Christian Chabbert

TRPV4 channels mediate the infrared laser-evoked response in sensory neurons.

Infrared laser irradiation has been established as an appropriate stimulus for primary sensory neurons under conditions where sensory receptor cells are impaired or lost. Yet, development of clinical applications has been impeded by lack of information about the molecular mechanisms underlying the laser-induced neural response. Here, we directly address this question through pharmacological characterization of the biological response evoked by midinfrared irradiation of isolated retinal and vestibular ganglion cells from rodents. Whole cell patch-clamp recordings reveal that both voltage-gated calcium and sodium channels contribute to the laser-evoked neuronal voltage variations (LEVV). In addition, selective blockade of the LEVV by micromolar concentrations of ruthenium red and RN 1734 identifies thermosensitive transient receptor potential vanilloid channels as the primary effectors of the chain reaction triggered by midinfrared laser irradiation. These results have the potential to facilitate greatly the design of future prosthetic devices aimed at restoring neurosensory capacities in disabled patients.

Salicylate Enables Cochlear Arachidonic-Acid-Sensitive NMDA Receptor Responses

Currently, many millions of people treated for various ailments receive high doses of salicylate. Consequently, understanding the mechanisms by which salicylate induces tinnitus is an important issue for the research community. Behavioral testing in rats have shown that tinnitus induced by salicylate or mefenamate (both cyclooxygenase blockers) are mediated by cochlear NMDA receptors. Here we report that the synapses between the sensory inner hair cells and the dendrites of the cochlear spiral ganglion neurons express NMDA receptors. Patch-clamp recordings and two-photon calcium imaging demonstrated that salicylate and arachidonate (a substrate of cyclooxygenase) enabled the calcium flux and the neural excitatory effects of NMDA on cochlear spiral ganglion neurons. Salicylate also increased the arachidonate content of the whole cochlea in vivo. Single-unit recordings of auditory nerve fibers in adult guinea pig confirmed the neural excitatory effect of salicylate and the blockade of this effect by NMDA antagonist. These results suggest that salicylate inhibits cochlear cyclooxygenase, which increased levels of arachidonate. The increased levels of arachidonate then act on NMDA receptors to enable NMDA responses to glutamate that inner hair cells spontaneously release. This new pharmacological profile of salicylate provides a molecular mechanism for the generation of tinnitus at the periphery of the auditory system.

Molecular diversity of voltage-gated sodium channel alpha subunits expressed in neuronal and non-neuronal excitable cells

In order to investigate the role of molecular diversity of voltage-activated sodium channel alpha-subunits in excitability of neuronal and non-neuronal cells, we carried out patch-clamp recordings and single-cell RT-PCR on two different types of mammalian excitable cells i.e. hippocampal neurons and non-neuronal utricular epithelial hair cells. In each cell type, multiple different combinations of sodium channel alpha-subunits exist from cell to cell despite similar sodium current properties. The mRNA isoforms, Nav1.2 and Nav1.6, are the most frequently detected by single cell analysis in the two cell types while Nav1.3 and Nav1.7 are also moderately expressed in embryonic hippocampal neurons and in neonatal utricular hair cells respectively. By investigating the particular alternate splice isoforms of Nav1.6 occurring at the exon 18 of the mouse orthologue SCN8A, we revealed that this subunit co-exist in the two cell types under different alternative spliced isoforms. The expression of non-functional isoforms of Nav1.6 in utricular epithelial hair cells excludes the involvement of this subunit in supporting their excitability. Thus, from a functional point of view, the present results suggest that, at the single cell level, both neuronal and non-neuronal excitable cells expressed different and complex patterns of sodium channel gene transcripts but this diversity alone cannot explain the sodium current properties of these cell types.

