Contents :

Presentation
Main Research Fields
Collaborations and Partners
Conclusion
Selection of Publication

Presentation

The team gathers scientists and clinicians who share common interest: setting new therapeutic strategies against vertigos of peripheral origin. Indeed, a large majority of vertigos (more than 80%) results from vestibular dysfunction. Yet pharmacological tools efficient in attenuating the dizziness sensation that characterizes these vertigos remain very limited. This is mainly due to the lack of knowledge of the molecular mechanisms that shape the vestibular sensory information within the inner ear.

The aim is to characterize these molecular mechanisms in order to set up targeted therapeutic strategies against these vertigos.

We therefore developed several experimental models (explants and slices of vestibular organs, organotypic cultures) and new methods combining morphological techniques (immunocytochemistry; electron microscopy), functional imaging (confocal and two-photon microscopy), electrophysiology (patch-clamp, intracellular recordings, ion-selective electrodes) and in vivo behaviour tests of vestibular function. With these animal models and methods of investigation we can study the setting and transfer of the vestibular sensory information either during the development, in the adult or under pathophysiological conditions.

Main Research Fields

In the inner ear, the endolymph is an extracellular medium that baths the apical side of both vestibular and auditory mechanoreceptor cells. This fluid contains a high amount of potassium (K+); this ion provides the driving force for the mechanotransduction (the encoding by sensory cells of mechanical inputs – motions of the head – into bioelectrical output). Hence, the ionic homeostasis of the endolymph needs to be finely regulated (Fig.1) in order to prevent dysfunction of the sensory perception. Indeed, alterations in the endolymph ionic composition are believed to be involved in, if not at the origin of, the Menière’s disease. This pathology is the second most common cause of vertigo in human beings and lacks of efficient therapy.

Establishing therapeutic strategies against such vertigo requires the identification of mechanisms of ion secretion in the endolymph, together with the understanding of their regulatory pathways. Thanks to a murine organotypic model of utricle able to regenerate the endolymphatic compartment in vitro, we can record the dynamics of the K+ secretion within the endolymph by means of K+ selective electrodes.

The pharmacological characterization of ionic channels, pumps and transporters, which contribute to the formation and maintenance of the endolymph, is under process in wild-type as well as in transgenic mice. This molecular approach combined to integrated functional investigations (such as ocular motor reflex recordings) should lead to noticeable advances in the identification of the regulatory mechanisms controlling ion secretions in the main inner ear fluid.

Figure 1: Schematic drawing of the endolymphatic compartment bathing the vestibular sensory epithelium. The boundary of this compartment is composed of several cell types: sensory cells (or hair cells), supporting cells, transitional cells, dark cells and canal/wall cells. Each cell type is involved in different ion fluxes that result in the ion homeostasis of the endolymph. The high K+ concentration (+/- 140 mM) is necessary for the genesis of the vestibular sensory information.

Figure 2: Organotypic culture of murine utricle (the sensor of linear acceleration – gravity). In our culture conditions, endolymph compartment regenerates in few days. Various electrodes, (arrow in A) driven, across the neoformed wall of the utricle allow the recording of several features: utricular endolymphatic potential (B), K+ concentration (C), resting potential of sensory cells (E). DIV: days in vitro; UP: utricular potential; [K+] cyst: K+ endolymphatic concentration; RMP: resting membrane

A: Direct recording of synaptic activity at calyx terminal
B: Calcium imaging of vestibular neurotransmission
C: Molecular bases of electrical activities in vestibular primary neurons

The molecular bases of the fast physiological signals at vestibular synapses remain largely unknown. This is mostly due to the confined location of the vestibular epithelia and their extreme fragility, which have until now restrained their functional characterizations. In the vestibulus, the sensory cells are innervated by two types of contacts: classical bouton terminals and calyx terminals that embed the basolateral part of the sensory hair cell. These synapses, which are mainly glutamatergic, are sensitive to the toxic effect of the glutamate, occurring during ischemia. We recently developed three original approaches to explore the molecular and pharmacological properties of the vestibular synapses.

A: Direct recording of synaptic activity at calyx terminal
The development of electrophysiological recordings by the loose patch clamp technique on explants (preserving the integrity of the calyx terminal) allowed pharmacological characterization of the cellular mechanisms (receptors and ionic channels) which encode and control the transmission of the sensory signal. This approach provided access to electrical activity (at rest or evoked by natural stimulation of the sensory hair cells) at post-synaptic terminals, which directly resulted of the synaptic transmission. These pharmacological studies specified the respective role of the AMPA and NMDA type receptors (both present at calyx synapse) in the origin of the synaptic events (Fig. 3, Bonsacquet et al., 2005).

