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High-resolution microCT imaging of black mollies Poecilia sp. (Poeciliidae, Teleostei) and Steatocranus tinanti (Cichlidae, Teleostei) stained with phosphotungstic acid (PTA) resulted in good tissue contrast, enabling us to perform a reliable 3D reconstruction of all three sensory maculae of the inner ears. Comparison with maculae that have been 3D reconstructed based on histological serial sections and phalloidin-stained maculae showed high congruence in overall shape of the maculae studied here.
Teleosts show several different orientation patterns, and these patterns have been interpreted with regard to or correlated with acoustic abilities (e.g. sound source localization) of fishes (e.g., [1]). For evaluation of patterns, maculae are flattened during preparation for further analysis using scanning electron or confocal microscopes (e.g., [2]). This in turn means that orientation patterns are obtained, shown, and compared between species in a purely two dimensionally way irrespective of the natural curvature of the maculae in the ear. As has already been stated by Platt and Popper (1981) [3], orientation patterns should preferably take into account the three dimensional structure of sensory epithelia. Until now, the reliable reconstruction of maculae in their original 3D curvature was difficult and has to our knowledge only rarely been performed (zebrafish: [4]; Atlantic molly, Poecilia mexicana: [2]). These reconstructions based on histological serial sections were labor-intensive and results were in part uneven as maculae were prone to distortion or artifacts such as disruption during dehydration or embedding procedures.
Validation of the 3D reconstructed maculae based on microCT imaging. 3D reconstructed macula sacculi (A) and macula lagenae (B) of the herein studied black molly are compared with a corresponding reconstruction from the Atlantic molly Poecilia mexicana in a previous study [2]. Reconstructions of the maculae of P. mexicana (C-E) based on histological serial sections (thickness of sections: 1 μm). The shape of the respective macula types of black molly and Atlantic molly show a high degree of similarity, especially with regard to the macula sacculi, indicating the reliability of 3D reconstructed sensory epithelia based on microCT imaging. Similar features of the maculae sacculi are marked by the arrow and circle. The yellow area in (C) represents reconstructed non-sensory epithelium surrounding the macula sacculi. All maculae are shown in lateral view with their dorsal side up and anterior to the right. Scale bars, 100 μm.
In order to derive reliable results for the 3D reconstruction of the maculae, it is necessary to incorporate prior knowledge of the shape of sensory epithelia and the inner ear anatomy of the species under study based on dissections or scanning electron microscopic investigations. Moreover, we carried out macula labeling in the 3D images with the aid of the borders of the furrows of the otolith housing the macula (in the case of the saccular and lagenar otoliths; Figure 3A-B), in part by using otolith dimensions (utricular otolith), and sites of innervation by the VIIIth cranial nerve. Although histological serial sections have the advantage that single cells can be identified, determination of macula delimitations is still difficult because of the smooth transition between macula and non-sensory epithelium (Figure 3C). In addition, as otoliths have to be dissolved during sample preparation for histology, the organic remains of the otolith can only in part be used as guideline for the identification of macula dimensions. In the case of the macula utriculi, delimitations of this sensory epithelium were easier to identify (Figure 8).
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