Development of an in vivo model system of sensory afferent neuron regeneration

Project Summary The afferent neurons of the eighth cranial nerve transmit information from the cochlea and vestibular organs to the brainstem. These neurons can be lost following damaging noise exposure, ototoxic injury, or as a consequence of normal aging. Once lost, afferent neurons do not regenerate, and their depletion can result in

permanent hearing and balance deficits. In the case of hearing, clinical strategies to restore sensory function, such as cochlear implants, require survival of a sufficient number of spiral ganglion neurons. Unfortunately, there is currently no practicable method for replacing lost spiral ganglion neurons. Although the mammalian

cochlea lacks the ability to regenerate neurons, the spiral ganglion contains a population of cells that express Nestin, which is widely regarded as a marker for neural stem cells. Studies conducted in mice have demonstrated that these Nestin-expressing cells do not divide once the cochlea has matured, and the signaling

molecules that regulate their proliferation are not known. One factor that hinders the study of neurogenesis in the cochlea is that the cell bodies of cochlear afferents and Nestin-expressing cells are housed within bone in the inner ear and are not visually or experimentally accessible in living mammals. The task of developing

methods for neuronal replacement in the inner ear would be greatly facilitated by the introduction of an appropriate model system to identify the basic cellular signals that permit ongoing addition and spontaneous regeneration of afferent neurons. The objective of the present proposal is to establish the posterior lateral line

(pLL) ganglion of larval zebrafish as a model system for the study of neuronal repair and replacement. The pLL ganglion is comprised of ~50 neurons, which can be easily visualized in living animals. Current data indicate that about five new neurons/day are added to the pLL ganglion, but the identities of neural precursor cells and

the factors that regulate their proliferation are not known. We have found that cells in larval pLL ganglion express two markers for neural precursors: Nestin and NeuroD. Moreover, expression of these two markers occurs in spatially distinct regions, suggesting two domains within the pLL ganglion that possess progenitor

cells. Our studies will determine the contributions of each population to the process of ongoing neurogenesis in the pLL ganglion and reveal whether proliferation of either (or both) cell types is enhanced following ablation of a subset of afferent neurons. We will also quantify expression levels of Nestin and NeuroD within the ganglion

as function of age to define the contribution of each cell type to the process of neuronal addition and regeneration. Finally, we will evaluate the function of macrophages in neurogenesis and regeneration of pLL ganglion neurons. Experiments will determine whether macrophages are activated by neuronal injury and will

resolve whether they help promote the proliferation of progenitor cells. The data generated by this project will identify the factors that regulate production of sensory afferents and may reveal potential strategies for inducing proliferation of resident progenitors and differentiation of new neurons in the mammalian inner ear.