 a molecular coupling, other mechanisms contribute to localized Ca2+ signalling. The presence of the mobile, proteinaceous Ca2+ buffers calretinin, calbindin and parvalbumin has now been documented in a variety of cochlear and vestibular hair cells (e.g., Edmonds et al., 2000; Heller et al., 2002; Hackney et al., 2005). Their relevance for presynaptic Ca2+ signalling has been inferred from recordings of Ca2+-activated large-conductance K+ channels (Fettiplace, 1992; Roberts, 1993; Tucker & Fettiplace, 1996; Edmonds et al., 2000), simulations (Roberts, 1994) and capacitance measurements (Moser & Beutner, 2000; Spassova et al., 2004). Most likely they spatiotemporally restrict the presynaptic Ca2+ domains and, hence, improve the timing of synaptic transmission. However, discrepancies regarding both the amount and kinetic properties of buffers in different hair cells (Roberts, 1993; Edmonds et al., 2000 vs Moser & Beutner, 2000) suggest that our understanding of calcium buffers at the hair cell active zone synapse is far from complete. Examination of hair cells from mutant mice lacking the major proteinaceous Ca2+ buffers promises further insight into this important question.
In addition to synchronous synaptic transmission, the molecular machinery needs to be suited for long-lasting transmitter release. By yet unknown mechanisms CaV1.3 currents in hair cells are much less inactivating than in heterologous expression systems (Koschak et al., 2001). Most likely this is due to interactions with a special combination of auxiliary subunits and proteins, such as the class of Ca2+ binding proteins (Haeseleer et al., 2004). None of these auxiliary determinants of hair cell Ca2+ current are known so far.
Once the neurotransmitter is released into the synaptic cleft, it activates an AMPA-like receptor residing within a receptor cluster at the postsynaptic density of afferent auditory nerve fibers. In a classical study, Ottersen and his colleagues showed the abundance of the glutamate receptor subtypes (GluR) 2, 3 and 4 by immunogold labelling (Matsubara et al., 1996). Interestingly, the distribution of the glutamate receptor was not homogenous in the postsynaptic density (PSD): the receptor numbers increase at the border of the PSD. Using patch-clamp recordings from the postsynaptic endings contacting rat IHCs, Glowatzki & Fuchs (2002) showed that excitatory postsynaptic currents of spiral ganglion neuron afferent fibres are mediated by AMPA receptors. They failed to detect NMDA or Kainate currents. Recently, Eybalin and colleagues studied the developmental expression changes of postsynaptic glutamate receptors using western blotting, RT-PCR and immunohistochemistry (Eybalin et al., 2004). They showed that GluR2 becomes expressed only around the onset of hearing, while GluR3 and 4 are present before. Little is known about the other molecular determinants of the PSD. Davies et al. (2001) report the presence of PSD-95 and PSD-93 in the PSD of auditory nerve fibres.
To avoid saturation and excitotoxicity due to an excess of glutamate in the synaptic cleft, glutamate is taken up by glutamate transporters (EAAT) into supporting cell transporters (Furness & Lehre, 1997; Furness & Lawton, 2003; Rebillard et al., 2003). Interestingly, glutamate-aspartate transporters (GLA ST) are more abundant on the pillar IHC side, where high spontaneous rate fibers are located, compared to the modiolar cell side, where low spontaneous rate fibres are located (Liberman, 1982; Furness & Lawton, 2003).
The active zone of the hair cell is characterized by an electron dense structure called synaptic body or ribbon. Most synaptic ribbons found in mature hair cells are anchored to the plasma membrane, one ribbon per active zone, and oppose a postsynaptic terminal. A small number of “floating” ribbons (<5 %) are observed, and probably reflect the turnover of these subcellular organelles (Zenisek et al., 2004; Khimich et al., 2005). Shape, size and number of the ribbons differ (see Table 1) between species as well as along the tonotopic axis, during development (Shnerson, Devigne & Pujol, 1981; Sobkowicz et al., 1982; Khimich et al., 2005) and even among different active zones within a single hair cell (Merchan-Perez & Liberman, 1996). For example, frog saccular hair cells display about 20 large, synaptic bodies irrespective of the cells’ location in the end organ (Roberts et al., 1990; Lenzi et al., 1999, 2002). Whereas in the chick cochlea, the number of synaptic bodies is constant in tall hair cells (functional homologue of the mammalian IHCs), the synaptic body diameter increases with increasing characteristic frequency (Martinez-Dunst, Michaels & Fuchs, 1997). Conversely, the number of ribbons varies along the tonotopic axis in many other species including turtle (plus small size differences: Sneary, 1988; Schnee et al., 2005), cat (Liberman, Dodds & Pierce, 1990), gerbil (Slepecky et al., 2000) and guinea pig (Hashimoto, Kimura & Takasaka, 1990).
