Calyx synapses glutamatergic homeostasis in the vestibular system : implication of several EAAT family glutamate transporters

L'homéostasie glutamatergique dans les fentes synaptiques régule la neurotransmission et préserve de l'excitotoxicité. Cela est particulièrement important dans l'oreille interne où il y a une libération soutenue de neurotransmetteur. Pour la plupart des cellules ciliées cochléaires et vestibulaires, la clairance du glutamate est assurée par les transporteurs du glutamate EAAT1 (GLAST) exprimés par les cellules de soutien. Un tel mécanisme n'est pas possible pour les cellules ciliées vestibulaires de type I car leur terminaison synaptique en calice empêche tout accès à la fente synaptique. Nous avons donc postulé qu'un ou plusieurs transporteurs du glutamate devaient être présents au niveau des cellules ciliées de type I ou du calice ou des deux.Grâce à des enregistrements électrophysiologiques, nous avons démontré qu'un courant anionique induit par le glutamate et bloqué par le DL-TBOA est présent dans les cellules ciliées de type I. Les techniques d'hybridation in situ et d'immunohistochimie ont révélé la présence d'EAAT4 et EAAT5. Ces deux transporteurs du glutamate, qui pourraient êtres à l'origine des courants enregistrés, sont exprimés par les cellules ciliées de type I et de type II. De plus, des expériences de RT-PCR et de microscopie électronique ont confirmé ces résultats et suggéré que ces transporteurs pourraient aussi être exprimés postsynaptiquement par le calice. Ces travaux de thèse montrent qu'EAAT4 et EAAT5, considérés respectivement comme spécifiques des tissus cérébelleux et rétiniens, ont une distribution plus large. Ces résultats posent la question des rôles potentiels de ces transporteurs dans l'homéostasie glutamatergique vestibulaire.Glutamate homeostasis in synaptic clefts shape neurotransmission and prevent excitotoxicity. This may be particularly important in the inner ear where there is a continually high rate of neurotransmitter release. In the case of most cochlear and vestibular hair cells, clearance involves the diffusion of glutamate to supporting cells, where it is taken up by EAAT1 (GLAST), a glial glutamate transporter. A similar mechanism is unlikely to work in vestibular type I hair cells because the presence of calyx endings separates supporting cells from the synaptic zone. Based on this arrangement, we postulated that a glutamate transporter must be present in the type I hair cell, the calyx ending, or both. Using whole-cell patch-clamp recordings, we demonstrated that a glutamate-activated anion current blocked by DL-TBOA is expressed in type I hair cells. In situ hybridization and immunohistochemistry revealed that EAAT4 and EAAT5, two glutamate transporters that could support the anion current, are expressed in both type I and type II hair cells. Furthermore, RT-PCR and immunogold investigations confirmed those results and added that although preferentially expressed presynaptically, the transporters may also be present in the postsynaptic calyx membrane. Previously thought to be exclusively expressed in the cerebellum and retina respectively, this thesis work shows that EAAT4 and EAAT5 have a wider distribution. The potential role of these transporters in the glutamatergic homeostasis of the calyx synapse is then discussed

Homéostasie glutamatergique des synapses en calice de l'appareil vestibulaire (implication de plusieurs transporteurs du glutamate de la famille des EAAT)

