Dopamine Modulates the Activity of Sensory Hair Cells

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.

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.

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.

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.

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.