Figure 1.
Transporter-mediated chloride conductance in partially isolated type I vestibular hair cells (VHCs).
(A) Differential interference contrast micrograph of a recording electrode tip approaching the base of an amphora-shaped, type I utricular hair cell. (B) Electrophysiological protocol used to identify type I hair cells (upper traces) versus type II hair cells (bottom traces). As previously described [27], only type I cells exhibit IKL, an outwardly rectifying current activated at rest that was evidenced by an instantaneous current upon stepping to higher voltages (leftmost arrow) and deactivated by hyperpolarization (rightmost arrow). (C) Inward current evoked in different type I VHCs voltage-clamped at −80 mV upon application of glutamate (uppermost trace); inward current enhanced by substitution of thiocyanate (SCN–) for chloride (second trace); the glutamate-evoked current was blocked following application of dl-TBOA (third trace). Application of glutamate did not evoke a current in type II VHCs (bottom trace). (D) 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 Mann-Whitney U comparisons (p<0.05 for glutamate, 100 µM vs 1 mM and 1 mM Cl- vs 1 mM SCN-); sample sizes (n) in parentheses. (E) 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. (F) Current-voltage relationship of glutamate-evoked responses obtained with ECl = −0.5 mV (n = 7) and ECl = −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.
Figure 2.
EAAT4 and EAAT5 mRNA expression in mouse tissue.
(A) 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, D) In situ hybridization using EAAT4- or EAAT5-specific antisense probes in the utricular macula. Both EAAT4 and EAAT5 mRNA are found in the hair-cell layer (HC), but not in the supporting-cell layer (SC). (C, E) Specific sense controls showed no labeling. Scale bars, 20 µm (B–E).
Figure 3.
EAAT4 (A, C, E) and EAAT5 (B, D, F) protein localization in mouse tissue.
Western blots using antibodies against EAAT4 (A, ∼61 kD) or EAAT5 (B, ∼62kD). EAAT4 is expressed in the cerebellum (Cbl), vestibular ganglia (VG) and vestibular organs (VE). EAAT5 is expressed in the retina (Rt), vestibular ganglia (VG) and vestibular organs (VE). Immunohistochemistry on utricular sections shows that EAAT4 (C) and EAAT5 (D) labeling (red, top and bottom panels) is conspicuous below the nuclei of both types of hair cells (I, II), where ribbon synapses are abundant. Calretinin labeling (green, top panels) marks type II hair cells (II) [33], [45], [46]. Immunohistochemistry of vestibular ganglion cells shows weak EAAT4 (E) and EAAT5 (F) labeling. Scale bars: 10 µm.
Figure 4.
In the EAAT4-eGFP mouse, the expression of eGFP is under the control of the EAAT4 promoter[23].
The GFP fluorescence is observed in type I (white arrows) and type II hair cells using an anti-eGFP antibody with phalloidin red labeling of hair bundles. Scale bar: 10 µm.
Figure 5.
EAAT4 and EAAT5 protein localization in mouse tissue.
Electron micrographs of EAAT4 (A) and EAAT5 (B) immunogold labeling particles on hair-cell membrane (arrows), calyx inner-face membrane (arrowheads) and calyx outer-face membrane (arrow, lower right in A). 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.
Table 1.
Immunogold densities, EAAT antibodies, type I hair cell and calyx ending.
Figure 6.
Schematic representation of EAAT4 and EAAT5 expression in the mammalian vestibular neuroepithelium.
EAAT4 and EAAT5 are expressed in type I (right) and type II (left) 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 [22].