Both the number and shape of the synaptic ribbons can also change with the onset of hearing. Mouse IHCs contain a large number (>20 in the apical cochlea, Nemzou et al., unpublished data) of small synaptic bodies (Shnerson et al., 1981; Sobkowicz et al., 1982) before the onset of hearing. After the onset of hearing only fewer but larger ellipsoid ribbons are observed (Shnerson et al., 1981; Sobkowicz et al., 1982; Francis et al., 2004; Khimich et al., 2005). An interesting feature described for cat IHCs is the spatial segregation of synapses with fibres of different spontaneous rates and thresholds. Thus, low-spontaneous-rate, high-threshold fibres contact the IHCs on their modiolar side, whereas high-spontaneous-rate, low-threshold fibres synapse on the pillar IHC side. EM analysis showed that the ribbon size and accordingly the number of ribbon-associated synaptic vesicles tend to increase with decreasing spontaneous rate of the contacted fibres (Liberman, 1980; Liberman et al., 1990; Merchan-Perez & Liberman, 1996).
Free cytosolic synaptic vesicles.
These vesicle populations have been quantified for hair cells of various species (Table 1). A variety of approaches have been used to assess the vesicle populations and accordingly, varying degrees of certainty surround these vesicle counts. The different approaches have included: precise three-dimensional EM counts based on few synapses (EM tomography: Fig 3, Lenzi et al., 1999; Lenzi et al., 2002; reconstructions based on ultrathin serial sections: Schnee et al., 2005); extrapolations from random ultrathin EM sections of tens of synapses (Khimich et al., 2005) and approximations based on ribbon surface areas and vesicle packing density estimates (Spassova et al., 2004; Khimich et al., 2005: more than 200 ribbons analyzed).
Morphology of synaptic ribbons. (a, b and c) Electron micrographs of inner hair cell ribbon synapses of 8-week-old mouse. Ribbons are attached to the plasma membrane and surrounded by a monolayer of synaptic vesicles. Colored dots in (b) illustrate the morphologically defined classes of synaptic vesicles: red dots indicate the docked vesicle associated with the ribbon; yellow marks ribbon- associated SV which are not docked and green indicates outlying cytoplasmic vesicles. (d) Slice through an osmium-stained frog saccular hair cell synapse, reconstructed by electron tomography. Note the round shape of the synaptic body in contrast to the elipsoid one of the mouse. (e) Three-dimensional structure of presynaptic organelles from the same reconstruction shown in (d). The synaptic body (blue) is open and lies adjacent to the hair cell’s plasma membrane in red. Synaptic body-associated vesicles (yellow) surround the SB; outlying vesicles (green) lie further out in the cytoplasm. Also visible are coated vesicles (gold) and cisterns (purple).
Docked vesicles at a conventional synapse are viewed as a homogeneous anatomical pool. However, at the hair cell ribbon synapse docked vesicles are found in a variety of anatomical states; this includes underneath the ribbon (docked and ribbon-associated), at the active zone without contact to the ribbon, and outside the active zone. Lenzi et al., (1999) termed these last two classes of docked vesicles as “outlying”. The functional implications of the different classes of docked vesicles are poorly understood. Obviously, proximity to a calcium channel increases the likelihood that a docked vesicle is of functional significance. Accordingly, Lenzi et al., (1999) attempted to correlate the positions of docked vesicles and Ca2+ channels at the active zone. While the “outlying” vesicles are substantial in number, they are located further from the presumed calcium channel containing presynaptic densities than are other docked vesicles. “Outliers” has been alternatively used in evanescent wave microscopic studies (Zenisek, Steyer & Almers, 2000) to describe the “docking” and fusion of synaptic vesicles outside of functionally defined active zones.
One fruitful way to investigate the compact afferent presynaptic terminal of the inner ear is to monitor exocytic fusion and endocytic retrieval of synaptic vesicle membrane as changes of the membrane capacitance (C m) using the patch-clamp technique (for review see Neher, 1998). During the fusion of the synaptic