Dopaminergic signaling in the cochlea: receptor expression patterns and deletion phenotypes

Abstract

Pharmacological studies suggest that dopamine release from lateral olivocochlear efferent neurons suppresses spontaneous and sound-evoked activity in cochlear nerve fibers and helps control noise-induced excitotoxicity; however, the literature on cochlear expression and localization of dopamine receptors is contradictory. To better characterize cochlear dopaminergic signaling, we studied receptor localization using immunohistochemistry or reverse transcriptase PCR and assessed histopathology, cochlear responses and olivocochlear function in mice with targeted deletion of each of the five receptor subtypes. In normal ears, D1, D2, and D5 receptors were detected in microdissected immature (postnatal days 10-13) spiral ganglion cells and outer hair cells but not inner hair cells. D4 was detected in spiral ganglion cells only. In whole cochlea samples from adults, transcripts for D1, D2, D4, and D5 were present, whereas D3 mRNA was never detected. D1 and D2 immunolabeling was localized to cochlear nerve fibers, near the first nodes of Ranvier (D2) and in the inner spiral bundle region (D1 and D2) where presynaptic olivocochlear terminals are found. No other receptor labeling was consistent. Cochlear function was normal in D3, D4, and D5 knock-outs. D1 and D2 knock-outs showed slight, but significant enhancement and suppression, respectively, of cochlear responses, both in the neural output [auditory brainstem response (ABR) wave 1] and in outer hair cell function [distortion product otoacoustic emissions (DPOAEs)]. Vulnerability to acoustic injury was significantly increased in D2, D4 and D5 lines: D1 could not be tested, and no differences were seen in D3 mutants, consistent with a lack of receptor expression. The increased vulnerability in D2 knock-outs was seen in DPOAEs, suggesting a role for dopamine in the outer hair cell area. In D4 and D5 knock-outs, the increased noise vulnerability was seen only in ABRs, consistent with a role for dopaminergic signaling in minimizing neural damage.

Figure 1.

Schematic of the synaptic interactions among hair cells, cochlear nerve afferents, and olivocochlear efferent fibers in the mammalian cochlea. The connections and AMPA receptor localization are known from ultrastructural studies (Liberman, 1980a,b; Matsubara et al., 1996); the nature of the cholinergic receptors on outer hair cells is known from knock-out studies (Vetter et al., 1999, 2007). Dopamine receptor localization (red and orange bars—see key) is inferred from the D1 and D2 receptor immunohistochemistry in the present study: solid versus dotted bars indicate unambiguous versus ambiguous localization, respectively. Only bright, punctate labeling localized to sites of possible synaptic interaction are considered in this schematic. RT-PCR evidence for expression of D1, D2, and D5 in outer hair cells is not schematized here. MOC and LOC refer to the lateral and medial olivocochlear system, respectively. ISB, Inner spiral bundle; HP, habenula perforata; OSL, osseous spiral lamina.

Figure 2.

A–C, RT-PCR (A, C) and qRT-PCR (B) analysis of dopamine receptor expression in wild-type mice. A, D1, D5, and D2 receptors were consistently expressed in OHCs and spiral ganglion neurons (SGNs), but not IHCs harvested from immature cochleas (P11–P13). D4 receptors were expressed only in SGNs. Primers for neurofilament (Nefh), otoferlin, and the nicotinic receptor subunit α10 (Chrna10) were used as markers for SGNs, IHCs, and hair cells, respectively. B, D1, D5, D2, and D4 were expressed in whole adult cochleas, but D3 receptors were never amplified. For the qRT-PCR (B), mean expression levels are normalized to 18S rRNA, after adjusting for primer efficiency, as described previously (Stankovic and Corfas, 2003). Error bars represent SEMs. C, As a positive control for the primers, we showed that all five dopamine receptors are expressed in adult brain. All bands in A and C appear at the expected location (Table 1), as calibrated by the ladder lane: dashed lines are positioned at 100, 200, and 300 base pairs. The 8-bit gel micrographs were adjusted digitally: after inversion, offset was set to 0 by subtracting the mean pixel value from an empty lane; gain was optimized by setting to 256 the mean pixel value from the 500 bp ladder band. Primer bands (<100 bp) are not shown.

Figure 3.