L'homéostasie glutamatergique dans les fentes synaptiques régule la neurotransmission et préserve de l'excitotoxicité. Cela est particulièrement important dans l'oreille interne où il y a une libération soutenue de neurotransmetteur. Pour la plupart des cellules ciliées cochléaires et vestibulaires, la clairance du glutamate est assurée par les transporteurs du glutamate EAAT1 (GLAST) exprimés par les cellules de soutien. Un tel mécanisme n'est pas possible pour les cellules ciliées vestibulaires de type I car leur terminaison synaptique en calice empêche tout accès à la fente synaptique. Nous avons donc postulé qu'un ou plusieurs transporteurs du glutamate devaient être présents au niveau des cellules ciliées de type I ou du calice ou des deux.Grâce à des enregistrements électrophysiologiques, nous avons démontré qu'un courant anionique induit par le glutamate et bloqué par le DL-TBOA est présent dans les cellules ciliées de type I. Les techniques d'hybridation in situ et d'immunohistochimie ont révélé la présence d'EAAT4 et EAAT5. Ces deux transporteurs du glutamate, qui pourraient êtres à l'origine des courants enregistrés, sont exprimés par les cellules ciliées de type I et de type II. De plus, des expériences de RT-PCR et de microscopie électronique ont confirmé ces résultats et suggéré que ces transporteurs pourraient aussi être exprimés postsynaptiquement par le calice. Ces travaux de thèse montrent qu'EAAT4 et EAAT5, considérés respectivement comme spécifiques des tissus cérébelleux et rétiniens, ont une distribution plus large. Ces résultats posent la question des rôles potentiels de ces transporteurs dans l'homéostasie glutamatergique vestibulaire.Glutamate homeostasis in synaptic clefts shape neurotransmission and prevent excitotoxicity. This may be particularly important in the inner ear where there is a continually high rate of neurotransmitter release. In the case of most cochlear and vestibular hair cells, clearance involves the diffusion of glutamate to supporting cells, where it is taken up by EAAT1 (GLAST), a glial glutamate transporter. A similar mechanism is unlikely to work in vestibular type I hair cells because the presence of calyx endings separates supporting cells from the synaptic zone. Based on this arrangement, we postulated that a glutamate transporter must be present in the type I hair cell, the calyx ending, or both. Using whole-cell patch-clamp recordings, we demonstrated that a glutamate-activated anion current blocked by DL-TBOA is expressed in type I hair cells. In situ hybridization and immunohistochemistry revealed that EAAT4 and EAAT5, two glutamate transporters that could support the anion current, are expressed in both type I and type II hair cells. Furthermore, RT-PCR and immunogold investigations confirmed those results and added that although preferentially expressed presynaptically, the transporters may also be present in the postsynaptic calyx membrane. Previously thought to be exclusively expressed in the cerebellum and retina respectively, this thesis work shows that EAAT4 and EAAT5 have a wider distribution. The potential role of these transporters in the glutamatergic homeostasis of the calyx synapse is then discussed.MONTPELLIER-BU Sciences (341722106) / SudocSudocFranceF

Glutamate transporters EAAT4 and EAAT5 are expressed in vestibular hair cells and calyx endings.

Glutamate is the neurotransmitter released from hair cells. Its clearance from the synaptic cleft can shape neurotransmission and prevent excitotoxicity. This may be particularly important in the inner ear and in other sensory organs where there is a continually high rate of neurotransmitter release. In the case of most cochlear and type II vestibular hair cells, clearance involves the diffusion of glutamate to supporting cells, where it is taken up by EAAT1 (GLAST), a glutamate transporter. A similar mechanism cannot work in vestibular type I hair cells as the presence of calyx endings separates supporting cells from hair-cell synapses. Because of this arrangement, it has been conjectured that a glutamate transporter must be present in the type I hair cell, the calyx ending, or both. Using whole-cell patch-clamp recordings, we demonstrate that a glutamate-activated anion current, attributable to a high-affinity glutamate transporter and blocked by DL-TBOA, is expressed in type I, but not in type II hair cells. Molecular investigations reveal that EAAT4 and EAAT5, two glutamate transporters that could underlie the anion current, are expressed in both type I and type II hair cells and in calyx endings. EAAT4 has been thought to be expressed almost exclusively in the cerebellum and EAAT5 in the retina. Our results show that these two transporters have a wider distribution in mice. This is the first demonstration of the presence of transporters in hair cells and provides one of the few examples of EAATs in presynaptic elements

Glutamate Transporters EAAT4 and EAAT5 Are Expressed in Vestibular Hair Cells and Calyx Endings

Glutamate is the neurotransmitter released from hair cells. Its clearance from the synaptic cleft can shape neurotransmission and prevent excitotoxicity. This may be particularly important in the inner ear and in other sensory organs where there is a continually high rate of neurotransmitter release. In the case of most cochlear and type II vestibular hair cells, clearance involves the diffusion of glutamate to supporting cells, where it is taken up by EAAT1 (GLAST), a glutamate transporter. A similar mechanism cannot work in vestibular type I hair cells as the presence of calyx endings separates supporting cells from hair-cell synapses. Because of this arrangement, it has been conjectured that a glutamate transporter must be present in the type I hair cell, the calyx ending, or both. Using whole-cell patch-clamp recordings, we demonstrate that a glutamate-activated anion current, attributable to a high-affinity glutamate transporter and blocked by DL-TBOA, is expressed in type I, but not in type II hair cells. Molecular investigations reveal that EAAT4 and EAAT5, two glutamate transporters that could underlie the anion current, are expressed in both type I and type II hair cells and in calyx endings. EAAT4 has been thought to be expressed almost exclusively in the cerebellum and EAAT5 in the retina. Our results show that these two transporters have a wider distribution in mice. This is the first demonstration of the presence of transporters in hair cells and provides one of the few examples of EAATs in presynaptic elements.</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

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 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