (±SEM) are shown for each group, normalized for each immunostain by dividing by the maximal mean value for that stain.

Fig. 5

Confocal image merging the TH signal (green) and the DIC image (gray scale) shows that TH immunoreactivity in the mouse cochlea is spotty: regions of the inner spiral bundle with high density of labeled terminals (solid green arrowhead) can be flanked by regions of low density (open green arrowheads) and, in turn, by regions with no immunostaining (white arrowheads). This image is the xy projection of a z-stack comprising 21 steps at 1.25-μm steps from the upper basal turn. Scale bar = 100 μm.

Fig. 6

Double immunostaining for TH (green) and VAT (red) in a control cochlea (A,C) and the opposite cochlea (B,D) after either section of the olivocochlear bundle (B) or injection of neurotoxin into the LSO (D). A is from a control cochlea showing a few TH-positive (green) swellings (e.g., arrow) among the VAT-positive (red) terminals in the inner spiral bundle (ISB). B is the place-matched cochlear region from the partially deefferented opposite ear, showing loss of VAT-positive terminals in ISB and outer hair cell (OHC) areas, loss of TH-positive terminals in the ISB area, and no obvious loss of TH-positive beaded fibers in the osseous spiral lamina (OSL). Both images are from the lower second turn and comprise xy projections of confocal z-series spanning 33–40 μm of focal depth in 0.5-μm steps. C shows TH and VAT immunostaining of the organ of Corti a control ear, whereas D shows the place-matched cochlear region from the opposite ear, which was ipsilateral to a neurotoxin injection that hit the LSO. Both images are from the upper basal turn and comprise xy projections of confocal z-series. Scale bars = 25 μm in A (applies to A,B); 40 μm in D (applies to C,D).

Fig. 7

TH-positive cell bodies (arrows) located within and around the LSO. An approximate outline of the LSO is indicated by the dashed line, and the locations of lateral and medial limbs are indicated. Image is an xy projection of a z-stack through one 15-μm section acquired with a conventional light microscope (0.5 μm/slice). Scale bar = 100 μm.

Fig. 8

Double staining (AChE, red; TH, green) of brainstem sections through the superior olivary complex suggests that the TH-positive innervation of the cochlea arises from a separate population of noncholinergic neurons, in the “shell” around the LSO. A shows an AChE-stained section of mouse brain: solidd arrows point to three of the many AChE-positive cholinergic neurons within the LSO. One of the many AChE-positive axons encircling the LSO is indicated by the open arrow. B: Three TH-positive cells (solid arrows) and one TH-positive axon (open arrow) are highlighted. C: Merge of images in A and B shows the complementary distribution of AChE and TH signal in both cell bodies and axons. The AChE was visualized with a histochemical stain producing a black reaction product; the TH was visualized with a fluorescent antibody. The AChE image is shown as red to simplify merging of the images. Scale bar = 100 μm.