Developmental changes in low and high voltage‐activated calcium currents in acutely isolated mouse vestibular neurons

1 The development of low voltage‐activated (LVA) and high voltage‐activated (HVA) calcium currents was studied in neurons acutely dissociated from mouse vestibular ganglia at embryonic stages (E)14, 15, 17 and birth using the whole‐cell patch‐clamp technique. 2 LVA current was present in almost all neurons tested at stages E14 to E17, although at birth this current was restricted to a few neurons. Two populations of neurons were characterized based on the amplitude of the LVA current. In the first population, LVA current densities decreased between E17 and birth by which time this current tended to disappear in most neurons. A second population of neurons with high density LVA current appeared at E17, and in this group the mean density increased during development. 3 Among HVA currents, the dihydropyridine‐sensitive L‐type current remained constant between E15 and birth. Over the same period, the density of N‐ and Q‐type currents continuously increased as shown using ω‐conotoxin‐GVIA (N‐type), and high concentrations of ω‐agatoxin‐IVA (Q‐type). The P‐type current, sensitive to low concentrations of ω‐agatoxin‐IVA, transiently increased between E15 and E17, and then both current density and its proportion of the global current decreased. 4 Our results reveal large modifications in the expression of voltage‐dependent calcium channels during embryonic development of primary vestibular neurons. The changes in the expression of LVA current and the transient augmentation of P‐type HVA current occur during a period characterized by massive neuronal growth and by the beginning of synaptogenesis. These results suggest a specific role of these currents in the ontogenesis of vestibular primary afferents.

Voltage‐Gated Na+ Channel Activation Induces Both Action Potentials in Utricular Hair Cells and Brain‐Derived Neurotrophic Factor Release in the Rat Utricle During a Restricted Period of Development

The mammalian utricular sensory receptors are commonly believed to be non‐spiking cells with electrical activity limited to graded membrane potential changes. Here we provide evidence that during the first post‐natal week, the sensory hair cells of the rat utricle express a tetrodotoxin (TTX)‐sensitive voltage‐gated Na+ current that displays most of the biophysical and pharmacological characteristics of neuronal Na+ current. Single‐cell RT‐PCR reveals that several α‐subunit isoforms of the Na+ channels are co‐expressed within a single hair cell, with a major expression of Nav1.2 and Nav1.6 subunits. In neonatal hair cells, 30 % of the Na+ channels are available for activation at the resting potential. Depolarizing current injections in the range of the transduction currents are able to trigger TTX‐sensitive action potentials. We also provide evidence of a TTX‐sensitive activity‐dependent brain‐derived neurotrophic factor (BDNF) release by early post‐natal utricle explants. Developmental analysis shows that Na+ currents decrease dramatically from post‐natal day 0 (P0) to P8 and become almost undetectable at P21. Concomitantly, depolarizing stimuli fail to induce both action potential and BDNF release at P20. The present findings reveal that vestibular hair cells express neuronal‐like TTX‐sensitive Na+ channels able to generate Na+‐driven action potentials only during the early post‐natal period of development. During the same period an activity‐dependent BDNF secretion by utricular explants has been demonstrated. This could be an important mechanism involved in vestibular sensory system differentiation and synaptogenesis.

Evaluation of the chemical model of vestibular lesions induced by arsanilate in rats.

Several animal models of vestibular deficits that mimic the human pathology phenotype have previously been developed to correlate the degree of vestibular injury to cognate vestibular deficits in a time-dependent manner. Sodium arsanilate is one of the most commonly used substances for chemical vestibular lesioning, but it is not well described in the literature. In the present study, we used histological and functional approaches to conduct a detailed exploration of the model of vestibular lesions induced by transtympanic injection of sodium arsanilate in rats. The arsanilate-induced damage was restricted to the vestibular sensory organs without affecting the external ear, the oropharynx, or Scarpas ganglion. This finding strongly supports the absence of diffusion of arsanilate into the external ear or Eustachian tubes, or through the eighth cranial nerve sheath leading to the brainstem. One of the striking observations of the present study is the complete restructuring of the sensory epithelia into a non sensory epithelial monolayer observed at 3months after arsanilate application. This atrophy resembles the monolayer epithelia observed postmortem in the vestibular epithelia of patients with a history of lesioned vestibular deficits such as labyrinthectomy, antibiotic treatment, vestibular neuritis, or Ménières disease. In cases of Ménières disease, aminoglycosides, and platinum-based chemotherapy, vestibular hair cells are destroyed, regardless of the physiopathological process, as reproduced with the arsanilate model of vestibular lesion. These observations, together with those presented in this study of arsanilate vestibular toxicity, suggest that this atrophy process relies on a common mechanism of degeneration of the sensory epithelia.

