Dopamine Modulates the Activity of Sensory Hair Cells

Abstract

The senses of hearing and balance are subject to modulation by efferent signaling, including the release of dopamine (DA). How DA influences the activity of the auditory and vestibular systems and its site of action are not well understood. Here we show that dopaminergic efferent fibers innervate the acousticolateralis epithelium of the zebrafish during development but do not directly form synapses with hair cells. However, a member of the D1-like receptor family, D1b, tightly localizes to ribbon synapses in inner ear and lateral-line hair cells. To assess modulation of hair-cell activity, we reversibly activated or inhibited D1-like receptors (D1Rs) in lateral-line hair cells. In extracellular recordings from hair cells, we observed that D1R agonist SKF-38393 increased microphonic potentials, whereas D1R antagonist SCH-23390 decreased microphonic potentials. Using ratiometric calcium imaging, we found that increased D1R activity resulted in larger calcium transients in hair cells. The increase of intracellular calcium requires Cav1.3a channels, as a Cav1 calcium channel antagonist, isradipine, blocked the increase in calcium transients elicited by the agonist SKF-38393. Collectively, our results suggest that DA is released in a paracrine fashion and acts at ribbon synapses, likely enhancing the activity of presynaptic Cav1.3a channels and thereby increasing neurotransmission.

Significance statement: The neurotransmitter dopamine acts in a paracrine fashion (diffusion over a short distance) in several tissues and bodily organs, influencing and regulating their activity. The cellular target and mechanism of the action of dopamine in mechanosensory organs, such as the inner ear and lateral-line organ, is not clearly understood. Here we demonstrate that dopamine receptors are present in sensory hair cells at synaptic sites that are required for signaling to the brain. When nearby neurons release dopamine, activation of the dopamine receptors increases the activity of these mechanosensitive cells. The mechanism of dopamine activation requires voltage-gated calcium channels that are also present at hair-cell synapses.

Keywords: D1 receptor; dopamine; efferents; hair cell; lateral-line organ; zebrafish.

Figure 1.

Innervation of lateral-line hair cells by dopaminergic fibers during early development. Efferent and afferent fibers were visualized in developing embryos by crossing two transgenic lines: Tg(slc6a3:EGFP) and Tg(neurod:tdTomato). A, Faint GFP expression is detected within the head at 1 dpf (arrowhead). B, tdTomato-positive fibers representing cranial and lateral-line afferent neurons at 1 dpf. Arrow indicates the lateral-line nerve extending down the trunk. C, Overlays of A (green) and B (magenta). C′, Overlay that includes the corresponding DIC image. D, Presence of a GFP-positive efferent fiber innervating the first trunk neuromast (L1) near the swim bladder at 2 dpf. E, Corresponding afferent fibers for the neuromast in D. F, F′, Overlays of D and E, respectively. G, Example of an efferent growth cone trailing after the afferent fibers (anterior posterior axis indicated by arrow). H, Corresponding afferent fibers for the same image shown in G (2 dpf). I, I′, Overlays of G and H, respectively. Scale bar: A–C′, 20 μm; D–F′, 5 μm; G–I′, 30 μm.

Figure 2.

Dopaminergic innervation at larval stages suggests that modulation by DA is global. A, Overview of the GFP signal in the head of a Tg(slc6a3:EGFP) larva (5 dpf). Autofluorescence of pigment cells is apparent in the skin and swim bladder. Arrowheads indicate GFP-positive descending fibers. A′, Higher-magnification view of the fibers in A. A total of two descending axons are present. B, Image of the GFP-positive fibers along the midline of the larval trunk with an example of a branch that innervates a posterior neuromast (arrow). Arrowheads indicate descending fibers. Additional GFP-labeled fibers are visible in the spinal cord. B′, B″, Higher-magnification view of the dopaminergic fibers within the neuroepithelium of the neuromast indicated by the arrow in B. C, A GFP-positive fiber extends from neuromast to neuromast near the eye. Also shown are numerous cell bodies in the midbrain region. Asterisks indicate two additional anterior neuromasts. C′, C″, Higher-magnification view of the neuromast indicated by the arrow in C. D, D′, Lateral view of a posterior crista within the inner ear of a 3 dpf larva. PTv, ventral posterior tuberculum; pc, posterior crista. Scale bars: A, 50 μm; A′, 10 μm; B, C, 20 μm; B′–D′, 10 μm.

Figure 3.

Dopaminergic fibers are located beneath the hair-cell layer in neuromasts. A–D, Top-down view of a neuromast. Hair cells are stably expressing tdTomato (magenta), whereas DA efferent fibers express GFP (green). E–H, Side view of a neuromast. Efferent fibers are closely positioned near the basal ends of hair cells but do not appear to make direct synapses. GFP-positive varicosities appear randomly with respect to hair-cell position. I–L, Top-down view of the extensive pattern of afferent fiber innervation (magenta) in contrast to the relatively sparse innervation by DA efferents (green). DA fibers track in parallel with a subset of afferent fibers. M–P, Immunolabel of the synaptophysin (Syp) synaptic vesicle marker indicates vesicles within the varicosities of the GFP-positive fibers (arrows) and also indicates clusters of vesicles within other types of efferents. Note the absence of juxtaposition of non-DA efferent vesicles (magenta only) and dopaminergic fibers. B–D, J–L, Maximum projections. F–H, 3D reconstructions of maximum projections. A, E, I, M–P, Single optical slices. Scale bars: A–P, 5 μm.

