EAAT4 and EAAT5 protein localization in mouse tissue.

<p>Electron micrographs of EAAT4 (<b>A</b>) and EAAT5 (<b>B</b>) immunogold labeling particles on hair-cell membrane <i>(arrows),</i> calyx inner-face membrane <i>(arrowheads)</i> and calyx outer-face membrane <i>(arrow</i>, lower right in <i>A).</i> In both panels from top to bottom, the darkened area is a hair-cell nucleus rimmed by hair-cell cytoplasm, hair-cell and calyx inner-face membranes. The lightened area with gray mitochondria is a calyx ending whose outer-face abuts supporting cells. Scale bars: 0.5 µm.</p

Schematic representation of EAAT4 and EAAT5 expression in the mammalian vestibular neuroepithelium.

<p>EAAT4 and EAAT5 are expressed in type I (<i>right</i>) and type II (<i>left</i>) hair cells and on the calyx inner face. EAAT4, but not EAAT5, is also expressed on the calyx outer face. EAAT5 may have a higher expression in type I versus type II hair cells. Both transporters are preferentially expressed in the subnuclear region of hair cells. EAAT1 is expressed in supporting cells <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046261#pone.0046261-Takumi1" target="_blank">[22]</a>.</p

In the EAAT4-eGFP mouse, the expression of eGFP is under the control of the EAAT4 promoter[23].

<p>The GFP fluorescence is observed in type I <i>(white arrows)</i> and type II hair cells using an anti-eGFP antibody with phalloidin red labeling of hair bundles. Scale bar: 10 µm.</p

EAAT4 (<i>A, C, E</i>) and EAAT5 (<i>B, D, F</i>) protein localization in mouse tissue.

<p>Western blots using antibodies against EAAT4 (<b>A</b>, ∼61 kD) or EAAT5 (<b>B</b>, ∼62kD). EAAT4 is expressed in the cerebellum (<i>Cbl</i>), vestibular ganglia (<i>VG</i>) and vestibular organs (<i>VE</i>). EAAT5 is expressed in the retina (<i>Rt</i>), vestibular ganglia (<i>VG</i>) and vestibular organs (<i>VE</i>). Immunohistochemistry on utricular sections shows that EAAT4 (<b>C</b>) and EAAT5 (<b>D</b>) labeling (red, top and bottom panels) is conspicuous below the nuclei of both types of hair cells (<i>I, II</i>), where ribbon synapses are abundant. Calretinin labeling (green, top panels) marks type II hair cells (II) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046261#pone.0046261-Lysakowski2" target="_blank">[33]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046261#pone.0046261-Desai1" target="_blank">[45]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046261#pone.0046261-Desai2" target="_blank">[46]</a>. Immunohistochemistry of vestibular ganglion cells shows weak EAAT4 (<b>E</b>) and EAAT5 (<b>F</b>) labeling. Scale bars: 10 µm.</p

EAAT4 and EAAT5 mRNA expression in mouse tissue.

<p>(<b>A</b>) Retina (Ret), vestibular epithelia (VE), or vestibular ganglion (VG) were pooled from ten mice. RT-PCR using substrate-specific primers show expression of both EAAT4 (first panel, 329 bp) and EAAT5 (second panel, 246 bp) in various tissues. Actin PCR was used as a control (third panel, 539 bp). (<b>B, D</b>) <i>In situ</i> hybridization using EAAT4- or EAAT5-specific antisense probes in the utricular macula. Both EAAT4 and EAAT5 mRNA are found in the hair-cell layer (<i>HC</i>), but not in the supporting-cell layer (<i>SC</i>). (<b>C, E</b>) Specific sense controls showed no labeling. Scale bars, 20 µm (B–E).</p

Transporter-mediated chloride conductance in partially isolated type I vestibular hair cells (<i>VHCs</i>).

<p>(<b>A</b>) Differential interference contrast micrograph of a recording electrode tip approaching the base of an amphora-shaped, type I utricular hair cell. (<b>B</b>) Electrophysiological protocol used to identify type I hair cells (<i>upper traces</i>) versus type II hair cells (<i>bottom traces</i>). As previously described <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046261#pone.0046261-Gaboyard1" target="_blank">[27]</a>, only type I cells exhibit <i>I_KL</i>, an outwardly rectifying current activated at rest that was evidenced by an instantaneous current upon stepping to higher voltages (<i>leftmost arrow</i>) and deactivated by hyperpolarization <i>(rightmost arrow)</i>. (<b>C</b>) Inward current evoked in different type I VHCs voltage-clamped at −80 mV upon application of glutamate <i>(uppermost trace)</i>; inward current enhanced by substitution of thiocyanate (<i>SCN^–</i>) for chloride <i>(second trace)</i>; the glutamate-evoked current was blocked following application of dl-TBOA <i>(third trace)</i>. Application of glutamate did not evoke a current in type II VHCs <i>(bottom trace)</i>. (<b>D</b>) Glutamate activated current under different conditions (glutamate or aspartate at various concentrations, chloride or thiocyanate anion intracellularly, and type I or type II VHCs). Each bar represents mean ± sd. Brackets with asterisk indicate <i>Mann-Whitney U</i> comparisons (<i>p</i><0.05 for glutamate, 100 µM vs 1 mM and 1 mM Cl^- vs 1 mM SCN^-); sample sizes (<i>n</i>) in parentheses. (<b><i>E</i></b>) Currents evoked by 1 mM glutamate application at various holding potentials on a type I VHC, from −120 to +20 mV in 20 mV steps. (<b>F</b>) Current-voltage relationship of glutamate-evoked responses obtained with <i>E</i>_Cl  =  −0.5 mV (n = 7) and <i>E</i>_Cl  =  −30.3 mV (n = 5). Peak currents at each holding potential were normalized to responses at −80 mV. Dots represent mean ± sem. When no bar is shown, the SEM was too small.</p