A–C, Immunostaining for D2 (red) shows receptor expression in cochlear nerve fibers, especially near the first nodes of Ranvier (red-filled arrows in A and C), where the spike initiation zone is located. Cochlear nerve fibers are immunopositive for Na⁺/K⁺ ATPase (green); olivocochlear terminals are immunopositive for synaptophysin (blue). A, In this xy projection (i.e., surface view of the sensory epithelium), cochlear-nerve peripheral axons are seen in the osseous spiral lamina (green-filled arrowhead), and their peripheral terminals are seen underneath the IHCs (green-filled arrow), where OC terminals contact them. B, C, Side views, i.e., zy projections, of the dashed subregion in A. In C the green channel has been eliminated to better view the small D2-positive puncta clustered near the two clouds of synaptophysin-positive OC terminals (red-fill arrows in B and C). The approximate outline of the IHCs in these zy projections is indicated by the dotted line in B, and efferents to the OHCs are indicated by the green-fill arrow. All images are maximum projections from a confocal z-stack obtained at the 8.0 kHz region of a wild-type. Scale bar in C applies to all panels. Numbered arrows in C show D2-positive puncta: arrow 1 points to the first nodes of Ranvier; arrows 2 and 3 point to two regions within the inner spiral bundle, below, and at the level of, the IHC bases, respectively.

Figure 4.

Immunostaining for D1 (red) shows receptor expression in cochlear nerve terminals, especially in the inner spiral bundle (ISB) area and the region of the first nodes of Ranvier, where synaptophysin-positive terminals of the OC system are concentrated. A, Confocal xy projection shows, in surface view, a 5 μm slab of the neuropil under the IHCs (i.e., maximum projection from 20 of the 80 z-slices, centered on the ISB, as indicated by the dashed lines in B and C. D1 immunolabel is seen in cochlear nerve terminals (green), which appear in cross section in the xy projection (red-filled arrows). Approximate size and location of an IHC is shown as a dotted circle. B, C, The zy and xz projections, respectively, of all 80 sections of the z-stack. The zy projection (B) shows the IHC area shown as in a cross-section through the epithelium (e.g., Fig. 1); the xz projection (C) shows the view from the tunnel of Corti. All images are from the 8.0 kHz region of a wild-type ear. Scale bar in A applies to all panels.

Figure 5.

The cochlear duct is histologically normal in adult mice (6–8 weeks old) lacking dopamine receptors, as seen in these light micrographs of osmium-stained plastic sections through the upper basal turn in a D1 and D2 knock-out line. Scale bar applies to both panels.

Figure 6.

Deletion of D1or D2 receptors enhanced or degraded cochlear sensitivity, respectively, whereas deletion of D3, D4, or D5 had no measurable effect. Each column shows a different mutant line compared with its wild-type littermates: numbers tested for each group are in Table 2. Group means (±SEMs) are shown: thresholds versus frequency in the top rows and amplitudes versus level in the bottom rows. The blue and red shading in the top rows identifies the frequencies (8.0 and 32 kHz) shown for amplitude versus level functions in the bottom rows. For ABR amplitudes, only wave 1 was measured. Symbol keys in the first column apply to all columns. Up-arrows on some threshold points indicate that some animals in the group had thresholds at that frequency >80 dB SPL, the maximum level routinely tested.

Figure 7.

Vulnerability to acoustic injury is increased by loss of D2, D4, and D5Rs as measured 6 h after exposure to an 8–16 kHz noise band (gray bars) at 94 dB for 15 min. The mean threshold shift increase (±SEM) observed in D2 knock-outs can be measured using both ABR and DPOAE metrics; however, the increase in sensitivity to acoustic injury is revealed in D4 and D5 knock-outs only when using ABR metrics. Symbol key in the D2 panel applies to all panels. Asterisks indicate statistical significance by two-way ANOVA.

Figure 8.

Cochlear effects of activating the medial olivocochlear efferents are undiminished by deletion of dopamine receptors, except for the D2R-nulls. To assay efferent effects, DPOAE amplitudes evoked by low-level tones are measured before, during, and after delivering a 70 s shock train to the olivocochlear bundle at the floor of the IVth ventricle. Top, One “run” of the assay shows DPOAE amplitudes, normalized to the mean pre-shock value. Efferent effect is defined as the difference between the pre-shock mean and the mean DPOAE amplitude for the first three measures taken after shock-train onset. Bottom, Mean efferent effect size (±SEM) for wild-type versus dopamine receptor-null ears for each of the 5 lines tested. Asterisk (D2 only) indicates statistical significance by two-way ANOVA.