The Origins of Spontaneous Morphological Activity in Kölliker’s Organ of the Developing Rat Cochlea

Abstract

Kölliker’s organ is an epithelial tissue in the developing cochlea of the inner ear that disappears following functional development of the auditory sensory organ, the organ of Corti. It is a large collection of columnar epithelial cells medial to the developing inner sensory hair cells (IHCs), and comprises a major part of the organ of Corti during stages of auditory development, before the sensory cells are sensitive to sound (“pre-hearing”). The role of this tissue is unclear, but it has been speculated that it is important for driving or modulating spontaneous auditory nerve activity before the onset of hearing. It is suggested that this involves a paracrine or autocrine action of ATP, acting via P2 receptors which initiates spontaneous electrical currents in epithelial cells and may influence the IHC and its primary type I auditory innervation. Associated with this, epithelial cells of Kölliker’s organ also undergo rhythmic morphological changes (crenation), the purpose and mechanisms of which is not yet known. These studies were undertaken to explore these morphological changes, in order to identify potential origins, generators, and mechanisms for this rhythmic activity. Using a live tissue imaging approach with wholemounts of developing cochlea, the crenation of a cluster of cells could be observed as a change in refractive index of the tissue, allowing optical detection of this activity in real time. Utilising this principle, and the dynamic nature of the activity, spontaneous morphological events in developing Wistar rat (P7-16) Kölliker’s organ were studied with realtime differential interference contrast (DIC) imaging. Tissue was superfused with artificial cerebrospinal fluid, and the involvement of P2 receptor activity was explored with the addition of P2 receptor manipulators (ATP, ATPγS and suramin). Initial studies verified the presence of spontaneous morphological events restricted to Kölliker’s organ of pre-hearing Wistar rats. These events were drastically inhibited with ATP depletion induced by perfusing tissue with oxygen and glucose depleted solution. Activity could not be restored with the introduction of P2 receptor agonists A TP a nd A TPγS during this time, demonstrating the requirement of periodic ATP release in generating these rhythmic volume changes. Further examination of the response to ATPγS revealed sustained crenation throughout Kölliker’s organ, and identified the border cells (defined in this thesis as the cells that surround IHCs in the developing cochlea) as the most affected. This crenation was unique to these border cells when tissue was exposed to a gap junction inhibitor carbenoxolone, outlining the requirement of tissue connectivity to induce changes in regions further medial from IHCs. In contrast to cell crenation with P2 receptor activation, inhibition of these receptors with suramin lead to tissue-wide swelling, again more prominent in the border cells, as confirmed by histological analysis of the sensory organ. These results suggest that cells of Kölliker’s organ (particularly border cells) are under tight volume regulation through P2 receptor activity, and its absence leads to cell swelling. Together with the higher frequency of events in this border cell region, these findings imply a generator role for border cells in spontaneous morphological activity, while underlying the role of P2 receptor activity in initiating cell volume changes. A model is proposed that ATP released from border cells activates Ca2+ dependent Cl- channels, which leads to Cl- and water efflux, causing crenation. Restoration of the intracellular Cl- may involve multiple channels, including NKCC1 for the inward drive of Cl-, coupled with Na-K-ATPase to remove Na+, and K+ channels (e.g. BK or KCNQ) to remove the excess intracellular K+. The role of these morphological events is speculated as a signal that connects IHC and the border cells which may be involved in the modulation of neural activity as well as signalling the maturation of hair cell activity and eventual atrophy of Kölliker’s organ.