Maturation of type I and type II rat vestibular hair cells in vivo and in vitro.

Vestibular sensory epithelia contain type I and type II sensory hair cells (HCI and HCII). Recent studies have revealed molecular markers for the identification of these cells, but the precise composition of each vestibular epithelium (saccule, utricle, lateral crista, anterior crista, posterior crista) and their postnatal maturation have not been described in detail. Moreover, in vitro methods to study this maturation are not well developed. We obtained total HCI and HCII counts in adult rats and studied the maturation of the epithelia from birth (P0) to postnatal day 28 (P28). Adult vestibular epithelia hair cells were found to comprise ∼65% HCI expressing osteopontin and PMCA2, ∼30% HCII expressing calretinin, and ∼4% HCII expressing SOX2 but neither osteopontin nor calretinin. At birth, immature HCs express both osteopontin and calretinin. P28 epithelia showed an almost adult-like composition but still contained 1.3% of immature HCs. In addition, we obtained free-floating 3D cultures of the epithelia at P1, which formed a fluid-filled cyst, and studied their survival and maturation in vitro up to day 28 (28 DIV). These cultures showed good HC resiliency and maturation. Using an enriched medium for the initial 4 days, a HCI/calretinin+-HCII ratio close to the in vivo ratio was obtained. These cultures are suitable to study HC maturation and mature HCs in pharmacological, toxicological and molecular research.

The vestibular calyceal junction is dismantled following subchronic streptomycin in rats and sensory epithelium stress in humans.

Hair cell (HC) loss by epithelial extrusion has been described to occur in the rodent vestibular system during chronic 3,3'-iminodipropionitrile (IDPN) ototoxicity. This is preceded by dismantlement of the calyceal junction in the contact between type I HC (HCI) and calyx afferent terminals. Here, we evaluated whether these phenomena have wider significance. First, we studied rats receiving seven different doses of streptomycin, ranging from 100 to 800 mg/kg/day, for 3-8 weeks. Streptomycin caused loss of vestibular function associated with partial loss of HCI and decreased expression of contactin-associated protein (CASPR1), denoting calyceal junction dismantlement, in the calyces encasing the surviving HCI. Additional molecular and ultrastructural data supported the conclusion that HC-calyx detachment precede HCI loss by extrusion. Animals allowed to survive after the treatment showed functional recuperation and rebuilding of the calyceal junction. Second, we evaluated human sensory epithelia obtained during therapeutic labyrinthectomies and trans-labyrinthine tumour excisions. Some samples showed abnormal CASPR1 label strongly suggestive of calyceal junction dismantlement. Therefore, reversible dismantlement of the vestibular calyceal junction may be a common response triggered by chronic stress, including ototoxic stress, before HCI loss. This may partly explain clinical observations of reversion in function loss after aminoglycoside exposure.

Decreased expression of synaptic genes in the vestibular ganglion of rodents following subchronic ototoxic stress.

The vestibular ganglion contains primary sensory neurons that are postsynaptic to the transducing hair cells (HC) and project to the central nervous system. Understanding the response of these neurons to HC stress or loss is of great interest as their survival and functional competence will determine the functional outcome of any intervention aiming at repair or regeneration of the HCs. We have shown that subchronic exposure to the ototoxicant 3,3'-iminodipropionitrile (IDPN) in rats and mice causes a reversible detachment and synaptic uncoupling between the HCs and the ganglion neurons. Here, we used this paradigm to study the global changes in gene expression in vestibular ganglia using RNA-seq. Comparative gene ontology and pathway analyses of the data from both model species indicated a robust downregulation of terms related to synapses, including presynaptic and postsynaptic functions. Manual analyses of the most significantly downregulated transcripts identified genes with expressions related to neuronal activity, modulators of neuronal excitability, and transcription factors and receptors that promote neurite growth and differentiation. For choice selected genes, the mRNA expression results were replicated by qRT-PCR, validated spatially by RNA-scope, or were demonstrated to be associated with decreased expression of the corresponding protein. We conjectured that decreased synaptic input or trophic support on the ganglion neurons from the HC was triggering these expression changes. To support this hypothesis, we demonstrated decreased expression of BDNF mRNA in the vestibular epithelium after subchronic ototoxicity and also downregulated expression of similarly identified genes (e.g Etv5, Camk1g, Slc17a6, Nptx2, Spp1) after HC ablation with another ototoxic compound, allylnitrile. We conclude that vestibular ganglion neurons respond to decreased input from HCs by decreasing the strength of all their synaptic contacts, both as postsynaptic and presynaptic players.