Molecular Assembly and Structural Plasticity of Sensory Ribbon Synapses-A Presynaptic Perspective

Abstract

In the mammalian cochlea, specialized ribbon-type synapses between sensory inner hair cells (IHCs) and postsynaptic spiral ganglion neurons ensure the temporal precision and indefatigability of synaptic sound encoding. These high-through-put synapses are presynaptically characterized by an electron-dense projection-the synaptic ribbon-which provides structural scaffolding and tethers a large pool of synaptic vesicles. While advances have been made in recent years in deciphering the molecular anatomy and function of these specialized active zones, the developmental assembly of this presynaptic interaction hub remains largely elusive. In this review, we discuss the dynamic nature of IHC (pre-) synaptogenesis and highlight molecular key players as well as the transport pathways underlying this process. Since developmental assembly appears to be a highly dynamic process, we further ask if this structural plasticity might be maintained into adulthood, how this may influence the functional properties of a given IHC synapse and how such plasticity could be regulated on the molecular level. To do so, we take a closer look at other ribbon-bearing systems, such as retinal photoreceptors and pinealocytes and aim to infer conserved mechanisms that may mediate these phenomena.

Keywords: cochlear development; cytoskeleton; molecular motors; peripheral auditory pathway; synapse maturation; synaptic sound encoding.

Figure 1

Structural overview of the peripheral auditory pathway. Light-sheet microscopic 3D- reconstructions of (A) the bony murine cochlea that harbors the organ of Corti and (B) the isolated peripheral auditory system, with afferent spiral ganglion neurons (SGNs) branching out towards the row of inner hair cells (IHCs), in the typical spiraling staircase anatomy. IHCs and SGNs are labeled with an antibody against the cytosolic Ca²⁺-buffer Calretinin. (C) Confocal maximum projection of the organ of Corti labeled for the cytosolic Ca²⁺-buffer Parvalbumin, displaying the three rows of outer hair cells (OHCs), the single row of IHCs and the afferent innervation (auditory nerve fibers; ANF) by the SGNs. (D) Innervation of IHCs (Calbindin; blue) by individual SGN neurites (a3-Na⁺/K⁺-ATPase; red), showing the presynaptic ribbons (CtBP2; cyan) in contact with the postsynaptic SGN boutons. (D’) Side view of the innervated IHC showing the basolateral position of the synaptic ribbon and connected bouton. (E,E’) STED microscopic images of IHC synaptic ultrastructure of (E) a developing IHC active zone distributed in several precursor spheres colocalizing with multiple clusters of the postsynaptic density (PSD) versus (E’) one large ribbon opposing one ellipsoid PSD in a mature preparation. (F,F’) Electron microscopic images of (F) immature multi-ribbon active zones with roundish profiles versus (F’) the wedge-shape of a mature IHC ribbon that is attached to the curved presynaptic membrane. Scale bars: A 300 µm; B 150 µm; C 50 µm; D-D’ 5 µm; E-E’ 250 nm; F-F’ 200 nm. (B,E,E’) with permission from Reference [4]; (D) with permission from Reference [5].

Figure 2

Ribbon synapse morphology and molecular composition differ between biological systems. Schematic drawings of stereotypic ribbon shapes from the indicated sensory or neuroendocrine system they are operating in, to illustrate gross morphological and molecular differences: Selected conserved (black) and non-conserved (magenta) ribbon-associated proteins are highlighted in the respective boxes. Insets show the approximate locations of the ribbon-bearing cell populations (green) in a larger context. Please note: Ribbon dimensions are not drawn to absolute scale. Zebrafish gene duplications are highlighted with an *, Piccolo a/b and Bassoon b are expressed on mRNA level (https://piotrowskilab.shinyapps.io/neuromast_homeostasis_scrnaseq_2018); however, protein localization to the ribbon remains to be demonstrated. ANF, afferent nerve fiber; BC, bipolar cell; HC, horizontal cell; IPN, intrapineal neuron; PC, pinealocyte; SGN, spiral ganglion neuron.

Figure 3

Synaptogenesis in murine cochlear IHCs. The formation of the synaptic connection between inner hair cells (IHCs) and spiral ganglion neurons (SGNs) starts in the late embryonic stages. Afferent contact formation precedes the arrival of pre- and postsynaptic proteins. In the late prenatal stages, ribbon precursors (cyan) are formed in the IHC cytoplasm and already tether synaptic vesicles (SVs). The precursors translocate to the developing active zone (AZ) within the basolateral compartment of the IHC. Dense core vesicles (black and gray), may play a role in AZ-directed transport of proteins not localizing to the precursors. In early postnatal stages ribbon precursors attach at the presynaptic membrane, where they fuse alongside their individual presynaptic densities (dark blue). Subsequently, ribbons grow and the size of the attached SVs decreases, while their number increases until hearing onset. Simultaneously, the postsynaptic densities convert from multi-cluster organization to one ellipsoid density per bouton. Developmental refinement of both pre- and postsynapse results in the innervation of predominantly one AZ per SGN and—in the majority of synapses—one single large, droplet-shaped and mature ribbon per AZ.

