Introduction 1 Figure 1 rib PSD arrowhead star  HASIC  ELEASE  ROPERTIES The synapse’s precision to code the temporal fine structure of acoustic stimuli is unparalleled. For example, our capacity to locate sound sources in space depends on interaural time differences in the arrival of sound of only tens to hundreds of microseconds. Unlike a conventional synapse that is driven by action potentials, the hair cell afferent synapse responds to graded changes in membrane potential. While the action potential results in a strong all-or-none stimulus-secretion coupling, the hair cell afferent synapse is faced with the challenge of encoding sounds of extremely different intensities and, hence, must rely on mechanisms that allow for variable stimulus-secretion coupling. At the same time, the hair cell afferent synapse must also maintain temporal fidelity. For example, the firing of an auditory nerve fiber can follow at a constant phase relationship the periodicity of tonal stimuli into the low kHz range of frequencies, and we infer that the hair cell neurotransmitter release must comply with this extraordinary timing. Such temporal precision in synaptic transmission is even observed with sub-threshold stimuli, where the temporal pattern of spike firing regularizes already before there is an overall increase in the discharge rate. ONIC  ELEASE  ROPERTIES As we all can tell from our own experience, the cochlear hair cell afferent synapse must be able to encode prolonged, ever-present sounds. For example, despite adaptation, the constant background noise of the room’s air handling system is faithfully reported by the peripheral auditory system. To meet this requirement of audition, sustained neurotransmitter release must be maintained and implicates the need for extensive synaptic vesicle cycling (both, vesicle exo- and endocytosis). The hair cell afferent synapse’s capacity for sustained signal has often been attributed to the presence of the synaptic ribbon, and apparent depot of synaptic vesicles. However, recent morphological and physiological studies suggest additional/different roles for the hair cell synaptic ribbon, and have fuelled the structure-function debate at the ribbon synapse. Molecular Anatomy and Physiology of the Hair Cell Synapse Safieddine & Wenthold, 1999 tom Dieck et al., 2005 Schmitz, Konigstorfer & Sudhof, 2000 Zenisek et al., 2003 Khimich et al., 2005 Khimich et al., 2005 tom Dieck et al., 2005 Schmitz et al., 2000 tom Dieck et al., 2005 Valente et al., 2005 see Gallop, Butler & McMahon, 2005 Khimich et al., 2005 Dick et al., 2001 Dick et al., 2003 Khimich et al., 2005 Altrock et al., 2003 Khimich et al., 2005 2+ 2+ V Brandt, Striessnig & Moser, 2003 unpublished data Safieddine & Wenthold, 1999 Xu et al., 1998 Sakaba et al., 2005 Favre et al., 1986 Safieddine & Wenthold, 1999 Sudhof, 2004 Safieddine & Wenthold, 1999 Safieddine & Wenthold, 1999 Virmani et al., 2003 unpublished data V 2+ Platzer et al., 2000 Brandt et al., 2003 Brandt, Khimich & Moser, 2005 V Bech-Hansen et al., 1998 Strom et al., 1998 Mansergh et al., 2005 L 2+ Rodriguez-Contreras & Yamoah, 2001 V Roberts, Jacobs & Hudspeth, 1990 Rodriguez-Contreras & Yamoah, 2001 2+ Issa & Hudspeth, 1994 Tucker & Fettiplace, 1995 Zenisek et al., 2003 Sidi et al., 2004 Brandt et al., 2005 2+ Roberts et al., 1990 see also Rodriguez-Contreras & Yamoah, 2001 Brandt et al., 2005 see 1 Table 1 Size and kinetics of release components related to the synaptic ribbon and to morphological vessel pools associated from different species  2+ Brandt et al., 2005 V Hibino et al., 2002 V Khimich et al., 2005 2 Figure 2 A red green white orange B 1 Safieddine & Wenthold, 1999 2 Furness & Lawton, 2003 3 Eybalin et al., 2002 4 Schmitz et al., 2006 5 Khimich et al., 2005 6 Platzer et al., 2000 7 Brandt, Striessnig & Moser, 2003 8 Brandt, Khimich & Moser, 2005 9 Matsubara et al., 1996 10 Eybalin et al., 2004  2+ 2+ Edmonds et al., 2000 Heller et al., 2002 Hackney et al., 2005 2+ 2+ + Fettiplace, 1992 Roberts, 1993 Tucker & Fettiplace, 1996 Edmonds et al., 2000 Roberts, 1994 Moser & Beutner, 2000 Spassova et al., 2004 2+ Roberts, 1993 Edmonds et al., 2000 Moser & Beutner, 2000 2+ V Koschak et al., 2001 2+ Haeseleer et al., 2004 2+ Matsubara et al., 1996 Glowatzki & Fuchs (2002) Eybalin et al., 2004 Davies et al. (2001) Furness & Lehre, 1997 Furness & Lawton, 2003 Rebillard et al., 2003 Liberman, 1982 Furness & Lawton, 2003 Relating Structural and Functional Vesicle Populations ORPHOLOGY Synaptic Ribbons Zenisek et al., 2004 Khimich et al., 2005 see 1 Shnerson, Devigne & Pujol, 1981 Sobkowicz et al., 1982 Khimich et al., 2005 Merchan-Perez & Liberman, 1996 Roberts et al., 1990 Lenzi et al., 1999 2002 Martinez-Dunst, Michaels & Fuchs, 1997 Sneary, 1988 Schnee et al., 2005 Liberman, Dodds & Pierce, 1990 Slepecky et al., 2000 Hashimoto, Kimura & Takasaka, 1990 Shnerson et al., 1981 Sobkowicz et al., 1982 Shnerson et al., 1981 Sobkowicz et al., 1982 Francis et al., 2004 Khimich et al., 2005 Liberman, 1980 Liberman et al., 1990 Merchan-Perez & Liberman, 1996 Synaptic vesicles Lenzi et al., 1999 3 Docked vesicles (no discernible space between vesicle and plasma membranes) Ribbon-associated vesicles Free cytosolic synaptic vesicles. 