Cloning and expression of Shaker alpha- and beta-subunits during inner ear development.

Sensory cells of the chicken cochlea exhibit different ion channels relative to their position along the epithelium. One of these channels conducts an A-type potassium current which is found primarily in 'short' hair cells. Here, we report the first full length cloning and developmental expression of Shaker genes from this endorgan. Clones were obtained by screening a chicken (Gallus gallus) cochlea cDNA library, using probes made from RHK1 (i.e., Kvalpha1.4) cDNA, a Shaker homologue isolated from rat heart, and hKvbeta1.2 cDNA, a beta homologue isolated from human heart. Sequence analysis revealed a chick homologue of Kvalpha1.4, with a deduced amino acid similarity of 76-79% to mammalian Kvalpha1.4, and a chick homologue of Kvbeta1.1, with a similarity of 95% to mammalian Kvbeta1.1. In addition, we isolated a variant of cKvalpha1. 4 (cKvalpha1.4(m)) that differs in its untranslated regions and shows complete similarity in its coding region, except for the deletion of a single nucleotide. During development of the inner ear, reverse transcription-polymerase chain reaction (RT-PCR) studies show that the beta-subunit is expressed as early as embryonic day 3, whereas alpha- and beta-subunits are coexpressed on embryonic days 7 to 10, 14, and in adult.

Neurotrophic factors modulate hair cells and their potassium currents in chick otocyst explants.

Neurotrophins, retinoids and their receptors are present in the sensory epithelia of the inner ear during development. We show that these factors modulate the proliferation of hair cells and their K+-currents when the embryonic day 3 (ED 3) presumptive inner ear (i.e. otocyst) is maintained in organ culture. All trans-retinoic acid (RA) increases hair cell differentiation and enhances the acquisition of outward currents, including a delayed rectifier and a fast activating, transient type, voltage-gated potassium current. In contrast, brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) decrease ionic current activity, and the addition of RA with the neurotrophins enhances this inhibitory response in an age-dependent manner. We measured the total number of cells per explant over time to determine precisely when and how these factors inhibit explant growth. We found that high concentrations of BDNF and NT-3 administered together, and low concentrations of both neurotrophins combined and administered with RA suppress otocyst cell numbers after 24 h in vitro. This suppressive response is induced by RA and NT-3, not by RA and BDNF. The suppressive or inhibitory influence of NT-3 and RA is the result of NT-3 binding to the low affinity receptor, p75NTR, not the result of RA increasing mRNA levels for the high affinity receptor, trkC. However, trk may act with p75NTR, as disruption of trk signalling alleviates the inhibitory response induced by NT-3 and RA. Our data suggest that various combinations and/or concentration gradients of these factors can differentially regulate inner ear development and hair cell excitability.

Patterns of synaptophysin expression during development of the inner ear in the chick.

The onset of active neural connections between the periphery and the central nervous system is integral to the development of sensory systems. This study presents patterns of synaptogenesis in the chick basilar papilla (i.e., cochlea) by examining the immunohistochemical expression of synaptophysin with a specific monoclonal antibody, SBI 20.10. The initial onset of synaptophysin expression occurs in nerve fibers and ganglion cell bodies at a time when neurites reach the basement membrane of the chick cochlea on embryonic day 6-7 (ED 6-7). By ED 8, synaptophysin positive fibers invade the neural side of the entire length of the cochlea, so that by ED 9-10, fibers are forming multiple terminals on the basolateral ends of retracting receptor or hair cells. In contrast, on the abneural side, immunoreactive terminals are seen first as small, punctate contacts and then as large, synaptophysin positive calyceal endings beneath short hair cells. These terminals are sparse during early development, more numerous by ED 17-19, but still incomplete after 2 weeks posthatching. In comparison, hair cells show synaptophysin immunoreactivity in both supra- and infranuclear regions by ED 11-12, a time when efferent innervation is incomplete. Thus, during development, synaptophysin is expressed at both synaptic and nonsynaptic sites, is relatively selective in its regional distribution, and is expressed in hair cells at a time when auditory function begins. Our results present a framework with which to understand the potential role of synaptophysin in early synaptogenesis of the cochlea.

Quantitative analysis of long-term survival and neuritogenesis in vitro: cochleovestibular ganglion of the chick embryo in BDNF, NT-3, NT-4/5, and insulin.

