Three-dimensional culture of newborn rat utricle using an extracellular matrix promotes formation of a cyst.
The vestibule is the end organ devoted to sensing of head movements in space. To function properly, its mechano-receptors require the presence of a unique apical extracellular medium, the endolymph. Numerous studies have elucidated the mechanisms involved in the production and homeostasis of this unique medium and the responses of sensory cells to stimulation. However, anatomical constraints have prevented direct and simultaneous studies of their relationships. The aim of this study was the development of an in vitro model that would allow concomitant investigations on maturation and physiological properties of both the hair cells and their endolymphatic compartment. A three-dimensional (3D) culture of newborn rat utricles using an extracellular matrix sustaining 3D cellular growth was developed during 3, 6, or 10 days in vitro (DIV). Using morphological and electrophysiological techniques, we describe the de novo formation of a cyst. It was composed of the sensory epithelium and non-sensory cells-canalar, dark and intermediate cells-that polarized so that their apical surface faced its lumen. During the time of culture, the utricular potential (UP) was steady (-1.1+/-5.0 mV) in oxygenated condition, while in anoxia, the UP significantly decreased to -8.4+/-1.0 mV at 8 DIV. Over the same period, the K+ concentration in the cyst increased up to 86.1+/-33.9 mM (versus 5.6+/-1.5 mM in the bath). These observations indicated that the mechanisms generating the UP and the K-secretory activity were functional at this stage. Concomitantly, the hair cells acquired mature and functional properties: the type 1 and type 2 phenotypes, a mean resting membrane potential of -68.1+/-4.6 mV and typical electrophysiological responses. This preparation provides a powerful means to simultaneous access the hair cells and their endolymphatic compartment, with the possibility to use multi-technical approaches to investigate their interdependent relationships.
Gaboyard
S
S
INSERM U583, Physiopathologie et Thérapies des Déficits Sensoriels et Moteurs, Hôpital Saint Eloi, Av. Augustin Fliche, 34295 Montpellier, France.
Chabbert
C
C
Travo
C
C
Bancel
F
F
Lehouelleur
J
J
Yamauchi
D
D
Marcus
D C
DC
Sans
A
A
D000818:Animals; D000831:Animals, Newborn; D020863:Central Nervous System Cysts / Q000473:pathology*; D004594:Electrophysiology; D016326:Extracellular Matrix Proteins / Q000378:metabolism*; D005456:Fluorescent Dyes; D006198:Hair Cells, Auditory / Q000502:physiology; D007150:Immunohistochemistry; D008208:Lymphatic System / Q000378:metabolism / Q000502:physiology; D008564:Membrane Potentials / Q000502:physiology; D018613:Microscopy, Confocal; D008855:Microscopy, Electron, Scanning; D046529:Microscopy, Electron, Transmission; D009924:Organ Culture Techniques; D015221:Potassium Channels / Q000502:physiology; D051381:Rats; D012444:Saccule and Utricle / Q000502:physiology*
D016326:Extracellular Matrix Proteins; D005456:Fluorescent Dyes; D015221:Potassium Channels