Salient properties of otoconia development & physical characteristics.

Data from the Fermin's labindicates that otoconia of the chick (Gallus domesticus) inner ear may have central cores in vivo. The data also show that the ultrastructural organization of fibrils fixed with aldehydes and histochemical stains follows trajectories that conform to the hexagonal shape of otoconia. These changes in direction may contribute to the formation of a central core. The existence of central cores is important for the in vivo buoyancy of otoconia. Packing of fibrils is tighter after phosphotungstic acid (PTA) stained otoconia than with other histochemical stains, which usually produce looser packing of fibrils and seemingly larger central core. TEM of tilted and untilted material showed that turning of fibrils occurs at the points where the face angles of otoconia form and where central cores exist. Video image processing of the images allowed reconstructing a template which, if assumed to repeat and change trajectories, would fit the pattern of fibrils seen in fixed otoconia. Since it is highly unlikely that aldehyde primary fixation or PTA stain caused such drastic change in the direction of fibrils, the template derived from these results may closely approximate patterns of otoconia fibrils packing in vivo. However, if the above is correct, the perfect crystallographic diffraction pattern of unfixed otoconia do not correspond to patterns of fixed fibrils.

The very first appearance of otoconia, may be initially independent of calcium integration into those fibrils (Kogaya, Haruna et al. 1989) . The initial stage(s) and duration of otoconia formation may be demarcated by organic fibrils seen first attached to the stereocilia (Cohen and Fermin 1985) . In the case of otoconia formation, organic fibrils later segment into presumptive units, away from the sterocilia (Fermin, Igarashi et al. 1987) . Segmentation away from the stereocilia allows otoconia flexibility from the very beginning of formation (Figure 1F). Such flexibility is further enhanced by the cementing substance between otoconia, which remains mostly un-mineralized. Both properties provide the elasticity required for otoconia and the otoconial membrane as a unit to behave as loaded springs and possibly have piezoelectric properties (Ross 1983).

Previous antimonate stain of chick otoconia indicates that fibrils which do not become part of otoconia units remain unmineralized. The structural integrity of fibrils cementing otoconia is similar to that displayed by immature otoconia of embryos prior to calcification of the hard components of the inner ear (Fermin and Igarashi 1985a) . Therefore, if formation and mineralization of otoconia organic template occur concomitantly as it has been suggested (Harada 1982) , the genesis and maturation of fibrils cementing otoconia together must be resolved. Two possibilities are attractive: 1) either fibrils form unmineralized templates that later mineralize, while the fibrils between each crystal remain unmineralized (Fermin and Igarashi 1986; Fermin, Igarashi et al. 1987) ; or 2) mineralized units appear (Ballarino, Howland et al. 1985; Ross 1979) and by a still unknown mechanism, give rise to cementing unmineralized fibrils between otoconia.

Furthermore, it is unlikely that aldehyde primary fixation induce changes that result in the core observed for each crystal, because otoconia contain glycoprotein which are probably cross-linked by the primary fixation. It is also unlikely that the aldehyde primary fixation used caused fibrils to turn abruptly at convergent points near or at the faces of the hexagon and central core. For these reasons and because of data presented in this study it is assumed that the ultrastructure of fibrils shown here represents a fairly close approximation of their packing in vivo, with minor distortions caused by preparatory conditions.

In a more likely scenario, fibrils are probably formed by glycoproteins like fibronectin and by glycosaminoglycans like keratan sulfate intertwined into a loosely packed mesh early in development (Fermin, Lovett et al. 1990; Sugiyama, Spicer et al. 1991) . As the ratio between these two groups of molecules changes, the fibrils become more organized and packed with intercalating calcium carbonate components that we see after potassium pyroantimonate staining. After fixation and demineralization, the fibrils become somewhat disorganized, with structural integrity shown in this report in chicken and in rodents (Lim and Rueda 1990; Salamat, Ross et al. 1980) . As indicated in the introduction, glycosaminoglycans are present in other parts of the inner ear, and are abundant in mineralizing cartilage and bone. Glycosaminoglycans may also be key component of otoconia formation and mineralization, because similar to teeth and bone, otoconia are composed of an organic matrix initially that is later mineralized.

The following should be addressed in future analyses of otoconia genesis: a) The relevance that glycosaminoglycans like Keratan sulfate play in otoconia formation, and maintenance after maturation; b) the coexistence of the utricular calcified macula and non-calcified cupula of the lateral canal crista. They are separated by less than 0.5 mm apart and are immersed in the same endolymphatic fluid.

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