B: Calcium imaging of vestibular neurotransmission
Pharmacological characterization of synaptic neurotransmission at bouton terminals was undertaken by developing an ex vivo explant preparation allowing to preserve the vestibular epithelium and its innervation. Two-photon microscopy was used to image local calcium events that participate to the synaptic transmission (Boyer et al., 2004). Pharmacological studies were currently developed during synaptic stimulations (mechanical stimulations of the sensory cells, applications of K+ or glutamate).

Figure 4: Two-photon imaging of calcium events at postsynaptic terminals in response to the hair cell stimulation. Terminals were charged with a Ca2+-sensitive dye by retrograde transport (A). Postsynaptic terminals were identifiable within sections of vestibular explants (B, arrow b: buttons; c: postsynaptic synapse of calyx terminal). Direct depolarization of the presynaptic element by ionophoretic applications of potassium on a sensory cell (C) induced an increase of the cytosolic calcium concentration in the terminal (D).

 
C : Molecular bases of electrical activities in vestibular primary neurons
Vestibular primary neurons transmit the sensory information elaborated in the sensory cell to the central nervous system. These neurons have various electric activities according to their ionic channels equipment. The characterization of these channels, using the patch-clamp technique allowed to demonstrate their implication in neuronal physiology (Chambard et al., 1999; Chabbert et., 2001a, b). During development, we carried out the molecular analysis of the different low voltage activated calcium current subunits (T-type currents) associated with the various electric activities by single cell RT-PCR (Autret et al unpublished). Using patch-clamp recordings, we showed that vestibular neurons displayed transiently a large T-type calcium current of type T carried by the Cav3.2/a1H subunit during the ontogenesis. This current appeared predominantly during the neuritogenesis that occurs in the mouse between embryonic day E14 and postnatal day 5 (Figure 5, Autret et al., 2005). Study of the role of the Cav3.2 calcium channel in neural growth is in progress.

Figure 5: Expression of the T-type calcium current (Cav3.2) by vestibular primary neurons during neuritogenesis. T-type calcium currents recorded at E17activated around -60 mV and peaked at -40 mV while High voltage-activated currents started around -20 mV and peaked at 0 mV (A). T-type calcium currents are preferentially expressed during the prenatal period during to the neuritic growth (B). At E17, vestibular neurons present sodium and calcium action potentials (C).

A: Molecular mechanisms that control the synaptogenesis in vestibular organs
B: Molecular mechanisms of synapses post-injury repair
C: Plasticity of vestibular sensory organs

A : Molecular mechanisms that control the synaptogenesis in vestibular organs
During the embryonic development, vestibular primary sensory neurons express a particular voltage-gated calcium channel involved in the neuritic outgrowth to the sensory hair cells. After birth, synaptogenesis process and leads to functional synapses between hair cells and sensory neurons. One of our ontogenetic studies was related to the presynaptic element: the hair cell. This latter acquired a transitory excitability, du to the expression of a neuronal-like tetrodotoxin (TTX)-sensitive voltage-gated sodium current (INa, characterisation by electrophysiology and single-cell RT-PCR), enabling these cells to generate sodium-driven action potentials (AP) (Fig 6. Chabbert et al., 2003a; Mechaly et al., 2005). We reported that INa is involved in the activity-dependent secretion of brain-derived neurotrophic factor (BDNF) in the neonate rat utricle (Chabbert et al., 2003a). This trophic factor activity is involved in the survival and the neurite outgrowth from embryonic vestibular ganglion neurons. This result brings insights into the understanding of the mechanisms that control the functional establishment of the vestibular peripheral innervation.

Figure 6: Voltage-gated sodium current expression in utricle sensory cells during the synaptogenesis process. Patch-clamp recording of hair cells permits the establishment of activation and inactivation curves (A -insert) of voltage-gated sodium currents recorded in voltage-clamp mode (B). The analysis of single-cell RT-PCR results revealed that this sodium current is mainly associated to the Nav1.2 subunit. During the first postnatal days, a high percentage of hair cells transiently express a large INa (C), enabling these cells to generate sodium gated action potentials (C- insert).

B : Molecular mechanisms of synapses post-injury repair
By the end of the first postnatal week, the voltage-gated sodium current INa almost disappears in sensory cells. We hypothesized that this down-regulation resulted from the establishment of synaptic contacts with the nerve endings of vestibular primary neurons. We then investigated whether impairment of these afferent nerve terminals in adult utricles (subsequently to synapse stabilization) would lead to the recovery of hair cell excitability. Ischemic damages to the inner ear are mediated by a massive glutamate release by hair cells, resulting in excitotoxic damage to auditory (Puel et al., 1994; Puel et al., 1995; Hakuba et al., 2003) and vestibular synapses (Liu, 1999; Shimogori and Yamashita, 2004). We developed a pharmacological in vivo approach to induce selective excitotoxic impairment to the nerve terminals (swellings) by application of a glutamatergic receptor agonist (Kainate) (Fig.7A). Following afferent terminal impairment (K+48h), the proportion of utricular hair cells expressing INa increased dramatically, and sensory cells recovered the ability to fire sodium-based action potentials (Whole-cell recordings Fig. 7B). These processes disappeared one week after kainic acid treatment and were prevented in presence of kainate receptors antagonist (DNQX, Fig 7B). Recovery of hair cell excitability may results from the release of cellular mechanisms that normally prevent membrane expression of INa once synaptogenesis has been completed. This process may be essential for the repair processes following synaptic impairments (Brugeaud et al., 2007).

