Differential Expressions and Functions of P2X1 and P2X4 in the Cochlea

Abstract

According to the World Hearing Organization, hearing loss affects 1.5 billion people globally. Currently, no effective pharmacological treatments for sensorineural hearing loss are available. Cochlear synaptopathy is the disruption of synapses between inner hair cells (IHCs) and spiral ganglion neurons (SGNs) in the cochlear that precedes hearing threshold shifts and hair cell loss. Therefore, preventing cochlear synaptopathy has emerged as a potential strategy for treating hearing loss. One of the molecular candidates that may be involved in cochlear synapse homeostasis is purinergic signalling. Previous studies have identified the expression of multiple P2X receptor subtypes in the cochlea. Of those, expressions of P2X4 and P2X1 have been found in hair cells and SGN, respectively. However, its precise localisations and functions are still unknown. This study aimed to characterise P2X1 and P2X4 expressions in IHC-SGN synapses, with the underlying hypothesis that these receptors are involved in regulating cochlear synapses homeostasis. Wistar rat (postnatal day 8- and 6–8-week-old, both genders) cochleae were collected, and P2X1 and P2X4 expression at the protein level was examined using immunohistochemistry with markers for presynaptic ribbon and type II SGN. P2X4 and presynaptic ribbon co-localisation was found in IHCs but not in outer hair cells (OHCs) in adult and P8 cochleae. Differential P2X1 expression was observed depending on the different subpopulations of SGNs, where some SGNs displayed a higher P2X1 signal than others. SGNs with high P2X1 signal co-expressed type II SGN marker (peripherin-1), suggesting P2X1 is expressed at a high level in type II SGNs and a lower level in type I SGNs. P2X1 expression was observed in dendrites of both type I and type II SGNs. Next, the effect of pharmacological manipulation of P2X signalling was tested in a cochlear organotypic culture prepared P8 Wistar rats. After four hours in culture, cochleae cultured in the presence of a broad P2X agonist, α, β- methyleneadenosine 5’-triphosphate (α,β-meATP ), showed some degree of IHC and OHC loss, but this was not statistically significant. Cochlea cultured in the presence of P2X4 antagonist, 5-(3-Bromophenyl)-1,3-dihydro-2H-Benzofuro[3,2-e]-1,4-diazepin-2-one (5-BDBD) showed significant IHC loss. Taken together, these results suggest, for the first time, that P2X4 is a presynaptic receptor in IHCs, and P2X1 is a post-synaptic receptor in type I and type II SGNs. Furthermore, a high level of P2X1 expression was found in type II SGNs subtype-specific manner. These results imply that P2X receptors coordinate synaptic transmission between IHC/OHC and different subtypes of SGNs.