Investigation Into the Dynamics of the Cupula in the Vestibular Organ of Adult Zebrafish Using Metabolic Glycoengineering.

Sudden loss of peripheral vestibular function is a common clinical disorder. The primary cause of this disorder is not known. Previous experiments in pigeons showed that an induced mechanical leakage in the cupula of a semicircular canal causes symptoms equivalent to those observed in humans after sudden loss of peripheral vestibular function. The cupula is an acellular membrane, which is critical for the detection of angular acceleration of the head. It consists of the matrix glycoprotein cupulin, which is secreted by supporting cells in the crista ampullaris. Currently, it is unclear whether cupulin is continuously produced resulting in permanent cupular neogenesis. Such a process could explain recovery of function observed in many patients. We applied metabolic glycoengineering to demonstrate the existence of cupulin renewal. Intraperitoneal injection of N-azidoacetylgalactosamine (GalNAz) into zebrafish leads to incorporation of this sugar in the cupula. Preparation of the cupula after various time intervals followed by fluorescence labeling by click chemistry, resulted in a distinct band within the cupula visible in fluorescence microscopy. Time-delayed double injection gave rise to two bands. A long-term experiment allowed to estimate that complete renewal of the zebrafish cupula occurs over a cycle of eight to ten weeks.