The mammalian inner ear contains the cochlea and vestibular organs, which are responsible for hearing and balance, respectively. The epithelia of these sensory organs contain hair cells that function as mechanoreceptors to transduce sound and head motion. The molecular mechanisms underlying hair cell development and differentiation are poorly understood. Math1, a mouse homolog of the Drosophila proneural gene atonal, is expressed in inner ear sensory epithelia. Embryonic Math1-null mice failed to generate cochlear and vestibular hair cells. This gene is thus required for the genesis of hair cells.

The type I and type II hair cells of mature amniote vestibular organs have been classified according to their afferent nerve terminals: calyx and bouton, respectively. Mature type I and type II cells also have different complements of voltage-gated channels. Type I cells alone express a delayed rectifier, gK,L, that is activated at resting potential. We report that in mouse utricles this electrophysiological differentiation occurs during the first postnatal week. Whole-cell currents were recorded from hair cells in denervated organotypic cultures and in acutely excised epithelia. From postnatal day 1 (P1) to P3, most hair cells expressed a delayed rectifier that activated positive to resting potential and a fast inward rectifier, gK1. Between P4 and P8, many cells acquired the type I-specific conductance gK,L and/or a slow inward rectifier, gh. By P8, the percentages of cells expressing gK,L and gh were at mature levels. To investigate whether the electrophysiological differentiation correlated with morphological changes, we fixed utricles at different times between P0 and P28. Ultrastructural criteria were developed to classify cells when calyces were not present, as in cultures and neonatal organs. The morphological and electrophysiological differentiation followed different time courses, converging by P28. At P0, when no hair cells expressed gK,L, 33% were classified as type I by ultrastructural criteria. By P28, approximately 60% of hair cells in acute preparations received calyx terminals and expressed gK,L. Data from the denervated cultures showed that neither electrophysiological nor morphological differentiation depended on ongoing innervation.

Calyx afferents, a group of morphologically and physiologically distinct afferent fibers innervating the striolar region of vestibular sensory epithelia, are selectively labeled by antibodies to the calcium-binding protein calretinin. In this study, the population of calretinin-stained calyx afferents was used to delineate and quantify the striolar region in six rodent species: mouse, rat, gerbil, guinea pig, chinchilla, and tree squirrel. Morphometric studies and hair cell and calyx afferent counts were done. Numbers of hair cells, area, length, and width of the sensory epithelium increase from mouse to tree squirrel. In the mouse and rat, calretinin is found in 5-9% of all type I hair cells, 20-40% of striolar type II hair cells, and 70-80% of extrastriolar type II hair cells. Numbers of calyx afferents increase from mouse to squirrel, with more complex calyx afferents in larger species. About 10% of calyx afferents are branched. Based on our counts of total numbers of calyx afferents in chinchilla maculae and in comparison to fiber counts in the literature, the proportion of calyx afferents is greater than previously described, constituting nearly 20% of the total. Because morphometric measures increase with body weight, we obtained additional data on vestibular end organ surface areas from the literature and used this to construct a power law function describing this relationship. The function holds for species with body weights less than approximately 4 kg. Greater than 4 kg, the surface area of the sensory epithelia remains constant even with increasing body weight.

BETA2/NeuroD1 is a bHLH transcription factor that is expressed during development in the mammalian pancreas and in many locations in the central and peripheral nervous systems. During inner ear ontogenesis, it is present in both sensory ganglion neurons and sensory epithelia. Although studies have shown that BETA2/NeuroD1 is important in the development of the hippocampal dentate gyrus and the cerebellum, its functions in the peripheral nervous system and in particular in the inner ear are unclear. Mice carrying a BETA2/NeuroD1 null mutation exhibit behavioral abnormalities suggestive of an inner ear defect, including lack of responsiveness to sound, hyperactivity, head tilting, and circling. Here we show that these defects can be explained by a severe reduction of sensory neurons in the cochlear-vestibular ganglion (CVG). A developmental study of CVG formation in the null demonstrates that BETA2/NeuroD1 does not play a primary role in the proliferation of neuroblast precursors or in their decision to become neuroblasts. Instead, the reduction in CVG neuron number is caused by a combination both of delayed or defective delamination of CVG neuroblast precursors from the otic vesicle epithelium and of enhanced apoptosis both in the otic epithelium and among those neurons that do delaminate to form the CVG. There are also defects in differentiation and patterning of the cochlear duct and sensory epithelium and loss of the dorsal cochlear nucleus. BETA2/NeuroD1 is, thus, the first gene to be shown to regulate neuronal and sensory cell development in both the cochlear and vestibular systems.

The chinchilla crista ampullaris was studied in 10 samples, each containing 32 consecutive ultrathin sections of the entire neuroepithelium. Dissector methods were used to estimate the incidence of various synaptic features, and results were confirmed in completely reconstructed hair cells. There are large regional variations in cellular and synaptic architecture. Type I and type II hair cells are shorter, broader, and less densely packed in the central zone than in the intermediate and peripheral zones. Complex calyx endings are most common centrally. On average, there are 15-20 ribbon synapses and 25-30 calyceal invaginations in each type I hair cell. Synapses and invaginations are most numerous centrally. Central type II hair cells receive considerably fewer afferent boutons than do peripheral type II hair cells, but have similar numbers of ribbon synapses. The numbers are similar because central type II hair cells make more synapses with the outer faces of calyx endings and with individual afferent boutons. Most afferent boutons get one ribbon synapse. Boutons without ribbon synapses were only found peripherally, and boutons getting multiple synapses were most frequent centrally. Throughout the neuroepithelium, there is an average of three to four efferent boutons on each type II hair cell and calyx ending. Reciprocal synapses are rare. Most synaptic ribbons in type I hair cells are spherules; those in type II hair cells can be spherical or elongated and are particularly heterogeneous centrally. Consistent with the proposal that the crista is concentrically organized, the intermediate and peripheral zones are each similar in their cellular and synaptic architecture near the base and near the planum. An especially differentiated subzone may exist in the middle of the central zone.