A key characteristic of every living individuum is the ability to interact with its environment.
Particularly in animals, this interaction does not only include the sensation of stimuli, but also communication with each other. For this purpose, environmental information is detected through sensory cells, e.g. retinal or inner ear cells, transduced into chemical or electrical signals, and transferred into the respective brain areas. Synapses formed between sensory cells and neurons, or between different neurons, form the basis for signal transmission within the sensory system. At these synapses, neurotransmitter-filled synaptic vesicles (SVs) undergo a cycle of release and recycling:
Exocytosis at the presynaptic active zone (AZ) is induced by cell depolarization and results in the integration of the SV membrane into the cellular plasma membrane. This process goes along with the release of neurotransmitters into the synaptic cleft formed between pre- and postsynapse.
Exocytosis is accompanied by SV recycling, which includes the internalization of membrane (endocytosis) and the reformation of new SVs. Endocytosis is not only required to balance the increase in the cell surface following exocytosis, but also to clear release sites at the AZ from vesicular proteolipids. Moreover, the endocytosed membrane material builds the basis for newly formed SVs.
These general steps within the SV cycle are conserved between different cell types and within different species. The molecular composition and architecture of synapses, though, are heterogenous within the animal body and individually adapted to the specific demands of each respective synapse.
One example for such a specialization are the so-called ribbon synapses within inner hair cells (IHCs) of the inner ear. In the first part of my introduction, I will summarize anatomical features of IHCs, followed by an extensive morphological and functional characterization of their ribbon synapses. In the third part of my introduction, I will compare synaptic transmission in neurons and in IHCs.
Especially in comparison to neurons, IHCs serve as an interesting model system for exo- and endocytic processes: here, synaptic transmission is adapted to the edge of synaptic capabilities.
Beyond that, understanding molecular processes in IHCs provides a basis for the development of therapeutic strategies for the treatment of hearing impairments associated with the inner ear.
1.1.1 T HE M AMMALIAN I NNER E AR
The mammalian ear consists of the outer ear, the middle ear, and the inner ear (Fig. 1-1A). While the outer ear is required for the bundling and filtering of incoming sound pressure waves, the middle
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ear, harboring the ear drum and the ossicles, matches the different impedance of sound conduction in the air and the fluid-filled inner ear. Within the inner ear, the mechanical information is transduced into electrical signals, which are transmitted via auditory nerves formed by the bipolar spiral ganglion neurons (SGNs) and perceived by the respective brain areas.
Fig. 1-1: The anatomy of the mammalian inner ear. (A) Overview of outer ear, middle ear and inner ear.
(B) Cross-section through the cochlea with the three fluid-filled cavities, the organ of Corti on top of the basilar membrane, and the auditory nerve. (C) The organ of Corti contains three rows of OHCs with stereocilia embedded in the tectorial membrane, and one row of IHCs, as well as different types of supporting cells. Images modified from Pearson Education 2012.
The inner ear is formed by the vestibular apparatus with its three semicircular canals and two macula organs, which are required for balance, and by the cochlea, which is a bony structure resembling a and displays the end-organ for hearing (Fig. 1-1A). The cochlea contains three fluid-filled compartments, which wind up along the cochlea: the scala media, scala vestibuli and scala tympani.
(Fig. 1-1B). These cavities contain solutions of different composition. The scala vestibuli and scala
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tympani contain low-K+ perilymph with a composition closely matching typical extracellular saline, whereas the scala media contains a high-K+ fluid called endolymph. When sound enters the inner ear, the basilar membrane, which borders the scala media, vibrates in form of a travelling wave. This wave reaches its maximal amplitude at a specific region along the longitudinal axis of the cochlea depending on the frequency of the sound. This frequency-place-relationship is called tonotopy and leads to low-frequency signals having their maxima at the apex of the cochlea and high-frequency signals reaching their maxima at the base according to the varying micromechanical properties of the basilar membrane along the tonotopic axis. At this specific position, the organ of Corti, which is the sensory epithelium of the cochlea and located on top of the basilar membrane, processes the given sound stimulus (Fettiplace, 2017). The organ of Corti is formed by three rows of outer hair cells (OHCs) and one row of IHCs, as well as by different types of supporting cells (Fig. 1-1C). OHCs are studded with stereocilia that project not only into the scala media but are, at least partially, also embedded into the tectorial membrane that covers the organ of Corti. Sound-induced movements of basilar membrane and tectorial membrane relative to each other induce deflections of the stereociliar hair bundles of the OHCs. The OHCs now contract and expand in response to the sound stimuli, which further amplifies the relative movements of the basilar membrane and tectorial membrane and also the resulting radial flux of the endolymph between the tectorial membrane and the surface of the organ of Corti (Fettiplace, 2017). The flux now causes displacements of hair bundles of the IHCs and eventually results in the opening of mechano-electrical transduction channels located within the membrane of the stereocilia (Assad et al., 1991; Howard and Hudspeth, 1988). These nonselective cation channels show ultrafast activation and closing kinetics and provide graded cell depolarization or hyperpolarization through K+ influx dependent on intensity and direction of stereociliar deflections (Beurg et al., 2006; Corey and Hudspeth, 1979; Fettiplace, 2017).