Figure 4.

Expression of DA receptor subtypes in zebrafish. A, RT-PCR of drd genes from adult inner ear cDNA that includes both epithelial and neuronal cell types. B, RT-PCR of drd genes in neuromast and whole larval cDNA. DNA markers are present in the first lane, followed by positive controls (gapdh and pcdh15a or cdh23). C, Diagram of D1b protein depicting the location of the amino acid sequences used to generate monoclonal antibodies. D, Immunolabel (SMGNNASMES mAb) of the head of a 5 dpf larva. The focal plane includes the dorsal region of the fish head, and signals are prominent within the forebrain, tectal, and hindbrain regions. Specific regions are indicated. Melanocytes partially occlude the labeling in deeper medial areas of the brain. OB, Olfactory bulb; OE, olfactory epithelium; OT, optic tectum; P, pallium; RL, rhombic lip. E–F, I–J, Immunolabel of neuromast epithelia with D1b mAbs. DIC image (single optical plane) and corresponding immunofluorescence for KKEDDSGIKT (E, F) and SMGNNASMES mAbs (I, J; maximum projections are shown). Colocalization with Ribeye a is shown in G, K. H, H′, L, L′, Peptide blocking experiments with KKEDDSGIKT and SMGNNASMES peptides using the corresponding D1b antibodies. Ribeye a antibody was included as a control in H′, L′. Scale bars: D, 75 μm; E–L′, 5 μm.

Figure 5.

Subcellular localization of D1b at ribbon synapses in inner ear and lateral-line hair cells (5 dpf). Punctate pattern of D1b immunolabel in a macular endorgan (utricle) of the inner ear in A, and lateral-line hair cells in D, G, J, M. B, E, Ribeye a immunolabel of hair cell ribbons. C, C′, F, F′, Overlap of D1b (green) and Ribeye a (magenta) staining in the inner ear and lateral-line organ. G–I′, D1b immunolabel (green) is juxtaposed to afferent fiber labeling (magenta). J–L′, D1b puncta (green) are restricted to the Vglut3-positive (magenta) basal half of hair cells. M–O′, Absence of juxtaposition of DA efferent fibers (green) with D1b puncta (magenta). A–C, Single optical slices; all other images are maximum projections. KKEDDSGIKT mAb was used for G–I′; all other panels are immunolabel obtained with SMGNNASMES mAb. Arrows indicate the D1b puncta shown in C′, F′, I′, L′, O′. Scale bars: A–O, 5 μm; C′, F′, I′, L′, O′, 0.5 μm.

Figure 6.

D1R modulators alter hair-cell activity. FM1–43 labeling of hair cells and microphonic potentials recorded from posterior lateral-line neuromasts were quantified to probe the effect of D1R activity on hair-cell function. For recordings, hair cells were stimulated with a 200 ms, 20 Hz sinusoidal wave. A, Percentage reduction of FM1–43 labeling in response to four concentrations of SCH-23390 antagonist. B, Normalized average microphonic amplitude for hair cells in control external solution (CON), in the presence of 100 nm D1R agonist SKF-38393 (SKF), and after replacement of SKF solution with control solution (WASH). Data from 7 fish and 8 sets of hair cells are shown (N = 8). C, As in B, except in the presence of 10 μm D1R antagonist SCH-23390. Data from 7 fish and 10 sets of hair cells are shown (N = 10). D, Average values from data in B, C. Significance between control and drug or wash was tested using one-way ANOVA with Dunnett's multiple-comparisons post test. *p = 0.01–0.05. **p = 0.001–0.01. ns, Not significant. B, C, Insets, Examples of averaged microphonics from a single neuromast. Calibration: 2 μV versus 100 ms.

Figure 7.

The increase in calcium transients elicited by the D1R agonist SKF-38393 is dependent upon Cav1.3a activity. Calcium transients were ratiometrically imaged in the hair cells of Tg(myo6b:D3) larvae. Hair cells were mechanically stimulated with a 2 s step stimulus. A, Averaged traces with shaded SE for DMSO control (blue), 100 nm SKF-38393 treated (purple), and agonist treated and washed (green) hair cells. Duration of the 2 s stimulus is indicated by gray area. B, Data from 19 individual hair cells. C, Averaged traces with shaded SE for DMSO control (blue), 10 μm isradipine treated (mint), and isradipine pretreated cells exposed to 100 nm SKF-38393 (purple). D, Data from 9 individual hair cells. E, Mean amplitudes (Maximum FRET Ratio) of evoked calcium transients in control and drug-treated hair cells. Con versus SKF, p = 0.0011; Con versus SCH, p = 0.0058; ISR versus ISR + SKF, p = 0.18. ** p < 0.01; ns, Not significant.