Histamine H4 receptor antagonists as potent modulators of mammalian vestibular primary neuron excitability.

BACKGROUND AND PURPOSE Betahistine, the main histamine drug prescribed to treat vestibular disorders, is a histamine H3 receptor antagonist.

AMPA type glutamate receptor mediates neurotransmission at turtle vestibular calyx synapse

Glutamate is thought to be the main neurotransmitter at the synapse between the type I vestibular hair cell and its cognate calyx afferent. The present study was designed to identify the type of glutamate receptors involved in neurotransmission at this unusual synapse. Immunocytochemistry showed that AMPA GluR2, NMDA NR1 and NR2A/B subunits of the glutamate receptors were confined to the synaptic contact. We then examined the electrical activity at calyx terminals using direct electrophysiological recordings from intact dendritic terminals in explanted turtle posterior crista. We found that sodium‐based action potentials support a background discharge that could be modulated by the mechanical stimulation of the hair bundle of the sensory cells. These activities were prevented by blocking both the mechano‐electrical transduction channels and L‐type voltage‐gated Ca2+ channels involved in synaptic transmission. Although pharmacological analysis revealed that NMDA receptors could operate, our results show that AMPA receptors are mainly involved in synaptic neurotransmission. We conclude that although both AMPA and NMDA glutamate receptor subunits are present at the calyx synapse, only AMPA receptors appear to be involved in the synaptic transmission between the type I vestibular hair cell and the calyx afferent.

Tmprss3, a transmembrane serine protease deficient in human DFNB8/10 deafness, is critical for cochlear hair cell survival at the onset of hearing

Mutations in the type II transmembrane serine protease 3 (TMPRSS3) gene cause non-syndromic autosomal recessive deafness (DFNB8/10), characterized by congenital or childhood onset bilateral profound hearing loss. In order to explore the physiopathology of TMPRSS3 related deafness, we have generated an ethyl-nitrosourea-induced mutant mouse carrying a protein-truncating nonsense mutation in Tmprss3 (Y260X) and characterized the functional and histological consequences of Tmprss3 deficiency. Auditory brainstem response revealed that wild type and heterozygous mice have normal hearing thresholds up to 5 months of age, whereas Tmprss3Y260X homozygous mutant mice exhibit severe deafness. Histological examination showed degeneration of the organ of Corti in adult mutant mice. Cochlear hair cell degeneration starts at the onset of hearing, postnatal day 12, in the basal turn and progresses very rapidly toward the apex, reaching completion within 2 days. Given that auditory and vestibular deficits often co-exist, we evaluated the balancing abilities of Tmprss3Y260X mice by using rotating rod and vestibular behavioral tests. Tmprss3Y260X mice effectively displayed mild vestibular syndrome that correlated histologically with a slow degeneration of saccular hair cells. In situ hybridization in the developing inner ear showed that Tmprss3 mRNA is localized in sensory hair cells in the cochlea and the vestibule. Our results show that Tmprss3 acts as a permissive factor for cochlear hair cells survival and activation at the onset of hearing and is required for saccular hair cell survival. This mouse model will certainly help to decipher the molecular mechanisms underlying DFNB8/10 deafness and cochlear function.

Characteristics of laser stimulation by near infrared pulses of retinal and vestibular primary neurons

The optical stimulation of neurons from pulsed infrared lasers has appeared over the last years as an alternative to classical electric stimulations based on conventional electrodes. Laser stimulation could provide a better spatial selectivity allowing single‐cell stimulation without prerequisite contact. In this work we present relevant physical characteristics of a non‐lethal stimulation of cultured mouse vestibular and retinal ganglion neurons by single infrared laser pulses.