Maturation of Heterogeneity in Afferent Synapse Ultrastructure in the Mouse Cochlea

Abstract

Auditory nerve fibers (ANFs) innervating the same inner hair cell (IHC) may have identical frequency tuning but different sound response properties. In cat and guinea pig, ANF response properties correlate with afferent synapse morphology and position on the IHC, suggesting a causal structure-function relationship. In mice, this relationship has not been fully characterized. Here we measured the emergence of synaptic morphological heterogeneities during maturation of the C57BL/6J mouse cochlea by comparing postnatal day 17 (p17, ∼3 days after hearing onset) with p34, when the mouse cochlea is mature. Using serial block face scanning electron microscopy and three-dimensional reconstruction we measured the size, shape, vesicle content, and position of 70 ribbon synapses from the mid-cochlea. Several features matured over late postnatal development. From p17 to p34, presynaptic densities (PDs) and post-synaptic densities (PSDs) became smaller on average (PDs: 0.75 to 0.33; PSDs: 0.58 to 0.31 μm2) and less round as their short axes shortened predominantly on the modiolar side, from 770 to 360 nm. Membrane-associated synaptic vesicles decreased in number from 53 to 30 per synapse from p17 to p34. Anatomical coupling, measured as PSD to ribbon distance, tightened predominantly on the pillar side. Ribbons became less spherical as long-axes lengthened only on the modiolar side of the IHC, from 372 to 541 nm. A decreasing gradient of synaptic ribbon size along the modiolar-pillar axis was detected only at p34 after aligning synapses of adjacent IHCs to a common reference frame (median volumes in nm3 × 106: modiolar 4.87; pillar 2.38). The number of ribbon-associated synaptic vesicles scaled with ribbon size (range 67 to 346 per synapse at p34), thus acquiring a modiolar-pillar gradient at p34, but overall medians were similar at p17 (120) and p34 (127), like ribbon surface area (0.36 vs. 0.34 μm2). PD and PSD morphologies were tightly correlated to each other at individual synapses, more so at p34 than p17, but not to ribbon morphology. These observations suggest that PDs and PSDs mature according to different cues than ribbons, and that ribbon size may be more influenced by cues from the IHC than the surrounding tissue.

Postsynaptic GluA3 subunits are required for the appropriate assembly of AMPA receptor GluA2 and GluA4 subunits on mammalian cochlear afferent synapses and for presynaptic ribbon modiolar-pillar morphological distinctions

“…These data show that ribbons on the modiolar side of GluA3 WT IHCs are elongated, while those on the pillar side are more round in shape (Fig. 3D, left and center), as previously shown for C57BL/6 mice at 5-weeks of age (Payne et al, 2021). Analysis of SV size showed that the SVs of modiolar-side synapses were larger (p= 0.0001, Mann-Whitney U test, U: 4975) than those of the pillar-side synapses (modiolar: 36 + 5 nm, CV: 0.13; pillar: 33 + 4 nm, CV: 0.14; Fig.…”

Sensory transduction is required for normal development and maturation of cochlear inner hair cell synapses

“…However, synapse counts were markedly decreased in P28 Tmc1 Δ/Δ ;Tmc2 Δ/Δ mice relative to WT mice and relative to P14 Tmc1 Δ/Δ ;Tmc2 Δ/Δ numbers at all frequencies (Figure 3C, Supplementary File 2C). Though synapse counts remain stable in WT mice post-hearing onset, changes in the distribution of ribbon sizes and continued reduction in the sizes of presynaptic/postsynaptic densities are observed until their full development around P34 (Payne et al, 2021). As with the abnormally elevated synapse counts at P7 in Tmc1 Δ/Δ ;Tmc2 Δ/Δ mice, the rapid loss of more mature ribbon synapses further suggests a role for sensory transduction in the development of synapses.…”

Reducing Auditory Nerve Excitability by Acute Antagonism of Ca2+-Permeable AMPA Receptors

“…To send information about sound to the brain, cochlear inner hair cells (IHCs) excite the auditory nerve by exocytosis of glutamate onto AMPA-type receptors at afferent ribbon synapses (Ruel et al, 1999;Glowatzki and Fuchs, 2002; Figure 1A). Each auditory nerve fiber (ANF, or spiral ganglion neuron) is driven by a single, large afferent synapse where the postsynaptic density (PSD) measured with electron microscopy is ∼ 850 nm in length, on average, and the two-dimensional surface area ranges 0.1-1.5 µm 2 [cat: Liberman, 1980;Merchan-Perez and Liberman, 1996;mouse: Meyer et al, 2009; see also Payne et al (2021) in this special issue]. Glutamatergic synapses in the brain are considerably smaller, where PSDs are ∼ 300 nm in length, on average, and most have surface area <0.1 µm 2 (Aoki et al, 2001;Qu et al, 2009;Santuy et al, 2018).…”