The dynamics of survival and growth were examined for cochleovestibular ganglion (CVG) cells maintained in long-term cultures. CVG cells were explanted from chick embryos after 90 h of incubation into a defined-medium containing BDNF, NT-3, or NT-4/5 and an insulin, transferrin, selenium, and progesterone supplement. Explant survival and neuritogenesis was measured for 23 to 24 days in vitro. All three neurotrophins prolonged CVG survival in a dose-dependent manner although insulin acted as a cofactor. In 0.872 microM insulin-containing medium the ED50 for BDNF and NT-3 was 100 pg/ml, whereas the ED50 for NT-4/5 was 600-1200 pg/ml. However, at later ages in vitro, survival decreased with concentrations of BDNF greater than 2 ng/ml. In insulin-free medium, concentrations of 5-200 ng/ml of BDNF or 30-200 ng/ml of NT-4/5 maintained the survival of explants at a rate that was equivalent to or less than the survival rate of cultures treated with insulin but not with neurotrophin. In contrast, NT-3-treated explants in insulin-free medium did not survive the duration of the experiment. Dose-dependent effects of BDNF and NT-3 on explant neuritogenesis were reflected as an initial delay in outgrowth, whereas NT-4/5 had no effect. Insulin regulation of neuritogenesis was suggested when outgrowth decreased in the presence of an antibody to the insulin receptor. These data suggest that while all three of these neurotrophins protect the CVG from death the long-term consequences of cofactors and certain dose levels should be considered when treating CVG cells in vivo.

Sensory cells of the chick cochlea express synaptophysin.

Previous evidence has shown expression of synaptophysin by sensory cells of the auditory and vestibular systems in the human, but not in other mammalian species. Using a monoclonal antibody, SBI 20.10, we investigated the expression of synaptophysin in the sensory cells of the avian cochlea. We present immunohistochemical data showing synaptophysin expression by cochlear hair cells in both late stage embryos and adult chickens. Immunoblotting of cochleae confirmed an antigen with an apparent molecular weight appropriate for synaptophysin that increases with development. Immunoreactivity in the apex of the cochlea occurred in hair cells on both neural and abneural sides, whereas immunoreactivity in the base of the cochlea was relegated to hair cells on the neural side. These observations indicate that, in the avian auditory system, like the human, synaptophysin is expressed in the sensory cells of both the embryo and adult.

Morphological and physiological development of vestibular hair cells in the organ-cultured otocyst of the chick.

The inner ear of the embryonic chick forms an oval-shaped sac or otocyst, on Embryonic Day 3, which contains presumptive sensory and support cells. After 3 weeks in organ culture the otocyst had sensory epithelia with an average of 325 +/- 41 hair cells. Using light and transmission electron microscopy most of these cells were identified morphologically as type II vestibular hair cells. Whole-cell tight-seal recordings, using potassium chloride-filled micropipetes, showed that mature cultured hair cells had four different types of K+ currents. These included: a voltage-gated delayed rectifier K+ current (IK), an inactivating K+ current (IA), a calcium-dependent K+ current (IK(Ca)), and a K+ inward rectifier (IIR). These currents were similar to those recorded from cristae ampullares cells isolated from 2- to 3-week-old posthatched chicks. We also determined the timing of K+ current acquisition in vitro. Initially, recordings showed that cells isolated from Embryonic Day 3 otocysts had no voltage-dependent outward currents at physiological membrane potentials. Eventually, K+ currents were acquired in the order of: IK and IIR after 9 days, IA after 12 days, and IK(Ca) after 17 days in vitro. In addition, recordings using cesium chloride-filled micropipetes showed that there were two types of inward currents that were elicited in response to membrane depolarizations. These two currents included a rapidly activating, noninactivating Ca2+ current and a tetrodotoxin-sensitive Na+ current. Both currents were elicited in hair cells grown in vitro for 13 days. Although INa was previously unreported in avians, both INa and ICa were also represented in hair cells isolated from the cristae ampullares of the posthatched chick. These results indicate that hair cells can acquire voltage-gated currents in vitro which are qualitatively similar to ionic currents found in crista ampullaris cells that differentiate in vivo. Thus, this organ culture system provides a means to study regulation of ionic currents in developing hair cells.

The acquisition during development of Ca-activated potassium currents by cochlear hair cells of the chick.

Voltage-clamp recordings were done on hair cells from a region of the chick's cochlea. In the adult, these cells have voltage-sensitive Ca currents and rapid, Ca-activated K currents that together support an electrical resonance, showing voltage oscillations at frequencies greater than 100 Hz. In embryos 14-days old (at one week before hatching) the same cells had a voltage-sensitive Ca current like that in adults, but a more slowly acting K current (of the delayed-rectifier type). In current-clamp they could generate only slowly repetitive action potentials. By two days before hatching, Ca-activated K currents were present. We suggest that the acquisition of Ca-activated K currents contributes to functional maturation of the chick's cochlea.

Auditory nerve rate-level functions for two-tone stimuli: possible relation to basilar membrane nonlinearity.

Rate versus level functions are presented for auditory-nerve fiber responses to best frequency (BF) tones presented alone and in the presence of a constant level suppressor tone. The paper focuses on units with sloping saturation rate functions for BF tones presented alone. Two-tone rate functions are generally parallel to BF functions at firing rates below those in the sloping saturation region of the BF function. At rates in the sloping saturation range the two-tone function grows with a greater slope than that of the BF function until the two functions meet. This result is discussed in terms of current knowledge of two-tone suppression on the basilar membrane.