1 3 Lenzi et al., 1999 Lenzi et al., 2002 Schnee et al., 2005 Khimich et al., 2005 Spassova et al., 2004 Khimich et al., 2005 Figure 3 a b c b red dots yellow green d e d blue red yellow green gold purple  Lenzi et al., (1999) Lenzi et al., (1999) 2+ Zenisek, Steyer & Almers, 2000 HYSIOLOGY C m see Neher, 1998 C m C m 1 V 2+ C m Coorssen, Schmitt & Almers, 1996 1 Moser & Beutner, 2000 Spassova et al., 2004 Rutherford & Roberts, 2006 Liley & North, 1953 Birks & MacIntosh 1961 Elmqvist & Quastel, 1965 Schnee et al., 2005 Khimich et al., 2005 Khimich et al., 2005 2+ Moser & Beutner, 2000 Spassova et al., 2004 Roberts et al., 1990 Issa & Hudspeth, 1994 Tucker & Fettiplace, 1995 Zenisek et al., 2003 Sidi et al., 2004 Brandt et al., 2005 Brandt et al., 2005 C m Beutner & Moser, 2001 Johnson, Marcotti & Kros, 2005 Glowatzki & Fuchs, 2002 Koutalos & Yau, 1996 Brenner, Bialek & de Ruyter van Steveninck, 2000 Holton & Weiss, 1983 Russell & Sellick, 1978 Furukawa & Matsuura, 1978 Moser & Beutner, 2000 Westerman & Smith, 1984 Yates, Robertson & Johnstone, 1985 Spassova et al., 2004 Griesinger, Richards & Ashmore, 2005 C m 1 Brandt et al., 2005 Griesinger et al., 2005 Edmonds, Gregory & Schweizer, 2004 2+ Brandt et al., 2005 Furukawa, Kuno & Matsuura, 1982 C m V C m Parsons et al., 1994 see 1 Schnee et al., 2005 2+ Heidelberger et al., 1994 Beutner et al., 2001 von Gersdorff et al., 1996 Khimich et al., 2005 Is it Possible at all to Relate Anatomically and Physiologically Defined Pools of Synaptic Vesicles? C m 2 Breckenridge & Almers, 1987 Lenzi et al., 1999 Khimich et al., 2005 Klyachko & Jackson, 2002 Sun, Wu & Wu, 2002 von Gersdorff & Matthews, 1994 Mennerick & Matthews, 1996 Neves & Lagnado, 1999 What Is the Anatomical Substrate of the Fast Kinetic Component in Hair Cell Exocytosis? Moser & Beutner, 2000 Khimich et al., 2005 Schnee et al., 2005 Rutherford & Roberts, 2006 Edmonds et al., 2004 Spassova et al., 2004 1 see Edmonds et al., 2004 Parsons & Sterling, 2003 Edmonds et al., 2004 Spassova et al., 2004 Glowatzki and Fuchs (2002) Glowatzki & Fuchs, 2002 1 Edmonds et al., 2004 Rutherford & Roberts, 2006 see see 1 see above 1 Zenisek et al. (2000) Griesinger et al. (2005) The apparent differences between the behavior of ribbon-tethered vesicles in these two types of ribbon synapses highlight the fact that it could be an oversimplification to assume that a common anatomical substrate underlies the first/fastest recorded kinetic component of hair cell exocytosis. The physical dimensions of hair cell active zones, shapes of synaptic ribbons, best stimulation frequency, and possibly endogenous calcium buffering vary dramatically across end organ, species and in some cases even within the same end organ of a given species. Thus, subsequent studies may reveal that different hair cells use different anatomical substrates for different functional pools of synaptic vesicles to meet their respective synaptic demands. What about the Anatomical Substrate of Subsequent, Slower Kinetic Components of Exocytosis? Moser & Beutner, 2000 Spassova et al., 2004 Edmonds et al., 2004 Griesinger et al., 2005 Schnee et al. (2005) C m Schnee et al., 2005 C m Khimich et al., 2005 Lenzi et al., 1999 2002 Beutner et al., 2001 Zenisek et al., 2000 C m Conclusion and Outlook The multidisciplinary approach to the hair cell ribbon synapse has for the first time quantitatively described important aspects of the synapse’s structure and function. Most importantly, despite all limiting uncertainty, we begin to relate molecules, structure and function. Although still debated, the concept of a readily releasable pool has been substantiated for the ribbon synapse of several hair cells. We have gained a few insights into the mechanisms underlying the incredible temporal precision of synapses that participate in the coding of sound. Moreover, in addition to learning about the molecular physiology, the availability of mouse mutants with defined synaptic lesions provided the possibility to study the properties of synaptopathic hearing impairment in greater detail. However, we are far from a comprehensive molecularly defined model of ribbon structure and function. The molecular dissection of the hair cell synapse is technically challenging due to the low amount of tissue and will require much more time and effort. Ear-specific genetic deletion also will be helpful to investigate synaptic protein function in hair cell sound coding. A more precise and direct biophysical analysis of single hair cell synapses will require combined pre- and postsynaptic recordings as well as optical measurements, such as evanescent wave microscopy and confocal techniques. The optical approach will be strongly facilitated by the generation of genetically targeted fluorescent vesicle tags.