1.1.2 I NNER H AIR C ELLS S HOW A P OLARIZED C ELLULAR O RGANIZATION
Like in OHCs, hair bundles of IHCs are located at the top of the cells, whereas voltage-gated CaV1.3 Ca2+-channels, which open upon depolarization of the cell, are exclusively found in the basal half of IHCs (see Fig. 1-2; Brandt et al., 2003; Platzer et al., 2000; Roberts et al., 1990). Generally, IHCs show a strong polarization along the apicobasal axis: Stereocilia at the apex of IHCs are anchored in the cuticular plate, which is an amorphous network of cytoskeletal proteins like actin (Slepecky and Chamberlain, 1985). Golgi complexes as well as LAMP1-positive structures likely displaying lysosomes have exclusively been detected in the apical half of IHCs (Revelo et al., 2014; Siegel and
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Brownell, 1986). Even though endosome-like vacuoles (ELVs) can be observed in all parts of IHCs, markers for early endosomes (EEA1) and late endosomes (syntaxin 16) have only been visible in the apical and nuclear region, but not beneath (Revelo et al., 2014).
Contrarily, synaptic transmission from IHCs to SGNs is restricted to the basal region of IHCs. Not only neurotransmitter release, but also stimulus-evoked endocytic processes and SV reformation were detected in the IHC base (Kamin et al., 2014; Kantardzhieva et al., 2013; Revelo et al., 2014).
Here, the specialized ribbon synapses are located to facilitate the Ca2+-induced release of glutamate (see Fig. 1-2C). Depending on the tonotopic position along the basilar membrane, individual cochlear IHCs harbor between 5 and 20 ribbon synapses (Meyer et al., 2009). Each of these synapses is thought to be innervated by a single afferent SGN (Liberman, 1978). After the onset of hearing at ~P12 in mice (Mikaelian and Ruben, 1965), usually one electron-dense structure named synaptic ribbon (see 1.2.2 Peculiarities of Inner Hair Cell Ribbon Synapses) can be detected per AZ, rarely two or even three (Sobkowicz et al., 1986; Wong et al., 2014). Interestingly, the size of synaptic ribbons and active zones as well as the Ca2+-influx and rates of spontaneous neurotransmitter release are heterogenous between individual AZs within the same IHC (Frank et al., 2009; Meyer et al., 2009; Ohn et al., 2016).
Likewise, SGNs that innervate the IHCs differ in their diameter (Liberman, 1982a; Merchan-Perez and Liberman, 1996; Ohn et al., 2016). Ribbon synapses containing a larger synaptic ribbon, more Ca2+-channels, and a high number of SVs are predominantly found at the modiolar side (facing the spiral ganglion), whereas smaller ribbons connected to a higher rate of spontaneous release are preferentially located at the pillar side (facing the OHCs) of IHCs (Frank et al., 2009; Meyer et al., 2009; Ohn et al., 2016). It has been suggested that the heterogeneity of ribbon synapses and SGNs is required for the encoding of different sound pressure levels (Liberman, 1982a; Liberman, 1982b;
Merchan-Perez and Liberman, 1996; Moser and Vogl, 2016).
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Fig. 1-2: Morphological characteristics of IHCs and ribbon synapses. (A) Overview of two IHCs. Cell borders are highlighted in blue, afferent (violet) and efferent (red) nerve terminals are shaded. Scale bar 2 µm. (B) Higher magnification of an IHC base containing a ribbon synapse. Scale bar 500 nm. (C) Cross-section of a synaptic ribbon (R) surrounded by SVs opposed to an afferent bouton. Scale bar 200 nm. (D-F) Schematic drawings of an IHC (D), a cross-section of a ribbon synapse (E) and the top view of an active zone without the ribbon (F). RA-SV ribbon-associated SV; MP-SV membrane-proximal SV; PD presysnaptic density; AMPA -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor.
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