Correlation between afferent rearrangements and behavioral deficits after local excitotoxic insult in the mammalian vestibule: a rat model of vertigo symptoms

Damage to inner ear afferent terminals is believed to result in many auditory and vestibular dysfunctions. The sequence of afferent injuries and repair, as well as their correlation with vertigo symptoms, remains poorly documented. In particular, information on the changes that take place at the primary vestibular endings during the first hours following a selective insult is lacking. In the present study, we combined histological analysis with behavioral assessments of vestibular function in a rat model of unilateral vestibular excitotoxic insult. Excitotoxicity resulted in an immediate but transient alteration of the balance function that was resolved within a week. Concomitantly, vestibular primary afferents underwent a sequence of structural changes followed by spontaneous repair. Within the first two hours after the insult, a first phase of pronounced vestibular dysfunction coincided with extensive swelling of afferent terminals. In the next 24 h, a second phase of significant but incomplete reduction of the vestibular dysfunction was accompanied by a resorption of swollen terminals and fiber retraction. Eventually, within 1 week, a third phase of complete balance restoration occurred. The slow and progressive withdrawal of the balance dysfunction correlated with full reconstitution of nerve terminals. Competitive re-innervation by afferent and efferent terminals that mimicked developmental synaptogenesis resulted in full re-afferentation of the sensory epithelia. By deciphering the sequence of structural alterations that occur in the vestibule during selective excitotoxic impairment, this study offers new understanding of how a vestibular insult develops in the vestibule and how it governs the heterogeneity of vertigo symptoms

Molecular Microdomains in a Sensory Terminal, the Vestibular Calyx Ending

Many primary vestibular afferents form large cup-shaped postsynaptic terminals (calyces) that envelope the basolateral surfaces of type I hair cells. The calyceal terminals both respond to glutamate released from ribbon synapses in the type I cells and initiate spikes that propagate to the afferent’s central terminals in the brainstem. The combination of synaptic and spike initiation functions in these unique sensory endings distinguishes them from the axonal nodes of central neurons and peripheral nerves, such as the sciatic nerve, which have provided most of our information about nodal specializations. We show that rat vestibular calyces express an unusual mix of voltage-gated Na and K channels and scaffolding, cell adhesion, and extracellular matrix proteins, which may hold the ion channels in place. Protein expression patterns form several microdomains within the calyx membrane: a synaptic domain facing the hair cell, the heminode abutting the first myelinated internode, and one or two intermediate domains. Differences in the expression and localization of proteins between afferent types and zones may contribute to known variations in afferent physiology

Molecular Microdomains in a Sensory Terminal, the Vestibular Calyx Ending

Many primary vestibular afferents form large cup-shaped postsynaptic terminals (calyces) that envelope the basolateral surfaces of type I hair cells. The calyceal terminals both respond to glutamate released from ribbon synapses in the type I cells and initiate spikes that propagate to the afferent’s central terminals in the brainstem. The combination of synaptic and spike initiation functions in these unique sensory endings distinguishes them from the axonal nodes of central neurons and peripheral nerves, such as the sciatic nerve, which have provided most of our information about nodal specializations. We show that rat vestibular calyces express an unusual mix of voltage-gatedNa and K channels and scaffolding, cell adhesion, and extracellular matrix proteins, which may hold the ion channels in place. Protein expression patterns form several microdomains within the calyx membrane: a synaptic domain facing the hair cell, the heminode abutting the first myelinated internode, and one or two intermediate domains. Differences in the expression and localization of proteins between afferent types and zones may contribute to known variations in afferent physiology

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

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