Degeneration of neural circuitry, either through synaptic loss or neural cell death, is an important cause of nervous system dysfunction, and afferent auditory nerve loss leading to deafness is a manifestation of these processes occurring in the cochlea. Our previous work has shown that neurons from transplanted stem cells can reinnervate hair cells of the organ of Corti after chemical de-afferentation, suggesting that this neural circuit can be reconstituted. In these studies we ask questions about repair of both types of neural degeneration: first, we investigate whether reinnervation and synaptogenesis with hair cells can be increased by manipulating axonal guidance and neurotrophic factors in in vitro and in vivo models of synapse damage; second, we study the effect of progenitor cell transdifferentiation to neurons and the regeneration of this circuit in a model of complete afferent neuron loss. Measurements of synaptic and neural repair include electrophysiological and histological analysis of new synapses in vitro and immunohistochemical as well as peripheral, central, and behavioral assessments of auditory function in vivo. Understanding the mechanisms underlying re-formation of neural connections to hair cells in the adult ear is important to therapeutic approaches for the treatment of neural dysfunction that causes hearing loss.
The Specific Aims comprise three inter-related experiments to probe key variables likely to influence the success of peripheral and central reinnervation of the auditory system.
In Aim 1 we assess the effect of inhibitors of axonal guidance as well as promoters of axonal growth on the reinnervation of hair cells in cochlear models with loss of the afferent synapse but preservation of ganglion cells. In the in vitro system (afferent synapses lost due to kainate administration) we use electrophysiological measurements to assess synaptic function: firing of action potentials and excitatory postsynaptic currents by the neurons after stimulation of hair cells, and inhibition of this synaptic function by pharmacological blockers of glutamatergic synapses. In the in vivo system (noise damage causing afferent synapse loss) metrics include synaptic analysis as well as auditory brainstem response and distortion product otoacoustic emissions. Assessments are based on the morphology and number of synapses formed with inner and outer hair cells determined immunohistochemically.
In Aim 2 we define the roles and optimal expression levels of Sox2 as well as Ngn1 for Schwann cell to neuronal conversion. We have isolated progenitor cells that can be converted to neurons from spiral ganglion, and we have shown that these cells arise from Schwann cells. We test the role of Sox2 and Ngn1 in this conversion. We assess conversion of Schwann cells to neurons in vivo using ouabain to produce a model of auditory nerve damage, and we stimulate the conversion of endogenous cells to spiral ganglion cells by modulating expression of the same genes.
In Aim 3 we record the spiking activity from single neurons in the auditory midbrain to sound stimuli delivered to the reinnervated and untreated ears. We compare markers of neural recovery to behavioral measures of improved sound discrimination.
The work described in this grant will lead to new approaches to replace degenerated nerves in the cochlea and a better understanding of the mechanisms of pathfinding and synapse formation by neurons that target auditory hair cells. Because of the clear correlation between loss of cochlear cells and sensorineural hearing loss, improvements in our ability to regenerate neurons and their afferent synapse with hair cells will lead to treatment for hearing loss.
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