Figure 7: Transient recovery of excitability in adult utricle hair cells following nerve terminals impairment. 48 hours after kainate application, both calyx and bouton terminals displayed characteristic swellings (A) and transiently express large sodium currents and recover the ability to fire action potentials (B). In control condition, such excitability properties don’t exit in sensory cells. In this excitotoxic model, the electrical properties disappeared one week after treatment and were prevented in presence of DNQX.

C : Plasticity of vestibular sensory organs
Developing organisms are highly subjected to plasticity influenced by genetic and epigenetic factors. Biochemical and electrical processes sustain this mechanism (see above).To investigate plasticity in the vestibular endorgans, we altered an epigenetic factor and search for potential modifications in the morphology or function of this neurosensory epithelium. Collaborating we the French National Space Agency (Centre National d’Etudes Spatiales, CNES), we developed experiments to alter one of the vestibular primary stimulus, gravity. Indeed, in our laboratory, a terrestrial centrifuge (Fig. 8) is used to subject rats to a hypergravitational environment (2g) during their pre- and postnatal development. We already showed some morphological (delay in synaptogenesis – Gaboyard et al., 2003) and functional (alterations of voltage gated currents, Chabbert et al. 2003b, Fig 10; Brugeaud et al., 2006) consequences of this stimulus alteration during development. Reversibility of such modifications is actually under study.

Figure 8. Terrestrial centrifuge provided by the CNES to host rodents in altered gravity. A constant rotation enables pregnant rat females to normally give birth and raise the pups under a gravitational force of 2g.

Figure 9. Immunohistrochemistry against synaptophysin (arrows) in cristae and utricles of 6 day-old rats. In normal gravity, synaptophysin locates at the apex of calyceal nerve endings in vestibular organs (A, crista; B, utricle). In rats developed in hypergravity, expression of synaptophysin in cristae (C) is similar to normal gravity. In opposition, in utricles of rats developed in hypergravity (D), synaptophysin locates in the basolateral part of the calyceal nerve endings in utricles, as it is observed in younger rats developed in normal gravity. Thus, hypergravity delays the process of synaptic maturation only in utricles, the vestibular epithelium specifically devoted to sense head movements in reference to gravity. Consequently, this observation suggests that in the vestibule, synaptic maturation depends on epigenetic factors specific to each sensory epithelium.

Figure 10. Development of rats in hypergravity (2g) strongly increases voltage-gated outward currents in vestibular hair cells of utricles. Depolarizing steps are expressed on the right of each record.

Conclusion

Identification of the molecular mechanisms involved in the setting of the vestibular sensory information and its transmission towards the brain stem is a pre-requisite for the setting of therapeutic strategies against vertigo. Our methodological approaches associating molecular and integrated studies should 1/ enlighten new targets for the pharmacological control of the vestibular sensory information, 2/ bring new methods to protect and repair synaptic contacts between the sensory cells and their cognate nerve fibres, 3/ bring new insights into the understanding of the mechanisms that regulates the ionic homeostasis in the endolymph. In our next orientations, we will use our experience in studying vestibular primary neurons to 1/ study their ability to survive in situ after impairment of the sensory epithelia, 2/ to define new neuroprotective strategies, and 3/ investigate potentialities to transplant vestibular organs for the rehabilitation of the vestibular function.

Collaborations:

J Goldberg School of Allied Health Sciences East Carolina University Chicago, USA
A Lysakowski Anatomy and Cell Biology, Univ. of Illinois at Chicago, USA
C Holt Department of Otolaryngology University of Texas UTMB Galveston, USA
T Jones School of Allied Health Sciences East Carolina University Greenville, USA
J Barhanin Institut de Pharmacologie du CNRS Sophia Antipolis Valbonne, France
J Llorens Departament de Ciencies Fisiologiques II Universitat de Barcelona, Spain
W Marcotti Department of Biomedical Science University of Sheffield Sheffield, UK
D Marcus University of Manhattan Kansas, USA

Partners:

Centre National des Etudes Spatiales (CNES), Laboratoires SERVIER, Laboratoires SOLVAY-PHARMA

Selection of publications :