GluA3 subunits are required for appropriate assembly of AMPAR GluA2 and GluA4 subunits on cochlear afferent synapses and for presynaptic ribbon modiolar–pillar morphology

Abstract

Cochlear sound encoding depends on α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors (AMPARs), but reliance on specific pore-forming subunits is unknown. With 5-week-old male C57BL/6J Gria3 knockout mice (i.e., subunit GluA3KO) we determined cochlear function, synapse ultrastructure and AMPAR molecular anatomy at ribbon synapses between inner hair cells (IHCs) and spiral ganglion neurons. GluA3KO and wild-type (GluA3WT) mice reared in ambient sound pressure level (SPL) of 55-75 dB had similar auditory brainstem response (ABR) thresholds, wave-1 amplitudes and latencies. Postsynaptic densities (PSDs), presynaptic ribbons, and synaptic vesicle sizes were all larger on the modiolar side of the IHCs from GluA3WT, but not GluA3KO, demonstrating GluA3 is required for modiolar-pillar synapse differentiation. Presynaptic ribbons juxtaposed with postsynaptic GluA2/4 subunits were similar in quantity, however, lone ribbons were more frequent in GluA3KO and GluA2-lacking synapses were observed only in GluA3KO. GluA2 and GluA4 immunofluorescence volumes were smaller on the pillar side than the modiolar side in GluA3KO, despite increased pillar-side PSD size. Overall, the fluorescent puncta volumes of GluA2 and GluA4 were smaller in GluA3KO than GluA3WT. However, GluA3KO contained less GluA2 and greater GluA4 immunofluorescence intensity relative to GluA3WT (3-fold greater mean GluA4:GluA2 ratio). Thus, GluA3 is essential in development, as germline disruption of Gria3 caused anatomical synapse pathology before cochlear output became symptomatic by ABR. We propose the hearing loss in older male GluA3KO mice results from progressive synaptopathy evident in 5-week-old mice as decreased abundance of GluA2 subunits and an increase in GluA2-lacking, GluA4-monomeric Ca2+-permeable AMPARs.

Female GluA3-KO mice show early onset hearing loss and afferent swellings in ambient sound levels

“…Next, we compared the PSD surface area and volume between modiolar side and pillar side and did not observe any difference in GluA3 WT (surface area p = 0.52; volume p = 0.10, unpaired t-test) (Figs. 7 and 8A top; Table 1). In contrast, male GluA3 WT PSDs were larger on the modiolar side (Rutherford et al, 2023). Data from single ultrathin sections showed that the PSD linear length was similar between the modiolarand pillar-side synapses (p = 0.93, Mann-Whitney test) (Fig.…”

“…Thus, GluA4-only synapses were seen only in GluA3 KO for females in ambient or quiet conditions. Although GluA4-only synapses were significantly greater in number in GluA3 KO males than WT raised in ambient conditions (Rutherford et al, 2023), the differences did not reach statistical significance for the females (All synapses ambient, Fig. 3E Next, we compared synapse size and protein expression based on immunofluorescence volume and intensity per synapse, displayed as means per image and per group after normalizing metrics from individual images to the respective WT mean for images from ambient or quiet conditions (Fig.…”

Complete Restoration of Hearing Loss and Cochlear Synaptopathy via Minimally Invasive, Single-Dose, and Controllable Middle Ear Delivery of Brain-Derived Neurotrophic Factor–Poly(dl-lactic acid-co-glycolic acid)-Loaded Hydrogel

“…The amplitude and latency of ABR wave I have been reported to reflect the function of cochlear ribbon synapses. ,, Thus, we further examined the changes in the ABR wave I amplitude and latency in this study. The NE group exhibited significantly decreased amplitudes of ABR wave I at all tested frequencies compared to the control group.…”

Diversity matters — extending sound intensity coding by inner hair cells via heterogeneous synapses

“…Furthermore, efforts are required also to elucidate the molecular and structural knowledge regarding the PSD of the postsynaptic SGNs. Immuno‐electron microscopy and confocal and superresolution immunofluorescence have established the notion that a ~ 0.5–1.5 μm sized dense array of glutamate receptors with a ring‐like structure likely surrounding the presynaptic AZ (Matsubara et al , 1996; Meyer et al , 2009; Wong et al , 2014; Rutherford et al , 2023) ensures efficient detection of glutamate. A number of postsynaptic density proteins such as PSD‐95, Shank, and Homer1 have been identified (e.g., Davies et al , 2001; Reijntjes et al , 2020).…”

“…Interestingly, opposing size gradients (decreasing in pillar–modiolar direction for AMPA receptor clusters, but in modiolar–pillar direction for ribbon size) have been described for IHCs of CBA mice (Liberman et al , 2011; Reijntjes et al , 2020), but while the presynaptic gradient was consistently found in two other mouse lines, the postsynaptic gradient was not (Reijntjes et al , 2020). A recent detailed study of the PSD of the IHC‐SGN synapse in C57BL/6J mice revealed a modiolar–pillar gradient of PSD size and showed that the GluA3 is required for modiolar–pillar gradient of ribbon size (Rutherford et al , 2023).…”