This proposal investigates the hypothesis that a heretofore unknown auditory pathway projects to unipolar brush cells (UBCs) of the cerebellar flocculus and paraflocculus (FL/PFL). This hypothesis is based upon three different and converging sets of preliminary data. The first line of evidence is provided by in vivo extracellular electrophysiological recordings from the gerbil flocculus showing a short latency, frequency-dependent response to acoustic stimulation. Interestingly, the firing pattern of the responder neuron closely resembles the peculiar firing pattern of UBCs. The second line of evidence shows that auditory stimulation leads to c-Fos expression in floccular neurons that have spatial distribution compatible with that of UBCs. The third evidence was obtained from injection of biotinylated dextran amine (BDA), an anterograde tracer dye, into the cochlear spiral ganglion. We found that BDA labeled fibers terminated in FL/PFL and contacted UBCs. Thus multiple sets of converging data strongly suggest that cerebellar UBCs receive auditory inputs. This finding is further supported by the fact that the only CNS region outside the cerebellar cortex where UBCs are expressed are the cochlear nuclei. The goal of this proposal is two-fold. We will perform further in-vivo electrophysiological recordings and examine auditory stimulation-induced c-Fos staining to confirm the existence and define the properties of the cerebellar responses to auditory stimulation (Specific Aim 1), and we will perform anatomical tracing experiments to determine the origin of the afferent cerebellar fibers carrying the auditory information (Specific Aim 2). Besides confirming this novel auditory pathway, our experiments will also determine the connectivity of cerebellar unipolar brush cells, which is still largely unknown. Cerebellar UBCs are classified into the less numerous type I and the more abundant type II neurons. Type I UBCs of the cerebellum are innervated by primary and secondary afferents from vestibular end organs and the vestibular nuclei, and, according to our preliminary finding, by primary auditory fibers. The afferents to type II UBCs are still completely unknown. Our experiments combining in vivo injections of anterograde and retrograde tracers, immuno-histochemical staining with cell-specific cell markers and electron microscopy will also determine the synaptic connectivity of these neurons. Successful completion of the proposed experiments will not only test the novel hypothesis that primary auditory fibers project to cerebellar UBCs, but will also help determine the connectivity of this still understudied cell type. Finally, because UBCs have the ability to generate action potentials in the absence of synaptic stimulation and their axons sprout and grow in response to cellular deafferentation, conclusive evidence that these UBCs are embedded in an auditory pathway would also suggest that they may originate electrophysiological activity that mediates phantom auditory perception after deafferentation. Therefore our studies may also open the way for new mechanistic studies of the generation of tinnitus.
Unipolar brush cells (UBCs) constitute the most recently identified neuronal class in the cerebellar cortex and little is known about their physiological role. Based on strong preliminary data, we hypothesize that cerebellar UBCs receive primary auditory inputs; therefore we will combine anatomical, cellular and electrophysiological techniques to test this hypothesis and to determine the connectivity of cerebellar UBCs and their responses to acoustic stimulation. Because of UBC functional properties, confirmation of our hypothesis may suggest that UBCs are critical elements in the generation of deafferentation-dependent tinnitus.