Figure 4

Molecular maturation of ribbon-type AZs. Molecular composition of a murine ribbon synapse between inner hair cells (IHCs) and spiral ganglion neurons (SGNs) and its differential constitution during early postnatal development (prior to hearing onset) and in matured stages (after hearing onset). Along with the growth, membrane attachment and fusion of precursors, the establishment of a mature ribbon synapse involves the spatial confinement of its molecular components: Ca_Vs localize in tight clusters underneath the ribbon, multiple presynaptic densities merge to one single ribbon anchor and postsynaptically one continuous elongated postsynaptic density is formed that stabilizes a ring-like organization of AMPA receptors. A developmental switch in the regulation of presynaptic exocytosis is present in the downregulation of Synaptotagmin (Syt) 1 and 2, which are likely absent from mature ribbons. Here, the IHC-specific multi-C2-domain protein Otoferlin becomes responsible for the regulation of synaptic vesicle release in the early post-natal stages, despite expression from embryonic ages. (#) Bassoon is part of ribbon precursor spheres in the retina. The here illustrated divergence in the illustrated protein expression between immature and mature preparations does not necessarily reflect absence of expression in the young tissue per se but rather results from the current lack of experimental evidence from pre-hearing preparations. For more detailed information please refer to the respective parts in the text. Font color indicates association with the correspondingly colored pre-/postsynaptic compartment.

Figure 5

Presynaptic structural plasticity at sensory ribbon synapses. (A) In retinal photoreceptors, ribbons can reversibly adjust in size upon exposure to light. (A’) Light-induced plasticity includes the detachment of spherical aggregates from the ribbon apex that continue to tether synaptic vesicles (SVs) and, upon prolonged light exposure, become free-floating in the cytoplasm. As a consequence, the membrane attached ribbon is smaller in the light phase than in the dark phase. When transitioning back to darkness, the ribbon regains material and rebuilds its size. Thereby, the largest ribbon size—thus largest SV tethering capacity—is coinciding with the highest activity state of photoreceptors during the dark phase. (B) In zebrafish lateral line neuromast hair cells, ribbons can show plastic changes in response to different levels of Ca²⁺ influx. (B’) Here, the prolonged opening of presynaptic Ca_Vs by BayK8644 application during early development (3 days post-fertilization, dpf) causes a decrease in ribbon size (upper panel), while the disruption of Ca_V function, either by genetic manipulation or isradipine treatment, causes an increase in ribbon size. Remarkably, in more matured hair cells (5 dpf; lower panel) the ribbons are unaffected by acute BayK8644 treatment, nevertheless ribbon size enhancement could be induced by long-term isradipine treatment. In direct contrast to the mammalian retina, the largest ribbon size—and hence largest vesicle pool—coincides with the lowest level of Ca²⁺ influx.

Figure 6

The role of cytoskeletal transport in IHC presynaptic AZ assembly. (A) The longitudinal microtubule network of murine inner hair cells (IHCs) is polarized in an apico-basal orientation. Shown is a super-resolution (STED) maximum projection of two p14 IHCs labeled against α-tubulin visualized with an intensity-coded look-up table, where warmer colors indicate higher intensities. (B) Representative STED images of cytoplasmically free-floating ribbon precursors (cyan) are located in close proximity to microtubule tracks (red) in IHCs prior to hearing onset (left panel) and colocalize with microtubule-based motor Kif1a (right panels). This suggests microtubule-based transport of ribbon material during development. (C,D) Developmental expression patterns of Kinesin motors (C) and Myosin motors (D), based on publicly-available RNA sequencing data of isolated murine IHCs replotted from the SHIELD database ([96]; https://shield.hms.harvard.edu/index.html). Illustrated are absolute expression patterns from embryonic day (e)18 to postnatal day (p)7 and the same data normalized to their maximum expression level over this time period to reveal temporal expression patterns of the individual targets. (*) indicates motor proteins linked with hereditary syndromic and/or non-syndromic hearing loss in humans (according to https://hereditaryhearingloss.org; October 2020). Scale bars: A 2.5 µm; B left panel: 250 µm, right panels: 200 µm. (B) with permission from Reference [4].