A computational model for rate-level functions from cat auditory-nerve fibers.

A computationally tractable form of the rate-level model proposed by Sachs and Abbas (1974) is presented. The first stage of the model is a compressive nonlinearity whose input-output function is chosen to reflect current data on basilar-membrane displacement. The output of this nonlinearity is converted to driven discharge rate by the saturating nonlinearity originally used by Sachs and Abbas (1974). In fitting the model to data four model parameters are chosen to minimize the mean squared error between rate functions generated by the model and the data. With parameters chosen in this way, the model provides good fits to the range of rate-level shapes from flat saturations to sloping saturations. One important parameter in the model is the 'threshold for compression'. For low- and medium-spontaneous rate fibers with similar best frequencies (BFs), the minimum mean squared error compression threshold is roughly constant at about 30 dB above the thresholds of the most sensitive (high-spontaneous rate) fibers at that BF.

Transmission electron microscopic study of the saccule in the embryonic, larval, and adult toadfish Opsanus tau.

The development of the sensory epithelium of the saccular macula of Opsanus tau was studied with transmission electron microscopy. In the 10-12 somite embryo all cells of the newly formed otocyst are morphologically undefined, having an apically placed cilium with an underlying basal body and parabasal body. Junctional complexes are characterized primarily by tight junctions and a few desmosomes. In the 17-somite embryo the sensory cells begin to differentiate and are definable by the development of microvilli, which lack a cuticular plate. When the embryo has approximately 25-30 somites, ganglion cells differentiate and send their nerve processes toward the thin, disrupted basal lamina and the developing rhombencephalon. Desmosomes are more definable in the sensory regions at this age. As the myotomes begin forming (approximately 5-8 days before hatching), the nerves invade the sensory epithelium, and the developing sensory cells contain dense bodies surrounded by clear, membrane-bound vesicles. Clear synapticlike vesicles are also found throughout the infranuclear region of the sensory cells. However, afferent fibers lack a postsynaptic density. Three to 6 days prior to hatching a cuticular plate begins forming under the ciliary bundles and support and peripheral cells begin to morphologically differentiate. Two to 4 days before hatching the cuticular plate is well formed, desmosomes are numerous, afferent synapses are complete, and the sensory cells are in the upper two-thirds of the epithelium. Seven to 10 days after hatching, sensory cells have efferent synapses and ganglion cells and nerves show a myelin coat. These results suggest that sensory cells begin their development prior to VIIIth nerve innervation, although the orientation and pattern development of these cells may be related to the formation of the cuticular plate, desmosomes, afferent innervation, and basal lamina formation.

Gross and ultrastructural development of the saccule of the toadfish Opsanus tau.

The development of the saccule of the inner ear in the toadfish was studied using light and scanning electron microscopy. Development was studied from the early embryo (2-3 days postfertilization), when the otocyst first forms, to the early-aged juvenile when the development of the inner ear approximates that of the adult (4 weeks postfertilization). The ultrastructural features examined included the morphological sequence of ciliary bundle growth, the development of orientation patterns of the ciliary bundles, and the relation of the ultrastructural development to overall gross development. Gross development may be divided into four distinct morphological stages. Stage I encompasses the time from initial formation of the otocyst until the start of stage II, which is the stage when the pars inferior begins migrating ventrally. In stage III the pars inferior continues to elongate ventrally. Stage IV starts when the pars inferior elongates in a rostral and caudal direction. The ear attains its adult shape in stage IV. The differentiation of the sensory cells begins during stage I. During the early part of stage I, a small cilium is found on the apical surface of each cell throughout the otocyst. In the middle and late periods of stage I, a few microvillous buds add to the surface of the cells that already have a kinocilium. These early ciliary bundles are clustered on the rostral-ventral and caudal walls of the otocyst. There is no clear patterning to the orientation of these ciliary bundles. In stage II the ventral stretching of the labyrinth wall causes a spreading of the clustered bundles along the ventral and medial walls of the pars inferior. The orientation of the ciliary bundles has no distinct pattern. In stage III the orientations of the ciliary bundles appear adultlike, although there are so few ciliary bundles that it is difficult to make a definite determination. During stage IV, hair cells with an adultlike horizontal and vertical orientation pattern are found on the rostral and caudal sections of the saccular macula, respectively. The transition region lying between these areas has ciliary bundles with various orientations.

Development of the otolith in embryonic fishes with special reference to the toadfish, Opsanus tau.

The development of the saccular otolith and the otolithic membrane was studied in the toadfish (Opsanus tau) using scanning and transmission electron microscopy. Development of the saccular otolith and its otolithic membrane in Opsanus begins with the formation of the primordia in embryos of 17-20 somite age. Calcification of the primordia begins shortly afterwards, although increased calcium layering and formation of the otolithic membrane corresponds to the development of a group of cells lying peripheral to the developing sensory epithelium. These cells contain an abundance of rough endoplasmic reticulum.